Encyclopedia of Genetics: New Research 1536144517, 9781536144512
This 8 volume encyclopedia set presents important research on genetics. Some of the topics discussed herein include the
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Contents
Preface
Chapter 1
Speciation in Diatoms: Patterns,
Mechanisms, and Environmental Change
Abstract
Introduction
Phylogenetic Relationships
Reproduction in Diatoms
Studying Speciation in Diatoms
Patterns of Speciation in Diatoms
Marine Records
Freshwater Records
Reproductive Isolation
Mate Discrimination
Temporal Isolation
F1 Hybrid Sterility and Inviability
The Genetics of Reproductive Isolation
Diatoms: A Model System to Study the Consequences of Climatic Change on Speciation
Paleontological History
Events Leading to Speciation
Conclusion
Acknowledgments
References
Chapter 2
Sympatric Speciation of Island Plants: The Natural Laboratory of Lord Howe Island
Abstract
Introduction
Allopatric and Peripatric Speciation
Sympatric and Parapatric Speciation
Empirical Studies of Speciation
Approaches Studying Sympatric Speciation
Causes of Reproductive Isolation during Sympatric and
Parapatric Speciation
Speciation-with-Gene-Flow
Homoploid Hybrid Speciation
Polyploid Speciation
Lord Howe Island: A Model System for Studying Speciation
Lord Howe Island Formation and Climate
The Vegetation of LHI and the Impact of Human Settlement
Why Study Speciation on LHI?
Conclusion
References
Chapter 3
Speciation of Arabian Gazelles
Abstract
Introduction
Major Clades of Gazelles
Extant Species of Smaller Gazelles
Parallel, Adaptive Speciation of Species Pairs
A Taxonomic Review of the Genus Gazella
Conclusion
Acknowledgments
References
Chapter 4
Chromosome Plasticity, Adaptation and Speciation in Malaria Mosquitoes
Abstract
Introduction
1. A Role of Chromosome Polymorphism in Ecological Adaptations of Malaria Mosquitoes
2. Ecological Heterogeneity of Malaria Mosquitoes is a Major Challenge to Vector Control
3. Chromosome Plasticity in Adaptation and Evolution of Malaria Mosquitoes
3.1. Chromosome Arms Evolve at Different Rates
3.2. Genomic Landscapes and Nonuniform Rates of Inversions Fixation
3.3. Polymorphic Inversions and Parallel Adaptations
3.4. Nuclear Architecture and Chromosome Plasticity
4. Heterochromatin and Speciation in Malaria Mosquitoes
5. Significance of High-Quality Genome Assemblies for Studying Mosquito Evolution
Conclusion
Acknowledgments
References
Chapter 5
Transcriptional Differentiation Across the Four Subspecies of Drosophila mojavensis
Abstract
Introduction
Analysis of Transcriptional Divergence
Ecological Divergence in the D. mojavensis Transcriptome
Transcriptional Divergence and Speciation
Conclusion
Acknowledgments
References
Chapter 6
Evolution of Male Courtship Songs in the Drosophila buzzatii Species Cluster
Abstract
Introduction
Phylogeny of D. buzzatii Cluster Species
Host Cactus, Biogeography and Ecology of D. buzzatii Cluster Species
Description of Courtship Songs and Comparative Methods
Phylogenetic Reconstruction
Mapping Song Traits onto the Phylogeny
Differences in Courtship Song Components
Character Mapping Analysis of Courtship Song
Conclusion
Evolution of Courtship Songs
Acknowledgments
References
Chapter 7
Abstract
Introduction
Nasonia Wasps
Courtship and Mating Behaviour
Patterns of Mate Discrimination: No Choice Experiments
Choice Experiments: Implications of Male-Male Competition
Genetics of Mate Discrimination
Other Traits Involved in Prezygotic Isolation
Reinforcement
Chemical Communication and Their Role in Species Discrimination
Conclusion
Acknowledgments
References
Chapter 8
Abstract
Introduction
A Systems Approach to Postmating, Prezygotic Phenotypes
What System Underlies Postmating, Prezygotic Phenotypes?
Cell Signaling and Phosphorylation as Mechanisms Underlying Postmating, Prezygotic Phenotypes
Using Phosphorylation Studies to Identify Ligand and Receptor Genes Underlying Postmating, Prezygotic Reproductive Isolation
Pathways Leading to Postmating, Prezygotic Reproductive Isolation
Membrane Channels and Reproductive Isolation Caused by Protein-Protein Interactions Outside the Female Reproductive Tract
Species-Specific, Receptor-Gated Membrane Channels
Species-specific, Male-female Protein Interactions within the Female Reproductive Tract
Mechanisms Triggering Phosphorylation within the Female Reproductive Tract
Conclusion
Acknowledgments
References
Chapter 9
The Molecular and Evolutionary Basis of Hybrid Sterility: From Odysseus to Overdrive
Abstract
Introduction
Odysseus
Overdrive
Conclusion
References
Chapter 10
The Role of Transposable Elements in Speciation
Abstract
Introduction
Mechanism I: Genomic Disease
TE Suppression by Small RNAs: A Molecular Mechanism for Speciation
Evidence for Mechanism I
Drosophila Hybrid Dysgenesis
TE Derepression in Interspecific Animal Hybrids
TE Derepression in Plant Hybrids
Mechanism II: Mechanical Incompatibility
Evidence for Mechanism II
Structural Variation Caused by TE Activity
Structural Variations Associated with Reproductive Isolation
A TE Induced Structural Variant That Causes Isolation
Mechanism III: Genomic Resetting
Conclusion
Acknowledgments
References
Chapter 11
Hybrid Incompatibility: Are Cellular Processes the Battlefields of Genomic Conflict?
Abstract
1. Hybrid Incompatibility
2. Conflict-Driven HI
3. Prdm9 and Other Recombination-Association Genes
4. The Centromere and Associated Structures
5. A Call for the Evolutionary Genetics of the Cell
References
Chapter 12
Processes in Organic and Cultural Evolution
Abstract
Introduction
Individual Development and Evolution
The Problem of Evolutionary Change
Aim
Evolutionary Developmental Biology
Heterochrony
Environment-Independent Natural Selection
Population Biology
A Mathematical Analysis
An Application to Bacteria
Development and Evolution
The Relationship between Development and Evolution
The Regularity of Condensation
Condensation and Terminal Addition
An Explication of the Regular Temporal Structure of Evolution
Empirical Support
Complexity
Common Views
The Assessment of Complexity
Accelerating Evolution
A Self-Reinforcing Feedback Process
Complexity and the Tree of Life
Some Authors’ Views of Evolution
Complexity and a New View of the Evolutionary Process
Competition for Room in the Complexity Space
The Tree of Life
The Appearance of Mankind
Cultural Evolution
Universal Darwinism
Steps of Cultural Evolution
Analogies between Biological and Cultural Evolution
Natural Selection in Cultural Evolution
The Process of Heredity in Cultural Evolution
Complexity in Cultural Evolution
The Evolution of Verbal Language
Condensation and Terminal Addition
Processes in the Evolution of Written Language
Processes in Scientific Evolution
The Human Species – a Unique Species
Conclusion
References
Chapter 13
Natural Selection and Diabetes Mellitus
Abstract
Introduction
Materials and Methods
Results
Discussion
Conclusion
References
Chapter 14
Genetic Drift among Native People from South American Gran Chaco Region Affects Interleukin 1 Receptor Antagonist Variation
Abstract
Text
Background
Uniparental Y Chromosome Variability
X Chromosome Variability
Uniparental Mitochondrial Variability
Autosome Non Coding Variation
Autosome Coding Variation in the Interleukine 1 Receptor Antagonist
Discussion
References
Chapter 15
Alternative Splicing and Neurological Disorders: Focus on Parkinson’s Disease
Abstract
1. Introduction
2. The Alternative Splicing Machinery
3. Alternative Splicing in the Nervous System and Neurological Diseases
4. Genetics of Parkinson’s Disease: Basic Concepts
5. Alternative Splicing of Genes Involved in the Dominantly Inherited Forms of Parkinson’s Disease
5.1. SNCA
5.2. LRRK2
6. Alternative Splicing of Genes Involved in the Recessively Inherited forms of Parkinson’s Disease
6.1. PARK2
6.2. PINK1
6.3. DJ1
6.4. ATP13A2
6.5. PLA2G6
6.6. FBXO7
7. Alternative Splicing of Other PD Genes
7.1. SNCAIP
7.2. MAPT
7.3. MAO-B
7.4. SRRM2
8. Whole-Genome RNA Expression Studies Reveal Overall Alternative Splicing Changes in PD
9. Epigenetic Regulation of PD Alternative Splicing
Conclusion
Acknowledgments
References
Chapter 16
Tau Alternative Splicing in Alzheimer’s Disease
Abstract
RNA Splicing
Alzheimer’s Disease
Conclusion
References
Chapter 17
The Role of Splicing Factors in Cancer Prognosis and Treatment
Abstract
Introduction
General Splicing Mechanism
Splicing Regulatory Factors
Alternative Splicing and Cancer
Mutations in the Core Splicing Machinery Related to Cancer
Cancer-Related Changes in Splicing Factors Expression
Splicing Regulators That Have Been Considered Oncogenes or Tumor Suppressors
Regulation of Proto-Oncogenes by Splicing Factors
The Role of Alternative Splicing in Cancer Progression
Therapeutic Tools Developed to Regulate Splicing Factors Activity and Cancer
Conclusion
References
Chapter 18
Nonsense-Mediated Decay and Human Disease
Abstract
Introduction
NMD Mechanism
NMD Factors Related to Particular Diseases
Genetic Diseases Linked to NMD
Relevance of NMD in Human Condition
Therapies for NMD-Related Diseases
Conclusion
References
Chapter 19
Alternative Splicing Alterations in Alzheimer’s Disease
Abstract
Introduction
Alternative Splicing of Pivotal AD Genes
Tau
APP (Amyloid Precursor Protein) Gene
Alternative Splicing of Genes involved in Aβ Processing
BACE 1 (β-site APP Cleaving Enzyme 1)
Presenilins
Nicastrin
APH-1
AIDA-1
Clusterin
FE65 Family Proteins
Apolipoprotein E (APOE)
Alternative Splicing of Other Candidate Genes
Acetylcholinesterase
Tissue Transglutaminase
Estrogen Receptor α
Brain Derived Neurotrophic Factor (BDNF)
Tropomyosin Receptor Kinase B (TrkB)
Excitatory Amino Acid Transporter 2 (EAAT2)
Ion Channels
Synapsin
Regulator of Calcineurin (RCAN1)
Glial Fibrillar Acidic Protein (GFAP)
Ubiquilin-1
CDKN1A Interacting Zinc Finger Protein 1 (CIZ1)
Differentially Expressed in Normal and Neoplastic Tissues MAPK-Activating, Death Domain Protein (DENN/MADD)
Diazepam-Binding Inhibitor (DBI)
Conclusion
References
Chapter 20
Plant RNA-Binding Proteins Implicate mRNA Processing in Abscisic Acid (ABA) Responses
Abstract
1. Introduction
2. Splicing Factors
2.1. snRNP Factors
2.2. SR and hnRNPs Proteins
2.3. Other Splicing Regulators
3. Non-Splicing Proteins
3.1. CAP Binding Proteins
3.2. RNA Helicases and Pentatricopeptide Repeat Proteins
3.3. Other RNA-Binding Proteins
Conclusion
References
Chapter 21
Comprehensive Analyses of Alternative Exons in Neuronally Differentiated P19 cells
Abstract
Introduction
Materials and Methods
Cell Culture and RNA Purification
Exon Arrays
Signal Estimations
Extraction of Candidate Exons and Genes
Semi-Quantitative RT-PCR
GO Analyses, Pathway Analyses, and Text Mining
Results
Extraction of Alternative Exon Candidates
Semi-Quantitative RT-PCR of the Candidate Exons
GO Analyses of the Candidate Exons
Extraction of Potentially Important Exons by Pathway Analyses and Text Mining
Informatics and Experimental Analyses of Splicing Isoforms
Discussion
Acknowledgments
References
Chapter 22
Identification of Genuine Alternative Splicing Variants for Rare or Long-Sized Transcripts
Abstract
1. Introduction
2. Synthesis of Full-Length cDNA Using the Vector-Capping Method
3. Analysis of Full-Length cDNA Clones
3.1. Retina-Derived Full-Length cDNA Libraries
3.2. Characterization of Eye-Specific Genes
3.2.1. Aryl Hydrocarbon Receptor Interacting Protein-Like 1 (AIPL1)
3.2.2. LIM Momeobox 3 (LHX3)
3.2.3. Neural Retina-Specific Leucine Zipper Protein (NRL)
3.2.4. OTX2 Antisense RNA 1 (OTX2-AS1)
3.3. Characterization of Long-Sized Genes
3.3.1. Very-Long-Sized Genes
3.3.2. Golgin B1 (GOLGB1)
3.3.3. Filamin A, Alpha (FLNA)
3.3.4. Filamin B, Beta (FLNB)
3.3.5. Eyes Shut Homolog (EYS)
4. Full-Length cDNA Libraries Derived from Other Species
5. Problems on Identification of AS Variants
5.1. AS Variants of Rare or Long-Sized Genes
5.2. Synthesis of Full-Length cDNA
(1) Oligo(dT) Priming
(2) Reaction Conditions
(3) PCR Step
(4) Restriction Enzyme Treatment
(5) Size Fractionation
5.3. Vector-Capping Method
Conclusion
References
Chapter 23
An Epigenetic View on Alternative Splicing
Abstract
1. Introduction
2. Alternative Splicing and RNA Pol II Elongation Rate
3. Nucleosomes and Alternative Splicing
4. Chromatin Remodeling and Alternative Splicing
5. Histone Post-Translational Modifications and Alternative Splicing
6. Intragenic DNA Methylation and Alternative Splicing
Conclusion
References
Chapter 24
Alternative RNA Splicing and Regulation of Nitric Oxide Signaling
Abstract
Abbreviations
Introduction
NO/cGMP Pathway and Its Role in Physiology
Neuronal Nitric Oxide Synthase (nNOS)
Endothelial Nitric Oxide Synthase (eNOS)
NO-Inducible Heterodimeric Soluble Guanylyl Cyclase (sGC)
cGMP-Dependent Protein Kinase G (PKGI)
cGMP Phosphodiesterases (PDEs)
Cyclic Nucleotide Gated Ion Channels (CNGCs)
Conclusion
References
Chapter 25
Alternative Splicing by Analyzing a Human mRNA Diversity Using Data of FLJ Human cDNAs
Abstract
Introduction
Identification of Transcription Products Produced by Variety of Tne Human mRNA
Construction of the Custom DNA Microarray for Analysis of mRNA Variation
Analysis of Alternative Splicing Using NT2 Cells
Induced Neuronal Differentiation by Retinoic Acid
Analysis of Relationship between mRNA Variation by Alternative Splicing and the Gene Function
Conclusion
References
Chapter 26
Alternative Splicing in Human Immune System and Autoimmune Diseases
Abstract
Alternative Splicing and Immune Cell Function
Alternative Splicing and Cytokine Response
Dysregulated Splicing in Autoimmune Diseases
Summary
References
Chapter 27
Poly(ADP-Ribosyl)ation Regulates Alternative Splicing
Abstract
Introduction
1. Regulation of Alternative Splicing by PARylation
1.1. Interaction of hnRNPs with Poly(ADP-ribose)
1.2. Inhibition of RNA-Binding Ability of hnRNP Proteins by PARylation
1.3. Splicing Regulation by PARylation of hnRNPs
1.4. Splicing Regulation by PARylation of S/R Proteins
2. Regulation of Splicing by PARP1 during the Transcription Process
2.1. Coupling Transcription with Splicing by Epigenetic Mechanisms
2.2. Chromatin Remodeling by PARP1
2.3. Linking Transcription with Splicing by PARP1
3. Perspectives
Acknowledgments
References
Chapter 28
Patent Roadmap for the Biosensor Space
Abstract
Introduction
Personalized Medicine
History of Personalized Medicine
Landscape of Personalized Medicine
“An Ounce of Prevention Is Worth a Pound of Cure” -Benjamin Franklin
The Exigent Need for Personalized Medicine
“Declare the Past, Diagnose the Present, Foretell the Future” -Hippocrates
Bioinformatics
Wearable Technology
Lifestyle Wearables
Medical and Healthcare Wearables
Diagnostic Wearables: Real-time Medicine
Therapeutic Implantables/Embeddables: The Future of Personalized Medicine
The Cloud and Home Automation as the Next Frontier: Analytics and Provisioning
Internet of Things Ecosystem
Challenges
The Broad-Sweeping Implications
Value of Patents
Patent Strategy
Step 1
Step 2
Step 3
Hypothetical Valuation Case Study
Step 1
Step 2
Step 3
Impact of Alice and Mayo on IP
Patent Prosecution
IP Due Diligence
Claim Drafting in the Wake of Bilski, Mayo, Myriad & Alice
Licensing
Enforcement
FDA’s Role in the ‘App’ World
Reimbursement
Can Intellectual Property and Open Source Co-Habitate?
Big Data and Privacy: Big Challenge of the Future
HIPAA and the Age of Big Data: Old Laws New Problems
Acknowledgments
References
Chapter 29
Avoidant/Restrictive Food Intake Disorder in a Female Patient Affected by Marfan Syndrome
1Centre for Inherited Cardiovascular Diseases, IRCCS,
Fondazione Policlinico San Matteo, Pavia, Italy
2Fondazione Istituto Neurologico Nazionale C. Mondino,
IRCCS, Pavia, Italy
Abstract
Introduction
1. Marfan Syndrome
2. Association between Marfan Syndrome and Psychiatric Disorders
3. Selective Feeding
4. L.’s Clinical Case
Conclusion
References
Chapter 30
Optimizing Oil Production in B. napus by Gene Stacking: Transgenic Co-Expression of DGAT1 and Partially-Suppressed mtPDCK Cumulatively Enhance Seed Lipid Deposition
Abstract
Introduction
Materials and Methods
Plant Material and Target Genes
Production of Transgenic Lines
A. Thaliana DGAT1 Lines
T. majus DGAT1 and T. majus SDM DGAT1 Lines
mtPDCK Suppression Lines
Two-Gene Stack Lines
Field Trials
Analysis of Seed Oil Content and Composition in Transgenic Lines
Protein Analyses
Results
DGAT1 Lines
Repressed Mitochondrial PDCK Lines
Increases in Brassica napus Seed Oil Content in Transgenic Lines Expressing the Two-Gene Construct Containing the SDM T. majus DGAT1 Plus A/S B. napus mtPDCK
Discussion
Single Gene Transfers for Improvements in Oil Content
Gene Stacking
Gene Stacking for Improvements in Oil Composition
Gene Stacking for Improvements in Oil Content
Effects on Seed Weight
Effects of the 2-Gene Stack on Protein Content
Future Studies and Conclusion
Acknowledgments
References
Chapter 31
Periodontitis: A Common Oral Disease the Genetic Susceptibility
Abstract
1. Periodontitis - A Brief Introduction
2. Review of Genetic Studies on Periodontitis
2.1. Periodontitis Associated with Other Genetic Disorders
2.2. Inheritability of Periodontal Disease: Family Aggregation and Twin Studies
2.3. Heredity Mode and Heterogeneity: Segregation Analysis and Early Linkage Study
2.4. Complex Common Disease Associated with Single Nucleotide Polymorphisms
2.4.1. Candidate Gene Association Study
2.4.2. Cytokine
2.4.2.1. Interleukins (IL)
IL-1
IL-4 and IL-13
IL-6
IL-10
2.4.2.2. Tumor Necrosis Factor Alpha
2.4.2.3. Cell Surface Receptors
The Fc Gamma Receptor Genes
Toll-Like Receptor
Vitamin D Receptor
2.4.2.4. Matrix Metalloproteinases
2.4.2.5. Summary of Candidate Gene Study
2.4.3. Genome-Wide Association Study (GWAS)
2.5. Future Direction
3. Functional Study
3.1. Observation at the mRNA and Protein Level
3.2. Annotation
3.2.1. Computational Annotation
3.2.2. Experimental Annotation
3.3. Epigenetic Regulation of Gene Expression and Gene-Environment Interactions
Conclusion and Future Direction
Acknowledgments
References
Chapter 32
Genomic Imprinting and the Brain: Neuron-Specific Switching of Gene Expression at Imprinting Regions
Abstract
Introduction
Overview of the SNRPN-UBE3A Domain
Bipartite IC of the SNRPN-UBE3A Domain
Developmental Regulation of the Bipartite IC
The Role of U Exon Transcription in Maternal Silencing of Snrpn
Regulation of Paternal Expression of Ube3a-ATS
Neuron-Specific Chromatin Decondensation and Localization of Paternal Transcripts to Their Transcription Sites
Model of Paternal Ube3a Silencing by Ube3a-ATS Transcripts
Overview of the DLK1-DIO3 Domain
Overview of the GRB10 Domain
Conclusion
References
Chapter 33
Pharmacogenomics Focusing on Phase Two Metabolizing Enzymes
Abstract
Introduction
UGT Gene Super Family Encodes Critical Phase Two Metabolizing Enzymes
Future Perspective
References
Chapter 34
Sexual Dimorphism in Insect Longevity: Insights from Experimental Evolution
Abstract
Introduction
1. The Role of Natural Selection in Shaping Insect Longevity
2. Sexual Conflict and Evolution of Sexual Dimorphism in Longevity
3. Mechanistic Causes of Sex-Specific Ageing
3.1. Neuroendocrine Regulation of Ageing
3.2. Metabolic Differences between Sexes and Longevity Consequences
3.2.1. Nutritional Basis of Ageing
3.2.2. Stress Resistance and Life-Span
3.2.3. The Roles of Mitochondria
Acknowledgments
References
Chapter 35
Evidence of Natural and Sexual Selection Shaping the Size of Nuptial Gifts among a Single Bush-cricket Genus (Poecilimon; Tettigoniidae): An Analysis of Sperm Transfer Patterns
Abstract
Introduction
Materials and Methods
Species and Sites
Spermatophore Consumption Time, Male Body Mass and Spermatophore Mass
Sperm Transfer
Analysis
Sperm Transfer and Spermatophore Consumption
Relative Spermatophore Mass and Proportion of Sperm Transferred
Phylogenetic Independent Comparisons
Results
Spermatophore Consumption and Sperm Transfer
Relative Spermatophore Mass and Proportion of Sperm Transferred
Discussion
Acknowledgments
References
Chapter 36
Mate Choice Copying in Both Sexes of the Guppy: The Role of Sperm Competition Risk and Sexual Harassment
Abstract
Introduction
Materials and Methods
Study Species and Animal Maintenance
Experiment 1
Experiment 2
Statistical Analysis
Experiment 1
Experiment 2
Results
Experiment 1: Mate Choice Copying in Males and Females
Preference for Large Mating Partners
Changes in Individual Preferences
Experiment 2: Preference for Different Copying Situations
Discussion
Acknowledgments
References
Chapter 37
Sexual Selection under Parental Choice: Mating Strategies and Reproductive Success
Abstract
Introduction
Parent-Offspring Conflict over Mating
The Model of Parental Choice
Sexual Selection under Parental Choice in Human Societies
Sexual Selection under Parental Choice in Pre-Industrial Societies
Sexual Selection under Parental Choice in Post-Industrial Societies
In-Law Preferences
Male Mating Strategies
Polymorphism in Mate Choice
The Evolution of Rape
Circumventing Parental Choice
Circumventing Female Choice
Conclusion
References
Chapter 38
Melon Germplasm Characteristics, Diversity, Preservation and Uses
Abstract
1. General Introduction: The Need for Germplasm Banks
2. Collection, Conservation, Characterization and Evaluation of Melon Resources
3. Melon Genetic Resources Management
3.1. Morphological Markers
3.2. Biochemical Markers: Isozymes
3.3. Genetic Markers
3.4. Core Collections
4. Uses of Melon Germplasm
5. Challenges and Prospects
References
Chapter 39
Preservation and Characterization of Woodland Grape (Vitis vinifera ssp. sylvestris GMEL.) Genotypes of the Szigetköz, Hungary
Abstract
Introduction
Materials and Methods
Results
Discussion
Acknowledgment
References
Chapter 40
A Mini Review on Morphological and Genetic Diversity of Sweet (Prunus Avium L.) and Sour Cherry (Prunus Cerasus L.) Cultivars
Abstract
Introduction
Morphological Traits
Isoenzyme Analysis
Genetic Diversity Studies with Molecular Markers
Sweet Cherry
Sour Cherry
S-Allele Genotyping
Conclusion
References
Chapter 41
Research on Purple Seed Stain of Soybean: Germplasm Screening and Genetic Resistance
Abstract
Introduction
Disease Symptoms
The Causal Agents
Disease Development and Epidemiology
Disease Management
Soybean Germplasm Screening for Resistance to Purple Seed Stain
Evaluation of Soybean Cultivars for Reaction to Cercospora Leaf Blight
Genetic Resistance
Summary
Acknowledgments
References
Chapter 42
Somatic and Gonadal Tissue Cryopreservation: An Alternative Tool for the Germplasm Conservation in Wild Mammals
Abstract
1. Introduction
2. The Testicular Tissue
2.1. Tissue Source
2.2. Tissue Preparations
2.3. Cryopreservation Techniques and Evaluations
Freezing Protocols
Addition of Cryoprotectant Agents
Tissue Analysis
2.4. Use of Testicular Tissue in the Conservation Programs
3. The Ovarian Tissue
3.1. Tissue Source and Preparation
3.2. Cryopreservation Techniques and Evaluations
3.3. Current Applications of Ovarian Tissue in Conservation Programs
4. The Somatic Tissue
4.1. Tissue Sources
Skin Biopsy
Others Sources of Somatic Tissues
4.2. Tissue Preparations
4.3. Cryopreservation Techniques and Evaluations
4.4. Use of Somatic Tissue in the Conservation Programs
5. General Considerations
References
Biographical Sketch
Chapter 43
Mitochondrial Gene Diversity of the Mega-Herbivorous Species of the Genus Tapirus (Tapiridae, Perissodactyla) in South America and Some Insights on Their Genetic Conservation, Systematics and the Pleistocene Influence on Their Genetic Characteristics
Abstract
Introduction
Materials and Methods
Molecular Procedures
Data Analyses
Genetic Diversity, Linkage and Heterogeneity Analyses
Phylogenetic Analyses
Demographic Changes
Spatial Structure
Results
Gene Diversity, Genetic Heterogeneity and Phylogenetic Considerations for the Latin American Tapir Taxa
Demographic Changes in Latin American Tapirs
Spatial Structure
Discussion
Genetic Conservation of the Latin American Tapirs
Systematics of the Neotropical Tapirs and the Case of the Alleged “T. Kabomani”
The Impact of the Pleistocene Changes on the Phylogeography of T. pinchaque and T. terrestris
Acknowledgments
References
Chapter 44
Strategies for Gene Prospecting of Plants in Response to Drought and Salinity
Abstract
1. Introduction
2. Genes Coding for Proteins Involved in Reponse to Drought and Salinity in Plants
2.1. Basic Leucine Zipper Proteins and Abscisic Acid
2.2. Late Embryogenesis Abundant Proteins
2.3. Calcineurin B-like proteins
2.4. Trehalose-6-Phosphate Synthases and Phosphatases
2.5. Pyrroline-5-Carboxylate Synthases and Reductases
2.6. Aquaporins
3. Approaches to Identification of Genes in
Response to Drought and Salinity
3.1. Differential Display Reverse Transcription-Polymerase Chain Reaction
3.2. cDNA-Amplified Fragment Length Polymorphism
3.3. Microarray
3.4. Suppression Subtractive Hybridization
3.5. Next Generation Sequencing
4. Plant Genetic Transformation
4.1. Agrobacterium-Mediated Genetic Transformation
4.2. Biolistic-Mediated Genetic Transformation
Conclusion
References
Chapter 45
Clinical Evidence and the Genetic Effects of Traditional Chinese Medicine for the Management of Proteinuria in Patients with Diabetic Nephropathy
Abstract
Introduction
Clinical Evidence of TCM for Proteinuria in DN Patients
What Are the Results of Randomized Controlled Trials Studying the Anti-Proteinuric Effects of TCM in Patients with DN?
TCM as a Monotherapy
TCM Extract
Tripterygium Glycoside
Formulae
Tangjiang Shenkang (TJSK)
Qiyao Xiaoke (QYXK)
Combination Therapy with TCM and Western Medicine
Astraglus
Other TCM
What Do the Systematic Reviews and Meta-Analyses Report in This Area?
TCM Extract
Breviscapine
Tripterygium Glycoside
Ligustrazine
Ginkgo Biloba Extract
Puerarin
Individual Traditional Chinese Medicinal Herb
Astraglus
Formulae
Xuezhikang
Danhong
Tongxinluo
Other TCM
What Are the Limitations with Respect to this Clinical Evidence?
The Effects of TCM on DN at the Genetic Level
What Are the Effects of TCM on DN at the Genetic Level in Animal Experiments?
TCM Extract
Gypenosides
Breviscapine
Artemisinin
Individual Traditional Chinese Medicinal Herb
Astraglus
Formulae
Shenxiao Kang (SXK)
What Are the Effects of TCM on DN at the Genetic Level in Humans?
The Association between the Clinical Evidence and the Genetic Effects of TCM
What Is the Association?
How to Relate Them?
Summarization of Current Literature
Clinical Evidence
Genetic Effects
Conclusion
Conflict of Interest
Acknowledgments
References
Chapter 46
Biochemistry and Genetics of Ansamycin Antibiotics
Abstract
Introduction
Maytansinoids
Benzenoid Ansamycins
Ansamitocin
Geldanamycin
Macbecin
Herbimycin
Mycotrienins (Ansatrienins), Trierixin and Quinotrierixin
Cytotrienin
Trienomycin
Naphthalenoid Ansamycins
Rifamycin
Rifampicin
Rifabutin
Rifapentine
Salinisporamycin
Hygrocin
New Bioactive Ansamycin
Chaxamycins
Juanlimycins
References
Chapter 47
BRCA Gene Mutations Mediate Particularly High TNBC Risk by Defective Estrogen Signaling
Abstract
Introduction
Interactions between Endogenous Estrogens and Well Functioning, Wild BRCA Genes
Defective Estrogen Signaling Associated with BRCA Gene Mutations
Decreased ER-Alpha Expression in Cells with BRCA Gene Mutation
Decreased Estradiol-Liganded Transcriptional Activity of ERs in BRCA Gene Deficient Cells
Defensive Counteractions against BRCA Gene Defects
Increased Aromatase Activity Associated with BRCA Gene Mutation
Hyperestrogenism as Compensatory Mechanism in Women with BRCA Gene Mutation
Increased Ligand-Independent Transcriptional Activity in Cells with BRCA1 Mutation
Hormonal Risk Factors for Overall Breast Cancer and TNBC
Correlations between Menopausal Status and TNBC Risk
Correlations between Reproductive History and the Risk of Different Breast Cancer Subtypes
Conclusion
References
Chapter 48
Genotype-Phenotype Relationships in Language Processes in Rett Syndrome
Abstract
1. Introduction
1.1. Rett Syndrome (RTT)
1.2. Standard Form of Clinical Features
1.3. Clinical Features of Variant Form
2. Genotype Characterization
2.1. Mutations in MECP2 Gene
2.2. MECP2 Proteine’s Structure
3. Phenotypes with Gene Mutation MECP2
4. Language in Rett Syndrome
5. Methods
5.1. Participants
5.2. Material
Cognitive Measures
Linguistic Measures
Respiratory Evaluation
5.3. Procedure
5.4. Results
Conclusion
References
Chapter 49
Marfan Syndrome: A Rare and Sometime Very Disabling Genetic Disorder
Abstract
Introduction
History of Marfan Syndrome
Genetic Causation and Frequency of Marfan Syndrome
Clinical Findings in Patients with Marfan Syndrome
Diagnostic Studies for the Study of Patients Suspected of Having Marfan Syndrome
Treatment of Patients with Marfan Syndrome
Discussion
References
Chapter 50
Marfan Syndrome and Periodontitis
Abstract
1. Marfan Syndrome: A Systemic Connective Tissue Disorder
2. Periodontitis: An Oral Connective Tissue Disorder
3. TGF-( Is a Common Critical Factor in MFS and Periodontitis
4. Clinical Observation of Periodontitis in MFS Patients
Conclusion
Acknowledgments
References
Chapter 51
Combined Pectus Correction and Aortic Valve Sparing Root Replacement in Marfan Patients
Abstract
Most of Chest Wall Deformities Are Pectus Excavatum (PE)
Operative Techniques for Pectus Excavatum
Methods
Results
Discussion
Conclusion
References
Chapter 52
Severe Periodontitis in Marfan Syndrome
Abstract
1. Cardiac problems and Angiotensin II Receptor Blocker (ARB) in Marfan Syndrome
2. Periodontal Disease in Marfan Syndrome
3. ARB and Periodontal Disease
References
Chapter 53
Preimplantation Genetic Diagnosis for Marfan Syndrome
Introduction
References
Chapter 54
Anuran Cytogenetics: An Overview
Abstract
1. Cytogenetics, Taxonomy and Phylogenetic Inferences
1.1. Inferences of Evolutionary Changes in Diploid Number
1.2. Inferences Related to Chromosomal Morphology or Chromosomal Markers
2. Cytogenetics as a Helpful Taxonomic Tool
3. How to Study Frog Chromosomes
4. PcP190 Satellite DNA
4.1. Satellite DNA as Chromosome Markers
4.2. PcP190 satDNA in Engystomops: Sequences and Chromosome Locations
5. Sex Chromosomes in Anura
5.1. Sex Determination in Anurans: More Questions Than Answers
5.2. Sex Chromosome Differentiation
5.3. Sex Chromosome Turnover and Occasional Recombination between Sex Chromosomes
5.4. New Technologies in the Study of Sex Chromosomes in Anurans
6. B Chromosomes in Anura: General Properties and Exceptions
References
Chapter 55
Human Cytogenetic Biomonitoring
Abstract
An Introduction to Human Biomonitoring
Cytogenetic Biomarkers in Human Biomoniroting
Chromosomal Aberrations (CAs): Mechanisms and Methodology
Micronucleus (MN): Mechanisms and Methodology
Sister Chromatid Exchanges (SCEs): Mechanisms and Methodology
CAs, MN, SCEs and Risk Predictivity of Human Diseases
Application in Other Human Cells
Conclusion
Acknowledgments
References
Chapter 56
Complex Karyotypes and Their Detection in Hematologic Malignancies: The Ongoing Role for Classical Cytogenetics
Abstract
Introduction
Genomic Instability and Chromosomal Aberrations
Methods of Detection of Chromosomal Aberrations
Complex Karyotype in Selected Hematologic Malignancies
Myelodysplastic Syndrome (MDS)
Adult Acute Myeloid Leukemia (AML)
Chronic Lymphocytic Leukemia (CLL)
Mantle Cell Lymphoma (MCL)
Multiple Myeloma (MM)
Conclusion
References
Chapter 57
Natural Hybridization between Chromosomal Discrepant Species and the Role of Hybrid Speciation in the Genus Astyanax
Abstract
Introduction
Material and Methods
Results
Discussion
Acknowledgments
References
Chapter 58
Trichorhinophalangeal Syndrome (TRPS) Revisited
Abstract
Introduction
Phenotype of TRPS Type I, II and III Patients
TRPS Type I Patients
TRPS Type II Patients
TRPS Type III Patients
Genetics
Diagnosis
Clinical Diagnosis
Genetic Diagnosis
Differential Diagnosis
Treatment
Genetic Counseling
References
Chapter 59
Epigenetic Modifications and Developmental Origin of Health and Diseases (DOHaD)
Abstract
Abbreviations
1. Introduction
2. What Is Epigenetics?
2.1. DNA Methylation
2.2. MicroRNAs
3. Evidence from Human Cohorts
3.1. Fetal Programming Hypothesis
3.2. DNA Methylation and Fetal Programming Hypothesis
3.3. MicroRNAs and Fetal Programming Hypothesis
4. Evidence from Animal Experiments
4.1. DNA Methylation and Animal Experiments
4.1.1. Maternal Nutrition
4.1.2. Paternal Nutrition
4.2. MicroRNAs and Animal Experiments
4.2.1. Maternal Nutrition
4.2.2. Paternal Nutrition
5. Developmental Plasticity and Reversibility
Conclusion and Further Perspective
References
Chapter 60
Effects of Oxidative Stress on Epigenetic Mechanisms
Abstract
Abbreviations
1. Epigenetic Mechanisms
1.1. DNA Methylation
1.2. Histone Post-translational Modifications
1.3. Micro RNAs and Epigenetic
2. Oxidative Stress
3. Interactions between Oxidative Stress and Epigenetic Mechanisms
3.1. Oxidative Stress - DNA Methylation
3.2. Oxidative Stress - Histone Post-Translational Modifications
3.3. Oxidative Stress – miRNAs
Conclusion
References
Chapter 61
Epigenetic Modifications and Their Potential Role in Tumorigenesis
Abstract
Abbreviations
1. Introduction
2. The Dysregulation of Epigenetic Factors in Various Cancers
2.1. The Deregulation of DNA Methylation in Cancer
2.2. The Deregulation of Histone Lysine Methylation in Cancer
2.3. The Deregulation of Histone Lysine Demethylases in Cancer
3. Histone Lysine Acetylation in Cancer
3.1. The Deregulation of Histone Lysine Acetyltransferases in Tumor Proliferation and Metastasis
3.2. The Deregulation of Histone Lysine Deacetylases (KDACs) in Cancer
4. Histone Acetylation “Code” as a Diagnostic Marker for Cancer Progression
5. Epigenetic Modifications in Oncogenes and Tumor Suppressor Genes
6. Epigenetic Modifications in Cellular Signaling Pathways
6.1. NF-(B Signaling Pathway
6.2. STAT3 Signaling Cascade
6.3. Wnt/(-Catenin Signaling Pathway
7. Epigenetic Modification in Cancer Stem Cells
8. Epigenetic Modifications Associated with Cancer Chemo- and Radio-Resistance
9. Pharmacological Inhibitors of Epigenetic Modifications
Perspectives and Conclusion
Acknowledgment
Conflict of Interest
References
Chapter 62
Alzheimer’s Disease: An Insight from Epigenetic Perspective
Abstract
Abbreviations
1. Introduction
2. Pathogenesis of Alzheimer's Disease
2.1. Aβ Hypothesis
2.2. Tau Hypothesis
2.3. Oxidative Stress in Alzheimer’s Disease
2.4. Metal Dyshomeostasis in Alzheimer’s Disease
2.5. Mitochondrial Dysfunction in Alzheimer’s Disease
2.6. Neuroinflammation in Alzheimer’s Disease
2.7. Mutations and Polymorphisms in Alzheimer’s Disease
3. Epigenetic Changes in Alzheimer's Disease
3.1. DNA Methylation
APP Gene
BACE, PS1 and DNMT Genes
Neprilysin (NEP)
3.2. Histone Modifications
3.3. MicroRNAs
4. Future Perspectives
References
Chapter 63
Epigenetic Mechanisms in Cardiovascular Diseases
Abstract
Abbreviations
1. Epigenetic Mechanisms
2. Epigenetic Mechanisms in Heart Development
2.1. DNA Base Modifications
2.2. Histone Code
2.3. MiRNAs and lncRNAs
3. Epigenetic Mechanisms in CVDs
3.1. DNA Base Modifications
3.2. Histone Code
3.3. MiRNAs and lncRNAs
4. Cardiovascular Risk Factors and Epigenetics
5. Epigenetics-Based Therapeutic Strategies in CVDs
Conclusion
References
Chapter 64
Insights from Epigenomics Analysis of Sperm: Sperm Development and Male Infertility
Abstract
Abbreviations
Introduction
1. Sperm Cell Development
2. Epigenetic Reprogramming of Male Germ Cells
2.1. DNA Methylation
2.2. Histone Modifications
2.3. Chromatin Remodeling
2.4. Small RNAs
3. Epigenetic Dysregulation: Male Infertility
3.1. Male Infertility
3.2. Genomic Imprinting
3.3. DNA Methylation
4. Epigenetics in ART (Assisted Reproductive Techniques)
5. Epigenetic Make-Up of Induced Pluripotent Stem Cells/PGC from Adult Cells
Conclusion
References
Chapter 65
Epigenetics, Inflammation and Inflammatory Related-Diseases:
A General Look
Abstract
Abbreviations
1. Introduction
1.1. DNA Methylation and Inflammation
1.2. Histone Modifications and Inflammation
1.3. Non-Coding RNAs and Inflammation
1.3.1. Long Non-Coding RNAs
1.3.2. Micro RNAs
2. Epigenetics and Inflammatory Disorders
2.1. Inflammatory Bowel Disease (IBD)
2.2. Rheumatoid Arthritis (RA)
2.3. Systemic Lupus Erythematous (SLE)
2.4. Multiple Sclerosis (MS)
2.5. Diabetes
2.6. Bacterial Infections
3. Epigenetics, Inflammation and Cancer
Conclusion
References
Chapter 66
A New Molecular Approach to Obesity: Epigenetic Perspective
Istanbul University Cerrahpasa Medical Faculty,
Department of Medical Biology, Istanbul, Turkey
Abstract
Abbreviations
1. Obesity and Its Molecular Pathogenesis
2. Epigenetics: Basic Mechanisms
3. Obesity: From an Epigenetic Perspective
3.1. Programmed Obesity and the Epigenome
3.2. Epiobesigenic Genes
Conclusion
References
Chapter 67
Genetics and Evolution of Entomopathogenic Fungi
Abstract
Entomopathogens Role in Nature
Biological Control
Plants Manipulate Fungal Entomopathogens
The Ecology of Fungal Entomopathogens in the Rhizosphere
Entomopathogenic Fungi and Their Interactions with the Insect Immune System
Classification
Important Species
Mode of Action
Adhesion and Germination
Penetration
Reproduction and Replication
Isolation of Entomopathogenic Fungi
Isolation by Serial Dilution
Direct Isolation of Entomopathogenic Fungi from Insects
Isolation of Entomopathogenic Fungi from Soil
Morphological Characteristics of Entomopathogenic Fungi
Beauveria bassiana
Metarhizium anisopliae
Paecilomyces lilacinus
Nomurae rileyi (Metarhizium rileyi)
Lecanicillium (=Verticillium) lecanii
Isaria fumosorosea
Hirsutella thompsonii
Aschersonia aleyrodis
Entomophthora muscae
Molecular Identification of Entomopathogenic Fungi
Genetic Diversity among Entomopathogenic Fungal Strains
Beauveria spp
Metarhizium spp
Paecilomyces spp
Genes Involved in Virulence of Entomopathogenic Fungi
Molecular Phylogeny of Entomopathogenic Fungi and Their Biogeographic Implications
Evolution of Pathogenicity Genes in Entomopathogens
Contraction and Expansion of Different Gene Families
Entomopathogen Fungi Pathogenicity versus Insect-Host Defense
Genetic Improvement of Entomopathogenic Fungi for Insect Biocontrol
Future Trends
Conclusion
Acknowledgments
References
Biographical Sketch
Chapter 68
Mitochondrial Population Genetics Inferences about the Phylogeography and Systematics of the Tayra (Eira barbara, Mustelidae, Carnivora)
Abstract
Introduction
Materials and Methods
Samples
Molecular Analyses
Data Analyses
Genetic Diversity
Phylogenetics Analyses
Heterogeneity Analyses
Demographic Changes
Results
Genetic Diversity and Phylogenetic Inferences
Genetic Distances and Genetic Heterogeneity among Putative Morphological Subspecies of Eira barbara
Demographic Evolutionary Changes in the Tayra
Discussion
Genetic Diversity
Genetic Heterogeneity, Gene Flow and the Systematics of the Tayra
Temporal Splits in the Tayras
Acknowledgments
References
Chapter 69
The Precision Medicine and Precision Public Health Approaches to Cancer Treatment and Prevention: A Cross-Comparison
Abstract
From the Human Genome Project to the Precision Medicine Initiative
Definitions of Precision Medicine and Precision Public Health
Pharmacogenomic Interventions and Cost-Effectiveness
The Precision Medicine Approach to Lung Cancer
The Precision Medicine Approach to Breast Cancer
The Precision Medicine Approach to Colorectal Cancer
Taking Precision Medicine Beyond the Personalized Level
Public Health Interventions and Cost-Effectivness
The Precision Public Health Approach to Lung Cancer
The Precision Public Health Approach to Breast Cancer
The Precision Public Health Approach to Colorectal Cancer
Conclusion: Continuity and Emergence in Precision Health Approaches
References
Chapter 70
Neuropsychological Profile of People with Williams Syndrome (WS)
Abstract
Introduction
Intellectual Ability
Perceptual Skills (Visual and Auditory)
Oral Language
Memory Abilities
Executive and Attentional Functions
Discussion and Perspectives
References
Chapter 71
Preimplantation Genetic Diagnosis (PGD) for Chromosome Rearrangements
Introduction
Embryo Biopsy
Pregnancy History of Chromosome Rearrangement Carriers
PGD for Balanced Reciprocal Translocation
PGD for Robertsonian Translocation
PGD for Complex Chromosome Rearrangement (CCR)
Application of Comprehensive Chromosome Screening (CCS) into PGD for Balanced Chromosome Rearrangements
Conclusion
References
Biographical Sketch
A. Positions
B. Selected Peer-Reviewed Publications
Chapter 72
A Family-Based Association Study between the BDNF Gene and Attention Deficit Hyperactivity Disorder in Mexican Children and Adolescents
Abstract
Introduction
Material and Methods
Patients and Participants
Ethic Considerations
Clinical Evaluation
Genotyping Tests
Statistical Analysis
Results
Discussion
Conclusion
Acknowledgments
Financial Support
Statement of Interest
Ethical Standards
References
Chapter 73
Petroleum Microbiology and Genetics
Abstract
1. Introduction
1.1. Formation of Petroleum in Relation to Geological Processes
1.1.1. The Origins of Oil
1.1.2. Determinant Factors of the Petroleum’s Occurrence in Sedimentary Basins
1.1.2.1. Generating Rock
1.1.2.2. Reservoir Rocks
1.1.2.3. Conversion of Kerogen into Oil
1.1.3. Petroleum Migration and Accumulation: Primary and Secondary Migration
1.2. Petroleum Composition
1.3. General Concepts Concerning Oil Industry: Exploitation, Recovery and Refining Process
1.4. Petroleum and the Environment: Pollution and Environmental Impacts
1.4.1. Acidification of the Oceans
1.4.2. Oil Spills
1.5. The Microbiome of Oil Reservoirs
1.6. Petroleum Degrading Genes and Metabolic Pathways
1.7. Biofilms in the Petroleum Industry
1.8. Metagenomics for Marine Microbial Communities
1.9. Microbiologically-Influenced Corrosion (MIC)
1.10. Microbial Control Methods Related to Oil Industry
1.11. Bioremediation of Organic and Hydrocarbon Pollutants
1.12. Reservoirs Souring
Conclusion
References
Chapter 74
Estrogen Activated ERs are the Chief Genetic Safeguards of Somatic and Reproductive Health in Mammalians
Abstract
Introduction
Fundamental Roles of ER Signaling in the Physiology of Mammalians
ER-Signaling Is in Strong Mutual Correlation with DNA Stabilizer Genes and Their Protein Products
Correlations between ER-Signaling and the BRCA System for Safe DNA Replication
The Circular Functional Mainstream of Three Crucial Genetic Units Involved in Maintaining Genomic Stability
I. Transcriptional Activities of ESR1 Promoter Regions
II. Transcriptional Activities of BRCA1 Promoter Regions
III. Transcriptional Activities of CYP19 Aromatase Promoter Regions
Upregulative and Down-Regulative Crosstalk between ER-Alpha and BRCA1 in Physiologic and Malignant Cell Proliferations
Tumor Cells Exhibit Remnants of the Machinery of Genome Stabilization Providing Circumstances for Self-Directed Domestication and Elimination
Clinical Data on Tumor Responses to Antiestrogen Treatment
Experimental Data on Tumor Responses to Antiestrogen Treatment
Conclusion
References
Chapter 75
Motor Abilities are Related to Specific Genotypes in Rett Syndrome
Abstract
1. General Introduction
1.1. Genetics and Clinical Features of RTT
1.2. MECP2 Mutations and Functions
1.3. CDKL5 and FOXg1 Gene Mutations
1.4. Severity and Genotype-Phenotype Relationships
2. Motor Features in Rett Syndrome
2.1. Motor Characteristics According to Rett Syndrome Stages
2.2. Neurological and Muscle Symptoms
3. Methods
3.1. Participants
3.2. Material
3.2.1. Motor Scale
3.3. Results
Conclusion
References
Chapter 76
A Longitudinal Study on the Development of a Boy with Fragile X Syndrome: Developmental Trends and Adaptive Changes in a Single Case Report
Abstract
1. Introduction
2. Method
2.1. Subject
2.2. Measures
2.3. Procedure
3. Results
4. Discussion
References
Chapter 77
Cornelia De Lange Syndrome: A Review
Abstract
Introduction
Clinical Features
Neuropsychiatric and Neurosensory
Craniofacial
Musculoskeletal
Cardiovascular and Pulmonary Findings
Gastrointestinal
Genitourinary
Pathogenesis and Causes
Diagnosis
Treatment
Support Services
Conclusion
References
Further Reading
Chapter 78
Cognitive-Behavioral Phenotype in Klinefelter Syndrome (KS) and Other Sex Chromosomal Aneuplodies (SCAs):
Clinical Variability
Abstract
1. Introduction
2. Present Study
3. Results
3.1. Developmental and Clinical History
3.2. DSM-IV Diagnosis
3.3. IQ and Adaptive Behavior
3.3.1. SCL-90
3.3.2. SCQ
3.4. Bem Sex Role Inventory
4. Discussion
Conclusion
References
Chapter 79
Transcranial Direct Current Stimulation (tDCS) and Cognitive Empowerment for the Functional Recovery of Diseases with Chronic Impairment and Genetic Etiopathogenesis
Abstract
1. Introduction
2. Current Study
2.1. Methods
2.1.1. Participants
2.2. Procedure
2.3. tDCS Stimulation Protocol
2.4. Results
2.4.1. Comparative Analyses
2.4.2. Statistical Analyses
2.4.3. Production of Vowels
2.4.4. Production of Consonants
2.4.5. Production of Words
2.4.6. Quantitative EEG Analysis
Conclusion
References
Chapter 80
Control of Force and Timing during Unimanual and Bimanual Tapping Movements of Adolescents with Down Syndrome
Abstract
Introduction
Method
Participants
Apparatus and Measurements
Procedure
Statistical Analysis
Results
Discussion
Conclusion
References
Chapter 81
Management of Executive Function Following Assisted Cycling Therapy
in Adolescents with Down Syndrome
Abstract
Introduction
Methods
Results
Discussion
Strengths and Limitations
Conclusion
References
Biographical Sketch
Chapter 82
Endocrine Features and Management of Endocrine Problems in Down Syndrome
Abstract
Introduction
Length, Height and Growth Hormone Axis
Weight and Nutritional Habits
Puberty and Fertility
Thyroid Function and Disorders
Bone Mineral Status and Metabolism
Autoimmune Diseases
Conclusion
References
Chapter 83
Autosomal Dominant Polycystic Kidney Disease: Pathophysiology and Treatment
Abstract
1. Introduction
2. Primary Cilia and Cystic Kidney
3. Liver Cysts
4. Pancreatic Cysts
5. Diverticulitis
6. Pain
7. Hypertension
7.1. Endothelin
7.2. Primary Cilia and Nitric Oxide
7.3. Angiotensin
8. Left Ventricular Hypertrophy
9. Aneurysm
10. Therapeutic Treatments for Cardiovascular Complications
11. Modern Therapies to Halt Progression of Renal Cysts
11.1. cAMP
11.2. mTOR
11.3. EGFR
11.4. Other Potential Targets
Summary
References
Chapter 84
Hereditary Haemorrhagic Telangiectasia or Rendu-Osler-Weber Syndrome
Abstract
Introduction
HHT Is a Rare Disease in Spite of Its Autosomal Dominance
HHT Clinical Diagnostic Criteria
Clinical Symptoms
Epistaxis
Mucocutaneous Telangiectases
Pulmonary AVMs
Hepatic AVMs
Brain AVMs
Spinal AVMs
Gastrointestinal Bleeding
HHT Genetics
Structure of Endoglin and ALK1
The TGF-β Pathway
Functional Implications of Endoglin and ALK1
General Clinical Recommendations for Management of HHT Patients
Treatment
Epistaxis
Pulmonary AVMs
Liver AVMs
Brain AVMs
Pregnancy
Pediatrics and HHT
References
Chapter 85
Osteogenesis Imperfecta
Abstract
Introduction
Classification and Genetics
Biochemical Causes
Clinical Features
Fractures
Dental Abnormalities
Sclerae
Premature Deafness
Cardiovascular Problems
Excessive Sweating
Joint Laxity
Neurological Problems
Impaired Growth
Temperament
Life Expectancy
Radiology and Densitometry
Densitometry
Clinical Biochemistry
Differential Diagnoses
Management
Drug Therapy
Pain Relief
Orthopaedic Surgery
Surgery for Basilar Invagination
Deafness
Dental Care
Education
Acknowledgments
References
Chapter 86
Autosomal Dominant Disorders Associated to Breast Cancer
Abstract
Breast Cancer
Li-Fraumeni Syndrome
TP53
2. Cowden Syndrome
PTEN (Phosphatase and Tension Homolog)
3. Peutz-Jeghers Syndrome
STKT11 Gene
References
Chapter 87
Fragile X Syndrome
Abstract
Introduction
Fragile X Syndrome
FMRP (Fragile X Mental Retardation Protein)
Animal Models for FXS
Genetic Counseling
Newborn Screening (NBS)
Treatment
Conclusion
References
Chapter 88
Fragile X-Associated Primary Ovarian Insufficiency (FXPOI)
Abstract
Introduction
Defining the Disorder
Diagnosis and Genetic Counselling
Mechanisms Producing FXPOI
Mouse Models
Conclusion
References
Chapter 89
Fragile X-Associated Tremor/Ataxia Syndrome
Abstract
Introduction
Fragile X-Associated Tremor/Ataxia Syndrome
FXTAS Clinical and Cognitive Overview
FXTAS Neuroimaging and Neuropathological Profiles
Molecular Genetics Overview
FXTAS Treatment
Conclusion
References
Chapter 90
Psychiatric Aspects in Fragile X Syndrome and Related Phenotypes
Abstract
Introduction
Fragile X Syndrome
Autism Disorders
Hyperactivity Disorders
Mood Disorders
Anxiety Disorders
Premutation Carriers
Fragile X Associated Tremor Ataxia Syndrome (FXTAS)
Conclusion
References
Chapter 91
Clinical Features Associated with FMR1 Premutation Carriers
Abstract
Introduction
Clinical Features Associated with FMR1 Premutation Carriers
Neuropathy
Immune-Mediated Disorders
Migraines
Neurocognitive Features
Current Understanding of the Molecular Mechanisms Underlying Premutation-Associated Pathologies
Conclusion
References
Chapter 92
Genetic Counseling of FMR1
Abstract
Introduction
Genetic Counseling in FMR1-Associated Disorders
Assessment Risk Based on CGGs Expansion
Normal Range and Intermediate Alleles
Premutation Alleles
Full Mutation Alleles
Genetic Counseling Based on FMR1 Alleles
Intermediate Alleles
Premutation Alleles
Full Mutation Alleles
FMR1 Point Mutations/Deletions
Reproductive Options for Premutation/Full Mutation Carriers
Prenatal Diagnosis for FXS
Preimplantational Diagnosis for FXS
Offspring Renouncement and Adoption
Donor Germline Cell
FMR1 Testing Guidelines
Conclusion
References
Chapter 93
Treatment of Fragile X Spectrum: FXS, FXTAS and FXPOI
Abstract
Introduction
Fragile X Syndrome
The FMRP Protein and the Synapse
The Glutamate Receptor (mGluR) Hypothesis
The GABA Hypothesis
Therapeutic Approaches in FXS
MGluR5 Inhibitors
GABA-A Agonists
GABA-B Agonists
AMPA Agonists
NMDA Receptor Antagonists
Additional Treatments
Final Remarks
Fragile X-Associated Tremor-Ataxia Syndrome (FXTAS)
Fragile X-Associated Primary Ovary Insufficiency (Fxpoi)
Conclusion
References
Chapter 94
Privacy and Progress in Whole Genome Sequencing(
Executive Summary
Basic Ethical Principles for Assessing Whole Genome Sequencing
Recommendations
Strong Baseline Protections while Promoting Data Access and Sharing
Recommendation 1.1.
Recommendation 1.2.
Data Security and Access to Databases
Recommendation 2.1.
Recommendation 2.2.
Recommendation 2.3.
Consent
Recommendation 3.1.
Recommendation 3.2.
Recommendation 3.3.
Recommendation 3.4.
Facilitating Progress in Whole Genome Sequencing
Recommendation 4.1.
Recommendation 4.2.
Public Benefit
Recommendation 5.1.
Introduction
The Potential of Whole Genome Sequencing
The Challenge of Privacy
Terminology
The Promise of Whole Genome Sequencing
Privacy Concerns
Policy and Governance
Ethical Principles
Respect for Persons
Public Beneficence
Responsible Stewardship
Intellectual Freedom and Responsibility
Democratic Deliberation
Justice and Fairness
The Commission’s Process
About This Report
Work of Previous Commissions
Section 1. Ethical Principles
The Public Benefit of Whole Genome Sequencing
Privacy Concerns Raised by Whole Genome Sequencing
Privacy and the Law
The Meanings of Privacy
Restricted Access
Autonomy
The Value of Medical Privacy
Privacy in Whole Genome Sequencing
Privacy and the Ethical Principles
Reconciling Competing Ethical Claims
Newborn Screening
Conclusion
Section 2. Policy and Governance
Privacy Concerns about Genetic and Whole Genome Sequence Data
Current Sharing of Specimens and Whole Genome Sequence Data
U.S. Federal Agency Activity
International Biorepositories
Commercial Genetic Testing Companies
Privacy Regulations
U.S. Privacy Regulations
Identifying Information under HIPAA
International Approaches to Regulating Genetic Information
Legal Protections of Genetic and Whole Genome Sequence Data
Conclusion
Section 3. Analysis and Recommendations
Strong Baseline Protections While Promoting Data Access and Sharing
An Example of Computational Access
Recommendation 1.1.
Data Protections That Move with the Data
Recommendation 1.2.
Data Security and Access to Databases
Recommendation 2.1.
Recommendation 2.2.
Recommendation 2.3.
Consent
Recommendation 3.1.
BioVU: An Opt Out Database
Recommendation 3.2.
Recommendation 3.3.
Recommendation 3.4.
Facilitating Progress in Whole Genome Sequencing
Recommendation 4.1.
Recommendation 4.2.
Public Benefit
Recommendation 5.1.
Appendix I: Glossary of Key Terms
Appendix II: Genetic and Genomic Background Information
Understanding Basic Genetic Architecture
Mendelian Genetics
Why It Is Good to Have Two Copies of Each Chromosome
Gene-Environment Interactions
Genetic Variation
Sickle Cell Anemia
How Genetic Variants Translate into Disease
Sequencing Strategies
The Challenges of Analyzing Whole Genome Sequence Data and Identifying Disease Associations
Appendix III: Guest Presenters to the Commission Regarding Privacy and Whole Genome Sequencing
Appendix IV: U. S. State Genetic Laws*
End Notes
Chapter 95
Genetic Testing: Scientific Background for Policymakers(
Abstract
Introduction
Fundamental Concepts in Genetics
Cells Contain Chromosomes
Chromosomes Contain DNA
DNA Codes for Protein
Genotype Influences Phenotype
Genetic Tests
What Is a Genetic Test?
Policy Issues
How Many Genetic Tests are Available?
What Are the Different Types of Genetic Tests?
Policy Issues
The Genetic Test Result
Policy Issues
Characteristics of Genetic Tests
Analytical Validity
Clinical Validity
Clinical Utility
Policy Issues
Coverage by Health Insurers
Policy Issues
Regulation of Genetic Tests by the Federal Government
End Notes
Chapter 96
Evolution of Human Genome Analysis: Impact on Diseases Diagnosis and Molecular Diagnostic Labs
Abstract
1. Increased Human Genome Knowledge through Progress in Sequencing Technology
2. The Genetic Diagnosis: A Multi Technical Approach for Neurodevelopmental and Metabolic Disorders
2.1. Mendelian Single Gene Disorders: At the Mutation or Gene Level
2.1.1. Targeted Mutations
2.1.2. Targeted Gene
2.2. Genetic Disease Panels: The Intermediate Decision
2.3. The “Omic” Approach, a Comprehensive Interrogation
3. The Future of Molecular Diagnostic Lab: What Will Be Kept and What Will Bot
4. The Non-Targeted Genetic Strategy Generates New Ethical Challenges
4.1. Consent Form
4.2. Data Storage and Sharing
4.3. Incidental Findings
Conclusion and Future
Acknowledgments
References
Chapter 97
Improvements in HSV-1- Derived Amplicon Vectors for Gene Transfer
Abstract
HSV-1 Background
HSV-1 Epidemiology and Clinical Features
HSV-1 Virion Structure
HSV-1 Genome
HSV-1-Derived Vectors
Amplicon Vectors
Amplicon Vector Properties
Amplicon Production Systems
Helper DNA-Based Packaging Methods
Helper Virus-Based Packaging Methods
LaL and LaL(J HSV-1 Helper System
Further Improvements of Amplicon Vectors Technology
Elimination of Bacterial DNA from the Amplicon Genome
Modification of the Complementing Cell Lines
VCre4 Cell Line
Amplicon Vectors and Cancer
Amplicon Vectors and Vaccines
Amplicon Vectors and Behaviour
Amplicon Vectors and Neurodegenerative Diseases
Ataxias
Alzheimer Disease
Parkinson Disease
Narcolepsy
Amplicon Vectors and Nervous System Damage
Ischemia
Neurotoxicity
Neurotrophins
Neuropathic Pain
Acknowledgments
References
Chapter 98
Advances in Viral Genome Research of Papillomaviruses
Abstract
Introduction
Strategies Used to Identify Papillomavirus Genomes
PCR-Based Methods
Metagenomic Methods
A Model for Research in Papillomavirus: Advances in Bovine Papillomavirus Knowledge
References
Chapter 99
Synthetic Synthesis of Viral Genomes
Abstract
Introduction
Essential Elements of Viral Genomes
Essential Genes
Organization and Modulation of Viral Genomes
Methodologies for Genomic Manipulation
Genomic Sequencing
Genetic Manipulations
Synthesis of New Viral Genomes
Genetic Circuits
Monitoring the Expression of Genetic Elements
Colorimetric Assays
Green Fluorescent Protein (GFP)
Luminescence
Aptamers
Detection of Cellular mRNAs
Debugging
Examples of Projects on Synthetic Genomes
Prospects
Conflict of Interest
Acknowledgment
References
Chapter 100
Novel Bioinformatics Method to Analyze More Than 10,000 Influenza Virus Strains Easily at Once: Batch-Learning Self Organizing Map (BLSOM)
Abstract
Introduction
Materials and Methods
Viral Genome Sequences
Batch-Learning Self-Organizing Map (BLSOM)
Result
Oligonucleotide BLSOM for All Virus Genome Sequences
Oligonucleotide BLSOM for Influenza Virus Genomes
Diagnostic Tetranucleotides Responsible for Host-Dependent Separation
Chronological Changes Visualized for Human Viruses
H17N10 Strains Isolated from Bat
BLSOM for Codon Usage
BLSOM for Amino-Acid and Peptide Composition
Conclusion
Acknowledgments
References
Chapter 101
Oncogenes: Classification, Mechanisms of Activation, and Roles in Cancer Development Role of Oncogenes in Gynecological Pathology
Abstract
Introduction
Oncogenes in Gynecological Cancers
Breast Cancer
Ovarian Cancer
Endometrial Cancer
Cervical Cancer
Gestational Trophoblastic Disease
Oncogenes and Cancer Therapy
Oncogenes in Reproductive Endocrinology
Endometriosis
Conclusion
References
Chapter 102
The Classification, Mechanisms of Activation and Roles in Cancer Development of Oncogenes
Centro de Investigación Biomédica de Occidente, Instituto Mexicano del Seguro Social, Guadalajara, Jalisco, México
Abstract
Introduction
Oncogenes
Oncogene Classification
Growth Factors and Growth Factor Receptors
Transcription Factors
Chromatin Remodelers
Signal Transducers
Apoptosis Regulators
Chronic Inflammation as Activation Mechanism
Reprogramming of Metabolic Pathways in Cancer
Metabolic Activity in Cell Proliferation
PI3K/Akt/mTOR Signaling Pathway
Regulation of Glycolysis by HIF-1
Metabolic Changes and Cancer Cells
Cancer Initiation and Progression
Oncogenes as Therapeutic Targets
MicroRNA Genes
References
Chapter 103
Dicer and MicroRNAs: Oncomirs Are the Next Frontier of Oncogenes Affecting Cancer Etiology and Tumor Progression
Abstract
Introduction
MicroRNA and Cancer
MicroRNA-Based Therapies in Cancer
MicroRNA Biogenesis and Degradation
Let-7 Family
Let-7 in Cell Differentiation
Let-7 and Cancer: Ras, HMGA2 and the Cell Cycle
Regulation of Let-7
MiR-17-92 Family and Organization of miRNA Clusters
Regulation of the miR-17-92 Family and Its Roles Cancer
Causes of Alterations in miRNA Expression
Chromosomal Instability
Epigenetic Regulations
Abnormalities in miRNA Biogenesis Machineries
Drosha
DGCR8
Exportin-5
The Structure of Dicer
Dicer in Early Development
The Role of Dicer in Cancer
Regulation of Dicer
Argonautes
References
Chapter 104
The Role of the Epidermal Growth Factor Receptor as a Therapeutic Target in Glioblastoma and Other Malignancies
Abstract
Introduction
The EGFR and Its Role in Tumorigenesis
Epithelial-to-Mesenchymal Transition
Extracellular Matrix and Microenvironment
Mechanisms of Resistance to Current Therapy of GB
Conclusion
References
Chapter 105
Tumor Suppressors Involved in DNA Repair and Carcinoprevention
Abstract
Abbreviations
1. Introduction
2. Relationship between DNA Repair and Carcinogenesis
3. Signal Transduction of p53 and BRCA1 in DNA Repair Pathway
4. Protein Interaction and Functional Interplay between PTEN and p53
5. Perspective
Acknowledgments
Competing Interests Statement
References
Chapter 106
Molecular Pathogenesis of Neurofibromatosis Type 1
Abstract
Introduction
Epidemiology of NF1
Clinical Features of NF1
Skin Manifestations
Skeletal and Osseous Abnormalities
Vascular Lesions
Psychiatric Disorders and Cognitive Dysfunction
Rare Manifestations
RASopathies
Dysplasia and Developmental Defects
Molecular Genetics of NF1
Genetic Diagnosis
Tumors Associated with NF1
Disease Modifier Genes in NF1
Molecular Pathogenesis of NF1
Using Systems Approaches to Study NF1
Current Treatment and Disease Management
Conclusion
References
Chapter 107
Imaging of Plexiform Neurofibromas in Neurofibromatosis Type 1
Abstract
Introduction
Imaging Appearance of Plexiform Neurofibromas
Plexiform Neurofibroma Types
Volumetric Analysis
Cranial Nerves
Spinal Nerves
Neck, Mediastinum, Chest
Abdomen, Pelvis
Malignant Peripheral Nerve Sheath Tumors (MPNSTs)
Conclusion
References
Chapter 108
Surgical Treatment of Giant Neurofibromas
Abstract
Introduction
Surgical Techniques
Discussion
Conclusion
References
Chapter 109
Neurofibromatosis Type 1-Associated
Nervous System Tumors
and Current Treatment Strategies
Abstract
Introduction
Central Nervous System Tumors in Children with NF1
Low Grade Glioma: Clinical Presentation
Low Grade Glioma: Imaging Characteristics
Low Grade Glioma: Prognosis
Low Grade Glioma: Treatment Modalities
1. Role of Surgery
2. Radiation Therapy
3. Chemotherapy
4. Targeted Therapy
RAS-MAPK Pathway
Peripheral Nervous System Tumors in Children with NF1
Plexiform Neurofibromas: Clinical Presentation
Plexiform Neurofibromas: Imaging Characteristics
Plexiform Neurofibromas: Prognosis
Plexiform Neurofibromas: Treatment Modalities
1. Role of Surgery
2. Radiation Therapy
3. Chemotherapy
4. Targeted Therapy
a) mTOR Pathway
Interferon Treatment
b) Anti-angiogenic Treatment
c) Tyrosine Kinase Inhibitor Treatment
Malignant Peripheral Nerve Sheath Tumor (MPNST): Clinical Presentation
MPNST: Imaging Characteristics
MPNST: Prognosis
MPNST: Treatment Modalities
1. Role of Surgery
2. Radiation Therapy
3. Chemotherapy
4. Targeted Therapy
a) Tyrosine Kinase Inhibitor Treatment
b) Angiogenic Pathways
c) Cytoskeletal Remodeling
Conclusion
References
Chapter 110
Outcome Measures for Optic
Pathway Gliomas
Abstract
Introduction
Epidemiology of OPGs
Presentation of OPGs
Clinical Symptoms
Diagnostic Imaging
Pathology
Management of OPGs
Clinical Course
Surveillance
Treatment
Outcome Measures
Anatomical Outcomes (MRI)
Visual Outcomes
Alternative Outcome Measures
Diffusion and Perfusion Imaging
Diffusion Tensor Imaging
Positron Emission Tomography
Visual Evoked Potentials
Optical Coherence Tomography
Conclusion
References
Chapter 111
Neurofibromatosis Type 1 Bone
Disease: Diagnosis and Management
Abstract
Introduction
Spinal Manifestations : Scoliosis
Management of Scoliosis
Dural Ectasia
Long Bone Dysplasia
Leg Length Inequality/Segmental Hypertrophy
Sphenoid Wing Dysplasia
Pectus Deformities
Generalized Bone Bisorders
Osteopenia/Osteoporosis
Fractures
Bone Cysts; Non-Ossifying Fibromas
Others: Ossifying Subperiosteal Hematoma
Muscle Involvement in NF1
Conclusion
References
Chapter 112
Cognition and Behaviour in Neurofibromatosis Type 1: Pathogenesis and Emerging Therapies
Abstract
Introduction
1. NF1 Cognitive Phenotype
1.1. Intelligence
1.2. Visuospatial Function
1.3. General Language
1.4. Learning and Memory
1.5. Attention
1.6. Executive Functions
1.7. Academic Achievement and Learning Disability
2. The Social and Behavioral Phenotype
3. Neurodevelopmental Aspects
4. Pathogenesis of Cognitive Impairments
4.1. Human Neuroimaging Studies
4.1.1. Structural
4.1.2. Functional
4.2. Insights from Animal Models
5.0. Neurocognitive Therapies in NF1
Conclusion
References
Chapter 113
Communication Disorders Associated with Neurofibromatosis Type 1
Abstract
Introduction
NF1 and Manifestations of the Head and Neck
NF1 and Voice Disorders
NF1 and Resonance/Velopharyngeal Functioning
NF1 and Fluency
Speech Production Problems Associated with NF1
Expressive and Receptive Language and Intelligence in NF1
Gaps and Opportunities
Assessment
Conclusion
References
Appendix 1: Prevalence of Communication Disorders Observed in the Population of Individuals with NF1 and the General Population (in percent)
Appendix 2: Glossary of Terms
Chapter 114
Children with Neurofibromatosis Type 1: Functioning in the Classroom
Abstract
Introduction
The International Classification of Functioning, Disability and Health– Children and Youth version (ICF-CY) (WHO, 2007)
Conceptual Framework of the ICF-CY
The Ecological Validity of Cognitive Assessments Tools
Evaluation of Cognitive and Functional Problems
The Use of Virtual Reality Environments and Questionnaires to Evaluate Day-to-Day Cognitive Functioning
Virtual Reality (VR)
Functional Questionnaires
Describing the Attention Deficit profile of Children with NF1 Using a Virtual Classroom Environment (Gilboa, Rosenblum, Fattal-Valevski, Toledano-Alhadef, Rizzo, & Josman, 2011a; Gilboa, Rosenblum, Fattal-Valevski, Toledano-Alhadef, Rizzo, & Josman, 2...
The EF of Children with NF1 and its Relation to their Academic Skills
The Activity of Handwriting
The Handwriting Performance of Children with NF1 (Gilboa, Josman, Fattal-Valevski, Toledano-Alhadef, & Rosenblum, 2010a)
Jonathan: Case Study
Referral to Occupational Therapy
Major Findings and Discussion
Executive Function
Attention
Writing
Recommendations
Conclusion
References
Index
Blank Page
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GENETICS - RESEARCH AND ISSUES
ENCYCLOPEDIA OF GENETICS NEW RESEARCH (8 VOLUME SET) VOLUME
No part of this digital document may be reproduced, stored in a retrieval system or transmitted in any form or by any means. The publisher has taken reasonable care in the preparation of this digital document, but makes no expressed or implied warranty of any kind and assumes no responsibility for any errors or omissions. No liability is assumed for incidental or consequential damages in connection with or arising out of information contained herein. This digital document is sold with the clear understanding that the publisher is not engaged in rendering legal, medical or any other professional services.
GENETICS - RESEARCH AND ISSUES Additional books and e-books in this series can be found on Nova’s website under the Series tab.
GENETICS - RESEARCH AND ISSUES
ENCYCLOPEDIA OF GENETICS NEW RESEARCH (8 VOLUME SET) VOLUME
HEIDI CARLSON EDITOR
Copyright © 2019 by Nova Science Publishers, Inc. All rights reserved. No part of this book may be reproduced, stored in a retrieval system or transmitted in any form or by any means: electronic, electrostatic, magnetic, tape, mechanical photocopying, recording or otherwise without the written permission of the Publisher. We have partnered with Copyright Clearance Center to make it easy for you to obtain permissions to reuse content from this publication. Simply navigate to this publication’s page on Nova’s website and locate the “Get Permission” button below the title description. This button is linked directly to the title’s permission page on copyright.com. Alternatively, you can visit copyright.com and search by title, ISBN, or ISSN. For further questions about using the service on copyright.com, please contact: Copyright Clearance Center Phone: +1-(978) 750-8400 Fax: +1-(978) 750-4470 E-mail: [email protected]. NOTICE TO THE READER The Publisher has taken reasonable care in the preparation of this book, but makes no expressed or implied warranty of any kind and assumes no responsibility for any errors or omissions. No liability is assumed for incidental or consequential damages in connection with or arising out of information contained in this book. The Publisher shall not be liable for any special, consequential, or exemplary damages resulting, in whole or in part, from the readers’ use of, or reliance upon, this material. Any parts of this book based on government reports are so indicated and copyright is claimed for those parts to the extent applicable to compilations of such works. Independent verification should be sought for any data, advice or recommendations contained in this book. In addition, no responsibility is assumed by the publisher for any injury and/or damage to persons or property arising from any methods, products, instructions, ideas or otherwise contained in this publication. This publication is designed to provide accurate and authoritative information with regard to the subject matter covered herein. It is sold with the clear understanding that the Publisher is not engaged in rendering legal or any other professional services. If legal or any other expert assistance is required, the services of a competent person should be sought. FROM A DECLARATION OF PARTICIPANTS JOINTLY ADOPTED BY A COMMITTEE OF THE AMERICAN BAR ASSOCIATION AND A COMMITTEE OF PUBLISHERS. Additional color graphics may be available in the e-book version of this book.
Library of Congress Cataloging-in-Publication Data
ISBN: HERRN
Published by Nova Science Publishers, Inc. † New York
CONTENTS VOLUME 1 Preface Chapter 1
Chapter 2
xv Speciation in Diatoms: Patterns, Mechanisms, and Environmental Change Joshua T. Cooper and John P. Masly Sympatric Speciation of Island Plants: The Natural Laboratory of Lord Howe Island Alexander S. T. Papadopulos, William J. Baker and Vincent Savolainen
Chapter 3
Speciation of Arabian Gazelles Hannes Lerp, Torsten Wronski, Thomas M. Butynski and Martin Plath
Chapter 4
Chromosome Plasticity, Adaptation and Speciation in Malaria Mosquitoes Igor V. Sharakhov
Chapter 5
Chapter 6
Transcriptional Differentiation Across the Four Subspecies of Drosophila mojavensis Luciano M. Matzkin and Therese A. Markow Evolution of Male Courtship Songs in the Drosophila buzzatii Species Cluster Cássia C. Oliveira, Maura H. Manfrin, Fábio de M. Sene and William J. Etges
Chapter 7
Prezygotic Isolation in the Parasitoid Wasp Genus Nasonia Maartje C. W. G. Giesbers, Sylvia Gerritsma, Jan Buellesbach, Wenwen Diao, Bart A. Pannebakker, Louis van de Zande, Thomas Schmitt and Leo W. Beukeboom
Chapter 8
Where to Look for Speciation Genes When Divergence Is Driven by Postmating, Prezygotic Isolation Jeremy L. Marshall
1
37
59
83
119
135
161
189
vi Chapter 9
Contents The Molecular and Evolutionary Basis of Hybrid Sterility: From Odysseus to Overdrive Nitin Phadnis and Harmit S. Malik
Chapter 10
The Role of Transposable Elements in Speciation Eric T. Watson and Jeffery P. Demuth
Chapter 11
Hybrid Incompatibility: Are Cellular Processes the Battlefields of Genomic Conflict? Norman A. Johnson
203 223
247
Chapter 12
Processes in Organic and Cultural Evolution Börje Ekstig
257
Chapter 13
Natural Selection and Diabetes Mellitus Svetlana Shtandel
301
Chapter 14
Genetic Drift among Native People from South American Gran Chaco Region Affects Interleukin 1 Receptor Antagonist Variation Cecilia Inés Catanesi and Laura Angela Glesmann
331
VOLUME 2 Chapter 15
Alternative Splicing and Neurological Disorders: Focus on Parkinson’s Disease Velia D’Agata, Valentina La Cognata and Sebastiano Cavallaro
345
Chapter 16
Tau Alternative Splicing in Alzheimer’s Disease Gonzalo Emiliano Aranda-Abreu, María Elena Hernández Aguilar, Fausto Rojas Durán, Sonia Lilia Mestizo Gutiérrez and Jorge Manzo Denes
373
Chapter 17
The Role of Splicing Factors in Cancer Prognosis and Treatment Rebeca Martínez-Contreras and Nancy Martínez-Montiel
383
Chapter 18
Nonsense-Mediated Decay and Human Disease Nancy Martínez-Montiel and Rebeca Martinez-Contreras
407
Chapter 19
Alternative Splicing Alterations in Alzheimer’s Disease T. A. Ishunina and D. F. Swaab
427
Chapter 20
Plant RNA-Binding Proteins Implicate mRNA Processing in Abscisic Acid (ABA) Responses Raquel F. Carvalho, Vera S. Nunes and Paula Duque
453
Comprehensive Analyses of Alternative Exons in Neuronally Differentiated P19 cells Hitoshi Suzuki and Toshifumi Tsukahara
475
Identification of Genuine Alternative Splicing Variants for Rare or Long-Sized Transcripts Seishi Kato
495
Chapter 21
Chapter 22
Contents
vii
Chapter 23
An Epigenetic View on Alternative Splicing Gabriele A. Fontana, Aurora Rigamonti and Silvia M. L. Barabino
515
Chapter 24
Alternative RNA Splicing and Regulation of Nitric Oxide Signaling Iraida G. Sharina
533
Chapter 25
Alternative Splicing by Analyzing a Human mRNA Diversity Using Data of FLJ Human cDNAs Takao Isogai and Ai Wakamatsu
551
Alternative Splicing in Human Immune System and Autoimmune Diseases Xiang Guo
569
Chapter 26
Chapter 27
Poly(ADP-Ribosyl)ation Regulates Alternative Splicing Yingbiao Ji and Alexei V. Tulin
583
Chapter 28
Patent Roadmap for the Biosensor Space Mohamed C. Azeez, Unisha Patel and Dennis Fernandez
595
Chapter 29
Avoidant/Restrictive Food Intake Disorder in a Female Patient Affected by Marfan Syndrome A. P. Verri, A. Serio, M. Grasso, I. Brega and F. Clerici
Chapter 30
Optimizing Oil Production in B. napus by Gene Stacking: Transgenic Co-Expression of DGAT1 and Partially-Suppressed mtPDCK Cumulatively Enhance Seed Lipid Deposition Elizabeth-France Marillia, Tammy Francis and David C. Taylor
Chapter 31
Periodontitis: A Common Oral Disease the Genetic Susceptibility Ying Zheng, You-Qiang Song and W. Keung Leung
Chapter 32
Genomic Imprinting and the Brain: Neuron-Specific Switching of Gene Expression at Imprinting Regions Masamitsu Eitoku and Hidenori Kiyosawa
Chapter 33
Pharmacogenomics Focusing on Phase Two Metabolizing Enzymes Kung-Hao Liang
649
657 689
715 739
VOLUME 3 Chapter 34
Chapter 35
Sexual Dimorphism in Insect Longevity: Insights from Experimental Evolution Jelica Lazarević, Biljana Stojković and Nikola Tucić Evidence of Natural and Sexual Selection Shaping the Size of Nuptial Gifts among a Single Bush-cricket Genus (Poecilimon; Tettigoniidae): An Analysis of Sperm Transfer Patterns J. McCartney, M. A. Potter, A. W. Robertson, K-G. Heller and D. T. Gwynne
747
777
viii Chapter 36
Chapter 37
Contents Mate Choice Copying in Both Sexes of the Guppy: The Role of Sperm Competition Risk and Sexual Harassment Claudia Zimmer, Alexandros S. Gavalas, Benjamin Kunkel, Janina Hanisch, Sara Martin, Svenja Bischoff, Martin Plath and David Bierbach Sexual Selection under Parental Choice: Mating Strategies and Reproductive Success Menelaos Apostolou and Marianna Zacharia
Chapter 38
Melon Germplasm Characteristics, Diversity, Preservation and Uses C. Mallor and A. Díaz
Chapter 39
Preservation and Characterization of Woodland Grape (Vitis vinifera ssp. sylvestris GMEL.) Genotypes of the Szigetköz, Hungary Gizella Jahnke, Zóra Annamária Nagy, Gábor Koltai, Edit Hajdu and János Májer
Chapter 40
Chapter 41
Chapter 42
Chapter 43
Chapter 44
A Mini Review on Morphological and Genetic Diversity of Sweet (Prunus Avium L.) and Sour Cherry (Prunus Cerasus L.) Cultivars Ioannis Ganopoulos, Panagiotis Madesis, Filippos A. Aravanopoulos, Athanasios Tsaftaris, Thomas Sotiropoulos and Konstantinos Kazantzis
795
813 825
845
859
Research on Purple Seed Stain of Soybean: Germplasm Screening and Genetic Resistance Shuxian Li, Pengyin Chen, Bo Zhang and Grover Shannon
871
Somatic and Gonadal Tissue Cryopreservation: An Alternative Tool for the Germplasm Conservation in Wild Mammals Alexsandra Fernandes Pereira, Alexandre Rodrigues Silva, Gabriela Liberalino Lima and Andréia Maria da Silva
881
Mitochondrial Gene Diversity of the Mega-Herbivorous Species of the Genus Tapirus (Tapiridae, Perissodactyla) in South America and Some Insights on Their Genetic Conservation, Systematics and the Pleistocene Influence on Their Genetic Characteristics Manuel Ruiz-García, Armando Castellanos, Luz Agueda Bernal, Diego Navas, Myreya Pinedo-Castro and Joseph Mark Shostell Strategies for Gene Prospecting of Plants in Response to Drought and Salinity Deyvid Novaes Marques, Nicolle Louise Ferreira Barros, Diehgo Tuloza da Silva, Fabiano Melo de Brito and Claudia Regina Batista de Souza
909
959
Contents Chapter 45
Clinical Evidence and the Genetic Effects of Traditional Chinese Medicine for the Management of Proteinuria in Patients with Diabetic Nephropathy Xiang Tu, XueFeng Ye, Quan Hu, ChunGuang Xie, ChengShi He, Ming Chen, James B. Jordan and Sen Zhong
ix
993
Chapter 46
Biochemistry and Genetics of Ansamycin Antibiotics Amit Kumar Jha and Jae Kyung Sohng
Chapter 47
BRCA Gene Mutations Mediate Particularly High TNBC Risk by Defective Estrogen Signaling Zsuzsanna Suba
1045
Genotype-Phenotype Relationships in Language Processes in Rett Syndrome Alessandra Falzone, Antonio Gangemi and Rosa Angela Fabio
1061
Marfan Syndrome: A Rare and Sometime Very Disabling Genetic Disorder Henry J. Mankin and Keith P. Mankin
1079
Chapter 48
Chapter 49
Chapter 50
Marfan Syndrome and Periodontitis Jun-ichi Suzuki and Yasunobu Hirata
Chapter 51
Combined Pectus Correction and Aortic Valve Sparing Root Replacement in Marfan Patients Jean-Philippe Verhoye and Jacques Tomasi
1017
1093
1099
Chapter 52
Severe Periodontitis in Marfan Syndrome Naoto Suda
1107
Chapter 53
Preimplantation Genetic Diagnosis for Marfan Syndrome Anver Kuliev and Svetlana Rechitsky
1113
VOLUME 4 Chapter 54
Anuran Cytogenetics: An Overview Cíntia Pelegrineti Targueta, Stenio Eder Vittorazzi, Kaleb Pretto Gatto, Daniel Pacheco Bruschi, Ana Cristina P. Veiga-Menoncello, Shirlei M. Recco-Pimentel and Luciana Bolsoni Lourenço
1117
Chapter 55
Human Cytogenetic Biomonitoring Armanda Teixeira-Gomes, Solange Costa and João Paulo Teixeira
1157
Chapter 56
Complex Karyotypes and Their Detection in Hematologic Malignancies: The Ongoing Role for Classical Cytogenetics Helena Urbankova
1177
x Chapter 57
Contents Natural Hybridization between Chromosomal Discrepant Species and the Role of Hybrid Speciation in the Genus Astyanax Francisco de Menezes Cavalcante Sassi, Snaydia Viegas Resende, Rubens Pazza and Karine Frehner Kavalco
Chapter 58
Trichorhinophalangeal Syndrome (TRPS) Revisited N. Selenti, M. Tzetis, E. Tsoutsou and H. Fryssira
Chapter 59
Epigenetic Modifications and Developmental Origin of Health and Diseases (DOHaD) Jia Zheng and Xinhua Xiao
1193
1203
1211
Chapter 60
Effects of Oxidative Stress on Epigenetic Mechanisms Yildiz Dincer and Onur Baykara
1225
Chapter 61
Epigenetic Modifications and Their Potential Role in Tumorigenesis Muthu K. Shanmugam, Frank Arfuso and Gautam Sethi
1241
Chapter 62
Alzheimer’s Disease: An Insight from Epigenetic Perspective Yildiz Dincer and Zeynep Sezgin
1291
Chapter 63
Epigenetic Mechanisms in Cardiovascular Diseases Mehmet Güven and Bahadir Batar
1323
Chapter 64
Insights from Epigenomics Analysis of Sperm: Sperm Development and Male Infertility Dheepa Balasubramanian, Eisa Tahmasbpour and Ashok Agarwal
1361
Epigenetics, Inflammation and Inflammatory Related-Diseases: A General Look Müge Sayitoğlu and Yücel Erbilgin
1383
Chapter 65
Chapter 66
A New Molecular Approach to Obesity: Epigenetic Perspective Burcu Bayoglu and Mujgan Cengiz
1419
VOLUME 5 Chapter 67
Genetics and Evolution of Entomopathogenic Fungi A. Garcia, M. Michel, S. Villarreal, F. Castillo, E. Osorio, M. T. García-Ruiz, F. Veana, A. C. Flores and R. Rodríguez
Chapter 68
Mitochondrial Population Genetics Inferences about the Phylogeography and Systematics of the Tayra (Eira barbara, Mustelidae, Carnivora) Manuel Ruiz-García, Nicolás Lichilín-Ortíz, Yolanda Mejia, Juan Manuel Ortega and Joseph Mark Shostell
1491
The Precision Medicine and Precision Public Health Approaches to Cancer Treatment and Prevention: A Cross-Comparison Stephen M. Modell, Sharon L. R. Kardia and Toby Citrin
1525
Chapter 69
1447
Contents Chapter 70
Chapter 71
Chapter 72
Neuropsychological Profile of People with Williams Syndrome (WS) Anne-Sophie Pezzino, Nathalie Marec-Breton and Agnès Lacroix
1547
Preimplantation Genetic Diagnosis (PGD) for Chromosome Rearrangements Chun Kyu Lim
1569
A Family-Based Association Study between the BDNF Gene and Attention Deficit Hyperactivity Disorder in Mexican Children and Adolescents Alan López-López, Miriam Margot Rivera-Angles, Carlos Alfonso Tovilla-Zárate, Roberto Molina-Solís, Araceli Valencia-Hernández, Luis Gómez-Valencia, Thelma Beatriz González-Castro, Isela E Juárez-Rojop, María Lilia López-Narváez, Ana Fresan and Yazmin Hernández-Díaz
Chapter 73
Petroleum Microbiology and Genetics Joana Montezano Marques, Carla Thais Moreira Paixão, Gabriel Villar Monte Palma Pantoja, Artur Luiz da Costa da Silva and Diego Assis das Graça
Chapter 74
Estrogen Activated ERs are the Chief Genetic Safeguards of Somatic and Reproductive Health in Mammalians Zsuzsanna Suba
Chapter 75
Motor Abilities are Related to Specific Genotypes in Rett Syndrome R. A. Fabio, T. Caprì, M. Lotan, G. E. Towey and G. Martino
Chapter 76
A Longitudinal Study on the Development of a Boy with Fragile X Syndrome: Developmental Trends and Adaptive Changes in a Single Case Report María Auxiliadora Robles-Bello, Nieves Valencia-Naranjo and David Sánchez-Teruel
Chapter 77
Cornelia De Lange Syndrome: A Review Elizabeth A. M. Frost
Chapter 78
Cognitive-Behavioral Phenotype in Klinefelter Syndrome (KS) and Other Sex Chromosomal Aneuplodies (SCAs): Clinical Variability A. P. Verri, C. D’Angelo, A. Cremante, F. Clerici, A. Mauri and C. Castelletti
Chapter 79
xi
Transcranial Direct Current Stimulation (tDCS) and Cognitive Empowerment for the Functional Recovery of Diseases with Chronic Impairment and Genetic Etiopathogenesis Antonio Gangemi, Tindara Caprì, Rosa Angela Fabio, Paola Puggioni, Alessandra Maria Falzone and Gabriella Martino
1589
1601
1633 1649
1669
1683
1697
1715
xii
Contents VOLUME 6
Chapter 80
Chapter 81
Chapter 82
Chapter 83
Chapter 84
Control of Force and Timing during Unimanual and Bimanual Tapping Movements of Adolescents with Down Syndrome Nobuyuki Inui and Junya Masumoto Management of Executive Function Following Assisted Cycling Therapy in Adolescents with Down Syndrome Shannon D. R. Ringenbach, Simon D. Holzapfel, Madeline Richter and Jay L. Alberts Endocrine Features and Management of Endocrine Problems in Down Syndrome Stefano Stagi, Elena Sandini, Elisabetta Lapi, Perla Scalini and Maurizio de Martino Autosomal Dominant Polycystic Kidney Disease: Pathophysiology and Treatment Ashraf M. Mohieldin, Viralkumar S. Upadhyay, Albert C. M. Ong and Surya M. Nauli Hereditary Haemorrhagic Telangiectasia or Rendu-Osler-Weber Syndrome Roberto Zarrabeitia, Cristina Amado, Virginia Albiñana and Luisa-María Botella
1727
1741
1757
1783
1807
Chapter 85
Osteogenesis Imperfecta Colin R. Paterson
1831
Chapter 86
Autosomal Dominant Disorders Associated to Breast Cancer Nelly Margarita Macías-Gómez
1849
Chapter 87
Fragile X Syndrome M. Milà
1859
Chapter 88
Fragile X-Associated Primary Ovarian Insufficiency (FXPOI) Maria-Isabel Tejada
1873
Chapter 89
Fragile X-Associated Tremor/Ataxia Syndrome L. Rodriguez-Revenga
1883
Chapter 90
Psychiatric Aspects in Fragile X Syndrome and Related Phenotypes E. Mur and M. Milà
1895
Chapter 91
Clinical Features Associated with FMR1 Premutation Carriers M. I. Alvarez-Mora and L. Rodriguez-Revenga
1911
Chapter 92
Genetic Counseling of FMR1 I. Madrigal
1927
Chapter 93
Treatment of Fragile X Spectrum: FXS, FXTAS and FXPOI F. J. Ramos and M. P. Ribate
1941
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VOLUME 7 Chapter 94
Privacy and Progress in Whole Genome Sequencing Presidential Commission for the Study of Bioethical Issues
1955
Chapter 95
Genetic Testing: Scientific Background for Policymakers Amanda K. Sarata
2045
Chapter 96
Evolution of Human Genome Analysis: Impact on Diseases Diagnosis and Molecular Diagnostic Labs Julie Gauthier, Isabelle Thiffault, Virginie Dormoy-Raclet and Guy A. Rouleau
Chapter 97
Improvements in HSV-1- Derived Amplicon Vectors for Gene Transfer Matias E. Melendez, Alejandra I. Aguirre, Maria V. Baez, Carlos A. Bueno, Anna Salvetti, Diana A. Jerusalinsky and Alberto L. Epstein
2057
2079
Chapter 98
Advances in Viral Genome Research of Papillomaviruses A. C. Freitas, A. L. S. Jesus, A. P. D. Gurgel and M. A. R. Silva
2115
Chapter 99
Synthetic Synthesis of Viral Genomes Monique R. Eller, Roberto S. Dias, Rafael L. Salgado, Cynthia C. da Silva and Sérgio O. De Paula
2131
Chapter 100
Novel Bioinformatics Method to Analyze More Than 10,000 Influenza Virus Strains Easily at Once: Batch-Learning Self Organizing Map (BLSOM) Yuki Iwasaki, Toshimichi Ikemura, Kennosuke Wada, Yoshiko Wada and Takashi Abe
Chapter 101
Chapter 102
Oncogenes: Classification, Mechanisms of Activation, and Roles in Cancer Development Role of Oncogenes in Gynecological Pathology Ciro Comparetto and Franco Borruto The Classification, Mechanisms of Activation and Roles in Cancer Development of Oncogenes Patricio Barros-Núñez, Mónica Alejandra Rosales-Reynoso and Clara Ibet Juárez-Vázquez
2149
2163
2193
VOLUME 8 Chapter 103
Chapter 104
Dicer and MicroRNAs: Oncomirs Are the Next Frontier of Oncogenes Affecting Cancer Etiology and Tumor Progression Ha T. Nguyen and Mandi M. Murph
2227
The Role of the Epidermal Growth Factor Receptor as a Therapeutic Target in Glioblastoma and Other Malignancies Andrej Pala, Christian Rainer Wirtz and Marc-Eric Halatsch
2255
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Contents
Chapter 105
Tumor Suppressors Involved in DNA Repair and Carcinoprevention Yasuko Kitagishi and Satoru Matsuda
2267
Chapter 106
Molecular Pathogenesis of Neurofibromatosis Type 1 Ming-Jen Lee, Alton Etheridge, David J. Galas and Kai Wang
2279
Chapter 107
Imaging of Plexiform Neurofibromas in Neurofibromatosis Type 1 Eva Dombi, Nicholas Patronas and Brigitte Widemann
2299
Chapter 108
Surgical Treatment of Giant Neurofibromas Stamatis Sapountzis, Ji Hoon Kim, Abid Rashid and Hung-Chi Chen
2317
Chapter 109
Neurofibromatosis Type 1-Associated Nervous System Tumors and Current Treatment Strategies Nilika Shah Singhal and Sabine Mueller
2329
Chapter 110
Outcome Measures for Optic Pathway Gliomas Peter M. K. de Blank, Grant T. Liu and Michael J. Fisher
Chapter 111
Neurofibromatosis Type 1 Bone Disease: Diagnosis and Management Elizabeth K. Schorry and Alvin H. Crawford
2375
Cognition and Behaviour in Neurofibromatosis Type 1: Pathogenesis and Emerging Therapies Jonathan M. Payne, Natalie A. Pride and Kathryn N. North
2391
Chapter 112
Chapter 113
Chapter 114
Communication Disorders Associated with Neurofibromatosis Type 1 Heather L. Thompson, David A. Stevenson, Sean M. Redmond, Bruce L. Smith and David H. Viskochil Children with Neurofibromatosis Type 1: Functioning in the Classroom Yafit Gilboa, Sara Rosenblum, Aviva Fattal-Valveski and Naomi Josman
2349
2421
2441
Index
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Related Nova Publications
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PREFACE This 8 volume encyclopedia set presents important research on genetics. Some of the topics discussed herein include the speciation of Arabian gazelles, tau alternative splicing in Alzheimer’s disease, Cornelia de Lange syndrome and autosomal dominant polycystic kidney disease. Chapter 1 - Diatoms represent one of the most speciose groups of organisms within the microeukaryota— they occupy almost every aquatic niche worldwide, provide important functions within ecosystems as a major contributor of carbon dioxide fixation from the atmosphere, and also serve as a major contributor to the base of the aquatic food web. Despite this striking example of biodiversity and their ecological importance, the author know relatively little about the speciation processes at work in this group of organisms. In this chapter, the author review the patterns of speciation in marine and freshwater diatoms with respect to their paleontological, environmental, and genetic evolutionary histories. The author summarize the evidence for sex and hybridization among diatom species, and discuss recent work on understanding the mechanisms of reproductive isolation that limit gene flow in the microeukaryota. The author also discuss aspects of diatom genome evolution that provide clues to the genetic basis of speciation in this group. Finally, the author end with a discussion of a natural model system that presents a unique opportunity to address some of the outstanding questions in diatom speciation. Chapter 2 - The evolution of new species without geographic isolation has received a great deal of attention over the past decade. The mechanisms that can lead to speciation of vascular plants in biogeographic sympatry fall broadly into three categories; speciation with ongoing gene flow (which is often, but not always, synonymous with ecological speciation), hybrid speciation and polyploid speciation. Here, with a particular focus on plant species, the author briefly review the concepts and research on sympatric speciation and its causes. Hybrid speciation and polyploid speciation have occasionally been dismissed as special (“simple”) cases of sympatric speciation and the author consider that, although they are distinctive, there are significant obstacles to overcome before new species can be generated by these mechanisms. As such they are deserving of as much research attention as is currently afforded to speciation with gene flow. The author argue that it remains important to continue identifying cases of speciation in sympatry, in order to gain a better understanding of how all of these mechanisms can drive speciation in restricted areas (rather than simply to quantify sympatric speciation per say) and the author elaborate on the usefulness of Lord Howe Island as a model system for speciation research with this in mind.
