Exploration of Host Genetic Factors associated with Malaria 9813347600, 9789813347601

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Tabish Qidwai

Exploration of Host Genetic Factors associated with Malaria

Exploration of Host Genetic Factors associated with Malaria

Tabish Qidwai

Exploration of Host Genetic Factors associated with Malaria

Tabish Qidwai Faculty of Biotechnology, Institute of Biosciences and Technology Shri Ramswaroop Memorial University Barabanki, Uttar Pradesh, India

ISBN 978-981-33-4760-1    ISBN 978-981-33-4761-8 (eBook) https://doi.org/10.1007/978-981-33-4761-8 © Springer Nature Singapore Pte Ltd. 2021 This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. The publisher, the authors, and the editors are safe to assume that the advice and information in this book are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty, expressed or implied, with respect to the material contained herein or for any errors or omissions that may have been made. The publisher remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. This Springer imprint is published by the registered company Springer Nature Singapore Pte Ltd. The registered company address is: 152 Beach Road, #21-01/04 Gateway East, Singapore 189721, Singapore

Contents

1 Human Genetics and Infectious Disease������������������������������������������������    1 1.1 Introduction������������������������������������������������������������������������������������    2 1.2 Hypothesis��������������������������������������������������������������������������������������    3 1.3 Predisposition to Disease����������������������������������������������������������������    3 1.3.1 Genetic Predisposition to Infections Due to Mendelian Traits ������������������������������������������������������������    4 1.4 Mendelian Resistance ��������������������������������������������������������������������    5 1.5 Genome Variability, Natural Selection, and Infectious Diseases������������������������������������������������������������������    6 1.5.1 Single Nucleotide Polymorphisms (SNPs)������������������������    7 1.5.2 Copy Number Variations (CNV)����������������������������������������    7 1.6 Malaria, an Infectious Parasitic Disease Shapes Human Genome������������������������������������������������������������������������������   10 1.7 Conclusion��������������������������������������������������������������������������������������   11 References��������������������������������������������������������������������������������������������������   12 Part I RBC Disorders 2 Sickle Cell Gene����������������������������������������������������������������������������������������   17 2.1 Introduction������������������������������������������������������������������������������������   17 2.2 Hypothesis��������������������������������������������������������������������������������������   18 2.3 Balanced Polymorphisms����������������������������������������������������������������   18 2.4 Sickle Cell Disease ������������������������������������������������������������������������   19 2.5 Epidemiology of Sickle Cell Hemoglobin��������������������������������������   19 2.6 Genetic Origin of Sickle Cell Disease��������������������������������������������   21 2.7 Malaria and Sickle Cell Hemoglobinopathy (HbS)������������������������   21 2.7.1 Hemoglobin C (HbC) ��������������������������������������������������������   22 2.7.2 Mechanism of Protection by Sickle Cell Hemoglobin ������   22 2.7.3 Hemoglobin Degradation by Plasmepsins��������������������������   24 2.8 Other Natural Protective Mechanisms��������������������������������������������   24 2.8.1 Knops Blood Group System ����������������������������������������������   25 2.8.2 Adhesion Molecules ����������������������������������������������������������   25 2.9 Conclusions������������������������������������������������������������������������������������   25 References��������������������������������������������������������������������������������������������������   26 v

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3 Alpha-Thalassemia����������������������������������������������������������������������������������   29 3.1 Introduction������������������������������������������������������������������������������������   29 3.2 Mechanism��������������������������������������������������������������������������������������   30 3.3 Epidemiology����������������������������������������������������������������������������������   31 3.4 Types of Alpha-Thalassemia����������������������������������������������������������   32 3.4.1 Alpha-Thalassemia Trait����������������������������������������������������   32 3.4.2 Silent Carrier State of Alpha-Thalassemia ������������������������   33 3.4.3 Hemoglobin H (Hb H) Disease������������������������������������������   33 3.4.4 Hb Bart’s Hydrops Fetalis Syndrome ��������������������������������   34 3.5 Molecular Basis of Thalassemia ����������������������������������������������������   34 3.6 Inheritance Pattern��������������������������������������������������������������������������   34 3.7 Diagnosis����������������������������������������������������������������������������������������   35 3.7.1 Hematological Test ������������������������������������������������������������   35 3.7.2 Molecular Diagnosis����������������������������������������������������������   36 3.8 Therapy ������������������������������������������������������������������������������������������   39 3.8.1 Blood Transfusion��������������������������������������������������������������   39 3.8.2 Transplantation of Bone Marrow and Cord Blood ������������   39 3.8.3 Gene Therapy����������������������������������������������������������������������   39 3.9 Alpha-Thalassemia and Malaria ����������������������������������������������������   39 3.9.1 Alpha-Thalassemia Trait and P. falciparum Malaria Protection����������������������������������������������������������������������������   40 3.10 Conclusion��������������������������������������������������������������������������������������   41 References��������������������������������������������������������������������������������������������������   41 4 Beta-Thalassemia ������������������������������������������������������������������������������������   43 4.1 Introduction������������������������������������������������������������������������������������   43 4.2 Molecular Biology and Epidemiology of Beta-Thalassemia����������   45 4.2.1 Molecular Biology of Disease��������������������������������������������   45 4.2.2 Epidemiology of Beta-Thalassemia������������������������������������   45 4.3 Hemoglobinopathies Selected as Balancing Trait Against Malaria������������������������������������������������������������������������������   47 4.4 Current Treatment Options ������������������������������������������������������������   47 4.4.1 Transfusion ������������������������������������������