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Chapter 3 - Gazelles are distributed across Africa and Asia and are adapted to arid and semi-arid environments. In this chapter, the author discuss potential factors promoting the divergence of lineages within this group (i.e., speciation events). The most recent common ancestor of gazelles is thought to have emerged during the Miocene (12-14 Ma) and to have split into the extant genera Nanger and Eudorcas (both endemic to Africa), Antilope (endemic to Asia), and Gazella (present in Africa, the Middle East and Asia). Within Gazella, two major clades are thought to have evolved allopatrically: (1) a predominantly Asian Clade (G. bennettii, G. subgutturosa, G. marica, G. leptoceros, and G. cuvieri) and (2) a predominantly African Clade (G. dorcas/ G. saudiya, G. spekei, G. gazella, and G. arabica). At present, both clades meet in North Africa and, especially, in Arabia. Other splits in this group are better explained by adaptive speciation in response to divergent ecological selection. In both clades, parallel evolution of sister species pairs (a desert-adapted form and a humid mountain-adapted form) can be inferred; desert-dwelling G. dorcas in Africa and G. saudiya in Arabia have a sister group relationship with mountain-dwelling G. gazella in the Levant and G. arabica in Arabia. This relationship exists within Africa between the desert-dwelling slender-horned gazelle (G. leptoceros) and the mountain-dwelling Cuvier’s gazelle (G. cuvieri) of the Atlas Mountains. A third species pair occurs in Asia; desert-dwelling goitred gazelle (G. subgutturosa) and mountain-dwelling chinkara (G. bennettii). These (ecological) speciation events correlate with ecology and behavior: the mountain forms being browsers, sedentary, territorial, and living in small groups, while the desert forms are grazers, migratory/ nomadic, non-territorial, and living in herds. Furthermore, cryptic sister species (G. gazella, G. arabica), with strikingly similar phenotypes, exist within presumed ‘G. gazella’, alluding to a possible allopatric origin of this divergence following an isolation of humid mountain regions during hyper-arid phases. On the other hand, phenotypes within G. arabica tend to be variable, but are difficult or impossible to distinguish genetically. Chapter 4 - Nonrandom distribution of rearrangements is a common feature of eukaryotic chromosomes that is not well understood in terms of genome organization and evolution. In malaria mosquitoes, chromosomal inversions—genome rearrangements that flip chromosomal segments by 1800—are often highly nonuniformly distributed among five chromosomal arms. These rearrangements are associated with epidemiologically important adaptations and, possibly, speciation in mosquitoes. A fundamental question is whether the genomic content of the chromosomal arms is associated with inversion polymorphism and fixation rates. This chapter highlights important differences in evolutionary dynamics of the sex chromosome and autosomes and reviews data about association between characteristics of the genome landscape and rates of chromosomal evolution. Recent studies suggest that a unique combination of various classes of genes and repetitive DNA in each arm, rather than a single type of repetitive element, is likely responsible for arm-specific rates of rearrangements. Additional factors, such as spatial constrains imposed by the nuclear architecture, may be responsible for the nonuniform distribution of rearrangements. Another important question is whether polymorphic inversions on homologous chromosomal arms of distantly related mosquito species nonrandomly share similar sets of genes. The available data indicate that natural selection favors specific gene combinations within polymorphic inversions when distant species are exposed to similar environmental pressures. This knowledge could be useful for the discovery of genes responsible for an association of inversion polymorphisms with phenotypic variations in multiple species. In this chapter, I also review the literature about a possible role of heterochromatin in speciation of malaria mosquitoes. The existing data demonstrate the
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elevated evolutionary plasticity of the heterochromatic portion of the mosquito genome. Finally, I discuss the importance of high-quality genome assemblies for reconstructing a gene order-based phylogeny and studying mosquito evolution. Chapter 5 - Local adaptation can play a fundamental role in the isolation of populations. While less well-studied than differentiation in sequence variation, changes in transcriptional variation during speciation also are fundamental to the evolutionary process. Drosophila mojavensis offers an unprecedented opportunity to examine the role of transcriptional differentiation in local adaptation. Drosophila mojavensis is a cactophilic fly composed of four ecologically distinct subspecies that inhabit the deserts of western North America. Each of the four subspecies utilizes necrotic tissue of different cactus host species characterized by distinct chemical profiles. The subspecies in Baja California, Mexico uses Stenocereus gummosus (Agria), in mainland Sonora it uses S. thurberi (Organ Pipe), in the Mojave Desert the host is Ferocactus cylindraceus (Red Barrel) and in Santa Catalina Island, USA, Opuntia littoralis (Prickly Pear) is the host. In this chapter the author examine how the adaptation to the different environmental conditions across the four subspecies have shaped their transcriptional profiles. Using complete D. mojavensis genome microarrays the author examined the transcriptome of third instar larvae from all four subspecies reared in standard laboratory media free of necrotic cactus-derived compounds. This experimental strategy focused on differences between constitutively expressed genes and not genes induced by necrotic cactus-derived compounds. The subspecies exhibited significant differential expression of genes that likely underlie the adaptation to different cactus hosts, such as detoxification genes (Glutathione S-transferases, Cytochrome P450s and UDP-Glycosyltransferases) and chemosensory genes (Odorant Receptors, Gustatory Receptors and Odorant Binding Proteins). Chapter 6 - Acoustic signals produced to attract mates before, during, and after courtship are frequently involved with sexual selection, sexual isolation, and reproductive isolation in Drosophila spp. and other animals, yet few studies have revealed how courtship songs evolve in a larger phylogenetic context. Therefore, the author mapped different acoustic components of courtship songs in the monophyletic Drosophila buzzatii species cluster onto an independently derived period (per) gene + chromosome inversion phylogeny to assess the concordance of courtship song evolution with species divergence. These cactophilic flies are distributed throughout several biomes in southern South America and include the sibling species D. buzzatii, D. koepferae, D. serido, D. borborema, D. seriema, D. antonietae, and D. gouveai. All seven species produced two song types; primary and secondary pulse songs, except for D. borborema and D. gouveai that produced no secondary songs. Courtship songs were characterized by analyzing six commonly studied acoustic components including burst duration (BD), carrier frequency (CF), pulse length (PL), pulse number (PN), inter-burst interval (IBI), and inter-pulse interval (IPI). Significant intra- and inter-specific song variation was observed for BD, PN, and IBI, while CF, PL, and IPI varied in a more species-specific manner, albeit with some overlap. Thus, some song components may be better species recognition signals than others. Multivariate clustering analyses resolved all species into distinct, non-overlapping groups. Mapping individual song traits (BD, IBI, and IPI) as well composites of these song variables onto our (per) gene + chromosome inversion phylogeny revealed no phylogenetic signal when different comparative mapping methods were used. Hence, the evolution of courtship songs in D. buzzatii cluster species was uncorrelated with the degree of species divergence. These findings reinforce previous observations that courtship songs evolve rapidly enough to erase any signature of evolutionary affinity between closely related animal species.
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Chapter 7 - Nasonia (Hymenoptera: Pteromalidae) are small haplodiploid parasitoids of flesh- and blowfly pupae that have become model organisms for speciation research. The genus consists of four closely related species that harbor species-specific Wolbachia bacteria that cause postmating reproductive isolation. Antibiotic curing allows for interspecific crosses and genetic exchange between species which, together with haploidy of males, facilitates genetic analysis of fitness traits. In this chapter the author synthesize the current knowledge on the different prezygotic isolation factors that act in the Nasonia genus, and on the genetic basis of these traits. A major prezygotic isolation factor is courtship behaviour. Species differ in male courtship behaviour, and there is large variation in interspecific mate discrimination depending on species pair. The author summarize data on the strength of prezygotic isolation barriers between all possible species pairs and present new data on mate discrimination in choice and no-choice experiments. In tests of reinforcement, the author found no stronger female mate discrimination of N. vitripennis strains occurring in microsympatry with N. giraulti compared to that of allopatric N. vitripennis strains. Additionally, the author present data on the significance of cuticular hydrocarbon profiles for assortative mating in males and discuss other factors that may be involved in prezygotic isolation, including pheromone communication, within-host-mating and sneaking behaviour. Chapter 8 - Not all genes are equally important during the process of speciation. This premise underlies a basic question in evolutionary biology – “Does divergence at any one gene, or set of genes, play a particularly important role in speciation?” The answer to this question may appear to be no for some forms of reproductive isolation, but there may indeed be ‘kinds of genes’ that routinely play a role in speciation when divergence is driven by postmating, prezygotic traits. Here, I outline the kinds of molecular pathways and interactions that underlie postmating, prezygotic phenotypes which ultimately points to the kinds of genes where the author should look for species-specificity. Interestingly, it is only when the author consider the entire system of interacting sex proteomes, cell structure, membrane dynamics, and physiological pathways that a picture of where species-specific interactions likely occur becomes clear. While this approach points to several kinds of pathways and gene types, a notable finding is that cell membrane receptors (like G-protein coupled receptors and receptor tyrosine kinases) that line the inside of the female reproductive tract and trigger post-copulation cell signaling should be considered the kinds of genes that routinely contribute to reproductive isolation and speciation when divergence is driven by postmating, prezygotic phenotypes. Chapter 9 - Speciation, the process by which one species splits into two, involves the evolution of reproductive barriers such as the sterility or inviability of hybrids between previously interbreeding populations. One of the earliest intrinsic barriers to gene flow to evolve between geographically isolated populations is the sterility of hybrids of the heterogametic sex. The Dobzhansky-Muller model describes how hybrid incompatibilities that underlie intrinsic postzygotic reproductive barriers such as hybrid sterility may evolve. A major goal in speciation research is to identify the genes that underlie hybrid incompatibilities. The identification of such genes opens the door to understanding the molecular pathways which, when tinkered by evolution within species, lead to sterility in hybrids, and promises to reveal the biological forces that drive the evolution of hybrid sterility genes. While very few hybrid sterility genes have been identified so far, the idea that the evolution of DNA-protein interfaces driven by intragenomic conflict may cause hybrid sterility is gaining wider acceptance. Here the author describe how the molecular and evolutionary insights from two hybrid sterility genes
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– Odysseus and Overdrive – have illuminated the role of heterochromatin in the molecular basis of hybrid sterility and the role of genetic conflict as a driving force in speciation. Chapter 10 - With innovative DNA sequencing technologies has come a new appreciation for the content of animal and plant genomes. Overwhelmingly, a picture has been painted in which mobile and repetitive elements dominate the genomic landscape. Mobile genetic elements have recently been shown to contribute coding and regulatory sequences during their proliferation, leading to functional and regulatory novelty as well as element-mediated rearrangements coinciding with speciation events. Additionally, dormant elements occasionally erupt in bouts of excision and transposition in interspecific hybrids, resulting in a suite of maladaptive traits. The potential for mobile elements as key players in the evolution and diversification of genomes and species is immense yet in many respects transposable elements still remain the “dark matter” of the genome. This is particularly true of their role in speciation, and in order to fully appreciate their role, much work is still needed. In this chapter, the author investigate the evidence for transposable elements as drivers of diversification and speciation. Chapter 11 - Recent studies suggest that much hybrid incompatibility results as a consequence of conflict between different components of the genome (genomic conflict). In this chapter, I argue that the battlegrounds for much of the conflict-driven hybrid incompatibility consist of various cellular structures and processes. These include the recombination machinery, centromeres and kinetochores, and heterochromatin and its associated proteins. I conclude with a call to integration between cell biology and evolutionary biology in the study of the evolutionary genetics of hybrid incompatibility. Chapter 12 - In the present work, I give an all-embracing macroevolutionary perspective on processes of the evolution of life and culture on earth. First, I investigate a complementary form of natural selection that diverges from the traditional form in that it is acting independently of the external environment. This form of natural selection is found as a result of a mathematical analysis of the conditions for population growth. I extend my investigation as well to other evolutionary processes than the organic, such as the evolution of human language and the evolution of science, thereby suggesting other possible forms of underlying explanatory processes. I examine the concept of complexity and show that it implies new insights into the ways natural selection has been acting in forming the evolutionary and developmental processes. Especially, complexity is found to be growing in an accelerating mode, a process that is explained as the combined result of natural selection and a self-reinforcing feedback process. The use of the concept of complexity opens the possibility of construction of a new form of a Tree of Life, which, in contrast to traditional forms, combines complexity and time. Such an illustration of the evolutionary process explains the observation that most species live without great changes over vast periods of time. For species at the highest level of complexity there is no competition from species at still higher levels and these species can therefore, if conditions are beneficent, form new species at still higher levels. The process explains the emergence of new species and the general trend of evolution towards cumulatively higher complexity levels. The cumulative addition of species with successively higher complexity implies that the latest appearing species is the one of the highest level of complexity. At present, this species is the human species. Chapter 13 - Advances in modern medicine enable a change in the tension of intragroup selection in human populations. Thus, implementation of insulin for type 1 diabetes mellitus (DM) treatment considerably lowered the selection tension for this symptom and converted it from the sub-lethal to the one with a lowered adaptability. Increasing variety of type 1 DM and
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type 2 DM is being observed recently in different populations. Moreover, recently the heterogeneity of type 1 DM and type 2 DM has also been observed. The investigation was aimed to study the influence of the selection on the evolution of DM clinical forms. Global implementation of insulin therapy into type 1 DM treatment caused the prevalent increase of this disease. Currently, there is a positive selection trend for type 2 DM, which is the original cause for the prevalence increase within the population, and the negative one for type 1 DM determines its prevalence within the population approximately on the same level. Intrapopulation change of gene frequencies, susceptible for type 1 and 2 DM, predetermined the development of such DM clinical forms as LADA (latent autoimmune diabetes in adults). It also resulted in the increasing number of patients with an absolute insulin deficiency of type 2 DM, which is a more complicated form of this DM type. Polymorphisms association, changing the immune response and forming the susceptibility to type 1 (С1858Т of the gene PTPN22, A49G of the gene CTLA4) and type 2 (Е23К of the gene KNJ11 participating in insulin insufficiency formation) with different DM forms illustrates the result of decreasing the selection tension against type 1 DM after insulin therapy implementation into the public health services practice. Chapter 14 - Genetic variation is generally responsible for ethnic differences in certain diseases, including inflammatory processes. The antagonist of cytokine IL-1, IL-1Ra, has been widely studied among Caucasian and African populations for genetic polymorphisms, and interethnic differences have been documented. However, the variation and genotype distribution of polymorphisms from these genes among South American Amerindians are thus far unknown. The author present the results for a VNTR located in the IL-1Ra second intron, in a sample of 169 individuals belonging to 5 Native American populations from Argentina and Paraguay, identified as native according to their self designation, and their geographic location. The author also compare this data with the results obtained from a sample of non-native Argentinian people. Among the five known alleles of the VNTR, the author found only two (alleles 1 and 2) in the native populations from Gran Chaco, and heterozygosity was 19%. The allele 2 which is considered proinflammatory (IL-1Ra * 2) has been found in homozygosity at a considerable frequency among native individuals. However, the association of this allele with inflammatory disease previously demonstrated for other populations of the world, might not be acting in the same way for native people, probably due to local adaptation. This would indicate that the allele 2 will probably not have a negative influence on individuals of native origin who have homozygous genotype 2-2. On the contrary, few records on inflammatory disease are available for the native people. It seems that the increment on allele 2 is not related to any adaptive process but to genetic drift, that changes randomly the allele frequencies of different genetic regions along the genome. The effect of genetic drift has already been demonstrated with genetic markers located in autosomes, X and Y chromosomes. These results indicate that the author must be very cautious when studying populations that passed a process of genetic drift, which can become a confounding factor in epidemiological studies. This information will contribute to a future understanding of the association of this polymorphism with disease, and its incidence in different ethnic groups. Chapter 15 - Alternative splicing (AS) is a fundamental mechanism of gene expression regulation that extremely expands the coding potential of genomes and the cellular transcriptomic and proteomic diversity. This dynamic and finely-tuned machinery is particularly widespread in the nervous system and is critical for both neuronal development and functions. Alternative splicing defects, therefore, frequently underlie neurological disorders. In
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this chapter, the author will focus on Parkinson’s disease (PD), the second most common neurodegenerative disorder worldwide. The author will provide a current overview on the impact of alternative splicing in PD by representing the multiple splicing transcripts produced from the major PD-linked genes and their regulation in PD states. Furthermore, the author will review the studies describing global splicing expression changes revealed by whole-genome transcriptomic approaches. The author will also summarize the current knowledge about the alternative splicing modulation in PD through non-coding RNAs (miRNA and lcnRNA) molecules. Assessing the role of alternative splicing on PD pathobiology may represent a central step toward an improved understanding of this complex disease. Chapter 16 - The microtubule-associated protein (MAP) tau is essential for the development of neuronal cell polarity. Tau protein is preferentially localized in the axon, whereas MAP2, another neuron-specific microtubule-associated protein, is localized in the somatodendritic domain. Previous studies have demonstrated that the localization of these proteins depends, at least in part, on mRNA subcellular localization - of tau mRNA into the axon and MAP2 mRNA into the dendrite. Tau protein plays a pivotal role in the pathophysiology of Alzheimer’s disease, in which its hyperphosphorylation promotes aggregation and microtubule destabilization. Tau undergoes alternative splicing, which generates six isoforms in the human brain, due to the inclusion/exclusion of exons 2, 3 and 10. Dysregulation of the splicing process of tau exon 10 is sufficient to cause tauopathy, and has been shown to be influenced by b -amyloid peptides, while there has been less research conducted on the splicing of other exons. This study found that the effects of -amyloid (42) on the alternative splicing of tau exon 2/3 and 6 caused formed cell processes to retract in differentiated cells and altered the expression of exon 2/3 in cell culture. Expression of exon 6 was repressed under -amyloid treatment. Although the molecular mechanism for this amyloidtau interaction remains to be determined, it may have potential implications for the understanding of the underlying neuropathological processes in Alzheimer’s disease. Chapter 17 - Alternative splicing is a co-transcriptional mechanism that regulates eukaryotic gene expression that affects almost 90% of the human genes. In this mechanism, different combinations of exons and introns can be identified and removed from the pre-mRNA, allowing multiple mRNA configurations of joined sequences to arise from a single gene, increasing the coding potential of the genome. Alternative splicing events are catalyzed by a large complex known as the spliceosome, which is conformed by more than 300 proteins and ribonucleoproteins. At the catalytic core of the spliceosome, the small nuclear ribonucleoproteins (snRNPs) U1, U2, U4, U5 and U6 are found. The auxiliary factors responsible for the fine regulation of this mechanism include two major groups: the SR proteins and the hnRNP family. Malfunctions of alternative splicing events can affect the natural expression of a large number of transcripts, including several factors involved in apoptosis or cell survival, molecular processes intimately associated with cancer evolution. In many cases, specific splicing factors or mutated components of the splicing machinery are linked to an anomalous event. Moreover, a switch in specific splicing factors occurs in particular types of cancer where the concomitant outcome is the production of non-functional proteins with added, deleted, or altered domains affecting tumorigenesis. With all this evidence, several strategies have been developed to regulate alternative splicing in which central or auxiliary splicing factors are the target of modulatory molecules. Given the combination of elements needed to regulate alternative splicing, the mechanisms underlying the functional and physiological
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implications of these tools are also diverse. Collectively, these strategies are intended to improve cancer prognosis, therapeutic and treatment. Chapter 18 - The precise control of protein production is essential for the appropriate cell physiology and survival. However, single mutations or accidentally introduced errors can occur during the flow of genetic information. In eukaryotic cells, some messenger RNA (mRNA) molecules leave the nucleus after splicing but further mechanisms are involved in the ultimate outcome of the correspondent protein. One of such systems corresponds to the mRNA surveillance network that includes nonsense-mediated mRNA decay (NMD), an important quality control system that ensures the accuracy of transcripts, to maintain a healthy cellular homeostasis. NMD eliminates anomalous mRNAs harboring premature termination codons (PTCs) to prevent the production of potentially harmful truncated proteins, but it can also regulate the steady state of many physiological mRNAs. Targets for NMD are sometimes linked to mutations or introduced errors but the vast majority can be generated as a result of alternative splicing. The key components required for NMD include the UPF and SMG proteins. These factors interact with a set of proteins (the exon junction complex or EJC) that are deposited just upstream of exon-exon junctions after mRNA splicing and orchestrate a regulated mechanism in order to identify PTCs and either sequester these mRNAs or target them for degradation. Some of these factors are linked to particular disorders and could be modulated in order to correct the defect. The NMD pathway is physiologically and medically important, because an escape from NMD can result in severe clinical phenotypes. Consistent with this, it is estimated that more than 60% of human genes have alternatively spliced products that generate at least one PTC isoform and that approximately 30% of inherited genetic disorders are caused by nonsense mutations or by frameshifts that generate nonsense codons. Initially associated as a genetic cause for beta-thalasemia, the NMD process has expanded from the haematopoietic system and it has reached different kind of human disorders, including cystic fibrosis, Duchenne muscular dystrophy, Hurler syndrome, recessive spinal muscular atrophy and polycystic kidney disease. Finally, it is predicted that some cancers are aided by NMD and the repression of this mechanism may be a potential target for the treatment of certain cancers. In this regard, pharmacological agents have been developed for the treatment of diseases caused by premature stop mutations, including aminoglycosides, but the whole therapeutic potential of NMD targets to correct genetic disorders remains to be exploited. Chapter 19 - Alzheimer’s disease (AD) is the most common type of dementia in the elderly population with a higher prevalence in women. Memory impairment and cognitive decline in AD are primarily linked to its neuropathological hallmarks, i.e., cholinergic neuronal atrophy and death, presence of intraneuronal neurofibrillary tangles and accumulation of amyloid β (Aβ) deposits within the extracellular senile plaques. Consequently, genes encoding Aβ, tau and proteins involved in Aβ procession received the most careful scientific attention. However, a growing body of evidence suggests that AD pathogenesis is not limited to the changes of AD genes’ expression, but also depends on their alternative splicing. Therefore, the present review focuses on data concerning alternative splicing changes in AD. First of all, pivotal AD genes (APP (amyloid precursor protein), tau, presenilin 1 (PS-1) and 2 (PS-2) and apolipoprotein E (APOE)) have a number of splice variants with divergent functions that are differentially expressed in AD and normal brain tissues. Second, alternative splicing of genes involved in Aβ processing and metabolism is also affected in AD. This group includes BACE-1 (β-site APP cleaving enzyme 1), nicastrin and APH-1 which are components of the γ-secretase complex, AIDA-1 (protein that binds to the intracellular domain of AβPP following cleavage by γ-
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secretase; FE65 (binds to the cytoplasmic tail of βPP). Finally, splice variants of such candidate AD genes as acetylcholinesterase (cholinergic deficit is one of the central mechanisms in AD development), estrogen receptor α (ERα) (estrogen deficiency is one of the predisposing to AD factors), BDNF (brain derived neurotrophic factor that is crucial for neuronal survival and synaptic transmission) and its receptor TrkB, excitatory aminoacid transporter 2 (EAAT2) (colocalized with tau in dystrophic neurons), genes of the ion channels and neurotransmitter receptors, synapsin, RCAN-1 (regulator of calcineurin), neuronal GFAP, ubiquilin-1 (related to the proteasomal degradation of proteins and interaction with PS-1 and PS-2) and genes involved in the regulation of the cell cycle and apoptosis (CIZ1, DENN/MADD) should be considered as important elements of the AD pathogenesis. Together, these data show that a lot of changes in alternative splicing are intimately linked to AD, which should be, thus, considered as a disorder with compromised and disregulated splicing events. Chapter 20 - Posttranscriptional control of gene expression is crucial for biological processes. In particular, alternative splicing allows the same gene to produce multiple proteins and is thus a key generator of functional complexity. This posttranscriptional regulatory mechanism is a prevalent feature of eukaryotic genomes, being currently estimated to occur in over 50% of plant genes. RNA-binding proteins (RBPs) are known to control many aspects of RNA metabolism, from pre-mRNA splicing to the transport and stability of mRNA transcripts. The Arabidopsis and rice genomes contain about 200-250 genes predicted to encode RBPs, but few of these proteins have been characterized in plants. As sessile organisms, plants are continuously exposed to environmental challenges that affect their growth and development. The phytohormone abscisic acid (ABA) is crucial in the coordination of plant responses to various abiotic stress factors. Interestingly, several RNA-metabolism genes have recently been implicated in the ABA pathway, providing a link between mRNA processing and ABAmediated plant stress responses. Indeed, the loss- or gain-of-function of genes encoding different classes of RBPs directly involved in constitutive and alternative pre-mRNA splicing, such as snRNP factors or SR and hnRNP proteins, has been shown to result in striking ABAresponse plant phenotypes. Functional roles in ABA biosynthesis or signaling have also been reported for other RBPs, including cap-binding proteins, RNA helicases, pentatricopeptide repeat proteins or poly(A) processing enzymes. Taken together, these data support the notion that posttranscriptional networks act as central coordinators of plant stress responses, namely by targeting key components of ABA signal transduction machinery. Future identification of the direct targets of these RBPs should uncover the molecular mechanisms underlying the mode of action of these proteins in the regulation of the ABA pathway. Chapter 21 - Alternative splicing occurs in most human genes and contributes to protein diversity by producing multiple mRNAs from each gene. Many of these alternative isoforms are expressed in a spatio-temporal manner and play important functional roles in many biological processes including neuronal events. Here, neuronal-specific splicing was comprehensively investigated by using P19 cells. GeneChip Exon Array analyses were performed of total RNA purified from cells during different stages of the differentiation process. Nine filtering conditions were used to efficiently and readily extract alternative exon candidates. A total of 262 candidate exons (236 genes) were obtained. Semi-quantitative RTPCR results of 30 randomly selected candidates suggested that the expression levels of 87% of the candidates were at least 2-fold different between undifferentiated and differentiated cells. Gene ontology and pathway analyses also showed that many of these 236 candidate genes played important roles in neuronal events. These results suggested that this novel method to
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determine alternative exons was successful and efficient. In addition to the known neuronal functions, informatics analyses demonstrated that alternative splicing events of 11 candidate genes played important cell cycle functions. These results suggest that this novel method also provides a way to determine the functional roles of previously unknown alternative splicing events. Chapter 22 - Only sequence analysis of full-length transcripts can identify genuine alternative splicing variants. However, it was difficult to obtain full-length cDNAs for rare or long-sized transcripts. Recently, the author have developed a powerful method, named a vectorcapping method, to construct a size-unbiased full-length cDNA library containing rare or verylong-sized cDNA clones with >10kbp inserts. The characteristic of the full-length cDNA contained in this library is that the intactness of the 5’-end capped site sequence of the cDNA can be assured by the presence of an additional dG at its 5’ end. Since this full-length cDNA is derived from a single mRNA, this library enables us to perform in-depth analysis of genuine alternative splicing variants. Using the vector-capping method, the author prepared full-length cDNA libraries from human retina-derived cell lines and analyzed the full sequence of the clones. As a result, the author found many novel alternative-splicing variants for rare or longsized transcripts. In this chapter, I show the examples of these variants including very-longsized transcripts with >7kbp that were identified by us for the first time. Chapter 23 - Genome-wide analysis indicate that alternative splicing of mRNA precursors (pre-mRNAs) affects the vast majority of human genes. Alternative splicing provides a fundamental mechanism to increase transcriptome complexity, allowing the production of two or more mRNA variants that often encode proteins with different, sometimes opposite functions. Its importance is underscored by the observation that misregulated alternative splicing can lead to human diseases. Pre-mRNA splicing has long been known to be regulated by cis-acting sequence elements and trans-acting protein factors. In higher eukaryotes, it mostly occurs co-transcriptionally so that it is not surprising that a role for chromatin and epigenetic factors in the regulation of exon inclusion is now emerging. In this review, the author will discuss the most recent findings on the roles played by chromatin structure on the modulation of the cotranscriptional splicing reactions. In particular, the author will focus our attention on how the modulation of the transcribing RNA polymerase II, the changes in nucleosome architecture and the presence of different histone modifications contribute to the regulation of the splicing process. Chapter 24 - Alternative splicing expands transcriptome diversity and allows cells to meet the requirements of an ever-changing extracellular environment. It has been more than 30 years since nitric oxide (NO), a gaseous free radical, was recognized as a critical physiologic signaling molecule. Since then the list of known NO-directed functions has grown substantially to include regulation of smooth muscle function in vascular and gastrointestinal systems, inhibition of platelet aggregation and adhesion, neurotransmission and neuromodulation, regulation of cellular respiration and cytotoxicity, mitochondrial biogenesis and, immune defense. However, the importance of alternative splicing in regulation of enzymatic components of NO signaling pathway started to emerge only recently. Our understanding of the mechanisms governing this process remains very limited and awaits systematic investigation. In this chapter the author will attempt to summarize the available information on alternative splicing of major enzymes mediating canonical NO transduction through the secondary messenger cGMP. The author will highlight evidence accumulated from different laboratories that suggest splicing of enzymes in the NO/cGMP pathway, including nitric oxide
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synthases, heterodimeric soluble guanylylcyclase and cGMP-dependent protein kinase, is very complex and strongly affects NO signaling in response to various environmental cues. Future studies will certainly bring new, exciting insights into the role that alternative splicing plays in NO/cGMP biology. Chapter 25 - Genes could produce multiple protein-coding transcripts by alternative splicing (AS). It was known that AS is related to several diseases in generating biological and functional diversity. So, analysis of the mRNA diversity of genes would be important for understanding gene function. Previously, we obtained 1.46 million human full-length cDNAs (FLJ cDNA) and sequenced their 5’-ends. The author selected approximately 55 thousand cDNAs from FLJ cDNA and sequenced completely. Our FLJ cDNAs were constructed by an optimized oligo-capping method. Thus, by using 5’-EST data, a lot of valuable information was obtained regarding the diversity of the transcription start site (TSS) and amino acid sequences at the N-terminal ends of proteins. The author found that alternative TSSs were utilized for tissue-specific expression. Using this data, the author constructed FLJ Human cDNA Database ver. 3.2, http://flj.lifesciencedb.jp. But, a lot of AS-related information still remains to be extracted from our 1.4 million cDNA resources. From a huge number of human sequence information, the author selected only the reliable cDNAs for the analysis of the mRNA diversity. And, the author developed probes which can analyze the mRNA diversity. As a result, by comparing our constructed 5,784 pairs of independent probes, the author are able to detect the expression profiles of splicing patterns in 3,413 genes. Moreover, using these probes, the author analyzed the mRNA diversity of genes after inducing neuronal differentiation in human NT2 teratocarcinoma cells using all-trans retinoic acid (RA). Analyses of NT2 cells identified 452 RA-responsive genes. The mRNA diversity analysis revealed that the rate of genes that showed AS in their N-terminus, internal region, C-terminus, is almost the same, respectively. Chapter 26 - Alternative splicing of pre-messenger RNA is nearly universal, involving more than 90% of human genes. It’s an essential step in gene expression and responsible for much of the proteome diversity in mammalian genomes. The immune system requires a great diversity of functional proteins and immune cells need to respond to various foreign invasions rapidly. Alternative splicing provides one more layer of regulation that is essential for the function of human immune system. Many immune genes have been found to undergo alternative splicing, which plays an important role in the regulation of immune cell activation and function. Dysregulated splicing has been shown to be involved in various immune disorders, such as systemic lupus erythematosus (SLE) and rheumatic arthritis (RA). It may be a direct cause of the disease, or a modifier of disease susceptibility and severity. Further understanding of how alternative splicing may be used as a general mechanism in immune response is essential for our research in the pathophysiology of autoimmune diseases and development of new therapeutics for those diseases. This chapter provides an updated review about alternative splicing in human immune system as well as the relationship between dysregulated splicing and autoimmune diseases, particularly SLE and RA. Chapter 27 - Poly(ADP-ribosyl)ation is a major post-translational modification performed by Poly(ADP-ribose) Polymerases (PARP1). PARP1 utilizes NAD as the substrate to synthesize a long linear and branching poly(ADP-ribose) (pADPr), ranging in length from 2 to 200 units of ADP-ribose. PARP1 can modify a variety of proteins by attaching pADPr to the target proteins in either a covalent or noncovalent manner. In this way, Poly(ADP-ribosyl)ation is involved in a number of biological processes, including transcription regulation, stress responses, and apoptosis. Recent studies have demonstrated that several splicing factors,
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including hnRNPs and S/R proteins, are poly(ADP-ribosyl)ated via noncovalent binding by PARP1. Here, the author describe how poly(ADP-ribosyl)ation regulates alternative splicing by modulating the activities of these splicing factors. In addition, the author discuss a possible role of PARP1 in coupling transcription with splicing. Chapter 28 - Genetic and behavioral data mining will revolutionize health/lifestyle delivery and outcomes, in much the same way the Internet delivered meaningful social outcomes by providing the infrastructure for information to be collected and connections to be made in ways that had not been possible hitherto. Genetic databases and lifestyle correlations via the cutting edge field of bioinformatics, wearable technology and personalized medicine will be the next echelon of information to be reconnoitered and used through the Internet to enrich our lives. Thanks to smart phones and wearable technology, it will all commence from the palm of our hands. In this review, the author will spotlight the emerging fields of bioinformatics, personalized medicine and biosensor technology: historical insights, current issues, and future trends. In particular, the author will embark on an in-depth look into the sub-fields of personalized medicine and wearable systems for health and lifestyle management. The healthsector, being information intensive, will exploit IT-led platforms that capture personalized health data in real-time and have connectivity with a cloud backbone to intelligently process the information to deliver real-time analytics and actions. Various roadmaps are presented offering aggressive offensive and defensive IP strategies and solutions to companies in the bioinformatics and biosensor space. How does current patent law and policy in the wake of the AIA reforms and Supreme Court decisions in Alice and Mayo impact on this field and on the strategic guidepost? What’s more, the author will reopen the patent troll reform debate: will it create a sweeping sea change, or be just another talking point. How do the sweeping provisions of Obamacare touch upon the field of wearable health systems and bioinformatics? Moreover, how can the author reconcile proprietary value with the growing trends of the open-source movement and the big health data initiatives that lie at the intersection of private and non-profit sectors? Finally, all of this big health data begs an inquiry into issues of privacy and a host of other ethical concerns. With the right strategies in their IP toolkit, bioinformatics and wearable startup companies will not only bolster their current patent position, but also be able to leverage it into a significant competitive advantage. More importantly, the broader public will be able to avail themselves of the utility of these flash-bulb popping innovations that promise to capture personalized health data in real-time to deliver targeted and improved health outcomes. Chapter 29 - Marfan syndrome is a heritable disorder of connective tissue that is transmitted as an autosomal dominant trait and is characterized by the mutation in the fibrillin 1 gene (FBN1). At present, the diagnosis of Marfan syndrome, based on Ghent criteria, considers both clinical evaluation (in which it is possible to observe alterations in eye, osteoarticular apparatus and cardiovascular system) and genetic evaluation (that reveals a FBN1 gene mutation). Moreover, the author take into account the presence of affected relatives suffering of the same syndrome. The diagnosis of Marfan syndrome is often complex because of the evolution of the phenotype with age and because of the inter-individual variation in the clinical presentation even among the affected family members. According to a recent review, Marfan syndrome is often associated with a range of psychiatric problems like anxiety disorders, depressive disorders, schizophrenia, neurodevelopmental disorders (autism spectrum disorder and attention deficit/hyperactivity disorder) and eating disorders. The author report a 16 years old female patient (MF) (kg 43, H 165 cm, BMI 16.9) who came to our outpatient service for a selective feeding successively diagnosed as Avoidant/Restrictive Food Intake
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Disorder (ARFID; DSM 5). “Selective feeding” refers to children who restrict the ingestion of food to a limited number of “favourite” foods, typically five or six different foods. ML., indeed, eats only pasta, sweets and a few other foods. Beyond the altered feeding behaviour, she was diagnosed with a mild intellectual disability (IQ=57; VIQ=61; PIQ=62) and reduced adaptive levels in the socialization and communication domains of the Vineland Adaptive Behavior Scales (VABS). This patient presented ligamentous laxity, long-limbed body habitus suggestive for Marfan syndrome. Therefore, she was addressed to the cardiologist: the echocardiographic evaluation showed mild dilatation of aortic root, with rectilinear sinotubular junction and enlarged ascending aorta (z score=2). Z-scores of the aortic root at the level of the aortic annulus, sinuses of Valsalva, sinotubular junction, and ascending aorta measured from the parasternal long axis in diastole using leading-edge-to-leading-edge technique. The eye examination was normal. Sanger sequencing of the FBN1 gene identified the c.7501G>A (p.Val250lle) variant/mutation. The missense mutation/varianti is not reported in UMD, Enseble, Exome variant server and dbSNP. The SIFT, PoliPhen2, Mutation Tester (software tool) for predicting damaging of missense mutations and variants gave the following results: PoliPhen2: benign; Mutation Tester: disease causing, SIFT: tolerant. Her mother carries the same mutation. ML. is affected by ARFID, (mild) intellectual disability and adaptive disorders often associated with Marfan syndrome, confirming and extending the results obtained in the review previously described. Moreover, the author underline the importance in multidisciplinary diagnosis and care (also) in these cases. Chapter 30 - The author have previously shown that introduction of single genes encoding diacylglycerol acyltransferases (DGAT1s) or partially-silenced mitochondrial pyruvate dehydrogenase kinase (mtPDCK), each had the capacity to enhance seed oil content in Arabidopsis. In the current study, the author report the cumulative effects of expressing a twogene stack: a site-directed mutagenized DGAT1 from Tropaeolum majus (TmDGAT1 Ser197to-Ala197) and an anti-sense mtPDCK from B. napus (A/S mtPDCK) introduced into B. napus cultivar DH12075 to alter seed oil content. Compared to plasmid-only controls, the best lines of the two-gene construct showed, on average, a 23.6% proportional increase (11.6% net increase as % of DW) in oil content which is near-additive to the best results obtained in transgenic experiments with the single genes. These findings demonstrate the utility of stacking two specific transgenes controlling the key steps in two very different metabolic streamsmitochondrial carbon flux (mtPDCK; “push”) and triacylglycerol assembly via the Kennedy pathway (DGAT1; “pull”), to bring about significant increases in oil content in B. napus. This approach holds promise for similar use in other oilseed crops. Chapter 31 - It is well known that genetic variations can in part affect human oral health. Periodontitis is a common dental noncommunicable disease (NCD). According to the World Health Organization, periodontitis affects 20% of the world population. Periodontal inflammation could eventually induce alveolar bone resorption, causing tooth mobility and tooth loss, which ultimately affects oral function, oral health and the individual’s quality of life. Family study, twins study and linkage analysis in the early years revealed that genetic influence contributes to periodontal disease. Since the first single nucleotide polymorphisms (SNP) study on chronic periodontitis in 1997, many genetic loci were found to be associated with periodontitis, including IL-1, Fc gamma receptor and complement component 5 genes. Population structure or earlier investigations employing a less desirable sample size may lead to various limitations or bias and therefore diverse results. Meta-analysis reports have proved the association between periodontitis and SNPs in some regions, such as IL1 and HLA. With
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development of the technologies, multiple genes can be analyzed in a single assay, and even whole genome SNPs can be screened. Seven genome-wide association studies (GWASs) investigated the potential genetic risk locus for periodontitis and found GLT6D1 is significantly associated with aggressive periodontitis. In the future, whole genome sequencing, together with replication in multiple populations and functional studies, could potentially disclose the nature of periodontitis as an NCD. Chapter 32 - Imprinted regions of the mammalian genome are commonly composed of one or more paired paternally and maternally imprinted genes and differentially methylated regions (DMRs). These DMRs are methylated in an allele-specific manner during germ-line or early embryonic development, and they regulate allele-specific gene expression. Although genes in most imprinted regions are expressed ubiquitously among tissues, some imprinted regions manifest tissue-specific gene expression patterns. The brain is a major site of tissue-specific gene expression from imprinted regions in embryonic tissues. The author summarize several imprinted regions that show neuron-specific gene expression patterns. First, the author describe the SNRPN-UBE3A domain, which contains three brain-specific imprinted genes, SNORD115, UBE3A-ATS and UBE3A, as well as one imprinted gene with a brain-specific promoter, SNRPN. Second, the author discuss the DLK1-DIO3 domain, which contains the braindominant imprinted genes SNORD112, SNORD113 and SNORD114 as well as numerous brainspecific imprinted miRNAs. Third, the author provide an overview of Grb10, which also has a brain-specific promoter and switches the imprinted allele during early neurogenesis. Our chapter reviews these imprinted regions and describes the similarities and differences of the neuron-specific imprinting switch in each region. Chapter 33 - Treatment of human diseases in general has dual goals: to treat effectively, at the cost of minimal adverse side effects. However, person-to-person differences on drug efficacy and adverse reactions have been frequently observed. Pharmacogenomic studies intend to address such differences based on personal genomic variants such as single nucleotide polymorphisms. In the past, success has been achieved on the identification of major histocompatibility complex genomic variants which are associated to immune-mediated adverse drug effects. Additionally, genomic variants on the direct drug targets have been found to be associated to drug efficacy. Besides immunological and drug-target factors, one other critical factor is on the drug metabolism mediated by various xenobiotic metabolizing and detoxification enzymes. They determine how quickly the drugs can be metabolized and then excreted from the body, thereby affecting drug efficacy and toxicity. Human xenobiotic metabolizing enzymes are categorized by their phases in the metabolozing processes: the modification phase (phase 1), the conjugation phase (phase 2), and further modification and excretion (phase 3). Previously, genes involved in phase 1 have been extensively studied in pharmacogenomics. In comparison, genes in phase 2 received less attention, despite the fact that they are no less important. These genes include Uridine DiphosphoGlucuronosyltransferases and others. This Chapter presents the genomic variants on phase 2 genes which can account for the drug efficacy and adverse reactions. Chapter 34 - In species with separate sexes, gender differences in longevity are widespread and the extent and direction of these differences varies tremendously among taxa. To understand sexual dimorphism in longevity and explain how different forms of selection shape longevity and other fitness-related traits within and among species, it is important to obtain information on the genetic architecture (the number of genes and degree of inter- and intragenic interactions) and various mechanistic causes which underlie mortality variation between
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the sexes. Here the author review recent empirical studies on gender differences in longevity in insect species, from both mechanistic and evolutionary perspective. Whenever it was possible, the author focus on data obtained from the laboratory evolution experiments because the study of evolution under controlled conditions may provide valuable evidence not only for the effects of natural and sexual selection in shaping sex-specific longevities and mortality rates but also it offers novel insights into the mechanistic basis of these differences. Chapter 35 - During mating, male bush-crickets transfer a complex spermatophore to the female. The spermatophore is comprised of a large nuptial gift which the female consumes while the sperm from the ejaculate-containing ampulla are transferred into her. Two main functions of the nuptial gift have been proposed: the ejaculate protection hypothesis and the parental investment hypothesis. The former, founded on sexual selection theory, predicts that the time to consume the gift is no longer than necessary to allow for full ejaculate transfer. The latter maintains that gift nutrients increase the fitness or quantity of offspring and hence the gift is likely to be larger than is necessary for complete sperm transfer. With an aim to better understanding the primary function of nuptial gifts, the author examined sperm transfer data from field populations of five Poecilimon bush-cricket taxa with varying spermatophore sizes. In the species with the largest spermatophore, the gift was four times larger than necessary to allow for complete sperm transfer and is thus likely to function as paternal investment. Species with medium and small gifts were respectively sufficient and insufficient to allow complete sperm transfer and are likely to represent, to various degrees, ejaculate protection. The author also found that species that produce larger spermatophores transfer greater proportions of available sperm than species producing smaller spermatophores, and thus achieve higher paternal assurance. Chapter 36 - Mate choice copying was mostly described as a strategy employed by females to assess the quality of potential mates, but also males can copy other males’ mate choice. In both cases, focal individuals show an increased propensity to copulate with a potential mating partner they could observe interact sexually with another individual (the ‘model’). Sexual interactions, however, convey additional—partly conflicting—information to the choosing individual: females may try to avoid sexually active males due to a rougher courtship or coercive mating attempts, and males may respond to an increased sperm competition risk (SCR). How do females and males respond to different copying situations, in which the model individual either resides in the vicinity of a potential mating partner without physical contact (i.e., no harassment, and low SCR), or interacts sexually with the potential mating partner? Do individuals copy less in the latter situation? The author investigated these questions in the guppy (Poecilia reticulata), a livebearing fish with internal fertilization, strong SCR among males, and frequent sexual harassment of females. Focal individuals could choose to associate with a large or a small stimulus fish, and mate choice tests were repeated after the previously non-preferred stimulus fish could be seen associating (low harassment and low SCR) or physically interacting (high harassment and high SCR) with a model individual. In a control treatment, no model was presented. The author found both males and females to copy similarly in both copying treatments, while no response was observed in the control. This contrasts with a study reporting that Atlantic molly (P. mexicana) males copy less under elevated SCR. Even though the author lack a compelling explanation as to why both congeners might differ, the author are tempted to argue that strong(er) benefits arising from copying may have selected guppies to copy in a broader range of contexts, including situations where the choosing individual incurs harassment or SCR.
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Chapter 37 - Across pre-industrial human societies mating is regulated, with arranged marriage, in which male parents select spouses for their female relatives, being the primary mode of long-term mating. This chapter reviews the model of sexual selection under parental choice that offers a good account for these patterns of human mating. This model postulates that parent-offspring conflict over mating induces parents to control the mate choices of their children, and the spouses they select for them are individuals who conform best to their preferences. By doing so, parents become a significant evolutionary force affecting the course of sexual selection. The model is applied in order to achieve a comprehension of the evolution of specific mating strategies. In particular, it is argued that in a context where mate choice is regulated, at least three mating strategies can thrive: addressing parental choice, addressing female choice and circumventing parental and female choice by force. Chapter 38 - The need for germplasm banks that safeguard the melon genetic resources is more than justified by the genetic erosion aggravated in the last few decades, not only in the cultivated materials, but also in traditional landraces and wild relatives. The classical and new technologies employed in all stages, from the sample prospecting to resources management, with the conservation and evaluation of the plant resources in between, are described. An added value to these germplasm collections is the use of the genetic resources there preserved in the melon breeding programs. The access to wild genetic resources make possible to exploit them, for instance, for germplasm enhancement incorporating disease resistances in elite varieties. In this sense, conventional breeding methods have rendered unquestionable benefits to agriculture, which can be accelerated by the appearance of new biotechnological tools and genomic resources as a result of the increasing number of vegetable species whose genomes have been or are being sequenced. All germplasm banks, and those preserving vegetable resources like melon in particular, will have to face up to the challenge of characterizing genetically their collections in order to maximize their use in breeding strategies assisted by molecular tools, like molecular markers. At the same time, the use of molecular markers can help to efficiently manage the resources by the creation of core collections. This would alleviate the problems derived from the high number of entries in many of them that is compromising some of the purposes for which they have been created, the conservation and the use of the genetic diversity originally prospected. The advantages in terms of labor and economical investment of preserving, characterizing and using a reduced subset of samples without a considerable sacrifice of the genetic diversity, are undeniable. Chapter 39 - The evolution of cultivated plants played important role in the ascent of humanity. A large number of theories exist about the evolution of the European grapevine (Vitis vinifera ssp. sativa L.), it is supposed, that woodland grape itself, or crossing with other species could be the progenitor. The woodland grape (Vitis vinifera ssp. sylvestris GMEL.) in Hungary is a protected species. The quest and preservation of its populations are significant in terms of nature conservation and reserve of biodiversity as well. In the years of 2010-2015 32 woodland grape genotypes were collected in the Szigetköz, Hungary and ex-situ preserved in the genebank National Agricultural Research and Innovation Centre, Research Institute for Viticulture and Enology, in Badacsonytomaj, Hungary. In 2015-2016 these genotypes were characterised by SSR analysis and were compared with 20 grape rootstocks and 16 Vitis vinifera ssp. sativa cultivars to ensure the true-to-typeness. Based on the results dendogram was constructed. In the dendogram the Vitis vinifera ssp. sylvestris accessions form an distinct group, but are closer to the Vitis vinifera ssp. sativa cultivars, than to the rootstocks. This raises the probability, that these accessions are true-to-type woodland grapes.
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Chapter 40 - Two major cherry species are grown for their fruit, the diploid sweet cherry and the tetraploid sour cherry. Cherries are characterized by high genetic diversity mainly due to their self-incompatibility propagation system. Estimation of the species phenological and genetic diversity has been performed using a number of different traits and marker systems including morphological and anatomical characteristics, as well as isoenzyme and molecular markers. Different molecular markers have been used, spanning from restriction fragment length polymorphisms (RFLPs) to single nucleotide polymorphisms (SNPs), simple sequence repeat (SSR) markers being the most frequently. Moreover, molecular markers have also been used to mark and trace specific agronomic traits, such as self-(in)compatibility (S-alleles) or fruit weight thus developing functional markers. The markers reviewed herein will be useful not only for monitoring the genetic diversity in cherry breeding programs, but also for gene conservation, while these or other markers may permit marker-assisted selection for favorable agronomic traits. Chapter 41 - Soybean purple seed stain (PSS) causes seed decay and purple seed discoloration, resulting in overall poor seed quality and reduced market grade and value. It is a prevalent soybean disease that also affects seed vigor and stem establishment. PSS is caused by the fungus Cercospora kikuchii and other Cercospora spp. The most common symptom of this disease occurs on the seed. Infected seeds may appear healthy or have discoloration in seed coat varying from pink to light or dark purple spots with range in sizes from a small speck to the entire seed coat. Warm and humid environments favor pathogen growth and disease development. Management strategies for this disease include crop rotation with non-legume or non-host crops, fungicides applications, and tilling the soil to disrupt spore dissemination. Along with these strategies, the use of resistant cultivars may provide more reliable and economical control of PSS, especially when environmental conditions are conducive for disease development. In this chapter, general information about the PSS and an overview of research on germplasm screening and genetic resistance are presented and discussed. Chapter 42 - The tissue cryopreservation represents an interesting tool for the conservation of animal biodiversity. The establishment of tissue banks has been indicated as a practical approach to the preservation of species and, associated with other biotechniques, it could provide the rescue or multiplication of endangered species. In general, a large number of wild species have been having their gonadal and somatic tissue cryopreserved and for this purpose, the vitrification is the method routinely used. There is a diversity in cryobiological properties and requirements among cell types within tissues, presenting a challenge for its procedure. Nevertheless, even with those obstacles, studies have shown satisfactory results in many wild mammalian species. The gonadal tissue use involves the possibility for the reestablishment of endocrine functions of the testes and ovary allowing the preservation and posterior use of spermatozoa and/or spermatogonial stem cell and oocytes for other assisted techniques. Recent developments in the autografting and xenografting of testes and ovary clearly demonstrated the potential value of cryopreserving gonadal tissue. Already on the somatic tissue, skin samples have been widely utilized because of the possibility of sampling a large group of animals, without a dependency of limitations regarding gender or age. Moreover, this tissue can be obtained quite easily at using a simple methodology with a reduced cost. In this sense, this chapter highlights the importance of applying tissue cryopreservation to wild mammals conservation at showing the most recent studies in this area and the perspectives for its use in conservative programs.
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Chapter 43 - The author sequenced mitochondrial genes (COI, COII, Cyt-b) of accepted Latin America tapir species (Tapirus pinchaque, T. terrestris and T. bairdii) as well as an alleged new species, T. kabomani. The mountain tapir (T. pinchaque) is a relatively rare large mammal species. Some population censuses indicate that no more than 2,000 mountain tapirs are left in the wilderness areas of Colombia, Ecuador and Peru. Our results showed that the gene diversity levels are medium to low with respect to other mammals sequenced for the same or similar genes. However, these gene diversity levels are not impoverished, which means that the genetic situation of this species is not as critical as its population censuses suggest. It will be crucial to determine the gene diversity levels in certain populations not included in the current work (the eastern and, possibly, western Andean Cordilleras in Colombia as well as the Tabaconas Namballe National Sanctuary in Peru), because they are probably the smallest populations of this species. On the other hand, the lowland tapir (T. terrestris), the species with the largest geographical distribution in Latin America, showed the highest gene diversity levels of all the other tapir species studied. Additionally, the genetic structure of T. terrestris is clearly more robust than that of T. pinchaque. Different geographic populations of both species showed different demographic trends throughout time. Our results including five samples of T. kabomani showed this taxon to be a haplogroup within T. terrestris, reducing the likelihood of T. kabomani being a new full species. Finally, the author also analyzed the influence of diverse Pleistocene climatic changes on the mitochondrial haplotype diversification of T. terrestris and T. pinchaque. The Pleistocene Refugia and the Recent Lake hypotheses probably played integral roles in the evolutionary history of T. terrestris. In contrast, the Pleistocene Refugia hypothesis involving the Andes, which probably played an important part in the genetic diversification of other mammals, did not have a significant impact on T. pinchaque. Chapter 44 - Plants are responsible for a significant part of food supply for the entire world, and through agriculture they play an extremely important socio-economic role for the mankind. Therefore, the development of genetically improved crops becomes even more relevant for it aims at an everlasting enhancement of agronomic traits of interest. For many years, plant genetic improvement program has been based in empiric selection of the target traits; however, significant advances were obtained in the last years. Many tools, allowing crops to be improved with greater optimization of the time needed to reach the necessary modifications, are currently available. Regarding the methods used in the genetic improvement, molecular studies have been essential to identify which genes are important for each specific agronomic trait, such as those related to tolerance to abiotic stress. Such studies contribute not only to a better understanding of the endogenous defense mechanisms of plants at molecular level by which these organisms adapt when facing hostile conditions, but also contribute to the generation of stress-tolerant crops by genetic engineering. These programs aim a significant productivity and sustainability that can be reached through soil preservation that is directly related to less necessity of farm inputs. Better adapted crop cultivars make it possible, as well as better use and decontamination of water resources. In this chapter the author attempt at providing an overview regarding strategies that have been used for prospection of genes related to the response of plants to abiotic stress. The combination of biotechnological and bioinformatics tools used in the identification of stress-related genes and development of genetically engineered crops by silencing and/or over-expression of specific genes will be presented in this chapter. Emphasis will be given to the drought and salinity that represent a major part of abiotic
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stresses by which the plants are often exposed, leading to serious production losses in many important crops worldwide. Chapter 45 - Proteinuria is the hallmark of diabetic nephropathy (DN) and vastly increases the incidence of cardio-vascular disease and mortality. Traditional Chinese Medicine (TCM) has been used for diabetes and its complications for thousands of years and appears to be promising in the treatment of proteinuria in DN patients. Clinical trials evaluating TCM for proteinuria, either used as a monotherapy or in combination with western medicine, has produced positive results. Although a large number of clinical studies have been conducted, the clinical evidence with regard to TCM for proteinuria in patients with DN remains inconclusive. The recent progression of evidence will be introduced in this chapter. Current basic research has disclosed that TCM might affect a variety of genes regarding DN etiology. This chapter will analyze recent achievements in this area and address the issue of the association between clinical evidence and the genetic effects of TCM. Chapter 46 - Ansamycins are composed of secondary metabolites possessing a high degree of activity against numerous types of Gram-positive and Gram-negative bacteria. Structurally, ansamycins are characterized by the presence of core structures including an aromatic moiety (benzene or naphthalene derivative) and an aliphatic chain. Most ansamycins were isolated and characterized from Actinomycetes, while a few were mined in higher plants, for example, maytansine and colubrinol. Due to the development of microbiological techniques, genetic engineering and recombinant proteins, a wealth of different types of ansamycins have been mined along with their biosynthetic gene clusters. This resulted in developed strategies for enhancement of ansamycin production as well as synthesis of novel structure derivatives. In this chapter, the author will describe the biochemistry and genetics of the most important members of ansamycin antibiotics and their applications as cytotoxic, anti-tumor, anti-parasitic and anti-bacterial agents. Thereafter, the future of ansamycins will be discussed to outline the most critically applied aspects in the last. Chapter 47 - Ubiquitously expressed protein products of BRCA1 and BRCA2 genes are implicated in processes fundamental to all cells, including DNA repair and recombination, checkpoint control of cell cycle, and transcription. BRCA gene mutations lead to disruption of BRCA proteins in mutation carrier cases and induce susceptibility to specific types of cancer. Among women with germline BRCA mutations near 50% of mammary malignancies are triple negative breast cancer (TNBC) presenting with a high grade histologically. Among women with breast cancer, TNBC was established in 57.1% of BRCA1-mutation positive and in 23.3% of BRCA2-mutation positive cases, whereas in only 13.8% of BRCA-proficient women. Although BRCA gene mutation carrier women usually exhibit clinical symptoms of defective estrogen receptor (ER) signaling; such as anovulatory infertility and early menopause, the serum estrogen levels of these patients are consequently elevated. In these cases, a compensatory feedback mechanism aims to break through the inherited or acquired ER resistance by increased estrogen synthesis so as to maintain the cellular estrogen surveillance. The higher the estrogen overproduction of BRCA-mutation positive cases, the higher the possibility of tumor-free survival. In conclusion, BRCA1 and BRCA2 gene mutations seem to increase the breast cancer risk, particularly that of TNBC, in case of insufficient compensation of defective ER signaling. Upregulation of these genes by means of elevated estrogen levels of high parity, artificial hormonal cycle created by oral contraceptives or a pregnancy mimicking high estrogen dose may decrease the excessive cancer risk of BRCA mutation positive women.
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Chapter 48 - Rett syndrome (RTT) is a neurodevelopmental disorder mainly caused by mutations in the MECP2 gene affecting around 1 in 10,000 female births. Mutations in the MECP2 gene have been associated with the onset of RTT. Clinical manifestations include severe linguistic and motor impairments that are the core of phenotype symptoms. Some patients show a moderate level of conservation of linguistic functions while others lose the use of functional verbal communication. The objectives of the present chapter are to study in depth the latest theoretical approaches to the link between linguistic processes and the specific RTT genotype. This chapter begins with a theoretical overview on cognitive alterations and then focuses on linguistic specific impairments characterized by the loss of articulation or the production of few functional sounds. A restricted sample shows the presence of verbal speech (Preserved Speech Variant). Renieri et al. (2009) proposed the term “Zappella variant” rather than “preserved speech variant” to describe milder forms of RTT, because other aspects, besides speech, are involved. The second part proposes a preliminary research which analyses the correlation between linguistic phenotype and specific genotype. Chapter 49 - Marfan Syndrome was originally described by Antoine Bernard-Jean Marfan in 1896 and is an uncommon inherited connective tissue abnormality which occurs as an autosomal dominant genetic disorder with frequent mutations. The patients have normal mentation but have a characteristic increase in height, abnormally long limbs, arachnodactyly, joint hypermobility, distinctive facial features, scoliosis, ectopia lentis, dural ectasia and array of aortic and cardiac abnormalities that are often life threatening. The genetic cause of the disease has been identified. Although some medical and surgical treatments are currently in practice they are not always helpful. The orthopaedic problems are often significant and sometimes require complex surgical interventions. Chapter 50 - Marfan syndrome (MFS) is a systemic connective tissue disorder that is caused by mutations in the extracellular matrix protein fibrillin-1. While MFS is considered to be at high risk of dental disorders and cardiovascular disease (CVD), little causal relationship has been provided to date. In this article, the author reviewed the prevalence of periodontitis in patients with MFS to assess the relationship between periodontal bacterial burden and CVD in MFS patients. Chapter 51 - Introduction: Pectus deformities can coexist with cardiovascular diseases. This association is well known in tissue conjonctive disorders such as Marfan's syndrom. Combined procedures can be performed safely and represent an interesting alternative in such situations. Valve sparing aortic root replacement has excellent long term outcomes and has become an increasigly popular alternative to aortic root replacement especially in young marfan patients to avoid lifelong anticoagulation. The author present our serie of single-stage pectus correction and cardiac surgery, and emphasize the role of aortic valve sparing interventions in such situations by a review of the literature. Methods: A retrospective review was conducted of patients who underwent chest deformity repair and cardiac surgery at the same time from January 2007 to May 2014. All datas were collected propestively in our data base. A review of literature was conducted to collect all published cases of combined valve sparing root replacement and correction of a chest wall deformity. Results: Including our serie (4 patients) 12 patients underwent a combined Tirone David and chest wall deformity correction. 10 patients underwent a Nuss procedure, 2 patients a modified Ravitch procedure. Conclusion: Combined technique of valve sparing aortic root replacement and correction of a chest wall deformity especially by Nuss technique is safe and effective. This strategy has excellent midterm results for both aortic and chest wall pathologies.
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Chapter 52 - Marfan syndrome frequently causes cardiac complications such as, aneurysm and dilatation of the aortic root. Many Marfan syndrome patients have these cardiovascular problems, and the surgical replacement of aortic and mitral valve, and aortic roots is frequently required. In addition, cases are associated with severe periodontitis, which is a chronic inflammation of the gingiva, periodontal ligament, and alveolar bone. Because of the surgical replacement, it is essential to prevent dental infection, such as infectious endocarditis caused by the periodontitis. In Marfan syndrome, an unfavorable oral hygiene due to the crowded teeth and narrow dental arch had been thought as a cause of severe periodontitis. However, clinical and basic studies have highlighted the genetic background as a pathogenesis of the severe periodontitis. It is suggested that cell alignment and tissue architecture of periodontal ligament are impaired in the model mice of Marfan syndrome. The model mice were more susceptible to alveolar bone resorption after the infection of Porphyromonas gingivalis, which is known to cause chronic periodontitis. It is likely that activated TGF-β signaling upregulates IL-17 and TNF-α levels, resulted in the increased alveolar bone resorption. In this review, the perspective of the dental management and the effect of angiotensin II receptor blocker are discussed. Chapter 53 - Preimplantation genetic diagnosis (PGD) was introduced 24 years ago with the purpose of performing genetic testing before pregnancy, in order to establish only unaffected pregnancies and avoid the need for pregnancy termination, which is the major limitation of traditional prenatal diagnosis. Despite the requirement for ovarian hyperstimulation and in vitro fertilization (IVF), needed to perform genetic testing of oocyte or embryo prior to transfer, PGD has been accepted in most parts of the world. Thousands of PGD cycles have now been performed for single gene disorders, with PGD presently offered for some indications that have never been practiced in prenatal diagnosis, such as late onset diseases with genetic predisposition, and preimplantation HLA typing. The present paper describes our experience on PGD for Marfan syndrome, caused by FBN1 gene, which was performed in 38 cases, as part of our PGD experience of 2,860 cycles for single gene disorders, which is the world’s largest PGD experience. Chapter 54 - The order Anura currently encompasses over 6,800 amphibian species distributed in 56 families and is an interesting group for cytogenetic studies. Whereas some groups of species present conservative karyotypes, others are highly variable in diploid number or number/location of diverse chromosomal markers, such as nucleolus organizer regions, heterochromatic bands and specific satellite DNA sites. In some cases, karyotypic variation overcomes morphological diversification, turning cytogenetics into a useful tool for taxonomy. Special variation is observed with respect to sex chromosomes and sex determination systems, with both female and male heterogameties observed. Although most species already karyotyped do not show sex chromosome heteromorphism, distinct levels of differentiation are observed between the sex chromosomes of several species, which makes this group particularly interesting for studies of sex chromosome evolution. In this chapter, the author explore the use of cytogenetic data for studies of frogs as well as the insights that hypotheses of phylogenetic relationships have added to this issue. In addition, the author provide a brief review of PcP190 satellite DNA (with new data for the genus Engystomops), sex chromosome systems and B chromosomes found in Anura. Chapter 55 - Humans are daily exposed to a variety of potentially harmful agents in the air they breathe, liquids they drink, food they eat and products they use. Long-standing evidence of the bond between health and environment has led to the recognition for the need of sustainable development. On the other hand, there is an increasing global awareness of the
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inevitable limits of individual health care and the need to complement such services with effective public health strategies. According to World Health Organization (WHO), cancer is a leading cause of death worldwide. Additionally, at least 200 000 people die every year from occupational or work-related cancers. Exposure assessment aims at prevention. Establishing the health effects of various activities and exposures requires information about the levels of exposure and the biological effects resulting from the interaction between the organism and the chemical agent. The resulting data provides a basis for designing effective prevention and mitigation strategies. Cytogenetic endpoints have long been applied in surveillance of human genotoxic exposure and early effects of genotoxic carcinogens. Assays measuring chromosomal aberrations (CAs), micronucleus (MN) and sister chromatid exchange (SCE) in lymphocytes are well-established techniques extensively used in human biomonitoring studies to assess DNA damage at the chromosomal level. The relevance of cytogenetic alterations as a cancer risk biomarker is further supported by epidemiologic data linking CAs and MN with cancer risk in human populations. Thus, the use of cytogenetic markers in human biomonitoring is of paramount importance due to its predictability regarding deleterious effects resulting from the exposure to environmental stressors. Chapter 56 - The great era for classical cytogenetics started sixty years ago with the description of the twenty-three pairs of human chromosomes and the discovery of the Philadelphia chromosome, the first known chromosomal defect associated with a specific type of cancer. Since then many recurrent chromosomal aberrations linked to specific hematological malignancies have been detected. The vast majority of these abnormalities can be detected by modern molecular genetic methods so it might seem there is no longer a need for microscopy. However, there is still a significant portion of hemato-oncologic patients for which the classical cytogenetic investigation is appropriate, i.e., cases with complex karyotypes. Complex karyotypes are characterized by the presence of three or more unrelated chromosomal aberrations co-existing in a single clone. Their occurrence is associated with adverse outcomes across the entire spectrum of hematologic malignancies. These aberrations are also a powerful diagnostic indicator for molecular targeted therapies, allogeneic stem cell transplantation or other generally more aggressive treatment strategies. Since the presence of complex karyotypes would be missed when using only molecular genetic methods, this highlights the irreplaceable role of classical cytogenetics as a first-tier analysis for the evaluation of complex structural chromosomal abnormalities in hemato-oncologic patients. Ideally, classical cytogenetics is then followed by more precise molecular genetic methods to identify specific chromosomal aberrations more deeply. Here the author focus on the complex karyotype issues in the myelodysplastic diseases, leukemias, lymphomas and multiple myelomas as seen daily in our center. Chapter 57 - Initially identified as Astyanax schubarti on the basis of morphological characteristics, its chromosomal analysis revealed a unique diploid number in the genus. With 42 chromosomes and the impossibility of homologous pairing, the karyotype of the individual was compared to a set of haploid complements of Astyanax schubarti (2n = 36 chromosomes) and Astyanax fasciatus (2n = 48 chromosomes). Natural hybrids are rare. The viability of a hybrid between species with such chromosomal discrepancy may offer important hypotheses to explain the morphological, molecular, and cytogenetic diversity of the genus. Chapter 58 - There are three distinct subtypes of Trichorhinophalangeal syndrome (TRPS); TRPS type I, TRPS type II and TRPS type III. Features common to all three subtypes include sparse, slowly growing scalp hair, laterally sparse eyebrows, a bulbous tip of the nose (pear-
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shaped), and protruding ears. The diagnosis of TRPS is based in typical clinical and radiographic features, as well as in the identification of causative mutations in the TRPS1 gene (TRPS type I and III) and in loss of functional copies of the TRPS1 and EXT1 genes (TRPS type II). The mode of inheritance in TRPS I and III is autosomal dominant, however de novo deletions of TRPS1 and EXT1 genes are the main defect of TRPS II. Parental balanced chromosomal rearrangements are an important cause of interstitial aberrations in TRPS. In cases of cytogenetically-invisible alterations, parental FISH analysis as well as aCGH should be considered as part of the clinical baseline testing. Treatment of clinical problems of TRPS types is mainly supportive and includes ectodermal and skeletal issues. The number of distinct syndromes as TRPS is rapidly increasing and their confirmation is necessary especially in cases that typical features are absent. Clinical geneticists should provide information for the families and advise them how to overcome problems. Chapter 59 - It is generally believed that genotype and adult lifestyle elements are primary risks of some metabolic diseases such as insulin resistance, obesity and diabetes mellitus in later life. However, increasing evidence demonstrates that early life malnutrition during the period of gestation and/or lactation may increase our susceptibility to such metabolic diseases in later life. The underlying mechanism is still not very clear. Recently, epigenetics is hypothesized to be the important molecular basis of the imbalanced early life nutrition and glucose metabolism disorders, which is known as "Developmental Origin of Health and Diseases" (DOHaD). Currently, there are substantial epidemiological studies and experimental animal models that have demonstrated nutritional disturbances during the critical periods of early life development can significantly impact the predisposition to developing some metabolic diseases in later life. The fundamental mechanism is that early developmental nutrition can regulate epigenetic modifications of some genes associated with development and metabolism. DNA methylation is the first discovered and one important epigenetic modification. MicroRNAs are recognized as an important epigenetic modification and they are a major class of small non-coding RNAs (about 20-22 nucleotides) which can mediate posttranscriptional regulation of target genes with cell differentiation and apoptosis. Recent studies suggest that DNA methylation and microRNAs maybe the crucial modulators of fetal epigenetic programming in nutrition and metabolic disorders. This chapter will focus on how early life nutrition can alter the epigenome, produce different phenotypes and alter disease susceptibilities, especially for impaired glucose metabolism. Chapter 60 - Oxidative stress is a state in which production of reactive oxygen species exceeds the capacity of antioxidant systems. Reactive oxygen species have one or more unpaired electrons, making them highly reactive with other cellular molecules such as protein, lipid, and nucleic acid. The peroxidation of polyunsaturated fatty acids in biological membranes results in impaired membrane integrity. Oxidatively modified proteins lose their capacity to carry out the physiological functions and they may form intracellular aggregates. Attacks of reactive oxygen species to DNA results in strand breakages and base oxidation. Major DNA oxidation product is 8-hydroxydeoxyguanosine which has a pro-mutagenic potential. Due to these damaging effects, oxidative stress plays an important role in various pathologies such as cancer, diabetes, chronic inflammatory diseases and neurodegenerative disorders. Epigenetic changes are regular and natural events which regulate gene expression without changing base sequences on DNA. Dysregulation of regular epigenetic mechanisms is a contributory factor for many of human pathologies. Recently, reactive oxygen species have been shown to cause epigenetic dysregulations that play a pivotal role in human disorders. The basic epigenetic
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mechanisms and their dysregulation by reactive oxygen species have been reviewed in this chapter. Chapter 61 - Cancer is one of the deadliest malignancies that have plagued mankind for decades. Epigenetic mechanism(s) play a central role in the homeostasis of normal cell proliferation and differentiation. Global epigenetic modifications are frequently associated with cancer initiation, progression, as well as metastasis. These changes include DNA methylation, histone lysine methylation/demethylation, acetylation/ deacetylation, including methylation and acetylation of non-histone proteins, and can alter the expression of various oncogenic signaling cascades, which in-turn can lead to uncontrolled proliferation. In this chapter, the author primarily focus on the major epigenetic changes that occur in oncogenes, tumor suppressor genes, transcription factors and cancer stem cells, which in turn mediate tumor growth. These modifications are controlled by regulatory enzymes such as DNA methyltransferase, histone acetyltransferases, histone deacetylase, lysine acetyltransferase, and arginine and lysine methyl transferases. In addition, the author also describe a few selected pharmacological agents that can modulate the action of these enzymes and display significant potential for cancer therapy. Chapter 62 - Alzheimer's Disease is one of the most common neurodegenerative disorders. Many efforts have been directed to prevent AD due to its rising prevalence and the lack of an effective curative treatment. Epigenetic changes are involved in regulation of gene expression, and may mediate various pathologies. Epigenetic changes are reversible so that can be easily modulated. Modulation of dysregulated epigenetic mechanisms is a promising therapeutic approach for many diseases. There is some evidence for epigenetic dysregulation at various levels contributing to AD pathogenesis. Despite the recent rapid accumulation of knowledge about AD pathogenesis, the role of epigenetic modifications has not been understood exactly. This chapter provides a brief overview about the role of epigenetic changes that are linked to AD pathogenesis and emerging targets for new therapeutic strategies in this field. Chapter 63 - Cardiovascular disease (CVD) is not a single condition, but an umbrella term used to describe a range of common diseases affecting the heart and the circulatory system. The term commonly includes diseases of the cardiac muscle and of the vascular system supplying the heart, brain, and other vital organs. Many of these conditions can be life-threatening. CVD is the leading cause of death in the world, affecting all populations, irrespective of demographic or socioeconomic differences and is responsible for one third of all deaths. In the present chapter, the author focus on the epigenetic control of embryonic cardiac development and the role of epigenetic mechanisms in CVD observed from results in human, animal and cell culture studies’ approaches. The author discuss the main epigenetic mechanisms involved in heart development and major CVDs such as coronary heart disease (CHD), heart failure (HF), myocardial infarction (MI), hypertension, stroke, arrhythmias, cardiomyopathy and cardiac hypertrophy (CH). Additionally, this chapter also focuses on the epigenetic modifiers that are involved in the development of CVD, and the potential utility of epigenetics-based therapeutic strategies in CVD. Chapter 64 - Extensive characterization has been performed on the genes and genetic mutations that are involved in spermatogenesis and male infertility, but the vast role of the sperm’s transcriptome and epigenome in male reproduction has yet to be completely explored. Recent research has established that epigenetic remodeling of the sperm is necessary for development and for its function following fertilization. The histone- retained regions of the sperm have been recently shown to carry the bivalent marks of activating histone H3 lysine K4
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trimethylation and the repressive H3 lysine K27 trimethylation. These bivalent histone marks facilitate the dynamic changes in stage-specific gene expression during sperm development. The paternal epigenome bears unique and important epigenetic modifications determined to be potentially important to the developing embryo, but the complete scope of its contribution is still emerging. Additionally, advances in assisted reproductive techniques have also suggested that alterations in the epigenetic profile in infertile men are transmitted to the developing embryo. This review will highlight the latest advances in epigenome profiling of the chromatin modifications during the development of immature to a mature sperm as well as provide a glimpse into the future role of epigenetic mechanisms in the generation of new germ cells/gametes from induced pluripotent stem cells, to treat male infertility. Chapter 65 - Epigenetic is defined as the study of mitotically and/or meiotically heritable changes in the gene function without changing DNA sequence and playing crucial roles both in normal development and human diseases. The molecular basis of epigenetic process consists of histone modifications, DNA methylation, positioning of histone variants, and non-coding RNAs. Genome-wide patterns of DNA and chromatin modifications together called as ‘epigenomes’. Epigenomes undergo precise, coordinated, reversible changes through the developmental stages and so contributes to the lineage and tissue specific expression of genes. In addition to the tissue specific impact, environmental factors such as nutrients, toxins, infections and hypoxia can also influence epigenomes. Distinct or global changes in the epigenetic landscape are hallmarks of chronic inflammation associated diseases. Alteration in methylation status of CpG sites, monoallelic silencing, and other epigenetic regulatory mechanisms have been observed in key inflammatory response genes. Epigenetic changes including DNA methylation, histone modification and noncoding RNA expression, were found associated with acute and chronic inflammatory disorders. Recently, therapies targeting epigenetic mechanisms are trending options for the treatment of chronic and degenerative disorders. Epigenetic drugs that are applied on animal models and some clinical trials are displaying positive therapeutic effects. Not only mono therapies but also combined usage of HDAC or DNMT inhibitors could be the next step for the epigenetic therapeutic modulations. Scope of this chapter is to provide an overview of the epigenetic modifications in inflammation and inflammation driven diseases. Chapter 66 - Obesity is a public health problem leading to morbidity and mortality throughout the world. It arises from the interactions between genetics and environmental factors. In recent years, susceptibility to obesity has been also linked to epigenetic factors. Epigenetics, is the study of heritable changes in gene expression which do not involve in the underlying DNA sequence. The epigenetic mechanisms include DNA methylation, covalent histone modifications, chromatin folding, miRNAs, and polycomb group repressive complexes. Both dietary factors and individual behaviors affect obesity development via epigenetic mechanisms. Epigenetic mechanisms are also linked to programmed changes in gene expression as a result of early environmental exposures during pregnancy which alter offspring growth and development. There is evidence that nutrient and environmental exposures during pregnancy may affect fetal/newborn development and result in offspring obesity or metabolic syndrome which is a cluster of metabolic abnormalities. Obesity related genes, epiobesigenes, display methylation patterns playing important roles in the development of obesity which are potential future epigenetic biomarkers of obesity. The susceptibility genes have been reported as FGF2, PTEN, CDKN1A, and ESR1, functional in adipogenesis; SOCS1 and SOCS3, functional in inflammation, and COX7A1, LPL, CAV1, and IGFBP3 which are functional in
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fat metabolism and insulin signaling. It is important to prevent this era’s epidemic, obesity, since it leads to chronic diseases including hypertension, atherosclerosis, insulin resistance, and moreover metabolic syndrome. It may be easier to prevent the progress of the disease by revealing the epigenetic mechanisms especially methylation profiles of the susceptibility genes. Chapter 67 - Entomopathogenic fungi have been mentioned as one of the best alternatives for insect pest control. These fungi cause insects death. More than 750 fungal species have been described infecting insects, some of the most utilized for insect control are: Beauveria bassiana and Metarhizium anisopliae. This is evidence of cosmopolitan distribution of entomopathogenic fungi and its evolutionary success, this type of fungi is related to a serie of interactions among fungi, plants, and insects. In this chapter, the main objective is to review and discuss the most recent information on genetics and evolution of entomopathogenic fungi. In this chapter are covered the following themes: Entomopathogens role in nature, Entomopathogenic fungi and their interactions with the insect immune system, Isolation and identification of entomopathogenic fungi, Genetic diversity among strains of entomopathogenic fungus, Genes involved in virulence of entomopathogenic fungi, Molecular phylogeny of entomopathogenic fungi and their biogeographic implications, Evolution of entomopathogenicity in fungi, Genetic improvement of entomopathogenic fungi for insect biocontrol, Future trends and Conclusions. Chapter 68 - The author sequenced the mitochondrial (mt) ND5 gene of 100 specimens of Eira barbara (Mustelidae, Carnivora). The samples represented six out of the seven putative morphological subspecies recognized for this Mustelidae species (E. b. inserta, E. b. sinuensis, E. b. poliocephala, E. b. peruana, E. b. madeirensis, and E. b. barbara) throughout Panama, Colombia, Venezuela, French Guiana, Brazil, Ecuador, Peru, Bolivia, Paraguay, and Argentina. The main results show that the genetic diversity levels for the overall samples and within each one of the aforementioned putative taxa were very high. The phylogenetic analyses showed that the ancestor of the Central and South-American E. barbara originated during the Miocene or Pliocene (6.3-4 millions of years ago, MYA). Furthermore, the ancestors of some geographical groups, (we detected at least four) originated during the Pliocene (3.7-2.5 MYA). These four groups (or lineages) were placed in the Cesar-Antioquia Departments (northern Colombia), Bolivia and northwestern Argentina, northern-central Peru, and in the trans-Andean area of Ecuador. However, during the Pleistocene, this species experienced a strong population expansion and many haplotypes expanded their geographical distributions. They became superimposed on the geographical areas of older geographical groups that originally differentiated during the Pliocene. Until new molecular studies are completed, including those with nuclear markers, the author proposed the existence of only two subspecies of E. barbara (E. b. inserta in southern Central America, and E. b. barbara for all South America). All of the demographic analyses showed a very strong population expansion for this species in the last 400,000 YA during the Pleistocene. Chapter 69 - Like other forms of diagnostics, genetic testing comes with a retinue of costs and benefits. Significant benefits in terms of morbidity and mortality have accrued to individuals tested for more prevalent genetic conditions like cystic fibrosis and sickle cell disease, including persons seen in the emergency room or identified through public health surveillance. These benefits do not mitigate the drawbacks of genetic testing, false and missed diagnoses and sheer cost among them. Both medicine and public health have aimed at means of maximizing genetic test benefits in the interventions that they apply. The President’s Precision Medicine Initiative (PMI) holds promise in that its results could be used to tailor
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medical treatments to the individual characteristics of patients, “precision” implying a more accurate and precise regimen overall. The National Cancer Institute (NCI) has already launched the NCI-MATCH precision medicine trial, which assigns targeted treatments based on the genetic abnormalities in a tumor, regardless of cancer type. Other trials, such as the NCI Pediatric MATCH trial, are yet to happen. The efficacy of cancer treatments also intersects public health concerns. The Evaluation of Genomic Applications in Practice and Prevention (EGAPP) Working Group has evaluated the use of UGT1A1 genotyping to determine the best dose of irinotecan to prevent side effects when treating patients for metastatic colorectal cancer. Analytic validity does not always equate with improved patient outcomes, however, thus the public health emphasis on development of a suitable evidence base for precision medical and public health efforts. The public health approach to precision medicine, or “precision public health”, differs from the medical approach in several important ways: (1) population-based with attention to at-risk populations, as opposed to being strictly individualized; (2) focus on primary and secondary prevention, rather than frank disease (tertiary prevention); and (3) prioritizing interventions that have already demonstrated readiness for large-scale implementation, in contrast to the undertaking of novel clinical trials. Precision public health is exemplified in the Centers for Disease Control and Prevention’s emphasis on the implementation of Tier 1 genetic tests that have passed systematic review for analytic and clinical validity and utility – the use of family history for referral for hereditary breast and ovarian cancer genetic testing (BRCA1/2 mutations), and hereditary nonpolyposis colorectal cancer cascade screening (Lynch syndrome MLH1, MSH2, MSH6 mutations). This paper will cross-compare the precision medical approach to cancer based on pharmacogenomic regimens using companion diagnostics, and the public health approach to precision management of hereditary cancer for 3 cancer types – lung, breast, and colorectal. It will describe methods of early detection and consider how lives can be saved through precise management – from predictive testing and cancer monitoring of the at-risk population, to tailored chemoprevention that fits the needs of the individual. In the population context, a cascade screening “multiplier effect” exists in that relatives can also be assessed and followed for mutations identified in the proband. Cost-benefit analyses (T4 translational research) of medical and public health approaches will be closely examined and compared. Points of commonality between the two approaches will also be discussed, since primary/secondary and tertiary disease prevention represent a continuum. These analyses point to the value of allocating resources towards the health of at-risk populations. Questions remain if particular forms of genetic testing are to become “universalized”, and if the needs of all atrisk groups, including racial-ethnic, are to be addressed. Chapter 70 - Williams syndrome (WS) is a genetic neurodevelopmental disorder (prevalence close to 1 in 20,000-30,000 births) resulting from the deletion of 16-25 genes on the long arm of Chromosome 7. Individuals with WS have an intelligence quotient of 40-70. Theirs is a unique neuropsychological profile, characterized by an apparent dissociation between cognition and language, as language is relatively well preserved, compared with other cognitive skills. However, a more complex profile is now emerging, with good lexical, shortterm memory (especially auditory-verbal) and face processing skills, but visuospatial (especially local processing of information), executive (planning and inhibition), memory (working memory and long-term) and attentional deficits. Individuals with WS also have specific auditory-perceptual cognitive skills (hyperacusis), category-specific perception of speech sounds, and musical skills that exceed their cognitive level. Other behavioral characteristics include hypersociability. In this chapter, the author provide a review of the
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literature on the specific neuropsychological profile of individuals with WS, from a cognitivebehavioral and neuroanatomical point of view. Early studies of the neuropsychological profile in WS focused on the dissociation between cognition and language. Since then, research has shown this syndrome to be more complex. Our aim is to highlight the heterogeneity of the cognitive profiles observed in this syndrome, and to identify the factors that might explain this heterogeneity. The complexity and specific features of the neuropsychological profile in WS need to be understood in order to develop therapeutic and learning methods adapted to the developmental pace of individuals with WS. Chapter 71 - Chromosome rearrangements are the most common genetic abnormalities in humans. Abnormal chromosomal configurations are formed among non-homologous chromosomes to allow full synapsis of homologous chromosomes at meiosis I of chromosome rearrangement carriers. These abnormal chromosomal configurations result in the production of gametes with various chromosomal complements due to malsegregation of derivative chromosomes or recombination. Most of the gametes have unbalanced chromosomal complements and only a small number of gametes have normal or balanced chromosomal complements. Carriers of balanced chromosome rearrangements are phenotypically normal but they are at an increased risk of abnormal pregnancies due to the unbalanced gametes. However, chromosome rearrangement carriers who cannot have babies or experience repeated abortions or abnormal pregnancies can have healthy babies after introduction of PGD. Balanced reciprocal translocation is the most common chromosome rearrangement. Thirty-two types of gametes can be produced in meiosis of reciprocal translocation carrier. According to the results that analyze the meiotic segregation of embryos from PGD cycles of reciprocal translocation carriers, 2:2 segregation is the main segregation mode. The meiotic segregation might be affected by the gender of carriers. The frequency of balanced embryos was not different between female and male carriers. However, the frequencies of 2:2 segregation, especially adjacent-1 segregation, 3:1 and 4:0 segregation were significantly different between female and male carriers. Robertsonian translocation is also common chromosome rearrangement in humans. Gametes with eight different chromosomal complements can be produced in Robertsonian translocation carriers. The frequency of balanced embryos was higher in male carriers than in female carriers. Carriers of complex chromosome rearrangements (CCR), very rare chromosome rearrangements, can achieve pregnancy by PGD although the number of normal or balanced embryos was extremely low. Although it is very difficult to estimate the rate of normal or balanced embryos, the rate is estimated to be less than 10% in PGD for CCR carriers. The 3:3 segregation and chaotic segregation (meiotic segregation whose meiotic segregation cannot be defined) are prevalent segregation modes in carriers of three-way translocation. In PGD cycles of CCR carriers, cycle cancellation is very frequent due to the absence of the normal of balanced embryos. Therefore, it is important that a large number of embryos are obtained in one cycle. Occasionally, abnormalities of chromosome rearrangementunrelated chromosomes are observed in embryos or abortuses although chromosome rearrangement-related chromosomes are normal or balanced. At present, PGD that diagnose all 24 chromosomes using array-CGH, SNP array or NGS is carried out worldwide. In those PGD cycles, the risk that embryo transfer is cancelled might be increased. However, the rate of normal or balanced embryos is unexpectedly increased and pregnancy rate is improved. Surely, PGD is the very effective assisted reproductive technique in achieving pregnancy of chromosome rearrangement carriers by preventing repeated abortions that chromosome rearrangement carriers usually experience. In the field of PGD for chromosome
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rearrangements, the next stage will be the development of technique that can discriminate between normal embryos and balanced embryos. Chromosome rearrangements, such as translocation (reciprocal or Robertsonian), inversion and complex chromosome rearrangements, are the most common genetic abnormalities in humans. Chromosome rearrangements are classified as balanced or unbalanced. Carriers of balanced chromosome rearrangements have the normal chromosomal complements but carriers of unbalanced chromosome rearrangements have additional or missing chromosomal material. Generally, the incidence of balanced chromosome rearrangements is about 0.19% of newborns. Carriers of balanced chromosome rearrangements are phenotypically normal because they have all the genetic materials. However, they are at an increased risk of implantation failure, repeated abortions or birth of chromosomally unbalanced offspring. Of course, not all carriers of balanced chromosome rearrangements experience the abnormal pregnancies. The abnormal pregnancies are resulted from the unbalanced gametes generated during meiosis of chromosome rearrangement carriers. Abnormal chromosomal configurations are formed to allow full synapses of homologous chromosomes during meiosis of balanced chromosome rearrangement carriers. The unbalanced gametes are produced as a results of malsegregation of these abnormal chromosomal configuration. Most of the gametes produced during meiosis of chromosome rearrangement carriers have unbalanced chromosomal complements. These unbalanced gametes result in implantation failure, repeated abortions or birth of chromosomally unbalanced offspring. After the first successful clinical application of preimplantation genetic diagnosis (PGD) in 1990, PGD has been widely used worldwide to select normal or balanced embryos in in vitro fertilization and embryo transfer (IVF-ET) programs of balanced chromosome rearrangement carriers. Cleavage-stage fluorescence in situ hybridization (FISH) has been widely used to PGD for carriers of balanced chromosome rearrangements till the early 2010s and PGD based on array comparative genomic hybridization (CGH) or next generation sequencing (NGS) is widely applied nowadays. The only abnormalities of rearranged chromosomes can be diagnosed in FISH-based PGD and the abnormalities of other chromosomes cannot be diagnosed. However, abnormalities of all 24 chromosomes can be diagnosed after the application of array CGH or NGS into PGD. Chapter 72 - Aims: Attention deficit hyperactivity disorder (ADHD) is a multifactorial psychiatric and neurobehavioral disorder. The brain-derived neurotrophic factor gene (BDNF) has been proposed as a strong candidate for this pathology. The aim of this study was to determine a family-based association between three polymorphisms of the BDNF gene and the ADHD in a Tabascan-Mexican population. Methods: The author analyzed the rs6265, rs12273363 and rs11030119 polymorphism of the BDNF gene through a family-based association study. A total of 105 individuals grouped in family-trios (mother, father and ADHD patient) were studied. Allelic and haplotypic transmission were assessed through transmission disequilibrium test (TDT), using HaploView software. Results: No statistically significant association was observed between the BDNF gene polymorphisms and the ADHD etiology in Tabascan-Mexican families: rs6265 (χ2 = 1.33; p = 0.24); rs12273363 (χ2 = 1.33; p = 0.24); rs11030119 (χ2 = 0.66; p = 0.41). Furthermore, no preference of transmission was observed for any of the haplotypes. Conclusions: It was not possible to prove any association between the BDNF gene polymorphic variants and ADHD in a Mexican population. Future studies comprising larger samples are necessary to determine the potential role of the BDNF gene in ADHD.
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Chapter 73 - Petroleum can be characterized as an oily, flammable substance, less dense than water, with a distinctive scent and color ranging from black to dark brown. The petroleum industry includes some global processes that can be highlighted by the environmental risks they present. At offshore platforms, the danger of a spill is aggravated by the density of the oil, which floats and is carried quickly through sea currents. Thus, in addition to the marine fauna, coastal fauna and flora (e.g., mangroves and estuaries) can also be affected by a leakage. Petroleum is predominantly composed of hydrocarbons, these can be degraded by several species of bacteria, which are of great interest for the bioremediation of contaminated environments. Among these species the author can mention Pseudomonas, Sphingomonas, Mycobacterium, Microbacterium and Gordonia. The success of a bioremediation process depends on numerous factors such as microbial biomass, population diversity, enzymatic activity, pH, temperature, and carbon source. Moreover, the strains have their genetic potential for bioremediation investigated through methods of analysis concerning genes related to the degradation of aliphatic and aromatic hydrocarbons mostly by oxygenases, such as the polycyclic aromatic hydrocarbon ring-hydroxylating dioxygenases (PAH-RHDα), and alkB (for n-alkane degradation) genes. The study of autochthonous microbial communities is of crucial importance for the understanding of the genetic and biotechnological potential of bioremediation in environments close to the areas of extraction and susceptible of contamination; it is also of great interest for industry and biotechnological development. This chapter covers the main aspects of petroleum, regarding its exploitation aspects, the process of geological formation, and environmental impacts. It will include topics in microbiology and genetics; for instance, metabolic pathways of hydrocarbon biodegradation, biofilm dynamics associated to the oil industry, metagenomics of communities in marine environments, corrosion influenced by microorganisms (CIM), microbial control methods related to the petroleum industry, and bioremediation will be discussed. Chapter 74 - In healthy individuals, the incessant activity of regulatory genetic mechanisms ensures the metabolic and proliferative equilibrium of cellular activity. In case of accidental defects occurring in any part of the system, a coordinated counteraction of numerous mediators may successfully help in the restoration of physiologic processes by means of either overexpression or hyperactivity. Even serious defects of genome stabilizer mechanisms may be kept in balance for a long duration, showing the clinical signs of good health. By contrast, due to the exhaustion of the compensatory processes, DNA defects may develop and lead to the clinical manifestations of diseases. Estrogen activated estrogen receptors (ERs) are the primary initiators and organizers of the up-regulatory circle of genome stabilization in correlation and crosstalk with aromatase enzyme and genome safeguarding proteins, such as BRCAs. The promoter regions of ESR1, BRCA1, and CYP19 aromatase genes exhibit strong triangular partnership for the harmonized regulation of the synthesis of ERs, BRCA proteins and aromatase enzyme, which can be reconstructed from the meticulous details of earlier scientific results. Considering the extreme capacities of ER-signaling for self-restoration, it is obvious that antiestrogen treatment, either ER binding by a false ligand or inhibition of estrogen synthesis, may provoke extreme compensatory actions in genetically proficient cases. Analyses of the results of genetic studies on tumor cells have shown that upregulation of ER-signaling induced by natural estrogen or antiestrogen is a beneficial defensive process even in tumor cells, promoting their domestication and elimination. A schematic representation of the main stream of upregulative genome stabilizing circle visualizes the possibilities for extreme counteractions against the toxic effects of antiestrogens. The presented complex genome
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stabilizer mechanisms reveal that cancer cells may preserve their residual capacity for the upregulation of ER-signaling, even if the efforts are not satisfactory. Chapter 75 - Rett syndrome (RTT) is a rare, neurodevelopmental genetic disorder that develops in early childhood and influences many functions within neurobehavioural domains. The core of phenotype symptoms includes severe linguistic and motor impairments. The onset of RTT is characterised by a gradual or sudden loss of speech and hand function followed by a slow decrease in acquired gross motor skills with subsequent severe functional dependence. RTT is associated primarily with mutations in MECP2, a gene located on the long arm of the X chromosome (Xq28). The severity of impairments depends not only on genotype, but on the extent of X inactivation. However, CDKL5 and FOXg1 gene mutations have been also identified in girls affected by atypical RTT. Despite sharing neurological features, subjects with RTT present considerable clinical variability. Research on effects of genotype of RTT is expanding in many directions. The current chapter, will discuss the correlations between genotype and motor abilities in subjects with RTT. The main aim of this chapter is to relate functional outcomes, in particular motor impairments, to mutation type in patients with RTT. This chapter begins with a theoretical overview on genetic alterations in RTT and then focuses on motor specific impairments. In the second part of this chapter, the author propose a preliminary research which analyzes the correlation between motor phenotype and specific genotype. Chapter 76 - Fragile X syndrome (FXS) is the most common inherited cause of intellectual disability. However, findings reported in cross-sectional studies on this population are heterogeneous. This chapter focuses on the longitudinal assessment of a boy with FXS from 12 months through to 6 years of age using two tests: one that assesses psychomotor development and another that assesses neuropsychological maturity. The child had attended an Early Childhood Development Intervention Center-ECDIC since 12 months old. He was administered the Brunet-Lézine Revised Scale of Psychomotor Development in Early Childhood until 36 months and underwent CUMANIN neuropsychological testing from age 3 to 6. The results obtained allow us to observe trends in the boy’s psychomotor development and neuropsychological maturity over time. Significant commonalities between these results and those of previous cross-sectional studies are discussed. Furthermore, some conclusions are drawn that may prove valuable to professionals and researchers interested in this syndrome. Chapter 77 - Cornelia de Lange syndrome (CdLS), also known as Bushy syndrome, Amsterdam dwarfism and Brachmann- de Lange syndrome is a genetic multi system disorder, usually caused by spontaneous mutation. Although present from birth, it may not always be immediately diagnosed. The estimated incidence is about 1:10,000-30,000 births. Both sexes are affected. Mortality is high early in life and neurosensory, craniofacial, musculoskeletal, cardiac and gastrointestinal abnormalities are all apparent. There is no known cure and treatment is supportive, requiring a team support system. Chapter 78 - Background: SCAs are the most frequently occurring chromosomal abnormalities with an incidence of 1 in 400 births. As the number of X chromosomes increases, the phenotypic severity increases as well and it is estimated that cognitive abilities decrease by 10–15 IQ points for each additional X chromosome. Aim of this paper is to illustrate clinical variability of cognitive-behavioral phenotype in the different SCAs. Design and Methods: The sample was composed by 53 subjects (mean age = 21.16 yrs, range: 13-54) with karyotype 47, XXY (73%), 49, XXXXY (7%), 48, XXYY (9%), mosaicism 47, XXY/48, XXXY (2%), 47, XYY (5%), 48, XXXY (2%), 49, XXXYY (2%). Only 5 subjects have been diagnosed
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prenatally (4 KS and 1 XXYY). Primary caregivers completed a comprehensive questionnaire detailing birth, medical, developmental and psychological history. Cognitive and behavioral assessment was performed with clinical interviews using DSM 5 criteria and psychometric questionnaires (WISC-R, WAIS-R, CPM, Token Test, VABS, SCL90, SCQ). Twenty-one sex and age matched subjects karyotypically normal were also evaluated from the behavioural point of view. Results: Mean IQ in typical KS was 87.45 ± 2 ds (sd = 20.12) range 45-123, VIQ 91.74 (sd = 19.55) range 50-130 and PIQ 86.87 (sd = 20.87) range 50-126. Mean IQ in other SCAs was 68.71 (sd = 20.81) range 45-106, VIQ 69.36 (sd = 21.97) range 47-113 and PIQ 74.72 (sd = 21.70) range 45-112. In CPM KS subjects scored 27.75 (range 13-36) and 31.50 in the Token Test (range 21-35) while in CPM the other SCAs subjects scored 22.27 (range 10-35) and 22.50 (range 9-31) in the Token Test (p94% NJ bootstrap support, supporting the notion that these are three new reducing PKS clades. As fungal polyketides from these entomopathogens represent valuable natural product resources, these PKSs phylogeny and expression data would be important for further studies of valuable polyketides in the future. Metarhizium is a frequently isolated from soil environments. However, profound knowledge of natural occurrence and distribution, genetic diversity and community structure of the species is required to evaluate consequences of biocontrol initiatives (Meyling and Eilenberg, 2007). In 2009, Bischoff et al., provided a multilocus phylogeny of the Metarhizium anisopliae (Metschn.) Sorokin lineage and revised the taxonomy recognizing nine species within the M. anisopliae lineage, including several cryptic species. Given the cryptic diversity within the M. anisopliae lineage, discrimination of species cannot solely be based on morphology but requires the use of molecular methods for accurate identification. For species discrimination, 5’ end of the elongation factor 1a has been highlighted as a reliable marker (Bischoff et al., 2009). However, this region does not provide sufficient resolution for identification of genotypes within species. Thus, for genotyping and assessing within species diversity, simple sequence repeat (SSR) markers are currently among the most suitable markers (Enkerli and Widmer, 2010). Enkerli et al., (2005) and Oulevey et al., (2009) have developed SSR markers for strain-level genotyping within the M. anisopliae lineage, which have successfully been used to investigate molecular diversity of isolates collection (Velasquez et al., 2007; Oulevey et al., 2009). In 2014, Steinwender et al., evaluated Metarhizium community in soil from an agricultural field in Denmark using Tenebrio molitor as bait insect. By sequence analysis of 5’ end of elongation factor 1a and their genotypic diversity characterized by multilocus simple sequence repeat (SSR) typing, 123 isolates were identified. In this study, Metarhizium brunneum was most frequent (78.8%) followed by M. robertsii (14.6%), while M. majus and M. flavoviride were infrequent (3.3% each). It revealed co-occurrence of at four Metarhizium species in the soil of the same agro ecosystem with a single M. brunneum multilocus genotype being highly prevalent. Abundance of a single genotype could be due to variability among Metarhizium genotypes in response to biotic and abiotic factors prevalent in the ecosystem (Bidochka et al., 2005). Moreover, five genotypes of M. brunneum and six genotypes of M. robertsii were identified among the isolates based on SSR fragment length
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analysis, demonstrating to be highly effective in detecting genotypic diversity beyond what could be found by sequencing 5’ EF-1α within the M. anisopliae lineage. Additionally, the entomopathogenic hyphomycete Paecilomyces fumosoroseus is a promising candidate for biocontrol of the Silverleaf Whitefly Bemisia tabaci-argentifolii (Hemiptera: Aleyrodidae), which is considered as a major insect pest in field and greenhouse crops (Lacey et al., 1996). Since 1995, Tigano-Milani et al., demonstrated intraspecific genetic variation in this fungus by RAPD-PCR and tRNA-PCR. However, the analysis of 28S-rDNA sequences did not provide enough information for differentiation of P. fumosorosesus isolates. Fargues et al., (2002) investigated the genetic variability in 48 isolates of this fungus by analyzing the RFLPs and sequence data of the internal transcribed spacer sequences ribosomal RNA gene (rDNA-ITS). Digestion with six endonucleases (AluI, HaeIII, Hin6I, HpaII, NdeII, and SmaI) allowed their separation into three distinct groups. The group 1 was composed of strains isolated only from the host B. tabaci-argentifolii. By contrast, the group 3 included strains from various insect host and geographical origins. These data were strongly supported by phylogenetic analysis of rDNA-ITS sequence that recognized three monophyletic groups within the P. fumosoroseus complex. Thus, these molecular tools could be useful to assess genetic relatedness of these species into the monitoring of such biocontrol products. Genetic variations has been also suggested in P. farinosus according to the broad geographic and host origins and morphological variation correlated with one or more distinguishing adaptations. In the study of Chew et al., (1997) the genetic relatedness of twenty isolates of P. farinosus collected from seven insect species in eastern Canada was determined by RAPD analysis. All P. farinosus isolates were clearly distinguished from three other entomopathogenic fungi, including P. fumosoroseus; however, RAPD banding patterns did not correlate with ecological backgrounds or morphological phenotypes. These observations support the conclusion that P. farinosus from eastern Canada is not composed of strains which can be separated on the basis of the ecological or morphological criteria selected.
EVOLUTION OF PATHOGENICITY GENES IN ENTOMOPATHOGENS Entomopathogenous fungi have devolped production of different enzyme and metabolites to parasitize susceptible insect hosts, among them, hydrolytic, assimilatory, and/or detoxifying enzymes such as lipase/esterases, catalases, cytochrome P450s, proteases, and chitinases; and (b) secondary metabolites which facilitate infection (Ortiz-Urquiza and Keyhani, 2013). In B. bassiana bacterial-like toxins and effector-type proteins were reported (Xiao et al., 2012). Evolution of the genes codifying for these enzymes has been in a convergent way. Phylogenomic studies of different species in the Cordyceps/Metarhizium genera suggest that have evolved into insect pathogens independently of each other, and that their similar large secretomes and gene family expansions are due to convergent evolution (Zheng et al., 2011). Sequencing of B. bassiana genome confirmed that ascomycete entomopathogenicity is polyphyletic and convergent evolution to insect pathogenicity (Xiao et al., 2012). Lipases are the first enzymes synthesized by the entomopathogenic fungi, in Beauveria bassiana and Metarhizium robertsii was reported a cytochrome P450 subfamily, these enzyme break down long-chain alkenes and fatty acids (Sanchez-Perez et al., 2014). Then, proteases
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are synthesized. The catalytic function of proteases is to hydrolyze proteins releasing amino acids which could be used for fungal nutrition (Ventura-Sobrevilla et al., 2015). One of the protease produced are subtilisin type (Pr1) which are considered as virulence indicators. This type of enzyme are regulated by a signal transduction mechanism activated by the protein kinase A (PKA) mediated by AMPc (Sanchez-Perez et al., 2014). Other important group of enzyme produced by the entomopathogen fungi is chitinases which are used to break down the insect chitin. Entomopathogens fungi descend from plant–asociated fungi. Quiroz-Velasquez et al., (2014), studying the transcriptome of Lagenidium giganteum which is an oomycete entomopathogenic mentioned that alignments of the cellulose synthase sequence indicated that this fungus appears to be evolved from a phytopathogenic ancestor. In addition, these fungal species have retained genes indicative of plant associations, and may share similar cores of virulence factors. Members of the glycoside hydrolase family 5 subfamily 27 (GH5_27) have been proposed as putative virulence factors which may be active on the host insect cuticle, these genes are shared by different entomopathogenic fungi. These virulence factors may be very host specific with a very low risk of attacking non-target organisms or beneficial insects (Shahid et al., 2012). Metarhizium and Beauveria are also found as plant symbionts. Recent studies showed that these fungi are more closely related to grass endophytes and developed genes for insect pathogenesis while maintaining an endophytic lifestyle. Some genes for insect pathogenesis may have been co-opted from genes involved in endophytic colonization. In contrast, other genes may be multifunctional and serve in both lifestyles (Barelli et al., 2015).
Contraction and Expansion of Different Gene Families Different entomopathogenic fungi such as Ophiocordyceps polyrhachis-furcata, Beauveria bassiana, Metarhizium robertsii, Metarhizium acridum, Cordyceps militaris, and Ophiocordyceps sinensis have similar genes implicated in pathogenicity and virulence. In B. bassiana, many species-specific virulence genes and gene family expansions and contractions correlate with host ranges and pathogenic strategies (Xiao et al., 2012). Contractions of some gene families in this type of fungi are implicated in narrow host-range insect species (specialists), some of these genes are cuticle-degrading genes and families of pathogen-insect interaction (PHI) genes. In some of the most specialized entomopathogens such as O. polyrhachis-furcata, for many genes-families has the least number of genes found (Wichadakul et al., 2015). Reduction in gene family sizes was reported in other entomopathogen (Hirsutella thompsonii) (Agrawal et al., 2015). The loss of different genes involved with pathogenicity result in a reduced capacity to exploit larger ranges of insect hosts and therefore in the different level of host specificity. Specialization is associated with retention of sexuality and rapid evolution of existing protein sequences (Hua et al., 2014). It is reported a co-evolution between entomopathogens and insect-host which in some case followed different patterns. Jensen et al., (2009) indicated that because of a high diversification over time among dipteran insects, the insect pathogenic fungi associated with these insects have also diversified. On the other hand, expansions of genes involved in 1) the production of bacterial-like toxins, and 2) retrotransposable elements have been mentioned in O. polyrhachis-furcata, in comparison to other entomopathogenic fungi. Expansions of gene families suggest an adaptation to particular environments or sophisticated mechanisms underlying pathogenicity
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through retrotransposons (Wichadakul et al., 2015). Similar pattern of evolutionary expansion of gene families such as chitinases, lipases, and proteases has been reported for Beauveria bassiana, Cordyceps militaris, and Metarhizium anisopliae which could be attributed to their insect killing strategies and host ranges (Agrawal et al., 2015). Generalist entomopathogens attack a wide range of insects; this condition has been associated with protein-family expansion, loss of genome-defense mechanisms, genome restructuring, horizontal gene transfer, and positive selection that accelerated after reinforcement of reproductive isolation. Generalists evolved from specialists via transitional species with intermediate host ranges and that this shift paralleled insect evolution (Hua et al., 2014). Species from the Metarhizium and Beauveria genera are recognized as generalists infecting a range of insects (Meyling et al., 2011).
Entomopathogen Fungi Pathogenicity versus Insect-Host Defense Insects have evolved different mechanisms in response to pathogens attack, some of these mechanisms are: production of (epi) cuticular antimicrobial lipids, proteins, and metabolites; (b) shedding of the cuticle during development; and (c) behavioral-environmental adaptations (Ortiz-Urquiza et al., 2013). After 25th generations under constant selective pressure from Beauveria bassiana, the larvae of Greater wax moth, Galleria mellonella, exhibited significantly enhanced resistance, which was specific to this pathogen, and not to another insect pathogenic fungus, Metarhizium anisopliae (Dubovskiy et al., 2013). It was hypothesized by the same authors that insects developed a transgenerationally primed resistance which was achieved not by compromising life-history traits but rather by prioritizing and re-allocating pathogen-species-specific augmentations to integumental front-line defenses that are most likely to be encountered by invading fungi. However, there is a coevolution between entomopathogen virulence factors and host defense molecules of insects. Virulence is thought to co evolve as a result of reciprocal selection between pathogens and their hosts. Because of shorter generation times and smaller genomes, microbes exhibit a high evolutionary adaptability in comparison with their hosts. In contrasts, insects can only compete with its pathogens if they develop mechanisms providing a comparable genetic plasticity (Vilcinskas, 2010). During B. bassiana infection, Galleria mellonella systemic immune defenses are suppressed in favor of a more limited but targeted repertoire of enhanced responses in the cuticle and epidermis of the integument (Dubovskiy et al., 2013). If a diversification of fungal proteinases for pathogenesis arose, an expansion of host proteinase inhibitors subsets contributing to insect innate immunity may occur. For example, the spectrum of proteolytic enzymes encompasses thermolysin-like metalloproteinases and this is associated with the pathogen-virulence. This spectrum putatively promoted the evolution of corresponding host inhibitors of these virulence factors (Vilcinskas, 2010). The same authors mentioned other molecular adaptations for host insect’s defense such as sensing and feedback-loop regulation of microbial metalloproteinases. The genetic events behind the countermeasures in host defense effectors are gene or domain duplication and shuffling by recombination. The entomopathogens also develop different strategies in order to avoid the host insect’s defenses. It has been proposed that M. anisopliae and B. bassiana survive to insect phagocytic haemocytes which are analogous to the mammalian macrophages as consequence of adaptations that have evolved in order to avoid predation by soil amoebae (Bidochka et al., 2010).
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GENETIC IMPROVEMENT OF ENTOMOPATHOGENIC FUNGI FOR INSECT BIOCONTROL Mycoinsecticides are being used for the control of many insect pests as an environmentally acceptable alternative to chemical insecticides uses (Leger et al., 1996; Hussain et al., 2014; Ortiz, et al., 2015). From 1960s, a substantial number of mycoinsecticides have been developed worldwide (Sardul et al. 2012). All groups of insects may be affected and over 700 species of fungi from around 90 genera are pathogenic to insects (Khachatourians and Sohail, 2008). Basically, the fungi pathogen activity depends on the ability of its enzymatic equipment, consisting of lipases, proteases and chitinases, which are in charge of breaking down the insect’s integument (Sardul et al., 2012). Besides exoenzymes, the entomopathogenic fungi are reported to secrete toxin proteins and metabolites in vitro and sometime in vivo as well. There are a number of toxic compounds in the filtrate of entomopathogenic fungi such as small secondary metabolites, cyclic peptides and macromolecular proteins (Khan et al., 2012). To improve both commercial and technical efficiency of these entomopathogen fungi, a large number of studies had been conducting to improve their virulence which can be achieved by understanding the mechanisms of fungal pathogenesis and genetically modifying targeted genes. In response, Fan et al., (2010) in order to accelerate penetration speed and have better target protein-chitin on the cuticle, they constructed a hybrid protease (CDEP-BmChBD) by fusion of a chitin binding domain BmChBD from Bombyx mori chitinase to the C-terminal of CDEP1, a subtilisin-like protease from B. bassiana. After comparing studies, the hybrid protease was able to bind chitin and released greater amounts of peptides/proteins from insect cuticles. The insecticidal activity of B. bassiana was improved by including proteases, CDEP-1 or CDEP: BmChBD produced in Pichia pastoris, as an additive, however, the improved effect of CDEP: BmChBD was significantly higher than that of CDEP-1. Expression of the hybrid protease in B. bassiana also significantly increased fungal virulence compared to wild-type and strains overexpressing the native protease. These results demonstrated that rational design of virulence factor is a potential strategy for strain improvement by genetic engineering. Recently, a cytochrome P450 subfamily, referred as CYP52XI and MrCYP52 has been identified in Beauveria bassiana and Metarhizium robertsii, respectively (Sanchez-Perez et al., 2014). Entomopathogens such as M. anisopliae and B. bassiana are well characterized in respect to pathogenicity to several insects and have been used as myco-biocontrol agents for biological control of agriculture pests worldwide (Sardul et al., 2012). Lenger et al., (2012) reported the development of a genetically improved entomopathogenic fungus because integration of copies from the gene encoding a regulated cuticle degrading protease (Prl) which were inserted into the genome of M. anisopliae. Prl was constitutively overproduced in the hemolymph of Manduca sexta, activating the prophenoloxidase system. The combined toxic effects of Prl and the reaction products of phenoloxidase caused larvae challenged with the engineered fungus to exhibit a 25% reduction in time of death and reduced food consumption by 40% compared to infections by the wildtype fungus. In addition, infected insects were rapidly melanized, and the resulting cadavers were poor substrates for fungal sporulation. Fang et al., (2009) found that overexpression of a subtilisin-like protease (Pr1A) or a chitinase (Bbchit1) resulted in increased virulence of M. anisopliae and B. bassiana,
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respectively. In this study, they found that a mixture of the B. bassiana Pr1A homolog (CDEP1) and Bbchit1 for degradation of insect cuticle were in vitro more efficient than either CDEP1 or Bbchit1 alone. Based on this, they constructed three plasmids; (1) Bbchit1, (2) CDEP1, and (3) a fusion gene of Bbchit1 linked to CDEP1 each under the control of the constitutive gpd promoter from Aspergillus nidulans. B. bassiana transformants secreting the fusion protein (CDEP1:Bbchit1) which penetrated the cuticle significantly faster than the wild type or transformants overexpressing either Bbchit1 or CDEP1. Compared to the wild type, the transformant overexpressing CDEP1 showed a 12.5% reduction in LT (50), without a reduction in LC (50). The LT (50) of the transformant expressing CDEP1:Bbchit1 was reduced by 24.9%. Strikingly, expression of CDEP1:Bbchit1 resulted in a 60.5% reduction in LC (50), more than twice the reduction obtained by overexpression of Bbchit1 (28.5%). This work represents a significant step towards the development of hypervirulent insect pathogens for effective pest control.
FUTURE TRENDS It is very important to investigate more about the relation of entomopathogenic fungi to grass endophytes and how they developed genes for insect pathogenesis while maintaining an endophytic lifestyle. Although, there is known that some genes for insect pathogenesis may have been co-opted from genes involved in endophytic colonization, it is important to understand the protein changes that lead this adaptation. On the other hand, it has been mentioned that other genes may be multifunctional and serve in both lifestyles, but still there lack of knowledge about the genes modification and protein changes to have this dual activity. Advances in molecular tools for phylogeny analysis could lead to significant new insights that should allow us a better understand of the ecology of fungal entomopathogens as well as their additional roles in nature, including as plant endophytes, antagonists of plant pathogens, beneficial rhizosphere-associates and possibly even plant growth promoters. Although some entomopathogen fungi are well known, some of the fungi of the Entomophtorales order such as Entomophtora, Erynia and Pandora species are poorly investigated because, the culture medium used to propagate entomopathogens such as Beauveria, Metarhizium, and Lecanicillium, are not the most adequate for these especial entomopathogens.
CONCLUSION Different fungal entomopathogenic species such as B. bassiana, M. acridum, M. anisopliae, and Metarhizium brunneum have showed commercial potential for insect control, and are considered as friendly mycoinsecticide. The complete genome of some entomopathogen fungi such as B. bassiana has been sequenced, which has revealed multiples gene associated with virulence. In addition, many bacterial-like toxins and effector-type proteins were also discovered. Entomopathogenic fungi are able to adapt to different environments by activating well-define gene sets. During infection entomopathogenic fungi, many genes are involved in seven steps: host adhesion, germination, cuticle degradation,
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growth as blastospores, host colonization and killing, immune response interactions and hyphal extrusion and conidiation.
ACKNOWLEDGMENTS A. Garcia, M. Michel, and S. Villarreal want to thank to the National Council of Science and Technology of Mexico (CONACYT) for the financial support during their postgraduate studies.
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BIOGRAPHICAL SKETCH Name: Raúl Rodríguez-Herrera Affiliation: School of Chemistry- Universidad Autónoma de Coahuila Education: Agriculture minor Horticulture. 1981. Universidad Autónoma Agraria Antonio Narro. México M. S. Plant Breeding. 1986. Universidad Autónoma Agraria Antonio Narro. México Ph. D. Plant Breeding 1999. Texas A&M University. USA. Post-doctorate and sabbatical training 1999, 2014. United States Department of Agriculture. USA Address: School of Chemistry Universidad Autónoma de Coahuila Blvd. V. Carranza e Ing. José Cárdenas S/n Col. Republica Saltillo Coahuila 25280 México Research and Professional Experience: 1999-to date Full professor- Universidad Autónoma de Coahuila- México 1999 Post-doctorate training Texas A&M University-USDA USA 1997-1998 Graduate Assistant Texas A&M University-USA 1986-1998 Researcher-National Research Institute for Forestal, Agricultural and LivestockMexico 1982-1984 Administrator- Ministry of Agrarian Reform-Mexico Professional Appointments: Mexican Society of Biotechnology and Bioengineering. México Directive Committee Symposium “Genomes and Proteomes in XXI Century.” México Institute of Food Technologists. USA Mexican Association of Food Science (AMECA). Mexico Honours 1981. Graduated with honours from the UAAAN. 1988. Award to outstanding research. PCCMCA Congress. San José, Costa Rican. 1990. Award to outstanding research. PCCMCA Congress. San Salvador, El Salvador. 2001. Distinguished as National Researcher- Level 2 Mexico. 2003. National Prize on Food Science and Technology. CONACYT-Coca Cola México, D. F. 2005. Coahuila State Prize for outstanding Food Research Project. Saltillo Coahuila, Mexico. 2005. National Agro-Bio Prize on Agricultural Biotechnology-Mexico Publications Last 3 Years: Selected Publications (Total 216 refereed publications, 60 divulgation papers, 51 book chapters, 5 books, 6 patents, 8 bulletins, recent refereed publications are listed).
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Meléndez Rentería, NP., Aguilar, CN., Rodríguez Herrera, R., Nevárez Moorillón, GV., 2013. Microbiological effect of fermented Mexican oregano (Lippia berlandieri Schauer) waste. Waste and Biomass Valorization. DOI 10.1007/s12649-013-9222-2 M. Cruz-Requena, H. De La Garza-Toledo, C. N. Aguilar-González, A. Aguilera-Carbó, H. Reyes-Valdés, M. Rutiaga and R. Rodríguez-Herrera. 2013. Chemical and molecular properties of sotol plants (Dasylirion cedrosanum) of different sex and its fermentation products. International Journal of Basic and Applied Chemical Sciences 3 (1):. 41-49. Diana B. Muñiz-Márquez, Guillermo C. Martínez-Ávila, Jorge E. Wong-Paz, Ruth Belmares-Cerda, Raúl Rodríguez-Herrera, Cristóbal N. Aguilar. 2013. Ultrasoundassisted extraction of phenolic compounds from Laurus nobilis L. and their antioxidant activity.” Ultrasonics Sonochemistry 20: 1149–1154. doi: 10.1016/j.ultsonch.2013. 02.008. Juan A. Ascacio-Valdés, José J. Buenrostro, Reynaldo De la Cruz, Leonardo Sepúlveda, Antonio F. Aguilera, Arely Prado, Juan C. Contreras, Raúl Rodríguez and Cristóbal N. Aguilar. 2014. Fungal biodegradation of pomegranate ellagitannins. Basic Journal of Microbiology. 54:28-34. DOI 10.1002/jobm.201200278. Ascacio-Valdés, Juan, Burboa, Edgardo, Aguilera-Carbo Antonio F., Aparicio Mario, Pérez-Schmidt Ramón, Rodríguez Raúl, Aguilar Cristóbal N. (2013). An antifungal ellagitannin isolated from Euphorbia antisyphilitica Zucc. Asian Pac J Trop Biomed. 3(1): 41–46. doi: 10.1016/S2221-1691(13)60021-0. IF = 0.371. T. López, A. Prado-Barragán, G.V. Nevárez-Moorillón, J. C. Contreras, R. Rodríguez and C.N. Aguilar. 2013. Incremento de la capacidad antioxidante de extractos de pulpa de café por fermentación láctica en medio sólido CyTA – Journal of Food, [Increase in antioxidant capacity of coffee pulp extracts by lactic acid fermentation in solid medium CyTA - Journal of Food], 11(4):359-365. dx.doi.org/10. 1080/19476337.2013.773563. Luis C. Mata, Catalina Chavez, Raúl Rodríguez-Herrera, Daniel Hernández-Castillo and Cristobal N. Aguilar. 2013. Growth inhibition of some phytopathogenic bacteria by cellfree extracts from Enterococcus sp. British Biotechnology Journal, 3(3): 359-366. Emilio Ochoa-Reyes, Gabriela Martínez-Vazquez, Saul Saucedo-Pompa, Julio Montañez, Romeo Rojas-Molina, Miguel A. de Leon-Zapata, Raúl Rodríguez-Herrera and Cristóbal N. Aguilar. 2013. Improvement of shelf life quality of green bell peppers using edible coating formulations. Journal of Microbiology, Biotechnology and Food Sciences, 2 (6) 2448-2451 Tanya Cecilia Espinoza-Hernández, Raúl Rodríguez-Herrera, Cristóbal Noé AguilarGonzález, Faustino Lara-Victoriano, Manuel Humberto Reyes-Valdés and Francisco Castillo- Reyes. 2013. Characterization of three novel pigment-producing Penicillium strains isolated from the Mexican semi-desert. African Journal of Biotechnology 12(22): 3405-3413. Lorena Lara-Fernández, Heliodoro de la Garza-Toledo, Jorge E. Wong-Paz, Ruth Belmares, Raúl Rodríguez-Herrera and Cristóbal N. Aguilar. 2013. Separation conditions and evaluation of antioxidant properties of boldo (Peumus boldus) extracts. Journal of Medicinal Plant Research, 7(15): 911-917. Delgado-García, M. Cruz Hernández, M.A., De la Garza-Rodríguez, I., Balagurusamy N, Aguilar, C., Rodríguez-Herrera, R. 2013. Characterization of halophilic microorganisms isolated from Mexican saline soils, ARPN Journal of Agricultural and Biological Science, 8(6):457-464.
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A. Garcia, M. Michel, S. Villarreal et al. Ascacio-Valdés, J., Aguilera-Carbo Antonio F., Rodríguez Raúl, Aguilar Cristóbal N. (2013). Análisis de ácido elágico en algunas plantas del semidesierto mexicano. [Analysis of ellagic acid in some plants of the Mexican semi-desert]. Revista Mexicana de Ciencias Farmacéuticas. 44(2): 36-40. Y.N. Mora, J.C. Contreras, C. N. Aguilar, P. Meléndez, I. De la Garza, R. Rodríguez, 2013. Chemical composition and functional properties from different sources of dietary fiber. American Journal of Food and Nutrition. 1(3):27-33. DOI:10.12691/ajfn-1-3-2. Mondragon-Preciado, G., Escalante-Minakata, P., Osuna-Castro, J.A., Ibarra-Junquera, V., Morlett-Chavez, J.A., Aguilar-Gonzalez, C.N., Rodriguez-Herrera, R., 2013. Bacteriocinas: características y aplicación en alimentos. [Bacteriocins: characteristics and application in food]. Investigacion y Ciencia 59: 64-70. M. Cruz-Requena, C.N. Aguilar-González, J. Espinoza-Velázquez, M.H. Reyes-Valdés and R. Rodríguez-Herrera. 2013. AFLPs loci associated with polyembryonic maize using selective genotyping analysis. Israel Journal of Plant Sciences. 61(1-4):46-50. DOI: 10.1080/07929978.2014.950045 Jorge E. Wong-Paz, Diana B. Muñiz-Márquez, Pedro Aguilar-Zárate, Raúl RodríguezHerrera and Cristóbal N. Aguilar. 2013. Microplate quantification of total phenolic content from plant extracts obtained by conventional and ultrasound methods. Phytochem. Anal. DOI 10.1002/pca.2512. D.B. Muñiz-Márquez, R. Rodríguez, N. Balagurusamy, M.L. Carrillo, R. Belmares, J.C. Contreras, G.V. Nevárez & C.N. Aguilar (2014) Phenolic content and antioxidant capacity of extracts of Laurus nobilis L., Coriandrum sativum L. and Amaranthus hybridus L., CyTA - Journal of Food, 12:3, 271-276, DOI: 10.1080/19476337 .2013.847500 V. Padilla-García, F. Castillo-Reyes, C. N. Aguilar-González, A. Cuenca-Arana, A. Téllez-Jurado, M. H. Reyes-Valdez and R. Rodríguez-Herrera. 2014. Molecular characterization of six tannase-producers Aspergillus strains using PCR-RFLP of ITS and IGS regions and RAPD´s. International Journal of Molecular Biology 8(12): 451459. GCG Martinez-Avila, A.F. Aguilera, S. Saucedo, R. Rojas, R. Rodriguez and CN Aguilar. 2014. Fruit wastes fermentation for phenolic antioxidants production and their application in manufacture of edible coatings and films, Critical Reviews in Food Science and Nutrition. 54(3):303-11 DOI:10.1080/10408398.2011.584135 ISSN 1040-8398 (Print), 1549-7852 (Online). JCR FI= 4.510. Salvador Eduardo Vásquez Rivera, Mayra Alejandra Escobar-Saucedo, Diana Morales, Cristóbal Noé Aguilar and Raúl Rodríguez-Herrera. 2014. Synergistic effects of ethanolic plant extract mixtures against food-borne pathogen bacteria. African Journal of Biotechnology 13(5): 699-704. Norma P. Meléndez, Virginia Nevárez-Moorillón, Raúl Rodríguez-Herrera, José C. Espinoza and Cristóbal N. Aguilar. 2014. A microassay for quantification of 2,2diphenyl-1- picrylhydracyl (DPPH) free radical scavenging. African Journal of Biochemistry Research 8(1):14-18. Dulce A. Flores-Maltos, Solange I. Mussatto, Juan C. Contreras Esquivel, Juan J. Buenrostro, Raúl Rodríguez, José A. Teixeira and Cristóbal N. Aguilar. 2014. Typical Mexican agroindustrial residues as supports for solid-state fermentation. American Journal of Agricultural and Biological Sciences 9 (3): 289-293.
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Reynaldo De la Cruz Quiroz, Sevastianos Roussos, Daniel Hernández, Raúl Rodríguez, Francisco Castillo, and Cristóbal N. Aguilar. 2014. Challenges and opportunities of the bio-pesticides production by solid-state fermentation: filamentous fungi as a model. Critical Reviews in Biotechnology. (doi:10.3109/07388551.2013.857292). Veana F., Fuentes-Garibay J.A., Aguilar C.N., Rodríguez-Herrera R., Guerrero-Olazarán M., Viader-Salvadó J. M. (2014). Gene encoding a novel invertase from a xerophilic Aspergillus niger strain and production of the enzyme in Pichia pastoris. Enzyme and Microbial Technology 63: 28-33. M. Delgado, J. Mendez, R. Rodríguez-Herrera, C.N. Aguilar, M. Cruz-Hernández & N. Balagurusamy (2014): Characterization of phosphate-solubilizing bacteria isolated from the arid soils of a semi-desert region of north-east Mexico, Biological Agriculture & Horticulture: An International Journal for Sustainable Production Systems, 30(3):211– 217. DOI: 10.1080/01448765.2014.909742 Jorge E. Wong-Paz, Juan C. Contreras-Esquivel, Diana Muñiz-Marquez, Ruth Belmares, Raul Rodriguez, Patricia Flores and Cristobal N. Aguilar. 2014. Microwave-assisted extraction of phenolic antioxidants from semiarid plants. American Journal of Agricultural and Biological Sciences 9 (3): 299-310. ISSN: 1557-4989doi:10.3844/ ajabssp.2014. 299.310. K. Cruz-Aldaco, C. N. Aguilar, A. Ilyina, R. Rodríguez-Herrera and J. L. MartínezHernández. 2014. Surface adhesion fermentation for lipase production by Mucor griseocyanus Micol. Apl. Int., 26(1): 9-16. J.A. Borrego-Terrazas, F. Lara-Victoriano, A.C. Flores-Gallegos, F. Veana, C.N. Aguilar and R. Rodríguez-Herrera. 2014. Nucleotide and amino acid variation of tannase gene from different xerophylic Aspergillus strains. Canadian Journal of Microbiology. 60: 509–516. 10.1139/cjm-2014-0163. Chávez-González, Mónica L.; Guyot, Sylvain; Rodríguez-Herrera, Raúl, PradoBarragán, Arely; Aguilar, Cristóbal N. 2014. Production profiles of phenolics from fungal tannic acid biodegradation in submerged and solid-state fermentation. Process Biochemistry. 49(4):541-546. Ayerim Hernández-Almanza, Julio Montanez-Sáenz, Cristian Martínez-Ávila, Raúl Rodríguez-Herrera, Cristóbal N. Aguilar. 2014. Carotenoid production by Rhodotorula glutinis YB-252 in solid-state fermentation. Food Bioscience 7:31–36. Hernández-Almanza, Ayerim; Cesar Montanez, Julio; Aguilar-González, Miguel A.; Martínez-Ávila, Cristian; Rodríguez-Herrera, Raúl; Aguilar, Cristóbal N. 2014. Rhodotorula glutinisas source of pigments and metabolites for food industry. Food Bioscience. 5(3):64-72. Burboa E.A., Ascacio V., J.A., Zugasti C., A., Rodriguez H., R., Aguilar G., C.N. 2014. Capacidad antioxidante y antibacteriana de extractos de residuos de candelilla. [Antioxidant and antibacterial capacity of candelilla waste extracts]. Revista Mexicana de Ciencias Farmaceuticas. 45(1):51-56. Sepúlveda, L.; Buenrostro -Figueroa, J. J.; Ascacio-Valdés, J A.; Aguilera-Carbó, A. F.; Rodríguez -Herrera, R.; Contreras-Esquivel, J. C.; Aguilar, C. N. 2014. Submerged culture for production of ellagic acid from pomegranate husk by Aspergillus niger GH1. Micología Aplicada International, 26(2): 27-35.
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Aguilar, C.N., R. Rodríguez H., S. Saucedo P., D. Jasso C. 2008. Phyto-chemicals from Mexican semi-desert: from plant to natural chemicals and biotechnology. Ed. Path Design. Saltillo Coahuila México 579 p (in Spanish). Soto-Cruz, O., P.M. Ángel, A. Gallegos-Infante and R. Rodríguez-Herrera. 2008. Advances in Food Science and Food Biotechnology in Developing Countries. AMECA. Saltillo Coahuila México 330 p. Rodríguez H., R., O. Soto C., J.L. Martínez, C.N. Aguilar. 2009. Proteomes and Genomes in the XXI century: Environmental Biotechnology. Ed. Valle del Candamo, Monclova Coah. México 356 p. (in Spanish). Rodríguez Herrera, R., Aguilar González, C.N., Simpson-Williamson, J.K., Gutiérrez Sánchez, G.. 2011 Phytopathology in the omics era. Signpost Research. Kerlala India. 366p. ISBN 978-81-308-0438-5. Flores AC., González VM., Aguilar CN., Rodríguez R., 2014. Microbial Biofertilizers. Ed. Plaza y Valdes. 434p. ISBN: 978-607-402-177-1. Mexico (in Spanish).
In: Encyclopedia of Genetics: New Research (8 Volume Set) ISBN: 978-1-53614-451-2 Editor: Heidi Carlson © 2019 Nova Science Publishers, Inc.
Chapter 68
MITOCHONDRIAL POPULATION GENETICS INFERENCES ABOUT THE PHYLOGEOGRAPHY AND SYSTEMATICS OF THE TAYRA (EIRA BARBARA, MUSTELIDAE, CARNIVORA) Manuel Ruiz-García1,, Nicolás Lichilín-Ortíz1, Yolanda Mejia1, Juan Manuel Ortega1 and Joseph Mark Shostell2 1
Laboratorio de Genética de Poblaciones-Biología Evolutiva, Unidad de Genética. Departamento de Biología, Facultad de Ciencias. Pontificia Universidad Javeriana, Bogotá DC, Colombia 2 Department of Math Science and Technology, University of Minnesota Crookston, Crookston, MN, US
ABSTRACT We sequenced the mitochondrial (mt) ND5 gene of 100 specimens of Eira barbara (Mustelidae, Carnivora). The samples represented six out of the seven putative morphological subspecies recognized for this Mustelidae species (E. b. inserta, E. b. sinuensis, E. b. poliocephala, E. b. peruana, E. b. madeirensis, and E. b. barbara) throughout Panama, Colombia, Venezuela, French Guiana, Brazil, Ecuador, Peru, Bolivia, Paraguay, and Argentina. The main results show that the genetic diversity levels for the overall samples and within each one of the aforementioned putative taxa were very high. The phylogenetic analyses showed that the ancestor of the Central and South-American E. barbara originated during the Miocene or Pliocene (6.3-4 millions of years ago, MYA). Furthermore, the ancestors of some geographical groups, (we detected at least four) originated during the Pliocene (3.7-2.5 MYA). These four groups (or lineages) were placed in the Cesar-Antioquia Departments (northern Colombia), Bolivia and northwestern
Corresponding Author’s Email: [email protected], [email protected].
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Argentina, northern-central Peru, and in the trans-Andean area of Ecuador. However, during the Pleistocene, this species experienced a strong population expansion and many haplotypes expanded their geographical distributions. They became superimposed on the geographical areas of older geographical groups that originally differentiated during the Pliocene. Until new molecular studies are completed, including those with nuclear markers, we proposed the existence of only two subspecies of E. barbara (E. b. inserta in southern Central America, and E. b. barbara for all South America). All of the demographic analyses showed a very strong population expansion for this species in the last 400,000 YA during the Pleistocene.
Keywords: Tayra, Eira barbara, mitochondrial ND5 gene, putative geographical subspecies, genetic diversity, phylogeography, population expansion during the Pleistocene
INTRODUCTION Tayra (Eira barbara) is a Mustelidae (Carnivora, Mammalia) with a long, and slender body. Its length varies from 56 to 71 cm, not including a 37 to 46 cm long bushy tail. Its weight ranges from 2.7 to 7 kg with males larger than females. This species has short, dark brown to black fur that is relatively uniform across the body, limbs, and tail, except for a yellow or orange spot on the chest. The fur on the head and neck is much paler, typically tan or greyish in color. The head has small, rounded, ears, long whiskers and black eyes with a blue-green shine. The feet have toes of unequal length with tips that form a strongly curved line when held together. The claws are short and curved, but strong, being adapted for climbing and running rather than digging. This species occurs from southern Veracruz (Mexico) throughout Central America and across South America to northern Argentina save for the high Andes and the Caatinga and Cerrado (eastern Brazil; Emmons and Feer 1990). It is one of the most common medium-size predators throughout its range (Emmons and Feer 1990). E. barbara is a diurnal, sometimes crepuscular species (González-Maya et al., 2015), with a solitary behavior and large home range (Sunquist et al., 1989). Emmons and Feer (1990) showed that the tayra inhabits tropical and subtropical forests, secondary rain forests, gallery forests, gardens, cloud forests, and dry scrub forests. Hall and Dalquest (1963) affirmed that it can live near human disturbed habitats. It frequently occurs in agricultural areas and along the edge of human settlements. Tayra usually inhabits areas below 1,200 m, but there are reports of it being in areas as high as 2,400 m (Eisenberg 1989, Emmons and Feer 1990) and it is common at 2,000 m (Cuarón et al., 2016). Its diet is omnivorous, including fruits, carrion, small vertebrates, insects, honey and small vertebrates such as marsupials, rodents, and iguanids among others (Cabrera and Yepes 1960, Hall and Dalquest 1963, Emmons and Feer 1990). This species is listed as Least Concern (Cuarón et al., 2016). Cabrera (1957) and Hall (1981) recognized seven morphological subspecies, two in Central America and five in South-America: 1- E. b. senex (Thomas in 1900). The type locality is Hacienda Tortugas, Jalapa, Veracruz, Mexico; 2- E. b. inserta (Allen in 1908), with the type locality in Ulse, Matagalpa, Nicaragua; 3- E. b. sinuensis (Humboldt in 1812), with the type locality for the Sinu River in the Bolivar Department in northern Colombia; 4- E. b. poliocephala (Traill in 1821), with type locality Demerara in Guyana; 5- E. b. madeirensis (Lonnberg in 1913), with type locality in Humaitá, Madeira River, Brazilian Amazon; 6- E. b.
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peruana (Nehring in 1886), with type locality in YuracYaku in the San Martin Department in Peru and 7- E. b. barbara (Linnaeus in 1758) with the type locality assigned by Lonnberg (1913) to Pernanbuco, Brazil. See Figure 1. Although it is a relatively common species, only one preliminary study on its molecular population genetics and infra-specific systematics has been published (Ruiz-García et al., 2013). Therefore, we expanded upon our initial molecular population genetics study with mitochondrial genes of the tayra with the following main aims: 1- To estimate the mitochondrial levels of genetic diversity in the overall tayra population and in some putative morphological subspecies; 2- To determine if there is a correlation between the molecular clades obtained in the phylogenetic analyses with the traditional putative morphological and geographical subspecies of tayras; 3- To estimate the possible temporal splits in the mitochondrial diversification within the evolution of the tayra; and 4- To determine if demographic evolutionary changes have characterized the natural history of the tayra.
MATERIALS AND METHODS Samples We sequenced 100 tayras at the mt ND5 gene. The samples came from 11 countries and represent seven of the eight putative morphological subspecies (Table 1 & Figure 1). They are: 1- Argentina, eight individuals (putative E. b. barbara); 2- Bolivia, 16 specimens (putative E. b. barbara); 3- Brazil, nine exemplars (four putative E. b. barbara; five putative E. b. madeirensis); 4- Colombia, 12 individuals (three putative E. b. madeirensis; nine putative E. b. sinuensis); 5- Ecuador, 27 specimens (four putative E. b. sinuensis; 23 putative E. b. madeirensis); 6- French Guiana, five exemplars (putative E. b. poliocephala); 7- Panama, one individual (putative E. b. inserta); 8- Paraguay, four specimens (putative E. b. barbara); 9Peru, 17 exemplars (nine putative E. b. peruana; eight putative E. b. madeirensis); 10- Trinidad and Tobago, one individual (putative E. b. poliocephala). Thus, these samples represent six out of the seven putative morphological subspecies recognized for this species. The DNA of some of the tayra individuals we analyzed was extracted from hairs obtained from animals found alive in diverse Indian communities throughout Central and South America. Another fraction of the DNA was obtained from skins, bones, and teeth of hunted individuals of E. barbara. We requested permission to collect biological materials from these skins, bones, and teeth that were already present in the Indian communities. In the case of the skins, we sampled 1-2 cm2. Communities were visited only once. All sample donations were voluntary, and no financial or other incentive was offered for supplying specimens for analysis. For more information about sample permissions, see the Acknowledgment section.
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Figure 1. Map with the approximate geographical distributions of the six putative geographical tayra’s subspecies (Eira barbara) sequenced at the mitochondrial ND5 gene. X represent localities where samples were obtained.
Molecular Analyses The DNA from skins and bones was extracted using the phenol-chloroform procedure (Sambrook et al., 1989), whereas DNA samples from hairs and teeth were extracted with 10% Chelex resin (Walsh et al., 1991). Primers and PCR conditions for the ND5 gene (265 bp) were brought to a volume of 25 l with 13.5 l of Mili-Q H2O, 3 l of MgCl2 1 mM, 1 l of dNTPs 0.2 mM, 1 l of each primer (0.1 M), 2.5 l of buffer 10X, and one unity of Taq Polymerase with 50-100 ng of DNA. We used the primers L12673 and H12977 (5’GGTGCAACTCCAAATAAAAGTA -3’ and 5´- AGAATTCTATGATGGATCATGT 3’; Waits et al., 1999). The PCR temperatures were 95° for 5 minutes followed by 10 cycles of 1 minute at 95°C, 1 minute at 64°C and 1.5 minute at 72°C, 25 cycles of 1 minute at 95°C, 1 minute at 60°C and 1.5 minute at 72°C and one final extension of 15 minutes at 72°C. All amplifications, including positive and negative controls, were checked in 2% agarose gels. Those samples that amplified were purified using membrane-binding spin columns (Qiagen). The PCR products were sequenced in both directions using the Big Dye™ kit in an ABI 377A automated DNA sequencer. A consensus of the forward and reverse sequences was determined using the Sequencher program. It is possible that some of the sequences represent numts (mitochondrial DNA fragments inserted into the nuclear genome) rather than true mtDNA (Chung and Steiper, 2008). However, we note that all amino acid translations of the obtained sequences showed the presence of initial start and terminal stop codons and the absence of premature stop codons. Protein translation was also checked to evaluate the possible presence of numts. Nevertheless, the mutations we observed were synonymous changes, thus suggesting that there were no numts in the sequences.
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Table 1. Samples of taya (Eira barbara), by countries, localities and putative geographic subspecies, sequenced at the mitochondrial ND5 gene for this work Country Panama Colombia
Number of samples studied 1 12
Localities
Putative geographic subspecies E. b. inserta 9 E. b. sinuensis 3 E. b. madeirensis
Trinidad & Tobago French Guiana Ecuador
1
1 Chiriqui 2 Agustín Codazzi-Cesar 1 PNN Los Katios-Chocó 1 Zaragoza-Antioquia 1 Yarumal-Antioquia 1 PNN Tama, Norte de Santander 2 El Tuparro-Vichada 1 San Martín-Meta 1 Playa Blanca-Guainia 1 Puerto Arara-Amazonas 1 Leticia-Amazonas 1 Rio Claro
5
5 Camopi River
E. b. poliocephala
27
4 E. b. sinuensis 22 E. b. madeirensis
Peru
17
3 Yarinacocha-Pastaza 2 Sucua-Morona Santiago 2 La Perla-Santo Domingo de Tsáchilas 2 Miashi-Zamora 2 Pillaro-Tungurahua 2 Canelos-Pastaza 2 Sarayaku-Pastaza 1 Coca-Napo 1 Misahuallí-Napo 1 La Bonita-Napo 1 Macuma-Morona Santiago 1 Loreto-Napo 1 Hushafindi-Napo 1 Pichincha 1 Miazal-Morona Santiago 1 Yangana-Loja 1 Cononaco-Pastaza 1 Tinigua-Napo 1 Nuevo Rocafuerte-Napo 2 Iquitos-Loreto 1 Caballococha-Loreto 1 Nauta-Loreto 1 Luceropata-Loreto 1 Puerto Venus-Loreto 1 Lamas-San Martin 1 Moyobamba-San Martin 1 Rioja-San Martin 1 Nuevo Cajamarca-San Martin 1 Bagua Grande-Amazonas 1 Oxamarca-Cajamarca 1 Puerto Bermudez-Pasco
E. b. poliocephala
8 E. b. madeirensis 9 E. b. peruana
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Country
Number of samples studied
Bolivia
16
Brasil
9
Paraguay
4
Argentina
8
Localities 1 Bolognesi-Ucayali 1 Seshea-Ucayali 1 Manu-Madre de Dios 1 Marcapata-Cusco 3 Ballivian-Beni 1 Piso Firme-Beni 1 Nicolas Suarez-Pando 1 Sena-Pando 1 Franz Tamayo-La Paz 1 Coripata-La Paz 1 Cajuata-La Paz 1 Totora-Cochabamba 1 Pojo-Cochabamba 1 Vila vila-Cochabamba 1 Julpe River-Cochabamba 1 El Cerro-Santa Cruz 1 Puerto Pailas-Santa Cruz 1 San Jose de Chuiquitos-Santa Cruz 3 Foz de Iguazu-Parana 3 Novo Airao-Negro RiverAmazonas 1 Moora-Negro River-Amazonas 1 Paumari-Yavari River-Amazonas 1 Tres Rios-Rio de Janeiro 2 Los Cedrales 2 Loma Grande 2 Salta 1 Abra Pampa-Jujuy 1 Humahuaca-Jujuy 1 La Cocha-Tucuman 1 Burruyacu-Tucuman 1 El Dorado-Misiones 1 San Javier-Misiones
Putative geographic subspecies
E. b. barbara
4 E. b. barbara 5 E. b. madeirensis
E. b. barbara E. b. barbara
Data Analyses Genetic Diversity The statistics used to determine the genetic diversity in the overall tayra sample and within the five South American putative tayra subspecies were as follows: the haplotypic diversity (Hd), the nucleotide diversity (), the average number of nucleotide differences (K), and the statistic by sequence. These statistics were obtained using the DNAsp 5.1 software (Librado and Rozas, 2009).
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Phylogenetics Analyses The sequence alignments were carried out manually as well as with the DNA Alignment program (Fluxus Technology Ltd.). MrModeltest v2.3 software (Nylander, 2004) and Mega 6.05 software (Tamura et al., 2013) were applied to determine the best evolutionary mutation model. The Akaike and Bayesian information criteria (AIC and BIC; Akaike, 1974; Schwarz, 1978) were used to determine the best evolutionary nucleotide model in the overall sequence set of E. barbara. Phylogenetic trees were constructed by using two procedures: Maximum Likelihood (MLT) and Bayesian analysis (BI). The ML trees were obtained using the RAxML v.7.2.6 software (Stamatakis, 2006). To select the best fitting model, 50 independent iterations were run using three data partitions (codon 1, codon 2, and codon 3). Additionally, 50 iterations were run using two data partitions (codons 1+2 combined, and codon 3). For each sequence data set, the GTR + G model (General Time Reversible + gamma distributed rate variation among sites; Tavaré, 1986) was used to search for the ML tree and topologic support was estimated with 500 bootstrap replicates using GTR. A BI tree was completed with the BEAST v. 1.8.1 program (Drummond et al., 2012). Four independent iterations were run using three data partitions (codon 1, codon 2, and codon 3) with six MCMC chains sampled every 10,000 generations for 30 million generations after a burn-in period of 3 million generations. We checked for convergence using Tracer v1.6 (Rambaut et al., 2013). We plotted the likelihood versus generation and estimated the effective sample size (ESS > 200) of all parameters across the four independent analyses to determine convergence and optimal results. The results from different runs were combined using LogCombiner v1.8.0 and TreeAnnotator v1.8.0 software (Rambaut and Drummond, 2013). A Yule speciation model and a relaxed molecular clock with an uncorrelated log-normal rate of distribution (Drummond et al., 2006) were used. Posterior probability values provide an assessment of the degree of support of each node on the tree. The tree was visualized in FigTree v. 1.4 software (Rambaut, 2012). This BI tree was used to estimate the time to most recent common ancestor (TMRCA) for the different nodes. We used a prior of 24.0 + 1 MYA (95% confidence interval: 26.24-22.36 MYA) for the split between the ancestors of Eira and one Procyonidae, as Potos flavus. This prior followed the results of Koepfli et al., (2008). Following Pennington and Dick (2010), the previous BI temporal estimates belong to one of two different approaches for inferring divergence times. The first approach is based on fossilcalibrated DNA phylogenies. The second approach is named “borrowed molecular clocks” and uses direct nucleotide substitution rates inferred from other taxa. For this second approach, we used a median joining network (MJN) with the help of Network 4.6.10 software from Fluxus Technology Ltd (Bandelt et al., 1999). The statistic (Morral et al., 1994) was estimated and transformed into years of divergence among the haplotypes studied. To determine the temporal splits, it is necessary to estimate a mutation rate at the mt ND5 gene. We used a nucleotide divergence of 1.22% per each million years (Culver et al., 2000), which yielded one mutation each 309,310 years. This estimate was obtained for Felidae. In this work, we assumed that this mutation rate could be similar in Mustelidae. The networks are more appropriate for intraspecific phylogenies than tree algorithms because they explicitly allow for the co-existence of ancestral and descendant haplotypes, whereas trees treat all sequences as terminal taxa (Posada and Crandall, 2001).
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Heterogeneity Analyses Several procedures were carried out to estimate the genetic heterogeneity among the diverse putative tayra subspecies analyzed. To determine the overall genetic heterogeneity in E. barbara, we used the statistics GST, ST, NST and FST (Nei, 1973; Hudson et al., 1992). Additionally, we relied on the HST, KST, KST*, Z, Z*, and Snn tests (Hudson, 2000), and the chi-square test on the haplotypic frequencies with permutation tests using 10,000 replicates to measure genetic heterogeneity. Also, we estimated the genetic heterogeneity by subspecies pairs within E. barbara. For this task, we used three procedures: 1- Exact tests with Markov chains, 10,000 dememorizations parameters, 20 batches, and 5,000 iterations per batch; 2Indirect gene flow estimates (Nm) from the FST statistic with a n-dimensional island model (Slatkin, 1985; Ruiz-García, 1993, 1994, 1997, 1999; Ruiz-García and Álvarez, 2000); and 3Kimura 2P genetic distances (Kimura, 1980). These genetic heterogeneity statistics were completed with DNAsp 5.1 (Librado and Rozas, 2009) and Arlequin 3.5.1.2 (Excoffier and Lischer, 2010). Demographic Changes We relied on three procedures to detect possible historical population changes in the tayra: 1- We used the Strobeck's S statistic (Strobeck, 1987), Fu and Li D* and F* tests (Fu and Li, 1993), the Fu FS statistic (Fu, 1997), the Tajima D test (Tajima, 1989) and the R2 statistic (Ramos-Onsins and Rozas, 2002). A 95% confidence interval and probabilities were obtained with 10,000 coalescence permutations. 2- The mismatch distribution (pairwise sequence differences) was obtained following the method of Rogers and Harpending (1992) and Rogers et al., (1996). We used the raggedness rg statistic to determine the similarity between the observed and the theoretical curves. 3- A Bayesian skyline plot (BSP) was obtained by means of the BEAST v. 1.8.1 and Tracer v1.6 software. The Coalescent-Bayesian skyline option in the tree priors was selected with four steps and a piecewise-constant skyline model with 30,000,000 generations (the first 3 million discarded as burn-in), kappa with log Normal [1, 1.25] and Skyline population size with uniform [0, infinite; initial value 80]. In the Tracer v1.6, the marginal densities of temporal splits were analyzed and the Bayesian Skyline reconstruction option was selected for the trees log file. A stepwise (constant) Bayesian skyline variant was selected with the maximum time as the upper 95% high posterior density (HPD) and the trace of the root height as the treeModel.rootHeight. To determine the time range for possible demographic changes for E. barbara, we consider that the evolution of this taxon occurred during the last 4 MY.
RESULTS Genetic Diversity and Phylogenetic Inferences The BIC showed that the best nucleotide substitution model was T92 + G (7,649.51). In contrast, the AIC detected GTR + G + I (5,881.29) as the best model. The genetic diversity levels in the overall studied sample of tayra were very high. For the 100 individuals analyzed, we found 70 different haplotypes with Hd = 0.983 + 0.006, = 0.0422 + 0.0048 and k = 11.175 + 5.117. The genetic diversity for four out of five South American putative morphological subspecies were very similar, all of them with very high genetic
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diversity levels (Hd = 0.991-0.960 and = 0.0562-0.0308). The genetic diversity of E. b. poliocephala was somewhat lower (Hd = 0.600 and = 0.0176), although the sample size for this putative subspecies was the lowest (Table 2). The MLT and BI can be seen in Figures 2 and 3. Both phylogenetic trees showed that the first diverging branch represented the animal sampled in northcentral Panama (putatively, E. b. inserta) (MLT: low bootstrap 28%; BI: p = 1). All of the South American specimens we analyzed were placed in the remaining cluster. However, although putatively animals from five different subspecies were included, very few significant clades were observed, and only partially related to the morphological subspecies. The first diverging cluster in the South American animals was one composed by three animals from northern Colombia (Cesar and Antioquia Departments; 50% and p = 0.97, respectively), which corresponded with the putative E. b. sinuensis. Nevertheless, a Bolivian exemplar and many other specimens “a priori” classified as E. b. sinuensis by their geographical origins that did not belong to this cluster, were present in the BI, within this clade. Henceforth, there was only a partial correspondence between this clade and E. b. sinuensis. There were other interesting clades in both phylogenetic trees. 1- One was composed of three individuals from the Pacific area of trans-Andean Ecuador (61% and p = 1, respectively), which also partially corresponded with E. b. sinuensis; 2Another cluster was composed of individuals from different areas of Bolivia and mainly by individuals from northwestern Argentina (Salta, Jujuy and Tucuman provinces) in MLT (41%). In the BI, this group was only composed of five individuals from northwestern Argentina (p = 0.96). This cluster was partially correlated with E. b. barbara. In the BI there was another cluster with several Bolivian and Argentinian specimens (p = 0.84). It was separated from the first Argentinian cluster we aforementioned. However, as we commented for E. b. sinuensis, this relationship was incomplete because other individuals “a priori” classified as E. b. barbara were dispersed by other clusters; 3- Another cluster of certain relevance was detected in the MLT and BI. It was composed of individuals of central Peru and one individual from the northern Peruvian Amazon (80% and p = 0.72, respectively). This cluster also partially supported the existence of the morphological subspecies E. b. peruana; 4- Small clusters of animals from the Ecuadorian and Colombian Amazon were present. One of them contained two animals from the Ecuadorian and Colombian Amazon (62% and p = 1, respectively) and other three from the Ecuadorian Amazon (73% and p = 1; 89% and p = 1; 28% and p = 0.8, respectively). These very locally restricted clusters were inside the putative morphological subspecies, E. b. madereinsis. Many other individuals of E. b. madereinsis were distributed in clusters with other individuals “a priori” considered different morphological subspecies. Table 2. Genetic diversity in the overall sample of Eira barbara and in the five putative South American morphological subspecies at the mt ND5 gene represented by the number of haplotypes (NH), the haplotype diversity (Hd), the nucleotide diversity (), and the average number of nucleotide differences (K) Eira barbara taxa Overall Sample E. b. sinuensis E. b.poliocephala E. b. peruana E. b. madeirensis E. b. barbara
NH 70 12 14 13 26 25
Hd 0.983 + 0.006 0.987 + 0.065 0.600 + 0.147 0.989 + 0.063 0.960 + 0.009 0.991 + 0.012
0.0422 + 0.0048 0.0562 + 0.0311 0.0176 + 0.0093 0.0517 + 0.0299 0.0308 + 0.0071 0.0412 + 0.0092
K 11.175 + 5.117 14.884 + 8.344 4.667 + 2.556 13.703 + 7.324 8.162 + 3.233 10.928 + 4.111
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Figure 2. (Continued).
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Figure 2. (Continued).
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Figure 2. Maximum likelihood tree with the 100 specimens of tayra (Eira barbara) sequenced at the mitochondrial ND5 gene. The number in the nodes are the bootstrap percentages. The procyonidae, Potos flavus, was employed as outgroup. In different colors, some relevant clusters which showed a limited correspondence with some putative morphological geographic subspecies of E. barbara.
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Figure 3. (Continued).
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Figure 3. (Continued).
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Figure 3. Bayesian tree with the 100 specimens of tayra (Eira barbara) sequenced at the mitochondrial ND5 gene. The three numbers in the nodes are the posteriori probabilities, estimated temporal splits in the nodes in millions of years, and the 95% high posterior density of these temporal splits. The procyonidae, Potos flavus, was employed as outgroup. In different colors, some relevant clusters which showed a limited correspondence with some putative morphological geographic subspecies of E. barbara.
The BI temporal split estimate showed an initial divergence of the ancestors of the Panamanian individual (E. b. inserta) and South-American specimens around 6.26 MYA (95%: 5.4-8.49 MYA; Miocene divergence). The ancestor of the clade from northern Colombia split around 3.7 MYA (1.55-6.37 MYA). The ancestor of the animals from northwestern Argentina diverged around 3.15 MYA (1.01-4.23 MYA). In contrast, the ancestors of animals of northcentral Peru, trans-Andean Ecuador and one of the Ecuadorian Amazonas clusters diverged 2.88 MYA (1.23-4.2 MYA), 2.35 MYA (0.31-2.7 MYA), and 1.49 MYA (0.42-3.74 MYA), respectively.
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Figure 4. Median Joining Network (MJN) for the haplotypes detected in 100 tayra (Eira barbara) sequenced at the mitochondrial ND5 gene. In light blue, one haplotype of E. b. inserta; in pink, haplotypes of E. b. barbara; in green, haplotypes of E. b. peruana; in yellow, haplotypes of E. b. madeirensis; in black, haplotypes of E. b. sinuensis; and in brown, haplotypes of E. b. poliocephala. Therefore, the five putative geographical subspecies of South American tayras and one Central America subspecies were represented in this analysis. Little red circles are extinct or not found haplotypes.
The MJN analysis revealed a view very similar to the phylogenetic trees (Figure 4). The major fraction of haplotypes were distributed irrespective of the geographical distribution of the morphological subspecies. For instance, the most frequent haplotypes (H1, H30, H7, H49, H19 and H9) included individuals of different putative subspecies: H1 contained exemplars classified “a priori” as madereinsis, sinuensis and barbara; H30 and H7 were composed of madereinsis and peruana individuals; H49 enclosed individuals of sinuensis; H19 consisted of specimens of sinuensis, peruana, madereinsis, and barbara, whilst H9 included poliocephala and peruana. Therefore, some haplotypes were widely distributed, which agrees quite well with extensive gene flow of this species across all of South America. Many of these main haplotypes presented other small haplotypes in star-like form, which is highly related to possible population expansions across the entire South American range of the tayra. Nevertheless, the MJN, as the phylogenetic trees, detected some haplotype clusters to be well delimited geographically. For example, there were the cases of the Central American individual (H66),
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the Cesar-Antioquia cluster (H65, H67, and H68), the trans-Andean Ecuadorian cluster (H45, H63, and H64), and the central Peruvian cluster (H11, H13, H18, and H20). The MJN temporal splits were slightly less than that those obtained with BI, but relatively similar. The temporal splits among these haplotypes and the main groups can be seen in Table 3. Some of these time splits are interesting. The divergence between the Panamanian individual and H7 was estimated to occur around 4.02 + 0.31 MYA. The temporal divergence between clusters from the areas of northwestern Argentina, Cesar-Antioquia area (northern Colombia), trans-Andean Ecuador, and north-central Peru in reference to H7 were 3.73 + 0.65 MYA, 3.29 + 0.57, 1.04 + 0.31 MYA, and 2.89 + 0.47 MYA, respectively. Therefore, the phylogenetics tree and the MJN analyses showed that the ancestor of Central and South American tayras originated during the Miocene or Pliocene. Also, the ancestors of some geographical groups, at least four of certain relevance, originated during the Pliocene and first part of the Pleistocene. However, during the Pleistocene (as we will show later), tayra experienced a strong population expansion and many haplotypes expanded their geographical distributions. They superimposed onto the geographical areas of these older and geographical groups that originally differentiated during the Pliocene.
Genetic Distances and Genetic Heterogeneity among Putative Morphological Subspecies of Eira barbara The Kimura 2P genetic distances among all of the comparison pairs of the six putative morphological subspecies of tayra are shown in Table 4. The differentiation between the Central American subspecies (E. b. inserta) and the five South American subspecies was elevated (5.5% - 7.8%), which confirmed that the Central America taxon is, at least, a different subspecies. It is interesting to note that the less differentiated South American taxon with regard to the Central American one was E. b. sinuensis (5.5%). It was the South American taxon closest geographically. The genetic distances with the other four South American taxa ranged from 7.1%-7.8%. In contrast, the genetic distances among the five South American subspecies were very small. They ranged from 0.1% to 1.2%. The pairs of subspecies with the greatest genetic distances were E. b. poliocephala-E. b. sinuensis (1.2%) and E. b. poliocephala-E. b. barbara (0.9%). The overall genetic heterogeneity for all five South American tayra subspecies taken together was significant (Table 5), but the genetic heterogeneity was relatively small. For example, the FST and the ST statistics showed values of 0.095 and 0.109, respectively. Their respective gene flow estimates of 4.75 and 4.10, were relatively high among the putative SouthAmerican subspecies. The analysis of subspecies pair comparisons with exact probability tests (Table 6) only showed two significant pairs: E. b. madereinsis-E. b. barbara (p = 0.0066 + 0.0017) and E. b. madereinsis-E. b. poliocephala (p = 0.0135 + 0.0034). In this analysis, the Central American taxon was deleted because only one sequence was analyzed. The estimates of Nm by subspecies pair comparisons (Table 7) clearly yielded that the values lower than 1 (which is considered the limit for low gene flow; Wright, 1943) always implied the Central American taxon: E. b. inserta-E. b. peruana (Nm = 0.584), E. b. inserta-E. b. barbara (Nm = 0.459), E. b. inserta-E.
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b. madereinsis (Nm = 0.286), E. b. inserta-E. b. poliocephala (Nm = 0.137). Only a South American taxon, E. b. sinuensis, (Nm = 1.202) had a more substantial gene flow with E. b. inserta. This agrees quite well with that determined in the phylogenetic trees and in the genetic distance analysis. The gene flow estimates among the South American taxa were all above 1, ranging from 2.469 to 11.847. These values strongly correlate to elevated historical gene flows among the populations of tayra throughout South America. Table 3. Temporal splits among different Eira barbara’s lineages estimated by means of a Median Joining Network (MJN). Values of temporal splits are in millions of years. SD = Standard deviation Lineages compared
+ SD
Between the Panamanian haplotype (inserta) and H49 (sinuensis) Between the Bolivian and northern-western Argentinian haplotypes and H7 (madereinsis, peruana) Between the Cesar-Antioquia (northern Colombia) haplotypes and H7 (madereinsis, peruana) Between the trans-Andean Ecuadorian haplotypes and H7 (madereinsis, peruana) Between the northcentral Peru and H7 (madereinsis, peruana) Between H9 (poliocephala, peruana) and H19 (sinuensis, peruana, madeirensis, barbara) Between H9 (poliocephala, peruana) and H7 (madereinsis, peruana) Between H19 (sinuensis, peruana, madeirensis, barbara) and H7 (madereinsis, peruana) Between H49 (simuensis) and H7 (madereinsis, peruana) Between H30 (sinuensis) and H7 (madereinsis, peruana) Between H9 (poliocephala, peruana) and H1 (madeirensis, sinuensis) Between H19 (sinuensis, peruana, madeirensis, barbara) and H1 (madeirensis, sinuensis) Between H49 (sinuensis) and H1 (madeirensis, sinuensis) Between H7 (madereinsis, peruana) and H1 (madeirensis, sinuensis) Between H30 (sinuensis) and H1 (madeirensis, sinuensis)
13.000 + 1.000
Temporal divergence 4.021 + 0.309
12.077 + 2.097
3.735 + 0.648
10.625 + 1.829
3.286 + 0.565
3.375 + 1.008
1.043 + 0.311
9.333 + 1.523
2.886 + 0.471
1.000 + 0.500
0.309 + 0.154
1.363 + 0.454
0.422 + 0.140
0.454 + 0.454
0.141 + 0.141
1.429 + 0.714
0.441 + 0.220
0.625 + 0.625
0.193 + 0.193
2.400 + 0.600
0.742 + 0.185
1.200 + 0.600
0.371 + 0.185
2.454 + 0.818 0.643 + 0.643
0.759 + 0.253 0.198 + 0.198
1.500 + 0.790
0.463 + 0.231
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Table 4. Kimura 2P genetic distance (Kimura, 1980) in percentages (%) among six different morphological subspecies of Eira barbara (Mustelidae) (below main diagonal) and standard deviations in percentages (%) (above main diagonal) at the mt ND5 gene. 1 = E. b. barbara; 2 = E. b. peruana; 3 = E. b. madeirensis; 4 = E. b. poliocephala; 5 = E. b. sinuensis; 6 = E. b. inserta; 7 = Potos flavus (Procyonidae) 1 1 2 3 4 5 6 7
0.2 0.2 0.9 0.3 7.1 27.5
2 0.1 0.1 0.5 0.5 7.7 27.5
3 0.1 0.1
4 0.4 0.2 0.4
0.8 0.4 7.4 28.5
5 0.1 0.1 0.1 0.5
1.2 7.8 24.6
5.5 26.5
6 1.4 1.5 1.5 1.5 1.2
7 3.5 3.5 3.7 3.2 3.5 3.5
24.9
Table 5. Overall genetic heterogeneity and gene flow (Nm) statistics for the five putative South American subspecies of Eira barbara at the mt ND5 gene. * P < 0.05; ** P < 0.01 Estimated Genetic Differentiation 2 = 313.351 df = 272 HST = 0.0236 KST = 0.0559 KST* = 0.0362 ZS = 2279.890 ZS* = 7.360 Snn = 0.511
P
Gene flow
0.0429* 0.0001** 0.0001** 0.0001** 0.0001** 0.0001** 0.0001**
GST = 0.0336 γST = 0.0951 NST = 0.1108 FST = 0.1086
Nm = 14.40 Nm = 4.75 Nm = 4.01 Nm = 4.10
Table 6. Exact probability tests (P) (below main diagonal) and standard deviations (above main diagonal) among six different putative morphological subspecies of Eira barbara by means of the mt ND5 gene. 1 = E. b. barbara; 2 = E. b. peruana; 3 = E. b. madeirensis; 4 = E. b. poliocephala; 5 = E. b. sinuensis; 6 = E. b. inserta. * = Significant probability 1 1 2 3 4 5 6
1.0000 0.0066* 0.2307 0.4044 ---------
2 0.0000 0.0679 0.3472 0.4796 ----------
3 0.0017 0.0071 0.0135* 0.1032 ----------
4 0.0138 0.0132 0.0034 0.0893 ---------
5 0.0197 0.0088 0.0201 0.0036 -----------
6 ----------------------------------------
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Demographic Evolutionary Changes in the Tayra All of the demographic change statistics indicated population expansion in the tayra (Strobeck's S statistic, P = 0.0001; Tajima D = -2.258, p = 0.0040; Fu & Li D* = -3.175, p = 0.0115; Fu & Li F* = -3.335, P = 0.0052; Fu’s Fs = -52.632, P = 0.00001; and R2 = 0.037, P = 0.0041). Table 7. Gene flow (Nm) estimates (below main diagonal) among six different putative morphological subspecies of Eira barbara by means of the mt ND5 gene. 1 = E. b. barbara; 2 = E. b. peruana; 3 = E. b. madeirensis; 4 = E. b. poliocephala; 5 = E. b. sinuensis; 6 = E. b. inserta
1 2 3 4 5 6
1
2
3
4
5
9.8245 9.2893 2.6879 7.1919 0.4597
11.8471 5.3236 5.8729 0.5843
2.2686 4.8435 0.2862
2.4691 0.1373
1.2019
6
Figure 5. Historical demographic analysis by means of the mismatch distribution procedure (pairwise sequence differences) for the mitochondrial ND5 gene studied in the overall sample of Eira barbara. The analysis showed a clear population expansion of this species during the Pleistocene.
The mismatch distributions also indicated population expansion (rg = 0.0040, P = 0.00280) (Figure 5). Assuming one year as one generation in the tayra, the population expansion began 343,586 YA, during the Pleistocene. The BSP analyses also determined a strong female population expansion during the Pleistocene for the tayra (Figure 6). The analyses showed the beginning of the expansion around 400,000 YA, very similar to the temporal estimate previously showed. Therefore, there is incontrovertible evidence that the tayra experienced a strong population expansion during the Pleistocene, as was previously suggested by the MJN analysis.
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Figure 6. Bayesian skyline plot analysis (BSP) to determine possible demographic changes across the natural history of the overall sample of tayra (E. barbara) sequenced at the mitochondrial ND5 gene. The analysis showed a clear female population expansion during the Pleistocene. On the x-axis, time in millions of years; on the y-axis, log effective population size of females.
DISCUSSION Genetic Diversity The levels of nucleotide diversity found in E. barbara ( = 0.0422) were quite high. They were higher than those found in many other neotropical carnivores. For example, they were higher than three fox species (Lycalopex culpaeus: = 0.008, Ruiz-García et al., 2013a; Lycalopex sechurae: = 0.015, Ruiz-García et al., 2013a; Cerdocyon thous: = 0.019, Tchaika et al., 2006), three otter species (Lontra felina: = 0.005, Vianna et al., 2010; Lontra longicaudis: = 0.011, Ruiz-García et al., 2017a; Pteronura brasiliensis: = 0.0086, RuizGarcía et al., 2017a), and two vulnerable Neotropical cats (Leopardus jacobita: = 0.0047, Ruiz-García et al., 2013b; Leopardus guigna: = 0.00461, Napolitano et al., 2013). The values of E. barbara were similar to those found in certain Neotropical cats, which are characterized by very elevated genetic diversity levels (Puma yaguaroundi: = 0.0661; Ruiz-García and Pinedo-Castro, 2013 and Ruiz-García et al., 2017b; Leopardus pajeros: = 0.0513, RuizGarcía et al., 2013b; Leopardus pardalis: = 0.068, Eizirik et al., 1998; Leopardus wiedii: = 0.035-0.074, Ruiz-García et al., 2017c). These high levels of genetic diversity in “a priori” neutral markers, as that we studied, could be related with the fact that many other genes in the genome of a given species contains enough variability for the action of natural selection (Kimura, 1986). This could be the origin of the great morphological variation and behavior plasticity found in the tayra. Emmons and Freer (1990) determined that tayras could live in a wide variety of habitats such as tropical and subtropical forests, primary rain forest (as throughout the Amazonian forest in Brazil, Peru,
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Colombia and Ecuador), secondary rain and gallery forests (as in the Llanos of Venezuela and Colombia), gardens, plantations, and cloud forests. They also inhabit dry scrub and deciduous forests (as in the Pantanal in Brazil, Paraguay and Bolivia) and tall grass savannas (as in Argentina, Bolivia, and Paraguay). Sunquist et al., (1989) showed that the extreme plasticity of this species for habitat preferences, activity periods, and diet preferences may reduce interspecific competition between E. barbara and other carnivores. This could also be the explanation why Konecny (1989) found no significant habitat preference for this mustelid in Belize. The abundance of the tayra throughout much of Central and South America may be a consequence of its ecological flexibility compared to sympatric carnivores. Associated with this, the tayra is a generalist predator, consuming a variety of fruits, carrion, small and medium vertebrates, insects, and honey (Cabrera and Yepes, 1960; Galef et al., 1976; Hall and Dalquest, 1963; Konecny, 1989; Sunquist et al., 1989).
Genetic Heterogeneity, Gene Flow and the Systematics of the Tayra Our results clearly showed that the specimen sampled in Central America was highly divergent from all of the individuals sampled in South America. However, the genetic heterogeneity among the putative morphological subspecies of South American tayras, although significant, is very small as we found with the FST statistic, exact probability tests, and genetic distances. The indirect gene flow estimates were clearly higher than 1. Wright (1943) stated that in an island model, if Nm > 1, then gene flow is important enough to erase the genetic heterogeneity among populations. In a stepping-stone model, this amount must be larger than 4 (Trexler, 1988). In both models, Nm < 0.5 means that the populations are highly disconnected from a reproductive point of view. For instance, the gene flow estimates between E. b. inserta and E. b. poliocephala (Nm = 0.137), E. b. inserta and E. b. madereinsis (Nm = 0.286) and E. b. inserta and E. b. peruana (Nm = 0.459) showed that the Central American taxon is completely isolated from these three South American taxa. However, recall that certain genetic relatedness was detected between the Central American taxon and the most northern South American taxon (E. b. sinuensis). Additionally, we found several gene flow comparison pairs between South American taxa, such as E. b. madereinsis-E. b. barbara (Nm = 9.289) and E. b. madereinsis-E. b. poliocephala (Nm = 2.168). They were elevated, although these comparison pairs showed significant heterogeneity. This might be explained according to Alledorf and Phelps (1981), who argued that the most correct interpretation of Nm > 1 is that the populations share the same alleles, although not necessarily with the same allele frequencies. By means of simulations, these authors showed that significant allele divergence occurred in 50% of the generations with a gene flow of Nm = 50. Significant allele divergence happened on most occasions when Nm = 10. The tayra seems to have a strong dispersion capacity, which could be related with these high gene flow estimates detected for all the putative South American subspecies. For instance, in the Venezuelan and Colombian Llanos, tayras are usually found along gallery forests. However, tayras cross these extensive grasslands at night, presumably moving from one forest to another covering long distances (Defler, 1980). Taking into consideration all these facts, we suggest that the six putative morphological subspecies analyzed could be reduced to two different subspecies: E. b. inserta for southern Central America and E. b. barbara for all South America. The name should be E. b. barbara
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because it was given by Linneus in 1758 versus E. b. sinuensis given by Humboldt in 1812, E. b. peruana given by Nehring in 1886, E. b. madeirenis given by Lonnberg in 1913 and E. b. poliocephala given by Traill in 1821. In reference to the putative northern Central America subspecies (E. b. senex), we cannot add any comment on its systematics because no individual of this putative subspecies was analyzed. Therefore, it is essential to sample tayras from this putative subspecies to determine its relationships with other tayra taxa. Here we suggest another alternative point of view. As we showed, the first tayra’s splits originated during the Miocene-Pliocene and beginning of the Pleistocene. However, during the Pleistocene, tayra experienced a strong population expansion and many haplotypes expanded their geographical distributions and they became superimposed on the geographical areas of older geographical groups that originally differentiated during the Pliocene. We suggest that future studies analyze nuclear genes to determine if there was hybridization between the older geographical groups (northern Colombia, part of Bolivia and northwestern Argentina, transAndean area of Ecuador, and north-central Peru) and the tayra’s population that expanded throughout South America during the Pleistocene. If data support this, then our view of a unique tayra’s subspecies in South America should be valid. On the contrary, if there was little or no hybridization between the original groups and Pleistocene colonizers in sympatry, then the number of subspecies in South America could be higher. Therefore, the northern Colombian population (Cesar, Antioquia and possibly nearby areas) should be named E. b. sinuensis and the northern central Peruvian population should be named E. b. peruana. Also, the Bolivian and especially the northwestern Argentinian population should be defined as a new subspecies (tentatively E. b. saltensis). The trans-Andean Ecuadorian population should be defined as a new subspecies (tentatively E. b. aequatorialis). The remaining populations of tayra in South America should be named as E. b. barbara. Additionally, the range distribution of E. b. sinuensis and E. b. peruana should be more restricted than traditionally accepted (see Presley, 2000). Even, if some reproductive isolation mechanism had emerged between the Central and the South America tayras due to the old split estimated during the Miocene-Pliocene, both tayra populations should be consider two different species (E. barbara and E. inserta; this last should be E. senex if both Central American forms of tayra were genetically undifferentiated because senex was firstly named by Thomas in 1900 and inserta was named by Allen in 1908). Only future nuclear genetic studies can clarify which of the two points of view is more acceptable.
Temporal Splits in the Tayras Our results showed that the divergence between the Central and the South American tayras occurred around 6.3 to 4 MYA (Miocene or Pliocene periods depending of the temporal estimation). Johnson and O´Brien (1997) and Johnson et al., (2006) showed that seven of the eight primary lineages of felids radiated in the early part of the Late Miocene (10.8-6.2 MYA). There was a noteworthy cooling of the global climate near the end of the Middle Miocene. This period of cooling coincides with formation of a permanent Antarctic ice sheet in the Middle and in the Late Miocene and an Arctic ice sheet in the Pliocene. A large peak of diversification in many vertebrate taxa occurred during the Pliocene epoch. The cold and dry climate during the Pliocene, coincides with the onset of high latitude glacial cycles, causing an explosive expansion of low-biomass vegetation, including grasslands and steppe at mid-latitudes and
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development of taiga at high latitudes of Eurasia and North America. These changes were correlated with the diversification of prey species such as muroid rodents and passerine birds that exploited these new habitats, which in turn provided new niches for little or medium carnivores, such as the tayra. Additionally, this Pliocene period agrees quite well with the last phase of rising of the Andes as shown by Dollfus (1974) and Clapperton (1993) (see, for instance, the rising of the “tablazos” of Piura, Peru) and very high volcanic activity in the Andes, with replacement of rainforests with steppe and grassland environments. Our initial divergence estimates in the tayra agrees relatively well with other molecular studies in the reconstruction of the phylogenetic relationships in the Mustelidae (Bryant et al., 1993; Dragoo and Honeycutt, 1997; Koepfli and Wayne, 1998, 2003; Sato et al., 2003, 2004; Flynn et al., 2005; Fulton and Strobeck, 2006; Koepfli et al., 2008). Two molecular studies are fundamental to understanding the phylogenetics of the Mustelidae (Koepfli and Wayne, 2003; Koepfli et al., 2008). In the first study, the authors used five nuclear gene segments and the mt Cyt-b gene. The genes APOB, FES, GHR, RH01 and mtCyt-b clustered E. barbara together with Martes americana, Martes pennanti and Gulo gulo. On the other hand, CHRNA1 clustered E. barbara with Meles meles and Arctonyx collaris. The major part of the trees generated by these authors showed that Mustelinae and Melinae were polyphyletic within the Mustelidae, whereas Lutrinae was monophyletic. The authors of the second study analyzed 22 nuclear and mitochondrial gene segments and determined Mustelidae to consist of seven primary groups. These groups include four major clades and three monotypic lineages. It also included Eira barbara clustered into the subfamily Martinae, together with Martes and Gulo, the most divergent taxa within this subfamily (100% of bootstrap and posterior probability of 1). In that study, the branch of E. barbara diverged from the other Mustelidae around 6.7-7.7 MYA (calculated using the mean values), which agrees with our estimate. These molecular results are not in conflict with the fossil record we know and understand for Mustelidae in America. Many species of Mustelidae appeared in North America during the Late Miocene-Early Pliocene. For instance, Cernictis hesperus, from the Pinole Tuff Local Fauna of California, has been dated radiometrically to have lived 5.3-5.5 MYA (Tedford et al., 2004; Baskin, 2011). Other cases of extinct genera appearances are Trogonictis and Sminthosinis as well as extant genera such as Lutra and Mustela during the Hemphilian period (4.7-5.9 MYA, Tedford et al., 2004). There is also Legionarictis fortidens from the Barstovian (Middle Miocene) marine Temblor Formation in California (Tseng et al., 2009). This form has shown a very close resemblance to other Miocene Mustelidae genera such as Dehmictis, Eirictis, Iberictis, and Trochictis, all from the Old World. The form also closely resembles Sminthosinis, Trigonictis (all from the New World), and, especially, with two extant genera, Galictis and Eira (Ray et al., 1981; Ginsburg and Morales, 1992; Baskin, 1998; Ginsburg, 1999). In fact, the cladistic analysis of Tseng et al., (2009) determined that this genus could be an evolutionary basal stage closely related to Eira. At the Longdan Fauna of the Gansu Province in China, a similar fossil to Eira was found by Qiu et al., (2004) from the Late Pliocene (Eirictis robusta; 2.58-2.15 MYA). However, the cladistic analysis of Tseng et al., (2009) determined that this mustelid was not very close to Eira and it is probably younger than Eira. Two possible fossil species of Eira have been described from post-Pliocene deposits of Maryland and Virginia under the names Galera macrodon and G. perdicida. However, the former has been assigned to Trigonictis based on additional material collected from deposits of the Blancan land mammal age from Washington, Idaho, Nebraska, Kansas, Texas, North Carolina, and Florida (Ray et al., 1981). Trigonictis is considered an intermediate form between Galictis and Eira
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and could be ancestral to both. The second species may be Mephitis (Alston, 1882). In addition, extinct species of Eira were noted from the Pliocene of the Eastern Hemisphere, but specific names were not given (Scott, 1937). Hershkovitz, (1972) claimed that Eira and other endemic monotypic mustelid genera, such as Lyncodon and Pteronura, may have evolved in South America and moved north as part of the north and south American interchange across the Panamanian land bridge. In contrast, fossil records suggest that Eira may have a North American origin (Ray et al., 1981). Therefore, the process of diversification within Eira could be at the end of the first mustelid diversification peak or at the beginning of the second mustelid diversification peak detected by Koepfli et al., (2008). In either case, this mitochondrial diversification process occurred before the Panamanian land bridge (3-2.5 MYA). Therefore, Eira’s could have radiated in North America before South America in concordance with the fossil record (Ray et al., 1981) and against the view of Hershkovitz (1972). If so, tayras arrived in South America before the complete formation of the Panamanian land bridge, coinciding with the Choco-Panama island bridge (Galvis, 1980), which could have been used by the ancestors of the current E. barbara to colonize northern South America from Central America. During the upper Pliocene orogeny, the present Tuira, Atrato and Sinu river basins and the nearby lowlands were raised above sea level. Thus, the mountains of southern Central America and of the northern Andes were uplifted to about their present elevation (Van der Hammen, 1961). Although the Nicaraguan, Panamanian and Colombian portals remained open (upper Miocene-middle Pliocene), numerous volcanic islands existed from the lower Atrato Valley and the Tuira River Basin of eastern Panama to the Nicaraguan portal. They could have been used by Eira’s ancestor to migrate southward. The Cuchillo Bridge of the Uraba region, connecting the Tertiary Western Colombian Andes with the Panamanian islands was probably above sea level during this period. Henceforth, tayras could be another “island hopper” species (Simpson 1950, 1965, 1980). Our results could also be considered as indirect evidence of a Miocene origin of the Isthmus of Panama (Montes et al., 2012, 2015). Indeed, the Isthmus of Panama formation began earlier and seems to be associated with the Northern Andean uplift, around 24 MYA (Farris et al., 2011). Therefore, the tayra could have arrived in South America before other Mustelidae. Koepfli et al., (2008) claimed that genera and species of mustelids found in South America today are largely descended from North American immigrants that arrived as part of the GABI following the rise of the Panamanian isthmus, 3.0-2.5 MYA. Several informational points support the statement of Koepfli et al., (2008). 1-For example, there is the clade of New World otters where Lutra canadensis is sister to Lontra felina, and Lontra longicaudis. The latter two species are found in South America and are estimated to have diverged 2.8–3.4 MYA (95% HPD: 1.6–5.2 MYA) overlapping with the formation of the Panamanian land bridge. 2-The long-tailed weasel, Mustela frenata, ranges from North America to northern South America. In addition, Mustela africana and Mustela felipei are endemic to South America. Fossil evidence clearly indicates that Mustela colonized South America from the north, apparently well after the Panamanian isthmus was in place. 3- Fossils of the current Lyncodon patagonicus, (and a fossil form very related as Lycodon bosei) and Stipanicicia sp (closely related to the extant Galictis cuja) have been registered in the Ensenadean and Bonaerense periods of the Argentinian Pleistocene (Forasiepi et al., 2007). However our results could be ratified by other results provided by the same authors (Koepfli et al., 2008). They found that Pteronura, Galictis and Eira could have a Eurasian origin for each genus with posterior diversification in North
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America. For example, Pteronura may be related to the extinct genus Satherium from the Pliocene of North America. Additionally, Eira may be related to Trigonictis and Legionarictis, also North American fossils (Tseng et al., 2009). Fossil evidence suggests mustelids colonized the New World across Beringia during different intervals when the land bridge between Eurasia and North America was open. Multiple genera of mustelids migrated into North America during the Late Miocene (around 11.2-5.3 MYA), prior to the first opening of the Bering Strait 5.4– 5.5 MYA, which severed the route across Beringia. Many genera that colonized North America during the Late Miocene or earliest Pliocene became extinct. However, Eira could be one of the surviving genera that began to diversify in North America and also in South America if we accept that they arrived in South America before the complete formation of the Panamanian land bridge. The mitochondrial diversification of the oldest groups of South American tayra occurred 3.7-2.3 MYA. This coincided with the climatic changes that originated from the completion of the Panamanian land bridge (3.1–2.8 MYA; Marshall et al., 1979, 1982; Marshall, 1985, 1988; Webb, 1985, 1997; Coates and Obando, 1996) in the Last Pliocene. Diversification in the tayra occurred close to the Gelasian period (2.5–1.8 MYA), a period characterized by the last stages of a global cooling trend that led to the quaternary ice ages (International Commission on Stratigraphy 2007). Around 2.5 MYA, the Andean forests were transformed into open cold dry savannah (‘paramo’), which could have potentially isolated populations of different species. They could have crossed the Northern Andes coming from Central America. Van der Hammen (1992) demonstrated that the mean temperature in the Colombian Andes was 4 ° C lower than today. He also stated that the rain level descended below the level reported for today (500– 1,000 mm). At 2,500 meters above sea level (masl), the temperature was 10 °C lower than it is today. Tayra’s diversification could have been affected by the rapid uplift that resulted in a significant elevation of the Northern Andes. The mountain range’s height climaxed around 2.7 MYA when the northern Colombian Andes reached its present day elevation (GregoryWodzicki, 2000). This also coincides with the last formation of the Central Andes. All of the Andean chain between Cajamarca and Huancavelica in Peru appeared by volcanism in this period. Much of the mitochondrial diversification process of the typical Pleistocene colonizer haplotypes occurred around 1.5-0.8 MYA. This divergence could have been initiated by the pre-Pastonian glacial period (1.3-0.8 MYA), which had the highest glacial peak of the first Quaternary glacial period (Günz). This glacial period was extremely dry, and there was a great degree of forest fragmentation. This period was a time for haplotype diversification. It was also a time of separation for many carnivores as it was previously determined for the Pampas cat (Cossíos et al. 2009), and for the foxes of the Lycalopex genus (Ruiz-García et al. 2013a). Around 1.3 MYA, the Buenos Aires’s fauna transformed into a typical semi-arid Patagonian fauna, represented by the guanaco, Lestodelphys and Lyncodon. Therefore, the climate was considerably colder and drier than today and could have influenced the mitochondrial fragmentation within the tayra. The strong population expansion detected around 0.4 MYA for the tayra agrees well with an interglacial period (0.39-0.20 MYA, West 1967) characterized by higher temperatures and humidity and forest expansions (Hoxniense in the British Islands, Yarmouth in North America, Holstein in northern Europe and Mindel-Riss interglacial period in central Europe). Future analyses with nuclear markers are needed as well as samples from Central America (especially, southern Mexico, Guatemala, Belize and Honduras), the Pacific areas of Colombia
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and Ecuador, other areas of Central Peru, and the Guyana shield. These markers and additional samples will help us to determine the exact number of subspecies or ESUs (Moritz, 1994). This information is crucial for the development of effective conservation plans for this species.
ACKNOWLEDGMENTS Special thanks to Dr. Diana Álvarez, Dr. Francois Catzeflis, Pablo Escobar-Armel, and Lina Arguello for their help in obtaining samples of tayras throughout Panama, Colombia, Trinidad, French Guiana, Peru, Ecuador, Brazil, Bolivia, Paraguay and Argentina. Thanks also go to the Instituto von Humboldt (Colombia), the Ministry of Environment, Consejo Nacional del Ambiente and the Instituto Nacional de Recursos Naturales in Peru, to the Colección Boliviana de Fauna (Dr. Julieta Vargas), to CITES in Bolivia and to the Ministerio del Ambiente in Coca and Santo Domingo de Tsáchilas (Ecuador) for their role in facilitating the obtainment of the collection permits in Colombia, Peru, Bolivia and Ecuador. Also thanks go to the Panamanian and Argentinian Ministries of Environment for their roles in obtaining permissions in these countries. Additional thanks to the many people of the diverse Indian tribes in Peru (Bora, Ocaina, Shipigo-Comibo, Capanahua, Angoteros, Orejón, Cocama, Kishuarana, and Alamas), Colombia (Jaguas, Ticunas, Huitoto, Cocama, Tucano, Nonuya, Yuri, Yucuna, Curripacos, and Desano), and Ecuador (Kichwa, Huaorani, Shuar, and Achuar) for their help in obtaining tayra samples.
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Kimura, M, (1986). DNA and the neutral theory. Philosophical Transactions of the Royal Society of London, series B, 312: 343-354. Koepfli, K-P., and Wayne, R. K. (1988). Phylogenetic relationships of otters (Carnivora: Mustelidae) based on mitochondrial cytochrome b sequences. Journal of Zoology (London), 246, 401-416. Koepfli, K-P., and Wayne, R. K. (2003). Type-1 STS markers are more informative than cytochrome b in phylogenetic estimation of the Mustelidae (Mammalia: Carnivora). Systematic Biology, 52, 571-593. Koepfli, K. P., Deere, K. A., Slater, G. J., Begg, C., Begg, K., Grassman, L., Lucherini, M., Veron, G., and Wayne, R. K. (2008). Multigene phylogeny of the Mustelidae: Resolving relationships, tempo and biogeographic history of a mammalian adaptive radiation. BMC Evolutionary Biology, 6, 10. Konecny, M. J. (1989). Movement patterns and food habits of four sympatric carnivore species in Belize, Central America. In K. Redford and J. Eisenberg (Eds.), Advances in Neotropical Mammalogy (pp. 243-264). Gainesville, Florida: Sandhill Crane Press. Librado, P., Rozas J. (2009). DnaSP v5: A software for comprehensive analysis of DNA polymorphism data. Bioinformatics, 25, 1451-1452 | doi: 10.1093/bioinformatics/btp187. Lönnberg, E. (1913). Mammals from Ecuador and related forms. Arkiv Zoologi, 16, 1–36. Marshall, L. G. (1985). Geochronology and land-mammal biochronology of the transamerican faunal interchange. In F. G. Stehli and S. D. Webb (Eds.), The Great American Interchange (pp. 49-85). New York, New York: Plenum Press. Marshall, L. G. (1988). Land mammals and the great American interchange. American Science, 76, 380–388. Marshall, L., Butler, R., Drake, R., Curtis, G., and Tedford, R. (1979). Calibration of the Great American Interchange. Science, 204, 272-279. Marshall, L. G., Webb, S. D., Sepkoski Jr., J. J., and Raup, D. M. (1982). Mammalian evolution and the great American interchange. Science, 215, 1351–1357. Montes, C., Bayona, G., Cardona, A., Buchs, D. M., Silva, C. A., Morón, S., Hoyos, N., Ramírez, D. A., Jaramillo, C. A., and Valencia, V. (2012). Arc-continent collision and orocline formation: Closing of the Central American seaway. J. Geophysical Research, 117, B04105, doi:10.1029/2011JB008959. Montes, C., Cardona, A., Jaramillo, C., Pardo, A., Silva, J. C., Valencia, V., Ayala, V. C., PérezAngel, L. C., Rodriguez-Parra, L. A., Ramirez, V., et al., (2015). Middle Miocene closure of the Central American sea way. Science, 348, 226–229. Moritz, C. (1994). Defining evolutionary significant units for conservation. Trends in Ecology and Evolution, 9, 373-375. Morral, N., Bertrantpetit, J., and Estivill,. X (1994). The origin of the major cystic fibrosis mutation (delta F508) in European populations. Nature Genetics, 7, 169-175. Napolitano, C., Sanderson, J., Johnson, W. E., O’Brien, S. J., Rus Hoelzel, A., Freer, R., Dunstone, N., Ritland, K., and Poulin E. (2013). Population Genetics of the felid Leopardus guigna in Southern South America: Identifying intraspecific units for conservation. In Ruiz-García, M and Shostell, J. M. (Eds.), Molecular Population Genetics, Phylogenetics, Evolutionary Biology and Conservation of the Neotropical Carnivores. New York, New York: Nova Science Publishers. Nei, M. (1973). Analysis of gene diversity in subdivided populations. Proc Natl Acad Sci USA, 70, 3321–3323.
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In: Encyclopedia of Genetics: New Research (8 Volume Set) ISBN: 978-1-53614-451-2 Editor: Heidi Carlson © 2019 Nova Science Publishers, Inc.
Chapter 69
THE PRECISION MEDICINE AND PRECISION PUBLIC HEALTH APPROACHES TO CANCER TREATMENT AND PREVENTION: A CROSS-COMPARISON Stephen M. Modell1,*, Sharon L. R. Kardia2 and Toby Citrin1 1
Department of Health Management and Policy, 2 Department of Epidemiology University of Michigan School of Public Health, Ann Arbor, MI, US
ABSTRACT Like other forms of diagnostics, genetic testing comes with a retinue of costs and benefits. Significant benefits in terms of morbidity and mortality have accrued to individuals tested for more prevalent genetic conditions like cystic fibrosis and sickle cell disease, including persons seen in the emergency room or identified through public health surveillance. These benefits do not mitigate the drawbacks of genetic testing, false and missed diagnoses and sheer cost among them. Both medicine and public health have aimed at means of maximizing genetic test benefits in the interventions that they apply. The President’s Precision Medicine Initiative (PMI) holds promise in that its results could be used to tailor medical treatments to the individual characteristics of patients, “precision” implying a more accurate and precise regimen overall. The National Cancer Institute (NCI) has already launched the NCIMATCH precision medicine trial, which assigns targeted treatments based on the genetic abnormalities in a tumor, regardless of cancer type. Other trials, such as the NCI Pediatric MATCH trial, are yet to happen. The efficacy of cancer treatments also intersects public health concerns. The Evaluation of Genomic Applications in Practice and Prevention (EGAPP) Working Group has evaluated the use of UGT1A1 genotyping to determine the best dose of irinotecan to prevent side effects when treating patients for metastatic colorectal cancer. Analytic validity does not always equate with improved patient
* Corresponding
Author’s Email: [email protected] (Center for Public Health and Community Genomics, University of Michigan School of Public Health, 4628 SPH Tower, 1415 Washington Hts., Ann Arbor, MI 48109-2029; Tel: (734) 615-3141; Fax: (734) 764-1357).
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outcomes, however, thus the public health emphasis on development of a suitable evidence base for precision medical and public health efforts. The public health approach to precision medicine, or “precision public health”, differs from the medical approach in several important ways: (1) population-based with attention to at-risk populations, as opposed to being strictly individualized; (2) focus on primary and secondary prevention, rather than frank disease (tertiary prevention); and (3) prioritizing interventions that have already demonstrated readiness for large-scale implementation, in contrast to the undertaking of novel clinical trials. Precision public health is exemplified in the Centers for Disease Control and Prevention’s emphasis on the implementation of Tier 1 genetic tests that have passed systematic review for analytic and clinical validity and utility – the use of family history for referral for hereditary breast and ovarian cancer genetic testing (BRCA1/2 mutations), and hereditary nonpolyposis colorectal cancer cascade screening (Lynch syndrome MLH1, MSH2, MSH6 mutations). This paper will cross-compare the precision medical approach to cancer based on pharmacogenomic regimens using companion diagnostics, and the public health approach to precision management of hereditary cancer for 3 cancer types – lung, breast, and colorectal. It will describe methods of early detection and consider how lives can be saved through precise management – from predictive testing and cancer monitoring of the at-risk population, to tailored chemoprevention that fits the needs of the individual. In the population context, a cascade screening “multiplier effect” exists in that relatives can also be assessed and followed for mutations identified in the proband. Cost-benefit analyses (T4 translational research) of medical and public health approaches will be closely examined and compared. Points of commonality between the two approaches will also be discussed, since primary/secondary and tertiary disease prevention represent a continuum. These analyses point to the value of allocating resources towards the health of at-risk populations. Questions remain if particular forms of genetic testing are to become “universalized”, and if the needs of all at-risk groups, including racial-ethnic, are to be addressed.
Keywords: lung cancer, breast cancer, colorectal cancer, Lynch syndrome, genetic testing, cascade screening, universal screening, cost-effectiveness, precision medicine, pharmacogenomics, public health, race, ethnicity
FROM THE HUMAN GENOME PROJECT TO THE PRECISION MEDICINE INITIATIVE If the twentieth century is known for its success in mapping the human genome, the twentyfirst century is becoming equally well known for scientists’ attempts at making genetic interventions “precise” – appropriately chosen, and delivered to the right person and physical target within the human body. The Precision Medicine Initiative (PMI), launched by the Obama administration in January 2015 with a $215 million outlay in the President’s 2016 Budget and continuing onward in the current administration, brought medicine closer than ever to the ability to tailor medical regimens to the needs of the individual patient [1]. An article by Francis Collins, Director of the U.S. National Institutes of Health (NIH), and Harold Varmus, former Director of the U.S. National Cancer Institute (NCI), which appeared shortly after the announcement, broke the Initiative into two stages: a near-term focus on cancers, and a longerterm aim to yield new knowledge applicable to the broader range of health and disease [2]. Muin Khoury, Director of the Office of Public Health Genomics at the U.S. Centers for Disease Control and Prevention (CDC-OPHG), and Sandro Galea, Dean of Boston University School
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of Public Health, followed-up with the affirmation that the PMI could, in time, be used to develop, evaluate, and deliver health interventions with greater “precision” for both individuals and populations [3]. The distance between a strictly individualized approach to precision medicine and one that is population-oriented, fitting the right intervention to the right population, is transcended by a common denominator shared by medicine and public health – cost-effectiveness. Translational research spans several distinct territories, from basic genome-based discovery as it yields candidate health applications (e.g., new genetic tests and therapeutic interventions) - T1 translational research, to evaluation of real-world outcomes (e.g., morbidity and mortality, costeffectiveness, and quality-of-life indicators) - T4 translational research [4]. In this piece we take the position that whatever the molecular genetic or behavioral approach used, it must make sense dollar-wise such that the interventions are being mustered in an effective and economical way. Our analysis will show that the criterion of cost-effectiveness naturally shifts the prospect of a national precision medicine effort in the population-oriented direction. Though the two may seem like strange bedfellows, both the pharmacogenomics industry and public health community are in agreement that the PMI must be a sustainable effort, so that the real question becomes, “What direction(s) can the PMI effectively take?”
DEFINITIONS OF PRECISION MEDICINE AND PRECISION PUBLIC HEALTH The definition of a particular medical intervention illustrates both the basic actions to be taken and its scope. Jameson and Longo define “precision medicine” as “treatments targeted to the needs of individual patients on the basis of genetic, biomarker, phenotypic, or psychosocial characteristics that distinguish a given patient from other patients with similar clinical presentations” [5]. Implicit in this definition is the goal of improving clinical outcomes for individual patients, while avoiding unnecessary side-effects that could be incurred by ignoring patients’ individual characteristics. The authors admit that medicine to date has more or less employed such an approach. Hemophilia requires the administration of an appropriate clotting factor, be it factor VIII or factor IX (these days in recombinant form), to stop the bleeding. Clinicians need to undertake a thorough work-up in order to arrive at a precise diagnosis of the hemophilia, which will enable the appropriate therapy to be administered. Choice of antibiotic is another example. For the antibiotic to take hold, the right type of antibiotic must be given for the particular bacterial infection. The latter example suggests that targeted approaches, aimed at particular persons for specific conditions, could actually have population-level applicability, since antibiotics and vaccinations are given on a widespread basis. They are the very opposite of “orphan drugs”, designed to cater to the needs of very rare cases. Shen and Hwang point out that despite the commonality with past precedent, a substantive shift in methodology between the old medicine and the new medicine is occurring [6]. The practice of medicine has so far remained largely “empirical”. Physicians typically rely on a combination of patient and occasionally family medical history, physical examination, and laboratory data to secure a diagnosis and choose a drug. Treatments are based on a provider’s experience with similar patients. Drugs administered are most often “blockbuster” drugs designed to accommodate the “typical” patient. If the wrong analgesic, antibiotic, or
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antiarrhythmic is given, the patient will be weaned off the current drug, and a new one tested until the right drug and dosage are chosen. The idea behind precision medicine is to rely on new biomarkers and genomic tests to “deliver the right treatment to the right patient at the right time” [6]. It is a personally tailored, as opposed to “one size fits all” approach. The fit is “precise” – one person; one drug. Classic pharmacogenomic examples readily demonstrate this novel approach. Warfarin (brand name Coumadin) dosing allows the frequently used anticoagulant to be titrated to the needs of the patient susceptible to clotting events associated with atrial fibrillation and deep vein thrombosis [7]. Two genes are known to be involved in warfarin therapeutic outcomes – CYP2C9, which codes for an enzyme primarily responsible for warfarin metabolism, and VKORC1, which codes for the warfarin drug target. Genotyping can yield information useful to guide a person’s initial warfarin dose and allow the clinician to readily stabilize his or her prothrombin measures, a process which usually takes several weeks. Cost-effectiveness studies of genotype-guided dosing have concluded that considerable potential exists for cost savings, but that it cannot be realized until test costs decrease and the uncertainty concerning effectiveness is reduced. The U.S. Centers for Medicare and Medicaid Services (CMS) has consequently adopted a provisional “coverage with evidence development” approach [7]. A physiologic measure, the ratio of the patient’s prothrombin time to a control or “normal” sample, continues to be the professional standard for warfarin dosing. Since the patient is followed with this measure, its use may be considered a tool in “personalized” or individuallytailored medicine. Imatinib (Gleevec) for chronic myelogenous leukemia (CML) and gastrointestinal stromal tumors is another prime example of the tailoring in drug regimens that may take place. Medical researchers developed this drug over a multi-decade period to inhibit the function of a translocation-related “fusion” gene, BCR-Abl, which produces an abnormal tyrosine kinase that is not properly regulated [8]. In initial trials, all of 31 research participants experienced complete remission. The five year survival rate for CML has increased from 31% (1993) to 59% (2003 – 2009) [9]. The drug is administered to patients who are Ph+ (Philadelphia chromosome positive), with effectiveness monitored by white blood cell and platelet counts. Like warfarin, Gleevec targets a particular type of patient and has a very specific chemical target – the ATP-binding site on a particular kinase – and is a drug that can be closely monitored. Gleevec’s cost is about $3,500 per month, which may evade some patients’ pocketbooks. However, it is covered by Medicare Part D and Medicare Advantage Plans. Both of the above examples describe “personalized medicine” – separating patients into different groups then individually tailoring the treatment to the patient. Though many authors use the two terms interchangeably, Khoury distinguishes “personalized medicine” from “precision medicine” in that the latter inculcates multiple determinants of health, genetics being one rung, thus can absorb the notion of social determinants as easily as it can molecular determinants. The President’s PMI plan is data intensive in a way that would allow the recording of multiple determinants for large numbers of people, ostensibly through a planned million-person cohort: Participants will be involved in the design of the Initiative and will have the opportunity to contribute diverse sources of data – including medical records; profiles of the patient’s genes, metabolites (chemical makeup), and microorganisms in and on the body; environmental and lifestyle data; patient generated information; and personal device and sensor data. [1]
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Collins and Varmus write that the numerous clinical trials stemming from the PMI and its large-scale cohort will require the building of a “cancer knowledge network” to store all the resulting molecular and medical data in digital form and make it readily deliverable to providers and patients [2]. Simonds and Khoury illustrate this idea with the example of a cancer clinical trials and effectiveness research infrastructure developed by the H. Lee Moffitt Cancer Center. The system is part of its personalized cancer care initiative started in 2006 – Total Cancer Care. The program: (1) integrates data from multiple sources (electronic medical records, biospecimen databases, and molecular data); (2) makes resultant information available to patients by providing active feedback about their health and upcoming appointments and expanded electronic health record; and (3) affords data interfaces for researchers and clinicians [10]. It is to be hoped that the streamlining of cancer clinical trials and centralization of incoming data will reduce costs and increase efficiency over standard medical practices. According to Cancer Research U.K., between 2003 and 2007 cancer trials were accompanied by a 75% increase in administrative costs, a figure in need of remedy [11]. Cost-effectiveness and clinical utility will enter into assessments of the PMI. Public health efforts in the U.S. have to date assumed a twin duty – assessment of the cost-effectiveness of clinical programs, at the same time that a vision of precision medicine’s meaning in terms of population health is being formulated. This vision departs from the medical model of precision health in a number of important respects: (1) it is population-based, with attention to at-risk populations, as opposed to being strictly individualized; (2) the focus is on primary and secondary prevention, rather than frank disease (tertiary prevention); and (3) the emphasis is on interventions that have already demonstrated readiness for large-scale implementation, in contrast to the undertaking of novel clinical trials [12]. To glimpse the future, it is helpful to visit recent experience with precision medicine in the cancer arena. This inspection will take our trek from the individuallyfocused domain of clinical medicine to the population-oriented territory of public health. The next section will focus on three major cancer categories of mutual interest to clinical medicine and public health – lung, breast, and colorectal cancer – from the medical pharmacogenomics point of view.
PHARMACOGENOMIC INTERVENTIONS AND COST-EFFECTIVENESS The Precision Medicine Approach to Lung Cancer Lung cancer is the second most common cancer in both men and women, and is the leading cause of cancer-related death in both genders [13, 14]. Of note, the lung cancer incidence rate for black women is roughly equal to that of white women, despite the fact that they smoke fewer cigarettes [13]. The two major forms of lung cancer are non-small cell lung cancer (NSCLC), and small cell lung cancer (SCLC). NSLC comprises ~85% of all lung cancers; SCLC ~10-15% [15]. The 5-year survival rate for NSCLC is 21%, suggesting progress that has been and that is yet to be made [16]. Targeted therapies aimed at cancers harboring very specific genetic alterations are becoming more and more common in oncogenomics. A 2012 review of the role of pharmacogenomics in moving genetic association studies from bench to bedside describes the
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use of EGFR tyrosine kinase inhibitors (TKI) in the treatment of lung cancer and HER2 (tyrosine kinase ERBB2)-directed therapies in the treatment of HER2 (human epidermal growth factor 2)-positive early-stage breast cancer as prime examples of success in the area of cancer pharmacogenomics [17]. A 2016 review of precision medicine approaches in oncology cites ALK (anaplastic lymphoma kinase) fusion oncogene and EGFR (epidermal growth factor receptor (EGFR)) mutations as the main molecular predictive biomarkers supporting NSCLC treatment [15]. Molecular testing for ALK fusion genes has proven valuable. Abbott Molecular already offers a multiplexed assay, the Vysis ALK Break Apart FISH Probe Kit, endorsed by the 2016 National Comprehensive Cancer Network (NCCN) NSCLC practice guidelines [15]. Though ALK fusion gene rearrangements are relatively rare (< 5% of NSCLC cases), clinical responses to targeted inhibitors (e.g., crizotinib) can be quite dramatic [5]. In favor of the precision medicine approach, excluding patients without these mutations can also minimize the exposure of patients to costly and potentially toxic therapies unlikely to benefit them. EML4-ALK is the specific biomarker used in determining patient efficacy for the choice of a TKI agent such as crizotinib in the tertiary or after-the-cancer-has-arisen management of NSCLC [5]. A 2014 cost-effectiveness study on the use of EML4-ALK fusion oncogene testing in first-line crizotinib treatment for patients with advanced NSCLC reveals the complexity inherent in this precision medicine approach [18, 19]. The investigative team found that EML4ALK testing to govern therapeutic decisions improved patient outcomes by an average of 0.011 quality-adjusted life-years (QALYs) while adding extra costs of $2,725 per patient, of which only $60 was attributable to the molecular assay itself. The overall interpretation of the costbenefit calculus changes dramatically, however, when the cost of the companion drug (crizotinib) is considered. The incremental cost-effectiveness ratio is defined as the difference in cost between two alternative interventions, divided by the difference in their effect or impact. The incremental cost effectiveness ratio for administration of the drug itself was $250,632 per QALY gained. The investigators concluded that the regimen is “likely not considered costeffective in the current setting” [19]. This assessment was unaltered when the model was subjected to a sensitivity analysis of alternative costs for the molecular testing. States one reviewer, “Where companion diagnostic precision medicine is considered, these assays are by nature tightly coupled to the cost of the specific associated drug” [18]. Assays for EGFR inhibition may be used when other TKI inhibitors, such as gefitinib and osimertinib, are being considered for NSCLC therapy [15]. The U.S. Food and Drug Administration (FDA) has approved two multiplex assay kits, also NCCN endorsed, for this purpose – the therascreen EGFR RGQ PCR Kit (for use with gefitinib), and the cobas EGFR Mutation Test (for use with osimertinib). A cost-effectiveness analysis performed by an Australian team compared the use of combined multiplex testing and targeted therapy with NSCLC chemotherapy without testing, and thirdly with best supportive care without testing [20]. The combined strategy resulted in an additional 0.009 life-years (LYs) gained, compared to 1.458 LYs gained in the case of each of the other two strategies. The combined strategy resulted in an incremental cost-effectiveness ratio of $485,199 (Australian)/QALY comparing combined and best supportive care strategies, and $489,338 (Australian)/QALY comparing combined and chemotherapy only strategies. Decreasing test and test interpretation costs by half reduced the ratios, but they still remained greater than $200,000 (Australian)/QALY. The authors concluded that multiplex testing and targeted therapy is not cost-effective as a fourthline treatment in metastatic lung cancer when first-line treatments such as chemotherapy without pharmacogenomics testing can be employed.
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Other predictive biomarkers for NSCLC treatment are on the horizon. For example, KRAS mutations can suggest lack of therapeutic efficacy to EGFR targeted therapies. The FDA has cleared KRAS mutation detection assays for use in colorectal cancer, but such assays have not yet been approved for use in NSCLC [15].
The Precision Medicine Approach to Breast Cancer Breast cancer is the most common cancer among American women. It is the second leading cause of cancer death in women, exceeded only by lung cancer [21]. About 85% of breast cancer cases occur in women with no family history of breast cancer. These cases are due to somatic cell mutations which occur as a result of aging, various exposures (such as pre- and postmenopausal hormone therapy), and other life events. Precision medicine efforts for breast cancer due to somatic mutations fall into at least four categories, two of them – endocrine therapy and HER2 therapy – being mainstay treatment categories. HER2-directed therapies, for which trastuzumab (Herceptin) is often used, are one of the two major pharmacogenomics successes in the cancer area cited by Ritchie [17]. Tamoxifen is a major drug of choice in the category of endocrine therapies, itself being a selective estrogen receptor modulator [15]. Herceptin has proven utility in reducing risk for cancer recurrence after surgery for earlystage HER2-positive (HER2+) breast cancer, and improving survival in late-stage (metastatic) HER2+ breast cancer, but it also poses serious side-effects such as heart damage. Herceptin therapy can also cost a sizable amount, up to $50,000 per year [22]. Overexpression of the HER2 gene (HER2+ status) is associated with rapid tumor growth and negative diagnostic and prognostic indicators. A systematic review and meta-analysis performed in Canada compared seven different strategies employing immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) to determine HER2 status, thus appropriateness of using Herceptin [22]. Each strategy utilized an IHC score (0, 1+, 2+, 3+) alone or coupled with FISH confirmation to form this inference. The incremental cost-effectiveness ratio was lowest when cases with an IHC score of 2+ or 3+ (as opposed to 0 or 1+) were confirmed by FISH, which yielded a ratio of $3,351 (Canadian) (minimum) to $12,230 (Canadian) (maximum) per accurately determined case. The cost-effectiveness analysis is favorable given that accurate assessment of HER2 status is capable of reducing the cost of Herceptin therapy by $0.6 million per year, and saving $12 million per year in women who are HER2-, thus can be kept off Herceptin [22]. Estrogen-focused therapies have been a part of standard care for more than thirty years, and have displayed an evolution in policy analysts’ thoughts about the gold standard for precision management [15]. The Evaluation of Genomic Applications in Practice and Prevention (EGAPP) Working Group was launched by the CDC Office of Public Health Genomics in 2004 to conduct systematic, evidence-based reviews of burgeoning genetic tests and other applications of genetic technologies in transition from research to clinical and public health practice. EGAPP has produced two systematic reviews of the use of gene expression profiling to improve therapeutic outcomes in women with breast cancer. EGAPP’s 2009 review examined the validity and utility of three tests – Oncotype DX, MammaPrint, and the H:I (normalized gene expression) ratio [23]. These tests have been designed to go beyond the standard estrogen/progesterone receptor status indicator to predict tumor recurrence risk for women on tamoxifen, for whom alternative therapies might be considered. The EGAPP
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Working Group found adequate evidence from the NSABP B-14 randomized controlled trial to support the association between Oncotype DX recurrence scores and 10-year distant metastases in estrogen receptor positive (ER+) patients, and adequate evidence to support the association between RS score and overall chemotherapy benefit, particularly for patients in the high risk category [23]. Recent publication of interim results for the TAILORx study has shown a further association between recurrence score and 5-year disease-free survival and distant recurrence in patients at low risk [24]. A follow-up systematic review by the EGAPP Working Group published in 2016 confirmed its previous findings but also noted the lack of direct evidence that the use of Oncotype DX improves clinical outcomes [25]. It also highlighted contradictory costeffectiveness results in studies performed in the U.S. (for which $2,000 in cost savings per patient were due to a decrease in post-testing chemotherapy use) and the U.K. (for which an incremental cost-effectiveness ratio of 26,940 £/QALY gained were due to an increase in chemotherapy use) [25]. The U.K. National Institute for Health and Care Excellence (NICE) Diagnostics Advisory Committee concluded that, under the assumption of equal chemotherapy benefit for all Oncotype DX risk categories, the test is not cost-effective at its current pricing level. In the first EGAPP review, data were adequate to support an association between the MammaPrint Index and 5- to 10-year metastasis rates, but the efficacy relative to classical clinical factors was unclear. More conclusive results await the completion of the MINDACT trial. For the H:I ratio test, populations studied were quite heterogeneous, and the test’s commercial offering was based on a single study in women with primary, untreated breast cancer.
The Precision Medicine Approach to Colorectal Cancer Colorectal cancer (CRC) is the third most common cancer in both men and women in the U.S. [26]. It is the third leading cause of cancer deaths when men and women are considered separately, and the second leading cause in both sexes combined. Considerable effort has been placed into targeted therapies and companion diagnostics for CRC. About 70-80% of patients present with resectable localized disease, treated by surgery and often followed by adjuvant therapy [27]. CRC patients with advanced disease may receive first-line chemotherapy, or chemotherapy and radiation before surgery is considered. The EGAPP Working Group published in 2009 a systematic review of one first-line chemotherapeutic agent, irinotecan, which may be accompanied by UGT1A1 genotyping to check for ability to adequately clear the drug [27]. Such genotyping aids decisions to either increase drug dose for more aggressive therapy, or switch to common alternate drugs, bevacizumab or cetuximab, for instance, in individuals with reduced clearance at risk for adverse events. EGAPP had two major findings: (1) unless patients receive a certain threshold dose of irinotecan, the increase in risk for toxicity is not significant, thus testing may not be warranted; and (2) reducing the dose of irinotecan (personalized dosing) to avoid adverse events may lead to more cases of unresponsive tumors than instances of adverse events avoided. It is inconclusive that benefits outweigh harms in the application of a precision approach here. The alternate drugs mentioned above are positioned among the three main classes of targeted therapies approved for metastatic CRC: (1) multikinase inhibitors; (2) angiogenic inhibitors; and (3) anti-EGFR antibodies [15]. Up to 50% of CRC patients respond to anti-
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EGFR therapy, which includes cetuximab [28]. KRAS wild-type status is considered an important factor in achieving a clinical response from this category of therapy [29]. However, 40-60% of patients with wild-type status do not respond to such therapy. Data suggest that BRAF gene status also plays a role in anti-EGFR response, and that the BRAF V600E mutation, present in 5-10% of CRC tumors, can lead to a positive response. To date the FDA has cleared two genetic testing kits, the therascreen KRAS kit from Qiagen, and the cobas KRAS Mutation Test from Roche [15]. The American Society for Clinical Oncology (ASCO) and three other professional organizations released new consensus guidelines in 2015 strongly recommending KRAS mutational testing for patients being considered for anti-EGFR therapy, and that BRAF V600 testing also be considered. Table 1. Pharmacogenomic/Companion diagnostic approach – 3 cancers Condition Non-small cell lung cancer (NSCLC)
HER2+ Breast cancer ER+ Breast cancer Metastatic colorectal cancer
Therapy Tyrosine kinase inhibitors
Biomarkers EML4-ALK
Cost-Effectiveness $255,970/QALY gained
References Djalalov et al. [19]
EGFR
$489,338 (Austr.)/QALY gained $3,351 – 12,230 (Can.) saved per case 26,940 £ (Brit.) cost/QALY gained ambivalent results – reducing toxicity vs. reducing tumor burden $7,500 – 12,400 saved per case
Doble et al. [20]
Herceptin
IHC, FISH
Endocrine
21-gene assay
Topoisomerase 1 inhibitor (TOP1) – irinotecan
UGT1A1 genotyping
Anti-EGFR
KRAS
$180,000/QALY gained
Dendukuri et al. [22] EGAPP [25]; Ward et al. [33] EGAPP [27]
EGAPP [30]; Vijayaraghavan et al. [31] EGAPP [30]; Shiroiwa et al. [32]
The EGAPP Working Group conducted a systematic review of companion diagnostic use of KRAS and BRAF mutation testing in anti-EGFR therapy in 2013 [30]. The systematic review looked at multiple pooled assessments, each consisting of up to seven studies, which together indicated statistically significant increased response rates to cetuximab and other anti-EGFR drugs, reduced risk of disease progression, and enhanced overall survival with KRAS wild-type status. EGAPP cited a cost-effectiveness study of KRAS testing in metastatic CRC patients in the U.S. and Germany [30, 31]. For the U.S. patients, use of KRAS testing to select patients for EGFR inhibitor therapy saved $7,500 to $12,400 per case. A second cited study from Japan displayed an incremental cost-effectiveness ratio for cetuximab with KRAS testing to be $180,000 per QALY gained compared to therapy without testing [32]. Due to this cost, the investigators concluded that the protocol was not cost-effective, even when treatment was limited to patients with wild-type KRAS. Two of three studies looking at BRAF testing found associations similar to the KRAS studies in terms of progression-free and overall survival, but these findings, however, were not contingent on whether or not cetuximab was included in the combination therapy. Thus, while the review supported recommendations for a precision
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approach using KRAS mutation testing, it found insufficient evidence to link BRAF V600E mutation testing with treatment response independent of prognostic association. Table 1 summarizes the cost-effectiveness and descriptive findings for the pharmacogenomics or companion diagnostic precision medicine approach to the three cancers.
TAKING PRECISION MEDICINE BEYOND THE PERSONALIZED LEVEL One argument against the informativeness of the above studies is that they represent a personalized medicine approach, but not a precision medicine approach in its full capacity [3]. The electronic storage of information, which can be multifactorial, including relevant lifestyle, would seem to be crucial to maximizing benefits and minimizing cost. The investigative team looking at the infrastructure characteristics of the Lee Moffit Cancer Center “Total Cancer Care” program also examined six other major healthcare programs engaged in cancer care comparative effectiveness research in genomic and precision medicine [10]. Four of the researched programs – at Duke University, Kaiser Permanente, University of Pennsylvania, and the Fred Hutchinson Cancer Center – had established a complex infrastructure (with data and biospecimen registries and multidisciplinary research teams), were engaged in “knowledge generation” (via randomized controlled trials and observational studies), and had reached the stage of “knowledge synthesis” (horizon scanning, evidence synthesis, and decision modeling). These programs display a depth of information and range of multidisciplinary expertise that is reflective of the type of knowledge synthesis of which the PMI’s million person cohort will be capable. The Duke University and Kaiser Permanente teams noted a number of strategic challenges to the amassing of cancer study data – limited data quality, large variation in genomic methodology used, and poor demonstration of clinical utility for the genomic tests supplying the data [10]. These observations point out challenges that could foreseeably face PMI investigators attempting to collate information from the expansive precision medicine cohort. The Kaiser Permanente group was able to show that in screening for Lynch syndrome, a hereditary form of CRC, microsatellite instability testing (MSI) was preferable compared to immunohistochemical staining (IHC). In the treatment phase, the investigators found that screening for KRAS and BRAF mutations improved the cost-effectiveness of anti-EGFR therapy, but that the cost of the therapy surpassed generally accepted cost-effectiveness thresholds of $100,000/quality-adjusted life-year. These findings allude to the capability of the PMI to develop new data on useful oncogenomic screening, mutational testing, and therapeutic procedures, and to the correspondingly likely possibility that many of the discoveries, while being individually beneficial, could elude effectiveness for the clinical population as a whole. The PMI will no doubt be carried in new directions that have not yet been quantified, however. NCI is engaged a new type of clinical trial called “NCI-MATCH” [34]. In this innovative program, adult cancer patients are assigned to targeted treatments based on the genetic abnormalities in their tumors, regardless of the type of cancer they have. This concept represents the therapeutic end of what has been happening with diagnostics. KRAS and EGFR mutation testing is now occurring for multiple cancer types. Why should cancer therapies be restricted to just one cancer type? New management models may also evolve. The data
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infrastructure supporting precision medicine can and is being used to develop procedural algorithms that combine genetic testing with genetically targeted therapies. These algorithms, if tailored to the lay person, can then be used by both medical providers and patients in collaboration, improving the effectiveness of the prescribed regimen. Shen and Hwang use CYP2C9 (they cite CYP450) testing for warfarin dose performed at home first by nurses then by patients as an example [6]. This linkage of “big” or “rich” data and the development of new, useful algorithms is cited by multiple authors [2, 5, 35]. The issue is whether providers of various types, and consumers, can understand the test results and appreciate the connection to one-size-does-not-fit-all therapeutic management [36]. Authors discussing the translation of big data into usable guidelines and algorithms are not just limiting the payoff to individualized treatment, however. Jameson and Longo, for instance, speak of not just a pharmacogenomic future, but one in which “guideline-based screening”, e.g., colonoscopy, can be targeted on the basis of age and family history [5]. Family health history is one of several tools, including individualized genetic testing and family cascade testing, fueling the public health drive towards precision interventions [37, 38]. The public health approach to precision medicine, or “precision public health”, is highly evidence-based. Precision public health is exemplified in the Centers for Disease Control and Prevention’s emphasis on the implementation of Tier 1 genetic tests that have passed systematic review for analytic and clinical validity and utility – family history-based hereditary breast and ovarian cancer (HBOC) genetic testing (BRCA1/2 mutations in relatives), and hereditary nonpolyposis colorectal cancer genetic testing (Lynch syndrome MLH1, MSH2, MSH6, PMS2, EPCAM mutations) [39]. Indeed, genetic counseling and testing for HBOC are already incorporated into healthcare reform as services not requiring co-pay for individuals deemed at risk by their providers [40]. The PMI promises much more, however, and part of the vision of public health is that precision techniques can be used to direct genetic preventive strategies to those subsets of the population that will derive maximal benefit [41].
PUBLIC HEALTH INTERVENTIONS AND COST-EFFECTIVNESS The Precision Public Health Approach to Lung Cancer It has been remarked that “although personalized treatments can help save the lives of sick people, prevention applies to all” [3]. This comment applies especially to lung cancer, which can be tackled as we have seen individually and after it has manifested, or by using a preventive, population-based approach. Initial genome-wide association studies (GWAS) published by several investigative teams in 2008 were suggestive of lung cancer susceptibility genes being situated on the long arm of chromosome 15 [42]. The studies were all large (3,500 to 14,000 participants) and replicated, yielding strong evidence for an association between SNP variations at 15q24/15q25.1 and lung cancer. Thorgeirsson et al. found a highly significant association (P = 1.5 x 10-8) between a common variant in the nicotinic acetylcholine receptor gene cluster on chromosome 15q24 and smoking frequency, with an odds ratio of 1.31 (1.19 – 1.44, 95% C.I.) between nicotine dependent cases and low quantity smokers plus population controls [43]. Studies by Hung et al. [44] and Amos et al. [45] found odds ratios between 1.21 and 1.77 for associations between the nicotinic acetylcholine receptor regions 15q25 and 15q25.1 and lung
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cancer in ever smokers, the former accounting for 14% (attributable risk) of the lung cancer cases in the first study. The second study examined 2,724 NSCLC cases, the same type of cancer being treated in later stages by pharmacogenomics regimens. More recent GWAS in never-smoking Asian females have pointed out genetic associations independent of smoking status. Case-control studies of never-smoking Asian females funded through the National Institutes of Health [46] and the Mayo Foundation [47] have identified genetic variants in the 3q28 and 13q31.3 regions associated with risk for lung cancer (N = 754 to 7,254 participants). These associations show both statistical significance (P = 10-6 to 10-8) in terms of odds ratios and allelic risk, and biological plausibility (association with the regulation of cell proliferation and division). These two lines of discovery – lung cancer in connection with nicotine dependence and independent of it – could beneficially lead to both personalized smoking-cessation interventions, and to increased screening of people at risk for lung cancer [41]. The difficulty is that the association studies are at the primary research (T1) stage and do not yet imply clinical validity. Precision medicine in public health terms involves targeting groups at risk. Various professional societies have developed guidelines for lung cancer screening, generally beginning at age 55 [48]. The NCCN lung cancer screening guidelines recommend screening individuals age 50-55 years, those who have between 20 and 30 pack-years of exposure, and who exhibit one additional risk factor, such as family history. The relative risk of developing lung cancer is 1.8 if the individual has at least one first-degree relative with lung cancer, and 3.0 given two first-degree relatives with the condition [49]. The positive and negative predictive values for lung cancer appearing in a proband’s first-degree relatives are 89.9% and 99.1%, respectively [50]. In the Utah Family High Risk Program, the cost of taking a family health history varied between $10 and $25 depending upon receipt of follow-up educational interventions [51]. These figures indicate cost-effectiveness for the use of family history in risk assessment for lung cancer. Public health programs are especially concerned with the rights and welfare of underserved groups, an aim that is built into public health codes of ethics [52]. Surprisingly, of the 58,160 lung disease studies published between 1993 and 2013, less than 5% reported the inclusion of minority participants [53]. NIH is presently consulting researchers adept at recruiting underrepresented groups into studies as part of PMI Research Cohort formation. An admixture study of 1812 African Americans performed by the Karmanos Cancer Institute in Detroit, MI demonstrates what can come out of the use of the PMI Cohort. Excess African ancestry was observed on chromosome 3q among ever smokers with NSCLC, a chromosomal region identified by previous studies with mostly persons of European ancestry [54].
The Precision Public Health Approach to Breast Cancer About 10 to 15% of women diagnosed with breast cancer have germline mutations in the BRCA1 or 2 genes. Between 10 and 30% of women under age 60 diagnosed with triple-negative breast cancer (cancer which does not have receptors for estrogen, progesterone, or HER2/neu) display a BRCA1/2 mutation [55]. Ashkenazi Jewish ancestry confers an increased risk, though such mutations are by no means relegated to just one group. Like lung cancer, the public health approach to hereditary breast and ovarian cancer (HBOC) focuses on primary prevention before disease has appeared. Primary prevention can be conducted through a variety of means, several
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of which fit under a public health precision model. A study out of the Cleveland Clinic Genomic Medicine Institute compared two methods for cancer risk assessment – “family history-based risk assessment” and “DTC [Direct-to-Consumer] personal genomic screening,” the latter using a variety of risk alleles [56]. Of 22 high risk females appearing in clinic, family history classified eight individuals as being at risk for breast cancer, but only one of the eight was classified as high-risk through personal genomic screening method. Family history is a quick way of identifying risk, and has value in this instance as it does with lung cancer. Valdez et al. note that the relative risk of developing breast cancer is 1.8 and ovarian cancer 2.9 if the individual has at least one first-degree relative with these conditions [49]. It is 3.0 and 14.7 given two affected first-degree relatives. The positive and negative predictive values for these cancers appearing in a proband’s first-degree relatives are 89.1% and 98.9% for breast cancer, and 76.1% and 99.3% for ovarian cancer [50]. The U.S. Preventive Services Task Force (USPSTF) has conducted evidence reviews of genetic testing for the key mutations involved in HBOC, BRCA1 and 2. It recommends: Primary care providers screen women who have family members with breast, ovarian, tubal, or peritoneal cancer with 1 of several screening tools designed to identify a family history that may be associated with an increased risk for potentially harmful mutations in breast cancer susceptibility genes (BRCA1 or BRCA2). Women with positive screening results should receive genetic counseling and, if indicated after counseling, BRCA testing. [57]
The CDC-OPHG classifies the use of family history of known breast/ovarian cancer with deleterious BRCA mutations as Tier 1 [39]. CDC defines Tier 1 genetic interventions as those supported by clinical practice guidelines based on thorough systematic review. These modalities are ready for implementation not just on the individual but on the level of the at-risk population as well [38]. Family history, however, is part of a train of diagnostic interventions, including genetic counseling and genetic testing. The latter can lead to annual screening via MRI or to surgery. A 2012 cost-effectiveness analysis of BRCA1/2 testing of women >= 35 years at elevated risk of carrying a mutation, considering the eventual use of these MRI and surgery, determined genetic testing to be cost-effective if testing cost were 200 CGG), premutated individuals (55-200 CGG) and intermediate alleles (45-54 CGG). In 1984, Fryns [1] studied a large population of fragile X males, describing a behavioral phenotype of hyperactivity and impaired attention, marked anxiety with poor eye contact, affective liability, aggression, self-injurious behavior (especially the characteristic hand biting), and autistic features reported as repetitive, perseverative and stereotypic behaviors. This basic formulation of the fragile X behavioral phenotype has remained intact to the present day, with substantial confirmation of these basic findings in subsequent studies.
FRAGILE X SYNDROME Fragile X patients suffer maladaptive behaviors and emotional disturbance with an enormous functional impairment; symptoms are a frequent reason for families to seek treatment and can lead to institutionalization in more severe cases.
Autism Disorders Autistic disorder is the most debilitating subgroup of a larger category known as pervasive developmental disorders (American Psychiatric Association) characterized by impairment in social interaction and verbal and non-verbal communication, and restricted, repetitive and stereotypic patterns of behavior, interests and activities. Although there is considerable variability in individual symptoms, core deficits in social communication and restricted and repetitive behaviors are hallmarks of the disorder [2]. FXS is the leading known monogenic
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cause of autism, accounting for approximately 5% of autism cases [reviewed in 3] and autism is one of the most recognized and severe behavioral abnormalities observed in males with FXS [4-8]. In individuals with the full mutation, approximately 30–50% meet full DSM-IV-TR criteria for autism with 60–74% fulfilling criteria for an autism spectrum disorder (ASD) [913]. Over 90% of individuals with FXS display some form of atypical behavior characteristic of autism, including social interaction (e.g., avoidance of eye contact, social withdrawal, and social anxiety) and repetitive and stereotyped behaviors [14]. Co-morbid FXS and autism are indicative of worse developmental outcomes [15-17], and greater impairment in cognition and adaptive behavior skills are more severe aberrant behavior than FXS without autism [18]. It has been suggested that autistic behaviors increase slowly but significantly over time, as do associated social avoidance behaviors [19]. The main dilemma arises when considering the pathogenesis of clinical autism spectrum in patients diagnosed with the FXS. Some authors consider that the clinical autism spectrum in these patients is a consequence of pathophysiological alterations resulting from the mutation of the FMR1 gene, while other authors consider that this clinical autism spectrum in these patients has an etiopathological mechanism similar to idiopathic autism. These different points of view are generated due to the fact that diagnostic criteria for autism spectrum disorders are purely clinical. Likewise, Harris (2011) [20] has proposed a focus on brain-behavior relationships targeting advancement of behavioral phenotyping in neurogenetic disorders precluding the application of DSM-IV diagnostic behavioral criteria to identify disorders such as FXS. He proposed that FXS is a neural model and phenocopy of autism and should not be considered a genetic model for autism. On the other hand, a number of investigators have reported findings indicating highly similar profiles between individuals with FXS and autism versus idiopathic autism and apply categorical or dimensional ratings of autism in FXS [9,16,21-23]. These authors agree with the concept that the autism phenomenon represents a range of behaviors, and perhaps also of other neurologic features [24]. While there is clear consensus regarding the shared phenomenology between FXS and idiopathic autism, there is great debate regarding diagnostic issues. Two of the primary debates on FXS center around questions of whether autism in FXS represents a continuum, with only those most severely affected meeting criteria for autism, and whether autism in FXS is the same as or different from idiopathic autism [9]. Although the literature on the co-morbidity of FXS and autism is extensive, few published studies have longitudinally examined early indicators of autism in infants. Study of the emerging characteristics in FXS is critical for understanding if early features in infants with FXS are associated with later autistic behaviors as reported in idiopathic autism. To date, the studies that have been conducted lend evidence to the fact that indicators of autism are present at as early as 12 months of age in males with FXS, and replicate findings in idiopathic autism that implicate difficulties, with disengagement of attention and shifts in behavior at 6 to 12 months of age in the later development of autism [25-27]. Clearly, brain–behavior relationships in idiopathic autism and FXS are complex, and there is currently insufficient evidence to resolve these debates. In summary, however, despite all the controversies generated in the diagnosis of clinical ASD, at present the FMR1 gene is considered the most common monogenic cause of ASD, and therefore, the molecular diagnosis of FXS should be ruled out in all ASD [28].
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Hyperactivity Disorders Attention deficit and hyperactivity (ADHD) is the most common diagnosable condition in FXS patients, with most males meeting formal criteria at some point in their lives. This condition is typically not stable over time in any given individual [1]. Using DSM-V, several of the ADHD symptoms must be present in individuals prior to the age of 12 years, compared to 7 years as the age of onset in DSM-IV. This change is supported by substantial research published since 1994 that found no clinical differences between children identified at 7 years versus later in terms of the course, severity, outcome, or treatment response. DSM-5 includes no exclusion criteria for people with ASD, since symptoms of both disorders co-occur. The higher inattentiveness, restlessness, fidgetiness and impulsivity in ADHD in children with FXS is suggestive of the ADHD inattentive sub-type compared to ADHD in the general population; these features do not necessarily improve with age [29]. The signature of the FXS is strong unsustained attention, but poor capacity to switch between tasks and weaker inhibitory control [reviewed in 30]. Very young children with FXS are often noted to be physically hypoactive, with somewhat impaired attention. Preschool children can display dramatic increases in activity levels, leading to markedly disruptive behavior. As children grow, hyperactivity declines with increasing body mass, while problems with attention continue throughout life. This can be seen as similar to the course of ADHD in the normal population, though the degree of hyperactivity in FXS is impressive. There is also evidence that the attention deficit seen in males with FXS has a specific profile [31], which is distinct from other causes of developmental disorders, suggesting that the attention problems seen in the course of FXS may represent more than nonspecific immaturity. There is evidence that ADHD symptoms in FXS respond to stimulants [32,33].
Mood Disorders There has been considerable controversy regarding the behavior phenotype of FXS and the nature of behavioral and emotional problems associated with it. As described by Backes and colleagues (2000) [34], males with FXS rarely meet formal criteria for a diagnosis of a major mood disorder as defined in DSM-V. Diagnoses such as major depression or bipolar disorder require periods of abnormal mood that are sustained, whereas individuals with FXS typically exhibit labile mood, irritability, self-injurious behavior, and aggressive outbursts of a more fleeting and episodic nature, not meeting the conventional duration criteria. These episodes are typically adaptive, related to environmental stressors and are less frequent in familial or more structured settings. However, affective symptoms can be severe and disruptive, and psychopharmacologic intervention is needed. Selective serotonin reuptake inhibitors are a commonly employed treatment strategy for affective symptoms, along with other antidepressants, anticonvulsants, and atypical antipsychotics in more severe cases [33]. There have been no clinical trials of any size, open or controlled, of antidepressants or anticonvulsants for the treatment of affective symptoms of FXS. Curiously, the study of valproic acid by Torrioli and collaborators (2010) [35] focused exclusively on ADHD symptoms in boys and they considered that this treatment could be considered as an alternative to treating symptoms with stimulants the efficacy of which needs
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to be confirmed by further studies in patients with FXS. Behavioral problems in FXS improve from childhood to young adulthood [36].
Anxiety Disorders Children with neurodevelopmental disorders, such as FXS, have a higher risk of presenting anxiety. Patients with FXS display a broad range of anxiety symptoms, but these symptoms often do not fit into the established categories of major anxiety disorders employed by the DSM. Cordeiro and coworkers (2011) [37] examined the prevalence of anxiety disorders in a FXS population using DSM-IV-TR criteria. They found that 83% of participants (n=97, ages 5.5– 33.3 years) met criteria for any anxiety disorder, and 58% exhibited multiple anxiety disorders. In addition to the high proportion of anxiety disorders in FXS, an important part of individuals with FXS display autistic symptoms as mentioned previously. Symptoms of anxiety and autism overlap and interrelate within FXS, although these disorders have been distinguished through behavioral and physiological profiles. Kaufmann, Budimirovic and colleagues have published a series of papers characterizing anxiety and autism as distinct social interaction disorders in FXS [38-39]. These authors propose that social anxiety emerges from a combination of lower non-verbal abilities and moderate social withdrawal, whereas autism (either alone or in conjunction with social anxiety) is characterized by a more complex constellation of severe social withdrawal and lower adaptive socialization or verbal skills. Negative affect is one of the most commonly studied predictors of problem behaviors in non-clinical [40-41] and clinical [42] populations. Negative affect is composed of several specific dimensions of temperament; including fear, approach, soothability, sadness, anger, discomfort, and motor activity [43] and predicts anxiety in preschool boys with FXS [44]. Multilevel models indicate associations between elevated anxiety and higher fear and sadness, lower soothability, and steeper longitudinal increases in approaches in the FXS population. A minority of males with FXS fulfill formal criteria for the diagnosis of obsessivecompulsive disorder (OCD), while “compulsive symptoms” have been noted in several studies in a large majority of subjects with FXS. In most cases of FXS, individuals exhibit symptoms strongly reminiscent of obsessions and compulsions, but which do not meet the precise psychiatric definitions for these symptoms. Often, pleasure is derived from repetitive and “compulsive” behaviors, in contrast to the ego-dystonic nature of true obsessions and compulsions. Hoarding, counting, and the need for symmetry are all typical symptoms of OCD frequently seen in FXS. Similarly, younger children with FXS meet the criteria for separation anxiety disorder in a small minority of cases [34], while symptoms of separation anxiety, social phobia, panic, and agoraphobia are seen clinically at a much higher rate. The psychiatric drugs currently available can provide significant symptomatic relief of the hyperactivity, anxiety disorders, and affective disturbances often seen in the course of FXS. However, patients with this syndrome may be especially susceptible to the psychiatric side effects of these medications, requiring particular care in their prescription.
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PREMUTATION CARRIERS For many years, premutation carriers of the FMR1 gene were considered asymptomatic, but various clinical disorders have been associated in both men and women, being Fragile X tremor –ataxia syndrome (FXTAS) one of the most severe. Individuals with carrier premutation are those that have alleles between 55-200 CGG. Some of the clinical features have been related to the CGG number; in general, more length in the CGG track implies more severe clinical manifestations. There is a growing body of evidence suggesting that FMR1 premutation carriers may have increased vulnerability of presenting psychiatric disorders [45]. However, the presence of neuropsychological and behavioral impairment among FMR1 premutation carriers remains controversial, as these features were initially thought to be associated with the stress of raising children with FXS [reviewed in 46]. Altered neurobehavioral profiles including variation of phenotypes associated with mood and anxiety may be expected among younger premutation carriers. An increased risk of anxiety and mood disorders among premutation carriers has not been established. Some studies have reported a lack of phenotype [47-48], while others have described repeat length associations with psychiatric symptoms [49-50]. Hessl and coworkers (2005)[51] found that the FMR1 transcript level, but not repeat length or Fragile X Mental retardation Protein (FMRP) levels, was significantly associated with increased severity of psychiatric symptoms in males, independently of FXTAS status. These results suggest that premutation carriers may be at risk of presenting emotional morbidity; however, phenotypic differences were subtle and of a small CGG effect size. The largest and most recent study of life-time mood and anxiety in the premutation population was completed by Bourgeois and colleagues (2009) [52]. In this study, the prevalence of anxiety disorders in carriers with and without FXTAS was compared with a very large age-matched national dataset. In terms of all anxiety disorders, only those with FXTAS demonstrated a higher prevalence. Upon separation, this was similarly true for panic disorder, post-traumatic stress disorder and specific phobia. Generalized anxiety disorder and OCD failed to demonstrate any difference between carriers and controls. Only social phobia was found to have higher levels in premutation carriers without FXTAS compared to controls. Chronic anxiety has also been associated with radiological signs on MRI; specifically, the higher the anxiety score the smaller the size of the hippocampus in women with the premutation [53]. Rodriguez-Revenga and collaborators (2008) [54] examined psychiatric and depressive symptoms in 34 FMR1 premutation carrier mothers of children with FXS in comparison with two control groups (39 mothers with a non-FXS intellectual disability child and 39 mothers from the general population). Both groups of mothers with a child with intellectual disability showed greater susceptibility to psychological problems than the control group without a mentally retarded child, but FMR1 premutated mothers evidenced a higher tendency to depression. These results suggest that, despite the stress of caring for a child with mental retardation, the premutation by itself could be responsible for some psychiatric traits. In a screening study of individuals from families with FXS, roughly 14% of boys and 5% of girls with the premutation were found to also have an ASD [10]. Even among those carriers not diagnosed with ASD, related psychological traits are more common among carriers
Psychiatric Aspects in Fragile X Syndrome and Related Phenotypes
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compared to controls without the premutation. A recent study examined a broad range of pragmatic language skills as well as related behavioral features of the broad autism phenotype among women with the premutation compared with mothers of children with autism, and mothers of typically developing children with no family history of FXS, autism, or language impairment [55]. In this study, conversational samples from a semistructured videotaped interview were used to assess pragmatic language using the Pragmatic Rating Scale (PRS) [56]. This study replicated previous findings in the autism parent group and also showed that women with the premutation exhibited similarly elevated rates of pragmatic language problems relative to the controls. The presence of broad autism phenotype traits was associated with greater expression of autism symptoms in their children with FXS. Other studies have also found increased rates of both social aloofness [49] and a rigid perfectionism [57] among carrier women. Given its relative rarity in the general population, psychosis has been challenging to study in premutation carriers. Initial linkage analysis failed to show a clear relationship of schizophrenia to the FMR1 gene [58]. Prevalence studies have found the overall rate of psychotic disorders to be low [49]. There have, however, been several case reports of combined psychotic illnesses and the premutation, including schizoaffective disorder [59] and with combined schizophrenia and schizoid personality disorder [60]. Interestingly, as opposed to frank psychotic disorders, multiple studies have found an increased prevalence of schizotypal personality traits in the carrier population [49,61]. Attention regulation difficulties have been proposed to be a problem in people with the premutation. Notably, when compared with their control siblings, premutation carriers had significantly more issues related to attention than their noncarrier siblings [62]. Inattention and impulsivity amongst FMR1 carriers can be problematic through adulthood [63], although hyperactivity was not noted to be increased in prevalence. Dysthymia and bipolar disorder have generally failed to demonstrate significant levels in carriers compared to controls [52].
FRAGILE X ASSOCIATED TREMOR ATAXIA SYNDROME (FXTAS) FXTAS is an X-linked neurodegenerative disorder affecting up to 45.5% of males and 16.5% of females carrying a premutation in the FMR1 gene [64-65]. There is a growing body of literature suggesting that the neuropsychiatric features of FXTAS follow a fronto-subcortical pattern with primary impairments in executive function and increased vulnerability to mood and anxiety disorders [51,53, 66-69] Increased rates of psychiatric symptoms may represent early markers of neurodegenerative diseases such as Parkinsons Disease (PD) [70] and Huntington’s Disease [71], which have also been reported among FMR1 premutation carriers without FXTAS, including obsessivecompulsive symptoms [51], social phobia [72], depression [50], schizotypal features [73] and abnormalities in social cognition [74]. Three studies have examined the psychiatric features of FXTAS: (1) Bacalman et al. (2006) [67]; (2) Adams et al. (2010) [53]; and (3) Hashimoto et al. (2011) [69]. They showed no significant association between FXTAS and different psychiatric diagnoses. The studies compared the prevalence of neuropsychiatric symptoms between individuals with FXTAS and
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matched controls with normal FMR1 alleles (cohort 1 and 3), and between asymptomatic FMR1 premutation carriers in cohort 2. Symptoms of depression were significantly elevated among males with FXTAS when measured using the informant-rated NPI in cohort 1; however, self-reported symptoms measured using the SCL-90-R in cohort 3 were comparable to controls. Women with FXTAS (cohort 2) tended to report higher rates of depression than controls with normal FMR1 alleles; however, this difference did not withstand correction for multiple comparisons. Anxiety was significantly elevated among older males with FXTAS compared to controls in cohort 2; however, in cohort 1 the group difference (50% FXTAS group vs. 0% control group) was not significant, possibly due to the small sample size (n=14). Male carriers with FXTAS exhibited elevated rates of anxiety in cohort 3 (Cohen’s d=0.69) and comparable scores in cohort 2. Females with FXTAS (cohort 2) exhibited higher rates of anxiety and obsessivecompulsive symptoms compared to controls with normal FMR1 alleles and asymptomatic FMR1premutation carriers. Males with FXTAS (cohorts 2 and 3) reported significantly higher rates of obsessive compulsive symptoms compared to asymptomatic carriers (Cohen’s d=0.92, cohort 7), but not controls (cohort 2). Symptoms of anxiety among asymptomatic male and female FMR1 premutation carriers were compared to controls with normal alleles in cohort 2 and no group differences were detected. Additional informant-rated behavioral disturbances found to be significantly elevated among males with FXTAS (cohort 1) compared to controls included apathy, irritability, disinhibition, and agitation/aggression (Cohen’s d=3.53) [67]. Increased obsessive-compulsive and depressive symptoms (but not anxiety) were also associated with decreased left amygdala volume among males with FXTAS in cohort 3 (all ps90% 30-50% 60-74%
Affective disorders
22% Females 12% Males 83%
Anxiety
PM without FXTAS
14% Males 5% females 30% Females 14% Males
30% Females 60% Males
References [14] [11-13] [11-12,57] [62]
65%
55.7% 34.2%
[49,62,72]
52%
35-40%
[37,49,72,]
CONCLUSION FMR1 is the first monogenic cause of autism disorder. Approximately 30–50% of FXS meet full DSM-IV-TR criteria for autism, 60–74% fulfilling criteria for an ASD and over 90% of individuals display some form of atypical behavior characteristic of autism. Molecular diagnosis of FXS should be ruled out in all ASD. Increased rates of psychiatric symptoms may represent early markers of neurodegenerative diseases which have been reported among FMR1 premutation carriers with and without FXTAS; including obsessive-compulsive symptoms, social phobia, depression, schizotypal features, and abnormalities in social cognition. Altered neurobehavioral profiles including a variation of phenotypes associated with mood and anxiety may be expected among younger premutation carriers. The trend is to present higher rates of anxiety and mood disorders in premutation carriers, but no study has found significant differences between carriers and the control population.
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fragile X premutation carriers. J. Clin. Psychiatry. 2011 Feb;72(2):175-82. Epub 2010 Aug 24. Sobesky WE, Hull CE, Hagerman RJ. Symptoms of schizotypal personality disorder in fragile X women. J. Am. Acad. Child Adolesc. Psychiatry. 1994 Feb;33(2):247-55. Cornish K, Kogan C, Turk J, Manly T, James N, Mills A, Dalton A. The emerging fragile X premutation phenotype: evidence from the domain of social cognition. Brain Cogn. 2005 Feb;57(1):53-60. Hocking DR, Kogan CS, Cornish KM. Selective spatial processing deficits in an at-risk subgroup of the fragile X premutation. Brain Cogn. 2012 Jun;79(1):39-44. doi: 10.1016/j.bandc.2012.02.005. Mitchell RJ, Holden JJ, Zhang C et al: FMR1 alleles in Tasmania: a screening study of the special educational needs population. Clin. Genet. 2005; 67: 38–46. Crawford DC, Meadows KL, Newman JL et al: Prevalence and phenotype consequence of FRAXA and FRAXE alleles in a large, ethnically diverse, special education-needs population. Am. J. Hum. Genet 1999; 64: 495–507. Patsalis PC, Sismani C, Hettinger JA et al: Frequencies of ‘grey-zone’ and premutationsize FMR1 CGG-repeat alleles in patients with developmental disability in Cyprus and Canada. Am. J. Med. Genet 1999; 84: 195–197. Mornet E, Chateau C, Simon-Bouy B, Serre JL: The intermediate alleles of the fragile X CGG repeat in patients with mental retardation. Clin. Genet 1998; 53: 200–201. Otsuka S, Sakamoto Y, Siomi H et al: Fragile X carrier screening and FMR1 allele distribution in the Japanese population. Brain Dev. 2010; 32: 110–114. Madrigal I, Xunclà M, Tejada MI, Martínez F, Fernández-Carvajal I, Pérez-Jurado LA, Rodriguez-Revenga L, Milà M. Intermediate FMR1 alleles and cognitive and/or behavioural phenotypes. Eur. J. Hum. Genet. 2011 Aug;19(8):921-3.
In: Encyclopedia of Genetics: New Research (8 Volume Set) ISBN: 978-1-53614-451-2 Editor: Heidi Carlson © 2019 Nova Science Publishers, Inc.
Chapter 91
CLINICAL FEATURES ASSOCIATED WITH FMR1 PREMUTATION CARRIERS M. I. Alvarez-Mora1,2 and L. Rodriguez-Revenga1,2,3,* 1
Centre for Biomedical Network Research of Rare Disease (CIBERER), Barcelona, Spain 2 Biochemistry and Molecular Genetics Department, Hospital Clinic, Barcelona, Spain 3 IDIBAPS (Institut d’Investigacions Biomèdiques August Pi i Sunyer). Barcelona, Spain
ABSTRACT The expansion of the CGG trinucleotide located within the 5’UTR of the FMR1 gene is involved in a growing number of diseases; the most well-established are Fragile X Syndrome (FXS), Fragile X Tremor/Ataxia Syndrome (FXTAS) and Fragile X Primary Ovarian Insufficiency (FXPOI). Whereas full mutation alleles (>200CGGs) are responsible for the FXS, smaller expansions called premutation alleles (55-200CGGs) are associated with FXTAS and FXPOI. Numerous evidence have currently been reported suggesting that premutation alleles give rise to an increased risk for carriers of these alleles in relation to additional medical, psychiatric and cognitive features which occur at a greater frequency than what would be expected for the general population. In this chapter, we review the clinical features including peripheral neuropathy, immune-mediated disorders, migraines and neurocognitive involvement which have been suggested to be associated with premutation alleles. In addition, the current understanding of the pathogenic molecular mechanisms that give rise to the spectrum of FMR1 premutation associated disorders is also reviewed. Although further research is needed in order to shed light on the factors underlying the common incomplete penetrance applicable to all phenotypes associated with the premutation, it is likely that a combination of environmental and genetic factors with differences in intrinsic susceptibility may modulate the appearance and the severity of these disorders.
Keywords: FMR1 premutation, fibromyalgia, thyroid disease, peripheral neuropathy
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INTRODUCTION In the last years, there has been intense interest in identifying and characterizing the Fragile X premutation-associated phenotypes from the perspective not only of basic science but also of public health given its high prevalence affecting 1:250 females and 1:800 males among the general population [1]. Historically, carriers of FMR1 premutation (PM) alleles were considered to be clinically unaffected, since the gene is generally not methylated and these individuals do not present intellectual disabilities (ID). The significance of these alleles was generally thought to be their propensity for expansion to the full mutation range (>200 CGG repeats) during maternal transmission resulting in transcriptional silencing of FMR1 gene, absence of the encoding fragile X mental retardation 1 protein (FMRP) and manifestation of fragile X syndrome (FXS) [2,3]. Although, the features of this expansion are not fully understood, it is currently accepted that they depend on the size of the maternal allele and also on the number of the AGG interruptions within the CGG-repeat track [4]. The AGG interruptions are likely to have stabilizing effects during transmission by decreasing the risk of DNA polymerase slippage during DNA replication [5]. In PM male carriers there is a relative stable transmission, and thus, the risk of expansion to a full mutation is negligible [reviewed in 6]. Despite the belief that PM carriers do not present signs of clinical involvement, prior to the discovery of the gene FMR1 Cronister and collaborators (1991) [7] reported, higher rates of premature ovarian failure (POF) among women heterozygous for X-chromosome fragility. Later, Allinghan-Hawkins and colleagues (1999) [8] established PM alleles as a significant risk factor for POF based on the study of 760 women in which 16% of PM carriers were affected with POF whereas none of the full mutation carriers and just one (0.4%) of the controls presented with POF [8]. Currently, the association between ovarian deficiency and PM female carriers, namely Fragile X-Primary ovarian Insufficiency (FXPOI), is well-established, presenting an incidence of around 20% in these women and estimated at around 1% among the general population. Chapter 2 describes the features of FXPOI. Ten years after, the first description of a neurodegenerative disorder named Fragile X Tremor Ataxia Syndrome (FXTAS) associated to older adults PM carriers was made by Hagerman and colleagues (2001) [9]. This syndrome is characterized by white matter changes and global brain atrophy, presenting with core features of intention tremor and gait ataxia. Details of FXTAS are described in Chapter 3. Over the past 10-15 years, an increasingly broad spectrum of clinical manifestations has been related to individuals who are carriers of PM alleles (Table 1). Domains of clinical involvement seen in some, but not all carriers of PM encompass the presence of medical, emotional and cognitive manifestations which have been widely reported to occur more frequently among PM carriers than in the general population. Some of these features have recently been classified as being ‘definitely related’, ‘probably related’, ‘possibly related’ or ‘not likely related’ to the molecular changes associated with an FMR1 expansion based on clinical and previously reported data [reviewed in 10]. Coffey and collaborators (2008) [11] reported the first evidence of an expanded clinical phenotype of women with the PM. In this study, PM female carriers without core features of FXTAS showed significantly more complaints of chronic muscle pain, persistent paraesthesias in the extremities, and a history of tremor than controls. Furthermore, a significantly greater
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presence of medical co-morbidity was detected in females with definite or probable FXTAS, with an increased prevalence of thyroid disease, hypertension, seizures, peripheral neuropathy, fibromyalgia compared with controls [11]. In addition, some of the comorbidites associated with FXTAS beyond central nervous system involvement, specifically peripheral neuropathy [11,12] and neuroendocrine dysfunction [11,13-15] have also been associated with PM carriers without FXTAS. Moreover, there is increasing evidence that young PM carriers present increased rates of neurodevelopmental phenotypes such as attention-deficit hyperactivity disorder (ADHD), autism spectrum disorder (ASD) and seizures [reviewed in 16]. Furthermore, increased rates of psychiatric involvement, particularly depression and anxiety have also been associated with FXTAS among adult PM carriers [reviewed in 17]. Neuropsychiatric aspects are discussed in Chapter 4. Table 1.Clinical manifestations associated with some FMR1 premutation carriers Cohort studied* Immune mediated disorders Fibromyalgia PM females Thyroid disease PM females Irritable bowel syndrome PM females Neurodevelopmental Phenotypes Working memory deficiencies PM males Language dysfluencies PM females Spatiotemporal processing impairment Young-adult PM carriers Arithmetic weaknesses PM females Developmental Delay PM carriers Reproductive features Ovarian Insufficiency (FXPOI) PM females Obstetric and perinatal difficulties PM females Estrogen-deficiency related conditions FXPOI PM females Autonomic dysfunction Impotence FXTAS PM males Hypertension FXTAS PM both Bowel and bladder incontinence FXTAS PM both Neurocognitive and phychiatric Involvement Depression PM females Anxiety disorders PM females Mood disorders PM females Seizures PM male children ASD PM male children ADHD PM females Other clinical manifestations Migraine PM carriers both Peripheral neuropathy PM carriers both
References [11-15] [11,13,15] [15] [27,28] [29] [32] [30,31] [75] [reviewed in 10] [56] [11,67] [18,68]
[69-71] [72] [72,73] [74] [25] [11,76]
CLINICAL FEATURES ASSOCIATED WITH FMR1 PREMUTATION CARRIERS Neuropathy Peripheral neuropathy is characterized by a damaging of peripheral nerves which carry information to and from the brain as well as to and from the spinal cord to the rest of the body.
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Depending on the type of nerve affected it may promote impaired sensation, movement, gland or organ function. The association between neuropathy and FMR1 alleles was first reported in PM carriers with FXTAS [9,18]. Thereafter, Berry-Kravis and collaborators (2007) [12] reported the first evidence that signs of neuropathy on clinical examination are associated with PM carrier status based on neurological examination data from 207 unrelated individuals. Results from this study revealed that the degree of clinical involvement strongly correlated with the CGG repeat length in males since these individuals presented significantly higher mean scores in the neuropathy screening scale score (P=0.0014), the vibration score (P=0.0015), and the reflex score (P=0.0014) than sex-matched controls suggesting that PM male carriers present higher impairment of both distal vibratory sense and reflexes [12]. The lack of significant differences among PM female carriers presumably reflects the broad variation in clinical involvement among carriers as a result of variation in the X-chromosome activation ratio as well as the decreased penetrance of the clinical manifestation due to the protective effect of the second non-mutated X chromosome [12]. Afterwards, Coffey and collaborators (2008) [11] demonstrated that females with PM alleles also presented significantly higher signs of neuropathy based on findings from 128 PM female carriers without FXTAS. In this study it was shown that these women presented a significant rate of numbness and tingling and muscle pain in the extremities, albeit evidence of neuropathy is increased in PM female carriers presenting with FXTAS [11]. It has been suggested that neuropathic symptoms in PM female carriers are manifested together with the emergence of FXTAS disease [reviewed in 10].
Immune-Mediated Disorders Numerous reports support elevated rates of immune-mediated disorders (IMD) in PM female carriers, particularly regarding hypothyroidism and fibromyalgia [11, 13-15]. In contrast, IMDs have not been evidenced among males with the premutation, likely due to the relative rarity of these disorders among the general male population. In a recent study, Winarni and colleagues (2012) [15] examined the relative likelihood of large clinical manifestations among 344 female carriers of PM alleles and 72 controls including autoimmune thyroid disorder, multiple sclerosis, Sjögren syndrome, rheumatoid arthritis, systemic lupus erythematosus, Raynaud’s phenomenon, irritable bowel syndrome and optic neuritis. The results of this study evidenced that among women over 40 years of age 46.54% of PM females without FXTAS experienced one or more of the IMDs surveyed, and the prevalence increased to about 72.73% for those with FXTAS compared to 31.58% for the control group [15]. With respect to FXPOI, both groups of PM females carriers present higher odds ratios of IMDs compared to controls, and similarly, when considering FXTAS symptoms, the odds ratio of IMDs among PM female carriers presenting with FXPOI is about 2.4-fold higher when compared to those without FXPOI. Moreover, these authors found that an autoimmune thyroid disorder was the most common IMD followed by fibromyalgia and irritable bowel syndrome [15]. Increased penetrance for both thyroid disease and fibromyalgia has been broadly reported among PM female carriers [13], although the penetrance of these disorders is highly variable among the general population since it increases with age. For thyroid disease, it has been suggested that the association with PM alleles may be more relevant in older women [10] due to the lack of statistical significance when considering women between 18 and 50 years of age
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[19]. Nonetheless, in the general population the penetrance estimated for thyroid disorders is of around 10% [20] and around 2-4% for fibromyalgia whereas it has been estimated to be around 15.9% and 24.4%, respectively, among PM female carriers [13]. Conversely, in 700 unrelated Spanish patients with fibromyalgia the frequency of PM alleles did not significantly differ from the estimated rate in the general population [21]. In contrast, another study found a higher incidence of PM alleles among a Spanish female fibromyalgia cohort. Indeed, the incidence of PM alleles was 1 of 88, being 1 of 250 in females in the general population [22]. These controversial results are likely to be caused by a sample size effect since the data reported by Martorell and colleagues (2012) [22] were based on the screening of 353 females whereas the data presented by Rodriguez-Revenga and coworkers (2013) [21] were based on 700 samples. Nonetheless, it has been shown that the pathophysiology of fibromyalgia involves hyperexcitability of central neurons through several synaptic and neurotransmitter/ neurochemical mechanisms [reviewed in 23] suggesting that it could arise through an alteration of pain neurotransmissor mechanisms among PM female carriers [14]. Finally, it has been recently demonstrated that individual carriers of PM alleles present an immune dysregulation and decreased immune responses when compared with healthy controls [24]. Moreover, it has been found that PM carriers present a reduction in the levels of cytokine production which is negatively associated with CGG repeat length, mainly with IL-12 production [24]. Furthermore, these women have also been shown to present a decrease in the relative levels of the surface marker CD25 in T cells suggesting potential differences in the activation of T-cells that regulate immune response [24].
Migraines Au and collaborators (2013) [25] have recently reported that PM carriers show increased rates in the prevalence of migraines based on physical and medical examination of 315 PM carriers (203 females and 112 males) and 154 controls (83 females and 71 males). Migraine is a neurologic disorder characterized by light and sound sensitivity and pulsatile pain, which is thought to have a polygenic and mutlifactorial etiology. The prevalence of migraine among the general population is estimated around 27.3% in women and 9.7% in men [26], reaching up to 54.2% and 26.79% among female and male PM carriers, respectively, both resulting in statistical significant differences [25]. In addition, this significance was obtained considering both those affected with and also those without FXTAS, adjusted for age. However, the risk of migraine headaches was not correlated with either CGG repeats or FMR1 mRNA expression [25].
Neurocognitive Features The expanded range of clinical involvement associated with PM carriers also includes an alteration of various cognitive domains including executive function, working memory and arithmetic skills which become apparent even in young individuals, with a usually more progressive course in PM individuals than in the general population [reviewed in 10]. However, neurocognitive deficits are reportedly more frequent in male than in female carriers of PM alleles. Results reported by Kogan and collaborators (2008) [27] revealed that the CGG
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expansion confers a significant risk for working memory difficulties based on a controlled study of 40 PM male carriers without manifest symptoms of FXTAS. In addition, Cornish and colleagues (2009) [28] reported neuropsychological measures in PM males regarding core subcomponents of working memory such as verbal memory, visual–spatial memory, and central executive memory revealing that PM males present specific vulnerability in executive control of memory including tasks requiring simultaneous manipulation and storage of new information, regardless of the presence of FXTAS symptoms. These authors revealed an impairment of the central executive working memory among PM male carriers without FXTAS, which was significantly correlated with larger CGG repeat expansions, whereas FXTAS patients demonstrated a more general impairment in terms of phonological working memory in addition to central executive working memory [28]. Moreover, language dysfluencies associated with deficits in organization and planning have been evidenced among PM female carriers [29]. Past research has demonstrated that language dysfluencies are an indicator of executive functioning deficits which are characteristic of other neurodegenerative disorders such as Parkinson and Alzheimer diseases. Regarding arithmetic skills it has been suggested that PM female carriers show weaknesses in mathematical tasks [30] and this has recently been supported by other groups [31]. Furthermore, it has been demonstrated that PM carriers from 19 to 45 years of age show impairment in spatiotemporal processing which may underlie the impairments observed in arithmetic skills among these individuals since the representations of space and time provide the foundation for an understanding of numbers [32]. Although further research is needed, it has been suggested that determining whether cognitive impairments are detectable in PM carriers without FXTAS should be prudent since it may not only be an early indicator of cognitive decline in PM carriers [29] but could also be used as a biomarker of disease progression if these features precede motor impairment [32].
CURRENT UNDERSTANDING OF THE MOLECULAR MECHANISMS UNDERLYING PREMUTATION-ASSOCIATED PATHOLOGIES Premutation-associated phenotypes have been mainly attributed to a pathogenic mechanism involving a gain-of-function toxicity of the expanded FMR1 mRNA, a process entirely distinct from the FMRP deficiency responsible for the FXS phenotype [reviewed in 33]. This observation was based on the restriction of these clinical phenotypes to the premutation range in which the molecular signature is a 2-8 fold increase in the expression of the PM mRNA and, paradoxically, a slight reduction in Fragile X Mental Retardation Protein (FMRP) levels [34]. The mechanisms underlying the increased transcriptional activity of the PM alleles remain unclear, however, it has been reported that these alleles use differential transcriptional start sites leading to different expression compared to non-expanded FMR1 alleles [35]. On other hand, it has also been suggested that the reduction of FMRP in PM carriers may promote an added contribution to the clinical involvement observed in both children and adults related to phenotypes associated with reduced cognition and disturbed behavior [36]. However, the FMRP deficiency cannot be a driving factor in the PM-associated disorders since FXTAS and FXPOI are not experienced by full mutation carriers. Despite wide reports that the levels of FMRP expression are only slightly decreased in patients with FXTAS, most of these
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measurements have been performed using lymphocytes or whole blood samples rather than brain tissue, in which changes are likely to be more robust [reviewed in 37]. The efficiency of FMRP translation is associated with the length of the CGG expansion in light of impairment in translation of larger CGG tracks by interference with ribosomal scanning through the 5’UTR, thereby preventing appropriate loading of the expanded FMR1 mRNA into polyribosomal complexes [38-40]. It is currently considered that the dual mechanism of involvement, the excess of PM mRNA expression and the decrease in FMRP translation, is a double hit which may promote phenotypic features of FXS and PM associated disorders [reviewed in 33, 41]. Notwithstanding, the reduction of FMRP synthesis, the phenotype of PM carriers, is different from carriers of full mutation alleles since milder protein deficiency among PM carriers usually leads to mild developmental problems with these individuals having higher IQs and less severe behavioral problems than those with FXS [reviewed in 42]. A FMRP deficit has been correlated with lowered activity of the amygdala among PM male carriers compared to controls on functional magnetic resonance imaging whereas increased levels of expanded mRNA have been strongly associated with obsessive–compulsive symptoms and psychoticism in PM male carriers [43,44]. It has also been described that RNA toxicity leads to the up-regulation of the heat shock proteins Hsp70 and αB-crystallin, which may stimulate immune dysregulation [15]. Regarding migraines, their association with mitochondrial dysfunction is well established. Interestingly, there is evidence pointing to a deregulation of mitochondrial function among PM carriers [45-47], therefore the increased prevalence of migraines in PM carriers may be the result of RNA toxicity leading to mitochondrial deregulation [25]. Likewise, RNA toxicity has also been proposed to shed light on the high rate of thyroid dysfunction among females with the PM by causing a direct effect on the hypothalamic-pituitary-adrenal axis or on the thyroid gland. Additionally, RNA toxicity has also been proposed to promote an autoimmune mechanism or apoptosis in thyroid cells [11]. However, data reported by Cunningham and coworkers (2011) [48] show that the presence of PM alleles promotes migration defects in the neocortex and altered expression of neuronal lineage markers among embryonic PM mice. These results support the hypothesis that the role of the RNA toxicity may be restricted to the initial triggering events since many features of the neuronal and astrocytic cellular phenotype observed in FXTAS patients are already present in the neonatal period, suggesting that the clinical involvement among children carriers of PM alleles may be manifestations of this early, non-degenerative process [reviewed in 33]. The sequestration hypothesis of RNA toxicity was first proposed and established for myotonic dystrophy type 1 caused by an expansion of a CTG repeat in the 3’UTR of DMPK gene [reviewed in 49]. Particularly, in Fragile X PM the model hypothesized that expanded CGG repeats form hairpin loops which are sticky and recruit an excess of specific RNA-binding proteins, resulting in a functional insufficiency of the sequestered proteins and leading to cell dysfunction and death [50]. Although the initial triggering events in these disorders are based on RNA toxicity, this model does not provide evidence regarding cell sickening and death. In this way, there are several candidate downstream pathways, although alterations of both mitochondrial function and calcium regulation are emerging as core mediators of cellular deregulation and dysfunction [reviewed in 31, 39] The increased expression of the expanded FMR1 mRNA is thought to be the main cause of clinical involvement in PM carriers since the FMR1 mRNA and the sequestered proteins form aggregates leading to intranuclear inclusions present in several tissues. Interestingly, the expanded mRNA of FMR1 is detected within the
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inclusion whereas FMRP is not present [51]. Furthermore, proteomic analysis of these inclusions revealed a large number of proteins presented in the aggregates, including the RNA binding protein hnRNP A2/B1, the nuclear envelope protein lamin A/C, the small heat shock protein αB-crystallin [52], the splicing factor Sam68 [53] and part of the microRNA processor complex DGCR8 [50]. Indeed, it has recently been demonstrated that the sequestration of DGCR8 promotes the deregulation of microRNAs biogenesis, suggesting a central role of DGCR8 as an inductor of RNA toxicity by leading to cell dysfunction and cell loss [50]. Intranuclear inclusions, which represent the neuropathological hallmark of FXTAS [52], have been also detected among PM carriers through different cell types including the central and peripheral nervous system and other tissue including the adrenal glands, the testes, pancreas and heart [54-58]. Recently, Hunsaker and colleagues (2011) [58] reported autopsy findings from ten PM carriers with FXTAS in which intranuclear inclusions were detected throughout multiple tissues including the hypothalamic-pituitary-adrenal axis, pineal gland, cardiac conduction system, peripheral nerves and autonomic ganglia, the thyroid gland, the digestive system, the testes and pancreas. The broad distribution of these inclusions suggests that many organ systems may be affected by RNA toxicity. Nevertheless, it is necessary to study the processes underlying inclusion formation in depth to address whether they themselves are toxic or reflect cellular dysfunction [58]. Furthermore, additional mechanisms have been proposed as possible triggering events in the PM-associated disorders, although most evidence support CGG-repeat mediated protein sequestration [reviewed in 33,37]. These mechanisms include a RNA-mediated protein aggregation model whereby the CGGs contained within the FMR1 mRNA might promote a conformational transition in proteins with prion-like domains that may initiate a cascade of protein aggregation similar to what occurs in amyloid plaque formation in Alzheimer’s disease. Moreover, the production of a toxic polyglycine peptide has also been proposed as an alternative toxicity model by a non-AUG-initiated (RAN) translation [59]. In addition, a specific splicing isoform is detected exclusively with transcripts of PM alleles suggesting a possible role of antisense transcripts generated at the FMR1 locus [60]. Otherwise very little attention has been focused on the other end of the spectrum, the socalled “low-normal” numbers of CGG repeats, up to 23 trinucleotide repeats. In this framework, data based on genotype–phenotype correlations have recently been reported suggesting that this range of CGG repeats may have substantial implications for cognitive functioning, cancer, and the odds of having children with neurodevelopmental or neuropsychiatric conditions [61]. Despite these range of CGG number not being associated with altered FMRP synthesis, Chen and co-workers (2003) [62] reported that the efficiency of FMRP translation was based on the number of CGG repeats, conferring the greatest efficiency of protein synthesis to the allele of 30 repeats. Thus, inefficient translation may be related to the clinical manifestations associated with the low numbers of CGG repeats. Likewise, Ramocki and Zoghbi (2008) [63] suggested that imbalances in homeostatic controls of multiple genes including FMR1 may partially promote the appearance of neurodevelopmental and neuropsychiatric disorders. At this point, the difficulty in understanding PM-associated pathologies lies in the inability of prediction of which PM carriers will develop any of these phenotypes. The incomplete penetrance across the phenotypic spectrum is likely to be associated with a combination of genetic and environmental factors which may confer specific vulnerability to PM carriers (Figure 1).
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Figure 1. Diagram of the potential players contributing to the clinical involvement associated to PM carriers.
Genetic factors that may contribute to the PM-associated disorders include CGG repeat length, expression levels of the expanded FMR1 mRNA, aberrant translation of the repeat sequence as well as genomic changes in other regions of the genome. Within this framework, specific polymorphisms of the CRHR1 gene have been associated with female clinical involvement (rs7209436), particularly with depression and anxiety mainly as this gene regulates the expression and release of ACTH from the anterior pituitary gland which, in turn, stimulates the release of cortisol from the adrenal cortex [64]. Furthermore, risk factors for other neurodegenerative disorders such as allele ε4 of the APOE gene may also influence the risk of FXTAS as a higher frequency of these alleles has been reported in PM carriers with compared to those without FXTAS [65]. Moreover, it has recently been suggested that
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individuals who are carriers of PM alleles presenting with ID, seizures or ASD are likely to have a second hit since PM carriers show a significant enrichment (P=2.27e-07) of CNVs compared to controls [66]. Furthermore, these authors found an association between the presence of rare CNVs (not detected in 8000 controls) among PM carriers with either autistic traits or neurological involvement, suggesting that they may have a possible role in this phenotypic variability [66]. Regarding environmental factors, it has been suggested that smoking, prolonged surgery with anesthesia, drug and alcohol abuse or the stress of carrying a child with FXS are also puzzling factors which may act as additional determinants for the phenotypic variability among PM individuals [reviewed in 41,42]. Finally, further longitudinal studies are required to determine the context in which any of the PM-associated phenotypes are developed and what protective factors might reduce the risks of more negative outcomes [reviewed in 10].
CONCLUSION Overall, it is currently accepted that PM alleles led to multiple distinct clinical features which are present at a greater frequency among PM carriers than what would be expected in the general population. However, the association of the PM with the phenotypes reviewed in this chapter is less well established than its association with FXTAS and FXPOI. Although further research is needed in order to shed light on the factors underlying the common incomplete penetrance applicable to all phenotypes associated with the PM, a combination of environmental and genetic factors with differences in intrinsic susceptibility likely modulate the appearance and the severity of these disorders.
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Cronister A, Schreiner R, Wittenberger M, Amiri K, Harris K, Hagerman RJ. Heterozygous fragile X female: historical, physical, cognitive, and cytogenetic features. Am. J. Med. Genet. 1991 Feb-Mar;38(2-3):269-74. Allingham-Hawkins DJ, Babul-Hirji R, Chitayat D, Holden JJ, Yang KT, Lee C, Hudson R, Gorwill H, Nolin SL, Glicksman A, Jenkins EC, Brown WT, Howard-Peebles PN, Becchi C, Cummings E, Fallon L, Seitz S, Black SH, Vianna-Morgante AM, Costa SS, Otto PA, Mingroni-Netto RC, Murray A, Webb J, Vieri F, et al. Fragile X premutation is a significant risk factor for premature ovarian failure: the International Collaborative POF in Fragile X study--preliminary data. Am. J. Med. Genet. 1999 Apr 2;83(4):322-5. Hagerman RJ, Leehey M, Heinrichs W, Tassone F, Wilson R, Hills J, Grigsby J, Gage B, Hagerman PJ. Intention tremor, parkinsonism, and generalized brain atrophy in male carriers of fragile X. Neurology. 2001 Jul 10;57(1):127-30. Wheeler AC, Bailey DB Jr, Berry-Kravis E, Greenberg J, Losh M, Mailick M, Milà M, Olichney JM, Rodriguez-Revenga L, Sherman S, Smith L, Summers S, Yang JC, Hagerman R. Associated features in females with an PM. J Neurodev Disord. 2014;6(1):30. Review. Coffey SM, Cook K, Tartaglia N, Tassone F, Nguyen DV, Pan R, Bronsky HE, Yuhas J, Borodyanskaya M, Grigsby J, Doerflinger M, Hagerman PJ, Hagerman RJ. Expanded clinical phenotype of women with the PM. Am. J. Med. Genet. A. 2008 Apr 15;146A(8):1009-16. Berry-Kravis E, Goetz CG, Leehey MA, Hagerman RJ, Zhang L, Li L, Nguyen D, Hall DA, Tartaglia N, Cogswell J, Tassone F, Hagerman PJ. Neuropathic features in fragile X premutation carriers. Am. J. Med. Gene.t A. 2007 Jan 1;143A(1):19-26. Rodriguez-Revenga L, Madrigal I, Pagonabarraga J, Xunclà M, Badenas C, Kulisevsky J, Gomez B, Milà M. Penetrance of FMR1 premutation associated pathologies in fragile X syndrome families. Eur. J. Hum. Genet. 2009 Oct;17(10):1359-62. Leehey MA, Legg W, Tassone F, Hagerman R. Fibromyalgia in fragile X mental retardation 1 gene premutation carriers. Rheumatology (Oxford). 2011 Dec;50(12):22336. Winarni TI, Chonchaiya W, Sumekar TA, Ashwood P, Morales GM, Tassone F, Nguyen DV, Faradz SM, Van de Water J, Cook K, Hamlin A, Mu Y, Hagerman PJ, Hagerman RJ. Immune-mediated disorders among women carriers of fragile X premutation alleles. Am. J. Med. Genet. A. 2012 Oct;158A(10):2473-81. Besterman AD, Wilke SA, Mulligan TE, Allison SC, Hagerman R, Seritan AL, Bourgeois JA. Towards an Understanding of Neuropsychiatric Manifestations in Fragile X Premutation Carriers. Future Neurol. 2014 Mar;9(2):227-239. Hunter JE, Abramowitz A, Rusin M, Sherman SL. Is there evidence for neuropsychological and neurobehavioral phenotypes among adults without FXTAS who carry the PM? A review of current literature. Genet Med. 2009 Feb;11(2):79-89. Jacquemont S, Hagerman RJ, Leehey M, Grigsby J, Zhang L, Brunberg JA, Greco C, Des Portes V, Jardini T, Levine R, Berry-Kravis E, Brown WT, Schaeffer S, Kissel J, Tassone F, Hagerman PJ. Fragile X premutation tremor/ataxia syndrome: molecular, clinical, and neuroimaging correlates. Am. J. Hum. Genet. 2003 Apr;72(4):869-78. Hunter JE, Rohr JK, Sherman SL. Co-occurring diagnoses among FMR1 premutation allele carriers. Clin. Genet. 2010 Apr;77(4):374-81.
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[20] Canaris GJ, Manowitz NR, Mayor G, Ridgway EC. The Colorado thyroid disease prevalence study. Arch. Intern Med. 2000 Feb 28;160(4):526-34. [21] Rodriguez-Revenga L, Madrigal I, Blanch-Rubió J, Elurbe DM, Docampo E, Collado A, Vidal J, Carbonell J, Estivill X, Mila M. Screening for the presence of PM alleles in women with fibromyalgia. Gene. 2013 Jan 10;512(2):305-8. [22] Martorell L, Tondo M, Garcia-Fructuoso F, Naudo M, Alegre C, Gamez J, Genovés J, Poo P. Screening for the presence of FMR1 premutation alleles in a Spanish population with fibromyalgia. Clin. Rheumatol. 2012 Nov;31(11):1611-5. [23] Yunus MB. Role of central sensitization in symptoms beyond muscle pain, and the evaluation of a patient with widespread pain. Best Pract. Res. Clin. Rheumatol. 2007 Jun;21(3):481-97. Review. [24] Careaga M, Rose D, Tassone F, Berman RF, Hagerman R, Ashwood P. Immune dysregulation as a cause of autoinflammation in fragile X premutation carriers: link between FMRI CGG repeat number and decreased cytokine responses. PLoS One. 2014 Apr 9;9(4):e94475. [25] Au J, Akins RS, Berkowitz-Sutherland L, Tang HT, Chen Y, Boyd A, Tassone F, Nguyen DV, Hagerman R. Prevalence and risk of migraine headaches in adult fragile X premutation carriers. Clin Genet. 2013 Dec;84(6):546-51. [26] Lipton RB, Stewart WF, Diamond S, Diamond ML, Reed M. Prevalence and burden of migraine in the United States: data from the American Migraine Study II. Headache 2001: 41 (7): 646–657. [27] Kogan CS, Turk J, Hagerman RJ, Cornish KM. Impact of the Fragile X mental retardation 1 (FMR1) gene premutation on neuropsychiatric functioning in adult males without fragile X-associated Tremor/Ataxia syndrome: a controlled study. Am. J. Med. Genet. B Neuropsychiatr Genet. 2008 Sep 5;147B(6):859-72. [28] Cornish KM, Kogan CS, Li L, Turk J, Jacquemont S, Hagerman RJ. Lifespan changes in working memory in fragile X premutation males. Brain Cogn. 2009 Apr;69(3):551-8. [29] Sterling AM, Mailick M, Greenberg J, Warren SF, Brady N. Language dysfluencies in females with the PM. Brain Cogn. 2013 Jun;82(1):84-9. [30] Lachiewicz AM, Dawson DV, Spiridigliozzi GA, McConkie-Rosell A. Arithmetic difficulties in females with the fragile X premutation. Am. J. Med. Genet. A. 2006 Apr 1;140(7):665-72. [31] Semenza C, Bonollo S, Polli R, Busana C, Pignatti R, Iuculano T, Maria Laverda A, Priftis K, Murgia A. Genetics and mathematics: FMR1 premutation female carriers. Neuropsychologia. 2012 Dec;50(14):3757-63 [32] Wong LM, Goodrich-Hunsaker NJ, McLennan Y, Tassone F, Harvey D, Rivera SM, Simon TJ. Young adult male carriers of the fragile X premutation exhibit genetically modulated impairments in visuospatial tasks controlled for psychomotor speed. J. Neurodev Disord. 2012 Nov 13;4(1):26. [33] Hagerman P. Fragile X-associated tremor/ataxia syndrome (FXTAS): pathology and mechanisms. Acta Neuropathol. 2013 Jul;126(1):1-19. [34] Tassone F, Beilina A, Carosi C, Albertosi S, Bagni C, Li L, Glover K, Bentley D, Hagerman PJ. Elevated FMR1 mRNA in premutation carriers is due to increased transcription. RNA. 2007 Apr;13(4):555-62.
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[35] Tassone F, De Rubeis S, Carosi C, La Fata G, Serpa G, Raske C, Willemsen R, Hagerman PJ, Bagni C. Differential usage of transcriptional start sites and polyadenylation sites in FMR1 premutation alleles. Nucleic Acids Res. 2011 Aug;39(14):6172-85. [36] Tassone F, Hagerman RJ, Taylor AK, Mills JB, Harris SW, Gane LW, Hagerman PJ (2000) Clinical involvement and protein expression in individuals with the FMR1 premutation. Am. J. Med. Genet. 91(2):144–152 [37] Renoux AJ, Todd PK. Neurodegeneration the RNA way. Prog. Neurobiol. 2012 May;97(2):173-89. [38] Primerano B, Tassone F, Hagerman RJ, Hagerman P, Amaldi F, Bagni C. Reduced FMR1 mRNA translation efficiency in fragile X patients with premutations. RNA. 2002 Dec;8(12):1482-8. [39] Ludwig AL, Raske C, Tassone F, Garcia-Arocena D, Hershey JW, Hagerman PJ. Translation of the FMR1 mRNA is not influenced by AGG interruptions. Nucleic Acids Res. 2009 Nov;37(20):6896-904. [40] Ludwig AL, Hershey JW, Hagerman PJ. Initiation of translation of the FMR1 mRNA Occurs predominantly through 5'-end-dependent ribosomal scanning. J. Mol. Biol. 2011 Mar 18;407(1):21-34. [41] Hagerman R, Hagerman P. Advances in clinical and molecular understanding of the FMR1 premutation and fragile X-associated tremor/ataxia syndrome. Lancet Neurol. 2013 Aug;12(8):786-98. [42] Polussa J, Schneider A, Hagerman R. (2014) Molecular Advances Leading to Treatment Implications for Fragile X Premutation Carriers. Brain Disord Ther 3:2. [43] Hessl D, Tassone F, Loesch DZ, Berry-Kravis E, Leehey MA, Gane LW, Barbato I, Rice C, Gould E, Hall DA, Grigsby J, Wegelin JA, Harris S, Lewin F, Weinberg D, Hagerman PJ, Hagerman RJ. Abnormal elevation of FMR1 mRNA is associated with psychological symptoms in individuals with the fragile X premutation. Am. J. Med. Genet. B Neuropsychiatr Genet. 2005; 139B (1):115–121. [44] Hoem G, Raske CR, Garcia-Arocena D, Tassone F, Sanchez E, Ludwig AL, Iwahashi CK, Kumar M, Yang JE, Hagerman PJ. CGG-repeat length threshold for FMR1 RNA pathogenesis in a cellular model for FXTAS. Hum. Mol. Genet. 2011; 20 (11):2161– 2170. [45] Ross-Inta C, Omanska-Klusek A, Wong S, Barrow C, Garcia-Arocena D, Iwahashi C, Berry-Kravis E, Hagerman RJ, Hagerman PJ, Giulivi C. Evidence of mitochondrial dysfunction in fragile X-associated tremor/ataxia syndrome. Biochem. J. 2010 Aug 1;429(3):545-52. [46] Napoli E, Ross-Inta C, Wong S, Omanska-Klusek A, Barrow C, Iwahashi C, GarciaArocena D, Sakaguchi D, Berry-Kravis E, Hagerman R, Hagerman PJ, Giulivi C. Altered zinc transport disrupts mitochondrial protein processing/import in fragile X-associated tremor/ataxia syndrome. Hum. Mol. Genet. 2011 Aug 1;20(15):3079-92. [47] Mateu-Huertas E, Rodriguez-Revenga L, Alvarez-Mora MI, Madrigal I, Willemsen R, Milà M, Martí E, Estivill X. Blood expression profiles of fragile X premutation carriers identify candidate genes involved in neurodegenerative and infertility phenotypes. Neurobiol. Dis. 2014 May;65:43-54. [48] Cunningham CL, Martínez Cerdeño V, Navarro Porras E, Prakash AN, Angelastro JM, Willemsen R, Hagerman PJ, Pessah IN, Berman RF, Noctor SC. Premutation CGG-
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M. I. Alvarez-Mora and L. Rodriguez-Revenga repeat expansion of the Fmr1 gene impairs mouse neocortical development. Hum. Mol. Genet. 2011 Jan 1;20(1):64-79. Todd PK, Paulson HL. RNA-mediated neurodegeneration in repeat expansion disorders. Ann. Neurol. 2010 Mar;67(3):291-300. Review. Sellier C, Freyermuth F, Tabet R, Tran T, He F, Ruffenach F, Alunni V, Moine H, Thibault C, Page A, Tassone F, Willemsen R, Disney MD, Hagerman PJ, Todd PK, Charlet-Berguerand N. Sequestration of DROSHA and DGCR8 by expanded CGG RNA repeats alters microRNA processing in fragile X-associated tremor/ataxia syndrome. Cell Rep. 2013 Mar 28;3(3):869-80. Tassone F, Iwahashi C, Hagerman PJ. FMR1 RNA within the intranuclear inclusions of fragile X-associated tremor/ataxia syndrome (FXTAS). RNA Biol. 2004 Jul;1(2):103-5. Iwahashi CK, Yasui DH, An HJ, Greco CM, Tassone F, Nannen K, Babineau B, Lebrilla CB, Hagerman RJ, Hagerman PJ. Protein composition of the intranuclear inclusions of FXTAS. Brain. 2006 Jan;129(Pt 1):256-71. Sellier C, Rau F, Liu Y, Tassone F, Hukema RK, Gattoni R, Schneider A, Richard S, Willemsen R, Elliott DJ, Hagerman PJ, Charlet-Berguerand N. Sam68 sequestration and partial loss of function are associated with splicing alterations in FXTAS patients. EMBO J. 2010 Apr 7;29(7):1248-61. Louis E, Moskowitz C, Friez M, Amaya M, Vonsattel JP. Parkinsonism, dysautonomia, and intranuclear inclusions in a fragile X carrier: a clinical-pathological study. Mov. Disord. 2006 Mar;21(3):420-5. Greco CM, Berman RF, Martin RM, Tassone F, Schwartz PH, Chang A, Trapp BD, Iwahashi C, Brunberg J, Grigsby J, Hessl D, Becker EJ, Papazian J, Leehey MA, Hagerman RJ, Hagerman PJ. Neuropathology of fragile X-associated tremor/ataxia syndrome (FXTAS). Brain. 2006 Jan;129(Pt 1):243-55. Greco CM, Soontrapornchai K, Wirojanan J, Gould JE, Hagerman PJ, Hagerman RJ. Testicular and pituitary inclusion formation in fragile X associated tremor/ataxia syndrome. J. Urol. 2007 Apr;177(4):1434-7. Gokden M, Al-Hinti JT, Harik SI. Peripheral nervous system pathology in fragile X tremor/ataxia syndrome (FXTAS). Neuropathology. 2009 Jun;29(3):280-4. Hunsaker MR, Greco CM, Spath MA, Smits AP, Navarro CS, Tassone F, Kros JM, Severijnen LA, Berry-Kravis EM, Berman RF, Hagerman PJ, Willemsen R, Hagerman RJ, Hukema RK. Widespread non-central nervous system organ pathology in fragile X premutation carriers with fragile X-associated tremor/ataxia syndrome and CGG knockin mice. Acta Neuropathol. 2011 Oct;122(4):467-79. Todd PK, Oh SY, Krans A, He F, Sellier C, Frazer M, Renoux AJ, Chen KC, Scaglione KM, Basrur V, Elenitoba-Johnson K, Vonsattel JP, Louis ED, Sutton MA, Taylor JP, Mills RE, Charlet-Berguerand N, Paulson HL. CGG repeat-associated translation mediates neurodegeneration in fragile X tremor ataxia syndrome. Neuron. 2013 May 8;78(3):440-55. Ladd PD, Smith LE, Rabaia NA, Moore JM, Georges SA, Hansen RS, Hagerman RJ, Tassone F, Tapscott SJ, Filippova GN. An antisense transcript spanning the CGG repeat region of FMR1 is upregulated in premutation carriers but silenced in full mutation individuals. Hum. Mol. Genet. 2007 Dec 15;16(24):3174-87.
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[61] Mailick MR, Hong J, Rathouz P, Baker MW, Greenberg JS, Smith L, Maenner M. Lownormal FMR1 CGG repeat length: phenotypic associations. Front Genet. 2014 Sep 9;5:309. [62] Chen LS, Tassone F, Sahota P, Hagerman PJ. The (CGG)n repeat element within the 5' untranslated region of the FMR1 message provides both positive and negative cis effects on in vivo translation of a downstream reporter. Hum. Mol. Genet. 2003 Dec 1;12(23):3067-74. Epub 2003 Sep 30. [63] Ramocki MB, Zoghbi HY. Failure of neuronal homeostasis results in common neuropsychiatric phenotypes. Nature. 2008 Oct 16;455(7215):912-8. Review. [64] Hunter JE, Leslie M, Novak G, Hamilton D, Shubeck L, Charen K, Abramowitz A, Epstein MP, Lori A, Binder E, Cubells JF, Sherman SL. Depression and anxiety symptoms among women who carry the PM: impact of raising a child with fragile X syndrome is moderated by CRHR1 polymorphisms. Am. J. Med. Genet. B Neuropsychiatr Genet. 2012 Jul;159B(5):549-59. [65] Silva F, Rodriguez-Revenga L, Madrigal I, Alvarez-Mora MI, Oliva R, Milà M. High apolipoprotein E4 allele frequency in FXTAS patients. Genet Med. 2013 Aug;15(8):63942. [66] Lozano R, Hagerman RJ, Duyzend M, Budimirovic DB, Eichler EE, Tassone F. Genomic studies in fragile X premutation carriers. J. Neurodev Disord. 2014;6(1):27. [67] Hamlin A, Liu Y, Nguyen DV, Tassone F, Zhang L, Hagerman RJ. Sleep apnea in fragile X premutation carriers with and without FXTAS. Am. J. Med. Genet. B Neuropsychiatr Genet. 2011;156B (8):923–928. [68] Leehey MA. Fragile X-associated tremor/ataxia syndrome: clinical phenotype, diagnosis, and treatment. J. Investig. Med. 2009 Dec;57(8):830-6. Review. [69] Rodriguez-Revenga L, Madrigal I, Alegret M, Santos M, Milà M. Evidence of depressive symptoms in fragile-X syndrome premutated females. Psychiatr Genet. 2008 Aug;18(4):153-5. [70] Bourgeois JA, Seritan AL, Casillas EM, Hessl D, Schneider A, Yang Y, Kaur I, Cogswell JB, Nguyen DV, Hagerman RJ. Lifetime prevalence of mood and anxiety disorders in fragile X premutation carriers. J. Clin. Psychiatry. 2011 Feb;72(2):175-82. [71] Hunter JE, Leslie M, Novak G, Hamilton D, Shubeck L, Charen K, Abramowitz A, Epstein MP, Lori A, Binder E, Cubells JF, Sherman SL. Depression and anxiety symptoms among women who carry the FMR1 premutation: impact of raising a child with fragile X syndrome is moderated by CRHR1 polymorphisms. Am. J. Med. Genet. B Neuropsychiatr Genet. 2012 Jul;159B(5):549-59. [72] Chonchaiya W, Au J, Schneider A, Hessl D, Harris SW, Laird M, Mu Y, Tassone F, Nguyen DV, Hagerman RJ. Increased prevalence of seizures in boys who were probands with the FMR1 premutation and co-morbid autism spectrum disorder. Hum. Genet. 2012; 131 (4):581–589. [73] Farzin F, Perry H, Hessl D, Loesch D, Cohen J, Bacalman S, Gane L, Tassone F, Hagerman P, Hagerman R. Autism spectrum disorders and attention-deficit/ hyperactivity disorder in boys with the fragile X premutation. J. Dev. Behav. Pediatr. 2006; 27 (2 Suppl):S137–144. [74] Kraan CM, Hocking DR, Georgiou-Karistianis N, Metcalfe SA, Archibald AD, Fielding J, Trollor J, Bradshaw JL, Cohen J, Cornish KM. Impaired response inhibition is associated with self-reported symptoms of depression, anxiety, and ADHD in female
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FMR1 premutation carriers. Am. J. Med. Genet. B Neuropsychiatr Genet. 2014 Jan;165(1):41-51. [75] Bailey DB Jr, Raspa M, Olmsted M, Holiday DB. Co-occurring conditions associated with FMR1 gene variations: findings from a national parent survey. Am. J. Med. Genet. A. 2008; 146A (16): 2060–2069. [76] Hagerman RJ, Coffey SM, Maselli R, Soontarapornchai K, Brunberg JA, Leehey MA, Zhang L, Gane LW, Fenton-Farrell G, Tassone F, Hagerman PJ. Neuropathy as a presenting feature in fragile X-associated tremor/ataxia syndrome. Am. J. Med. Genet. A. 2007 Oct 1;143A(19):2256-60.
In: Encyclopedia of Genetics: New Research (8 Volume Set) ISBN: 978-1-53614-451-2 Editor: Heidi Carlson © 2019 Nova Science Publishers, Inc.
Chapter 92
GENETIC COUNSELING OF FMR1 I. Madrigal Biochemistry and Molecular Genetics Department, Hospital Clínic and IDIBAPS Centre for Biomedical Research on Rare Diseases (CIBERER), ISCIII, Barcelona, Spain IDIBAPS (Institut d’Investigacions Biomèdiques August Pi i Sunyer), Barcelona, Spain
ABSTRACT Fragile X syndrome is the most common form of inherited intellectual disability, with an estimated incidence of 1 in 4,000 males and 1 in 6,000 females. Each diagnosis of an FMR1 mutation has far reaching clinical and reproductive implications for the extended family. Until now genetic counseling was offered based on the expansion risk in premutation carrier women, but the description of FMR1-associated disorders has increased the complexity of the genetic counseling for FXS families, especially FMR1 premutation carriers. Male individuals carrying full mutated alleles present with intellectual disability while the penetrance is incomplete in females (30-50%). Premutation allele carriers are intellectually unaffected, but several FMR1 premutation-related disorders have been described. The most prevalent are fragile X-associated primary ovarian insufficiency and fragile X-associated tremor/ataxia syndrome, but behavioral features such as impaired executive function, social deficits or anxiety have also been related to several FMR1 premutation carriers. Premutation women have 50% of risk of transmitting a premutation/full mutation allele to their offspring, depending on the CGG expansion repeat and the presence of AGG interruptions. On the contrary, premutation men carriers will only transmit the premutation allele to their daughters. Some issues such as risk assessment for intermediate alleles and the clinical prognosis for females with full mutations still remain challenging. Genetic counselors must have an updated and solid understanding of this genetic condition and the FMR1-associated disorders in order to cover all the counseling aspects of these disorders.
Keywords: FMR1, FXS, premutation alleles, full mutation alleles, genetic counseling, intermediate alles
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INTRODUCTION The World Health Organization defines Genetic Counseling as the process through which trained genetic counselors inform about the genetic aspects of a particular genetic disease to those who are at an increased risk of developing the disorder or of passing it on to their unborn offspring. A genetic counselor must provide accurate information to the patient and their families about the clinical consequences of the conditions, the inheritance of illnesses and their risks of recurrence, management and/or treatment, quality of life, life expectancy and social aspects. These professionals must then work in a wide variety of fields such as general genetics, prenatal care or family planning. The counseling process consists of two stages: pre and the post-test genetic counseling. In pre-test genetic counseling individuals are informed about the purpose of the test, the clinical consequences of the conditions, including the phenotypic features, inheritance patterns, the reliability and limitations of the test and the possible psychological impacts to the counselees and their relatives. In post-test genetic counseling, after disclosure of the test results, the counselor should focus on the emotional impact on the counselee and the relatives involved. The counselees must be informed about implications to them and their close relatives, including management and/or treatment options, quality of life, life expectancy and available reproductive choices. Counselors must ensure the privacy and confidentiality of the results.
GENETIC COUNSELING IN FMR1-ASSOCIATED DISORDERS Fragile X syndrome (FXS, #300624) is the most common form of inherited intellectual disability (ID), with an estimated incidence of 1 in 4,000 males and 1 in 6,000 females [1]. The molecular basis of this syndrome is mainly the expansion of an unstable CGG repeat in the 5’ untranslated region of the fragile X syndrome gene (FMR1). The polymorphic CGG repeat of the FMR1 gene is distributed in the population in four allelic classes according to repeat length (Table 1). Alleles ranging from 6 to 44 CGG repeats are the most common in the general population and have a stable transmission to the next generation. Alleles within the 45–54 CGG repeat range are called intermediate alleles and most are stable. Individuals carrying intermediate alleles are phenotypically normal. Alleles about 55–200 repeats are called premutation alleles. These alleles are associated with a significant elevation of FMR1 mRNA levels [2-3] and are highly unstable during maternal transmission. Premutation carrier individuals do not present with ID, but they have a risk of developing fragile X-associated tremor/ataxia syndrome (FXTAS), premature ovarian insufficiency (FXPOI) or other psychological symptoms [4-7]. Finally, expansion of the repeat region to more than 200 CGG trinucleotide sequences is called full mutation. FMR1 is an X-linked gene regulated by methylation of the promoter during X inactivation in somatic cells of females. This leads to gene silencing and insufficient synthesis of the FMR1 protein (FMRP). Expansions over 200 CGGs lead to hypermethylation of the CpG island resulting in non-expression of the FMR1 gene and absence of the FMRP. The lack of FMRP is the direct cause of the FXS phenotype [8]. Besides CGG repeat expansions, it has been reported that almost 1% of individuals with FXS have a partial or full deletion, or point mutation of the FMR1 gene [9-12].
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Until now genetic counseling was offered based on the expansion risk in premutation carrier women, but the description of FMR1-associated disorders has increased the complexity of the genetic counseling for FXS families, particularly to FMR1 premutation carriers. Furthermore, some issues such as risk assessment for intermediate alleles and the clinical prognosis for females with full mutations still remain challenging. Most of the families who first receive a diagnosis of FXS have no prior knowledge of FMR1-premutation disorders, and it is essential that individuals under study receive pre and post genetic counseling, including information about possible outcomes and the implications of carrying full, premutation or intermediate alleles. Genetic counseling of FXS requires attention to a wide range of clinical manifestations including developmental, neurodegenerative, and reproductive symptoms that may vary in age of onset and severity. Counselors must have a solid understanding of this genetic condition, including the trinucleotide repeat instability and the phenotypic variability. Table 1 shows associated FMR1 allele phenotypes. Table 1. FMR1 alleles and associated phenotypes Allele Normal Intermediate Premutation
CGG repeat range 5-44 45-54 55–200
Full Mutation
>200 (methylated)
Phenotype Normal Normal Normal, FXTAS, FXPOI and others ID in 100% males and reduced penetrance in females (30-50%)
ASSESSMENT RISK BASED ON CGGS EXPANSION Normal Range and Intermediate Alleles Alleles ranging from 6 to 44 CGG repeats, the most common in the general population, and most of the intermediate alleles have stable transmission to the next generation. Intermediate alleles may show some instability, including expansion to a full mutation in two generations [13-15].
Premutation Alleles Premutation alleles are highly unstable during maternal transmission and may expand to a higher CGG repeat size or even to a full mutation in only one generation. Even expansion of paternal premutation alleles is possible; the expansion from a premutation allele to a full mutation has only been observed through female meioses. Thus, all the daughters from a premutation male will inherit the premutation allele and will not manifest FXS. Table 2 shows the risk of expansion of the different allele types. The expansion risk of a FMR1 allele depends both on CGG repeat size and the presence of AGG interruptions. In 1994, Eichler et al. suggested that AGGs interspersed within the FMR1 repeat region may be linked to repeat stability and the risk of expansion [16]. In the general population, almost 95% of alleles have
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one or two AGG interruptions, which the most common allele pattern is two AGGs at positions 10 or 11 and 20 or 21 repeats. In contrast, alleles in FXS families contain no or few AGGs at the 5′ end and they contain long stretches of uninterrupted CGGs at the 3′ end [16,17]. Presumably maternal alleles with no AGG interruptions confer increased risk for unstable transmissions and thus, the inclusion of AGG genotype studies would be of benefit in clinical practice. AGG testing is expected to reassure premutation carriers with AGG interruptions while alerting premutation carriers with alleles without AGG interruptions who would have the highest risk for instability. Table 2. Risk of expansion of FMR1 alleles Allele Normal Intermediate
Premutation
Full Mutation
CGG repeat range 5-44 45-54 55–59 60–69 70–79 80–89 90–99 >100 >200 (methylated)
Risk of expansion to a full mutation for females* 0% 0% 3.70% 5.30% 31.10% 57.80% 80.10% 94–100%
*[68-70].
Full Mutation Alleles Full mutated males never transmit the full mutated allele to their daughters, only an allele in the premutation range. Full mutated females have a 50% of transmission risk of the full mutated allele to their offspring: all male descendants carrying the full mutation allele will be affected while a smaller percentage of women will present the syndrome.
GENETIC COUNSELING BASED ON FMR1 ALLELES Intermediate Alleles Intermediate alleles present a 45–54 CGG repeat range [18] and have a frequency in the general population of 1/35 to 1/57 females [19,20]. Intermediate allele carriers do not manifest ID, FXTAS or FXPOI, although some groups have suggested associations between intermediate alleles and some disorders such as Parkinson’s disease [21], primary ovarian insufficiency [22], and autism and cognitive disabilities [23,24]. However, these findings have not consistently been supported by other studies [25-27]. A frequent concern in FXS screening is the genetic counseling to intermediate allele carriers. Currently, the follow-up of individuals with no AGG interruptions is indicated, despite the low risk of expansion in the next generation.
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Premutation Alleles Individuals carrying premutation alleles are intellectually unaffected, but in the last decades several premutation-related disorders have been described. Among them the most prevalent are FXPOI and FXTAS [28,29]. In addition, behavioral features such as impaired executive function, social deficits or anxiety have been related to some FMR1 premutation carriers [30]. Premutation alleles contain trinucleotide expansions in the range of 55 to 200 CGG repeats. In Western populations premutation frequencies in females range from 1/151 to 1/259, but there is some evidence of ethnic and racial variability. In Israel, for example, the premutation frequency is around 1/113 and in Taiwan 1/837 [20,31-33]. Some authors state that the different reproductive aptitudes of female premutation carriers and the differences in mean maternal age may contribute to this variation in premutation frequencies [34]. In males the rate ranges from 1/468 to 1/813 [20,35]. Men carrying the premutation allele must mainly be counseled about the risk of FXTAS. This syndrome presents with incomplete penetrance even among premutation carriers with identical CGG-repeat lengths. Approximately 50% of premutation carrier males will develop neurodegenerative symptoms of FXTAS after the age of 50 [36]. The first clinical signs of the syndrome typically appear when patients are in their 50s and 60s, with a mean tremor onset at approximately 60 years and ataxia onset at 62 years [37]. The risk and severity of the disorder appear to be related to the CGG repeat size, with higher risk in larger repeats [38]; nevertheless, a biomarker that predicts the apparition of FXTAS or the protective factors in asymptomatic carriers has still not been identified. Other clinical signs associated with premutation male carriers are neuroendocrine dysfunction, including testosterone deficiency [39], hypertension [40] or bowel and urinary incontinence [41]. Genetic counseling of premutation carrier women must be addressed to the risk for premature ovarian insufficiency, besides the risk for expansion and the FXTAS syndrome. In women, FXTAS is much less common than in men, as many as 16% will develop FXTAS symptoms, it presents a later age of onset and is milder in presentation. The risk for FXPOI in these women increases around 20% with an age of onset before the age of 40 years [28]. Other pathologies associated with females premutation carriers are psychiatric disorders such as depression, anxiety or mood disorders [42-44], migraine [45], immune-mediated disorders, particularly hypothyroidism (15.9%) [36], and fibromyalgia (25%) [7,46,47]. The latter two in particular are even more common among women with FXTAS, with a frequency of 43% and 50%, respectively [7].
Full Mutation Alleles Full mutations are responsible for FXS, a spectrum of clinical features which includes physical, cognitive and behavioral aspects. All males carrying the full mutation will present with mild to severe intellectual disability and exhibit a variety of maladaptive behaviors overlapping those described for autism spectrum disorders [48,49]. On the contrary, only 50%70% of women manifest FXS symptoms, albeit in a milder form than men, and some appear to be completely unaffected or exhibit minor neurobehavioral features [50-52]. Despite a common genetic etiology, the clinical presentation of FXS is variable and this variability is related to residual levels of the FMRP due to the presence of CGG expansion size mosaicism, different
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methylation levels of the full mutation allele and X-inactivation that leads to a differential pattern of FMRP expression within tissues [51]. On the other hand, there are full mutation carrier males with a standardized IQ score of 70 or higher who are known as high functioning males. In normal males, the 5' CpG island containing the CGG trinucleotide repeat is not methylated, whereas a CGG triplet repeat expans to more than ~200 repeats, this site is methylated and the FMR1 is transcriptionally silenced [53]. Many mildly affected individuals show mosaic methylation at the FMR1 promoter. Regarding their offspring, their daughters will inherit an allele in the premutation range. High functioning males do not present with intellectual disability but recently, FXTAS has been described in a high functioning male [54] and in the case of an unmethylated mosaic (premutation-full mutation) male [55]. The latter suggests that the definition of FXTAS should also include those cases with an expanded allele the size and lack of methylation of which leads to RNA toxicity.
FMR1 Point Mutations/Deletions It has been suggested that patients with a clinical FXS-like phenotype but not carrying the FMR1 gene full mutation should be screened for FMR1 mutations [56,57]. It is estimated that FMR1 point mutations or deletions are responsible for up 1% of FXS cases. Nevertheless, the prevalence of mutations in the FMR1 coding region is still not well known since the standard FXS protocols only comprise the study of the CGG repeats size. These mutations can be de novo or inherited from a carrier mother. A male carrying a point mutation or deletion will always have carrier daughters, who might be affected depending on the X-chromosome inactivation. Carrier females have 50% of risk of transmitting the mutated allele. Within this 50%, all males will be affected and females will be variably affected, depending on the Xinactivation.
REPRODUCTIVE OPTIONS FOR PREMUTATION/FULL MUTATION CARRIERS Individuals at risk for passing on FXS mutations to their offspring have a variety of preand postconception options available. Regardless of the option they choose, women must be advised of any contraindication of these procedures [58]. When offering the reproductive options to a couple at risk, a fertility issue has to be considered since the risk of POF has significant reproductive implications. First of all, the onset of POF is difficult to predict. Secondly, the subtle endocrine perturbations (elevated follicle-stimulating hormone levels) observed in these women decreases the efficiency of ovarian stimulation required for preimplantational diagnosis and finally, there is evidence that premutation carriers may have hormonal changes suggestive of early ovarian aging despite regular menstrual cycles (See Chapter 5).
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Prenatal Diagnosis for FXS Prenatal diagnosis for FXS is possible using chorionic villi or amniotic fluid cells and it depends on several factors, including the physician performing the procedure and patient preference. When the establishment of the sizing of the expansion is sufficient to determine the genotype, the prenatal diagnosis on chorionic villi allows performing a therapeutic abortion at early stages in the pregnancy. On the contrary, the methylation pattern of a full mutation is established after the 14th week of pregnancy, thus the use of a methylation-sensitive method (i.e Southern Blot) is not suitable for early prenatal diagnosis on DNA from chorionic villi [59]. Moreover, the risk of miscarriage may be lower on amniotic fluid cell sampling. Table 3. Genetic counseling in FMR1 Progenitor allele and gender Normal male Normal female IA carrier male IA carrier female PM carrier male
% expected offspring No risk No risk Females IA range 50% NA range 50% IA range
Offspring outcome Males Normal Normal Normal
Females Normal Normal Normal
Normal
Normal
Females PM range
Normal
50% NA range 50%* PM/ FM*
Normal Normal, FXTAS, and others 100% ID
FM carrier male
females PM range
Normal
FM carrier female
50% NA range 50% FM
Normal 100% ID
PM carrier female
Normal, FXTAS, FXPOI and others Normal Normal, FXTAS, FXPOI and others 30-50% ID Normal, FXTAS, FXPOI and others Normal 30-50% ID
NA: normal allele, IA: intermediate allele; PM: premutation allele; FM: full mutation allele. *percentage varies according to the expansion risk indicated in Table 2.
Prenatal diagnosis should be offered to women with premutation or full mutations, but it is not intended for the pregnant partner of a premutation male carrier. These males, nevertheless, should receive genetic counseling about phenotypic risk to their daughters, who will inherit the premutation allele. Neither is prenatal testing intended for intermediate allele carriers. As indicated before, there are no reports of intermediate alleles expanding to full mutations in a single generation. The risk for a female premutation carrier of transmitting the expansion is 50%, and the risk for the maternal premutation to expand to full mutation is proportional to its size and AGG content. Table 3 summarizes the possible outcomes depending on the carrier state of each parent. The identification of a female fetus with the fragile X full mutation entails a significant challenge for professionals. The probability that a full mutation carrier fetus is affected is around 50-70%, but at present there are no biomarkers to predict this affectation. The diagnosis of a FXS patient requires extending the study to the mother in order to confirm her premutation carrier status.
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Preimplantational Diagnosis for FXS Preimplantation genetic diagnosis (PGD) is a technique based on the genetic analysis of an embryo obtained through in vitro fecundation. The most common option is to perform the genetic study on a day-3 embryo which is then transferred to the uterus. This approach increases the risk of embryo viability due to the biopsy. Further, the possibility of embryo mosaicism has been described at the cleavage stage and self-correction of aneuploidies between the cleavage and blastocyst stages [60]. Polar body biopsies can be used to avoid any misdiagnosis due to embryo mosaicism, although only maternal genetic information is obtained. The third option is to biopsy trophectoderm cells from blastocysts (5-6 day embryo), which is less invasive and has higher concordance between inner cell mass and trophectoderm cells [61]. PGD has several advantages over prenatal diagnosis. The diagnosis is performed in the embryo, avoiding the parental stress and the emotional trauma of a termination of pregnancy.
Offspring Renouncement and Adoption These two options are drastic and are usually rejected by the couples. Therefore they are currently seldom observed as the first choice option by couples at risk that wish to prevent the birth of an affected child.
Donor Germline Cell Another possible option is egg or sperm donation for female and male mutation carriers, respectively. Based on our experience, around 10% of FXS families chose for a germline cell donation [62]. At present, it is a very good option for premutation carrier females who have been involved in previous prenatal diagnosis and termination of pregnancies. Moreover, given the relatively high prevalence of FXS in the general population, potential gamete donors should be tested for this syndrome.
FMR1 TESTING GUIDELINES Several scientific societies such as the European Molecular genetics Quality Network (EMQN), the American College of Medical Genetics (ACMG), the National Society of genetic Counselors (NSGC) and the American College of Obstetrics and Gynecology (ACOG) have published best practice guidelines for the molecular genetic testing and diagnosis of FXS and other fragile X-associated disorders [18,63-66]. The best practice guidelines for genetic analysis and reporting in FXS, FXPOI, and FXTAS are listed in Table 4. Appropriate FMR1 molecular testing is very important for optimal genetic counseling in the fragile X-associated disorders due to the particular pattern and transmission of the CGG repeat. Although not being endorsed by current guidelines, screening women without known risk factors to FXS is increasingly being offered. In 2005 Musci and Caughey demonstrated the efficacy and cost effectiveness of prenatal population-based fragile X carrier screening [67].
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Table 4. FMR1 mutation testing recommendations FMR1 mutation testing is recommended for*: Individuals of either sex with intellectual disability, developmental delay or autism Individuals who have a family history of FXS Women with a family history of FMR1-related disorders, including FXPOI Women with reproductive or fertility problems associated with elevated levels of follicle stimulating hormone (FSH) Individuals with late onset tremor or cerebellar ataxia of unknown origin Given the relatively high prevalence of FXS in the general population, potential gamete donors should be tested for this syndrome *Sherman, 2005; ACOG, 20010.
CONCLUSION When first described, FXS was a rare X-linked disease responsible for intellectual disabilities in males and females in a lesser percentage. Research into FMR1 has brought to light the molecular and phenotypic complexity of FMR1-related diseases. The first issue to consider is that a FXS patient will always have a premutation carrier mother who should receive genetic counseling. Premutation carrier females have a 50% risk of transmitting a premutation/full mutated allele to their offspring, unlike carrier males who will only transmit the premutation allele. The expansion risk in females depends on the CGG repeats number and the AGG interruptions. Lastly, premutation carriers are at risk not only of having FXS offspring (females) but also of FXPOI (females), FXTAS and other disorders. The complexity of the issues surrounding genetic testing and the management of FMR1-associated disorders has increased, and investigation related to this gene covers many aspects, including molecular factors, epigenetic factors, emotional issues or targeted pharmaceuticals. As the knowledge regarding disease causing mechanisms evolves, genetic counselors must have an updated and solid understanding of this genetic condition and the FMR1-associated disorders in order to cover all the counseling aspects of these syndromes.
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[64] American College of Obstetricians and Gynecologists Committee on Genetics. ACOG Committee Opinion No. 469: Carrier screening for fragile X syndrome. Obstet. Gynecol. 2010;116:1008-10. [65] Finucane B, Abrams L, Cronister A, Archibald AD, Bennett RL, McConkie-Rosell A. Genetic counseling and testing for FMR1 gene mutations: practice guidelines of the national society of genetic counselors. J. Genet Couns. 2012;21:752-60. [66] Biancalana V, Glaeser D, McQuaid S, Steinbach P. EMQN best practice guidelines for the molecular genetic testing and reporting of fragile X syndrome and other fragile Xassociated disorders. Eur. J. Hum. Genet. 2014 Sep 17. doi: 10.1038/ejhg.2014.185 [67] Musci TJ, Caughey AB. Cost-effectiveness analysis of prenatal population-based fragile X carrier screening. Am. J. Obstet. Gynecol. 2005;192:1905-12. [68] Nolin SL, Brown WT, Glicksman A, Houck GE Jr, Gargano AD, Sullivan A, Biancalana V, Bröndum-Nielsen K, Hjalgrim H, Holinski-Feder E, Kooy F, Longshore J, Macpherson J, Mandel JL, Matthijs G, Rousseau F, Steinbach P, Väisänen ML, von Koskull H, Sherman SL. Expansion of the fragile X CGG repeat in females with premutation or intermediate alleles. Am. J. Hum. Genet. 2003;72:454-64. [69] Rifé M, Badenas C, Quintó L, Puigoriol E, Tazón B, Rodriguez-Revenga L, Jiménez L, Sánchez A, Milà M. Analysis of CGG variation through 642 meioses in Fragile X families. Mol. Hum. Reprod. 2004;10:773-6. [70] Lévesque S, Dombrowski C, Morel ML, Rehel R, Côté JS, Bussières J, Morgan K, Rousseau F. Screening and instability of FMR1 alleles in a prospective sample of 24,449 mother-newborn pairs from the general population. Clin. Genet. 2009;76:511-23.
In: Encyclopedia of Genetics: New Research (8 Volume Set) ISBN: 978-1-53614-451-2 Editor: Heidi Carlson © 2019 Nova Science Publishers, Inc.
Chapter 93
TREATMENT OF FRAGILE X SPECTRUM: FXS, FXTAS AND FXPOI F. J. Ramos1 and M. P. Ribate2 1
Unidad de Genética-Pediatría, GCV-CIBERER, Hospital Clínico Universitario “Lozano Blesa”, Facultad de Medicina, Universidad de Zaragoza, Spain 2 Facultad de Ciencias de la Salud, Universidad San Jorge, Zaragoza, Spain
ABSTRACT Fragile X Spectrum includes three different clinical conditions Fragile X Syndrome (FXS), Fragile X-Associated Tremor and Ataxia (FXTAS), and Fragile X-Associated Premature Ovarian Insufficiency (FXPOI). Treatment of FXS is mainly symptomatic and it is addressed to improve or make disappear some of the more dyscapacitating symptoms like hyperactivity, deficit attention disorder and behavioral problems of language anomalies. Clinical trials are in course focusing on new discovered therapeutical targets (i.e., mGluR). Treatment of FXTAS and FXPOI are also mainly symptomatic and should be individually prescribed and modified depending on the clinical evolution.
INTRODUCTION Fragile X spectrum includes Fragile X Syndrome (FXS), Fragile X-Associated Tremor Ataxia Syndrome (FXTAS) and Fragile X-Associated Premature ovarian Insufficiency (FXPOI). All three conditions are associated with the CGG trinucleotide expansion in the FMR1 gene on the X chromosome: FXS with more than 200 CGGs (full mutation) and FXTAS and FXPOI with 50 to 200 CGGs (premutation). Patients with FXS and FXTAS are mostly males, although females can also be affected. All three conditions are treated symptomatically, combining pharmacological and non-pharmacological therapies directed at alleviate the main disabling symptoms present in each condition.
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FRAGILE X SYNDROME Fragile X syndrome (FXS) is the most common inherited cause of intellectual disability that affects approximately 1 in 4,000 males and 1 in 8,000 females. Its genetic cause was identified in 1991 and consisted of the expansion of the number of CGG trinucleotide repeats at the FMR1 gene, located on the distal region of the long arm of chromosome X. The normal gene has less than 55 CGGs but when it has more than 55 CGGs it tends to expand when transmitted by the mother to the next generation. Individuals who have between 55- 200 CGGs (premutation) are considered carriers, and those with more than 200 CGGs (full mutation) and have a hypermethylated gene are affected. This methylation leads to the silencing of the FMR1 gene and the subsequent absence, partial or complete, of the FMRP (Fragile X Mental Retardation Protein), considered as the ultimate cause of FXS [1]. The vast majority of FXS males with the full mutation develop intellectual disabilities, which occurs in only 25% of females due to the presence of a normal X chromosome that produces FMRP. However, most females with FXS have learning disabilities and/or emotional and behavior problems [2]. The clinical presentation of FXS is highly variable. In addition to intellectual disability, affected patients have neurodevelopmental problems such as attention deficit hyperactivity disorder, disruptive behavior or autism spectrum disorders. The physical characteristics of FXS include dysmorphic facial features (long face, large and prominent ears and prominent chin), and macroorchidism after puberty [3].
The FMRP Protein and the Synapse FMRP is expressed ubiquitously in many tissues, predominantly in brain neurons. There it binds to different nuclear mRNAs, including the FMR1 gene mRNA, repressing the translation of the target mRNA to postsynaptic dendritic spines, where its activity is important for synaptic plasticity during transport. Dendritic spines are specialized and rapidly changing protrusions in the neurons whose creation and elimination are essential for learning and memory processing [4]. Microscopic analysis of brain samples of patients with FXS and Fmr1 knockout mice revealed no gross morphological abnormalities; however, in certain areas of the brain there are spindly dendritic spines, which are interpreted as immaturity and affect adversely the synaptic plasticity [5]. The discovery of a morphological spine phenotype indicates a possible defect in synaptic plasticity in FXS that could result in the intellectual disability phenotype. Whether the abnormal spine morphology is a cause or a consequence of altered signal transmission is currently unknown [6].
The Glutamate Receptor (mGluR) Hypothesis In the brain, there are two main types of neurotransmitter receptors in the synaptic membrane: metabotropic receptors and ionotropic receptors. Metabotropic receptors (mGluRs) have eight different subtypes (mGluR1-8) which in turn are divided into three groups (I, II and III) according to sequence similarities and pharmacological properties. Group I includes mGluR1 and mGluR5 receptors which activates the Gq protein coupled and phospholipase C.
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Group II receptors (mGluR2 and mGluR3) and Group III (mGluR4, mGluR6, and Glu7 Glu8) are coupled to the proteins Gi / Go by inhibiting adenylate cyclase. Ionotropic receptors are ligand-gated ion channels in which the incorporation of a specific ligand induces a conformational change leading to opening of the receiver [7]. The opened receiver allows ion flow across the cell membrane by changing the excitability of the neuron. The main dependent neuronal glutamate ionotropic receptors are: AMPA (isoxazolepropionic amino-3-hydroxy-5methyl-4-acid), NMDA (N-methyl-D-aspartic acid), and kainate receptors, whose activation produces a fast excitatory neurotransmission [8]. The mechanisms of long-term potentiation (LTP) and long-term depression (LTD) are the most important regulatory mechanisms of neuronal synaptic plasticity, defined as long-lasting changes in synaptic strength accompanied by abnormalities in the size and morphology of dendritic spines. The mechanism of strengthening of LTP produces a connection between presynaptic and postsynaptic neurons. The LTD mechanism is opposite to that of LTP and results in the weakening of synapses, mainly due to a reduction of glutamate-gated ionotropic AMPA-alpha in the postsynaptic membrane [9]. The ‘theory of glutamate receptor (mGluR)’, first published in 2004, tried to explain many aspects of clinical symptoms present in patients with FXS and in the Fmr1-KO mouse, including a higher density and immaturity of dendritic spines compared to normal individuals/mice. This theory stated that internalization of the AMPA receptor is excessive in FXS and likely caused by stimulation of group I mGluRs (mGluR1 and mGluR5). Such stimulation would induce local mRNA translation, resulting in the synthesis of new protein that triggers the internalization of the AMPA receptor, essential for the long-term plasticity of dendritic spines. FMR1 mRNA is also present in the postsynaptic compartment and FMRP is synthesized locally after mGluR activation [10]. On the other hand, FMRP seems to negatively regulate the translation of proteins that are important for the internalization of the AMPA receptor. The exact mechanism by which the FMRP local translation represses mRNA remains unknown. Moreover, phosphorylation and dephosphorylation of FMRP seems play a crucial role in the signaling cascade induced by stimulation of mGluR5 receptors, which stimulate local protein synthesis at the synapses [11]. Since the formulation of the mGluR theory, clinical researchers in FXS have sought the support of the pharmaceutical industry to collaborate in identifying therapeutic strategies aimed at mGluR5 in order to at least partially reverse some of the symptoms of FXS. The MPEP (2-methyl-6-(phenylethynyl)-pyridine), a negative modulator of mGluR5, can, in vitro, counter the excess of activity of the receptor and rescue the loss of AMPA receptors after the loss of FMRP. In FXS animal models, researches have got the pharmacological rescue of audiogenic seizures, behavioral phenotypes and alterations in dendritic spines by using several negative modulators of mGluR5. MGluR theory has focused research into the basic mechanisms underlying FXS, prompting the search for new therapeutic strategies [12].
The GABA Hypothesis Besides the mGluR´s hypothesis, researches have suggested that the signaling receptor gamma-aminobutyric acid (GABA) is altered in patients with FXS. GABA is the major inhibitory neurotransmitter in the central nervous system (CNS) and, as such, plays a key role in the modulation of neuronal activity in the brain. GABA mediates its action through two
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distinct systems, the ionotropic GABA-A and the metabotropic GABA-B receptors [13]. Many patients with FXS have epilepsy and sleep disorders, conditions associated with the signaling pathway of the GABA receptor. Interestingly, the mRNA encoding GABA-A receptor subunits are targets of FMRP. The Fmr1-KO mice have decreased levels of mRNA and protein in various subunits of GABA-A receptor compared to normal animals [14]. These studies support a model in which the FMRP mRNA would regulate the stability of the GABA-A receptor subunits, preventing its degradation by joining these mRNAs in vivo. In addition to GABA-A receptors, GABA-B receptors could also be related to FXS, therefore turning into another potential therapeutic target. The GABA-B receptor agonists inhibit the presynaptic release of glutamate and the postsynaptic signaling cascade downstream of mGluR5 [15]. All those studies have demonstrated a dysfunction in the mRNA and protein expression of several subunits of GABA receptors, mechanisms that would be likely involved in the occurrence of the FXS phenotype.
THERAPEUTIC APPROACHES IN FXS Currently, the treatment of patients with FXS is mainly symptomatic. The two most commonly used medications are stimulants used to improve attention and hyperactivity, and selective inhibitors of serotonin reuptake to reduce the risk of aggression associated with anxiety. FXS patients are not only treated with pharmacological agents, but also benefit from behavioral therapies for improving emotional and language problems. As already demonstrated in the mouse FXS behavior improves with an enriched environment and therefore this therapy might also be beneficial to humans [16]. Current therapeutic strategies, both pharmacological and non-pharmacological, are aimed at improving the symptoms but do not improve cognitive function. In recent years, new strategies have been developed for therapeutic interventions in FXS based on the theories of mGluR and GABA receptors. Several clinical trials using new designed drugs were initiated to correct the abnormal activity of the mGluR and GABA pathways in FXS [6]. Unfortunately, some of them have been recently discontinued due to the lack of demonstrable improvement.
MGluR5 Inhibitors The fenobam is a potent and selective antagonist of mGluR5 and was the first negative mGluR5 modulator tested in patients with FXS. To test whether it had significant effect on the FXS phenotype, fenobam was administered as a single oral dose to twelve affected patients (six males and six females) that the pre-pulse flicker noise (PPI -Pre-Pulse inhibition was measured inhibition of Acoustic Startle-) before and after drug administration. Generally, patients with FXS show decreased PPI compared to healthy control individuals. Six of the twelve patients with FXS showed improvement after treatment with PPI fenobam, with no significant adverse effects observed [17]. Although results were promising, it was difficult to draw definitive conclusions because of the lack of a controlled study with placebo, the fact that the patients received only a single dose
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of fenobam, and the low number of participants enrolled. Other inhibitors are the AFQ056 mGluR5, from Novartis; the STX107, developed by Merck and whose license was issued to Seaside Therapeutics, and the RO4917523, from Hoffman-La Roche [6]. Despite of the results of a preliminary clinical trial with AFQ056 (Mavoglurant) in which certain cognitive aspects improved in FXS patients with complete methylation of the FMR1 gene [18], the trial was discontinued by Novartis last April after results of phase IIb/III failed to show demonstrable improvement. Last month, Roche discontinued its trial as well because of negative phase II clinical results.
GABA-A Agonists GABA-A receptor agonists are currently used as anticonvulsants, antidepressants or anxiolytics. Benzodiazepines, which enhance the function of the GABA receptor, are the bestknown drugs of this group. Although their anxiolytic effects are useful in patients with FXS have unwanted side effects such as sedation or ataxia, and treatment discontinuation may cause withdrawal symptoms [6]. Besides the selective agonists of GABA-A receptor, neuroactive steroids that allosterically modulate them may be also effective, for example ganaxolone, which has a favorable safety profile and may be useful in patients with FXS [19]. The use of inhibitors of GABA-A receptors in patients with FXS is likely to be effective in reducing symptoms such as seizures or sleep disorders.
GABA-B Agonists Arbaclofen (STX209), a drug proven effective and safe for gastroesophageal reflux disease (GERD), has been used to treat autism spectrum disorders (ASD) in several studies. In an animal model of FXS, arbaclofen demonstrated a reversal of behavioral, neurological, and neuropathological features associated with the disease. Results from one double-blind, placebocontrolled study that treated children and adults with FXS have been promising, with signs of improvement in social function, especially in the most severely socially impaired individuals. Two studies, one open-label and one double-blind, placebo-controlled, were also conducted in children, adolescents, and young adults with ASD showing improvements in socialization [20, 21]. Approximately 13-18% of patients with FXS have seizures. The GABA-B and mGluR5 receptors are thought to be involved in the occurrence of audiogenic seizures in Fmr1-KO mice. Treatment of these mice with racemic baclofen reduced the incidence of seizures. Another positive effect of GABA-B receptor agonists could be the reduction of anxiety in patients with FXS. Treatment of Fmr1-KO mice with a GABA-B receptors agonist can inhibit audiogenic seizures, supporting the idea that GABA-B receptors are involved in the etiology of FXS [6].
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AMPA Agonists The increased internalization of AMPA receptors in neurons of Fmr1-KO mice plays a major role in altering the transmission of signals. CX516 is an ampakine which acts as a positive allosteric AMPA receptor modulator. This compound binds to the AMPA receptor-channel complex and induces a slower deactivation of the receptor, which results in a longer opening time, in a slower disappearance of the excitatory postsynaptic potential, and ultimately to an increased hippocampal LTP, compared to baseline state before administering the drug. Thus, CX516 enhances AMPA receptors after glutamate-mediated synaptic. CX516 has been tested in phase II, randomized, double-blind, placebo-controlled safety for four weeks in adult patients with FXS. Unfortunately no significant or cognitive measures and improvement in behavior were observed. This could be due to the use of low doses, to the CX516 short half-life in humans or, finally, to insufficient time of treatment. In the study, there were only minimal side effects and no serious adverse effects were observed [22]. Theoretically, treatments targeting AMPA receptors could improve behavior in FXS, although the beneficial effects of ampakines have yet to be convincingly demonstrated.
NMDA Receptor Antagonists Memantine is a non-competitive antagonist of NMDA receptors that can slow the progression of Alzheimer's disease and has been tested to treat Pervasive Developmental Disorders (PDD). In the presence of low levels of synaptic glutamate, binding of memantine blocks NMDA receptors, which are unblocked when glutamate levels increase. Besides excessive signaling through mGluR5 is linked to an increased AMPA receptor internalization and to the dysregulation of NMDA receptor activity [23]. Therefore, memantine may have positive effects on behavioral problems of patients with FXS. A clinical trial of memantine in six patients with FXS who had a comorbid diagnosis of PDD has been reported. Therapeutic effects were determined by the clinical evaluation of the “Clinical Global Impressions-Improvement” (CGI-I) scale during the time of treatment. The results did not show any significant improvement, although in four of the six patients certain signs of improvement were observed. Furthermore, the study was not a randomized placebocontrolled trial and, therefore, it was difficult to draw valid conclusions [24]. The NMDA receptor may be a target for pharmacological treatment of SXF because some brain regions of the Fmr1-KO mice showed impaired NMDA-dependent LTP. However, there is no convincing evidence that NMDA receptor antagonists have demonstrable beneficial effects on behavior [6].
Additional Treatments Lithium has been used for many years as a mood stabilizer. Most studies that have linked lithium with FXS have focused on the path of GSK3. Lithium inhibits the activity of GSK-3b, which in turn inhibits the phosphorylation of microtubule-associated protein 1B (MAP1B). The MAP1B is one of the major targets of the mRNA to which it binds and translationally regulates FMRP. In 2008, a clinical trial with open-label lithium was published in FXS patients; although
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the test was not randomized and placebo controlled, lithium appears that produced beneficial effects as decreased responses of aggression, abnormal vocalization improvement of self-harm and anxiety [25]. In conclusion, lithium appears to have beneficial effects on behavior and some cognitive functions but further research, such as long-term placebo-controlled studies are needed to check the effects of lithium in patients with FXS. Minocycline is a tetracycline analog which can inhibit matrix metalloproteinase-9 (MMP9) and reduce inflammation in the CNS. MMP-9 is an extracellular endopeptidase which breaks down the extracellular matrix proteins acting on synaptogenesis and morphology of dendritic spines. Minocycline is thought to act on the mGluR pathway and it has been tested in clinical trials to treat various neurological disorders (stroke, multiple sclerosis and autism). It has also been shown to have beneficial effects on the maturation of dendritic spines in cultured hippocampal neurons of Fmr1-KO mice [26]. A clinical trial of open-label studied the effects of minocycline in 50 patients with FXS was recently completed. The results were an improvement in language and behavior [27]. Acamprosate (calcium acetyl-homotaurine) is a commercially available drug used for the maintenance of abstinence from alcohol. This compound appears to have several mechanism of action: antagonist of mGluR5, weak antagonist of NMDA receptors and agonist of GABAA receptors. Although the mechanism of action of acamprosate is not fully understood, a small clinical trial was conducted in three patients with FXS to evaluate the response to treatment with acamprosate using the CGI-I scale. After a minimum of 16 weeks of treatment, all three patients improved. Surprisingly, they also showed improvement in language skills. Two subjects experienced nausea. Although these results appear promising, this trial was not placebo-controlled and the number of participants was too small to draw definitive conclusions [28]. Aripiprazole, an atypical antipsychotic that was approved in the USA for the treatment of children and adolescents with autism, has also been tested in patients with FXS. The effect of aripiprazole in patients with FXS was assessed by the CGI-I and ABC-I scales. All subjects who completed the study showed significant improvement in irritable behavior [29]. However, the study was not placebo-controlled and therefore the findings were difficult to interpret. Oxidative stress has been implicated in some psychiatric disorders, including autism. Studies in animal models suggested that in FXS there is an increased sensitivity to oxidative stress, which may alter neuronal and glial function. Indirect evidence was obtained by preclinical treatments of FXS performed using antioxidants. Indeed, chronic alpha-tocopherol treatment normalized free radical production and oxidative stress in the brain of FXS mice, in which it improved many of the behavioral and learning deficits of these mice, such as exploratory behaviors, habituation abnormalities, anxiety responses and contextual fear conditioning [30]. Recently, a phase II randomized, placebo-controlled trial was protocol was designed for children and adolescents with FXS [31]. Chronic melatonin treatment of FXS mice normalized the glutathione levels and prevented lipid peroxidation in the brain and testes of the mice. Moreover, melatonin improved some abnormal behaviors observed in FXS mice such as context-dependent exploratory and anxiety behaviors and learning abnormalities [32]. In a 4-week double-blind, placebo-controlled study with melatonin in 12 children with FXS, ASD or both, results showed a favorable effect (longer) in night sleep duration [33].
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Final Remarks FXS patients are treated with medications to alleviate the symptoms of the disease and/or the behavior problems such as hyperactivity or anxiety. Attempts to discover new drugs for the treatment of patients with FXS are becoming more promising with time due to improved understanding of the molecular mechanisms involved in the pathogenesis of the syndrome. Preliminary trials of new drugs in KO-mice models will be essential before they can be used in clinical trials in humans, although this does not guarantee their success. To date, several clinical trials have been carried out with promising preliminary results. Nevertheless, others had to be discontinued due to lack of positive results. Hopefully, more trials are currently being conducted or designed with improvements such as randomized double-blind methodology, sufficient number of patients and the use of outcome objective measurements to determine the therapeutic efficacy of the drug. For this, most clinical trials used one or more classic psychological questionnaires, however, the measurements of improvement were based on the likely subjective assessments made by relatives, caregivers and teachers. To ensure objectivity, it is important to develop reliable and objective outcome measurements (i.e.: questionnaires, scales or tests) to determine the true efficacy of the new drug [34]. Fortunately, and despite of some recent disappointing results, a number of clinicaltherapeutic trials for FXS and/or ASD are currently in course or being designed. It is remarkable that in less than 25 years since the discovery of the genetic defect that causes FXS, targeted therapeutic strategies have been already developed, with more or less success, to treat some of the most disabling symptoms of affected individuals. Although curative treatment seems to be far yet, it is likely that in the near future more effective symptomatic treatments will be available for FXS, especially for individuals with ASD. If expectations are met, they will significantly improve their quality of life and of their families.
FRAGILE X-ASSOCIATED TREMOR-ATAXIA SYNDROME (FXTAS) Fragile X-Associated Tremor-Ataxia Syndrome (FXTAS) is a progressive neurological disease that affects mainly males over 50 who carry a premutation allele (55-200 CGG repeats) in the fragile X gene FMR1. Affected individuals usually have intention tremor, ataxia, signs of parkinsonism, cognitive progressive decline and peripheral neuropathy. Besides, ancillary findings include autonomic dysfunction and psychiatric symptoms such as anxiety, depression and disinhibition [35]. As in FXS, there is no curative treatment for FXTAS, although there are a number of symptomatic therapies that can improve the clinical manifestations in affected individuals. Moreover, there is enough evidence regarding the efficacy of various medications for treatment of other diseases (i.e., Alzheimer diseases) that have important clinical overlap with FXTAS. The most common therapeutic interventions for FXTAS are listed in Table 1, which includes symptom-based treatments [36]. The drugs are used, basically, to alleviate symptoms related to tremor, equilibrium coordination, parkinsonism, sleep problems, anxiety, mood alterations, memory problems and pain.
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FRAGILE X-ASSOCIATED PRIMARY OVARY INSUFFICIENCY (FXPOI) Primary ovarian insufficiency or premature ovarian failure (POI/POF) is one of the causes of female infertility. POI consists in cessation of menstrual periods, increased levels of FSH and diminished levels of estrogens, all before the age of 40. POI occurs in about 1% of women between 30 to 40 years of age. Fertility of women with POI is severely diminished, but unlike menopause, POI may be accompanied with spontaneous ovarian activity and natural pregnancies [37]. The major causes of POI include autoimmunity, genetic and environmental factors. Among the most common genetic conditions that produce POI is Fragile X premutation, which is present in all female carriers. Treatment of POI includes hormone replacement therapy to reduce complications due to impaired endocrine function of ovaries, and fertility preservation therapies that include ovarian cortex, oocyte and embryo cryopreservation, oocyte or embryo donation and adoption in women without any ovarian function [38]. Recent research findings in animals and humans showed that neonatal and adult ovaries have some oogonial stem cells (OSCs) that can stably proliferate for months and produce mature oocytes in vitro. Studies on the isolation of OSCs form ovaries of aged animals and production of mature normal oocytes in ovaries of young adult animals lead to the recognition of the of importance of OSC niche and intraovarian environment on their differentiation to mature, normal oocytes. Therefore, cases of POI that result from defects in ovarian niche and its incapacity to support differentiation and growth of oocytes and also ovarian aging may be reversible in future [39]. These results have offered the opportunity for the application of OSCs as a target for POI therapy, restoration of ovarian function and, subsequently, restoration of normal fertility. However, clinical utility of these cells for treatment requires more evidence to confirm their safety, especially the effects from epigenetic changes during in vitro culture, and manipulation of produced oocytes and also resultant offspring. Table1. Symptom-based treatments for patients with FXTAS Symptoms Tremor Ataxia Parkinsonism Cognitive deficits and dementia Psychiatric problems Autonomic dysfunction Pain *
Treatment/Therapy Primidone, beta-blockers, benzodiazepines Amantadine and physical therapy Carbidopa/levodopa, pramipexole and eldepryl Donepezil, rivastigmine, galantamine, memantine Sertraline, citalopram, escitalopram, duloxetine, mirtazapine, venlafaxine and aripiprazole Bladder incontinency: Tricyclic antidepressants, muscarinic receptor antagonists, cytoscopy with injection of Botox Swallowing difficulties: pyridostigmine bromide Antidepressants, antiepileptics, topical analgesics, gabapentin and/or pregabalin
Modified from Capelli et al., (2010). [36]
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CONCLUSION There is no curative treatment for Fragile X Syndrome. Symptomatic paliative therapies include behavioral and cognitive interventions, speech therapy, occupational and physical therapy, as well as pharmacological treatments. All of them intended to improve intellectual abilities, familial interactions and social integration. Pharmacological treatments should be individualized depending on the age and the intellectual-cognitive level of the affected individual. Treatment for Fragile X-Associated Tremor and Ataxia is also symptomatic, and include physical therapy, psychological and/or psychiatric interventions, and pharmacological treatments. Fragile X Carrier females with Premature Ovarian Insufficiency should be treated by the appropriate specialists. Hormone replacement and fertility preservation therapies are the two main issues that should be addressed.
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[12] Nakamoto, M., Nalavadi, V., Epstein, M. P., Narayanan, U., Bassell, G. J., Warren, S. T. Fragile X mental retardation protein deficiency leads to excessive mGluR5-dependent internalization of AMPA receptors. Proc Natl Acad Sci USA 2007, 104, 15537-15542. [13] D’Hulst, C. and Kooy, R. F. The GABA(A) receptor: A novel target for treatment of fragile X? Trends Neurosci 2007, 30, 425-431. [14] Selby, L., Zhank, C., Sun, Q. Q. Major defects in neocortical GABAergic inhibitory circuits in mice lacking the fragile X mental retardation protein. Neurosci Lett 2007, 412, 227-232. [15] Tu, H., Rondard, P., Xu, C., Bertaso, F., Cao, F., Zhang, X., Pin, J. P., Liu, J. Dominant role of GABAB2 and Gbetagamma for GABAB receptor-mediated-ERK1/2/CREB pathway in cerebellar neurons. Cell Signal 2007, 19, 1996-2002. [16] Meredith, R. M., Holmgren, C. D., Weidum, M., Burnashev, N., Mansvelder, H. D. Increased threshold for spike-timing-dependent plasticity is caused by unreliable calcium signaling in mice lacking fragile X gene FMR1. Neuron 2007, 54, 627-638. [17] Hessl, D., Berry-Kravis, E., Cordeiro, L., Yuhas, J., Ornitz, E. M., Campbell, A., Chruscinski, E., Hervey, C., Long, J. M., Hagerman, R. J. Prepulse inhibition in fragile X syndrome: feasibility, reliability, and implications for treatment. Am J Med Genet B Neuropsychiatr Genet 2009, 150B, 545-553. [18] Jacquemont, S., Curie, A., des Portes, V., Torrioli, M. G., Berry-Kravis, E., Hagerman, R. J., Ramos, F. J., Cornish, K., He, Y., Paulding, C., Neri, G., Chen, F., Hadjikhani, N., Martinet, D., Meyer, J., Beckmann, J. S., Delange, K., Brun, A., Bussy, G., Gasparini, F., Hilse, T., Floesser, A., Branson, J., Bilbe, G., Johns, D., Gomez-Mancilla, B. Epigenetic modification of the FMR1 gene in Fragile X Syndrome is associated with differential response to the mGluR5 antagonist AFQ056. Sci Transl Med 2011; 3:64ra1. [19] Cornish, K., Turk, J., Hagerman, R. The fragile X continuum: New advances and perspectives. J Intellect Disabil Res 2008, 52, 469-482. [20] Cryan, J. F. and Kaupmann, K. Don’t worry ‘B’ happy! A role for GABA(B) receptors in anxiety and depression. Trends Pharmacol Sci 2005, 26, 36-43. [21] Erickson, C. A., Veenstra-Vanderweele, J. M., Melmed, R. D., McCracken, J. T., Ginsberg, L. D., Sikich, L., Scahill, L., Cherubini, M., Zarevics, P., Walton-Bowen, K., Carpenter, R. L., Bear, M. F., Wang, P. P., King, B. H. STX209 (arbaclofen) for autism spectrum disorders: An 8-week open-label study. J Autism Dev Disord 2014, 44, 958964. [22] Berry-Kravis, E., Krause, S. E., Block, S. S., Guter, S., Wuu, J., Leurgans, S., Decle, P., Potanos, K., Cook, E., Salt, J., Maino, D., Weinberg, D., Lara, R., Jardini, T., Cogswell, J., Johnson, S. A.; Hagerman, R. Effect of CX516, an AMPA-modulating compound, on cognition and behavior in fragile X syndrome: a controlled trial. J Child Adolesc Psychopharmacol 2006, 16, 525-540. [23] Shang, Y., Wang, H., Mercaldo, V., Li, X., Chen, T., Zhuo, M. Fragile X mental retardation protein is required for chemically-induced long-term potentiation of the hippocampus in adult mice. J Neurochem 2009, 111, 635-646. [24] Erickson, C. A., Mullett, J. E., McDougle, C. J. Open-label memantine in fragile X syndrome. J Autism Dev Disord 2009, 39, 1629-1635. [25] Berry-Kravis, E., Sumis, A., Hervey, C., Nelson, M., Porges, S. W., Weng, N., Weiler, I. J., Greenough, W. T. Open-label treatment trial of lithium to target the underlying defect in fragile X syndrome. J Dev Behav Pediatr 2008, 29, 293-302.
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[26] Bilousova, T. V., Dansie, L., Ngo, M., Aye, J., Charles, J. R., Ethell, D. W., Ethell, I. M. Minocycline promotes dendritic spine maturation and improves behavioural performance in the fragile X mouse model. J Med Genet 2009, 46, 94-102. [27] Utari, A., Chonchaiya, W., Rivera, S. M., Schneider, A., Hagerman, R. J., Faradz, S. M., Ethell, I. M., Nguyen, D. V. Side effects of minocycline treatment in patients with fragile X syndrome and exploration of outcome measures. Am. J Intellect Dev Disabil 2010, 115, 433-443. [28] Erickson, C. A., Mullett, J. E., McDougle, D. J. Brief report: Acamprosate in fragile X syndrome. J Autism Dev Disord 2010, 40, 1412-1416. [29] Erickson, C. A., Stigler, K. A., Wink, L. K., Mullett, J. E., Kohn, A., Posey, D. J., McDougle, C. J. A prospective open-label study of aripiprazole in fragile X syndrome. Psychopharmacology (Berl). 2011, 216, 85-90. [30] De Diego-Otero, Y., Romero-Zerbo, Y., El Bekay, R., Decara, J., Sánchez, L., Rodriguez-de-Fonseca, F., del Arco-Herrera, I. Alpha-tocopherol protect against oxidative stress in the fragile X knockout mouse: An experimental therapeutic approach for the Fmr1 deficiency. Neuropsycopharmacology 2009, 34, 1011-1026. [31] De Diego-Otero, Y., Calvo-Medina, R., Quintero-Navarro, C., Sánchez-Salido, L., García-Guirado, F., Del Arco-Herrera, I., Fernández-Carvajal, I., Ferrando-Lucas, T., Caballero-Andaluz, R., Pérez-Costillas, L. A combination of ascorbic acid and αtocopherol to test the effectiveness and safety in the fragile X syndrome: study protocol for a phase II, randomized, placebo-controlled trial. Trials 2014, 15, 345-360. [32] Romero-Zerbo, Y., Decara, J., el Bekay, R., Sanchez-Salido, L., Del Arco-Herrera, I., de Fonseca, F. R., de Diego-Otero, Y. Protective effects of melatonin against oxidative stress in Fmr1 knockout mice: a therapeutic research model for the fragile X syndrome. J Pineal Res 2009, 46, 224-234. [33] Wirojanan, J., Jacquemont, S., Diaz, R., Bacalman, S., Anders, T. F., Hagerman, R. J., Goodlin-Jones, B. L. The efficacy of melatonin for sleep problems in children with autism, fragile X syndrome, or autism and fragile X syndrome. J Clin Sleep Med 2009, 5, 145-150. [34] Berry-Kravis, E., Hessl, D., Abbeduto, L., Reiss, A. L., Beckel-Mitchener, A., Urv, T. K. and Outcome Measurements Working Groups. Outcome measures for clinical trials in Fragile X Syndrome. J Dev Behav Pediatr 2013, 34, 508-522. [35] Hagerman, R. J., Hall, D. A., Coffey, S., Leehey, M., Burgeois, J., Gould, J., Zhank, L., Seritan, A., Berry-Kravis, E., Olichney, J., Miller, J. W., Fong, A. L., Carpenter, R., Bodine, C., Gane, L. W., Rainin, E., Hagerman, H., Hagerman, P. J. Treatment of fragile X-associated tremor ataxia syndrome (FXTAS) and related neurological problems. Clin Intervention Aging 2008, 3, 251-262. [36] Capelli, L. P., Rodrigues-Gonçalves, M. R., Leite, C. C., Barbosa, E. R., Nitrini, R., Vianna-Morgante, A. M. The fragile X-associated tremor and ataxia syndrome (FXTAS). Arq Neuropsiquiatr, 2010, 68, 791-798. [37] Sadeghi, M. R. New hopes for the treatment of Primary Ovarian Insufficiency/ Premature Ovarian Failure. J Reprod Infertil 2013, 14, 1-2. [38] Blumenfeld, Z. Fertility treatment in women with premature ovarian failure. Expert Rev Obstet Gynecol 2011, 6, 321-330.
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[39] White, Y. A., Woods, D. C., Takai, Y., Ishihara, O., Seki, H., Tilly, J. L. Oocyte formation by mitotically active germ cells purified from ovaries of reproductive-age women. Nat Med 2010, 18, 413-421.
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Chapter 94
PRIVACY AND PROGRESS IN WHOLE GENOME SEQUENCING Presidential Commission for the Study of Bioethical Issues EXECUTIVE SUMMARY Over the course of less than a decade, whole genome sequencing has progressed from being one of our nation’s boldest scientific aspirations to becoming a readily available technique for determining the complete sequence of an individual’s deoxyribonucleic acid (DNA)—that person’s unique genetic blueprint. With this tremendous advance comes the accumulation of vast quantities of whole genome sequence data and complex questions of how—across a multitude of clinical, research, and social environments—to protect the privacy of those whose genomes have been sequenced. Collections of whole genome sequence data have already been key to important medical breakthroughs, and they hold enormous promise to advance clinical care and general health moving forward. To realize this promise of great public good ethically, individual interests in privacy must be respected and secured. Large-scale collections of genomic data raise serious concerns for the individuals participating. One of the greatest of these concerns centers around privacy: whether and how personal, sensitive, or intimate knowledge and use of that knowledge about an individual can be limited or restricted (by means that include guarantees of confidentiality, anonymity, or secure data protection). Because whole genome sequence data provide important insights into the medical and related life prospects of individuals as well as their relatives— who most likely did not consent to the sequencing procedure—these privacy concerns extend beyond those of the individual participating in whole genome sequencing. These concerns are compounded by the fact that whole genome sequence data gathered now may well reveal important information, entirely unanticipated and unplanned for, only after years of scientific progress. Another privacy concern associated with whole genome sequencing is the potential for unauthorized access to and misuse of information. For example, in many states someone could This
is an edited, reformatted and augmented version of the Presidential Commission for the Study of Bioethical Issues, dated October 2012.
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legally pick up a discarded coffee cup and send a saliva sample to a commercial sequencing entity in an attempt to discover an individual’s predisposition to neurodegenerative disease. The information might then be misused, for example, by a contentious spouse as evidence of unfitness to parent in a custody case. Or, the information might be publicized by a malicious stranger or acquaintance without the individual’s knowledge or consent in a social networking space, which could adversely affect that individual’s chance of finding a spouse, achieving standing in a community, or pursuing a desired career path. Realizing the promise of whole genome sequencing requires widespread public participation and individual willingness to share genomic data and relevant medical information. This, in turn, requires public trust that any whole genome sequence data shared by individuals with clinicians and researchers will be adequately protected. Current U.S. governance and oversight of genetic and genomic data, however, do not fully protect individuals from the risks associated with sharing their whole genome sequence data and information. In particular, a great degree of variation exists in what protections states afford to their citizens regarding the collection and use of genetic data. Only about half of the states, for example, offer protections against surreptitious commercial genetic testing. Currently, the majority of the benefits anticipated from whole genome sequencing research will accrue to society, while associated risks fall to the individuals sharing their data. This report focuses on reconciling the enormous public benefits anticipated from whole genome sequencing research with the potential risks to privacy of individuals, and the protections that must be foremost in our minds as we focus our policies to facilitate such privacy and progress.
Basic Ethical Principles for Assessing Whole Genome Sequencing Laws and regulations cannot do all of the work necessary to provide sufficient privacy protections for whole genome sequence data. The Commission has been mindful of how the five ethical principles set out in its first report, New Directions: The Ethics of Synthetic Biology and Emerging Technologies, apply to the ethics of whole genome sequencing. These principles—which f low from the ideal of respect for persons—are public beneficence, responsible stewardship, intellectual freedom and responsibility, democratic deliberation, and justice and fairness. This report, Privacy and Progress in Whole Genome Sequencing, enlists these principles along with those set forth in the Belmont Report (a landmark statement of ethics for research involving human participants). Privacy and Progress focuses on recommendations aimed at pursuing and securing the public benefits anticipated from whole genome sequencing while minimizing the potential privacy risks to individuals. These principles suggest ethically important and practically useful guidelines for whole genome sequencing. Chief among these is the principle of respect for persons, which requires strong baseline protections for privacy and security of data, while public beneficence requires facilitating ample opportunities for data sharing and access to data by clinicians, researchers, and other authorized users. Respect for persons further requires that any collection and sharing of individual data be based on a robust process of informed consent. Responsible stewardship calls for oversight and management of whole genome sequence information by funders, managers, professional organizations, and others. The principle of intellectual freedom and responsibility provides further support for pursuing whole genome sequencing and seeking models for broad data sharing by promoting regulatory parsimony. Democratic deliberation
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urges all parties to consider changes to policies and practices in light of the evolving science and its implications for enduring ethical values. Finally, justice and fairness requires that we seek to channel the benefits of whole genome sequencing to all who can potentially benefit, and to ensure that the risks are not disproportionately borne by any subset of the population, including vulnerable or marginalized groups.
Recommendations Currently we are in a period of intense transition with respect to integrating whole genome sequencing into clinical care, as well as facilitating access to and use of whole genome sequence data for research purposes. Moreover, the challenges we face today are not precisely the same challenges we will face in one, five, or ten years, as genomic technologies continue to develop and mature. Due to the rapid development of technology, we need to craft policies that are flexible and agile enough to ensure that we do not constrain our ability to adapt to evolving technology and social norms related to privacy and access. Recognizing that ethical obligations reach beyond what is legally enforceable, the Commission examines both the relevant ethical principles and the relevant legal requirements to offer guidance as to what (ethically) ought to be done and what (legally) must be done. This is the foundation on which the Commission builds its Privacy and Progress recommendations.
Strong Baseline Protections while Promoting Data Access and Sharing Presently, many national and state policies are in place to guard personally identifiable health information and records of participation in research. These policies should apply to all handlers of the data, from those who collect the data, to researchers who use them, to thirdparty storage and analysis providers (e.g., hosts of cloud computing services). Privacy protections should guard against unauthorized access to, and illegitimate uses of, whole genome sequence data and information while allowing for authorized users of these data to advance individual and public health. Recommendation 1.1. Funders of whole genome sequencing research; managers of research, clinical, and commercial databases; and policy makers should maintain or establish clear policies defining acceptable access to and permissible uses of whole genome sequence data. These policies should promote opportunities for models of data sharing by individuals who want to share their whole genome sequence data with clinicians, researchers, or others. Strong baseline privacy protections require a spectrum of policies starting with data handling through the protection of persons from future disadvantage and discrimination arising from misuse of their whole genome sequence data. It is critical, however, to ensure that privacy regulations allow individuals to share their own whole genome sequence data with clinicians, researchers, and others in ways that they choose.
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Recommendation 1.2. The Commission urges federal and state governments to ensure a consistent floor of privacy protections covering whole genome sequence data regardless of how they were obtained. These policies should protect individual privacy by prohibiting unauthorized whole genome sequencing without the consent of the individual from whom the sample came. Treating like data alike is crucial to ensuring consistent protections for whole genome sequence information across the United States. Although states should enact genomic policies that are most relevant and important to their constituents, bringing such protections to a minimum standard that addresses privacy—while still allowing individuals to share their own data—would provide just and fair protections regardless of where one happens to reside. Data Security and Access to Databases Data privacy requires data security. Data security requires ethical responsibility and accountability from all those who handle whole genome sequence data. It must further be supported by policies and infrastructure to protect safe sharing of data.
Recommendation 2.1. Funders of whole genome sequencing research; managers of research, clinical, and commercial databases; and policy makers should ensure the security of whole genome sequence data. All persons who work with whole genome sequence data, whether in clinical or research settings, public or private, must be: 1) guided by professional ethical standards related to the privacy and confidentiality of whole genome sequence data and not intentionally, recklessly, or negligently access or misuse these data; and 2) held accountable to state and federal laws and regulations that require specific remedial or penal measures in the case of lapses in whole genome sequence data security, such as breaches due to the loss of portable data storage devices or hacking. Many observe that absolute privacy is not possible in this, or many other realms. The greater potential for harm is not by virtue of authorized others knowing about one’s whole genome make-up, but rather through the misuse of data that have been legally accessed. Recommendation 2.2. Funders of whole genome sequencing research; managers of research, clinical, and commercial databases; and policy makers must outline to donors or suppliers of specimens acceptable access to and permissible use of identifiable whole genome sequence data. Accessible whole genome sequence data should be stripped of traditional identifiers whenever possible to inhibit recognition or re-identification. Only in exceptional circumstances should entities such as law enforcement or defense and security have access to biospecimens or whole genome sequence data for non health-related purposes without consent. The consent process should communicate limits on access to and use of genomic data to those having their whole genome sequenced in clinical care, research, and consumer-initiated contexts. These policies should apply to the original recipient of the data as well as to all parties who work with the data, from those who collect the sample or data through third-party storage and analysis service providers. Those who work with whole genome sequence data should remain current on regulations regarding data privacy and security.
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Recommendation 2.3. Relevant federal agencies should continue to invest in initiatives to ensure that thirdparty entrustment of whole genome sequence data, particularly when these data are interpreted to generate health-related information, complies with relevant regulatory schemes such as the Health Insurance Portability and Accountability Act and other data privacy and security requirements. Best practices for keeping data secure should be shared across the industry to create a solid foundation of knowledge upon which to maximize public trust. Whole genome sequence data not stripped of traditional identifiers are considered “protected health information” and are covered under the Health Insurance Portability and Accountability Act’s Privacy, Security, and Enforcement Rules and the federal Common Rule for protecting human research participants. The same regulations, policies, and ethical guidelines that protect such health information should also be in place to govern the sharing of whole genome sequence data with third-party storage and analysis service providers. Public and the private sector parties should share their lessons learned to promote efficiency and avoid duplicating efforts. Consent Not unique to whole genome sequencing, a well-developed, understandable, informed consent process is essential to ethical clinical care and research. To educate patients and participants thoroughly about the potential risks associated with whole genome sequencing, the consent process must include information about what whole genome sequencing is; how data will be analyzed, stored, and shared; the types of results the patient and participant can expect to receive, if relevant; and the likelihood that the implications of some of these results might currently be unknown, but could be discovered in the future. Respect for persons requires obtaining fully informed consent at the outset of diagnostic testing or research.
Recommendation 3.1. Researchers and clinicians should evaluate and adopt robust and workable consent processes that allow research participants, patients, and others to understand who has access to their whole genome sequences and other data generated in the course of research, clinical, or commercial sequencing, and to know how these data might be used in the future. Consent processes should ascertain participant or patient preferences at the time the samples are obtained. Recommendation 3.2. The federal Office for Human Research Protections or a designated central organizing federal agency should establish clear and consistent guidelines for informed consent forms for research conducted by those under the purview of the Common Rule that involves whole genome sequencing. Informed consent forms should: 1) briefly describe whole genome sequencing and analysis; 2) state how the data will be used in the present study, and state, to the extent feasible, how the data might be used in the future; 3) explain the extent to which the individual will have control over future data use; 4) define benefits, potential risks, and state that there might be unknown future risks; and 5) state what data and information, if any, might be returned to the individual.
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Each Common Rule agency has its own enforcement authorities to protect research participants. All agencies should work together as they develop clear and consistent guidelines for their informed consent forms. Clinical consent documents for whole genome sequencing will have to address a number of issues specific to whole genome sequencing: an explanation of the science, whether whole genome sequence data collected for clinical applications will be made available for research purposes, and what types of results will be produced through whole genome sequencing. For example, an important unsettled issue is the ethics of reporting incidental findings to individuals— that is, information gleaned from whole genome sequencing research or clinical practice that was not its intended or expected object.
Recommendation 3.3. Researchers, clinicians, and commercial whole genome sequencing entities must make individuals aware that incidental findings are likely to be discovered in the course of whole genome sequencing. The consent process should convey whether these findings will be communicated, the scope of communicated findings, and to whom the findings will be communicated. Recommendation 3.4. Funders of whole genome sequencing research should support studies to evaluate proposed frameworks for offering return of incidental findings and other research results derived from whole genome sequencing. Funders should also investigate the related preferences and expectations of the individuals contributing samples and data to genomic research and undergoing whole genome sequencing in clinical care, research, or commercial contexts. Individuals undergoing whole genome sequencing in research, clinical, and commercial contexts must be provided with sufficient information in informed consent documents to understand what incidental findings are, and to know if they will or will not be notified of incidental findings discovered as a result of whole genome sequencing. Facilitating Progress in Whole Genome Sequencing Currently, large amounts of patient data are being collected in the health care setting, stripped of traditional identifiers, analyzed, and fed into research that might one day improve clinical care. This “learning health system” model both translates advances in health services research into clinical applications and collects data during clinical care to facilitate further advances in research. Learning health system advocates and others support standardized electronic health record systems and infrastructure to facilitate health information exchange so that data can be easily aggregated and studied. Integrating whole genome sequence data into health records in the learning health system model can provide researchers with more data to perform genome-wide analyses, which in turn can advance clinical care.
Recommendation 4.1. Funders of whole genome sequencing research, relevant clinical entities, and the commercial sector should facilitate explicit exchange of information between genomic researchers and clinicians, while maintaining robust data protection safeguards, so that whole genome sequence and health data can be shared to advance genomic medicine.
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Current sequencing technologies and those in development are diverse and evolving, and standardization is a substantial challenge. Ongoing efforts are critical to achieving standards for ensuring the reliability of whole genome sequencing results, and facilitating the exchange and use of these data.
Recommendation 4.2. Policy makers should promote opportunities for the public to benefit from whole genome sequencing research. Further, policy makers and the research community should promote opportunities for the exploration of alternative models of the relationship between researchers and research participants, including participatory models that promote collaborative relationships. Respect for persons implies not only respecting individual privacy, but also respecting research participants as autonomous persons who might choose to share their own data. Public beneficence is advanced by giving researchers access to plentiful data from which they can work to advance health care. Regulatory parsimony recommends only as much oversight as is truly necessary and effective in ensuring an adequate degree of privacy, justice and fairness, and security and safety while pursuing the public benefits of whole genome sequencing. Therefore, existing privacy protections and those being contemplated should be parsimonious and not impose high barriers to data sharing. While the Commission supports the intellectual freedom this access will encourage, clinicians and researchers must also act responsibly to earn public trust for the research enterprise. Public Benefit Thousands of citizens have participated in whole genome sequencing research personally, and all citizens help to support government investment in whole genome sequencing through their general participation in and support of our political system. Therefore, all citizens should have the opportunity to benefit from medical advances that result from whole genome sequencing. Special caution should be taken on the part of researchers to ensure that their participants accurately reflect as much as possible the rich diversity of our population. Different groups have genomic variants at different frequencies within their populations, and sufficiently diverse data must be collected so that advances arising from whole genome sequencing can be used for the benefit of all groups.
Recommendation 5.1. The Commission encourages the federal government to facilitate access to the numerous scientific advances generated through its investments in whole genome sequencing to the broadest group of persons possible to ensure that all persons who could benefit from these developments have the opportunity to do so. Government investment in genomic research has resulted in public benefit through improved health care and in economic return on investment. The principle of justice and fairness requires that the benefits and risks of whole genome sequencing be distributed equitably across society. Research funded with taxpayer contributions should benefit all members of society. To these ends, researchers should be vigilant about including individuals from all sectors of society in their studies, so that research findings can be translated widely into improved clinical care. The federal government should follow through on its investment
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in research and assure that the discoveries of whole genome sequencing are integrated with clinical care to benefit the health of all.
INTRODUCTION The Potential of Whole Genome Sequencing In 1996, Retta Beery gave birth to apparently healthy twins Alexis and Noah.1 It soon became clear, however, that something was wrong; the twins cried nonstop and had developmental problems. Over the next two years, Retta and her husband Joe endured the physical, emotional, and financial costs of visiting numerous specialists, putting their young twins through countless tests, and having their children undergo surgery. None of these steps provided results or solutions. In 1998, the twins were diagnosed with cerebral palsy and a related course of treatment was outlined. Although the treatment yielded some symptomatic improvement, Retta felt that the diagnosis was incorrect. In 2002, the Beerys were starting to look at wheelchairs and feeding tubes when Retta, after four years of research, stumbled upon an article on DOPAresponsive dystonia (also known as Segawa’s dystonia) and suspected that this was the disease that the twins had. The Beerys contacted a specialist, and after a physiological test the twins were diagnosed with Segawa’s dystonia. They began a new course of treatment to increase brain dopamine, which yielded a dramatic improvement in their health. In 2009, Alexis developed breathing problems and was forced again to endure multiple emergency room visits and a battery of tests and visits to specialists. In August 2010, the Beerys went to Baylor College of Medicine for diagnostic whole genome sequencing. By November, Alexis’s and Noah’s whole genomes had been sequenced. Their data were compared to other whole genome sequences in databases, such as the Baylor Human Genome Sequencing Center’s database, to reveal what was unique about the Beery twins’ genomes. Clinicians now had answers for the family. The geneticists had uncovered an extremely rare and only recently recognized genetic cause of DOPA-responsive dystonia producing a deficiency of not only dopamine but also serotonin production in the brain. Armed with this new information, the Beerys returned to their neurologist, who amended the treatment regimen for Alexis and Noah with an over-the-counter supplement. Within a month, Alexis’s breathing problems disappeared. As a result of that final piece of the puzzle—the information provided by whole genome sequencing—Alexis is able to breathe normally and can now even compete in sports. Both children have a definitive diagnosis, and are expected to live long, healthy lives.
THE CHALLENGE OF PRIVACY Victoria Grove’s sisters struggled with a difficult genetic diagnosis: alpha-1 antitrypsin deficiency. The genetic illness meant that her sisters’ bodies did not make enough of a protein that protected their lungs and liver from damage, which could lead to emphysema and liver disease.
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Victoria wanted to help them, and in 2004 agreed to enroll in a research study of families with alpha-1 antitrypsin deficiency. “I just knew I didn’t have it, so I signed up for the study.” But the tests came back positive—Victoria had the same genetic mutation as her sisters. She did not yet have any symptoms, and wanted to keep her test results private, so she did not tell her doctor. In 2005, Victoria got tested again to confirm the research study results. She used a private company and had the results sent directly to her. Victoria’s second test came back positive, and she chose again not to send the results to her doctor, fearing that the information would be included in her medical record. Victoria worried that this information could lead her insurance company to drop her coverage or charge her higher rates. Victoria kept her genetic results private for nearly three years. The pivotal moment came when Victoria felt she was coming down with a bout of pneumonia but could not convince the nurse practitioner who saw her to order the X-ray necessary to prescribe antibiotics. Victoria went home without antibiotics, her condition worsened, and she called back a few days later. The nurse asked Victoria to come in again, but Victoria told them she could not drive across town in the snowstorm that had immobilized the city. She could, however, get to a pharmacy near her house if the office called in the antibiotics. The nurse on the phone insisted this was not possible. “My emotions just took hold and I cried ‘I have alpha-1 and I need that antibiotic,’” Victoria said. “At that point the cat was out of the bag.” Today Victoria gets regular treatments for her condition. She recognizes that fear kept her from providing her clinicians with crucial information. Still, she can’t convince either her brother or her son to get tested for alpha-1. Victoria says both men are aware that there is federal protection from discrimination in employment and health insurance, but fear that these laws will not provide sufficient protection. Her son already has to buy his own health insurance; he does not want any information in his medical record that could jeopardize his job or his access to health insurance. “I can imagine in a job situation, it’s expensive to take on someone if they’re ill. And you can always get rid of people for other reasons. I assume that’s going on.” Whole genome sequencing offers great promise of medical advances that could benefit all of society, but this promise is tempered by the concerns of individual privacy. This tension between medical progress and the risks to privacy from whole genome sequencing is the subject of this report. To use whole genome sequencing to discover the changes in deoxyribonucleic acid (DNA) that underlie disease, scientists and clinicians must have access to whole genome sequence data from many individuals (for the definitions of scientific terms used in this report, see Appendix I: Glossary of Key Terms). Continued advances therefore depend on large numbers of individuals who are willing to share their whole genome sequence data for research purposes. Further, scientists are better able to make connections between variations in whole genome sequence data and specific diseases when additional health and demographic information accompanies these data. But this additional information might make it easier to identify an individual and discover his or her private health information.2 Thus, while society stands to benefit from advances in improved medical treatment and diagnosis from whole genome sequencing, the privacy risks associated with sharing whole genome sequence data fall predominantly on the individuals themselves. Whole genome sequencing is a technique that determines the complete sequence of DNA in an individual’s cells (See Figure 1. For more information regarding the science of whole
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genome sequencing, see Appendix II: Genetic and Genomic Background Information). Whole genome sequencing reveals the genetic blueprint for a person, generating information on every gene in the nucleus of one’s cells. Each person’s DNA is unique, and changes in DNA can lead to disease. The ability to link variations in DNA with health and disease outcomes, a process still in its infancy, holds promise for substantial public benefit.3 These benefits have the potential to alter the way we treat cancer, heart disease, diabetes, Alzheimer’s disease, schizophrenia, and countless other illnesses. The Commission believes that the ethical principles and recommendations in this report should not be limited to whole genome sequencing. Whole genome sequencing is the focus of this report because of its current promise to advance health. The ideas in this report, however, apply broadly to all studies using large-scale genetic and genomic data and information, including whole exome sequencing, genome-wide SNP analysis, and other large-scale genomic studies. The tools used to decipher and study whole genomes are evolving rapidly, and it is not clear what additional technologies will emerge in the near future. What is clear is that all current genetic and genomic research can be measured against the ethical principles and recommendations described in this report, and the Commission is optimistic that its recommendations will specifically accommodate future advances in large-scale sequencing and analysis.
Figure 1. The Structure of DNA.
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TERMINOLOGY Whole genome sequencing: determining the order of nucleotide bases—As, Cs, Gs, and Ts—in an organism’s entire DNA sequence Whole genome sequence data: the file of As, Cs, Gs, and Ts that results from whole genome sequencing Whole genome sequence information: facts derived from whole genome sequence data, such as predisposition to disease Genomics: the study of all the DNA (the genome) in an individual, and how parts of the genome interact with each other and the environment Genetic test: a discrete test that examines a specific genetic location or a single gene, such as the test for Huntington’s disease Genotyping: analyzing a handful to thousands of discrete variants across the genome (i.e., more than a discrete genetic test, but less than whole genome sequencing) For additional terminology, please see Appendix I: Glossary of Key Terms and Appendix II: Genetic and Genomic Background Information.
Current clinical uses of DNA information are limited mostly to specific genetic tests. If clinicians suspect a particular disease with a known genetic cause, such as Huntington’s disease, they can order a genetic test looking at one specific gene among the more than 20,000 genes in the human genome. These test sex a mine only a few of the whole genome’s three billion pairs of building blocks. The price of sequencing a whole genome is dropping rapidly, however, and soon it will be less expensive to sequence an entire genome than to perform a few individual genetic tests. Once this happens, whole genomes might be sequenced in lieu of discrete genetic tests, and such information can be stored in a patient’s medical records. Then, if a clinician would like to find out something about a patient’s DNA in the future, he or she could examine the whole genome sequence data already stored in that patient’s record. For example, a patient’s response to a particular dose of warfarin, a drug that helps prevent blood clotting, is partly dependent on his or her genetic makeup. A clinician with access to a patient’s whole genome sequence can use it to identify drug sensitivity and reduce the time required to achieve the optimal dosage.4 The sheer amount of information contained in our genomes is what makes whole genome sequence data different from other medical information. Our whole genome sequence data can reveal predispositions to diabetes, cancer, or psychiatric conditions. The data can also reveal variations in DNA that are not yet understood. For example, an apparently healthy individual could be missing a small piece of DNA. The person seems healthy, but will that variant cause a problem in the future? Over 20,000 individual human genes have been identified. A major recent advance by the National Institute of Health’s (NIH) Encyclopedia of DNA Elements (ENCODE) project greatly enhanced our knowledge of the function of the genome through a flurry of scientific publications, finding that 80 percent of the genome has a “biochemical function.”5 For years,
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that number had stood at 10 percent.6 Eric Lander, president of the Broad Institute, compared the results of the Human Genome Project, which sequenced the first full human genome, to a picture of the Earth from space, and compared the ENCODE project to Google Maps. The ENCODE Project is a major step toward demonstrating the function of the whole genome sequence that was determined in the Human Genome Project, much like Google Maps can refine a snapshot of the Earth by showing traffic, alternate routes, and the location of landmarks.7 The function of 20 percent of the non-coding regions—regions of DNA that do not contain specific instructions for making proteins—is still unknown, but these regions might have functions that are yet to be determined. Unlike genetic testing—which looks at the specific parts of the genome to reveal a variant at a specific location of a single gene indicating a particular disease—whole genome sequencing reveals an individual’s entire genome, including all variants within the genome. These variants are changes in the DNA sequence and range in size from small changes like a single base pair change, to larger changes such as a deletion of a portion of the DNA strand. As more information about our genomes becomes available, variants that might be revealed by whole genome sequencing include: specific known disease variants; variants of unknown significance (e.g., an unknown variant in the region that increases risk for heart disease); nonmedical genetic traits, including hair and eye color; carrier status variants, including variants that do not cause disease in the individual but could be passed on, such as mutations for hemophilia or cystic fibrosis; susceptibility genes, such as those that slightly increase susceptibility to diabetes, heart disease, or some cancers; and genes for conditions with late onset that will not affect an individual until much later in life, such as Alzheimer’s disease and Huntington’s disease.8 Only a small number of the genetic variants that whole genome sequencing might reveal have yet been studied enough to substantiate their connection to disease.9 “And so having the genome may be not incredibly powerful right now, but it opens the door to outrageous rates of discovery, which I’m pretty certain are going to happen over the next five to ten years.” Leonard D’Avolio, Associate Center Director for Biomedical Informatics, Massachusetts Veterans Epidemiology Research and Information Center, Department of Veterans Affairs, Instructor, Harvard Medical School. (2012). Genomic Privacy, Data Access, and Health IT. Presentation to PCSBI, May 17, 2012. Retrieved from http:// bioethics.gov/cms/node/713.
Whole genome sequencing also raises many potential concerns for individuals. One might shoulder the burden of knowing medical in format ion regarding future adverse health conditions for which there is currently no treatment. Whole genome sequencing raises concerns about our privacy as well. Just as patients would not want to give anyone access to their medical record, many people might not want others to have access to their whole genome sequence data and information. With unauthorized access comes concerns about misuse of information. For example, someone could pick up a discarded coffee cup and send a sample of saliva—which contains DNA—from the rim of the cup to a commercial sequencing entity in an attempt to discover an individual’s predisposition to neurodegenerative disease. The information might then be misused by publicizing it in a social networking space, which could derail that
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individual’s chance of finding a spouse, achieving standing in a community, or pursuing a certain career path. To yield medically useful information, an individual’s genomic sequence data needs to be coupled with clinical information about disease and compared to other genomic sequence data. Further, genomic research is complex because each person’s DNA naturally has thousands of variants, and the vast majority of these variants do not cause disease. The research and clinical power of whole genome sequencing lies in being able to compare a large number of whole genome sequence data sets that are linked with relevant health and disease states. This type of study allows researchers to identify sequence variations and associations between whole genome sequence variations and disease. For this reason, scientists need whole genome sequence data to be linked to clinical, laboratory, and socio-demographic data. This linking can be done by entering only relevant information (e.g., disease state or symptoms) and excluding personally identifiable information, such as an individual’s name or address; but without access to relevant medical data, links between whole genome sequence variations and disease could not be identified. Recent technological advances have facilitated storing and sharing of whole genome sequence data. Whole genome sequence data and associated health information can now be stored in genomic databases and biorepositories that contain digital information and physical samples, respectively, from large numbers of persons. By using these resources, researchers will have the volume of data they need to advance medical understanding for the public good through genomics. However, this data storage and sharing raises its own questions: How does one securely store these huge data files? Who should have access to these data files? How can these data be used productively, and how might they be misused? What constitutes “misuse”? What should the penalties be for misusing these data? The summation of all these issues—the unknowns, privacy, consent, data security, and data storage involved in whole genome sequencing—will require careful and sustained ethical attention. This report delves into two crucial questions: What information about an individual’s whole genome should remain private, and when should it remain private? The Commission explores how, when, and why genomic information should remain subject to clear rules of confidentiality, secrecy, information security, decisional autonomy, and freedom from unwanted intrusion out of respect for individuals. Without trust in the confidentiality and security of the data, individuals could be less likely to participate in research. Conversely, with well-founded trust that their sense of privacy will be honored, individuals are treated with the respect to which they are entitled and might be more likely to contribute to the research enterprise that promises important public benefits. This report therefore aims to pursue and secure the public benefit anticipated from whole genome sequencing while minimizing the potential privacy risks to individuals. The recommendations draw upon the principles that flow from the ideal of respect for persons, and are set forth in the Belmont Report, a landmark statement of ethics for research involving human participants, and those outlined in the Commission’s first report New Directions: The Ethics of Synthetic Biology and Emerging Technologies.10
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The Promise of Whole Genome Sequencing Whole genome sequencing can help researchers and clinicians better understand the unique qualities of a disease, and, especially when combined with other information, might help select treatment methods.11 Researchers already have been able to help clinicians aid some children born with rare birth defects by sequencing and analyzing their whole genomes to diagnose and treat their illnesses.12 Researchers are also teaming up with clinicians in using whole genome sequence data to advance personalized medicine, including predicting an individual’s risk for a heart attack or determining the best dosage of medication for an individual.13 Researchers recently determined a fetal whole genome sequence using a blood sample from the mother, an innovation that could soon reach the clinic.14 And this is only the beginning of the whole genome sequencing era, which has the potential to revolutionize medicine. In 2000, the cost of sequencing a single human genome was estimated to be 2.5 billion dollars; it is anticipated that this cost will soon be $1,000. As the cost falls, whole genome sequencing will be increasingly integrated into clinical care. Clinicians can—and many will— incorporate whole genome sequence information into the clinic to promote the practice of personalized medicine.15 Nevertheless, little has been written about the ethical concerns of integrating whole genome sequencing into the clinical context, which is particularly problematic given the speed with which this could occur.16 The Commission therefore presents its recommendations mindful of the changing uses and implications of whole genome sequencing. Although this report focuses on issues related to privacy and sharing of whole genome sequence data, the Commission recognizes that another important unsettled issue is the ethics of reporting incidental findings to individuals—that is, information gleaned from whole genome sequencing research or clinical practice that was not its intended or expected object. The Commission plans to take up the issue of incidental findings in the future.
Privacy Concerns At age 13, Brian Hurley learned from an ophthalmologist that he had retinitis pigmentosa and that at some unknown point in his life he would go blind. During high school, Brian learned about careers in law and thought this was something he could do well, regardless of eyesight. When he started law school, however, he realized he did not like it. Brian needed to find something he could do and wanted to do—not just something a blind person was considered capable of doing. Brian felt tremendous pressure to resolve his career path before he lost his vision: “In the beginning of a career, you try to figure out what you are good at and hopefully enjoy, but I was more concerned about could I do it well when blind.” Brian spent hours online searching for careers that might work, and successful blind professionals that he could use as role models. “It was like having a time bomb inside of me,” Brian said. After college, Brian experienced a steady decline in his peripheral vision. At age 27, Brian stopped driving. During this time, Brian’s actual symptoms did not match the decline of his emotional state. Brian said he was so panicked that it took the joy out of his last few years before becoming legally blind. “If you took my mental condition, I might as well have been blind already.”
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Then, at 33, Brian lost the majority of his eyesight. “The irony is, anticipation was much worse than the actual loss. It was a relief to stop worrying when the loss would occur.” Today, at 39, and relieved of the anticipation, he enjoys his current role as a Public Affairs Program Director. Brian refuses to let his vision loss be an obstacle to his professional and personal goals. Brian recently learned of the eyeGENE® program at the National Institutes of Health. He wants to help with eye research—specifically research related to retinitis pigmentosa. He knows research is important and wants to contribute his data to help others. He does not, however, want his whole genome sequenced in the course of participating in research. Before enrolling in the eyeGENE® program, Brian spent three days with a lighted magnifier and 20 pages of consent forms to ensure that researchers would not sequence his entire genome and that they will not divulge findings about other diseases to him. Having lived with one time bomb, Brian understands its collateral damage. He never wants to carry that burden again. In his situation, Brian feels that having less information is better. With regard to whole genome sequence data, privacy concerns are more complex than a simple decision about whether to undergo whole genome sequencing and, if so, whether the data should be included with an individual’s medical record. Individuals might have good reason for wanting to share particular parts of their genomic data—such as for the purposes of research— but might also want to limit the extent to which others can access these data. The prevention of unauthorized use or disclosure of medical information about specific individuals has long been a serious ethical concern. Whole genome sequencing dramatically raises the privacy stakes because it necessarily involves examining and sharing large amounts of biological and medical information that is not only inherently unique to a single person but also has implications for blood relatives. Genomic information is inherited and determines traits like hair and eye color. Unlike a decision to share our hair or eye color, which does not reveal anything about our relatives that is not observable, a decision to learn about our own genomic makeup might inadvertently tell us something about our relatives or tell them something about their own genomic makeup that they did not already know and perhaps do not want to know. More than other medical information, such as X-rays, our genomes reveal something both objectively more comprehensive and subjectively (to many minds) more fundamental about who we are, where we came from, and the health twists and turns that life might have in store for us. The fact that whole genome sequence information is uniquely connected to our conceptions of self is what could cause the inappropriate disclosure or misuse of this information to be so harmful. In theory, whole genome sequence information could be used to deny financial backing or loan approval, educational opportunities, sports eligibility, military accession, or adoption eligibility.17 Disclosing genomic information could affect the opportunities available to individuals, subject them to social stigma, and cause psychological harm. The full extent of what whole genome sequencing can reveal is unknown, but we k now that having one’s whole genome sequenced today could reveal genetic variants that increase the risk for certain conditions such as Alzheimer’s disease, which many people either do not want to know about themselves or others to know about them.
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“[H]arm is not the act…of distributing data. Harm comes from actions that are taken once the data have been distributed.” John Wilbanks, Founder, Consent to Research; Senior Fellow, Kauffman Foundation; Research Fellow, Lybba. (2012). Privacy II – Control, Access and Human Genome Sequence Data. Presentation to PCSBI, February 2, 2012. Retrieved from http://bioethics. gov/cms/node/659.
It is understandable, therefore, that whole genome sequencing heightens concerns about how unauthorized disclosure can threaten one’s individual privacy. But determining what privacy requires in the whole genome context is not straightforward. In the legal context, privacy is multidimensional and includes physical, informational, decisional, proprietary, associational, and intellectual aspects.18 While there is no consensus definition of privacy, in this report we consider privacy to be a general concept that includes confidentiality, secrecy, anonymity, data protection, data security, fair information practices, decisional autonomy, and freedom from unwanted intrusion.19 Whole genome sequencing calls for serious consideration of each of these components and their related ethical concerns. It also is important to recognize at the outset that, in some significant respects, parts of our genomic information are not and cannot be wholly private. When we routinely provide a blood sample in a clinical exam, decide to submit a DNA sample to be used in research, or unintentionally leave behind traces of DNA on a coffee cup that we discard in a public waste bin, we are providing some other individuals the opportunity to learn something about us. While doing everything possible to prevent any use of whole genome sequence data certainly would provide strong privacy protection, it would fail to allow the anticipated public benefit that is to be achieved by sharing whole genome sequence data and advancing science. Because preventing all whole genome sequence data sharing would stifle potentially life-saving and life-enhancing medical progress, we must focus on how best to protect confidentiality of data, ensure security of information from unauthorized access and uses, preserve decisional autonomy as to possible uses, and guarantee the freedom of individuals from unwanted and unwarranted intrusion.
Policy and Governance “If you sequence people’s exomes you’re going to find stuff,” said Gholson Lyon, a physician and researcher previously at the University of Utah, now at Cold Spring Harbor Laboratory. As part of his research, Dr. Lyon worked with a family in Ogden, Utah. Over two generations, four boys had died from an unknown disease with a distinct combination of symptoms—an aged appearance, facial abnormalities, and developmental delay. Dr. Lyon sought to identify the genetic cause of this disease, and collected blood samples from 12 family members who had signed consent forms. The family members understood these forms to mean that they would have access to their results. Dr. Lyon conducted exon capture and sequencing of the X chromosome—a process that analyzes specific regions of the X chromosome and is a less expensive alternative to whole genome sequencing—to analyze the blood samples. Dr. Lyon and his colleagues identified a genetic mutation, and named the disease Ogden Syndrome after the family’s hometown.
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After Dr. Lyon and his team identified the genetic basis of Ogden Syndrome, one of the family members contacted him. This young mother of one daughter had submitted a blood sample for Dr. Lyon’s research. She had not been pregnant at the time, but was now four months pregnant with her second child. She knew that she was carrying a boy and wanted to know if she was a carrier of the mutation. She wanted to be able to mentally and emotionally prepare herself and her family. By reexamining his research data, Dr. Lyon was able to see that the expectant mother was a carrier of Ogden Syndrome. This meant that her son had a 50 percent chance of being born with the disease. Dr. Lyon could not, however, legally share this important information with the family because he had conducted the original sequencing in a research laboratory that had not satisfied federally mandated standards designed to ensure the accuracy of clinical genetic results. Instead, Dr. Lyon worked to have the mutation validated at a laboratory that satisfied those federal standards; this involved overcoming substantial bureaucratic hurdles and other obstacles that held up the process. During this time, the baby boy was born and died of Ogden Syndrome at four months of age. While knowing the results would not have changed the outcome, Dr. Lyon feels he should have been able to do more for the family. Dr. Lyon has become an outspoken advocate for conducting whole genome sequencing in laboratories that satisfy the federal standards so that researchers can return results to participants, if appropriate. Dr. Lyon wants clear guidance for laboratories conducting genetic research and clear language in consent forms that clarifies the results that participants should expect to have returned from the researchers. Realizing the promise of whole genome sequencing requires widespread public participation and individual willingness to share genomic data and relevant medical information. This requires public trust that any whole genome sequence data shared by individuals with researchers and clinicians will be adequately protected. Individuals must trust that their whole genome sequence data will not be either intentionally or inadvertently disclosed or misused. Current U.S. governance and oversight of genetic and genomic data, however, do not fully protect individuals from the risks associated with sharing their whole genome sequence data and information. The Genetic Information Nondiscrimination Act of 2008 (GINA) is the leading federal protection of genetic information, but it offers only prohibition of genetic discrimination in health insurance and employment. GINA does not regulate access, security, and disclosure of genetic or whole genome sequence information across all potential users, nor does it protect against discrimination in other contexts. U.S. state laws on genetic information vary greatly in their protections of individuals, and they also fail to provide uniform privacy protections. In an era in which whole genome sequence data are increasingly stored and shared using biorepositories and databases, there is little to no systematic oversight of these systems.
Ethical Principles Laws and regulations cannot do all of the work necessary to provide sufficient privacy protections for whole genome sequence data. Individuals who obtain their whole genome sequence data also have a responsibility to thoughtfully consider to what extent they ought to
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act to protect their own privacy beyond current legal protection when considering whether to share their data and information publicly. In its previous reports, the Commission established an ethical framework for considering the implications of scientific advances, including emerging technologies, that can be applied in similar situations. That framework outlines principles developed to apply particularly to emerging biotechnologies that do not directly involve human therapy or human experimentation. These guiding principles are 1) public beneficence, 2) responsible stewardship, 3) intellectual freedom and responsibility, 4) democratic deliberation, and 5) justice and fairness. As biomedical science has evolved over time, the lines between clinical care, human research, and research not involving human participants have become blurred. The principles developed by this Commission, which flow from the concept of respect for persons, are described in detail in New Directions: The Ethics of Synthetic Biology and Emerging Technologies, and also apply when considering the ethics of whole genome sequencing.20 As applied to the science of whole genome sequencing, these principles, along with the principle of respect for persons, guide us to focus on pursuing public benefit while minimizing both personal and public risk.
Respect for Persons Respect for persons provides a strong, enduring, and widely accepted foundation for this report’s recommendations for protecting individual privacy in the pursuit of public benefit. As set forth in the Belmont Report, respect for persons requires one to give great “weight to autonomous persons’ considered opinions and choices while refraining from obstructing their actions unless they are clearly detrimental to others.”21 The Belmont Report recognizes that not all persons can act as autonomous agents, and makes clear that there are special responsibilities to those who cannot. Public Beneficence Public beneficence asks us to pursue and secure public benefits and minimize personal and public harm. It encompasses society’s duty to promote activities that have great potential to improve the public’s well-being.22 Public beneficence also supports scientific enterprises that benefit society by increasing economic opportunities. Responsible Stewardship Responsible stewardship calls upon governments and societies to proceed prudently in promoting scientific advancement by taking into account the interests and needs of those who are not in a position to represent themselves such as children, the mentally ill, future generations, or individuals that may be unaware of risks. Responsible stewardship expresses a shared obligation to act in ways that demonstrate respect for such individuals. Emerging technologies present particularly profound challenges for responsible stewardship because our understanding of their potential benefits and risks is incomplete and uncertain.23 This makes it all the more important that we take great care not to make choices that have a substantial chance of causing irreversible harm to current or future generations.
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Intellectual Freedom and Responsibility Intellectual freedom grants scientists, acting responsibly, the right to use their creative abilities to advance science and the public good. Sustained and dedicated creative intellectual exploration produces much of our scientific and technological progress. Intellectual responsibility, the complementary part of this principle, calls upon scientists to adhere to the ideals of research; to avoid harm to others; and to abide by all applicable policies, rules, and regulations. Institutions, policies, and practices of a free society—along with the many citizens who support them—collectively provide the means for scientists to do their work, and the culture that recognizes and upholds intellectual freedom. As a result, scientists bear profound collective responsibility to society.24 The Commission endorses the principle of regulatory parsimony, which encourages fostering an achievable balance of intellectual freedom and responsibility. Regulatory parsimony calls for “only as much oversight as is truly necessary to ensure justice, fairness, security, and safety while pursing the public good.”25 In this spirit, policy makers are obligated to avoid restrictive rules that offer few benefits and hinder progress in science, medicine, and health care.26 Democratic Deliberation Democratic deliberation is an approach to collaborative decision making that embraces respectful debate of opposing views and active participation by citizens. Democratic deliberation warrants engaging the public and fostering dialogue among the scientific community, policy makers, and persons concerned with the issues raised by scientific progress.27 The principle of democratic deliberation acknowledges that while decisions must eventually be reached, those decisions need not (and often should not) be unalterable, particularly when subsequent developments warrant additional examination. It is in the spirit of democratic deliberation that the Commission was created, has undertaken its work in publicly open meetings, and offered all of its reports to the President and members of the public. Justice and Fairness The principle of justice and fairness relates to the distribution of benefits and burdens across society. A commitment to justice and fairness is a commitment to ensuring that the unavoidable burdens of technological advances do not fall disproportionately on any particular individual or group, and that the benefits are widely and equitably distributed.28 The principle of justice and fairness counsels that the numerous scientific advances stemming from investments in science and medicine should be made accessible to the broadest possible number of persons, consistent with the ability to advance science and medicine for the true benefit of the public.
The Commission’s Process In concert with the principle of democratic deliberation, the Commission invited experts from the public and private sectors to inform their deliberations. Over the course of four public meetings, speakers addressed issues of privacy, consent, data security, access to whole genome sequence data, views of the patient advocacy community, and relevant philosophical topics (for
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a complete list of Commission speakers, see Appendix III: Guest Presenters to the Commission Regarding Privacy and Whole Genome Sequencing). The Commission also posed a data call to the 18 Common Rule departments and agencies, asking them to identify relevant statutes, agency regulations, guidance documents, and policies that govern privacy and access to genetic information generally and whole genome sequence data specifically.29 Finally, a Request for Information was published in the Federal Register that elicited many thoughtful comments from individuals and professional societies.30 The Commission identified the field of whole genome sequencing as an important topic for consideration because this rapidly advancing technology raises many ethical issues that have not been fully addressed. After careful consideration of where it could make the greatest contribution at the present time, the Commission chose to focus on privacy rather than address ethical issues that are currently under consideration or have been addressed by other high-level groups or federal agencies, including commercial genetic testing and other important and controversial topics relevant to whole genome sequencing.31 In focusing on the potential risks to individuals’ privacy, the Commission also recognizes the anticipated societal benefit of the scientific and medical applications of advances in whole genome sequencing. Reconciling these goals means addressing the competing concerns of ensuring confidentiality of whole genome sequence data, granting access to and use of these data, and empowering participants who want to share their data without weakening privacy protections for others. The Commission reviewed rules and regulations already in place that protect privacy and prevent discrimination based on genetic information (currently there are no state or federal laws explicitly addressing whole genome sequence data), and heard testimony about the technological security systems used to protect whole genome sequence data. The Commission heard from experts about the ways whole genome sequencing is being, and will continue to be, integrated into clinical care. In addition, the Commission heard from the patient advocacy communities who expressed their wishes for more participatory models of research.
About This Report With its guiding principles in mind, the Commission sought to reconcile the anticipated societal benefit of the scientific and medical applications of advances in whole genome sequencing with the potential risks to individuals’ privacy. Recognizing that our ethical obligations reach beyond what is legally enforceable, the Commission examined both the relevant ethical principles and the relevant legal requirements to offer guidance as to what (ethically) ought to be done and what (legally) must be done.32 This is the foundation upon which the Commission builds its recommendations, which apply to both the public and private sectors. Accordingly, Section 1 deploys and applies the relevant ethical principles. Section 2 summarizes the legal framework governing whole genome sequencing and the legal protections provided for persons who decide to share their whole genome sequence data. Finally, Section 3 offers recommendations and guidelines that are aimed at reconciling the existing tension between minimizing risks to individuals and maximizing the anticipated future societal benefits of whole genome sequencing. The Commission intends that any changes resulting from these recommendations be prospective and not apply retrospectively to specimens already collected or stored in the research or clinical setting.
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WORK OF PREVIOUS COMMISSIONS Previous bioethics commissions have issued reports on topics related to genetics. In 1982, the President’s Commission for the Study of Ethical Problems in Medicine and Biomedical and Behavioral Research published a report, Splicing Life, which addressed the ethical and social implications of genetic engineering (http://bioethics.georgetown.edu/ documents/pcemr/ splicinglife.pdf). In 1983, the same commission issued a report on the ethical, social, and legal implications of genetic screening, counseling, and education programs, titled Screening and Counseling for Genetic Conditions (http://bioethics. georgetown.edu/pcbe/reports/past_ commissions/ geneticscreening.pdf). Genetic issues were not revisited until the National Bioethics Advisory Commission (NBAC) discussed the issue of human cloning in 1997, in its report Cloning Human Beings (http://bioethics.georgetown.edu/nbac/pubs/ cloning1/cloning.pdf). In 1999, NBAC issued Research Involving Human Biological Materials: Ethical Issues and Policy Guidance, which focused on research involving human biological materials (http://bioethics. georgetown. edu/nbac/hbm.pdf). In 2002, the President’s Council on Bioethics took up the issue of human cloning in its report, Human Cloning and Human Dignity: An Ethical Inquiry (http://bioethics. georgetown.edu/pcbe/reports/ cloningreport/pcbe_cloning_report.pdf). The Council also published The Changing Moral Focus of Newborn Screening, which sought to establish ethical principles to guide newborn genetic screening (http://bioethics. georgetown.edu/pcbe/reports/ newborn_ screening/Newborn Screening for the web.pdf).
SECTION 1. ETHICAL PRINCIPLES Whole genome sequencing offers the promise of tremendous public benefit, and is expected to change substantially our ability to assess risk, diagnose, and treat disease. Achieving this public benefit requires that researchers have access to large amounts of whole genome sequence data and associated medical information to assess correlations between underlying genomic variants and expressed disease. While many of the potential benefits arising from whole genome sequencing will accrue to the broader public, the risks associated with collecting and sharing whole genome sequence data will be borne disproportionately by the individuals whose data are being shared. Because whole genome sequencing begins with obtaining a sample from an individual, to reconcile anticipated public benefits with potential individual harms the Commission begins with the principle of respect for persons. Respect for persons is among the most enduring and widely accepted foundation s for protecting individual privacy in the pursuit of public benefit, and it is well formulated in the Belmont Report, a declaration of ethical principles regarding research involving human participants.33 Since biomedical science has evolved significantly since the Belmont Report’s publication in 1979 from clinically focused research to research for public benefit, the Commission also applies five additional ethical principles which flow from the principle of respect for persons—as outlined in New Directions: The Ethics of Synthetic Biology and Emerging Technologies—to the field of whole genome sequencing.34 These five principles—public beneficence, responsible stewardship, intellectual freedom and responsibility, democratic deliberation, and justice and fairness—apply well not only to
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emerging biotechnologies, but also to scientific advancement and innovation generally. The Commission’s five principles are thus a useful supplement to the Belmont principles for the purpose of assessing the ethics of whole genome sequencing. “The state of technology is that data acquisition is now… relatively inexpensive, and while the free access to genetic data has many positive benefits, we need to represent, of course, the tension of that with all of the other personal privacy issues...” Richard Gibbs, Wofford Cain Professor, Department of Molecular and Human Genetics; Director, Human Genome Sequencing Center, Baylor College of Medicine. (2012). Ethics and Practice of Whole Genome Sequencing in the Clinic. Presentation to PCSBI, February 2, 2012. Retrieved from http://bioethics. gov/cms/node/658.
In the case of whole genome sequencing, as is true for many emerging medical technologies, there are tensions between some of these principles. Two of the principles— public beneficence and intellectual freedom and responsibility— support the continued pursuit of whole genome sequencing research because of the promise of intellectual gains and substantial public benefit. Simultaneously, other principles—respect for persons, responsible stewardship, and justice and fairness—counsel the adoption of protections to minimize the privacy risks that could befall individuals. Drawing upon the process of democratic deliberation, the Commission sought to reconcile the potentially conflicting practical implications of these principles. It did so by taking into account various paths to the anticipated promise of this rapidly advancing technology, while respecting the ethical concerns of the increasing numbers of individuals facing the prospect of whole genome sequencing: concerns, for example, about confidentiality, information security, decisional autonomy, and freedom from unwanted intrusion into personal lives.
The Public Benefit of Whole Genome Sequencing Scientists predict that whole genome sequencing research will foster better understanding of the genetic factors that contribute to human health and diseases including cancer, heart disease, diabetes, and neuropsychiatric conditions, as well as many rare diseases. Further, whole genome sequencing is expected to usher in an era of personalized medicine, providing information that might allow clinicians to tailor treatments or manage the health of individuals based on their genomic profile. The Commission’s recommendations regarding the continued pursuit of whole genome sequencing to advance medical science are based primarily on the principles of public beneficence and intellectual freedom and responsibility. Public beneficence gives rise to a societal and governmental duty to promote individual activities and institutional practices, such as scientific and biomedical research, that have great potential to improve the public’s wellbeing.35 Public beneficence also supports scientific enterprises that advance the common good by increasing economic opportunities, a criterion that whole genome sequencing satisfies.36 The U.S. government invested billions of dollars in the Human Genome Project—a collaborative research project with the ambitious goal of sequencing the entire human genome. This
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investment has since generated $244 billion in personal income and $796 billion in overall economic impact.37 In 2010 alone, the human genome sequencing projects and associated research and industry activity directly and indirectly generated over 300,000 jobs and brought in tax revenue of $3.7 billion. While not unique to whole genome sequencing, increased economic productivity is often a positive by-product, consistent with public beneficence, of scientific and medical progress. Intellectual freedom grants scientists—acting responsibly—the right to use their creative abilities to advance science. Creative, sustained, and dedicated intellectual exploration is an essential aspect of scientific, technological, and clinical progress. At the same time, it serves to expand our general understanding of the world. However, both public beneficence and intellectual responsibility, the complement to intellectual freedom, caution against pressing forward with whole genome sequencing without regard to negative consequences. The principle of public beneficence requires both that public benefits be secured and that public harms be minimized. Likewise, intellectual responsibility calls upon all researchers and clinicians—including their staff and the institutions that support them—to adhere to the ideals of research, one component of which is avoidance of harm to others.38 Pursuing whole genome sequencing without considering potential harms would violate the clear and compelling mandates of public beneficence and intellectual responsibility.
Privacy Concerns Raised by Whole Genome Sequencing Respect for persons includes respect for the dignity and privacy of individuals. As a result, respect for privacy assumes special salience in discussions about ethics and genetics. Because whole genome sequence data provide important insights into the medical and related life prospects of individuals as well as their relatives (who most often did not consent to the sequencing procedure), whole genome sequencing poses real privacy concerns. These concerns are compounded by the fact that whole genome sequence data gathered now might reveal important information, entirely unanticipated and unplanned for, as science progresses. The potential power of the information contained in whole genome sequencing substantially raises the privacy stakes of medical information. “Public trust is fundamental to the ongoing support of these activities and to participant willingness to actually contribute to the research. And without the participant willingness to contribute to the research, we will not move forward at all.” Laura Lyman Rodriguez, Director of Office of Policy, Communications and Education, National Human Genome Research Institute. (2012). Presentation to PCSBI, August 1. Retrieved from http://bioethics.gov/cms/node/749.
Privacy and the Law Concerns about privacy are not new; worries about the proper boundaries between self, others, and government extend as far back in recorded human history as ancient Greece and Rome.39 The central role of privacy in U.S. culture and ethics is reflected in the tone of its laws. The word “privacy” does not appear in the U.S. Constitution. However, as American courts and scholars have observed, the Bill of Rights implicitly recognizes the value of privacy and
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rights of privacy through provisions guaranteeing: 1) freedom of speech, freedom of religious, political and personal association, and related forms of anonymity (First Amendment); 2) freedom from government appropriation of one’s home (Third Amendment); 3) freedom from unreasonable search and seizure of one’s body and property (Fourth Amendment); 4) freedom from compulsory self-incrimination (Fifth Amendment); 5) freedom from cruel and unusual punishment, including unnecessarily extreme deprivations of privacy (Eight Amendment); and 6) other personal freedoms (Ninth Amendment). In addition to the Bill of Rights, the Supreme Court and state courts have marshaled the due process clause and language of “liberty” of the Fourteenth Amendment to strike down laws interfering with autonomous medical, marital, sexual, and family decision making. “With advancing technologies it’s increasingly hard to keep secret our genetic information. There’s more data-sharing... but that doesn’t mean that we don’t have privacy interests here, it just means that we may need more explicit protections of those interests.” Sonia Suter, Law Professor at George Washington University. (2012). Presentation to PCSBI, August 1. Retrieved from http://bioethics.gov/cms/ node/748.
A number of U.S. states have explicit privacy protection provisions in their constitutions that apply to privacy violations by state and, in some cases, private entities. The common law of some states includes a breach of confidentiality tort. Most states recognize one or more right to privacy torts, first proposed in the 1890 article “The Right to Privacy” by Samuel Warren and Louis Brandeis. This seminal article persuasively argued that courts should recognize a “right to be let alone” against unwanted intrusion and publicity.40 Today personal injury suits can be brought alleging intrusion upon seclusion; publication of private facts; publication placing one in a false light; and appropriation of name, likeness, or identity. The United States takes a sectoral approach to regulating privacy, which means that the United States specifically regulates privacy concerns in particular settings as they arise. In the past four decades, in response to pervasive new technologies and related business practices, state and federal authorities have enacted many statutes and agency rules protecting the privacy of data related to health, education, finances, taxes, the federal census, video rentals, liedetection, motor vehicle records, library records, and electronic and telephonic communications. This sectoral approach means that a number of areas that have no specific laws currently do not receive even baseline privacy protections. By contrast, Europe regulates privacy comprehensively, providing privacy protections that are consistent across different types of data or information.41
The Meanings of Privacy Privacy and associated terms, including confidentiality, anonymity, choice, and data protection, refer to related concepts. Discussions about ethics and whole genome sequencing sometimes inappropriately use these terms interchangeably. To enable clear ethical analysis in this report, we provide basic definitions of the family of privacy terms applicable to our work and map their relationships. Scholars differ in their precise definitions of the terms we use, but the language we present in this report is consistent with a general consensus view. The following definitions are meant to show how the Commission uses these terms and to help
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guide future discussions regarding ethics and genetics. They should not be taken as formal arguments for precise definitions. Restricted Access The term privacy is used here (and in many ethical and legal contexts) broadly to mean states of affairs by virtue of which the accessibility of persons, personal information, or personal property is limited or restricted. What is valued as “personal,” “sensitive,” or “intimate” may be restricted by virtue of, for example, spatial distances, physical barriers, electronic passwords, social norms, or customs. In the United States and other developed societies, health information is widely considered personal, sensitive, or intimate, and genetic information especially so. The term informational privacy refers generically to restricted access to information or data. “Confidentiality,” “anonymity,” and “data protection” are specific ways to protect informational privacy in the broad sense, with special relevance in clinical and health research settings. Confidentiality is used to denote restricting access to information or data to groups of specifically authorized recipients. In the medical context, health information is often limited by custom to close family and friends and by law to health practitioners, insurers, and professional researchers. Patients and research participants may even choose to keep health conditions secret from intimate kin by deliberately concealing the information. Confidentiality is closely connected with trusting relationships. One can share private information with another person on the understanding that he or she can be trusted to keep that information secret (i.e., will not divulge it to others). Patients entrust clinicians with medical information provided that they have a “need to know,” and understanding that the clinician will keep the information confidential. In the context of whole genome sequencing, data must be kept confidential; databases must be secure and information must not be divulged to unauthorized users. Anonymity is used to denote restrictions on access to personally identifiable information pertaining to individuals or groups, achieved through intentionally disguising or removing identifiers. A health record can be made more anonymous, for example, by removing a patient’s name, address, or social security number. Data protection refers to measures designed to thwart deliberate or accidental disclosures of confidential or anonymous information. Health data that are electronically stored or transmitted can be protected with computer passwords and encryption. Health care providers employ technology to protect data, but ethical norms and business practices can also protect data from unauthorized access, use, and disclosure. Autonomy The term “privacy” has a second distinct use in ethics and law. Privacy is a rough synonym of autonomy with respect to self-regarding conduct and intimate relationships. Here, privacy denotes the absence of substantial government or other outside interference with individuals’ decisions and choices. In traditional bioethics, the “privacy” at issue in euthanasia, birth control, and consent to research is this second understanding of privacy, which involves the ability to make autonomous decisions. We note that there are other uses of “privacy,” some health-related, that do not play a major role in this report. Seeking greater precision and focus, privacy scholars and the courts commonly qualify the term “privacy” using descriptive adjectives. Indeed, they commonly
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speak of informational privacy in relationship to the collection, use, and sharing of information or data. They speak of physical privacy in relation to observing, concealing, and touching the human body, such as entering hospital rooms or respecting patient modesty. They refer to spatial, geographical, and locational privacy in relation to GPS and beeper technologies. They speak of associational privacy in relation to affiliation with like-minded people. They recognize decisional privacy in relation to independent decision-making. Less commonly, privacy scholars and the courts distinguish proprietary privacy in relation to repositories of personal identity and genetic ownership claims. And finally, they identify intellectual privacy in relation to interests in freedom of thought, conscience, and the right to read and access knowledge. There is ample debate and disagreement about the value of particular privacies and the basis for laws and policies promoting or regulating each type of privacy. In this report, the Commission focuses on informational and decisional privacy as they pertain to whole genome sequencing. We use the term “privacy” in reference to both limited access to genetic information and data, and to the absence of interference with decisions about the collection, use, and sharing of genetic information. A person whose whole genome is sequenced might have both decisional privacy concerns (about who is permitted to decide whether whole genome sequencing data are shared) and informational privacy concerns about whether such data will be shared in confidence, securely, or in de-identified form. Although the precise contours and content of privacy have changed substantially over time, with shifts in culture as well as technology, intense and widespread human interest in the protection of privacy is abiding, not only in the United States, but also around the world. Privacy protections promote a set of highly prized values. Although modern technology can facilitate unobserved and uninvited intrusions into homes, for example, what individuals choose to do in such a domain is generally valued as a matter of “privacy” and deemed legitimately “private,” unless that behavior violates particularly weighty ethical or legal limits. That is, there are constraints on what behavior can be considered legitimately private. In the inclusive understanding of what falls under the privacy umbrella we adopt here, what individuals choose to do at home is presumptively confidential, anonymous, intimate, secure, free from unwanted intrusion, and/or subject to decisional autonomy.42 Concern for privacy values (while additionally a means of enabling privacy at home and other vital privacies) also incorporates the increasingly elusive ideal of control over the flow of information regarding oneself, again subject to broad ethical and legal limits.43
The Value of Medical Privacy The Commission agrees that respect for patient and participant privacy can greatly benefit individuals and the general public. Under the principle of respect for persons, and for the sake of public beneficence and justice and fairness, those who collect, use, or share health data should employ practices that include confidentiality, anonymity, and informed consent to shelter clinical patients and research participants from the unwanted glare and control of others. It is important to ensure that respect for patient and participant privacy not be compromised, not only in clinical care and research, but also in the publication or archiving of medical lectures, scholarly articles, and personal papers. Medical privacy remains an important ethical principle, despite the recognition that many people voluntarily share their health information or data, including genetic information and data, and despite the practical reality that modern institutional practices presuppose that a great deal of sensitive health information can and will be lawfully shared among providers, insurers, researchers, and the government.
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Medical privacy has many varieties of recognized public value. First, medical privacy encourages individuals to seek medical care. Individuals will be more inclined to pursue medical attention if they believe they can do so on a confidential basis. Practicing confidentiality assures that, in most cases, a patient can choose when to disclose an illness, condition, or genetic status. Confidentiality and anonymity enable individuals to exercise constitutionally protected liberties of autonomous medical decision-making by safeguarding information they do not choose to share because it is embarrassing or would expose them to discrimination or disapprobation. “It just seemed safer to keep it to myself…I didn’t know what somebody would do with that information in the future…and I was very concerned about it.” Victoria Grove, introductory vignette, referring to her decision to keep secret her positive genetic test for alpha-1 antitrypsin deficiency.
Second, medical privacy encourages frank disclosures in clinical and research settings. Individuals seeking care can be open and honest if they can trust that facts reported to or uncovered by clinicians or researchers will not be broadcast to the world at large. People are often embarrassed by symptoms, histories, and prospects of illness. Individuals concerned about discrimination, shame, or stigma have an interest in controlling the flow of information about their health. Some patients and participants believe they own personal information about themselves, especially genetic information, and should be able to control its release. Third, if individuals believe they can decide whether to share data, information, and biospecimens under conditions of confidentiality, anonymity, and informed consent, they might be more likely to participate in research. In the context of health research, ethics committees and institutional review boards properly require researchers to protect the privacy of research participants and their medical records. Obligations of privacy may require the use of coded information rather than names or “de-identification” procedures such as data aggregation. Some have argued that researchers must publish genomic data in ways that obscure the identities of whole families. Even statistical use of individuals’ health data has raised privacy concerns, as some have argued that for cultural or social reasons individuals might have an ethical interest in the uses of data sets without personal identifiers that include data about them.44 Fourth, alleviating the concerns about exposure and discrimination that keep patients away from clinicians enhances confidentiality, which can further the goals of health care cost savings by ensuring that patients seek early medical care rather than waiting until their conditions worsen and require more dramatic medical intervention.45 The Commission recognizes that privacy, like most values, has ethical as well as practical limits. It is not an absolute public good. Certain diseases, conditions, and prescriptions must be reported to government to protect public health and safety. Health care providers and responsible adults are ethically obligated to report evidence of child neglect and abuse uncovered in treatment. Mental health providers have an ethical duty to warn police or potential victims of the credibly violent intentions of patients with mental illness. Situations arise in which medical confidentiality cannot be preserved because the media has a right to publish information or legal authorities have the authority to subpoena information for use in legal proceedings and investigations. Members of the military and civil servants serving in war zones may be also required to undergo mandatory genetic biobanking or testing for varied purposes.
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Privacy in Whole Genome Sequencing Currently, whole genome sequencing involves generating, storing, sharing, and analyzing large amounts of data. Although members of the public express general comfort with the idea of sharing genomic data in biorepositories, privacy ranks among participants’ highest concerns.46 Data also show that for many, privacy concerns are an important obstacle to participation in large cohort studies.47 Although 60 percent of people surveyed said they would participate in a study that involved storing data in biorepositories, 91 percent of those potential research participants would be concerned about privacy.48 Additional data indicate that although a large majority of survey participants trust clinicians and researchers, they are concerned that results of genetic tests could end up in the wrong hands and be used against them.49 Most of the people interviewed following enrollment in one sequencing study indicated that their primary concern was that they be informed if there was a possibility that their data would be shared with other researchers and that it was important they maintain some control over who could have access to their genomic data. The participants wanted insurance companies and employers to be excluded from access to these data, but were comfortable with data sharing within the research community.50 Informational and decisional privacy concerns about the unauthorized disclosure or misuse of whole genome sequence data are not only common and intensely important in the minds of potential research participants, they are also objectively linked to the potential for serious harms from such disclosure and misuse. Potential harms include the risk of lost opportunities in employment, long-term health care, disability and life insurance, loan approvals, education, sports eligibility, military accession, and adoption eligibility.51 In areas that are far less amenable to any legal protection or recourse, individuals could find themselves facing social stigma from disclosure of sensitive genomic information, and subsequent disruption of their home, family, and community life.52 Risks that are more internal to, and variable among, individuals include being subject to psychological harms upon learning information that can be difficult to bear, including that one has a predisposition to a disease such as cancer or Alzheimer’s disease. Because whole genome sequence information directly implicates relatives, psychological harms often are not limited to the person whose genome is voluntarily being sequenced and publicly disclosed. Even individuals who learn that they do not carry a harmful variant may experience “survivor’s guilt” if another family member is affected.53 To date, the number of documented cases of discrimination on the basis of genetic test results is small.54 This might be due to the relatively few conditions for which there are currently definitive genetic tests, coupled with the expense and difficulty of conducting these tests. As a result, genetic information is rarely available to third parties. Another reason for the small number of reported cases, now and potentially in the future, might be the difficulty of uncovering and documenting discriminatory use of data.55 It is also possible that such discrimination might not occur, either because there are other more definitive bases on which to make insurance or employment decisions, or because all individuals have some form of disease predispositions. Regardless, legitimate concerns remain about the potential for differential treatment of individuals based on their genomic information, even if legally prohibited discrimination rarely occurs. If individuals lack assurances against misuse of their genomic information, their privacy concerns might motivate them to not share their whole genome sequence data, which could harm the research enterprise that generates life-saving discoveries.
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Privacy and the Ethical Principles A robust set of ethical principles—respect for persons, responsible stewardship, and justice and fairness—supports the adoption of norms to minimize the privacy risks that could befall individuals while enabling research and clinical care for public benefit to continue. Respect for persons requires one to give great “weight to autonomous persons’ considered opinions and choices while refraining from obstructing their actions unless they are clearly detrimental to others.”56 Exercising autonomy includes self-determination, which requires that persons be allowed to make “important decisions about one’s life for oneself and according to one’s own values or conception of a good life.”57 Respect for persons highlights an individual’s autonomy and recognizes that we should respect individuals’ ability to decide for themselves what they value, and how and when to act on those values. For example, an autonomous person should be able to decide whether to undergo a medical procedure based on personal considerations of risks, benefits, costs, and cultural and religious views. Forcing an individual to undergo a procedure, even for their medical benefit, would violate that person’s autonomy and would fail to demonstrate respect for the individual as a person. Respect for persons also encompasses respect for the individual’s dignity and privacy. Therefore, violation of an individual’s privacy, such as the misuse or unauthorized disclosure of whole genome sequencing data, demonstrates a violation of the principle of respect for persons. Governments and societies that exercise responsible stewardship accept a duty to proceed prudently in promoting scientific advancement and emerging technologies. They recognize a shared duty to act in ways that demonstrate concern for all those who might be affected, and especially for those who are not in a position to represent themselves (e.g., children, the disenfranchised, vulnerable populations, and future generations). Rapidly advancing technologies such as whole genome sequencing present profound challenges for responsible stewardship because our understanding of the potential benefits and risks is largely incomplete and uncertain.58 This makes it important that governments and societies take great care not to make decisions that have a substantial chance of causing irreversible harm to current or future generations, and especially those who have little or no say over such decisions. Responsible stewardship advises against decisions that are entirely precautionary (no action without complete certainty of security) or entirely proactionary (no limitations on science). Heeding the principle of responsible stewardship therefore neither thwarts the development of new scientific enterprises nor lets science advance unchecked on the fallible assumption that it is safe. The principle of justice and fairness is, in important part, a commitment to ensuring that the unavoidable burdens of technological advances do not fall disproportionately on any individual or group, and that the benefits are widely distributed.59 The principle of justice and fairness encompasses the idea of fair distribution in that it demands society ensure that risks not be disproportionately borne by any particular group and strive for “the broadest distribution of beneficial technologies.”60 As such, the principle of justice and fairness entails protection for those who decide to share their whole genome sequence data to reduce the chances that they will be harmed by unauthorized disclosure or misuse. These three principles, taken together, suggest that individuals are entitled to privacy protections that prevent undue and disproportionate burden. But these protections are not absolute. Prohibiting all gathering and sharing of whole genome sequence data would protect privacy absolutely, but still would fail to adequately respect persons. A total prohibition prevents individuals from choosing to participate in whole genome sequence research, even if they consider themselves adequately protected; it also fails to take into account individuals’
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other interests, such as an interest in excellent medical care. Respect for persons demands respect both for individuals’ privacy and for their interest in benefitting themselves and others from medical advances. The Commission emphasizes that there is extremely good reason for individuals to choose to share information in a context where there is adequate protection for individual privacy: whole genome sequencing has the potential to be of substantial public benefit. The ability to share information is the sort of important decision that is central to autonomous action, which respect for persons commits us to recognize. Respect for persons supports giving persons the opportunity to share their whole genome sequence information for scientific advancement, subject to strong baseline privacy protections. At the same time, individuals have a responsibility to safeguard their privacy as well as that of others, by giving thoughtful consideration to how sharing their whole genome sequencing data in a public forum might expose them to unwanted incursions upon their privacy and that of their immediate relatives. To be indifferent to the implications of disclosure of sensitive data and information about one’s self is to act irresponsibly. That being said, it can be good and virtuous to share sensitive data about oneself in appropriate circumstances, for example, for the good of public health research or public education. To determine what baseline privacy protections should be, we need to distinguish between access to, use of, and possession of whole genome sequence data. To possess whole genome sequence data is to have a copy of the data file and, therefore, to have access to it at any time. Having access to data implies the ability to manipulate and work with the data files. It is possible to access data that one does not possess; a researcher might be allowed to access data files in a secure database to address research questions without keeping a copy of the data. One can have access to data even if one does not (and either ethically or legally cannot) use it, as when whole genome sequence data are stored on a server available to download, but one does not download them. The use of data refers to seeking answers to questions by analyzing the data. A researcher could use data in a protected database without having either access to or possession of the data by submitting a query to the database manager and then receiving the results of the query from the database manager. In these ways, it is possible to allow researchers to work with whole genome sequencing data through access to or use of the data while maintaining the security of the data themselves and protecting the privacy of the individuals who contributed to the database. The confidentiality of information or data about persons can be maintained through a number of means designed to prevent unauthorized access to the data: these means are collectively called informational security or data security. Examples of data security mechanisms include legal limitations, locked drawers, and computer firewalls. Presentations to this Commission indicated that whole genome sequence data could be used without actually possessing it: that is, technologies already are being developed to allow researchers to have limited computational access to select whole genome sequence data sets without physically transferring possession of all data files in the set.61 The researcher would be able to use the data for analysis, but would not maintain possession of the data. This means that possession of genomic information is neither necessary nor sufficient for its use. As with control of information, the use of information (including misuse and unauthorized use) in some cases will be of greater ethical salience than either access or possession.
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Reconciling Competing Ethical Claims The principles of public beneficence and intellectual freedom and responsibility support continued pursuit of whole genome sequencing to advance scientific understanding and medical progress. But these principles have components that suggest such pursuits should not be unrestrained. The positive argument for restraint is founded upon the principles of respect for persons, responsible stewardship, and justice and fairness, which together require implementing privacy protections and minimizing the chance of harm to individuals. But these principles do not suggest that privacy protections should erect absolute barriers to voluntary data sharing. In moving forward with whole genome sequencing, respect for persons requires informing individuals about the foreseeable consequences of their decision to share their genomic data, including who has access to their whole genome sequence data and how these data might be used in the future. Respect for persons also counsels individuals who collect samples to determine patient and research participant preferences at the time samples are obtained so that they can choose whether to participate, or whether feasible limits on the use of their whole genome sequence data can be agreed upon. Providing individuals who are choosing whether to share whole genome sequence data with the information necessary to make a fully informed decision about the potential consequences—including who can access the data and how the data will be used—allows individuals to make an autonomous decision. The principle of respect for persons applies to all whole genome sequence data regardless of whether they were obtained in a research or a clinical context. The Commission’s principle of regulatory parsimony calls for “only as much oversight as is truly necessary to ensure justice, fairness, security, and safety while pursing the public good.”62 Regulatory oversight is appropriate in certain contexts—for example, disallowing certain types of research or permitting other types of research only when certain conditions are met. But some aspects of research—including data security protections for whole genome sequence data—remain outside most regulatory frameworks. For otherwise unregulated aspects of research, informed consent is one mechanism by which individuals can protect their own privacy. By informing individuals about the potential risks and benefits of participation in whole genome sequencing, along with information about the security protections in place, individuals can autonomously choose whether to provide a biological sample for use in whole genome sequencing research. In this way, informed consent is one means of reconciling the public good that can come from whole genome sequencing with the potential harms to individual privacy. NEWBORN SCREENING In the case of Beleno v. Texas Department of State Health Services parents sued, claiming that the Texas Department of State Health Services collected and stored newborn blood samples, subsequently making them available for research purposes, without seeking parental consent. The parents argued that the lack of proper consent was a violation of privacy. The out-of-court settlement that was reached resulted in the destruction of 4 million similar specimens that had been collected without parental consent.
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Sources: Beleno v. Lakey, No. SA-09CA-188-FB (W.D. Tex. Sept. 17, 2009).; and Aaronson, B. (2010, December 8). Lawsuit alleges DSHS sold baby DNA samples. The Texas Tribune, TribBlog. Available at: http://www.texastribune. org/texas-state-agencies/departmentof-statehealth-services/lawsuitalleges-dshs-sold-baby-dna-samples/.
The Commission is also mindful of democratic deliberation, an approach to collaborative decision-making that embraces respectful debate of opposing views and active participation by citizens. Democratic deliberation warrants engaging the public and fostering dialogue among the scientific community, policy makers, and those concerned with the issues raised by whole genome sequencing.63 In this spirit, the Commission sought input from a broad range of voices, including members of the patient advocacy community calling for more participatory models of research and from researchers who feared further administrative burden. The principle of democratic deliberation acknowledges that while decisions (e.g., recommendations, policies, and guidance documents) must be reached in a timely manner, those decisions need not—and generally should not—be unalterable, particularly when relevant new information emerges. Modern societies change rapidly, especially in the domain of science and technology, and decisions in changing realms are best considered provisional rather than permanent. Researchers and clinicians must be particularly mindful of the deliberative value of provisionality, of being tentative or temporary, as whole genome sequencing moves from the realm of research and enters the broader clinical context.64 The transition is already raising new challenges, and the policies that were once created with the assumption that the research realm is clearly and cleanly separated from clinical contexts may no longer be either sustainable or desirable due to the reciprocal relationship that has developed between them. Clinical samples, stripped of identifiers and transferred to genomic databanks and biorepositories for broader use by researchers, contribute to the common good by making possible research that could not be done without large numbers of samples from which to generate data. Subsequently, medical benefits developed as a result of such research will be available to the broader population including the persons from whom the deidentified clinical samples were taken.
Conclusion The Belmont principles and the principles articulated by this Commission suggest ethically important and practically useful guidelines for whole genome sequencing. Chief among these is that the principle of respect for persons requires strong baseline protections for privacy and security of data, while public beneficence requires facilitating ample opportunities for data sharing and access to data by clinicians, researchers, and other authorized users. Respect for persons further requires that any collection and sharing of an individual’s data be based on a robust process of informed consent. The principle of responsible stewardship calls for oversight and management of whole genome sequence information by funders, managers, professional organizations, and others. The principle of intellectual freedom and responsibility provides further support for pursuing whole genome sequencing and seeking models for broad data sharing by promoting regulatory parsimony. Democratic deliberation is the foundation of the process that gave rise to this document, and others like it, and will continue to be the foundation moving forward. Democratic deliberation urges all parties to consider changes to policies and practices in light of the evolving science and its implications for enduring ethical values.
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Finally, the principle of justice and fairness requires that we seek to channel the benefits of whole genome sequencing to all who may potentially benefit, and ensure that the risks are not disproportionately borne by any particularly vulnerable or marginalized group.
SECTION 2. POLICY AND GOVERNANCE This section describes current policy and legal protections of genetic information and the ways in which genome sequence data are shared in the United States. There is no comprehensive federal law that protects genetic privacy. The Genetic Information Nondiscrimination Act (GINA) prohibits discrimination by employers and health insurers based on the results of genetic tests, but does not provide privacy protections. In addition, GINA does not address the complexity of large-scale genomic data. Many states have laws governing genetic information and some of these laws provide privacy protections, but the laws vary greatly from state to state. As a result, our laws lack the specificity required to encourage participation and secure public benefits from this emerging science, while still ensuring the protection of privacy. To gain the most benefit from recent innovations in whole genome sequencing, researchers need as much data as possible, derived from broad public participation in whole genome sequencing research. Widespread participation will be achieved only if participants trust the research enterprise and are comfortable that their privacy interests are protected. Currently, the patchwork of state and federal laws does not provide uniform protection of genomic data privacy. Protecting privacy interests of individuals requires a spectrum of conditions to be in place, including ethical and trustworthy behavior by researchers and clinicians, sufficient security of information technology, and policies and laws that hold violators accountable.
Privacy Concerns about Genetic and Whole Genome Sequence Data For as long as the nature of genetics and heritability has been understood, there have been concerns about misuse. During most of the 20th century, erroneous notions about genetics led to eugenic policies based on the idea that genetic “inferiority” should be eliminated. Since the launch of the Human Genome Project in 1990, scientific knowledge about genetic information has grown exponentially, especially in identifying genetic variations that cause disease. This new information has resulted in a heightened concern about privacy, and the implications of others knowing an individual’s genetic information. To draw meaningful conclusions and answer broad research questions, researchers aggregate and share whole genome sequence data from large numbers of individuals. To garner widespread participation in research and maintain trust in the enterprise, users and holders of whole genome sequence data must guide themselves according to at least three facets of privacy and confidentiality. The first facet, the individual, requires fostering ethical behavioral norms for researchers and clinicians. Participants, patients, and consumers must be assured that those who have contact with identifiable data intend to use them in an ethical manner—namely, only for those uses for which the participant, patient, or consumer has given consent. Many individuals trust researchers and medical professionals to consider their needs along with the
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greater good, despite substantial privacy concerns. A 2010 study of research participants’ views on genomic research indicated that, while individuals expressed concerns about privacy and data security, they also understood the value of sharing whole genome sequence information. Overall, concerns about privacy did not outweigh their sense of the importance of sharing genomic data in the interest of a larger social good.65 The second facet of privacy protection is information technology. Participants and patients must be assured that their data are secure. A 2006 survey queried the public’s wariness about health information technology systems and found that 80 percent of survey participants were concerned about identity theft and fraud, 77 percent about health information being used for marketing purposes, and 55 percent about health information being misused by insurers or employers.66 These concerns highlight the need for secure information technology systems tailored to sensitive biomedical information, including whole genome sequence data and information. These concerns build upon the need for fundamental trust in the ethical behavior of data users and in the security of the systems that store these data—participants and patients should be assured that they can rely on their consent to allow identified data to be used for certain purposes and not for others. The third facet of privacy protection is policy. Policy-level protection requires that systems be in place to provide clear institution-level expectations of training and preparation to handle whole genome sequencing data and information, to ensure an atmosphere of trust and an expectation of security, and to provide recourse should individual and information technology privacy protections fail. While rapid advancement of genomic science in the past decade has led to vast potential for valuable research and societal benefits through medical advances, privacy and confidentiality concerns persist. Without reliable protection from potential harms, perceived and real fears of privacy violation and discrimination could cause individuals to balk at sharing their whole genome sequence data, thus stifling scientific progress.
Current Sharing of Specimens and Whole Genome Sequence Data The past few years have seen the rise of sharing whole genome sequence data through biorepositories (facilities that store large numbers of physical biospecimens containing genetic material and associated data and information that researchers can access) and databases. Biorepositories are categorized generally into four groups: disease-specific (e.g., cancer databases); longitudinal population studies (e.g., the United Kingdom biorepository); isolated populations (e.g., the Faroe Islands); or twin registries, used to distinguish between genetic and non-genetic bases for disease.67 Biorepositories often have different missions and different governance structures and must reconcile the rights of individuals with potential societal benefit accordingly. Other organizations, such as academic institutions, government agencies, and private not-for-prof it entities, store data in databases—repositories that do not contain physical biospecimens, but rather electronic versions of genome sequence files. For many purposes, it is no longer necessary to maintain actual stored DNA from an individual once the genome sequence data have been collected, because it is easier to share electronic data files than physical specimens. Despite these differences, biorepositories and their associated databases share some commonalities. The collection of specimens and data and subsequent storage in biorepositories
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and databases give rise to risks that might include minor harm to the donor in obtaining the biospecimen (such as bruising upon blood withdrawal); nonphysical harms such as discrimination, stigmatization, and untoward psychological impact upon discovering unwelcome information; group harms, like those incurred by the Havasupai; and ethical harms that arise when individuals are not treated with respect and dignity.68 Various laws and regulations govern the ways that these data currently are collected, shared, and used in the United States and around the world.
U.S. Federal Agency Activity In order to inform this report, the Commission sought information about human whole genome sequencing research sponsored by the 18 U.S. Common Rule agencies, and related privacy protections of the data generated in the research they sponsor (see Table 1). The Commission supplemented these responses with publicly available information.69 Twelve of the responding agencies stated that they do not conduct research involving human genomics, have not advocated formally for policy changes, and do not anticipate policy changes related to genomics.70 Table 1. Human genomics research in federal common rule agencies
Department/Agency Agency for International Development (USAID) Central Intelligence Agency (CIA) Consumer Product Safety Commission (CPSC) Department of Agriculture (USDA) Department of Commerce (DOC) Department of Defense (DOD) Department of Education (ED) Department of Energy (DOE) Department of Health and Human Services (HHS) Department of Homeland Security (DHS) Department of Housing and Urban Development (HUD) Department of Justice (DOJ) Department of Transportation (DOT) Department of Veteran Affairs (VA) Environmental Protection Agency (EPA) National Aeronautics and Space Administration (NASA) National Science Foundation (NSF) Social Security Administration (SSA)
Conducts/ Sponsors Research Involving Human Genomics No No No No No Yes No No Yes Yes No Yes Yes Yes No No No No
Anticipates Proposing New Policies No No No Yes No Yes No No Yes No No No No As needed No As needed No No
Six agencies—the Department of Homeland Security (DHS), the Department of Defense (DOD), the Department of Justice (DOJ), the Department of Health and Human Services (HHS), the Department of Veterans Affairs (VA), and the Department of Transportation (DOT)—currently sponsor genetic and/or genomic studies, and five maintain or support biorepositories and databases.71 The confidentiality, privacy, and security of samples and data stored by federal agencies are governed by a baseline of laws and regulations, including the
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Health Information Technology for Economic and Clinical Health (HITECH) Act, the EGovernment Act, the Federal Information Security Management Act, the Health Insurance Portability and Accountability Act (HIPAA), the Privacy Act, and the Policy for Privacy Act Implementation and Breach Notification.72 Several agencies have additional mission or function-specific policies that govern the entities they fund that perform whole genome sequencing studies. DOD uses large-scale genomic data in the DNA Dog Tag program, a mandatory program that has collected and stored blood and tissue samples from every member of the Armed Forces since 1991. The program does not give service members the opportunity to opt out of this collection. DNA is extracted from the samples only if needed to assist in identifying human remains. Specimens stored in the repository are not used for any other purpose unless approved by the Assistant Secretary of Defense for Health Affairs. DOD has several policies for protecting and securing genetic information that address disclosure, medical records, and information systems.73 DOD expects to increase the use of whole genome sequencing for forensic applications related to human remains identification.74 Agencies within HHS routinely use or sponsor whole genome sequencing. The confidentiality and security of samples and data used by HHS are covered both by HHS-wide and agency-specific policies, laws, and regulations.75 For example, one HHS agency, the Centers for Disease Control and Prevention coordinates efforts to conduct whole genome sequencing of residual dried blood spots archived by states after newborn screening with parental consent.76 The Centers for Disease Control and Prevention also collects DNA specimens for its National Health and Nutrition Examination Survey, and the confidentiality of identifiable information collected is protected under the Public Health Service Act.77 Another HHS agency, the National Institutes of Health (NIH), devotes resources to studying the influence of genetic factors on human health and illness. NIH has established a number of genetic data repositories, most notably the database of Genotypes and Phenotypes (dbGaP).78 dbGaP stores various types of genetic information, including whole genome sequence data. Access to data stored in dbGaP is two-tiered: open access, which grants the public access to information about study design and aggregate phenotypic information; and controlled access, which grants researchers access to information including de-identified genotypes and phenotypes of individual study participants.79 Researchers who seek controlled access must submit formal research requests that are reviewed and approved by NIH Data Access Committees.80 NIH has implemented policies and procedures to which every researcher with access to dbGaP must adhere to protect the privacy and confidentiality of genetic and, specifically, whole genome sequence data.81 The Combined DNA Index System (CODIS) is a DNA database funded by the Federal Bureau of Investigation (FBI), a Department of Justice agency. CODIS consists of DNA profiles from the Convicted Offender Index, the Forensic Index, the Arrestee Index, the Missing or Unidentified Persons Index, and the Missing Persons Reference Index. The National DNA Index contains almost 11 million offender profiles.82 CODIS does not contain personally identifiable information, nor does it contain whole genome sequence data. To further protect the data in CODIS, access to computers containing CODIS software is limited to authorized users approved by the FBI. Unauthorized disclosure of DNA data in the National DNA database is subject to a criminal penalty.83 DHS uses genetic data, but not whole genome sequence data, in several ways. DHS collects DNA from individuals who are arrested, facing charges, or convicted of federal or military
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crimes. DHS also collects DNA from non-U.S. citizens who are detained under the authority of the United States. U.S. Citizen and Immigration Services can require genetic testing to establish familial relationships to determine immigration or refugee status. Finally, DHS is piloting a program for overseas refugees who request asylum for family members; refugees can be asked to voluntarily undergo familial relationship testing using a portable DNA testing device. DHS does not generally maintain or have access to the genetic information it collects from individuals; it sends DNA samples to the Department of Justice for processing and entry into CODIS.84 VA has active research and clinical genomics programs. In 2012, VA launched the Million Veteran Program, which aims to collect one million biospecimens from veterans to explore the role of genes in health and disease.85 VA treats genomic data as personally identifiable medical information protected under HIPA A, although it stores the biospecimens securely and without other traditional identifiers such as name. VA has applied for a Certificate of Confidentiality from NIH, and has several additional departmental policies to protect the privacy of identifiable medical information.86 The Million Veteran Program database is accessible only to authorized researchers for projects that have been approved by appropriate VA oversight committees.87 The Federal Aviation Administration, a Department of Transportation agency, is researching human factors related to aviation safety from a gene expression viewpoint (gene expression is the process by which genes are translated into proteins).88 Specifically, the Federal Aviation Administration is researching how alcohol use, fatigue, and cosmic radiation change gene expression and is correlating changes in gene expression to human performance to improve aviation safety. The Federal Aviation Administration’s intent is to have unique sets of molecular markers for these factors that are generally applicable across the broad human genetic spectrum with a high degree of specificity. Genetic data collected by the Department of Transportation are subject to a number of federal data security policies. INTERNATIONAL BIOREPOSITORIES While the United States has many publicly funded biorepositories of limited size, a number of countries have implemented or attempted to implement population-wide biorepositories. In the United Kingdom, for example, a half million volunteers are being recruited to donate genetic material to be linked to medical records in a biobank. The biobank will obtain informed consent from its participants and will allow for withdrawal from the database. Participants can request: 1) complete withdrawal and destruction of existing samples, 2) discontinued participation but continued use of existing data, or 3) no further contact, but continued use of existing data. Source: UK Biobank [website]. Retrieved from http://www.ukbiobank.ac.uk/.
Commercial Genetic Testing Companies Over the past few years, accessibility and availability of commercial genetic testing and genotyping has greatly expanded. Companies like 23andMe, Navigenics, and Ancestry DNA provide an array of services including paternity testing, testing for predisposition to certain diseases and traits, genealogy and ancestry information, pharmacogenomics (the influence of genomic factors on drug response), and even private forensic tests to establish profiles of suspects not included in the federal CODIS database.89 Most commercial genetic testing
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companies currently do not conduct whole genome sequencing. Instead, they analyze hundreds of thousands of single-nucleotide polymorphisms (SNPs) or discrete variants throughout the genome, which they describe as “genotyping.”90 Commercial genetic testing companies often conduct research that uses biospecimens submitted by their customers. Most recently, 23andMe patented one of their research discoveries, “polymorphisms associated with Parkinson’s disease.”91 Commercial entities face issues of data maintenance and storage similar to those of government-sponsored biorepositories and databases. They collect and analyze genetic and genotypic data and maintain electronic databases of consumer data. In addition, many commercial genetic testing companies have a research arm that conducts research on consumer data in biorepositories. Currently, there are no overarching federal or industry guidelines indicating how commercial genetic testing companies should operate, what privacy controls they should implement, or what limits they should put on the use of genetic data and information. Like government-sponsored biorepositories and databases, they can protect consumers by developing systems to promote ethical and trustworthy behavior of employees, strengthening the security of information technology systems, and developing company policies that hold violators accountable.
Privacy Regulations Individuals who share their genomic information, like those who share any medical data, accept risks to their privacy and confidentiality should the data be improperly shared or used. Rather than a broad framework that provides general privacy protections, the United States has developed a patchwork of subject-specific regulations to protect the privacy of different types of information.92 This system of subject-specific regulations includes, for example, regulations that protect census data, financial information, medical records, and video rental records, but does not include regulations that protect personally identifiable information that is not financial or medical, including name, address, occupation, affiliations, or internet activity.93 As a matter of respect for persons as well as justice and fairness, a government can institute laws and regulations that help mitigate risks to individuals who share whole genome sequence data, and it can protect individuals from unwillingly or unwittingly sharing their whole genome sequence data. But it cannot eliminate all privacy risks while still effectively encouraging scientific, economic, and social progress. Just as the Commission strongly supports effective protections of privacy, it also emphasizes that sharing whole genome sequence data for the sake of medical research holds great potential for public benefit. The principle of public beneficence strongly encourages this sharing in a setting that provides adequate protections of privacy.
U.S. Privacy Regulations The collection and protection of personally identifiable information is not new. The United States has collected personally identifiable information through the census and the tax systems since its early history. The government has recognized the importance of keeping this information secure and has implemented protections to ensure the privacy and security of these data.94 Privacy laws and regulations permit but regulate cross-agency matching of collected data, and establish precedent that personal data shared by an individual for one specific purpose
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should not be used to other ends, such as law enforcement or judicial proceedings, without their consent. In addition, traditional identifying information often is removed from the data files.95 The United States has made several sectoral legislative attempts to regulate the privacy and security of personal data. These laws include the Fair Credit Reporting Act; the Privacy Act of 1974; the Confidentiality of Alcohol and Drug Abuse Act; the Family Educational Rights and Privacy Act of 1974; the Electronic Communications Privacy Act of 1986; the Video Privacy Act of 1988; the Children’s Online Privacy Protection Act of 1998; and the Gramm-LeachBliley Act of 1999, also known as the Financial Services Modernization Act (requiring financial institutions to protect consumer privacy).96 The laws cited above generally comport with “fair information practice” principles and practices first set forth in the Department of Health, Education, and Welfare (the precursor of HHS and the Department of Education) report, “Records, Computers and the Rights of Citizens.”97 The practices include the following principles: 1) there must be no personal data record-keeping systems whose very existence is secret; 2) individuals must be able to find out what personal information about them is in a record and how it is used; 3) individuals must be able to prevent information obtained for one purpose from being used for other purposes without consent; 4) individuals must be able to correct or amend a record of identifiable information; and 5) any organization creating, maintaining, using, or disseminating records of identifiable personal data must assure the reliability of the data for their intended use and must take reasonable precautions to prevent misuse of the data.98 HIPA A, enacted in 1996, is the federal law most relevant to medical privacy.99 Pursuant to the authority of Title II, HIPA A sets forth policies, procedures, and guidelines for maintaining the privacy and security of personally identifiable health information.
Figure 2. U.S. Federal Privacy Laws.
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The HIPA A-mandated Privacy Rule was finalized in 2005. The Privacy Rule defines the circumstances in which an individual’s protected health information—including any identifiable in format ion — may be used or disclosed by a covered entity.100 A covered entity is a health plan, a health care clearinghouse, or a health care provider that transmits any health information in electronic form.101 Under HIPA A, health information is not “identifiable” if there is “no reasonable basis to believe that the information can be used to identify an individual” or if it is stripped of the HIPAA identifiers.102 An individual’s privacy rights under the Privacy Rule survive death.103 While it is clear that genetic information is health information under HIPA A, HHS has stated that it is only covered by the Privacy Rule to the extent that it meets the definition of protected health information.104 HHS has not clarified whether genetic or genomic information on its own is protected health information—that is, whether it falls under one of the HIPAA identifiers, such as “biometric identifier” or “any other unique identifying number, characteristic, or code.”105 IDENTIFYING INFORMATION UNDER HIPAA Names Address Dates Phone numbers Fax numbers Email addresses Social security numbers Medical record numbers Health plan beneficiary numbers Account numbers Certificate/license numbers Vehicle identifiers Device identifiers and serial numbers Web URLs Internet protocol (IP) addresses Biometric identifiers, including finger and voice prints Full face photographic images and any comparable images Any other unique identifying number, characteristic, or code (with certain exceptions)
A covered entity must disclose an individual’s protected health information to him or her when specifically requested, and to HHS in the event of a compliance investigation or enforcement action.106 A covered entity may disclose protected health information without consent in specifically enumerated circumstances, including for purposes related to treatment, payment, public health, and health care operations. A covered entity that discloses protected health information, however, must try to disclose only the minimum necessary to achieve its purpose.107 There are no restrictions on the use or disclosure of de-identified health information, which is information that neither identifies nor provides a reasonable basis with which to identify an individual.108
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HITECH updated and revised HIPAA to extend slightly its privacy protections. HITECH adds business associates of covered entities to the list of those who can be subject to liability for disclosure of protected health information. It also strengthens the accounting requirements for the protection of health information, and imposes new notification requirements for covered entities to comply with when a breach has occurred.109 The Office of the National Coordinator for Health Information Technology was created in 2004 through an Executive Order, and legislatively mandated in the HITECH Act. Its mission is to coordinate nationwide efforts to implement and use the most advanced health information technology and the electronic exchange of health information.110 While the requirements of HIPAA and HITECH apply only to “covered entities,” most academic institutions and federal agencies are required to follow the rules set forth for human research under the Common Rule. The Common Rule is a federal regulation governing human research in the United States that requires federally funded scientific research to be subjected to independent review by an institutional review board (IRB), have equitable subject selection, use procedures consistent with sound research design, minimize risks to participants, and obtain informed consent. Informed consent by participants must generally include, among other things, a description of the procedures in the research plan, an explanation of the risks and benefits to the participant, a description of the extent to which confidentiality of records will be maintained, and an explanation of the right to withdraw from the study.111 Currently, whole genome sequence data obtained in the clinical context can be stripped of traditional identifiers and used for research purposes without IRB review or additional consent. This is because whole genome sequence data, when stripped of traditional identifiers (such as name or address), are not considered readily identifiable under the Common Rule.112 The logic behind this is that while whole genome sequence data are unique to an individual, without a key that matches particular data to an identity, one could not readily ascertain which person the whole genome identifies. Similarly, while fingerprints are considered identifiable for law enforcement purposes, a fingerprint with no personal identifying information cannot point to whom that fingerprint belongs. In other words, a fingerprint does not have a name or address encoded directly in it. To discover the suspect’s identity, one must link the print to a database containing both traditional personal identifiers and fingerprints in order to know which person to arrest. Only research that uses data where the identity of the subject is, or may readily be, determined is considered human research under the Common Rule. Research using data stripped of traditional identifiers is not considered human research and therefore does not trigger Common Rule protections such as IRB review or consent. Research using whole genome sequence data that have not been stripped of traditional identifiers (e.g., readily identifiable information) is considered human research. Accordingly, this research is governed by the Common Rule, meaning that IRB approval and informed consent must be obtained or waived by an IRB before the research can occur. HHS recently published an Advanced Notice of Proposed Rulemaking (ANPRM), entitled Human Subjects Research Protections: Enhancing Protections for Research Subjects and Reducing Burden, Delay, and Ambiguity for Investigators, and collected comments on whether some types of genomic data should be considered identifiable. This ANPRM acknowledges that “there is an increasing belief that what constitutes ‘identifiable’ and ‘de-identified’ data is fluid” and that evolving technologies and the increasing accessibility of data could allow deidentified data to become re-identified.113 It also highlights the concern that “advances that have come in genetic and information technologies” might “make complete de-identification of
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biospecimens impossible and re-identification of sensitive health data easier.”114 This is an ongoing discussion. A change to the Common Rule pertaining to identifiability could impact the collection and subsequent use of whole genome sequence data.
International Approaches to Regulating Genetic Information The United States is not the only country deciding how best to prevent the misuse of genetic information. No international models yet exist regarding the misuse of and specific protections for whole genome sequence data. Some countries have enacted general privacy laws that encompass personal health information; patient rights’ acts that regulate, among other things, informed consent and confidentiality of medical information; and legislation that specifically regulates genetic information and genetic research. These laws differ from U.S. law, which is focused on prohibiting discrimination resulting from disclosure of genetic information rather than ensuring privacy of genetic information. Many countries and foreign bodies have broad laws that regulate the use of personal information.115 Some of these, such as the European Union’s Data Protection Directive, offer special protection for more sensitive data, including personal health information.116 These privacy laws are far reaching—covering private and public institutions and many types of data—and are often overseen by data commissions or commissioners.117 In addition to these general data protection laws, many countries also have enacted patient rights’ laws that prohibit discrimination and require confidentiality of patients’ health information. These laws often require informed consent for disclosure of personal health information.118 Some of these laws, like those in the United States, also require that patients have access to their own medical records.119 In recent years, some countries have enacted laws specifically regulating genetic information and research. For example, Chile enacted a law in 2006 regulating genetic research that prohibits discrimination on the basis of genetic heritage and requires informed consent for research, confidentiality of genetic information, and anonymization of genetic data.120 Some of these laws allow genetic testing only for individual health reasons or scientific research.121
Legal Protections of Genetic and Whole Genome Sequence Data In light of mounting concerns about genetic privacy at the onset of the Human Genome Project, the U.S. Congress adopted legislation protecting against genetic discrimination. In 2008, Congress passed GINA, which aims to prevent genetic discrimination in the health insurance market (Title I) and in employment decisions such as hiring, firing, job assignments, and promotions (Title II).122 GINA does not protect against discrimination in the context of life insurance, disability insurance, or long-term care insurance. GINA’s protections apply to asymptomatic individuals, not those who have “manifested disease.”123 Nor does it prescribe rules for genetic research.124 GINA also expanded HIPA A privacy protections by applying prohibitions against genetic discrimination to all health insurers.125
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“There is certainly more room for legislation about privacy… the Genetic Information Nondiscrimination Act…is only a start. There are many more protections that the patient community would like that are not present in GINA.” Greg Biggers, Council Member, Genetic Alliance; Chief Executive Officer, Genomera. (2012). Genomic Privacy, Data Access, and Health IT. Presentation to PCSBI, May 17, 2012. Retrieved from http://bioethics.gov/cms/node/713.
Under Title I of GINA, all health insurers are barred from: 1) using genetic information to determine coverage, eligibility, or premiums; 2) requesting or requiring genetic testing or genetic information for underwriting decisions; and 3) obtaining genetic information for underwriting purposes.126 Additionally, insurers may not, on the basis of genetic information, impose a preexisting condition exclusion.127 GINA extended HIPAA protections to cover persons purchasing individual, rather than group, health insurance policies.128 GINA substantially expanded protections from genetic discrimination in employment. Under Title II of GINA, an employer with more than 15 employees cannot use an individual’s genetic information when making employment decisions such as hiring, firing, job assignments, and promotions, nor can an employer request, require, or purchase genetic information about an individual employee or family member. Although GINA prohibits specific types of misuse of genetic information by health insurers and employers, it does not address the use of or access to genetic data. In other words, GINA is an anti-discrimination law; it does not provide comprehensive privacy protections. GINA provides a uniform federal law as a floor of protections against genetic discrimination, but also allows for state laws that provide additional safeguards.129 Slightly fewer than half of all U.S. states have laws providing additional protection against discrimination in aspects of life, long-term care, or disability insurance not present in GINA.130 About half of the U.S. states have policies governing genetic privacy. There is a great degree of variation, however, in what protections states afford their citizens regarding the collection and use of genetic data and, similar to the federal level, none have specific prohibitions for whole genome sequence data. Some states protect against the improper collection of genetic material without consent.131 Others protect against the improper disclosure of genetic information (and several of these states’ laws do not specify to whom the disclosure is prohibited, whether to the donor or another party).132 Still others protect against improper retention of genetic information without consent.133 The result of the variation in state laws is that there is no standard or comprehensive approach to the protection of genetic information in the United States, and the level of protection afforded to an individual’s genetic information differs widely from state to state (for more information regarding the diversity of state law genetic protections, see Table 2 and Appendix IV: U.S. State Genetic Laws). The U.S. Supreme Court has not established a constitutional right to informational privacy applicable to whole genome sequence data. Although the Supreme Court has addressed privacy rights of biomedical information in the context of the Fourth and Fourteenth Amendments, there is no case law addressing informational privacy in the context of whole genome sequencing.134 Legal protections might be afforded if individuals have state property rights over their biospecimens, though courts have generally favored scientists over individuals from whom the specimen was taken.135 The most famous case is Moore v. Regents of the University of
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California in which the Supreme Court of California held that individuals are entitled to informed consent, but do not maintain property or privacy rights over cells after they have been removed from their body.136 Table 2. Examples of State Genetic Privacy Laws State Arizona AZ Rev. Stat. §122801-4, §20-448.02; 21 Oklahoma Okl. St. § 1175 Hawaii HRS §§ 431:10A-118 Missouri § 375.1309 R.S.Mo. Rhode Island R.I. Gen. Laws §27-1852, 52.3, §27-19-44, 44.1, §27-20-39. 39.1, §27-41-53, 53.1 Vermont V.T. Stat. Ann. tit. 18, §9331 to 9335 Wyoming Wyo. Stat. Ann. §14-2701 to 710 Michigan Mich. Comp. Laws §§ 333.17020, 333.17520
Protections Requires informed consent for genetic testing performed by health care providers, but does not address whether a non-health care provider may collect or analyze genetic material. Provides privacy protections for genetic information obtained from newborns, but does not provide similar protections for adult genetic data. Prohibits disclosure of genetic information by insurers, but does not specify the same for health care providers, nor does it protect against unwanted analysis of genetic material. Prohibits the disclosure of genetic information by persons who hold such information “in the course of business,” but does not address persons who have obtained it for any other reason. Prohibits the unauthorized disclosure of genetic information by insurance companies, but does not prohibit unauthorized disclosure by anyone else who may have access to genetic information.
Prohibits people from performing genetic tests without consent and from disclosing the results of genetic tests without consent, but does not regulate the unauthorized obtaining or retention of genetic information. Prohibits the disclosure of genetic tests for paternity without consent, but does not address any other kinds of genetic tests. Prohibits the performing of a genetic test by a health care provider without consent, but does not address performance of genetic tests by any other party, and does not prohibit unauthorized obtaining, retaining, or disclosure of genetic tests by any party.
State contract law also may provide legal protection if an individual has signed an informed consent document. In the context of genetic databases, researchers and participants can contractually determine who can access or use the data and on what terms, and the penalties for misusing protected information.
Conclusion There is considerable concern in modern society about unauthorized or unintended disclosures of genetic information. While GINA prohibits genetic discrimination in the health insurance and employment contexts, it does not regulate use, access, security, or disclosure of genetic data, and does not specifically address whole genome sequence data or information. State-based privacy laws, consent forms, and IRBs collectively create a patchwork of privacy protections, but they neither comprehensively nor consistently protect the whole genome sequence data of individuals. In an era in which genomic data increasingly are stored and shared using biorepositories and genetic databases, there is little to no systematic oversight of these facilities.
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To address the complex privacy and data security issues that arise in this arena, we need each of three robust facets of privacy and confidentiality protections of whole genome sequence data: individual researchers and clinicians; information technology systems; and laws, regulations, and institutional policies. Protection of personally identifiable data requires attention at all three broad facets of responsibilities. Individuals who collect, handle, store, and use data must recognize the ethical imperative of protecting the privacy of persons from whom they collect data. The information technology systems should be designed to protect persons by prohibiting the unauthorized access and release (intentional or unintentional) of identifiable data and protecting databases from intrusion. Laws and policies must protect persons from negative consequences of disclosure of information (e.g., discrimination) as well as enforce accountability and consequences for unauthorized access or disclosure. It is clear that laws and regulations cannot do all of the work necessary to provide sufficient privacy protections for whole genome sequence data. Together with laws and regulations preventing misuse of data, individuals who receive such data have professional ethical obligations to protect the data that go beyond the limitations of the legal protections. Moreover, given how rapidly whole genome sequence technology is changing, it is in some ways preferable to adopt professional guidelines and policies rather than enact additional laws, since professional guidelines and policies are updated far more easily.137 Guidelines and policies also might help those affected consider more deeply the privacy considerations at issue rather than focusing entirely on compliance with the letter of the law.138
SECTION 3. ANALYSIS AND RECOMMENDATIONS For this report on whole genome sequencing, the Commission has been mindful of the five ethical principles set out in its first report, as described in detail in Section 1. These principles for assessing emerging biotechnologies are public beneficence, responsible stewardship, intellectual freedom and responsibility, democratic deliberation, and justice and fairness. The Commission’s principles complement and build upon the Belmont principles of respect for persons, beneficence, and justice. The Commission drew on both sets of principles to develop recommendations that facilitate responsible development of, access to, and use of whole genome sequencing. The Commission focused on the principle of respect for persons by seeking to minimize risks to individuals willing to share their whole genome sequence data. Although individual benefits of whole genome sequencing are emerging, they are more elusive than predicted a decade ago. Many of the benefits anticipated from advances in whole genome sequence research will accrue to society generally through, for example, improved diagnosis and public health resulting from efficient medical treatment. Related privacy risks, however, primarily fall to individuals willing to share their genomic information. Risks might also fall to blood relatives of these individuals who carry similar genomic variants, thereby raising the stakes of privacy concerns in whole genome sequencing compared with most other types of research. Strong privacy protections enable individuals to determine autonomously their preferred level of data and information sharing. When individuals have control and can govern sharing of their data at a level with which they are comfortable, they are more likely to have trust in the research or clinical enterprise, and are more likely to participate and share data, benefiting
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society generally. These privacy interests are served by robust informed consent, data security provisions, and systematic oversight. With the above in mind, the Commission identified the following areas for ethical analysis:
Standards that allow individuals, if they wish, to access and share their whole genome sequence data and information; Security of whole genome sequence data and information and standards of access to and use of whole genome sequence databases; Informed consent to whole genome sequencing in the contexts of clinical care and research; Oversight of collection, storage, access, and use of whole genome sequence data and information; and Distribution of benefits from medical advances resulting from whole genome sequencing.
The recommendations presented in this section apply to individuals and entities that have an interest in, and work with, whole genome sequence data and information, both in the public and private sectors. Whole genome sequence data collected in the clinical setting are indistinguishable from whole genome sequence data collected in the course of research, and data increasingly move back and forth between the clinical and research settings. Ethical principles providing guidance in this area are based on a shared morality. While the implementation of recommendations that follow might be different depending on the entity involved in the collection, storage, access, and use of whole genome sequence data, the ethical issues at stake are the same. The Commission’s recommendations are also based on the fact that whole genome sequence data are inherently unique, meaning there is only one person in the world with that specific sequence. If the identity of the donor is not apparent to the user of the data, that individual is not readily identifiable. However, whole genome sequence data are often most useful when linked with information about physical characteristics, environmental factors, and medical records. These additional pieces of information, in turn, might make whole genome sequence data readily identifiable. The Commission sees promise in the application of information technology to the field of whole genome sequencing. Information technology is able to tailor access to data with a degree of specificity not possible with traditional medical records, potentially making all types of whole genome sequence data more secure. Uses of whole genome sequence data are rapidly evolving, and some of these uses do not fit easily into the current regulatory framework. The Commission, therefore, has crafted its recommendations to call attention to areas where it believes that current laws, regulations, and policies need to be reconsidered to honor applicable ethical principles and ensure that whole genome sequencing is most effectively used to the benefit of society and its individuals.
Strong Baseline Protections While Promoting Data Access and Sharing Whole genome sequencing increasingly is being incorporated into clinical care and research. Presently, numerous national and state policies are in place to guard personally
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identifiable health information and records of participation in research.139 These policies should apply to all handlers of the data, from those who collect the data, to researchers, to third-party storage and analysis providers.140 Privacy protection is an essential component of oversight of the use of whole genome sequencing in research and clinical care. Privacy protections should guard against unauthorized access to, and illegitimate uses of, data and information while allowing for authorized users of these data to advance public health. For both ethical and practical purposes, it is important to carefully distinguish between access to, use of, and possession of whole genome sequence data. Access means being able to come in contact with the information, whether physically or electronically. It would be impossible to limit physical access to all sources of whole genome sequence data. We leave behind specimens containing our DNA in myriad public places—by discarding a coffee cup, for example—that could be used to perform whole genome sequencing. (It is more feasible of course to protect electronic access to whole genome sequence data in biorepositories and databases.) While individuals might have abandoned these genomic samples to public access, they nonetheless have a strong interest in whether the data they contain are collected and how they are used. On the other hand, sometimes persons might have authorized access to whole genome sequence data but misuse the information (e.g., by sharing information with a reporter). In certain cases, others simply have no right to know certain things about other people, no matter what they do with the information.141 Unauthorized access to data is not necessarily a problem in and of itself— despite having access to information, one can choose to not use it, and thereby not produce any harm. Misuse of information can therefore be more ethically significant than unauthorized access. Laws and regulations can prohibit unauthorized parties from accessing or misusing whole genome sequence data, but it is impossible to guarantee that this will not occur. Laws and regulations can, however, provide deterrents to inappropriate access or misuse (such as fines), and compensation for the individuals whose data have been inappropriately accessed or misused. Presentations to the Commission also indicated that authorized individuals can use data without having actual possession of those data.142 Technologies are being developed that allow “computational” access to data sets, which allow access and use without the user possessing the data set. In computational access, the data are possessed by a central party, but others can remotely perform analyses (i.e., use) of the data. AN EXAMPLE OF COMPUTATIONAL ACCESS Google, a major internet search engine, has collected data from its customers’ internet activity. Google views these data as a commercial asset and does not share possession of them. However, Google tools, such as Google Correlate and Google Trends, allow users to query Google’s collected data. A user can search for “stapler” to ascertain whether stapler and staple sales correlate, but users receive only the answer to their question (not access to the data mined by Google yielding the result). By using computational access, Google can give users access to answers, but not access to the data. Source: Google. (n.d.). Google Trends. Retrieved from http://www.google.com/ trends/.
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Developments in the science of whole genome sequencing, which are progressing quickly, will require ongoing ethical consideration and democratic deliberation. Individuals and groups have differing sensibilities toward the privacy and publicity of whole genome sequence data, which might be relevant to distinguishing between acceptable and unacceptable uses of data. Perceived misuses of whole genome sequence data vary between cultures and individuals. For example, some individuals might be open to having a secondary researcher use his or her whole genome sequence data for an ancestry study. Members of the Havasupai tribe, on the other hand, strongly disapproved of their samples being used in ancestry studies, because these studies contradicted their traditional origin beliefs.143 Some parents do not object to using Guthrie card newborn blood screening spots in future research without consent. Notable lawsuits in Minnesota and Texas, however, have indicated that some parents feel otherwise.144 Requiring consent for future uses of readily identifiable whole genome sequence data, and encouraging consent for future uses of any data, are important to appropriate use. However, it is difficult to consent to all specific future uses in rapidly advancing scientific technology. While privacy and confidentiality remain imperatives for protecting whole genome sequence data, it is also important to recognize that the American public, generally speaking, has become more open about communicating their health information. The development of online resources and communities reflects a shift in societal notions of what data should remain private (regardless of whether individuals would want to make it public). People now freely share information that was once considered inherently private or not suitable to be shared with a broad audience. The arrival of whole genome sequencing in health care has coincided with an era of greater openness about diseases that used to carry social stigma, such as HIV/AIDS, cancer, and mental health conditions. Many patients may choose to publicly share their stories, although others might not for privacy reasons. Social attitudes about privacy are changing. There have been shifts not only in what information is considered private but also in how entities can realistically be expected to protect that privacy. Technological advances can trigger the creation of new privacy policies, such as the National Institutes of Health’s (NIH) updated genome-wide association study policies.145 While policy makers continue to focus on genetic non-discrimination policies that protect those whose privacy has been compromised, they have also begun to focus on data security policies that protect the data in the first place.146 Finally, informed consent practices increasingly acknowledge that absolute privacy cannot be guaranteed.147 Policies likely will evolve as notions of privacy continue to change. Future policies need to be flexible so that they can adapt to such advances in data security and information technology.
Recommendation 1.1. Funders of whole genome sequencing research; managers of research, clinical, and commercial databases; and policy makers should maintain or establish clear policies defining acceptable access to and permissible uses of whole genome sequence data. These policies should promote opportunities for models of data sharing by individuals who want to share their whole genome sequence data with clinicians, researchers, or others. Strong baseline privacy protections require a spectrum of policies starting with data handling through the protection of persons from future disadvantage and discrimination arising from misuse of their whole genome sequence data. It is critical, however, to ensure that privacy regulations allow individuals to share their own whole genome sequence data with clinicians, researchers, and others in ways that they choose.
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Policy makers should also revisit efforts to strengthen protections against, and sanctions for, discrimination by treating the Genetic Information Nondiscrimination Act as a floor, not a ceiling, of protection. For example, because GINA does not cover symptomatic persons or address discrimination in life, disability, or long-term care insurance, persons with genetic diseases and predispositions are vulnerable to discrimination.148 Advances in information technology should be pursued that promote appropriate use of whole genome sequence data while safe-guarding access to data files. For example, computational models that limit access to data files without preventing researchers’ ability to analyze these data can be a valuable tool to protect privacy. DATA PROTECTIONS THAT MOVE WITH THE DATA The President’s Council of Advisors on Science and Technology advocates a “tagged data element approach [that] allows for a sophisticated, fine-grained model of implementing strong privacy controls (including honoring patient-controlled privacy preferences where applicable) and strong security protection.” This approach encourages privacy protections to move with the data across institutions, as opposed to changing protections based on the handler. Source: President’s Council of Advisors on Science and Technology. (2010, December). Report to the President Realizing the Full Potential of Health Information Technology to Improve Health care for Americans: The Path Forward, p. 52. Retrieved from http://www.whitehouse.gov/ sites/default/ files/microsites/ostp/pcast-health-itreport.pdf.
Last, policies regarding access to and use of data should take into consideration varying cultural, ethnic, and racial views about what might or might not constitute a misuse of data.149 Currently, about half of the U.S. states have laws or regulations governing genetic privacy that outline illegitimate uses of these data. However, there is tremendous variation in these laws. In some instances, it is difficult to determine whether a state prohibits surreptitious testing of genetic material from an unwilling donor because of unclear language in the statutes. Some states prohibit unauthorized acquisition or analysis of genetic information, while others prohibit only unauthorized disclosure (and it is often unclear to whom disclosure is prohibited). Some laws at the state level encompass all genetic information, while others address only healthrelated information, or information obtained or used in particular settings (e.g., employment or insurance discrimination).150 Therefore, whether genetic testing or whole genome sequencing without the consent of the donor is prohibited can depend on a combination of factors: who conducts the test, to whom the DNA belongs, what the test attempts to determine, how the results will be used, and in what state the testing takes place.151 Moreover, no states have laws or regulations specific to whole genome sequence data; some states have laws that include the words “DNA” and “genetic,” although it is unclear whether these laws might be interpreted to cover whole genome sequence data and information. Some of the topics specified in existing genetic laws could be used for whole genome sequencing laws as well. Types of regulations that would translate effectively into genomic protections include those regarding:
Defining restrictions on what information can be stored in a biorepository, biobank, or genomic research database;
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Presidential Commission for the Study of Bioethical Issues Sharing of whole genome sequencing data, and if clinical data are shared with researchers, what type of information can be shared (e.g., stripped of traditional identifiers or not), and penalties for violations; and Using whole genome sequence information for life, disability, or long-term care insurance.
When individuals are asked about their concerns with respect to online health information, most focus on illegitimate uses of the data. They also cite discrimination, such as unauthorized use by insurers or employers, or use of their data for marketing purposes.152 However, the existing patchwork of state protections—with some states having no laws and the others having an inconsistent potpourri of legal prohibitions—does not protect all individuals from unauthorized uses. These uneven protections might also affect the development of trust in contexts where individuals are asked to share their whole genome sequence data for the public benefit in the course of research, clinical care, or commerce. Like all medical information, whole genome sequencing data should be ensured baseline privacy protections in all jurisdictions.
Recommendation 1.2. The Commission urges federal and state governments to ensure a consistent floor of privacy protections covering whole genome sequence data regardless of how they were obtained. These policies should protect individual privacy by prohibiting unauthorized whole genome sequencing without the consent of the person from whom the sample came. Currently federal and state laws protect data dependent on who collected them (i.e., a clinician, researcher, or consumer). Although a whole genome sequenced in the clinic is the same as a whole genome sequenced during research, data collected in the course of clinical care are governed by the Health Insurance Portability and Accountability Act, while data collected in the course of research are governed by the federal Common Rule for human research. The exact same data are treated differently depending on who collected the sample. Clinical data are collected to benefit the patient, while research data are collected to advance science and health care generally. However, the blurring of clinical and research lines, particularly in the field of whole genome sequencing, compels reconsideration of the differences between how clinical and research data are protected. In addition, while the requirement for consent to whole genome sequencing is regulated in the clinical and research contexts (depending, to some extent, on whether or not traditional identifiers—such as name, address, or social security number—are attached to the sample), commercial genetic testing has opened a new loophole in privacy protections. One can now pick up a discarded coffee cup and send a saliva sample to a genetic testing company.153 The potential consequences of unauthorized surreptitious testing could be profound (e.g., revealing disease risks to sway the disposition of a custody battle).154 There are, of course, exceptions to this need for consent, such as use for legitimate law enforcement purposes. The Commission therefore recommends the prohibition of “unauthorized” whole genome sequencing—a term intended to carve out an exception for legitimate law enforcement. Data protections should be tied to the nature of the data, not who collects them. Widely shared norms of justice and fairness dictate that similar kinds of data should be treated in similar ways, no matter in which state or health facility they are sequenced. If protections are inherent to the data, they should follow the data and dictate appropriate use. For example, meta-data
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tags could be used to encode the level of security protections required for the data file and elements of consent (e.g., these data can/cannot be used in reproductive research). Using this approach, data will receive appropriately consistent use protections throughout their life span. More consistency in state protections of genetic and genomic data could also enhance privacy. Treating like data alike is crucial to ensuring consistent protections for whole genome sequence information across the United States. Although states should enact genomic policies that are most relevant and important to their constituents, bringing such protections to a minimum standard that addresses privacy—while still allowing individuals to share their own data—would provide just and fair protections regardless of where one happens to reside. Because the options for implementation of such protections are unclear, the Commission recommends that experts in federal law, state law, policy, and privacy be brought together to engage in further democratic deliberation regarding acceptable access to and permissible use of whole genome sequence data. The Commission will consider following up with stakeholders regarding: 1) suggested requirements to ensure a floor of protection of whole genome sequence data and data sharing in all states; and 2) the practical steps necessary to accomplish this goal, such as federal, state, or non-regulatory interventions.
Data Security and Access to Databases Respect for persons requires honoring data privacy. Data privacy requires data security. Data security requires ethical responsibility and accountability from all those who handle whole genome sequence data and information. It must further be supported by policies and infrastructure to protect safe sharing of data. Authorized users must have access to whole genome sequence databases to conduct research and make advances that will contribute to improved medical diagnostics and treatment for all. Security should allow only authorized individuals to access these data. However, breaches of unsecured protected health information have been publicized in the past, and can cause patients and research participants to doubt the security of their data. Unsecured health information can be accessed by unauthorized persons through means such as the loss or theft of unencrypted information on data storage devices, hacking of network servers, unauthorized disclosure, or improper disposal of paper records.155 In a recent case, the unencrypted health information of over 800 patients was inadvertently embedded in PowerPoint presentations that were posted online.156 In light of the possibility of data security breaches, it is important to address misuse of whole genome sequence data rather than wholly relying on preventing unauthorized access to these data. “Technology can help save privacy, it can change your thinking, whether [it is] with respect to setting norms, [or] whether [it is] with respect to changing the way you set up the platform so that the platform can do more [computational] analysis… versus sharing the data around.” Latanya Sweeney, Visiting Professor and Scholar, Computer Science Director, Data Privacy Lab, Harvard University. (2012). How Technology is Changing Views of Privacy. Presentation to PCSBI, August 1, 2012. Retrieved from http://bioethics. gov/cms/node/748.
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Presidential Commission for the Study of Bioethical Issues
When told of data hacking, some assume that the transition of private information to an electronic format makes it less safe. Quite the opposite might be true. In many respects, advances in information technology can be used to strengthen data security. For example, electronic files bear marks of who accessed them and when, allowing for more fine-tuned file tracking than is possible with paper records that may be surreptitiously accessed without a trace. In addition, current technology allows data files to be analyzed without the need to export the data files to other networks, that is, computational access can be allowed without data transfer. Even when individuals are willing to share their readily identifiable data and information for use in research, they might not want copies of their information saved on computers around the world. Access to and sharing of data files do not have to be one and the same. The Commission supports ongoing exploration and development of a set of best practice models that separate possession of, access to, and use of data.157
Recommendation 2.1. Funders of whole genome sequencing research; managers of research, clinical, and commercial databases; and policy makers should ensure the security of whole genome sequence data. All persons who work with whole genome sequence data, whether in clinical or research settings, public or private, must be: 1) guided by professional ethical standards related to the privacy and confidentiality of whole genome sequence data and not intentionally, recklessly, or negligently access or misuse these data; and 2) held accountable to state and federal laws and regulations that require specific remedial or penal measures in the case of lapses in whole genome sequence data security, such as breaches due to the loss of portable data storage devices or hacking. Absolute privacy, many observe, is not possible in this as in many other realms. The greater potential for harm is not by virtue of authorized others knowing about one’s whole genome make-up, but rather through the misuse of data that have been legally accessed.158 For example, a clinician with a celebrity client would have legally authorized access to their client’s whole genome sequence data for purposes of providing clinical care, but could not then sell that information to a tabloid. Researchers, clinicians, and others authorized to access whole genome sequence data should be guided by professional ethical standards so that they do not intentionally or inadvertently misuse these data. In the event that data are mishandled or lost, those responsible should be aware of federal and state policies that require specific remedial actions, such as the requirement under the Health Information Technology for Economic and Clinical Health Act Breach Notification Rule to report breaches to the Department of Health and Human Services within the required number of days.159 Those persons authorized to access whole genome sequence data should take part in regular training sessions to remain current on regulations governing whole genome sequence data privacy and security. Public and private entities have different policies governing access to whole genome sequence databases by those seeking to use data for purposes other than that for which they were originally collected. Some policies create absolute prohibitions on releasing data to outside parties and associated penalties for violation, and some are more flexible, relying on the discretion of the person who holds the data.160 Certificates of Confidentiality, for example, permit but do not require investigators to refuse access to research data by law enforcement officials and others.161 The use of Certificates of Confidentiality however, is limited; one study found that only 114 (0.04 percent) of 27,000 funded studies secured such a certificate.162
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Although empirical data on the use and effectiveness of these forms of privacy protection are not robust, scholars have questioned the strength of these protections, how well understood these protections are, and how they affect research participation.163 Besides researchers, parties who might be interested in accessing information already compiled in whole genome sequence databases and biorepositories include law enforcement officials and marketing agencies. While commercial advertising can be a valuable tool in educating at-risk populations, this technique is often viewed as invasive when used as a way to sell products, for example, to selectively market a statin to someone with a genomic predisposition to high cholesterol.164 In order to establish and maintain trust between members of the general public, clinicians, and the scientific research community, strong whole genome sequence data protections must be in place to secure data. Further, these limits on access must be communicated to those giving consent to have their whole genome sequenced in clinical, research, or consumer-initiated settings. Obtaining a whole genome sequence data file by itself yields information about, but does not definitively identify, a specific individual. The individual still has “practical obscurity,” as his or her identity is not readily ascertainable from the data. Practical obscurity means that simply because information is accessible, does not mean it is easily available or interpretable, and that those who want to find specific information must expend a lot of effort to do so. While some experts might be able to determine an individual’s hair color or specific cancer risk from whole genome sequence data (a file of 6 billion As, Cs, Gs, and Ts), these data are not interpretable by the vast majority of individuals. In addition, even if we know that a whole genome sequence is from one individual, we cannot know which of the over 7 billion people on Earth that person is without a key linking the whole genome sequence information with a single person or their close relative. Therefore, while whole genome sequence data are uniquely identifiable, they are not currently readily identifiable. Traditional identifiers have been stripped from samples or data in the clinical and research setting to mitigate the possibility of risks to the individual from whom the samples came. Removing traditional identifiers from samples and data can allow for research on samples previously collected for different purposes, deter users from illegitimately identifying individuals, and minimize the risk that users might recognize individuals and use this information subconsciously in their daily life.
Recommendation 2.2. Funders of whole genome sequencing research; managers of research, clinical, and commercial databases; and policy makers must outline to donors or suppliers of specimens acceptable access to and permissible use of identifiable whole genome sequence data. Accessible whole genome sequence data should be stripped of traditional identifiers whenever possible to inhibit recognition or re-identification. Only in exceptional circumstances should entities such as law enforcement or defense and security have access to biospecimens or whole genome sequence data for non health-related purposes without consent. The consent process should communicate limits on access and use to those having their whole genome sequenced in clinical care, research, and consumer-initiated contexts. These policies should apply to the original recipient of the data, as well as to all parties who work with the data, from those who collect the sample or data to third-party storage and analysis service providers.
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Presidential Commission for the Study of Bioethical Issues
An existing policy that could serve as a model is the Agency for Healthcare Research and Quality’s confidentiality statute.165 This statute was put in place to foster participation in research and provides a respected form of statutory protection for all identifiable data submitted to the Agency for Healthcare Research and Quality for research. The statute covers AHRQ, its grantees, and contractors. The statute also defines strict penalties for individuals who use these data for non-consented purposes. Whole genome sequencing and related analyses generate enormous data sets. As of March 2012, the 1000 Genomes Project contained the sequence data of 1,700 people. The project database contained 200 terabytes of data, or the equivalent of 30,000 standard DVDs. This data set is a tremendous resource for biomedical researchers. At the same time, these data might not be useful to medical scientists and researchers without the computing power required to work with such a large data set. Exploring options for making these data available to qualified researchers is critical so that innovation and research are not slowed simply because researchers’ computer networks cannot store these large data files. “The explosion of biomedical data has already significantly advanced our understanding of health and disease. Now we want to find new and better ways to make the most of these data to speed discovery, innovation, and improvements in the nation’s health and economy.” NIH Director Francis S. Collins, M.D., Ph.D., in a press release announcing the movement of the 1000 Genomes Project data set to the Amazon Web Services cloud. Retrieved from http://www.nih.gov/ news/health/mar2012/nhgri-29.htm.
The question of how best to handle large data sets has gained attention throughout the government. The federal Office of Science and Technology Policy recently announced a “Big Data Research and Development Initiative,” with the goal of “improving our ability to extract knowledge and insights from large and complex collections of digital data.”166 Six federal departments and agencies are part of the initiative. This initiative includes NIH, which recently made its 1000 Genomes Project public data set available on the Amazon Web Services cloud. NIH now expects that researchers can access and analyze the data at a fraction of the cost it would take to establish the computing capacity at their own institution.167 Making whole genome sequence data accessible to researchers and clinicians is a promising step toward advancing medicine for the betterment of society. Moving data to thirdparty storage and analysis service providers, however, complicates the protection of individual data. When data are moved to third parties, an expanded range of data handlers and administrators have access to the data. Currently, a wide range of federal regulations govern the conduct of entities that handle protected health information.168
Recommendation 2.3. Relevant federal agencies should continue to invest in initiatives to ensure that thirdparty entrustment of whole genome sequence data, particularly when these data are interpreted to generate health-related information, complies with relevant regulatory schemes such as the Health Insurance Portability and Accountability Act and other data privacy and security requirements. Best practices for keeping data secure should be shared across the industry to create a solid foundation of knowledge upon which to maximize public trust.
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2009
Whole genome sequence data not stripped of traditional identifiers are considered “protected health information” and are covered under the HIPAA Privacy, Security, and Enforcement Rules and the Common Rule. The same regulations, policies, and ethical guidelines that protect such health information should also be in place to govern the sharing of whole genome sequence data with third-party storage and analysis service providers (those otherwise not considered covered health entities under HIPA A). Entities within the public and private sectors have developed a range of practices for protecting privacy. For example, the National Institute of Standards and Technology, the Office of the National Coordinator for Health Information Technology, and the Office for Human Research Protections are developing policies concerning access to and use of data by third parties. The National Institute of Standards and Technology recently released guidance on “Security and Privacy in Public Cloud Computing” and the Office of the National Coordinator for Health Information Technology worked to strengthen protections of identifiable health information handled by third parties.169 Also, the Office for Human Research Protections issued guidance on research with coded private information or biological specimens.170 Parties from the public and the private sectors should share their lessons learned to promote efficiency and avoid duplicating efforts. Because of the expansive potential of information technology, special attention should be paid to those practices that leverage information technology to protect privacy. In order for the public to benefit as much as possible, best practices across the industry should be shared to ensure the privacy and security of whole genome sequence data and best gain the trust of those who have their whole genome sequenced in research, clinical, and consumer-initiated contexts. These best practices should include encrypting stored data and storing data without traditional identifiers when possible. Even when data are being accessed and used with informed consent, persons who access the data should be responsible and accountable for protecting the privacy of individuals and the confidentiality of the data. Respect for persons requires that these and other privacy protections do not become a competitive advantage for certain parties but rather serve, in both appearance and reality, as a reliable standard of individual protection.
Consent Although not unique to whole genome sequencing, a well-developed, understandable, informed consent process is essential to ethical clinical care and research. Conveying the complexities of whole genome sequencing to an individual, however, is likely more difficult than for the average diagnostic test. To make the issue more complex still, informed consent documents are often overly legalistic and written at a reading level beyond the capacity of the average research participant.171 Studies have demonstrated varying levels of comprehension of consent documents, including reports of persons signing consent forms who are later either unable to recall whether they signed a consent form or describe to what they had consented.172 To educate participants thoroughly about the potential risks associated with whole genome sequencing, the consent process must include in format ion about what whole genome sequencing is; how data will be analyzed, stored, and shared; the types of results the patient or participant can expect to receive, if relevant; and the likelihood that implications of some of these results might currently be unknown, but could be discovered in the future. As per usual consent protocol, permission to perform whole genome sequencing for a person who cannot
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Presidential Commission for the Study of Bioethical Issues
consent for him or herself should be obtained from an informed, legally authorized representative. “How does consent change when a person lacks genetic health literacy, [or] when the health condition does not yet exist, but is a future probability, and some of those may be non-treatable conditions? When a health condition does not have implications for you, but it does for your offspring, what are the terms of consent there, especially if your offspring have different views about what they want to know about genetics, and then lastly, for these incidental findings versus disease specific testing..? I’ll just leave you with those questions, as the first of many that you will engage.” Daniel Masys, Affiliate Professor, Biomedical and Health Informatics, University of Washington School of Medicine. (2012). Ethics and Practice of Whole Genome Sequencing in the Clinic. Presentation to PCSBI, February 2, 2012. Retrieved from http://bioethics.gov/cms/node/658.
Consent documents differ between research and clinical care. Research informed consent documents are often long and contain n elements such as a summary of the research, future uses of data, the option to opt out, potential risks of participation, conditions of compensation in case of injury, and potential benefits to the individual. Clinical consent documents contain some of the same elements but generally are shorter than, and not as detailed as, research consent forms. In fact, oral consent might be sufficient for low-risk clinical procedures. The reason clinical consent is less comprehensive is because clinical procedures are done for the direct benefit of the patient and thus pose less of a risk of conflicting interests. More substantive clinical written consent is required, however, for higher-risk procedures, such as those expected to produce pain, require anesthesia, or have a significant risk of complications. In the research world, public opinion polls have found that individuals believe that being asked for consent throughout the course of research with their specimens or data would make them feel “respected and involved.”173 Informed consent involves an autonomous decision to participate in research that results from a communication process between researchers and prospective research participants that describes the research and explains the risks and benefits associated with enrolling in the study. Respect for persons dictates that individual consent should be well-informed and honored, regardless of a person’s specific privacy preferences. Clinical written consent documents for whole genome sequencing need not be as detailed as research consent documents, but these documents should still adequately explain whole genome sequencing and its potential impact upon privacy interests. A clinician should not frame whole genome sequencing as “just another type of blood test.” Consent procedures for clinical whole genome sequencing should build on those consent procedures already in place for discrete genetic tests. In the clinical context, as in research, individuals being asked to consent to whole genome sequencing should understand the volume of data and information to be generated, as well as the risks, benefits, and implications of the results of whole genome sequencing. The Common Rule states that data and specimens collected in the clinic, when stripped of traditional identifiers, can be used in research without consent. Because consent requirements differ in clinical and research settings, researchers could theoretically seek out data and specimens collected in the clinic to bypass the more involved research consent requirements.
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While it is acceptable to use clinical data and specimens in research, the Commission does not condone researchers circumventing Institutional Review Board approval by seeking out clinical data and specimens for use in research when they could not otherwise obtain IRB approval. Whole genome sequencing involving minors raises additional ethical quandaries even when permission is properly obtained from an informed, legally authorized representative. First, federal privacy laws inconsistently define the age of consent—for the most part, the age of consent is 18 years old in the United States, yet in health care for certain contexts (e.g., mental health, contraception, or substance use), state laws allow consent by minors as young as age 14.174 Second, the potential future risks raised by the current unknowns of whole genome sequencing are compounded in children who will see advancement in the science during their lifetime. While the function of all genes is not currently known, researchers will continue to determine the function of more genes, and could feel compelled to re-contact these children, as adults, with results that they are not prepared to receive or do not want. Third, whole genome sequence data obtained from a minor already could have been widely shared before the minor reached an age at which they could determine preferred data sharing limits themselves, thereby decreasing their autonomy. Whole genome sequencing in children, therefore, raises a number of unique issues with regard to fully informed decision making.175 Some commentators are concerned that participants enrolled in research that requires especially large data sets, and who are given too much control over their data, will stifle the production of public benefits, such as improvements in clinical care, comparative effectiveness research, and epidemiological studies.176 If individuals can choose not to participate in certain types of studies, the amount of data available to clinicians and researchers upon which to base their conclusions will be limited to some extent. A range of consent frameworks are available that offer participants varying levels of control over their data. Most of these frameworks fall into four categories: 1) broad; 2) narrow; 3) tiered; and 4) participant-centric or dynamic approaches. Under broad consent, individuals are given the option to opt in or opt out of general, and often yet to be determined, future uses of their data. Narrow consent usually states that data will be used only by the research team carrying out a specific study or for a specific treatment in the clinic. Tiered consent processes allow individuals to specify acceptable and unacceptable uses of their sample and data at the outset of research. “We also, as a research community, need to get used to the fact that there are patient-driven research objectives now and [patients] are coming together to do [research].” Laura Lyman Rodriguez, Director, Office of Policy, Communications, and Education, National Human Genome Research Institute. (2012). Protection of Private and Public Genomic Databases. Presentation to PCSBI, August 1, 2012. Retrieved from http:// bioethics.gov/cms/node/749.
Other consent models use computer-based participant-centered consent processes, which generally give participants freedom to determine their specific data sharing preferences up front, with some allowing participants to monitor and modify their preferences on an ongoing basis through a computer interface.177 One prototype that has been implemented by a group called Consent to Research allows users to “attach” consent to the data they donate, and any researcher who can accommodate the provisions of that consent can use those data.178 Alternatively, as databases become more technologically flexible, those donating biospecimens
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can express preferences at the outset about permissible and impermissible uses that can be respected by future users of whole genome sequence data. Further, sample donors could electronically update their consent to encompass proposed new studies, with minimal hassle to the donor or the researcher. These models, however, can only be used by participants who have computer and internet access. Some data have been collected on participant views of consent forms for biorepository research. Biorepository specimens and data files can be collected in clinical or research settings, and include (among other things) medical waste, newborn blood spot cards, and biopsy specimens. In one pair of studies, when asked about an opt out consent process, over 90 percent of participants agreed or strongly agreed that “DNA biobank research is fine as long as people can choose not to have their DNA included.”179 Another study found that, despite privacy concerns, 60 percent of individuals surveyed would participate in a genetic biorepository, 48 percent of whom would prefer broad consent, while 42 percent would prefer project-specific consent with re-consent for each project.180 These studies indicate that the majority of individuals enrolled in research are willing to share their data when asked, and the limited data available suggest that individuals vary widely across this spectrum of preferred form of consent.181 More research is needed, however, including on minority and marginalized populations where research participation is not as high. The Common Rule, which governs most human research in the United States, requires that research consent be informed. Consent may be waived in some circumstances, and research with samples or data that are not readily identifiable is not considered human research (and thus does not fall under the Common Rule). Blanket authorization for all future uses of identifiable data, known and unknown, at the outset of a research study cannot legally satisfy the current requirements for informed research consent. However, the Common Rule Advanced Notice of Proposed Rulemaking (ANPRM) proposes a broad consent requirement that would give participants the opportunity to say “yes” or “no” to all future research uses of their data and specimens at the outset of research.182 The ANPRM also proposes that individuals could designate special categories of research in which they would not want their samples included, for example, reproductive research. By giving individuals the option to not participate in research to which they object, these individuals are respected as persons. Moreover, the option to not participate in a set of specific categories of research that one finds objectionable might actually encourage broader participation in research. Broad consent at the outset of research might be a more practical solution than re-consent, or obtaining informed consent from every donor for a new use. Re-consent is difficult or, in some cases, impossible, as individuals frequently change residences, clinicians, phone numbers, and email addresses. Researchers also maintain that obtaining consent for each future study is burdensome and could hinder research.183
Recommendation 3.1. Researchers and clinicians should evaluate and adopt robust and workable consent processes that allow research participants, patients, and others to understand who has access to their whole genome sequences and other data generated in the course of research, clinical, or commercial sequencing, and to know how these data might be used in the future. Consent processes should ascertain participant or patient preferences at the time the samples are obtained.
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Respect for persons requires obtaining fully informed consent at the outset of treatment or research. The informed consent process should cover the current proposed use of individuals’ data, convey who might have access to their data, and explain potential future uses of these data, as well as what research results and incidental findings, if any, will be returned to the patients or participants. Some patients might be surprised to discover that their whole genome sequence data obtained in the clinic could be used for research in the future without additional consent. With the blurring of the line between clinical care and research, data may be shared back and forth to improve clinical diagnosis and treatment.184 Patients in the clinic should thus be explicitly informed that their whole genome sequence data could be used in research. When possible, individuals should be given the option to withhold their data from certain types of future research to avoid inadvertent complicity with research goals to which they are opposed. The Commission acknowledges the complexity of integrating individual options into the research enterprise, but if a framework is in place that accommodates identifying specific participant preferences at the time of enrolling in research, such as proposed in the ANPRM, these preferences should be honored.185 As long as consent processes are equivalently effective in informing individuals about what they are consenting to, and as long as they do not unduly shape or undermine individuals’ ability to make genuinely voluntary choices, there is no philosophical or ethical imperative to use one kind of consent process over another. In cases where the public stands to benefit from an activity and the research consent is fully informed and consistent with the ability to make autonomous choices, it might be advantageous to use consent processes that make it easier for individuals to participate—but most definitely not “trick” them into participating—at higher rates. In other words, the most important issue in consent is not the type, but rather that the consent is properly informed and consistent with voluntary choice. Opt in consent policies assume that the default is not to go forward with some proposal, such as to consent to whole genome sequencing; the individual must actively consent to the proposal in order for anything to happen. Opt out consent means that, in the absence of a refusal, the default is participation, which tends to encourage higher rates of participation, a result particularly supportive of the public value of scientific and medical research that is otherwise ethically and legally sound. BIOVU: AN OPT OUT DATABASE Vanderbilt’s BioVU database, which has collected DNA samples from almost 150,000 individuals, is an opt out database. Unless patients check a box indicating that they do not want their DNA in the BioVU database, their samples are included. In this way, BioVU is able economically to collect a large number of samples. To protect the data in its database, the samples are coded before being entered in the database. The computer system can match the DNA with information in medical records, but researchers working with the data do not know to whom the data belong. Data are stripped of identifiers before being shared with secondary researchers. Vanderbilt University Medical Center. (2012). Vanderbilt BioVU. Retrieved from http://www. vanderbilthealth.com/main/25443.
2014
Presidential Commission for the Study of Bioethical Issues
Organ donation policies in Europe provide an example of opt out consent procedures. Austria, France, Hungary, Poland, and Portugal have opt out organ donation polices and all have organ donation consent rates above 99 percent.186 The United States, on the other hand, uses an opt in system. Polls show that about 90 percent of Americans support organ donation, but only about 44 percent of people in the United States opt in to be organ donors.187 This indicates that where Americans’ values dispose them in favor of consent to organ donation, the often cumbersome and anxiety-inducing procedures of an opt in policy make them reconsider, passively resist, or fail to follow through with the extra steps (like filling out extra forms) required to opt in.188 With some exceptions, federally funded research studies are required by law to obtain informed consent from all individuals enrolled in research or from their legally authorized representative.189 The informed consent document is one component of the informed consent process. Current federal regulation requires that informed consent documents include, among other things, a description of the procedures in the research plan, an explanation of the risks and benefits to the participant, a description of the extent to which confidentiality of records will be maintained, and an explanation of the right to withdraw from the study. By regulation, research participants can withdraw from research to which they consented at any time for any reason. However, complete destruction of whole genome sequence data is likely impossible. Although physical biospecimens and data files stored by the primary researchers can and will be destroyed at the time of withdrawal according to guidelines laid out in consent documents, the destruction of distributed copies of associated data files may not be feasible as distributed genome sequence data files can be stored on local computers or network servers. Therefore, those conducting whole genome sequencing research might not be able to promise complete withdrawal from a study.
Recommendation 3.2. The federal Office for Human Research Protections or a designated central organizing federal agency should establish clear and consistent guidelines for informed consent forms for research conducted by those under the purview of the Common Rule that involves whole genome sequencing. Informed consent forms should: 1) briefly describe whole genome sequencing and analysis; 2) state how the data will be used in the present study, and state, to the extent feasible, how the data might be used in the future; 3) explain the extent to which the individual will have control over future data use; 4) define benefits, potential risks, and state that there might be unknown future risks; and 5) state what data and information, if any, might be returned to the individual. Each government agency has its own enforcement authorities to protect research participants. For example, the Office for Human Research Protections has jurisdiction over human research conducted or supported by HHS, the Central Intelligence Agency has a Human Subject Research Panel, and the Department of Veterans Affairs uses a combination of Research Compliance Officers and its Office of Research Oversight. All these agencies should work together as each agency develops clear and consistent guidelines for their informed consent forms, enabling an individual to make a fully informed decision to participate in research. Looking forward, clinical consent documents for whole genome sequencing will have to address a number of issues specific to whole genome sequencing: an explanation of the science, what types of results will be produced through whole genome sequencing, and whether whole
Privacy and Progress in Whole Genome Sequencing
2015
genome sequence data collected for clinical applications will be made available for research purposes. Further, whole genome sequence data can provide information about many conditions, not just the condition under study. Acknowledging this, informed consent documents for studies involving whole genome sequencing should include which (if any) research results and incidental findings will be returned to individuals.190 “Now, on the other side of the ledger…are the findings… which the patient is not expecting…which are going to have a dramatic impact of known consequence to them, and then the set of things for which there is much less certain impact.” Richard Gibbs, Wofford Cain Professor, Department of Molecular and Human Genetics; Director, Human Genome Sequencing Center, Baylor College of Medicine. (2012). Ethics and Practice of Whole Genome Sequencing in the Clinic. Presentation to PCSBI, February 2, 2012. Retrieved from http:// bioethics.gov/cms/node/658.
In whole genome sequencing, many individuals might want, and even expect, access to data or results.191 From the perspective of many individuals, the inability to receive or access their data denies them a fundamentally important sense of control over information about their own genomic makeup. While some individuals wish to share their data broadly for the advancement of science, others want control over their data to maintain their privacy, control information shared with intimate relations, or protect their right not to know results that might be discovered during whole genome sequencing. Individuals who seek return of data or results often feel that if someone else knows something unique about them, such as their risk for a particular disease, they ought to know it as well.192 On the other hand, some experts have said that although participant or patient preferences should be considered in the return of results, individual preferences are not a sufficient reason for agreeing to return results because of the importance of ensuring that the results are accurately communicated to individuals. These experts argue that the decision of whether to return incidental findings and other data should be in the hands of those who can more fully understand the broad implications of returning those findings, and what needs to accompany the return of raw results. They call for criteria to be developed, for example, by return of results committees.193 There are, of course, reasons within our current research systems for not returning research results to individuals enrolled in research studies as well. First, by current law, only sequencing results from Clinical Laboratory Improvement Amendments (CLIA) compliant laboratories may be returned to individuals.194 This requirement came about in the 1980s as a result of stories in the media that raised concerns about the quality of laboratory results, especially the return of false-negative Pap smear results.195 This attention catalyzed the passage of CLIA in 1988, designed to improve quality and consistency in clinical laboratory testing. CLIA made it illegal to return to patients clinical results generated in a non-CLIA-certified laboratory.196 Currently, most research is not conducted in CLIA-certified laboratories, including those laboratories performing whole genome sequencing.197 In addition, researchers leading projects that are producing whole genome sequence data might not be qualified or trained to return sensitive, potentially devastating results directly to individuals, nor are grants usually structured to hire someone with the appropriate qualifications to do so.
2016
Presidential Commission for the Study of Bioethical Issues
Ethical analysis of whether and how individual research results and incidental findings should be returned is ongoing, and these questions are currently the subject of wide-ranging debate.198 Many agree that participants should have the option to opt out of receiving research results and data from a study. There is less consensus on what should be done in cases where individuals want to receive incidental research results and data but, for example, researchers or clinicians did not themselves collect the information, are not trained in interpreting incidental results, did not perform the sequencing in a CLIA-approved lab, or have no prior knowledge of or relationship to the individual to appropriately convey the results. Alternatively, in some cases, investigators might feel personally obligated to provide research results that could be clinically meaningful.199 One example that illustrates this dilemma is the Alzheimer’s risk associated with certain variants of the ApoE gene. Individuals who carry the ApoE4 variant have a higher risk of developing Alzheimer’s disease, but not everyone with this variant will develop Alzheimer’s disease. Suppose that whole genome sequencing is being performed on a young adult for a breast cancer research study he or she is involved in, and the ApoE4 variant is discovered. Should this finding be returned? The finding is not clinically actionable—meaning that there is not an effective treatment or cure—and it is not certain that individuals with the ApoE4 variant will develop Alzheimer’s disease. Some argue that the only acceptable reason to return an incidental finding is that the finding is clinically relevant and actionable, and the ApoE4 variant’s association with Alzheimer’s disease fails to cleanly meet these criteria.200 Others argue that it should be completely up to the individual whose whole genome is sequenced to make this decision.201 A number of frameworks for return of research results and incidental findings have recently been proposed by broadly constituted groups. A recent consensus paper authored by academic researchers, legal scholars, and patient advocates determined that researchers should offer to return individual research results that 1) are analytically valid; 2) are in compliance with CLIA; 3) the patient has consented to receiving; 4) are clinically actionable; and 5) present an “established and substantial risk of a serious health condition.”202 Another framework proposes grouping incidental findings into three “bins” including: “clinically actionable,” “clinically valid but not directly actionable,” (subdivided into low-, medium-, or high-risk incidental information groups), and “unknown or no clinical significance.”203 The bin into which the data fall in this model, in combination with other variants, determines if the result should be reported to the participant in a clinical context. Models also exist that are more finely tuned and consider multiple variables, such as participant preference (what results the participant does and does not want to know), significance of the result (analytic validity of the test and possibility for medical intervention), and communicability (literacy of the participant and clarity of the message).204 In contrast to these fine-tuned, multivariable return of results frameworks, many representatives of the patient advocacy community propose the wholesale return of whole genome sequence data to individuals. They argue that although universities or companies provide a service by performing whole genome sequencing, the individuals who supplied the samples should retain the right to control the use of the data, access to the data, and be able to share the data with whomever they choose (such as with researchers conducting other studies related to conditions affecting the individuals or the individuals’ families).205 There is a difference however between the return of “data” and “information” in the context of whole genome sequencing. Some have suggested that regardless of whether meaningful
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information (that is, analyzed data interpreted by experts) is made available, raw data might be valuable to individuals. Currently, the Food and Drug Administration is debating the classification of these data in the context of commercial genetic testing. If companies are returning results with clinical or medical significance, commercial genetic services might be subject to regulatory requirements; but if they are simply returning unanalyzed whole genome sequence data files, regulatory requirements might not apply.206 For example, the commercial genetic test company Lumigenix does not interpret medically relevant genetic variants in-house. Rather, it provides customers with raw whole genome sequence data, inviting the consumer to use free genome analysis software to discover and interpret clinically relevant information on their own.207 This is a mere sampling of the many complex and detailed issues that need to be addressed before reaching a comprehensive set of actionable recommendations about whether and when incidental findings from whole genome sequencing can and should be returned to individuals with their fully informed consent.
Recommendation 3.3. Researchers, clinicians, and commercial whole genome sequencing entities must make individuals aware that incidental findings are likely to be discovered in the course of whole genome sequencing. The consent process should convey whether these findings will be communicated, the scope of communicated findings, and to whom the findings will be communicated. Recommendation 3.4. Funders of whole genome sequencing research should support studies to evaluate proposed frameworks for offering return of incidental findings and other research results derived from whole genome sequencing. Funders should also support research to investigate the related preferences and expectations of the individuals contributing samples and data to genomic research and undergoing whole genome sequencing in clinical care, research, or commercial contexts. Individuals undergoing whole genome sequencing in research, clinical, and commercial contexts must be provided with sufficient information in informed consent documents to understand what incidental findings are, and to know whether they will be notified of incidental findings discovered as a result of whole genome sequencing.208 Users of whole genome sequence data should continue supporting research into the management of incidental findings and individual research results obtained in both CLIA and nonCLIA-certified laboratories. Previous research has generated many models and guidelines for returning incidental findings and other results obtained in clinical and basic research. In order to take the next step of translating these models into best practices for the return of results, additional data must be collected to inform the deliberations. In particular, research should be expanded to collect empirical data on participant, patient, researcher, and clinician opinions of each model, and the consequences and costs of implementing each model. These studies should examine the motivations of patients and participants enrolled in research, undergoing genome sequencing in the clinical context, or engaging in commercial whole genome sequencing to obtain their research results. Respect for patient and participant values is essential to guide the development of these tools ethically.
2018
Presidential Commission for the Study of Bioethical Issues
Facilitating Progress in Whole Genome Sequencing Current protections for research participants emerged from a series of lapses in research ethics uncovered in the 1960s and 1970s in which clinicians and scientists conducted research without the fully informed consent or even knowledge of the research participants.209 One outcome of this history was the drawing of a bright line between clinical care and research. But this distinction is no longer so clear. Currently, large amounts of patient data are being collected in the health care setting, stripped of traditional identifiers, analyzed, and fed into research that might one day improve clinical care. This learning health system model both translates advances in health services research into clinical applications and collects data during clinical care to facilitate further advances in research.210 With patient data increasingly being transitioned to electronic medical records, persons engaged in this type of research can also more easily access data to aggregate and analyze.211 Advocates of the learning health system model advocate encouraging intellectual freedom through clinical research and engaging in regulatory parsimony.212 Large amounts of data are essential for researchers to make correlations between genomic variants and disease states. Learning health system advocates and others call for standardized electronic health record systems and infrastructure to facilitate health information exchange so that data can be easily aggregated and studied.213 Integrating whole genome sequence data into health records within the learning health system model can provide researchers with more data to perform genomewide analyses, which in turn can advance clinical care. Several Institute of Medicine (IOM) working groups have supported these goals, outlining the desirability of establishing a universal health information technology system and learning environment that engages health care providers and patients. The IOM reports recommend that such a system include both genomic and clinical information, increased interoperability of medical records systems, and reduced barriers to data sharing.214 The President’s Council of Advisors on Science and Technology identified the lack of sharing electronic health records—with patients, with a patient’s health care providers at other organizations, with public health agencies, and with researchers—as a barrier to improved health care.215
Recommendation 4.1. Funders of whole genome sequencing research, relevant clinical entities, and the commercial sector should facilitate explicit exchange of information between genomic researchers and clinicians, while maintaining robust data protection safeguards, so that whole genome sequence and health data can be shared to advance genomic medicine. Performing all whole genome sequencing in CLIA-approved laboratories would remove one of the barriers to data sharing. It would help ensure that whole genome sequencing generates high-quality data that clinicians and researchers can use to draw clinically relevant conclusions. It would also ensure that individuals who obtain their whole genome sequence data could share them more confidently in patient-driven research initiatives, producing more meaningful data. That said, current sequencing technologies and those in development are diverse and evolving, and standardization is a substantial challenge. Ongoing efforts, such as those by the Standardization of Clinical Testing working group are critical to achieving standards for ensuring the reliability of whole genome sequencing results, and facilitating the exchange and use of these data.216
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In order for all persons to benefit from whole genome sequencing research, diverse populations must be involved in research. Consequently, it is incumbent upon the research community to earn and maintain the trust of individuals from a wide range of diverse populations across society. This trust is particularly important in minority and marginalized populations where levels of trust in the medical and research communities have been historically low. To encourage such trust, some scholars and advocates have proposed alternative models for the interactions between researchers and individuals enrolled in research that attempt to increase transparency and shift the balance of control between these two parties.217 As opposed to the traditional research model, in which there is usually little contact between the researcher and the individual enrolled in research beyond initial sample contribution, participant-centric initiatives put research participants at the center of the decision making, and are based on principles of respect and empowerment.218 The federal government has shown a n interest in giving patients a better understanding of disease, treatment, and care options through its establishment of the Patient-Centered Outcomes Research Institute.219 The challenges we face today in whole genome sequencing are not (or only partially overlap with) the challenges we will face in the coming years as technologies continue to develop and mature. For example, one current concern is the integration of data into electronic medical records; in 20 years or less, society might have to decide if every newborn should have their whole genome sequenced and added to their electronic medical record. Due to rapid technological developments, today’s policies must be crafted specifically enough to be actionable and targeted to address our current concerns, yet agile enough to ensure that we do not constrain our ability to adapt to evolving technology, research, and social norms related to privacy and sharing.220
Recommendation 4.2. Policy makers should promote opportunities for the public to benefit from whole genome sequencing research. Further, policy makers and the research community should promote opportunities for the exploration of alternative models of the relationship between researchers and research participants, including participatory models that promote collaborative relationships. Respect for persons implies not only respecting individual privacy, but also respecting research participants as autonomous persons who might choose to share their own data. Public beneficence is advanced by giving researchers access to plentiful data from which they can work to advance health care. Regulatory parsimony recommends only as much oversight as is truly necessary and effective in ensuring an adequate degree of privacy, justice and fairness, and security and safety while pursuing the public benefits of whole genome sequencing. Therefore, existing privacy protections and those being contemplated should be parsimonious and not impose high barriers to data sharing.221 While the Commission supports the intellectual freedom this access will encourage, clinicians and researchers must also act responsibly to earn public trust for the research enterprise.
2020
Presidential Commission for the Study of Bioethical Issues
Public Benefit The federal government has made a substantial investment in genetics research, including whole genome sequencing, and the benefit of this investment has been realized in two major ways. First, disease diagnosis and treatment have been advanced, and the functions of many genes have been and will continue to be discovered, which will further improve clinical care in the coming years. Incorporating knowledge gained through advances in whole genome sequencing into the clinic could improve diagnosis and treatment of diseases that have brought turmoil and tragedy into the lives of individuals and their families. We have already begun to see some benefits resulting from these advances; for example, genetic variants that can lead to adverse drug reactions have been identified. In the future, as the genetic variations that underlie common diseases are discovered, clinicians will, in some instances, be able to detect predispositions to disease before those diseases occur, and begin treatment or recommend lifestyle changes before a patient exhibits symptoms. Second, an indirect economic benefit has been realized. The U.S. government invested $3.8 billion in the Human Genome Project; it is estimated that this investment generated $244 billion in personal income and $796 billion in overall economic impact.222 These health and economic gains not only benefit the public through improved health care but also through increased economic opportunities. Thousands of citizens have participated in whole genome sequencing research personally, and all citizens help support government investment in whole genome sequencing through their participation in and support of our political system. Therefore, all citizens should have the opportunity to benefit from medical advances that result from whole genome sequencing. Special caution should be taken on the part of researchers to ensure that their participants reflect as much as possible the rich diversity of our population. Different groups have genomic variants at different frequencies within their populations, and sufficiently diverse data must be collected so that advances arising from whole genome sequencing can be used for the benefit of all groups.223
Recommendation 5.1. The Commission encourages the federal government to facilitate access to the numerous scientific advances generated through its investments in whole genome sequencing to the broadest group of persons possible to ensure that all persons who could benefit from these developments have the opportunity to do so. Government investment in genomic research has resulted in public benefit through improved health care and in economic return on investment. The principle of justice and fairness requires that the benefits and risks of whole genome sequencing be distributed across society. Research funded with taxpayer contributions should benefit all members of society. To these ends, researchers should be vigilant about including individuals from all sectors of society in their studies, so that research findings can be translated widely into clinical care. The federal government should follow through on its investment in research and assure that the discoveries of whole genome sequencing are integrated with clinical care that can be accessed by all.
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APPENDIX I: GLOSSARY OF KEY TERMS Allele: a form of a gene at a particular location on a chromosome. Biorepository: a stored collection of physical biological samples (e.g., blood or tissue) and associated data (e.g., medical information and policies). Sometimes called a biobank. Carrier: an individual who has one normal and one mutated version of a gene. Chromosome: X-shaped structure made of tightly wrapped DNA in the nucleus of the cell that carries genes from one generation to the next. Humans have 46 chromosomes (in 23 pairs). Clinical utility: an assessment of the risks and benefits associated with a clinical test and the likelihood that the test will result in improved patient outcome. Clinical validity: the degree to which a genetic test can predict clinical status, as measured by the strength of the association between the genotype and phenotype. Copy number variations (CNVs): DNA mutations that occur when large sections of DNA are inserted or deleted during cell division. Database: an organized collection of data or information (e.g., whole genome sequence data files and information). Deoxyribonucleic acid (DNA): the molecule that contains the instructions to develop and direct the biological and chemical activities of a living organism. DNA Sequencing: the process that identifies the order of the nucleotide bases in a strand of DNA. Exome Sequencing: DNA sequencing of only the parts of the genome that make proteins (exons). Exon: a stretch of DNA, part of a gene, that codes for a protein. Gene: a piece of DNA that contains the information required for making a product that will have a biological function. A full set of genes is called a genome. Gene-environment interaction: the environmental factors that can influence a gene’s expression and the resulting phenotype. Genetic test: a discrete test that examines a specific genetic location or a single gene, such as the test for Huntington’s disease. Genetic variation: differences in alleles of allele frequency between or among individuals or populations. Genomics: the study of all the DNA (the genome) in an individual, and how parts of the genome interact with each other and the environment. Genome: the full set of genes in an individual. Humans have about 20,000 to 25,000 genes in their genome. Genome-wide association study (GWAS): compares large amounts of genetic data from individuals with and without a specific condition to identify DNA variants that correlate with diseases. Genotype: the genetic make-up of an individual. Genotype/phenotype correlation: the association between a certain mutation (genotype) and the resulting physical characteristic (phenotype). Genotyping: analyzing discrete variants, from a handful to thousands, across the genome (i.e., more than a discrete genetic test, but less than whole genome sequencing).
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Presidential Commission for the Study of Bioethical Issues
Guthrie Card: piece of paper used to capture and store a few drops of blood collected from a newborn. DNA from the dried blood spot is then used to test for a range of genetic conditions and infections. Heterozygous: when the genes or alleles on the two chromosomes are different. Homozygous: when the genes or alleles on each of the two chromosomes are the same. Incidental finding: a finding discovered in the course of clinical care or research concerning a participant that is beyond the aims of the clinical test or research but has potential health importance. Individual research result: a finding discovered in the course of clinical care or research concerning a research participant that relates to the aims of the clinical test or research and has potential health importance. Intron: part of a gene present between exons that does not directly code for a protein. Locus: the location of a gene on a chromosome. Mutation: a change in the DNA sequence. Mutations can arise from mistakes during cell division or from an outside source (e.g., radiation from the sun). Nucleotide bases: the four chemical units that compose DNA. The bases are adenine (A), thymine (T), guanine (G), and cytosine (C). A always pairs with T on the opposite strand of DNA, and C always pairs with G. One A-T or G-C pair is called a base pair. Phenotype: the expression of an individual’s genotype. An individual’s phenotype consists of their physical characteristics. Public health utility: the likelihood that a clinical test will reduce disease burden and/or result in improved patient outcome in the population. Single nucleotide polymorphisms (or SNPs): variations in the genome that involve single base pairs. Structural variants: the insertion, deletion, duplication, translocation (the movement of DNA from one location to another on the same or another chromosome), or inversion (flipping over) of long DNA segments (greater than about 1,000 base pairs in length). Whole genome sequence data: the file of As, Cs, Gs, and Ts produced as a result of whole genome sequencing. Whole genome sequence information: facts derived from whole genome sequencing data, such as predisposition to disease. Whole genome sequencing: determining the order of nucleotide bases— As, Cs, Gs, and Ts— in an organism’s entire DNA sequence.
APPENDIX II: GENETIC AND GENOMIC BACKGROUND INFORMATION Understanding Basic Genetic Architecture Deoxyribonucleic acid (DNA) is the molecule that contains the instructions to develop and direct the biological and chemical activities of nearly all living organisms. DNA is a twisting pair of strands, called a double helix, made of four basic building blocks, or nucleotide bases. These bases are abbreviated A, T, C, and G. The As, Cs, Gs, and Ts are linked together in long strands. The A on one strand will link to at T on the other strand of the double helix, bringing
Privacy and Progress in Whole Genome Sequencing
2023
the two strands together at each point along the DNA strand, like rungs on a ladder. A always binds with T, and C always binds to G. One A-T or G-C pair is called a nucleotide base pair. If the DNA in a single human cell was stretched out, it would be about six feet long. If all the DNA in a human body was stretched out, it would reach almost 70 times from the earth to the sun and back.224 In order to fit this much DNA into cells, the long strands of DNA have to be stored compactly. In the cell, DNA is nearly always wrapped tightly into X-shaped structures called chromosomes, which prevent the long strands of DNA from tangling or being damaged. Chromosomes pass DNA from one generation to the next. MENDELIAN GENETICS Gregor Mendel, a 19th Century European monk, discovered the mechanism for trait inheritance in plants and animals. Mendel studied traits in peas, including flower color, stem length, seed shape, and seed color. Through selective pollination, he was able to observe how traits were expressed when two plants produced seed. He found that organisms have two copies of every inheritance “unit” (now called genes): one from each parent.
Chromosomes are located in the nucleus of a cell (a sub-compartment of the cell that stores DNA). Chromosomes are usually found in pairs, with one member of each pair coming from the individual’s genetic mother and the other from the genetic father (See Figure 3). Humans have 46 chromosomes, in 23 pairs. Of the 46 chromosomes, two are sex chromosomes (X and Y) that determine if an individual is male or female. In addition to the 22 pairs of chromosomes that all humans have, females inherit one X chromosome from each parent, making their 23rd pair XX, while a male inherits an X chromosome from his mother and a Y chromosome from his father, making his 23rd pair XY. A complete set of DNA, or a full set of 46 chromosomes in a human, is called his or her genome. In humans, the genome is made up of approximately 3 billion nucleotide base pairs (A-T and G-C pairs). Nearly every cell in the human body contains a complete copy of the genome. WHY IT IS GOOD TO HAVE TWO COPIES OF EACH CHROMOSOME Having two copies of each gene ensures that if one gene on one chromosome of a pair is damaged, the gene on the other chromosome of the pair might not be damaged. In most cases, having only one functional copy of a gene is sufficient for normal function. This could be compared to having two kidneys: if one kidney is damaged, the healthy one can function well enough so that the individual can lead a relatively normal life.
Genes are specific regions of DNA on chromosomes. Genes are the basic physical unit of inheritance, and are passed from parents to their children. There are approximately 20,00025,000 genes distributed over the 46 chromosomes. Together, these genes make up the blueprint for the body and how it functions. The location of a gene on a chromosome is called the locus, which is much like an address. For example, a gene can be found on chromosome 16 (e.g., the name of the street), on a particular end of the chromosome (e.g., the North or South end of a street), at a particular location (e.g., the house number).
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Presidential Commission for the Study of Bioethical Issues
Figure 3. The 23 pairs of human chromosomes.
Almost all genes come in pairs with one copy (or allele) from each parent. While the alleles might or might not be identical, the genes are the same (just like we all have ears, but our ears do not all look exactly alike). Every person has the same number of genes, although they might have different alleles from one another; that is, every person has the genes for cystic fibrosis (CFTR) and breast cancer (BRCA1/2), but most of us do not have disease-causing mutations in these genes. In order to go from the blueprint in the genes to a functioning human, information in DNA is turned into proteins. Genes contain the instructions for making proteins that make up the human body. Examples of proteins include collagen, which is a major component of our hair and skin; and enzymes, a special type of protein, some of which break down the food we eat. If the DNA coding for a protein is mutated, it could result in that protein not functioning. For example, if the enzyme that breaks down lactose (a protein found in milk) is not assembled properly, it cannot break down lactose effectively and an individual is said to be lactose intolerant. Not every single part of our DNA contains the instructions for making a protein, only certain parts of genes make proteins. These regions are called exons. The function of the regions of DNA that do not code for proteins, called introns, is unknown. Introns were once called “junk” DNA, but scientists are learning that introns are likely essential for the rest of the gene to function properly.
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2025
GENE-ENVIRONMENT INTERACTIONS Cystic Fibrosis is a recessive genetic disorder, which means that a child must inherit a mutated copy of the CFTR gene from each parent to have the disease. While the genetic cause is clear, the severity of disease is linked to environmental factors such as exposure to second hand smoke, stress, and poor nutrition. Smoke in particular has been shown to interact with the CFTR gene and a secondary gene as well, worsening lung function in the patient. Source: Collaco, J.M., et al. (2008). Interactions between secondhand smoke and genes that affect cystic fibrosis lung disease. Journal of the American Medical Association, 299(4), 417-424.
The term genotype refers to an individual’s collection of genes or to the two alleles inherited for a particular gene. The expression of the genotype, through making proteins, contributes to the individual’s outward characteristics, called their phenotype. The association between a certain mutation or mutations (genotype) and the resulting physical characteristics (phenotype) is called the genotype/phenotype correlation. This association is at the core of genetic testing and research. Gene-environment interaction refers to how environmental factors modify the expression of a gene and, therefore, the trait, or phenotype. Some phenotypic traits are strongly influenced by genes, while others are more strongly influenced by the environment. Most traits are influenced by one or more genes interacting in complex ways with the environment.
Genetic Variation A mutation is a change in a DNA sequence (see Figure 4). Mutations can come from mistakes that happen when DNA is copied during cell division, exposure to chemicals or harmful radiation (like UV rays from the sun), or infection with certain viruses. Some mutations occur in the cells of an individual’s body and are not passed on to offspring, such as DNA damage in the skin caused by sunburn. Other mutations occur in the eggs and sperm and can be passed on to offspring, such as a mutation in the gene for sickle cell anemia. The term genetic variation refers to differences in alleles and other genetic changes between or among individuals. Genetic variation can also refer to how often those differences in alleles occur between or among populations. Humans have about 99.9 percent our genetic information in common, but there is considerable genetic variation. The differences in our genomes can explain why we are diverse as individuals or populations in appearance, predisposition to specific diseases, and adaptation to our environment. Understanding genetic variation is at the heart of understanding the role of genetics in disease. Genetic variations involving only a single nucleotide base (an A, C, G, or T building block) are referred to as single nucleotide polymorphisms, or SNPs (pronounced “snips”). Most people have thousands of SNPs in their genomes, but they often occur in the parts of DNA that do not make proteins, so they do not cause disease. When SNPs occur within a gene, they might cause disease by affecting the gene’s function. Researchers have found SNPs that might help predict how an individual responds to certain drugs, their susceptibility to environmental factors such as toxins, and their risk of developing particular diseases. SNPs have been used extensively to study diseases that are passed from one generation to the next in families.
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Presidential Commission for the Study of Bioethical Issues
Figure 4. Types of Genetic Variations.
Genetic variation can also involve much longer stretches of DNA. Structural variants involve the insertion, deletion, duplication, translocation (the movement of DNA from one location to a not her on the same or another chromosome), or inversion (flipping over) of long DNA segments (greater than about 1,000 base pairs in length). One type of structural variation is a copy number variant. Copy number variations (CNVs) can occur when large sections of DNA are inserted or deleted during cell division. Scientists are trying to understand how copy number variation contributes to health and disease. Each person carries roughly 100 copy number variants, but many do not appear to have a disease linkage. SICKLE CELL ANEMIA Sickle cell anemia is caused by a SNP in the gene for hemoglobin, a protein in red blood cells that is responsible for carrying oxygen. If the hemoglobin gene is mutated on both alleles, an individual will have sickle cell disease, which leads to a shortened life span. If an individual has one normal hemoglobin allele and one mutated allele, however, they will not have sickle cell disease (because they also have one functional allele) and they will have some protection against malaria. Sickle cell disease is most common in populations who live in malaria-prone regions of the world, because carrying this mutation is actually protective against malaria. Source: CDC. (n.d.). Protective Effect of Sickle Cell Trait Against Malaria Associated Mortality and Morbidity. Retrieved from http://www.cdc.gov/ malaria/about/biology/sickle_cell.html.
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How Genetic Variants Translate into Disease Today, clinical genetic testing is used in individuals with a family history of disease; in other words, the tests are limited to those who are considered at risk of carrying known genetic variants that are linked to a particular disease. However, some clinical studies are evaluating the use of whole genome sequencing in regular clinical practice.225 In addition, individuals can try to bypass the traditional health care system and use the services of companies that offer consumers SNP analysis, whole exome sequencing, and more. Very few genetic variants are directly linked to a specific disease, however some examples include cystic fibrosis, sickle cell anemia, and Huntington’s disease. Targeted genetic tests have been developed for many of these diseases. Many other diseases are suspected to have a genetic component, but scientists have not determined which genetic variants might cause them. For example, heart disease could be caused by genetic mutations, but it is certainly not a simple case of one mutation in one gene. The genetic component of heart disease could be many mutations throughout the genome that interact with the environment to cause heart disease. Whole genome sequencing could reveal complex interactions between genes and disease, where a particular mutation on a certain gene, in conjunction with another mutation on another gene, or several other mutations on other genes, come together to cause disease. Some of the genetic variants discovered during whole genome sequencing will have clear links to disease, but the majority will be unknown. Based on how they translate to disease, genetic variants can fall into six categories:
Variants of unknown significance: An example of this might be when a piece of DNA has been cut out of one location on a chromosome and inserted into another location on the chromosome. The fact that the DNA is different is clear, but what that difference means, or how it will relate to disease, is unclear. Nonmedical genetic markers: These are genes that code for things such as eye color. If there were a mutation in one of these genes, it would not be something that would require medical treatment. Carrier status: An individual is a carrier of a variant if they have one normal and one mutated version of a gene. Most often, the individual is not affected by the disease, but they can pass the gene on to their children. An example is sickle cell disease, where individuals with one mutated version of the gene and one normal version of the gene do not have the full-blown disease themselves. Susceptibility genes: These are genes that make it more likely, but not certain, that an individual will develop a particular disease, i.e., they are “susceptible” to it. An individual might carry genes that make them susceptible to diabetes, but with proper diet and exercise, they will not necessarily develop diabetes. Late onset genetic conditions: Late onset conditions present later in life. Examples are Alzheimer’s disease, Huntington’s disease, and some degenerative eye diseases. Medical conditions found by current prenatal genetic tests: These are conditions that, if an individual has one or two copies of the gene, they will have the disease, and the disease will affect their health and quality of life throughout their life span. An example is phenylketonuria. Individuals with phenylketonuria cannot break down a particular amino acid and must follow a diet that is low in that amino acid.
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Sequencing Strategies DNA sequencing is the process of determining the exact order of the bases (nucleotides) in a strand of DNA. Since base pairing is predictable (A always pairs with T; G always pairs with C), knowing the sequence on one strand automatically reveals the sequence on the other strand. Sequencing technology has rapidly advanced in recent years, allowing scientists to make discoveries about the regulation, variability, and evolution of the human genome.226 A consequence of decreasing cost and increasing accessibility of sequencing technologies is the increasing use of whole genome sequencing. Whole genome sequencing is the process of sequencing all the DNA in an organism, in contrast to testing for only a handful of known mutations or sequencing a particular gene. Whole genome sequencing reads more than 95 percent of the genome, compared to SNP genotyping, which typically covers less than 0.1 percent of the genome. That said, knowing one person’s complete DNA sequence does not necessarily provide useful clinical information, because each person’s DNA is different from the DNA of others at millions of places. One goal of whole genome sequencing research is to create a reference catalog of all common and rare genetic variants in human populations so that the relationship between variants and disease can be studied. By comparing one person’s whole genome sequence with other whole genome sequences, reference sequences, and associated health information, one can find places in the genome where, for example, a group of people with the same DNA mutation at the same locus all have the same disease. Comparisons like this will hopefully lead to meaningful associations and ultimately guide clinical and personal health decisions. Exome sequencing might be an efficient alternative to whole genome sequencing in some cases. Exome sequencing selectively sequences only the part of the genome that make proteins (exons). An estimated 85 percent of disease-causing mutations are found in the exome.227 Therefore, sequencing only the exons, which make up about 1 percent of the genome, should be faster and less expensive than sequencing the entire genome, and is likely to identify most disease-causing mutations. Increasingly, exome sequencing is being used in clinical diagnostic testing. However, now that 80 percent of the genome has been found to have “biochemical function,” with non-coding regions of the genome influencing the activity of genes that are spatially distant, exome sequencing that does not find an answer could be complemented by targeted sequencing of non-coding regions. A genome-wide association study (GWAS) is a method that has been used heavily in recent years to identify links between specific genetic variations and specific diseases. The method involves studying the genomes of many people with and without a disease of interest and searching for genetic markers (e.g., SNPs) that can be used to predict the presence of a disease. GWASs alone cannot specify which genes cause disease; however, by looking at hundreds of thousands of SNPs, researchers can identify mutations that are more frequent in people with the disease than without. These mutations are therefore considered “associated” with the disease. Disease-associated SNPs are used as markers or pointers to the region of the genome where a disease-causing mutation is likely to be found. The Challenges of Analyzing Whole Genome Sequence Data and Identifying Disease Associations The primary goal of whole genome sequencing research is to describe the relationship between genotype (genetic variants) and phenotype (physical characteristics, including
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disease). Whole genome sequence data alone will not provide a complete understanding of disease. The data must be linked to phenotypic data, such as medical records. Environmental data will also be needed to fully understand gene-environment interactions. A challenge of whole genome sequencing research is the hard-to-detect relationship between genetic variant and phenotypic trait, such as disease risk. To interpret an individual’s disease risk, one must have reliable information about every validated genetic disease to use as a standard of comparison. Currently, there is no central, publicly available repository of all variants found to be associated with a clinically relevant trait or disease. Refinements must also be made to take into account the genomic diversity of the human population. While no “private” variants have been found only in one population and not in others, many variants occur at different frequencies in different populations (for example, a particular SNP might be common in one population and rare in another). Studying genetic variation across populations can provide some, but not all, clues to the causes of health disparities.228 Finally, even if a specific mutation is linked to a disease, the expression of that gene and environmental interactions can result in different phenotypic effects in different people. In other words, one person carrying a particular mutation might develop the disease and another person with the same mutation might not, or that person might exhibit the disease in a more or less severe form. Further, a single mutation in one gene rarely leads to the particular phenotype of an individual. The current clinical value of whole genome sequencing for linking genomic variants to disease remains challenging because of the many gene-gene and gene-environment interactions. Thus, the field continues to work toward establishing the clinical validity (future disease positive and negative predictive value stratified by exposure), clinical utility (targeted interventions to reduce disease risk among persons with the profile) and public health utility (comparing reduction of disease burden in the population based on genomic analysis) of whole genome sequence data.
APPENDIX III: GUEST PRESENTERS TO THE COMMISSION REGARDING PRIVACY AND WHOLE GENOME SEQUENCING George Annas, J.D., M.P.H. Chair, Health Law, Bioethics & Human Rights; William Fairfield Warren Distinguished Professor, Boston University School of Public Health
Richard Gibbs, Ph.D. Wofford Cain Professor, Department of Molecular and Human Genetics; Director, Human Genome Sequencing Center, Baylor College of Medicine
Retta Beery Mother of twins who benefitted from diagnosis made possible by whole genome sequencing
Jane Kaye, D.Phil., L.L.B. Director, Centre for Law, Health and Emerging Technologies (HeLEX),Oxford University
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Greg Biggers Council Member, Genetic Alliance; Chief Executive Officer, Genomera Ken Chahine, Ph.D., J.D. Senior Vice President and General Manager, Ancestry DNA, LLC Ellen Wright Clayton, J.D., M.D. Craig-Weaver Professor of Pediatrics; Professor of Law and Director, Center for Biomedical Ethics and Society, Vanderbilt University Leonard D’Avolio, Ph.D. Associate Center Director for Biomedical Informatics, Massachusetts Veterans Epidemiology Research and Information Center (MAVERIC), Department of Veterans Affairs; Instructor, Harvard Medical School James P. Evans, M.D., Ph.D. Clinical Professor and Bryson Distinguished Professor of Genetics and Medicine, Department of Genetics, University of North Carolina School of Medicine Madison Powers, J.D., D.Phil. Professor, Department of Philosophy; Senior Research Scholar, Kennedy Institute of Ethics, Georgetown University Laura Lyman Rodriguez, Ph.D. Director, Office of Policy, Communications, and Education, National Human Genome Research Institute, National Institutes of Health Mark A. Rothstein, J.D. Herbert F. Boehl Chair of Law and Medicine, University of Louisville School of Medicine
Bartha Knoppers, Ph.D. Director, Centre of Genomics and Policy; Canada Research Chair in Law and Medicine, McGill University Daniel Masys, M.D. Affiliate Professor, Biomedical and Health Informatics, University of Washington School of Medicine Amy McGuire, J.D., Ph.D. Associate Professor of Medicine and Medical Ethics; Associate Director of Research, Center for Medical Ethics and Health Policy, Baylor College of Medicine Melissa Mourges, J.D. Assistant District Attorney; Chief, Forensic Sciences/Cold Case Unit, New York County District Attorney’s Office Pilar Ossorio, Ph.D., J.D. Associate Professor of Law and Bioethics, University of Wisconsin-Madison Erik Parens, Ph.D. Senior Research Scholar, The Hastings Center Latanya Sweeney, Ph.D. Visiting Professor and Scholar, Computer Science; Director, Data Privacy Lab, Harvard University John Wilbanks Founder, Consent to Research; Senior Fellow, Kauffman Foundation; Research Fellow, Lybba Susan Wolf, J.D. McKnight Presidential Professor of Law, Medicine & Public Policy; Faegre & Benson Professor of Law; Professor of Medicine; Faculty Member, Center for Bioethics, University of Minnesota
Privacy and Progress in Whole Genome Sequencing Sonia Suter, M.S., J.D. Professor of Law, George Washington University
APPENDIX IV: U. S. STATE GENETIC LAWS*
* Map
and table current as of March 2012.
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Consent required to DISCLOSE genetic information
AL AK
X
X
X
AZ AR CA
CO
CT DC DE
16 Del. Code Ann.§§ 1201 through 1208 Fla. Stat. § 760.40 (2010) G.A. Code Ann. §§ 3354-1 through 33-54-8 (2011) Haw. Rev. Stat. §§ 431:10A-118 (2010)
FL GA HI ID IL IN IA KS KY LA ME MD MA MI MN MS MO MT
X
X
X
X
Mo. Rev. Stat. §375.1309 (2011)
ordinary course of business ordinary course of business ordinary course of business/ insurance
X
X
X
X
X
X
X
X
X
healthcare provider or insurance company no law on point no law on point comprehensive law
X X
X
X X
comprehensive law insurance
X
insurance
X
no law on point comprehensive law
La. Rev. Stat. Ann. § 22:1023, 40:1299.6 (2011) 22 Me. Rev. Stat. Ann. § 1711-C (2011)
X
no law on point comprehensive law
X
§ 225 Ill. Comp. Stat. 135/90, § 410 ILCS 513/1 through 513/91 (2011) Ind. Code Ann. § 1639-5-2 (LexisNexis 2011)
Mass. Ann. Laws ch. 111, § 70G (2010) Mich. Comp. Laws §§ 333.17020, 333.17520 (2011) Minn. Stat. § 13.386 (2010)
X
Application/ context limited to
Consent required to OBTAIN genetic information Consent required to RETAIN genetic information
Alaska Stat. §§ 18.13.010 through 18.13.100 (2011) Ariz. Rev. Stat. §§ 1-602, 12-2801 through 12-2804, & 20-448-02 (2011) Ark. Code. Ann. §§ 1643-1101, 2035-101 through 20-35-103 (2010) Cal. Civ. Code §§ 56.17, 56.265 (West 2010); Cal Ins Code 10123.35, 10148 through 10149.1 (2010) Colo. Rev. Stat. §§ 10-3-1104.6 & 103-1104.7 (2010)
Citation
Consent required to PERFORM genetic tests
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State
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X
insurance
X
no law on point no law on point no law on point insurance
X
X X
X
X
X X
X
X
X X
healthcare providers no law on point healthcare providers healthcare providers government entity no law on point ordinary course of business no law on point
NE
Neb. Rev. Stat. § 71551 (2010)
X
NV
Nev. Rev. Stat. Ann. §§ 629.141 through 629.201 (2010) N.H. Rev. Stat. Ann. 141-H:1 through 141H:6 (2010) N.J. Stat. Ann. §§ 10:5-44 through 10:5-49 (2011) N.M. Stat. Ann. §§ 24-21-1 through 2421-7 (2010) N.Y. Civ. Rights Law § 79-l (2011)
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X X
X X
X
NH
NJ NM
NY NC ND OH OK OR PA RI
SC SD TN TX UT VT VA WA
WV WI WY
21 Okla. Stat. § 1175 Or. Rev. Stat. §192.531 to 192.549 (SB 618) R.I. Gen. Laws §27-1852, 52.3, §2719-44, 44.1, §27-20-39. 39.1, §27-4153, 53.1 S.C. Code Ann. §3893-10 to §38-9390; S.C. Code Ann. § 16-1-10 S.D. Codified Laws §34-14-14 to -24
X
X
X X
X
insurance comprehensive law no law on point insurance
X
X
V.T. Stat. Ann. tit. 18, §9331 to 9335
X
X X X
X X
X
comprehensive law comprehensive law
X
Tex. Ins. Code Ann. §546.001 et squ.
Wis. Stat. §942.07 Wyo. Stat. Ann. §14-2701 to 710
healthcare providers comprehensive law comprehensive law
comprehensive law no law on point no law on point no law on point newborns comprehensive law no law on point insurance
X
V.A. Code Ann. §38.2508.4 Wash. Rev. Code §70.02.05 through 70.02.90; RCW 49.44.180
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Consent required to DISCLOSE genetic information
Consent required to RETAIN genetic information
Consent required to OBTAIN genetic information
Consent required to PERFORM genetic tests
Citation
State
Privacy and Progress in Whole Genome Sequencing
X X
no law on point comprehensive law insurance healthcare providers no law on point employment perform: comprehensive prohibition; disclose: paternity law
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End Notes 1
The individuals mentioned in these vignettes have given their permission to have their names included. For more detail, please see Section 2: Policy and Governance-Privacy Regulations. 3 The Genomics and Personalized Medicine Act of 2006 (S. 3822, 109th Cong. [2006]; subsequently reintroduced in the Senate in 2007 and in the House of Representatives in 2008 and 2010) encouraged accelerating genetics and genomics research and translating knowledge gained into clinical and public health applications. The proposed bill stated that “pharmaco-genetics has the potential to dramatically increase the efficacy and safety of drugs and reduce health care costs, and is fundamental to the practice of genome-based personalized medicine.” The Act identified several drugs that are more or less effective in people with particular genetic profiles, including the cancer drug Gleevac, the breast cancer drug Herceptin, and the acute lymphoblastic leukemia drug 6mercaptopurine. All four bills were referred to committee and were not enacted. 4 American Medical Association (AMA). (2007). Personalized Health Care Report 2008: Warfarin and Genetic Testing. Retrieved from http://www.ama-assn.org/ama1/pub/upload/ mm/464/warfarin-brochure.pdf. 5 Kolata, G. (2012, September 5). Bits of mystery DNA, far from ‘junk,’ play crucial role. New York Times, p. A1. Retrieved from http://www.nytimes.com/2012/09/06/science/far-from-junk-dna-dark-matter-proves-crucial-tohealth.html?_r=2&hp; Young, E. (2012, September). ENCODE: The rough guide to the human genome. Discover Magazine. Retrieved from http://blogs.discovermagazine.com/notrocketscience/2012/09/05/encodethe-rough-guide-to-the-human-genome/. 6 McGuire, A.L., and J.R. Lupski. (2010). Personal genome research: What should the participant be told? Trends in Genetics, 26(5), 199-201. 7 Kolata, G., op cit. 8 Donley, G., Hull, S.C., and B.E. Berkman. (2012). Prenatal whole genome sequencing: Just because we can, should we? Hastings Center Report, 42(4), 28-40. 9 Cirulli, E.T., and D.B. Goldstein. (2010). Uncovering the roles of rare variants in common disease through wholegenome sequencing. Nature Reviews Genetics, 11, 415-425. 10 The National Commission for the Protection of Human Subjects of Biomedical and Behavioral Research. (1978). The Belmont Report: Ethical Principles and Guidelines for the Protection of Human Subjects of Research. Washington, DC: Department of Health, Education, and Welfare, DHEW Publication OS 78-0012. Retrieved from http://www.hhs.gov/ohrp/ human-subjects/guidance/belmont.html; PCSBI. (2010, December). New Directions: The Ethics of Synthetic Biology and Emerging Technologies. Washington, DC: PCSBI. 11 Pokorska-Bocci, A. (2010). Early examples of clinical use of whole-genome sequencing. Retrieved from http:// www.phgfoundation.org/news/5708/. 12 Beery, R., Mother of twins who benefitted from improved diagnosis gained by whole genome sequencing. (2012). The Beery Family Whole Genome Sequencing Success. Presentation to the Presidential Commission for the Study of Bioethical Issues (PCSBI), February 2, 2012. Retrieved from http://bioethics.gov/cms/ node/658. 13 Topol, E. (2012). The Creative Destruction of Medicine: How the Digital Revolution Will Create Better Health Care. New York: Basic Books. 14 Fan, H.C., et al. (2012). Non-invasive prenatal measurement of the fetal genome. Nature, 487, 320-324; Motluk, A. (2012). Fetal genome deduced from parental DNA, Nature News, doi:10.1038. 15 Topol, E., op cit. 16 Donley, G., et al., op cit. 17 Geller, L.N., et al. (1996). Individual, family, and societal dimensions of genetic discrimination: A case study analysis. Science and Engineering Ethics, 2(1), 71-88; NOVA. (2012). Cracking Your Genetic Code, 46:30 et seq. Retrieved from http://video.pbs.org/ video/2215641935; Suter, S., Professor of Law, George Washington University. (2012). How Technology is Changing Views of Privacy. Presentation to PCSBI, August 1, 2012. Retrieved from http://bioethics.gov/cms/node/748. 18 Allen, A.L. (2011). Privacy Law and Society, 2nd ed. St. Paul, MN: Thomson Reuters. 19 Ibid; DeCew, J. (2012). Privacy. In E.N. Zalta (ed.), The Stanford Encyclopedia of Philosophy (Fall 2012 Edition). Stanford, CA: The Metaphysics Research Lab, Stanford University. Retrieved from http://plato. stanford.edu/archives/fall2012/entries/privacy/. 20 PCSBI. (2010, December). New Directions: The Ethics of Synthetic Biology and Emerging Technologies. Washington, DC: PCSBI. 21 National Commission for the Protection of Human Subjects of Biomedical and Behavioral Research. (1979). The Belmont Report: Ethical Principles and Guidelines for the Protection of Human Subjects of Research. Washington, DC: Department of Health, Education, and Welfare, DHEW Publication OS 78-0012. Retrieved from http://www.hhs.gov/ohrp/ humansubjects/guidance/belmont.html. 22 PCSBI. (2010, December). New Directions: The Ethics of Synthetic Biology and Emerging Technologies. Washington, DC: PCSBI, pp. 24-25, 113. 23 Ibid, pp. 25-27, 123-127. 24 Ibid, pp. 27-28, 141-145. 2
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Ibid. Ibid. 27 Ibid, pp. 28-30, 151. 28 Ibid, pp. 30-31, 161-163. 29 The Commission reached out to the heads of the 18 federal agencies and departments that have adopted the federal Common Rule for protecting human research participants. They are: Department of Agriculture; Department of Commerce; Department of Defense; Department of Education; Department of Energy; Department of Health and Human Services; Department of Homeland Security; Department of Housing and Urban Development; Department of Justice; Department of Transportation; Department of Veterans Affairs; Agency for International Development; Consumer Product Safety Commission; Environmental Protection Agency; National Aeronautics and Space Administration; National Science Foundation; Social Security Administration; and the Central Intelligence Agency. 30 Request for Comments on Issues of Privacy and Access With Regard to Human Genome Sequence Data, 77 Fed. Reg. 18, 247 (March 27, 2012). 31 For example, the American College of Medical Genetics and Genomics is taking the lead in considering which results ought to be returned. A number of federal agencies have looked into direct-to-consumer testing, including the Secretary’s Advisory Committee on Genetic Testing in its report “Enhancing the Oversight of Genetic Tests: Recommendations of the SACGT” (http://oba.od.nih.gov/oba/sacgt/reports/oversight_ report.pdf); its successor, the Secretary’s Advisory Committee on Genetics, Health, and Society in its report “U.S. System of Oversight of Genetic Testing: A Response to the Charge of the Secretary of Health and Human Services” (http://oba.od.nih.gov/oba/sacghs/reports/sacghs_oversight _report.pdf); and by the Government Accountability Office first in its 2006 report “Nutrigenetic Testing: Tests Purchased from Four Web Sites Mislead Consumers” (http://www.gao.gov/new.items/d06977t.pdf) and in its 2010 report “Misleading Test Results Are Further Complicated by Deceptive Marketing and Other Questionable Practices” (http://www.gao.gov/products/GAO10-847T). The Food and Drug Administration has also considered the issue of direct-to-consumer testing, but has not yet published any formal recommendations. And the American Heart Association recently published a report entitled “Genetics and cardiovascular disease: a policy statement from the American Heart Association” that discussed gene patenting and insurance coverage of genetic tests (Ashley, E.A. et al. (2012). Genetics and cardiovascular disease: A policy statement from the American Heart Association. Circulation, 126). 32 Gutmann, A., Chair, PCSBI. (2012). How Technology is Changing Views of Privacy. Addressing PCSBI, August 1, 2012. Retrieved from http://bioethics.gov/cms/node/748. 33 National Commission, op cit. 34 PCSBI, (2010, December), op cit. 35 Ibid, pp. 24-25, 113. 36 The distribution of both scientific knowledge and subsequently of economic opportunity in the field of genome sequencing has been debated within the legal system. In a recent example, Myriad Genetics attempted to patent the BRCA1 and BRCA2 genes, which are associated with breast and ovarian cancer. Myriad Genetics’ actions were contested by the American Civil Liberties Union, which argued that they were in violation of the First Amendment. In August 2012, a U.S. federal appeals court ruled that the company has the right to patent the two genes, stating that the patents would encourage innovation, but simultaneously denied the company’s attempt to patent methods comparing or analyzing DNA sequences. Reuters. (2012, August 16). Court reaffirms right of myriad genetics to patent genes. New York Times. Retrieved from http://www.nytimes. com/2012/08/17/ business/court-reaffirms-right-of-myriad-genetics-to-patent-genes.html?_r=1. 37 Battelle. (n.d.). $3.8 billion investment in Human Genome Project drove $796 billion in economic impact creating 310,000 jobs and launching the genomic revolution. Retrieved from http://www.battelle.org/media/ news/2011/05/11/$3.8-billion-investment-in-human-genome-project-drove-$796-billion-in-economicimpactcreating-310-000-jobs-and-launching-the-genomic-revolution. 38 PCSBI, (2010, December), op cit. 39 Aristotle, Politics, (Benjamin Jowett trans., Dover first ed. 2000) (350 B.C.); DeCew, J.W. (1997). In Pursuit of Privacy. Ithaca, NY: Cornell Press, p.10. 40 Warren, S.D., and L.D. Brandeis. (1890), The Right to Privacy, 4 Harv. L. Rev. 5. 41 Lowrance, W. (2012). Privacy, Confidentiality, and Health Research. New York: Cambridge University Press, p. 40-47. 42 Allen, A.L. (1997). Genetic privacy: Emerging concepts and values. In M. Rothstein (Ed.), Genetic Secrets: Protecting Privacy and Confidentiality in the Genetic Era (pp. 31-59). New Haven, CT: Yale University Press; Allen, A.L. (1999). Coercing Privacy, 40 Wm. & Mary L. Rev. 723 (1999); DeCew, J.W. (2004). Privacy and policy for genetic research. Ethics and Information Technology, 6, 5-14; Farahany, N.A. (2012). Searching Secrets, 160 U. Penn. L. Rev. 1239, 1255. 43 Fried, C. (1970). An Anatomy of Values. Cambridge: Harvard University Press; Moore v. Regents of the Univ. of Cal., 51 Cal. 3d 120 (Cal. 1990); Rachels, J. (1975). Why privacy is important. Philosophy & Public Affairs, 4(4), 323-333. 44 Newcombe, H.B. (1994). Cohorts and privacy. Cancer Causes and Control, 5(3), 287-291. 26
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Allen, A.L. (2009). Confidentiality: An expectation in healthcare. In Ravitsky, V., Fiester, A., and A.L. Caplan (Eds.), The Penn Center Guide to Bioethics. (pp.127-135). New York: Springer Publishing Co. 46 Goldstein, M., Associate Professor, Department of Health Policy, School of Public Health and Health Services, George Washington University, and McGraw, D., Director, Health Privacy Project, Center for Democracy and Technology. (2012). Comments submitted to PCSBI, May 25, 2012; Health Privacy Project. (2007). Health Privacy Stories. Retrieved from https://www.cdt.org/healthprivacy/20080311stories.pdf. 47 Williams, S., et al. (2009). The Genetic Town Hall: Public Opinion About Research on Genes, Environment, and Health. Washington, DC: Genetics and Public Policy Center. Retrieved from http://www.dnapolicy.org/pub. reports.php?action=detail&report_id=27. 48 Kaufman, D.J., et al. (2009). Public opinion about the importance of privacy in biobank research. The American Journal of Human Genetics, 85(5), 643-654. 49 Genetics and Public Policy Center. (2007, April 24). U.S. Public Opinion on Uses of Genetic Information and Genetic Discrimination. Retrieved from http://www.dnapolicy.org/ resources/GINAPublic_Opinion_ Genetic_Information_Discrimination.pdf. 50 McGuire, A.L., et al. (2008). DNA data sharing: Research participants’ perspectives. Genetics in Medicine, 10(1), 46-53. 51 For a survey of self-reported genetic discrimination, see Geller, L.N., et al. (1996). Individual, family, and societal dimensions of genetic discrimination: A case study analysis. Science and Engineering Ethics, 2(1), 71-88; NOVA. (2012, March 28). Cracking Your Genetic Code, 46:30 et seq. Retrieved from http://video.pbs.org/ video/2215641935 (Discussing potential harms from whole genome sequencing at birth). 52 Powers, M., Professor, Department of Philosophy, Senior Research Scholar, Kennedy Institute of Ethics, Georgetown University. (2012). Theory and Practice of a Right to Privacy. Presentation to PCSBI, May 17, 2012. Retrieved from http://bioethics.gov/cms/node/712. 53 Andrews, L.B. (2001). Future Perfect. New York: Columbia University Press. 54 For example in EEOC v. Burlington Northern and Santa Fe Railway, the Federal District Court for the Northern District of Iowa addressed the scope of protection from genetic discrimination under the Americans with Disabilities Act. In this case, the railway required that employees who complained of carpal tunnel syndrome undergo genetic testing for a genetic predisposition. No. C01-4013 (N.D. Iowa Feb. 9, 2001). 55 Sweeney, L., Visiting Professor and Scholar, Computer Science; Director, Data Privacy Lab, Harvard University. (2012). How Technology is Changing Views of Privacy. Presentation to PCSBI, August 1, 2012. Retrieved from http://bioethics.gov/cms/node/748. 56 The Belmont Report recognizes that not all persons can act as autonomous agents, and makes clear that there are special responsibilities to those who cannot. National Commission for the Protection of Human Subjects of Biomedical and Behavioral Research. (1979). The Belmont Report: Ethical Principles and Guidelines for the Protection of Human Subjects of Research. Washington, DC: Department of Health, Education, and Welfare, DHEW Publication OS 78-0012. Retrieved from http://www.hhs.gov/ohrp/humansubjects/guidance/ belmont.html. 57 Brock, D. (1999). A critique of three objections to physician-assisted suicide. Ethics, 109(3), 523-524. 58 PCSBI. (2010, December). New Directions: The Ethics of Synthetic Biology and Emerging Technologies. Washington, DC: PCSBI. 59 Ibid. 60 Ibid, p. 31. 61 Sweeney, L., Visiting Professor and Scholar, Computer Science, Director, Data Privacy Lab, Harvard University. (2012). How Technology is Changing Views of Privacy. Presentation to PCSBI, August 1, 2012. Retrieved from http://bioethics.gov/cms/node/748. 62 PCSBI, (2010, December), op cit, p.5. 63 Ibid. 64 Gutmann, A., and D. Thompson. (1996). Democracy and Disagreement. Cambridge, MA: Harvard University Press. 65 Trinidad, S.B., et al. (2010). Genomic research and wide data sharing: Views of prospective participants. Genetics in Medicine, 12(8), 486-495. 66 Lake Research Partners and American Viewpoint. (2006). National Survey on Electronic Personal Health Records, conducted by the Markle Foundation. Retrieved from http://www.markle.org/sites/default/files/ research_doc_120706.pdf. 67 Gottweis, H., and K. Zatloukal. (2007). Biorepository governance: Trends and perspectives. Pathobiology, 74(4), 206-211. 68 In the early 1990s, researchers obtained consent from the Havasupai Indian tribe to collect samples and conduct research on a genetic link to diabetes. The University of Arizona conducted the initial research, and later used the samples from the Havasupai to perform unrelated studies, including genetic and medical records analysis of inbreeding, schizophrenia, migration history, and genealogy. The Havasupai sued the University, alleging that it misused their genetic information, and that it conducted research for which it never obtained informed consent. The lawsuit resulted in a large settlement and destruction of the samples. Eriksson, S., and G. Helgesson. (2005).
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Potential harms, anonymization, and the right to withdraw consent to biobank research. European Journal of Human Genetics, 13(9), 1071-1076; Harmon, A. (2010, April 21). Indian tribe wins fight to limit research of its DNA. New York Times. Retrieved from http://www.nytimes.com/2010/04/22/us/22dna. html?pagewanted=all. 69 The Common Rule is a federal regulation regarding human subject research, adopted by 18 federal agencies; see footnote 29. 70 Those agencies are: 1) USAID; 2) CIA; 3) CPSC; 4) USDA; 5) DOC; 6) DOE; 7) ED; 8) EPA; 9) HUD; 10) NASA; 11) NSF; and 12) SSA. SSA does receive genetic information that it considers personally identifiable information and deals with the information accordingly. 71 USDA indicated that its Agricultural Research Service expects to conduct human research programs in the future that may include more extensive use of whole genome analysis or sequencing. 72 Chief Information Officer, Office of Information Services, Centers for Medicare & Medicaid Services. (2007). Policy for Privacy Act Implementation & Breach Notification. Retrieved from https://www.cms.gov/ SystemLifecycleFramework/downloads/privacypolicy.pdf; E-Government Act, 116 Stat. 2899 (2002); Federal Information Security Management Act (FIMSA), 116 Stat. 2899 (2002); Health Information Technology for Economic and Clinical Health (HITECH), 123 Stat. 115 (2009); Health Insurance Portability and Accountability Act (HIPAA), 110 Stat. 1936 (1996); Policy for Privacy Act Implementation and Breach Notification, 5 U.S.C.§ 552a. 73 DOD. (2002). DOD Directive 6025.18, Privacy of Individually Identifiable Health Information in DOD Health Care Programs. December 19, 2002. Retrieved from http://biotech.law.lsu.edu/blaw/dodd/corres/ html/602518.htm; DOD Regulation, DOD Directive 8580.02-R. (2007). DOD Health Information Security Regulation; DOD Regulation, DOD Directive 5400.11-R. (2007). Department of Defense Privacy Program. 74 Letter from Earl C. Wyatt, Deputy Assistant Secretary of Defense, and Rapid Fielding, DOD to Amy Gutmann, Chair, PCSBI. (April 27, 2012). 75 HITECH, Key provisions codified at 42 U.S.C. §§17931 et seq.; HIPAA, Key provisions codified at 42 U.S.C. §§ 1320d et seq.; FISMA, Key provisions codified at 44 U.S.C. §§ 3541 et seq. and 40 U.S.C. § 11331; Confidential Information Protection and Statistical Efficiency Act (CIPSEA), 44 U.S.C. § 3501 note, see also the OMB Implementation Guidance on CIPSEA, 72 Fed. Reg. § 33361 (2007); AHRQ general provisions, 42 U.S.C. § 299c-3(c); SAMHSA General Provisions, 42 U.S.C. § 290aa(n); Privacy Act of 1974, 5 U.S.C. § 552a; Confidentiality of information from health statistics and other activities, 42 U.S.C. § 242m(d). 76 Letter from Kathleen Sebelius, Secretary, HHS to Amy Gutmann, Chair, PCSBI. (May 16, 2012). 77 The Public Health Service Act, 42 U.S.C. §242m, part 301(d). 78 Letter from Kathleen Sebelius, Secretary, HHS to Amy Gutmann, Chair, PCSBI. (May 16, 2012). 79 Ibid. 80 Ibid. 81 NIH. (2007). NIH Policy for Sharing of Data Obtained in NIH Supported or Conducted Genome-Wide Association Studies (GWAS). Retrieved from http://grants.nih.gov/grants/ guide/notice-files/NOT-OD-07088.html; See updated data access policy at http://gwas.nih. gov/pdf/Data Sharing Policy Modifications.pdf. 82 Federal Bureau of Investigation (FBI). (2012, June). CODIS-NDIS Statistics [web post]. Retrieved from http://www.fbi.gov/about-us/lab/codis/ndis-statistics. 83 FBI. (n.d.). Frequently Asked Questions (FAQs) on the CODIS Program and the National DNA Index System [web post]. Retrieved from http://www.fbi.gov/about-us/lab/codis/ codis-and-ndis-fact-sheet/. 84 Collection and Use of DNA Identification Information from Certain Federal Offenders Act, 42 U.S.C. § 14135a; Collection of DNA Samples, 28 C.F.R. §28.12. 85 VA. (2011). The Million Veteran Program: VA’s Genomics Game-Changer Launches Nationwide [Press Release]. Retrieved from http://www.va.gov/opa/pressrel/pressrelease. cfm?id=2090. 86 VA has departmental policies, including the Notice of Privacy Handbook Practice 1605.04 and Determining Service Connection for Congenital, Developmental, or Hereditary Disorders. See Letter from Robert A. Petzel, Under Secretary for Health, VA to Amy Gutmann, Chair, PCSBI. (May 1, 2012). 87 VA. (2011). The Million Veteran Program: VA’s Genomics Game-Changer Launches Nationwide [Press Release]. Retrieved from http://www.va.gov/opa/pressrel/pressrelease. cfm?id=2090. 88 Letter from Judith S. Kaleta, Deputy General Counsel, DOT to Amy Gutmann, Chair, PCSBI. (August 1, 2012). 89 Bregman-Eschet, Y. (2006). Genetic Databases And Biobanks: Who Controls Our Genetic Privacy? 23 Santa Clara Computer & High Tech. L.J. 1. 90 23andMe. (2012, April 10). What is the difference between genotyping and sequencing? [Frequently Asked Questions web post]. Retrieved from https://customercare.23andme.com/ entries/21262606. 91 US Patent Number 8,187,811 (filed Nov. 30, 2010). 92 Lowrance, W. (2012). Privacy, Confidentiality, and Health Research. New York: Cambridge University Press, p. 48. 93 See e.g., Farahany, N.A. (2012).Searching Secrets, 160 U. Penn. L. Rev. 1277-83 (2012). 94 Confidentiality and Disclosure of Returns and Return Information, 26 U.S.C. § 6103(d) et. seq. 95 See e.g., Census Confidentiality Statute of 1954, 13 U.S.C. §9 (1954); HIPAA Privacy Rule, 45 C.F.R. § 164.514.
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Fair Credit Reporting Act, 15 U.S.C. § 1681 et seq.; Privacy Act of 1974, 5 U.S.C. § 552a; HHS. (2004). The Confidentiality of Alcohol And Drug Abuse Patient Records Regulation and the HIPAA Privacy Rule: Implications for Alcohol and Substance Abuse Programs. June. Retrieved from http://www.nj.gov/ humanservices/das/information/SAMHSA-Pt2-HIPAA.pdf; Family Educational Rights and Privacy Act of 1974, 20 U.S.C. § 1232g; Electronic Communications Privacy Act of 1986, 18 U.S.C. § 2510-22; Video Privacy Act of 1988, 18 U.S.C. § 2710; Children’s Online Privacy Protection Act of 1998, 15 U.S.C. §§ 6501–6506; and Gramm-Leach-Bliley Act, Pub. L. 106-102, 113 Stat. 1338. 97 Department of Health, Education, and Welfare (DHEW). (1973). Records, Computers and the Rights of Citizens: Report of the Secretary’s Advisory Committee on Automated Personal Data Systems. Retrieved from http://aspe.hhs.gov/datacncl/1973privacy/ tocprefacemembers.htm. The Electronic Communications Privacy Act of 1986 is not a fair information statute, but rather a set of rules regulating government and other access to the many modes of communications used in daily life. 98 DHEW. (1973). Records, Computers and the Rights of Citizens: Report of the Secretary’s Advisory Committee on Automated Personal Data Systems. Retrieved from http://aspe. hhs.gov/datacncl/1973privacy/ tocprefacemembers.htm. 99 HIPAA, Pub. L. 104-191, 110 Stat. 1936, enacted August 21, 1996. 100 HIPAA Privacy Rule, 45 C.F.R. § 164.501. 101 HIPAA Privacy Rule, 45 C.F.R. § 160, 164. 102 HIPAA Privacy Rule, 45 C.F.R. § 164.514. 103 On the other hand, deceased individuals are not considered “human subjects” under, and are therefore not covered by, the Common Rule. 45 C.F.R. § 46.102(f). 104 HIPAA Administrative Simplification: Standards for Privacy of Individually Identifiable Health Information, Proposed Rule, 74 Fed. Reg. 51698 (Oct. 7, 2009). 105 Institute of Medicine (IOM). (2009). Beyond the HIPAA Privacy Rule: Enhancing Privacy, Improving Health Through Research. Washington, DC: National Academies Press. 106 HIPAA Privacy Rule, 45 C.F.R. § 164.502. 107 HIPAA Privacy Rule, 45 C.F.R. § 164.502(b). 108 HIPAA Privacy Rule, 45 C.F.R. § 164.502. 109 HITECH, 42 U.S.C. § 300jj. 110 The Office of the National Coordinator for Health Information Technology [web post]. (n.d.). Retrieved from http://healthit.hhs.gov/portal/server.pt/community/healthit_hhs_gov__onc/ 1200. 111 Protection of Human Subjects, 45 C.F.R. § 46. 112 Office for Human Research Protections (OHRP). (2008). OHRP Guidance on Research Involving Coded Private Information or Biological Specimens. October 16. Retrieved from http://www.hhs.gov/ohrp/policy/ cdebiol.html. 113 Human Subject Research Protections: Enhancing Protections for Research Subjects and Reducing Burden, Delay, and Ambiguity for Investigators, 76 Fed. Reg. 44512, 44524. 114 Ibid. 115 See e.g., The Personal Information Protection and Electronic Documents Act, S.C. 2000, c. 5 (Canada, 2000); The Personal Data Act (523/1999) (Finland, 1999); The Federal Data Protection Act, BDSG 2003 (Germany, 2003); Act on the Protection and Processing of Personal Data, No. 77/2000 (Iceland, 2000); Personal Data Protection Code, Legisl. Ital. 196 (Italy, 2003); Data Protection Act, Cap. 440 (Malta, 2001); Personal Data Protection Act, 95/46/EC (Netherlands, 2001); Privacy Act, Public Act 1993 No 28 (New Zealand, 1993). 116 European Union, Directive 95/46/EC (1995). 117 See e.g., The Personal Information Protection and Electronic Documents Act, S.C. 2000, c. 5 (Canada, 2000); European Union, Directive 95/46/EC (1995). 118 See e.g., Patient Rights’ Act, Law No. 482 (Denmark, 1998); Patients’ Rights Act, No. 63 (Norway, 1999). 119 See e.g., Law on the Rights of Patients, (Belgium, 2002); Act on the Status and Rights of Patients, No.785/1992 (Finland, 1992); Patient’s Rights Act (Romania, 1996). 120 Law No. 20120 on scientific research on human beings, the human genome, and the prohibition of cloning (Chile, 2007); see also, Human Genome Research Act, RT I 2000, 104, 685 (Estonia, 2000); Rights of Users of Genetic Services Act (France, 2004); Law 14/2007 on Biomedical Research (Spain, 2007); Human Genetic Examination Act (Germany, 2009); Genetic Information Law, 5761-2000 (Israel, 2000). 121 See e.g., Law on the Rights of Patients, No. 283-IIS (Georgia, 2000); Human Genetic Examination Act (Germany, 2009). 122 Genetic Information Nondiscrimination Act (GINA), 122 Stat. 881-922 (2008). 123 Kang, P.B. (2011). Presymptomatic and early symptomatic genetic testing. Neurogenetics, 2, 343-6. 124 Kostecka, B.E. (2009). GINA Will Protect You, Just Not From Death: The Genetic Information Nondiscrimination Act and Its Failure to Include Life Insurance within Its Protections, 34 Seton Hall Legis. J. 93 (2009). 125 HIPAA, 29 U.S.C. §§1181-82, 42 U.S.C. §§300gg-41. 126 GINA, 122 Stat. 881-922 (2008). 127 Ibid.
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Rothstein, M.A. (n.d.). GINA’s beauty is only skin deep. Genewatch. Retrieved from http:// www.councilforresponsiblegenetics.org/GeneWatch/GeneWatchPage.aspx?pageId=184. 129 Kostecka, op cit. 130 See Appendix IV: State Law Tables. Also see Genome Statute and Legislation Database. (2010). Retrieved from http://www.genome.gov/PolicyEthics/LegDatabase/pubsearch.cfm 131 See e.g., Del. Code Ann. §§ 12.2.1220 to 12.2.1227. 132 See e.g., Ariz. Rev. Stat. Ann. §12-2801-4, §20-448.02. 133 See e.g., Colo. Rev. Stat. 10-3-1104.7. 134 Whalen v. Roe 429 U.S. 589 (1977); Sorrell v. IMS Health Inc., 131 S. Ct. 2653 (2011). 135 For a discussion on how Courts have generally disfavored a property-rights analysis in identifying information, see Farahany, op cit. 136 Moore v. Regents of the Univ. of Cal., 51 Cal. 3d 120 (Cal. 1990). 137 Rodriguez, L.L., Director, Office of Policy, Communications, and Education, National Human Genome Research Institute, NIH. (2012). Presentation to PCSBI, August 1, 2012. Retrieved from http://bioethics.gov/ cms/node/749. 138 Knoppers, B., Director, Centre of Genomics and Policy, Canada Research Chair in Law and Medicine, McGill University. (2012). Presentation to PCSBI, August 1, 2012. Retrieved from http://bioethics.gov/cms/node/740. 139 HITECH, Pub. L. 111-5, 123 Stat. 115 (2009); E-Government Act, Pub. L. 107-347, 116 Stat. 2899 (2002); HIPAA, Pub. L. 104-191, 110 Stat. 1936 (1996); Privacy Act, 5 U.S.C. § 552a; GINA, Pub. L. No. 110-233, 122 Stat. 881 (2008); PCSBI. (2012). Analysis of Responses to Common Rule Agency Data Call; See Appendix IV: U.S. State Genetic Laws. 140 An example of a third-party storage and analysis provider is cloud computing services, which include web-based systems of virtual servers. 141 Rachels, J. (1975). Why privacy is important. Philosophy & Public Affairs, 4(4), 323-333. 142 Sweeney, L.S., Visiting Professor and Scholar, Computer Science; Director, Data Privacy Lab, Harvard University. (2012). How technology is changing views of privacy. Presentation to PCSBI, August 1, 2012. Retrieved from http://bioethics.gov/cms/node/748. 143 Harmon, A. (2010, April 21). Indian tribe wins fight to limit research of its DNA. New York Times. Retrieved from http://www.nytimes.com/2010/04/22/us/22dna.html?pagewanted=all. 144 Bearder v. State, 788 N.W.2d 144 (2010); Settlement Agreement and Release, Beleno v. Tex. Dept. of State Health Servs., No. SA-09-CA-188-FB (W.D. Tex. 2009). Document obtained from plaintiffs’ attorney, Jim Harrington, at the Texas Civil Rights Project. 145 NIH. (2008). Policy for Sharing of Data Obtained in NIH Supported or Conducted Genome-Wide Association Studies (GWAS). Retrieved from http://grants.nih.gov/grants/guide/ notice-files/NOT-OD-07088.html. 146 GINA, Pub. L. No. 110-233, 122 Stat. 881 (2008). 147 Baruch, S., and K. Hudson. (2008). Civilian and military genetics: Nondiscrimination policy in a postGINA world. American Journal of Human Genetics, 83, 435-444; Greenbaum, D., et al. (2011). Genomics and privacy: Implications of the new reality of closed data for the field. PLoS Computational Biology, 7(12), e1002278; Hayden, E.C. (2012). A broken contract. Nature, 486, 312-314. 148 The Affordable Care Act of 2010 helps mitigate concerns about obtaining insurance with its prohibition on discriminating against individuals with pre-existing conditions. 149 Sweeney, L.S., Visiting Professor and Scholar, Computer Science Director, Data Privacy Lab, Harvard University. (2012). How Technology is Changing Views of Privacy. Presentation to PCSBI, August 1, 2012. Retrieved from http://bioethics.gov/cms/node/748. 150 Genetics and Public Policy Center. (2009, January 21). State laws pertaining to surreptitious DNA testing. Retrieved from http://www.dnapolicy.org/resources/State_law_summaries_ final_all_states.pdf. 151 Genomics Law Report. (2010). Surreptitious genetic testing: WikiLeaks highlights gap in genetic privacy law. Retrieved from http://www.genomicslawreport.com/index.php/ 2010/12/09/surreptitious-genetic-testingwikileaks-highlights-gap-in-genetic-privacy-law/. 152 Lake Research Partners and American Viewpoint. (2006). National Survey on Electronic Personal Health Records, conducted by the Markle Foundation. Retrieved from http://www.markle.org/sites/default/files/ research_doc_120706.pdf. 153 Aldhous, P., and M. Reilly. (2009). Special investigation: How my genome was hacked. New Scientist. Retrieved from http://www.newscientist.com/article/mg20127013.800-special-investigation-how-my-genome-washacked.html?page=1. 154 Green, R.C., and G.J. Annas. (2008). The genetic privacy of presidential candidates. New England Journal of Medicine, 359(21), 2192-2193. 155 HHS Health Information Privacy, HIPAA Breach Notification Rule, Breaches Affecting 500 or More Individuals. Retrieved from http://www.hhs.gov/ocr/privacy/hipaa/administrative/ breachnotificationrule/ breachtool.html. 156 Memorial Sloan-Kettering Cancer Center. (2012, June 15). Privacy Alert. Retrieved from http://www.mskcc.org/public-notices/privacy-alert. 128
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Sweeney, L.S., Visiting Professor and Scholar, Computer Science Director, Data Privacy Lab, Harvard University. (2012). How Technology is Changing Views of Privacy. Presentation to PCSBI, August 1, 2012. Retrieved from http://bioethics.gov/cms/node/748. 158 Terry, S.F., and P.F. Terry. (2001). A consumer perspective on informed consent and third party issues. Journal of Continuing Education in the Health Professions, 21, 256-264. 159 HHS Health Information Privacy, HIPAA Breach Notification Rule, Breaches Affecting 500 or More Individuals. Retrieved from http://www.hhs.gov/ocr/privacy/hipaa/administrative/ breachnotificationrule/ breachtool.html. 160 Dissemination of Information (AHRQ), 42 USC §299C-3; General Provisions Respecting Effectiveness, Efficiency, and Quality of Health Services (CDC), 42 USC §242M; Justice System Improvement Administrative Provisions, 42 USCS § 3789g. Confidentiality of Information; The Public Health and Welfare Act, 42 U.S.C. § 46. 161 NIH. (2002). Notice NOT-OD-02-037, NIH Announces Statement on Certificates of Confidentiality. March 15. Retrieved from http://grants.nih.gov/grants/guide/notice-files/NOT-OD-02-037.html. 162 Cooper, Z.N., Nelson, R.M., and L.F. Ross. (2004). Certificates of confidentiality in research: Rationale and usage. Genetic Testing, 8(2), 214-220. This study sampled three NIH institutes: The National Human Genome Research Institute (NHGRI); the National Heart, Lung, and Blood Institute (NHLBI); and the National Institute of Neurological Disorders and Stroke (NINDS). 163 Angrist, M. (2010). Urge overkill: Protecting deidentified human subjects at what price? Health Privacy in Research, 10(9), 17-18; Catania, J.A., et al. (2007). Research participants’ perceptions of the certificate of confidentiality’s assurances and limitations. Journal of Empirical Research on Human Research Ethics, 2(4), 53-59; Cooper, Z.N., Nelson, R.M., and L.F. Ross. (2004). Certificates of confidentiality in research: Rationale and usage. Genetic Testing, 8(2), 214-220; Hudson, K., and S. Devaney. (2008). Novel forensic technique highlights need for greater privacy protection for research participants [News release]. Retrieved from http://www.dnapolicy.org/news.release.php?action=detail& pressrelease_id=104; Lo, B., and M. Barnes. (2011). Protecting research participants while reducing regulatory burdens. Journal of the American Medical Association, 306(20), 2260-2261; Wolf, L.E., and J. Zandecki. (2006). Sleeping better at night: Investigators’ experiences with certificates of confidentiality. IRB, 28(6), 1-7. 164 Liang, B.A., and T. Mackey. (2011). Reforming direct-to-consumer advertising. Nature Biotechnology, 29(5), 397400. 165 Dissemination of Information (AHRQ), 42 USC §299c-3(c). The confidentiality statute that is part of AHRQ’s authorizing legislation, grounded in judicially recognized public policies intended to foster participation in and the conduct of research, provides a respected form of federal statutory protection for all identifiable data submitted to the Agency, its grantees and contractors, for research purposes and permits no disclosures or uses of it, other than those consented to by the suppliers of the data or by the research subjects. Memorandum from Susan Greene Merewitz, Senior Attorney, AHRQ to Nancy Foster, Coordinator for Quality Activities, AHRQ. (April 16, 2001). Statutory Confidentiality Protection of Research Data. Retrieved from http://www.ahrq.gov/fund/datamemo.htm. 166 Office of Science and Technology Policy (OSTP). (2012, March 29). Obama administration unveils “Big Data” initiative: Announces $200 million in new R&D investments [Press release]. Retrieved from http://www.whitehouse.gov/sites/default/files/microsites/ostp/big _data_press_release_final_2.pdf. 167 National Human Genome Research Institute (NHGRI). (2012). 1000 Genomes Project data available on Amazon Cloud [Press release]. Retrieved from http://www.nih.gov/news/ health/mar2012/nhgri-29.htm. 168 Breach Notification for Unsecured Protected Health Information, 74 Fed. Reg. 42,740 (Aug. 24, 2009) (codified at 45 C.F.R. §§ 160, 164); Health Breach Notification Rule; Final Rule, 74 Fed. Reg. 42,962 (Aug. 25, 2009) (codified at 16 C.F.R. § 318); HIPAA Administrative Simplification: Enforcement; Final Rule, 71Fed. Reg. 8,390 (Feb. 16, 2006) (codified at 45 C.F.R. §§ 160, 164); Standards for Privacy of Individually Identifiable Health Information; Final Rule, 65 Fed. Reg. 82,462 (Dec. 28, 2000) (codified at 45 C.F.R. §§ 160, 164); Health Insurance Reform: Security Standards; Final Rule, 68 Fed. Reg. 8,334 (Feb. 20, 2003) (codified at 45 C.F.R. §§ 160, 162, and 164). 169 HHS. (2010). HHS Strengthens Health Information Privacy and Security through New Rules [Press Release]. Retrieved from http://www.hhs.gov/news/press/2010pres/07/20100708c. html; National Institutes for Standards and Technology (NIST). (2012). Guidelines on Security and Privacy in Public Cloud Computing. Retrieved from http://csrc.nist.gov/publications/nistpubs/800-144/SP800-144.pdf. 170 OHRP. (2008). Guidance on Research Involving Coded Private Information or Biological Specimens. October 16. Retrieved from http://www.hhs.gov/ohrp/policy/cdebiol.html. 171 Paasche-Orlow, M.K., Taylor, H.A., and F.L. Brancati. (2003). Readability standards for informed-consent forms as compared to actual readability. New England Journal of Medicine, 348(8), 721-726. 172 PRIM&R. (McGuire, A.L.) (2012). Data sharing in genomic research: Participant attitudes and ethical issues. [Webinar]. 173 Kaufman, D.J., et al. (2009). Public opinion about the importance of privacy in biobank research. American Journal of Human Genetics, 85, 643-654.
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The Family Educational Rights and Privacy Act (FERPA), 20 U.S.C. § 1232g; 34 C.F.R. § 99; Children’s Online Privacy Protection Act of 1998, 18 U.S.C. §§ 6501-6506; Parent Initiated Alternatives Act of 2005, HB10582005-06 (Washington); Hickey, K. (2007). Minors’ rights in medical decision making. Journal of Nursing Administration Health Care Law, Ethics, and Regulation, 9(3), 100-104. 175 AMA. (1996). AMA Code of Medical Ethics, Opinion 2.138: Genetic testing of children. Retrieved from http://www.ama-assn.org/ama/pub/physician-resources/medical-ethics/code-medical-ethics/opinion2138. page; American Academy of Pediatrics. (2001). Ethical issues with genetic testing in pediatrics. Pediatrics, 107(6), 1451-1455; Donley, G., Hull, S.C., and B.E. Berkman. (2012). Prenatal Whole Genome Sequencing: Just Because We Can, Should We? Hastings Center Report, 42(4), 28-40. 176 Gostin, L.O. (2009). Privacy: Rethinking health information technology and informed consent. In M. Crowley (Ed.), Connecting American Values with Health Reform. Garrison, NY: The Hastings Center, pp. 15-17. 177 Kaye, J., et al. (2012). From patients to partners: Participant-centric initiatives in biomedical research. Nature Reviews Genetics, 13(5), 371-376; Nietfeld, J.J., Sugarman, J., and J.E. Litton. (2011). The Bio-PIN: A concept to improve biobanking. Nature Reviews Cancer, 11, 303-308; Time to open up. (2012). Nature, 486, 293. 178 Consent to Research (n.d.). About Us. Retrieved from http://weconsent.us/about. 179 Brothers, K.B., Morrison, D.R., and E.W. Clayton. (2011). Two large-scale surveys on community attitudes toward an opt-out biobank. American Journal of Medical Genetics, Part A, 155, 2982-2990. 180 Kaufman, D.J., et al. (2009). Public opinion about the importance of privacy in biobank research. American Journal of Human Genetics, 85, 643-654. 181 Valle-Mansilla, J.I., Ruiz-Canela, M., and D.P. Sulmasy. (2010). Patients’ attitudes to informed consent for genomic research with donated samples. Cancer Investigation, 28(7), 726-734. 182 Human Subjects Research Protections: Enhancing Protections for Research Subjects and Reducing Burden, Delay, and Ambiguity for Investigators, 76 Fed. Reg. 143, 44,513 (July 26, 2011). 183 Colditz, G.A. (2009). Constraints on data sharing: Experience from the Nurses’ Health Study. Epidemiology, 20(2), 169-171. 184 One potential result of this sharing of data between physicians and researchers is the publication of findings in academic journals. In the interest of making data sharing an element of the scholarly publication process, some journals require that, if public repositories have been established for a particular type of data (including whole genome sequence data), all data from which results were drawn should be deposited in open access databases before publication. 185 Knoppers, B., Director, Centre of Genomics and Policy, Canada Research Chair in Law and Medicine, McGill University. (2012). Consent and Return of Findings. Presentation to PCSBI, August 1, 2012. Retrieved from http://bioethics.gov/cms/node/740. 186 Johnson, E.J., and D. Goldstein. (2003). Do defaults save lives? Science, 302, 1338-1339. 187 Donate Life America. (2012). 2012 National Donor Designation Report Card Released [Press Release]. Retrieved from http://donatelife.net/2012-national-donor-designation-report-card-released/; Goldman, R. (2012, May 2). States see instant spike in organ donors following Facebook push. ABC News. Retrieved from http://abcnews.go.com/Health/states-instant-spike-organ-donors-facebook-push/story?id=16255979. 188 Sunstein, C.R., and R.H. Thaler. (2003). Libertarian paternalism is not an oxymoron. University of Chicago Law Review. Retrieved from http://www.law.uchicago.edu/ files/files/185.crs_.paternalism.pdf; Thaler, R.H., and C.R. Sunstein. (2008). Nudge: Improving Decisions About Health, Wealth, and Happiness. New Haven, CT: Yale University Press. 189 Human Subjects Research Protections: Enhancing Protections for Research Subjects and Reducing Burden, Delay, and Ambiguity for Investigators, 76 Fed. Reg. 143, 44,513 (July 26, 2011). 190 McGuire, A.L., and L.M. Beskow. (2010). Informed consent in genomics and genetic research. Annual Review of Genomics and Human Genetics, 11, 361-381. 191 Bollinger, J.M., et al. (2012). Public preferences regarding the return of individual genetic research results: Findings from a qualitative focus group study. Genetics in Medicine, 14(4), 451-457; Murphy, J., et al. (2008). Public expectations for return of results from large-cohort genetic research. American Journal of Bioethics, 8(11), 36-34; Terry, S.F., and P.F. Terry. (2011). Power to the people: Participant ownership of clinical trial data. Science Translational Medicine, 3(69), 1-3. 192 Murphy, J., et al. (2008). Public expectations for return of results from large-cohort genetic research. American Journal of Bioethics, 8(11), 36-43. 193 Maschke, K.J. (2012). Returning genetic research results: Considerations for existing no-return and future biobanks. Minnesota Journal of Law, Science, and Technology, 13(2), 559-573. 194 Clinical Laboratory Improvement Amendments of 1988 (CLIA), 42 U.S.C. § 263a (2006). 195 Sharp, S.E., and B.L. Elder. (2004). Competency assessment in the clinical microbiology laboratory. Clinical Microbiology Review, 17(3), 681-694. 196 CLIA, 42 U.S.C. § 263a (2006). 197 Hudson, K., et al. (2006). Oversight of U.S. genetic testing laboratories. Nature Biotechnology, 24(9), 10831091; Ledbetter, D.H., and W.A. Faucett. (2008). Issues in genetic testing for ultra-rare diseases: Background and introduction. Genetics in Medicine, 10(5), 309-313. 174
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Wolf, S.M. (2012). The past, present, and future of the debate over return of research results and incidental findings. Genetics in Medicine, 14(4), 355-357. 199 Green, R., et al. (2012). Exploring concordance and discordance for return of incidental findings from clinical sequencing. Genetics in Medicine, 14(4), 1-6. 200 GARNET. (2012). GARNET Statement on Incidental Findings and Potentially Clinically Relevant Genetic Results. May 18. Retrieved from https://www.garnetstudy.org/sites/ www/content/files/subcom/if/GARNET_ RORstatement_final.pdf. 201 Kohane, I.S., et al. (2007). Reestablishing the researcher-patient compact. Science, 316, 836-837. 202 Wolf, S.M., et al. (2012). Managing incidental findings and research results in genomic research involving biobanks and archived data sets. Genetics in Medicine, 14(4), 361-384. 203 Berg, J.S., Khoury, M.J., and J.P. Evans. (2011). Deploying whole genome sequencing in clinical practice and public health: Meeting the challenge one bin at a time. Genetics in Medicine, 13(6), 499-504. 204 Kohane, I.S., and P.L. Taylor. (2010). Multidimensional results reporting to participants in genomic studies: Getting it right. Science Translational Medicine, 2(37), 1-4. 205 Angrist, M. (2011). You never call, you never write: Why return of ‘omic’ results to research participants is both a good idea and a moral imperative. Personalized Medicine, 8(6), 651-657; Kohane, I.S., et al. (2007), op cit.; Terry, S., and R. Cook-Deegan. (2012, June 8). Your genome belongs to you. Health Affairs Blog. Retrieved from http://healthaffairs.org/ blog/2012/06/08/your-genome-belongs-to-you/; Time to open up. (2012). Nature, 486, 293. 206 Genomics Law Report. (2011, March 11). The FDA and DTC genetic testing: Setting the record straight. Retrieved from http://www.genomicslawreport.com/index.php/2011/03/11/ the-fda-and-dtc-genetic-testing-setting-therecord-straight/. 207 Vorhaus, D., MacArthur, D., and L. Jostins. (2011, June 16). DTC genetic testing and the FDA: Is there an end in sight on regulatory uncertainty? [Blog]. Retrieved from http://www.genomesunzipped.org/2011/06/ dtc-genetictesting-and-the-fda-is-there-an-end-in-sight-to-the-regulatory-uncertainty.php#more-3681. 208 The Commission plans to look into incidental findings in a future report. 209 Beecher, H.K. (1966). Ethics and clinical research. New England Journal of Medicine, 274(24), 1354-1360; Jones, J.H. (1993) Bad Blood: The Tuskegee Syphilis Experiment. New York: The Free Press; Advisory Committee on Human Radiation Experiments (ACHRE). (1996). Final Report of the Advisory Committee on Human Radiation Experiments. New York: Oxford University Press. 210 Friedman, C.P., Wong, A.K., and D. Blumenthal. (2010). Achieving a nationwide learning health system. Science Translational Medicine, 2(57), 1-3. 211 IOM. (2007). The Learning Health Care System: Workshop Summary (IOM Roundtable on Evidence-Based Medicine). Washington, DC: The National Academies Press. 212 Kass, N., Faden, R., and S. Tunis. (2012). Addressing low-risk comparative effectiveness research in proposed changes to U.S. federal regulations governing research. Journal of the American Medical Association, 307(15), 1589-1590. 213 Friedman, C.P., op cit. 214 IOM. (2011). Integrating Large-scale Genomic Information into Clinical Practice: Workshop Summary. Washington, DC: National Academies Press; IOM. (2012). Digital Data Priorities for Continuous Learning in Health and Health Care: An Institute of Medicine Workshop. National Academies Press: Washington, DC; IOM. (2011). Digital Infrastructure for the Learning Health System: The Foundation for Continuous Improvement in Health and Health Care. Washington, DC: National Academies Press; Nass, S.J., Levit, L.A., and L.O. Gostin. (2009). Beyond the HIPAA Privacy Rule: Enhancing Privacy, Improving Health Through Research. Washington, DC: National Academies Press. 215 President’s Council of Advisors on Science and Technology. (2010). Report to the President Realizing the Full Potential of Health Information Technology to Improve Health Care for Americans: The Path Forward. Retrieved from http://www.whitehouse.gov/sites/default/ files/microsites/ostp/pcast-health-it-report.pdf. 216 CDC. (2012). Next generation sequencing: Standardization of clinical testing (Nex-StoCT) working group. Retrieved from http://www.cdc.gov/osels/lspppo/Genetic_Testing_Quality _Practices/Nex-StoCT.html. 217 Kaye, J., Director of the Centre for Law, Health and Emerging Technologies, Oxford University. (2012). Privacy II – Control, Access and Human Genome Sequence Data. Presentation to PCSBI, February 2, 2012. Retrieved from http://bioethics.gov/cms/node/ 659. 218 Kaye, J., et al. (2012). From patients to partners: Participant-centric initiatives in biomedical research. Nature Reviews, 13, 371-376. 219 The Patient-Centered Outcomes Research Institute (PCORI). (2012). Retrieved from http://www.pcori.org/. 220 Wetterstrand, K.A. (n.d.). DNA Sequencing Costs: Data from the NHGRI Large-Scale Genome Sequencing Program Retrieved from www.genome.gov/sequencingcosts. 221 Brothers K.B., Morrison D.R., and E.W. Clayton. (2011). Two large-scale surveys on community attitudes toward an opt-out biobank. American Journal of Medical Genetics Part A, 155, 2982-2990; Human Subjects Research Protections: Enhancing Protections for Research Subjects and Reducing Burden, Delay, and Ambiguity for
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Investigators, 76 Fed. Reg. 143, 44,513 (July 26, 2011); Kaye, J., et al., op cit; HITECH, Pub. L. 111-5, 123 Stat. 115 (2009). 222 Battelle. (n.d.). $3.8 billion investment in Human Genome Project drove $796 billion in economic impact creating 310,000 jobs and launching the genomic revolution. Retrieved from http://www.battelle.org/media/ news/2011/05/11/$3.8-billion-investment-in-human-genome-project-drove-$796-billion-in-economicimpactcreating-310-000-jobs-and-launching-the-genomic-revolution. 223 Bustamante, D.D., Burchard, E.G., and F.M. De la Vega. (2011). Genomics for the world. Nature, 475, 163-165. 224 Length of a human DNA molecule. In The Physics Factbook. Elert, G. (Ed.). Retrieved from http:// hypertextbook.com/facts/1998/StevenChen.shtml. 225 NIH. (2012). Clinical sequencing exploratory research coordinating center (U01). RFA-HG-12-008. Retrieved from http://grants.nih.gov/grants/guide/rfa-files/RFA-HG-12-008.html. 226 Gimelbrant, A., et al. (2007). Widespread monoallelic expression on human autosomes. Science, 318(5853), 11361140; Mardis, E.R. (2011). A decade’s perspective on DNA sequencing technology. Nature, 470, 198-203; Lander, E. (2011). Initial impact of the sequencing of the human genome. Nature, 470, 187-197; Mason, C.E., and E. Onull. (2012). Faster sequencers, larger datasets, new challenges. Genome Biology, 13, 314. 227 Choia, M., et al. (2010). Genetic diagnosis by whole exome capture and massively parallel DNA sequencing. PNAS, 106, 19096-19101; Ng, S.B., et al. (2009). Targeted capture and massively parallel sequencing of 12 human exomes. Nature, 461, 272-276. 228 Ramos, E., and C. Rotimi. (2009). The A’s, G’s, C’s, and T’s of health disparities. BMC Medical Genomics, 2, 29.
In: Encyclopedia of Genetics: New Research (8 Volume Set) ISBN: 978-1-53614-451-2 Editor: Heidi Carlson © 2019 Nova Science Publishers, Inc.
Chapter 95
GENETIC TESTING: SCIENTIFIC BACKGROUND FOR POLICYMAKERS Amanda K. Sarata ABSTRACT Congress has considered, at various points in time, numerous pieces of legislation that relate to genetic and genomic technology and testing. These include bills addressing genetic discrimination in health insurance and employment; personalized medicine; the patenting of genetic material; and the quality of clinical laboratory tests, including genetic tests. The focus on these issues signals the growing importance of the public policy issues surrounding the clinical and public health implications of new genetic technology. As genetic technologies proliferate and are increasingly used to guide clinical treatment, these public policy issues are likely to continue to garner considerable attention. Understanding the basic scientific concepts underlying genetics and genetic testing may help facilitate the development of more effective public policy in this area. Most diseases have a genetic component. Some diseases, such as Huntington’s Disease, are caused by a specific gene. Other diseases, such as heart disease and cancer, are caused by a complex combination of genetic and environmental factors. For this reason, the public health burden of genetic disease is substantial, as is its clinical significance. Experts note that society has recently entered a transition period in which specific genetic knowledge is becoming critical to the delivery of effective health care for everyone. Therefore, the value of and role for genetic testing in clinical medicine is likely to increase significantly in the future.
INTRODUCTION Virtually all disease has a genetic component.1 The term “genetic disease” has traditionally been used to refer to rare monogenic (caused by a single gene) inherited disease, for example, This is an edited, reformatted and augmented version of a Congressional Research Service publication, CRS Report
for Congress RL33832, prepared for Members and Committees of Congress, from www.crs.gov, dated December 19, 2011.
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cystic fibrosis. However, we now know that many common complex human diseases, including common chronic conditions such as cancer, heart disease, and diabetes, are influenced by several genetic and environmental factors.2 For this reason, they could all be said to be “genetic diseases.” Considering this broader definition of genetic disease, the public health burden of genetic disease can be seen to be substantial. In addition, an individual patient’s genetic makeup, and the genetic make-up of his disease, will help guide clinical decision making. Experts note that “(w)e have recently entered a transition period in which specific genetic knowledge is becoming critical to the delivery of effective health care for everyone.”3 This sentiment is still broadly shared, despite the fact that the translation to practice has perhaps been slower than anticipated due to the lack of a comprehensive evidence base to inform clinical validity and utility determinations for many genomic technologies. Experts in the field note that, “[d]espite dramatic advances in the molecular pathogenesis of disease, translation of basic biomedical research into safe and effective clinical applications remains a slow, expensive, and failureprone endeavor.”4 Over time, as translational obstacles are addressed, the value of and role for genetic testing in clinical medicine is likely to increase significantly. As the role of genetics in clinical medicine and public health continues to grow, so too will the importance of public policy issues raised by genetic technologies. Science is beginning to unlock the complex nature of the interaction between genes and the environment in common disease, and their respective contributions to the disease process. The information provided by the Human Genome Project is helping scientists and clinicians to identify common genetic variation that contributes to disease, primarily through genome-wide association studies (GWAS).5 However, researchers have identified a significant translational gap between genetic discoveries and application in clinical and public health practice and note that “the pace of implementation of genome-based applications in health care and population health has been slow.”6 Efforts are underway to close this gap and expedite translation into practice, specifically the recent development of the NIH-CDC collaborative Genomic Applications in Practice and Prevention Network.7 Experts note that the moderate effect of many common variants, uncovered by GWAS, has helped to underscore the multifactorial etiology of complex disease, and that substantially greater research efforts will be required to detect “missing” genetic influences.8 GWAS efforts have identified 1,100 well-validated genetic risk factors for common disease; however, the potential for many of these factors to serve as drug targets is unknown.9 In addition, research conducted utilizing large population databases that collect health, genetic, and environmental information about entire populations will likely provide more information about the genetic and environmental underpinnings of common diseases. Many countries have established such databases, including Iceland, the United Kingdom, and Estonia. The knowledge of the potential relevance of genetic information to the clinical management of nearly all patients coupled with the lack of complete information about the genetic and environmental factors underlying disease creates a challenging climate for public policymaking. In many cases, the results of genetic testing may be used to guide clinical management of patients, and a particularly prominent role is anticipated in the realm of preventive medicine.10 For example, more frequent screening may be recommended for individuals at increased risk of certain diseases by virtue of their genetic make-up, such as colorectal and breast cancer. In some cases, prophylactic surgery may even be indicated. Decisions about courses of treatment and dosing may also be guided by genetic testing, as might reproductive decisions (both clinical and personal). However, many diseases with an identified molecular cause do not have any
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treatment available; specifically, therapies exist only for approximately 200 of the more than 4,000 conditions with a known molecular cause.11 In these cases, the benefits of genetic testing lie largely in the information they provide an individual about his or her risk of future disease or current disease status. The value of genetic information in these cases is personal to individuals, who may choose to utilize this information to help guide medical and other life decisions for themselves and their families. The information can affect decisions about reproduction; the types or amount of health, life, or disability insurance to purchase; or career and education choices. As genetic research continues to advance rapidly, it will often be the case that genetic testing may be able to provide information about the probability of a health outcome without an accompanying treatment option. This situation again creates unique public policy challenges, for example, in terms of decisions about the coverage of genetic testing services and education about the value of testing. Policymakers may need to balance concerns about the potential use and misuse of genetic information, as well as issues of genetic exceptionalism12 and genetic determinism13, with the potential of genetics and genetic technology to improve care delivery, for example by personalizing medical care and treatment of disease. In addition, policymakers face decisions about the extent of federal oversight and regulation of genetic tests, patients’ safety, and innovation in this area. Finally, the need for and degree of federal support for research to develop a comprehensive evidence base to facilitate the integration of genetic testing into clinical practice (for example, to support coverage decisions by health insurers) may be debated. This report will summarize basic scientific concepts in genetics and will provide an overview of genetic tests, their main characteristics, and the key policy issues they raise.
FUNDAMENTAL CONCEPTS IN GENETICS The following section explains key concepts in genetics that are essential for understanding genetic testing and issues associated with testing that are of interest to Congress.
Cells Contain Chromosomes Humans have 23 pairs of chromosomes in the nucleus of most cells in their bodies. These include 22 pairs of autosomal chromosomes (numbered 1 through 22) and one pair of sex chromosomes (X and Y). One copy of each autosomal chromosome is inherited from the mother and from the father, and each parent contributes one sex chromosome. Many syndromes involving abnormal human development result from abnormal numbers of chromosomes (such as Down Syndrome). Other diseases, such as leukemia, can be caused by breaks in or rearrangements of chromosome pieces.
Chromosomes Contain DNA Chromosomes are composed of deoxyribonucleic acid (DNA) and protein. DNA is comprised of complex chemical substances called bases. Strands made up of combinations of
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the four bases (adenine (A), guanine (G), cytosine (C) and thymine (T)) twist together to form a double helix (like a spiral staircase). Chromosomes contain almost 3 billion base pairs of DNA that code for about 20,000-25,000 genes (this is a current estimate, although it may change and has changed several times since the publication of the human genome sequence).14
DNA Codes for Protein Proteins are fundamental components of all living cells. They include enzymes, structural elements, and hormones. Each protein is made up of a specific sequence of amino acids. This sequence of amino acids is determined by the specific order of bases in a section of DNA. A gene is the section of DNA which contains the sequence which corresponds to a specific protein. Changes to the DNA sequence, called mutations, can change the amino acid sequence. Thus, variations in DNA sequence can manifest as variations in the protein which may affect the function of the protein. This may result in, or contribute to the development of, a genetic disease.
Genotype Influences Phenotype Though most of the genome is very similar between individuals, there can be significant variation in physical appearance or function between individuals. In other words, although we share most of the genetic material we have, we can see that there are significant differences in our physical appearance (height, weight, eye color, etc.). Humans inherit one copy (or allele) of most genes from each parent. The specific alleles that are present on a chromosome pair constitute a person’s genotype. The actual observable, or measurable, physical trait is known as the phenotype. For example, having two brown-eye color alleles would be an example of a genotype and having brown eyes would be the phenotype. Many complex factors affect how a genotype (DNA) translates to a phenotype (observable trait) in ways that are not yet clear for many traits or conditions. Study of a person’s genotype may determine if a person has a mutation associated with a disease, but only observation of the phenotype can determine if that person actually has physical characteristics or symptoms of the disease. Generally, the risk of developing a disease caused by a single mutation can be more easily predicted than the risk of developing a complex disease caused by multiple mutations in multiple genes and environmental factors. Complex diseases, such as heart disease, cancer, immune disorders, or mental illness, for example, have both inherited and environmental components that are very difficult to separate. Thus, it can be difficult to determine whether an individual will develop symptoms, how severe the symptoms may be, or when they may appear.
GENETIC TESTS What Is a Genetic Test? Scientifically, a genetic test may be defined as:
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an analysis performed on human DNA, RNA, genes, and/or chromosomes to detect heritable or acquired genotypes, mutations, phenotypes, or karyotypes that cause or are likely to cause a specific disease or condition. A genetic test also is the analysis of human proteins and certain metabolites, which are predominantly used to detect heritable or acquired genotypes, mutations, or phenotypes.15
Once the sequence of a gene is known, looking for specific changes is relatively straightforward using the modern techniques of molecular biology. In fact, these methods have become so advanced that hundreds or thousands of genetic variations can be detected simultaneously using a technology called a microarray.16
Policy Issues The way genetic test is defined can be very important to the development of geneticsrelated public policy. For example, the above scientific definition is very broad, including both predictive and diagnostic tests and analyses on a broad range of material (nucleic acid, protein, and metabolites), but this may not be the best way to achieve certain policy goals. It may sometimes be desirable to limit the definition only to predictive, and not diagnostic, genetic testing because often, predictive tests raise public policy concerns that diagnostic tests do not (see “What Are the Different Types of Genetic Tests?”). In other cases, it may be desirable to limit the definition to only analysis of specific material, such as DNA, RNA, and chromosomes, but not metabolites or proteins, for example, to help avoid capturing certain types of tests, such as some newborn screening tests, in the scope of a proposed law. Policies extending protection against discrimination may aim to be as broad as possible, whereas policies addressing the stringency of oversight of genetic tests may aim to be more limited (to predictive or probabilistic tests only, or to those for conditions with no treatment, for example).
How Many Genetic Tests are Available? As of December 2011, genetic tests are available for 2,492 diseases. Of those tests, 2,238 are available for clinical diagnosis, while 254 are available for research only.17 The majority of these tests are for single-gene rare diseases.
What Are the Different Types of Genetic Tests? Most clinical genetic tests are for rare disorders, but increasingly, tests are becoming available to determine susceptibility to common, complex diseases and to predict response to medication. With respect to health-related tests (i.e., excluding tests used for forensic purposes, such as “DNA fingerprinting”), there are two general types of genetic testing: diagnostic and predictive. Genetic tests can be utilized to identify the presence or absence of a disease (diagnostic). Predictive genetic tests can be used to predict if an individual will definitely get a disease in the future (presymptomatic) or to predict the risk of an individual getting a disease in the future (predispositional). For example, testing for mutations in the BRCA1 and/or BRCA2 genes provides probabilistic information about how likely an individual is to develop
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breast cancer in his or her lifetime (predispositional). The genetic test for Huntington’s Disease provides genetic information that is predictive in that it allows a physician to predict with certainty whether an individual will develop the disease, but does not allow the physician to determine when the onset of symptoms will actually occur (presymptomatic). In both of these examples, the individual does not have the clinical disease at the time of genetic testing, as they would with diagnostic genetic testing. Within this broader framework of diagnostic and predictive genetic tests, several distinct types of genetic testing can be considered. Reproductive genetic testing can identify carriers of genetic disorders, establish prenatal diagnoses or prognoses, or identify genetic variation in embryos before they are used in in vitro fertilization. Reproductive genetic testing, such as prenatal testing, may be either diagnostic or predictive in nature. Newborn screening is a type of genetic testing that identifies newborns with certain metabolic or inherited conditions (although not all newborn screening tests are genetic tests). Traditionally, most newborn screening has been diagnostic, but some states have chosen to add certain predictive genetic testing to their newborn screening panels (for example, Maryland includes testing for cystic fibrosis).18 Finally, pharmacogenomic testing, or testing to determine a patient’s likely response to a medication, may be considered either diagnostic or predictive, depending on the context in which it is being utilized (i.e., before administration of a medication to determine potential effectiveness, dosing levels, or potential adverse interactions or events vs. after administration and manifestation of a clinical event, for use in determining the basis of the specific event or outcome in the particular patient).
Policy Issues Generally, predictive genetic testing (both presymptomatic and predispositional), rather than diagnostic testing, raises more complex ethical, legal, and social issues. For example, issues surrounding insurance coverage and reimbursement for this type of test, especially if no treatment is available, are more complex than with diagnostic genetic testing. A private insurer may feel that paying for a test that predicts the onset of a disease with no treatment is not costeffective. Even more complicated are cases where the test only shows an increased probability of getting a disease. Another issue is the oversight of genetic tests. Decisions about the need for oversight of genetic testing may be based on whether the information they provide is probabilistic rather than diagnostic, and whether a treatment is available. Additionally, stronger regulation of direct-to consumer marketing of genetic tests, or direct access testing,19 may be desirable in cases where a test is probabilistic rather than diagnostic. Finally, issues of genetic discrimination may be different with predictive testing than they are with diagnostic testing. Genetic discrimination may be defined as differential treatment in either health insurance coverage or employment based upon an individual’s genotype. Discriminatory action based on the possibility of something happening in the future, or even the certainty of it happening in the future, might raise more concern than would action taken based upon diagnostic information. With probabilistic genetic information (generated by predictive testing, see above), the health outcome at issue may never manifest, or if it is certain to, may not manifest for decades into the future. An individual’s concern about the privacy of her genetic information may also be different if the information is probabilistic. For example, an individual who tests positive for being at increased risk of developing breast cancer in the future might believe unfavorable insurance or employment decisions based on this information
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in the present (when she does not have breast cancer) would be unfair. If this were in fact her belief, this individual may have heightened concern with keeping this information private from health insurers or employers.
The Genetic Test Result Genetic tests can provide information about both inherited genetic variations, that is, the individual’s genes that were inherited from their mother and father, as well as about acquired genetic variations, such as those that cause some tumors. Acquired variations are not inherited, but rather are acquired in DNA due to replication errors or exposure to mutagenic chemicals and radiation (e.g., UV rays). In contrast with most other medical tests, genetic tests can be performed on material from a body, and may continue to provide information after the individual has died, as a result of the stability of the DNA molecule. DNA-based testing of inherited genetic variations differs from other medical testing in several ways. These test results can have exceptionally long-range predictive powers over the lifespan of an individual; can predict disease or increased risk for disease in the absence of clinical signs or symptoms; can reveal the sharing of genetic variants within families at precise and calculable rates; and, at least theoretically, have the potential to generate a unique identifier profile for individuals. Genetic changes to inherited genes can be acquired throughout a person’s life (acquired genetic variation). Tests that are performed for acquired genetic variations that occur with a disease have implications only for individuals with the disease, and not family members. Tests for acquired genetic variations are also usually diagnostic rather than predictive, since these tests are generally performed after the presentation of symptoms. Pharmacogenomic testing may be used to determine both acquired genetic variations in disease tissue (i.e., acquired variations in a tumor) or may be used to determine inherited variations in an individual’s drug metabolizing enzymes. For example, with respect to determining acquired genetic variations in disease tissue, a tumor may have acquired genetic variations that render the tumor susceptible or resistant to chemotherapy. With respect to inherited genetic variation in drug metabolizing enzymes, an individual may, for example, be found to be a slow metabolizer of a certain type of drug (e.g., statins) and this information can be used to guide both drug choice and dosing.
Policy Issues In some cases, people feel differently about their genetic information than they do about other medical information, a sentiment embodied by the concept of genetic exceptionalism. This viewpoint may be based on actual differences between genetic testing and other medical testing, but also may be based on personal belief that genetic information is powerful and different than other medical information. For example, genetic information about an individual may reveal things about family members, and therefore decisions by an individual to share her own genetic information can potentially also affect her family. Partially as a result of these considerations, the 110th Congress passed the Genetic Information Nondiscrimination Act of 2008 (P.L. 110-233), and many states, beginning in the early 1990s, enacted laws addressing genetic discrimination in health insurance, employment, and life insurance. Whether genetic
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information is in fact different from other medical information and whether it deserves special protection are important public policy issues.20 Pharmacogenomic testing is important because it will help provide the foundation for personalized medicine. Personalized medicine is healthcare based on individualized diagnosis and treatment for each patient determined by information at the genomic level. Many public policy issues are associated with personalized medicine. For example, there is some uncertainty currently as to how health insurers will assess and choose to cover pharmacogenomic testing as it becomes available. In addition, there are issues surrounding the regulation of pharmacogenomic testing and the encouragement of the co-development of drugs and diagnostic genetic tests (companion diagnostics). Companion diagnostics guide the use of the drug in a given individual.
Characteristics of Genetic Tests Genetic tests function in two environments: the laboratory and the clinic. Genetic tests are evaluated based primarily on three characteristics: analytical validity, clinical validity, and clinical utility.
Analytical Validity Analytical validity is defined as the ability of a test to detect or measure the analyte it is intended to detect or measure.21 This characteristic is critical for all clinical laboratory testing, not only genetic testing, as it provides information about the ability of the test to perform reliably at its most basic level. This characteristic is relevant to how well a test performs in a laboratory. Clinical Validity The clinical validity of a genetic test is its ability to accurately diagnose or predict the risk of a particular clinical outcome. A genetic test’s clinical validity relies on an established connection between the DNA variant being tested for and a specific health outcome. Clinical validity is a measure of how well a test performs in a clinical rather than laboratory setting. Many measures are used to assess clinical validity, but the two of key importance are clinical sensitivity and positive predictive value. Genetic tests can be either diagnostic or predictive and, therefore, the measures used to assess the clinical validity of a genetic test must take this into consideration. For the purposes of a genetic test, positive predictive value can be defined as the probability that a person with a positive test result (i.e., the DNA variant tested for is present) either has or will develop the disease the test is designed to detect. Positive predictive value is the test measure most commonly used by physicians to gauge the usefulness of a test to clinical management of patients. Determining the positive predictive value of a predictive genetic test may be difficult because there are many different DNA variants and environmental modifiers that may affect the development of a disease. In other words, a DNA variant may have a known association with a specific health outcome, but it may not always be causal. Clinical sensitivity may be defined as the probability that people who have, or will develop a disease, are detected by the test.
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Clinical Utility Clinical utility takes into account the impact and usefulness of the test results to the individual and family and primarily considers the implications that the test results have for health outcomes (for example, is treatment or preventive care available for the disease). It also includes the utility of the test more broadly for society, and can encompass considerations of the psychological, social, and economic consequences of testing. Policy Issues These three above-mentioned characteristics of genetic tests, or analytic validity, clinical validity, and clinical utility, have important ties to public policy issues. For example, although the analytical validity of genetic tests is regulated by the Centers for Medicare and Medicaid Services (CMS) through the Clinical Laboratory Improvement Amendments (CLIA) of 1988 (P.L. 100- 578), the clinical validity of the majority of genetic tests is not regulated at all. This has raised concerns about direct-to-consumer marketing of genetic tests where the connection between a DNA variant and a clinical outcome has not been clearly established. Marketing of such tests to consumers directly may mislead consumers into believing that the advice given them based on the results of such tests could improve their health status or outcomes when in fact there is no scientific basis underlying such an assertion. This issue was the subject of a July 2006 hearing by the Senate Special Committee on Aging. In addition, clinical utility and clinical validity both figure prominently into coverage decisions by payers, but a lack of data often hinders coverage decisions, potentially leaving patients without coverage for these tests.
Coverage by Health Insurers Health insurers are playing an increasingly large role in determining the availability of genetic tests by deciding which tests they will pay for as part of their covered benefit packages. Many aspects of genetic tests, including their clinical validity and utility, may complicate the coverage decision-making process for insurers. While insurers require that, where applicable, a test be approved by the Food and Drug Administration (FDA), they also want evidence that it is “medically necessary;” that is, evidence demonstrating that a test will affect a patient’s health outcome in a positive way. This additional requirement of evidence of improved health outcomes underscores the importance of patient participation in long-term research in genetic medicine. Particularly for genetic tests, data on health outcomes may take a very long time to collect.
Policy Issues Decisions by insurers to cover new genetic tests have a significant impact on the utilization of such tests and their eventual integration into the health care system. The integration of personalized medicine into the health care system will be significantly affected by coverage decisions. Although insurers are beginning to cover pharmacogenomic tests and treatments, the high cost of such tests and treatments often means that insurers require stringent evidence that they will improve health outcomes. As mentioned previously, this evidence is often lacking. In addition, coverage of many genetic tests and services, which may be considered preventive in some cases, might be affected by the passage of the Patient Protection and
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Affordable Care Act of 2010 (ACA, P.L. 111-148). The ACA requires private health insurers, Medicare, and Medicaid to cover clinical preventive services (as specified in the law) and outlines cost-sharing requirements in some cases for these services.22 However, the ACA provisions in some cases tie coverage of clinical preventive services to determinations by the U.S. Preventive Services Task Force (USPSTF, located in the Agency for Healthcare Research and Quality), and these determinations are based on the quality of the evidence available to support a given clinical preventive service. For this reason, coverage of genetic tests and services (that are determined to be preventive clinical services) might be negatively affected by a lack of high-quality evidence to support their use.
Regulation of Genetic Tests by the Federal Government Genetic tests are regulated by the Food and Drug Administration (FDA) and CMS, through CLIA. FDA regulates genetic tests that are manufactured by industry and sold for clinical diagnostic use. These test kits usually come prepackaged with all of the reagents and instructions that a laboratory needs to perform the test and are considered to be products by the FDA. FDA requires manufacturers of the kits to ensure that the test detects what the manufacturer says it will, in the intended patient population. With respect to the characteristics of a genetic test, this process requires manufacturers to prove that their test is clinically valid. Depending on the perceived risk associated with the intended use promoted by the manufacturer, the manufacturer must determine that the genetic test is safe and effective, or that it is substantially equivalent to something that is already on the market that has the same intended use. Most genetic tests, however, are performed not with test kits, but rather as laboratory testing services (referred to as either laboratory-developed or “homebrew” tests), meaning that clinical laboratories themselves perform the test in-house and make most or all of the reagents used in the tests. Laboratory-developed tests (LDTs) are not currently regulated by the FDA in the way that test kits are and, therefore, the clinical validity of the majority of genetic tests is not regulated. The FDA does regulate certain components used in LDTs, known as Analyte Specific Reagents (ASRs), but only if the ASR is commercially available. If the ASR is made in-house by a laboratory performing the LDT, the test is not regulated at all by the FDA. This type of test is sometimes referred to informally as a “homebrew-homebrew” test. Any clinical laboratory test that is performed with results returned to the patient must be performed in a CLIA-certified laboratory. CLIA is primarily administered by CMS in conjunction with the Centers for Disease Control and Prevention (CDC) and the FDA.23 FDA determines the category of complexity of the test so the laboratories know which requirements of CLIA they must follow. As previously noted, CLIA regulates the analytical validity of a clinical laboratory test only. It generally establishes requirements for laboratory processes, such as personnel training and quality control or quality assurance programs. CLIA requires laboratories to prove that their tests work properly, to maintain the appropriate documentation, and to show that tests are interpreted by laboratory professionals with the appropriate training. However, CLIA does not require that tests made by laboratories undergo any review by an outside agency to see if they work properly. Supporters of the CLIA regulatory process argue that regulation of the testing process gives the laboratories optimal flexibility to modify tests
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as new information becomes available. Critics argue that CLIA does not go far enough to assure the accuracy of genetic tests since it only addresses analytical validity and not clinical validity.
End Notes Collins, F.S. and V.A. McCusick. (2001) “Implications of the Human Genome Project for Medical Science.” Journal of the American Medical Association 285:540-544. 2 Manolio, T.A. et al. (2009) “Finding the missing heritability of complex diseases.” Nature 461(8): 747-753. 3 Guttmacher, A.E. and F.S. Collins. (2002) “Genomic Medicine - A Primer.” New England Journal of Medicine 347(19): 1512-1520. 4 Collins F.S. (2011) “Reengineering Translational Science: The Time Is Right.” Sci Transl Med. 3(90):90cm17. 5 Genome-wide association studies are defined by the National Human Genome Research Institute as “...an approach used in genetics research to associate specific genetic variations with particular diseases. The method involves scanning the genomes from many different people and looking for genetic markers that can be used to predict the presence of a disease.” http://www.genome.gov/glossary/index.cfm?id=91 6 Khoury M.J. et al. (2009) “The Genomic Applications in Practice and Prevention Network.” Genetics in Medicine 11(7): 488-494. 7 For more information about the Genomic Applications in Practice and Prevention Network, see http://www.cdc.gov/ genomics/translation/GAPPNet/index.htm. 8 See note 2 at page 751. 9 Collins F.S. (2011) “Reengineering Translational Science: The Time Is Right.” Sci Transl Med. 3(90):90cm17. 10 Collins F.S. (2010) “Opportunities for Research and NIH.” Science 327: 36-37. 11 Collins F.S. (2011) “Reengineering Translational Science: The Time Is Right.” Sci Transl Med. 3(90):90cm17. 12 Genetic exceptionalism is the concept that genetic information is inherently unique, should receive special consideration, and should be treated differently from other medical information. For more information about genetic exceptionalism in public policy, see CRS Report RL34376, Genetic Exceptionalism: Genetic Information and Public Policy, by Amanda K. Sarata. 13 Genetic determinism is the concept that our genes are our destiny and that they solely determine our behavioral and physical characteristics. This concept has mostly fallen out of favor as the substantial role of the environment in determining characteristics has been recognized. 14 National Research Council, Reaping the Benefits of Genomic and Proteomic Research: Intellectual Property Rights, Innovation, and Public Health. Washington, DC: National Academies Press (2006); p. 19. The National Human Genome Research Institute at the National Institutes of Health reports that the estimated number of human genes is closer to 25,000. See http://www.genome.gov/11508982. 15 Report of the Secretary’s Advisory Committee on Genetic Testing (SACGT), “Enhancing the Oversight of Genetic Tests: Recommendations of the SACGT,” July 2000, at http://oba.od. nih.gov/oba/sacgt/ reports/ oversight_report.pdf. 16 Microarray technology is defined as “...a developing technology used to study the expression of many genes at once. It involves placing thousands of gene sequences in known locations on a glass slide called a gene chip. A sample containing DNA or RNA is placed in contact with the gene chip. Complementary base pairing between the sample and the gene sequences on the chip produces light that is measured. Areas on the chip producing light identify genes that are expressed in the sample.” See http://ghr.nlm.nih.gov/glossary= micro arraytechnology. 17 See http://www.genetests.org for information on disease reviews, an international directory of genetic testing laboratories, an international directory of genetics and prenatal diagnosis clinics, and a glossary of medical genetics terms. 18 Newborn Screening Home, Maryland Department of Health and Mental Hygiene. http://dhmh. maryland.gov/labs/ html/nbs.html. 19 For more information about direct-to-consumer genetic testing, see http://ghr.nlm.nih.gov/handbook/testing/ directtoconsumer. 20 For more information about characteristics of genetic information that may be viewed as unique and public perspectives on the differences between genetic and other medical information, see CRS Report RL34376, Genetic Exceptionalism: Genetic Information and Public Policy, by Amanda K. Sarata. 21 An analyte is defined as a substance or chemical constituent undergoing analysis. 1
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For more information about requirements relating to the coverage of clinical preventive services under the ACA, see CRS Report R41278, Public Health, Workforce, Quality, and Related Provisions in PPACA: Summary and Timeline, coordinated by C. Stephen Redhead and Erin D. Williams. 23 See http://www.cms.hhs.gov/CLIA/.
In: Encyclopedia of Genetics: New Research (8 Volume Set) ISBN: 978-1-53614-451-2 Editor: Heidi Carlson © 2019 Nova Science Publishers, Inc.
Chapter 96
EVOLUTION OF HUMAN GENOME ANALYSIS: IMPACT ON DISEASES DIAGNOSIS AND MOLECULAR DIAGNOSTIC LABS Julie Gauthier1,*, Isabelle Thiffault1, Virginie Dormoy-Raclet1 and Guy A. Rouleau1,2 1
Medical Biological Unit, Molecular Diagnostic Laboratory, Sainte-Justine University Hospital Center, Montreal, QC, Canada 2 Montreal Neurological Institute and Hospital, Montréal (Québec), Canada
ABSTRACT The advent of massively parallel sequencing has changed the interrogation process of the human genome and now provides a high resolution and global view of the genome which is beyond research applications. Together with powerful bioinformatics tools, these next generation sequencing technologies have revolutionized fundamental research and have important consequences for clinically actionable tests, diagnosis and treatment of rare diseases and cancers. Today, molecular testing is commonly used to confirm clinical diagnosis of specific diseases; it requires that a clinician specify the gene or mutation to test and, in return, will receive information only about this sequence. Despite relative successes, a large number of patients receive no accurate diagnosis, even after many expensive molecular investigations. A clear paradigm shift has taken place in the health network with the introduction of the exome sequencing in molecular diagnostic lab. In this chapter, the impact of the implementation of high throughput sequencing technologies on molecular diagnosis and on the practice of medicine, with an emphasis in paediatrics, is reviewed. We compared well-established genetic tests, using examples from our molecular diagnostic lab, to the recent exome sequencing applications. The genetic tests can fall into three main categories: 1) Mendelian Single Gene Disorder tests that include targeted mutation and targeted gene approaches 2) Genetic Disease Panels which are composed of a few to a dozen genes and 3) Exome or Genome approaches, which interrogate either the * Corresponding
Author’s Email: [email protected].
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entire coding sequences of the 22,333 human genes or the entire human genome. For each of these categories, advantages and limitations are discussed. We devoted a section on the future of molecular diagnosis and discuss which tests will subsist and which one may be soon abandoned. Massively parallel sequencing is transforming the molecular diagnostic field: it offers personalized genetic tests and generates new ethical challenges. Important questions like incidental findings and possible forms of discrimination are addressed. Finally, we conclude with a section on the future directions surrounding the application of these multimodal molecular approaches in general and their putative applications in neonatal intensive care units.
1. INCREASED HUMAN GENOME KNOWLEDGE THROUGH PROGRESS IN SEQUENCING TECHNOLOGY Sequencing the DNA of an organism is feasible since 1977 when the "dideoxy" chaintermination method for sequencing DNA molecules was introduced by Frederick Sanger and colleagues [1]. This technique allows for the sequencing of a single DNA fragment up to 1000 base pairs long. The original method used radiolabeled dNTP and the reading was done manually. Sanger sequencing has been the basis of several major gene discoveries transforming the field of molecular biology. For example, before DNA sequencing, proteins were sequenced directly, this is a laborious technique. Now, this is easily accomplished by sequencing a cDNA and translating the DNA sequence into the amino acid sequence of the protein. In the early 1990s, DNA sequencing was automated using a 4-channel capillary approach (basically the current Life Technologies ABI 3730 system) and ddNTP labeled with different color fluorescent dyes which allow fast DNA sequencing, with hundreds of fragments simultaneously. These sequencing systems were the first generation of sequencing technologies. Their high-throughput yield allows a single lab to sequence millions of base pairs, compared to thousands before their introduction. Commonly called the Sanger method, this sequencing technique is the gold standard in research and clinical diagnostic laboratories for genetic testing. This advance in technology led to science’s greatest achievement, the Human Genome Project. This project aimed to determine the sequence of the 3 billion nucleotide base pairs that constitute the human genome and to identify all 22,333 genes [2] in human DNA [3]. This 13-year project gave rise to the first draft of the human genome sequence in 2001. Decoding the human genome has opened unprecedented new avenues in research and increased our understanding of human health. Information from the human genome sequence enables us to understand how the genetic information drives the development and function of the human body. More importantly, the Human Genome Project accelerated the exploration of genetic variations predisposing to disease and thus revolutionizes medical practice and biological research. Since then, the availability of genomic information (gene and chromosome structure, polymorphisms, disease causing mutations, etc.), has continuously increased. Sanger sequencing helped the discovery of new genetic mechanisms. For example, by sequencing more than 400 synaptic genes in affected probands with sporadic schizophrenia or autism (that is, with no history of psychiatric disorders in the parents or the extended family), we and others have observed a significant excess of potentially deleterious de novo mutations in affected individuals [4]. A similar observation was done in intellectual disability cases [5].
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The most recent DNA sequencing development is the advent of massively parallel sequencing platforms leading to the “next generation sequencing” technologies. The development of ultra-high throughput sequencing technologies pushed forward at an unprecedented speed the identification of DNA variations associated with diseases. Ultrasequencing platforms can be combined with microarray technologies to capture and amplify the functional genome that encodes proteins, also known as the "exome", or to capture more comprehensively a subset of related genes. As technology progressed, next-generation DNA sequencing techniques have improved significantly and are now much faster, and to some extent less expensive than Sanger sequencing. What took over 10 years like the Human Genome Project can now be done in less than a month. It’s now feasible to decode multiple human genomes at once. All of these progresses gave birth to larger scale genetics. A remarkable increase of genetic and genomic data occurred in the last decade notably through the introduction of microarrays and next-generation sequencing. In fact, human genome array-comparative genomic hybridization and SNP arrays were developed to detect genetic aberrations or copy number variants (CNVs) at a higher resolution than traditional karyotyping. Comparative genomic hybridization, also called array CGH, is now currently used in diagnostic labs to identify small chromosomal deletions and duplicated regions. For example, conventional karyotyping has a resolution of 5-10 million bases compare to 100 000 for arrayCGH. Therefore, submicroscopic chromosomal alterations can now be detected. These technological progresses lead researchers to major discoveries in the understanding of genetic mechanisms of different neuropsychiatric conditions such as intellectual disabilities, autism and schizophrenia. De Vries et al. used array based comparative genomic hybridization (array CGH) to study 100 patients with unexplained intellectual disability. They were able to detect a potential causative chromosomal anomaly in 10% of their patients which represents a diagnostic yield of at least twice as high as that of conventional method [7]. In addition to the identification of the causative genetic factors implicated in pediatric and childhood complex disorders, microarrays initiated the discovery of new genetic mechanisms for a number of complex disorders, namely autism, intellectual disability and schizophrenia. Using these technologies, studies have highlighted the involvement of rare (2,500 being rare) have been characterized at the molecular level, and for over 3,500 of these the genetic causes are still unknown. Some genetic conditions are related to a single gene (e.g., cystic fibrosis), but most genetic diseases are characterized by a great heterogeneity, each of which can be caused by mutations in one of several genes (Intellectual disability > 300 genes, Deafness > 60 genes, Mitochondrial diseases >300 genes). The current molecular diagnosis of clinical phenotypic heterogeneity in genetic conditions is laborious and expensive because it involves the sequential analysis of several genes.
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2. THE GENETIC DIAGNOSIS: A MULTI TECHNICAL APPROACH FOR NEURODEVELOPMENTAL AND METABOLIC DISORDERS The advance of modern genomics has changed the health economic decisions concerning genetic screening, with costs- per-megabase for DNA sequencing falling at a faster rate than predicted. Although individually rare, Mendelian diseases collectively account for a significant percentage of infant mortality and pediatric hospitalizations [10]. The speed with which genomics is becoming clinically relevant and the increasing power of the new sequencing technologies led to its rapid implementation in clinical settings. Assuming that new technology automatically translates into improved patient care is idealistic. The ability to amplify DNA by polymerase chain reaction (PCR) has revolutionized our ability to test for genetic mutations. Many different assay systems are based on PCR for analyzing the amplified DNA or RNA. These techniques are sensitive, reliable and can be performed easily on different material: blood, skin or muscle biopsies, tumour, foetus, and even single cells from blastomeres and polar bodies. The aims of adapting new PCR-based strategies in clinical settings are improving the accuracy, speeding up the diagnosis, the time-consuming and costing without decreasing the sensitivity or specificity. Understanding the type of mutations, the incidence of de novo mutations or genetic rearrangements is essential to accurately select the best molecular technique for the detection of carrier in pre-natal diagnosis. Techniques involving PCR-based amplification are successfully used for mutation screening in the majority of diseases. It can be combined for the detection of the presence or absence of restriction sites, electrophoretic mobility shift or sizing analysis, as in single strand conformation polymorphism (SSCP) or in denaturing gradient gel electrophoresis (DGGE). Computer‐assisted highly sensitive mutation detection is also performed, for the above techniques, by means of fluorescent PCR for allelic specific discrimination. Recent advances in the development of quantitative real-time PCR (qPCR)-based diagnostic tools allow detection and quantification of gene or exon dosage. An alternative procedure to mutation‐directed protocols for complex genetic mutations is the use of fluorescent multiplex PCR. Indirect diagnosis performed by the use of polymorphic microsatellite markers, allowing identification of the pathogenic haplotype instead of the mutation [11, 12] as for instance in some spinal muscular atrophy or Duchenne muscular dystrophy families. For diseases involving a heterogeneous spectrum of identified mutations, such as cystic fibrosis, autosomal non-syndromic intellectual disability (SYNGAP1) or Rett Syndrome (MECP2), the development of a mutation‐based strategy is not practical and sequencing of the entire coding sequence is recommended to facilitate mutation detection. Multiplex ligation-dependent probe amplification (MLPA) is a variation of the multiplex polymerase chain reaction that permits multiple targets (exons) to be amplified with only a single primer pair [13]. Specific fluorescent probes consist of two unique oligonucleotides which recognise adjacent target sites on the DNA and each amplicon generates a fluorescent peak which can be detected by a capillary sequencer. Comparing the peak pattern obtained on a given sample with those obtained on various control samples, allow the relative quantity of each target to be determined. This technique is commonly used to detect chromosomal anomalies in cell and tissue samples, [14] detection of gene copy number [13], detection of duplications and deletions in disease-related genes such as DMD, MECP2, BRCA1, BRCA2, etc. It has replaced the need to use of southern blot in many diseases [15-17].
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Expansion repeat disorders such as of the glutamine codon (CAG) in spinocerebellar ataxias, or the trinucleotide repeat in non-coding regions, as the CGG in Fragile-X syndrome, the GAA in Friedreich ataxia or the CTG in myotonic dystrophy type I, are a special type of mutation. The unstable dynamic nature of those mutations requires the use of a multimodal approach [18]. Molecular tests for the diagnosis and carrier detection often combine PCR for the repeat expansion and triplet repeat primed PCR (TP-PCR) analysis of genomic DNA, as well as southern blot. The detection rate of the screening test, as well as the frequency of the mutation in the study population and the technical limitations of the procedure, will determine the usefulness of a positive or negative result. Preimplantation testing provides a paradigm for the ease of use of PCR-based testing, yet also underscores the problems encountered with genetic screening because of the multitude of possible mutations and the possible misinterpretation of results [12]. Molecular diagnostic testing is currently available for only a certain number of disorders and with the increasing number of new genes associated to human diseases, it is becoming more and more challenging to provide cost effective molecular testing [10]. Preconception screening, together with genetic counselling of carriers, has resulted in remarkable declines in the incidence of several severe recessive diseases in populations at risk [19, 20]. However, extensive preconception screening and molecular diagnostic testing has been limited to targeted population or family history–directed individual and is still impractical for many disorders [20].
2.1. Mendelian Single Gene Disorders: At the Mutation or Gene Level Single-gene disorders have a straight forward inheritance pattern, and the genetic causes can be traced to changes in specific individual genes. A particular disorder may be rare; however, as a group of disease-causing genes, single-gene disorders are responsible for a significant percentage of pediatric diseases [21]. About 1% of the approximately 4 million annual live births in the United States will have a single gene disorder that requires intensive clinical investigation, specific medical treatment and hospitalization [22, 23]. Based on the location of the relevant genes, single-gene traits can be divided into autosomal or sex-linked inheritance. Autosomal inheritance, depending on whether one or two mutant alleles are required to cause the disease phenotype, can be classified as autosomal dominant or autosomal recessive. Each of these single gene disorders, called Mendelian traits or diseases, are relatively uncommon. The frequency often varies with ethnic background, with each ethnic group having one or more Mendelian traits in higher frequency when compared to the other ethnic groups. For example, cystic fibrosis has a frequency of about one in 2,000 births in Americans descended from western European Caucasians [10] but is much rarer in African-Americans descent while sickle cell anemia has a frequency of about 1/600 births in African-American, but is rare in Caucasians [24]. Just to name a few, Mediterranean descent have a high frequency of thalassemia [25]; Eastern European Jews have a high frequency of Tay-Sachs disease [26, 27]; French Canadians from Quebec have a high frequency of tyrosinemia [28], all when compared to other ethnic groups. It has been estimated, regardless of the ethnicity, that each healthy individual is carrying between 1 and 8 mutations which, if found in the homozygous state would result in the expression of a Mendelian recessive disease [10]. Since each human genome has 22,333 genes it is unlikely that any two unrelated individuals would be carrying
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the same mutations, even if they are from the same ethnic background. This explains why most Mendelian diseases are rare, affecting about 1/10,000 to 1/100,000 live births [21, 29]. In Quebec, the distribution of Mendelian diseases is due to local founder effects caused by the stratification of the contemporary French Canadian gene pool. The migration of a small number of French individuals from France to Quebec created a founder effect. Subsequent inland migrations have created smaller regional founder effects [30, 31]. The limited size of the population favoured genetic drift, and the social context encouraged endogamy, only few unions were reported between French Canadians with English and other immigrants [31]. The French-Canadian population of Quebec, currently about 6 million people, descends from about 7,798 immigrant founders who arrived in Quebec between 1608 and 1759 [31]. Recent studies showed that the Quebec population structure through the analysis of the genetic contribution of the first French settlers can be partitioned in eight regions, and they contributed to over 90% of gene pools in seven out of those eight regions [32]. This particular local genetic effect highlights the importance of considering the geographic origin of samples in the design of genetic testing in Quebec [33]. The conditions under which the peopling of Quebec was made, have favoured changes in the frequency of certain alleles in comparison with the French original population. As a result, certain genetic diseases are specific or more prevalent to the Quebec population. The prevalence and distribution of genetic diseases in Quebec is an essential factor to consider in clinical practice and particularly in differential diagnostic to prioritize molecular investigations [20]. This founder effect has impacted our molecular diagnostic testing system and is still a key factor when developing new diagnostic test for a genetic disease. The prevalence of the disease and the nature of the mutations found in the Quebec population need to be taken into account. The performance of the test depends on how well it accounts for the particularities of the disease in the French Canadians but more specifically depending on the regional founder effect [31]. The current changes in the immigration and the increased admixture is bringing new challenges in the differential diagnosis and amelioration of our molecular genetic testing system. Over thirty Mendelian diseases have a high prevalence in the Quebec population [31, 34, 35]. Before the advances in sequencing technologies, it was believed that some of the disease were almost exclusive to the French Canadians, as autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS; MIM 270550), hereditary motor and sensory neuropathy with agenesis of the corpus callosum (ACCPN; MIM 218000) or French-Canadian-type Leigh syndrome (MIM 220111) [36-38]. Taking ARSACS as an example, after extensive resequencing of the responsible gene, SACS, it became evident that ARSACS was not limited to Quebec, and more than 100 different pathogenic mutations have now been identified worldwide [39, 40]. ARSACS is believed to be underdiagnosed in patients with atypical phenotypes and recent data on exome sequencing suggest a presumably high frequency of allele carriers around the world [39-42]. Admixture has more and more impact on the genetic characteristics of disease in French Canadians. Other ethnic groups have deep roots in the province. Many immigrant communities that are established mainly in and near Montreal now account for over 15% of the Quebec population. First Nations groups in Quebec have specific genetic diseases with three autosomal recessive conditions that have been well documented: Cree leukoencephalopathy [MIM 603896], Cree encephalitis [MIM 608505], North American Indian Childhood Cirrhosis [MIM 604901]. In Cree communities, the carrier frequency of Cree leukoencephalopathy is estimated at 1/10, 1/30 for Cree encephalitis and, 9/100 for the North American Indian Childhood
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Cirrhosis [43, 44]. However, universal screening is currently offered only in the neonatal period for phenylketonuria, tyrosinaemia type I, medium-chain acyl-coenzyme A dehydrogenase deficiency [MCAD] and congenital hypothyroidism in addition to selected inborn errors of metabolism by urine screening [45, 46]. Effective targeted carrier-screening programs are provided in some specific communities but are often limited to individuals with a family history of recessive diseases. For public health, the genetic structure of Quebec presents major challenge for genetic screening but brings also opportunities for gene identification studies, clinical genetics research and practice. In the next section, we will discuss our experience over time with the implementation of effective targeted genetic testing and carrier-screening programs.
2.1.1. Targeted Mutations The French-Canadian population of Quebec evolved as a mosaic of layered founder effects which has stimulated the development and feasibility of population-based carrier screening for at-risk individuals. For most of the Mendelian diseases in Quebec, the mutant founder alleles are characteristic and often unique. Usually one or two founder mutations account for 90% of French-Canadian alleles, depending on the region. However, founder mutations panels do exist for some disorders in Quebec such as for familial hypercholesterolemia, hereditary breast cancer, etc. A good example of a mutant founder allele in Quebec is the Cree encephalitis, a severe early-onset progressive neurological disorder in an inbred Canadian Aboriginal community [MIM 608505]. The symptoms appear within the first few weeks of life and children usually die in infancy or early childhood. The main neurological symptoms are acquired microcephaly, mental retardation, cerebral atrophy with white matter changes, cerebral calcification, and chronic cerebrospinal fluid lymphocytosis. Cree encephalitis shows phenotypic overlap with Aicardi-Goutières syndrome with elevated levels of IFN-a in cerebrospinal fluid [47, 48]. The gene responsible, named TREX1 (3-prime repair exonuclease 1, MIM 606609), has only one coding exon and is located on 3p21.31. The mutation causing Cree encephalitis is the p.Arg164Ter (c.490C>T) [49]. The molecular genetic diagnosis consists of determining the presence or absence of p.Arg164Ter in symptomatic patients. CREE encephalitis is among the leading causes of death of Cree infants. Cree carrier rates of this mutation are estimated to be 2-3 individual out of twenty meaning that about one in 300 births will be affected. In 2006, a genetic screening program was developed and managed by the Cree Health Board to identify carriers of the mutation p.Arg164Ter in the TREX1 gene. A second autosomal recessive condition is included in the Cree population genetic screening, Cree leukoencephalopathy [MIM 603896]. All patients are homozygous for the mutation p.Glu584Ala in the translation-initiation factor EIF2B5 gene causing childhood ataxia with central hypomyelination and vanishing white matter disease [CACH/VWM] [50]. Both tests are performed in our molecular diagnostic laboratory at CHU Sainte-Justine. To date, >500 individuals have been tested. Pregnant woman and teenagers in High School are the targeted population for this genetic test. In fact, the rate of teenage pregnancy is very high in Cree population (23% among those aged 45 known genes), spinocerebellar ataxia (> 15 known genes) and mitochondrial disease (> 300 known genes). The exploration of these assumptions was simply prohibitive as proposed by the clinical genetic testing targeted 20 genes and would have cost > $ 40,000. Exome sequencing, with a cost of ~$ 7000 rapidly led to the causative mutation/gene. The major hurdle of exome sequencing is the missing or suboptimal exons coverage mainly due to high GC content. One good example is the aortopathies characterized by aortic dilation, which can lead to life threatening aneurysms and/or dissections. An early diagnosis is critical,
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since timely initiation of pharmacological treatment can slow dilation and prophylactic surgery can prevent aortic dissection or rupture. Mortality rate is 20% for aortic dissections. Mutations in twelve different genes (for a total of 360 exons) are known to be responsible for these diseases. The commercial kit of exome capture SureSelect from Agilent is missing (or suboptimal) 11% exons of these genes. Fortunately, these weaknesses will be overcome with promised technological update.
3. THE FUTURE OF MOLECULAR DIAGNOSTIC LAB: WHAT WILL BE KEPT AND WHAT WILL BOT Routine DNA-based diagnostic sequencing for neurodevelopmental or metabolic disorders currently targets well-defined genes or sets of genes that address very specific clinical questions. Today, available services vary greatly depending on the diagnostic laboratory, including Sanger sequencing of complete genes harboring mutations with known, welldescribed clinical phenotypes or multi-gene testing panels or next-generation sequencing-based disease/phenotype panels. Test ordering depends on the ability of a physician to apply a differential diagnosis; it follows a paradigm where a patient with phenotype A will have a mutation in a known gene causing phenotype A, which will be sequenced. Today’s reality is that several genes are known to cause almost any particular phenotype, so gene panels can be used to sequence multiple genes simultaneously, or in a linear approach where the most commonly mutated genes are sequenced first, and if a negative result is obtained, the remainder are sequenced. The recent advances of next-generation sequencing enable the laboratory to simultaneously sequence a large number of genes at a significantly decreasing cost per gene. This raises multiple questions - is there a point at which additional sequencing diminishes the clinical utility of the resulting data? Is the proliferation of DNA testing panels enabled by nextgeneration sequencing beneficial for clinical diagnostics? What will be kept and what will be dropped? This type of linear strategy is generally very satisfying for the diagnosing clinician as each test in specific population, for example in micro founder effect of Cree subpopulation or Saguenay, has a known value for diagnostic purpose. It also utilizes a focused approach, which tends to prioritize the most common gene involved in a certain phenotype. The genetics field is still young, but the actual genes and associated casual mutations are often incrementally discovered. Newly discovered genes are reported weekly, but it is generally difficult to systematically add them to a laboratory test menu. The diagnostic value from a single, wellcharacterized gene is relatively high since it is easier to interpret and it minimizes the likelihood that a variant of unknown significance will be discovered [64]. Howeer, if a gene is reported only for a few cases, the diagnostic test may not be offered. Offering genetic testing for rare orphan disease has a high relative cost of test development and a low diagnostic yield [41, 63, 64]. As described in the previous section, some specific mutation panels will remains for at-risk population until the cost of next-generation sequencing decreases further. In addition, tripletrepeat diseases because of the unstable dynamic nature of those mutations will continue to require the use of a multimodal approach.
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Next-generation sequencing is really moving the field of towards comprehensive gene panels. Expanded comprehensive diagnostic gene panels have several advantages. First of all, the design of an expanded diagnostic panel is quite straightforward and begins by identifying all of the genes involved in the disease(s) being targeted. Adding genes to the list is simple and does not requir additional cost, unless enrichment is needed [63]. More comprehensive gene panel tests will simplify test ordering by consolidating all candidate genes into a single diagnostic test. By taking a more comprehensive approach, the sensitivity of the test increases and the rate of molecular under diagnosis decreases by including genes with low-frequency. It will also allow the identificaiton of in the causative gene in patients with an atypical phenotypic presentation. Moreover, with the advent of next-generation sequencing, an expanded neurodevelopmental and metabolic molecular diagnostic sequencing panel becomes economically feasible, allowing diagnosis of extremely rare orphan disease in one genetic test. Next-generation sequencing introduces some interpretative challenges which are not new to diagnostic testing, but the scale of incidental findings or number of variants of unknown significance will be far greater than any previous test. Sanger sequencing typically identifies three categories of mutations: known non-pathogenic or benign polymorphisms; known pathogenic mutations; and variants of unknown significance. Variants identified as known pathogenic mutations are straightforward to interpret as in most cases, there are clearly indicated for clinical action [76]. With time, and better variant databases, the experience gained using next-generation sequencing testing panels will provide more data to improve variant interpretation and, over time, reduce the total number of variants of unknown significance encountered during next-generation sequencing diagnostic test[76]. The molecular clinical approach should focus on immediate clinical benefit of next-generation sequencing, such as the ability to offer comprehensive gene panels, to provide a first-pass diagnostic yield, and to limit gene list to genes of known diagnostic value [64]. This will aid the interpretation of the data set in a way that the healthcare provider will be able to understand and utilize.
4. THE NON-TARGETED GENETIC STRATEGY GENERATES NEW ETHICAL CHALLENGES Particular emphasis has been placed in recent years on the identification of rare disease genes due to the availability of new genomic sequencing technologies. As seen earlier, these technologies greatly facilitate the search for causative disease mutations since they allow the analysis of the 22,333 genes at once in a short period of time for a reasonable cost. As a result, more genetic mechanisms involved in rare diseases are known, and a portion of these findings are already translated into diagnostic tools. Indeed, the clinical sequencing (referring to exome or genome sequencing) is rapidly being integrated into the practice of medicine. Although technically feasible and proven to be successful [77, 78], several challenges remain surrounding the use of clinical sequencing. Nonetheless molecular diagnosis using genomic and genetic methods such as microarray CGH, exome and genome sequencing have in common several ethical, legal and social concerns with other methods of medical investigation. Because of the unprecedentedly large amount of information generated by these comprehensive tests on an individual, the concerns (e.g., content of the consent form), incidental findings, returning results to the patient, etc. are amplified.
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4.1. Consent Form Consent forms need to be adapted to the new generation of sequencing clinical test. The main addition to the current medical consent form is the extent to which the patients would have control/access to the whole data (exome/genome), what results might be returned to the patient and what are the potential risks. The consent form should also inform the patient about the possible risk of discovering unwanted findings (unrelated to the original medical investigation). For instance, the exome analysis may reveal that the patient is likely to get a serious and untreatable disease.
4.2. Data Storage and Sharing Next-generation sequencing technologies generate terabytes of data, and storing this amount of data will constitute a challenge in itself but also raised ethical issues. The genetic information of individual genome that is derived from the clinical sequencing is an accessible and robust “login” into a patient identity. It can give information on the patient’s relatives, ethnic groups, and more. The Wellcome Trust and the National Institute of Health, two respective Institutions whose mission is to operate open access genetic and genomic databases for the benefits of researchers and patients, now restricted and controlled their web database access. They even formed specific committees to oversee the circulation of the data included in their databases. This was effective following the conclusion from the study of Homer and colleagues in 2008 demonstrating that genome-wide association derived data is sufficient to reidentify an individual that have participated in a study [79]. They concluded that anonymizing data was unsatisfactory to protect the confidentiality of research participants. Because the understanding, diagnosed and treatment of complex diseases such as cancer or pediatric disorders required comparison of genetic information from several hundreds and often thousands of individuals, genetic data sharing is critical for research and molecular diagnosis. To further illustrate the significance of protecting information derived from whole genome sequencing of individual, the Presidential Commission for the Study of Bioethical Issues published a 150-page report, on the Privacy and progress in the era of whole genome sequencing. They concluded “to realize the enormous promise that whole genome sequencing holds for advancing clinical care and the greater public good, individual interests in privacy must be respected and secured. As the scientific community works to bring the cost of whole genome sequencing down from millions per test to less than the cost of many standard diagnostic tests today, the Commission recognizes that whole genome sequencing and its increased use in research and the clinic could yield major advances in health care. However it could also raise ethical dilemmas. The Commission offers a dozen timely proactive recommendations that will help craft policies that are flexible enough to ensure progress and responsive enough to protect privacy.” Finally, researchers and policy-makers need to find methods to protect individuals’ genomic data while still being able to share information.
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4.3. Incidental Findings As mentioned in earlier sections, clinical exome and genome sequencing present several advantages for clinical practice and for the patient. The main advantage discussed is the nondiscrimination on the selection of candidate’s genes that will be analyzed. However, these methods can yield incidental medical information not related to the principal medical target. For instance, the genome of a child investigated for a rare form of deafness can lead to the discovery of a known mutation leading to a late onset neurodegenerative disorder. In addition, the patient can be found to be carrier of a recessive lethal disease. These “secondary” findings have been the subject of many discussions and recently the American College of Medical Genetics and Genomics whose mission is to improve health through medical genetics released it’s highly anticipated “Recommendations on Incidental Findings in Clinical Exome and Genome Sequencing”. In addition to fortuitous findings, whole genome sequencing raises great social concerns that still need to be resolved, such as possible forms of discrimination. Efforts have been made to overcome this phenomenon since the Genetic Information Nondiscrimination Act was signed in the USA in 2008 to protect individuals from improper use of genetics information with regard to health insurance and employment [69]. Emerging discoveries of susceptibility genes and gene variants associated with major neurological or psychiatric disorders are likely to challenge the existing ethical guidelines. It is up to researchers, health professionals and experts in the Ethical, Economic, Environmental, Legal, and Social aspects of genomics [GE3LS] to manage this growing scientific knowledge in a way that prioritizes protection of research participants and improves patient care.
CONCLUSION AND FUTURE The successful use of whole-genome and exome sequencing for diagnosis has been amply confirmed by numerous studies. Whether targeted gene, gene panel approaches and exome sequencing will be entirely replaced by whole-genome sequencing is still unknown. Compared to traditional methods, it is now well accepted that exome sequencing has fewer false positives, and a greater sensitivity due to the higher coverage achieved when focusing only on a small fraction of the genome. Exome and whole-genome sequencing allow the discovery of point mutations and small deletion. With advanced bioinformatics tool it is now feasible to detect larger insertions and deletions (CNVs), currently detected using the microarrays technologies. We think that these “cytogenetic” methods, including karyotyping and molecular diagnosis, will merge. We also think that the current modalities of next-generation sequencing will eventually be replaced by genome sequencing. Recently, Saunders and colleague showed the feasibility of using whole-genome sequencing in neonatal intensive care units to screen for genetic disease diagnosis [77]. They described a 50-hour delay in the diagnosis of genetic disorders using whole genome sequencing. Some challenges will subsist: mosaicism, balanced translocation and complex diseases (unknown mode of transmission). However, an important challenge in using whole-genome sequencing will be the interpretation of the data linking a genetic variation to the disease/phenotype and all ethical issues around it.
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The rapid development of sequencing is now positively impacting prenatal diagnosis. Since the discovery of cell-free fetal nucleic acids circulating in the blood of pregnant women, [80] combined with next-generation DNA sequencing technologies, it is now possible to do early, noninvasive prenatal genetic testing [81]. Clinical applications of these methods already include fetal sex determination and blood group typing [82]. Ongoing research is currently evaluating the use of this approach for noninvasive detection of trisomies [83]. Other uses being explored are the detection of single-gene disorders, chromosomal abnormalities and inheritance of parental polymorphisms across the whole fetal genome. Use of preimplantation genetic diagnosis and preimplantation genetic screening using nextgeneration sequencing can provide blastocyst preimplantation genetic diagnosis (PGD) results with high level of consistency with established diagnostic methods. Furthermore, single-gene disorder screening by next-generation sequencing could be performed in parallel with qPCRbased comprehensive chromosome screening or array SNP-CGH. Several studies showed that next-generation sequencing could serve as an essential PGD tools for further development of this important and emerging field [84]. Next-generation sequencing data provides a unique opportunity to evaluate multiple genomic loci and multiple samples on one experiment (e.g foetus can be tested in parallel with the parents). Next-generation sequencing might also be useful for simultaneous evaluation of aneuploidy, single-gene disorders, and translocations from the same biopsy without the need for multiple technological platforms [85]. Clearly, the fertility field has seen intensive efforts to significantly improve and validate better state-of-theart in vitro fecundation techniques. Martin J. et al. suggested that IVF techniques coupled with deep comprehensive diagnosis/screening methods using NGS should result in high implantation and live birth rates [85]. Nevertheless, there is always room for improvements; it is important to be prudent, recognize the limits of sequence depth necessary to maintain accuracy, and variation in sequencing depth across different genomic loci that is critical to its clinical application in PGD. We expect that whole-genome sequencing will allow the identification of genetic variants that will determine an individual’s risk for developing diseases, including neurological or psychiatric disorders. In fact, the continuing reduction in sequencing costs may lead to replacement of most of the other currently used approaches.
ACKNOWLEDGMENTS We are very grateful for the support of our Institution. We wish to thank our colleagues, Mélanie Lafleur, Françoise Couture, Sylvie Filiatrault, Josée Gauthier, Janique Ladouceur, Caroline Deschaînes, Marie-Josée Lassonde, Julie Ménard, Li Fan and Marie-Lyne Darveau for their technical and administrative work in the Molecular Diagnostic Lab. We also acknowledge the work/input of our collaborative clinicians: Drs. Jacques Michaud, Grant Mitchell, Jean-François Soucy, Dorothée Dal Soglio, Luc Oligny, George-Etienne Rivard and Sonia Cellot for their help in the discovery, development and for working in close collaboration to improve the use of genetics and genomic knowledge and tools in today’s medicine.
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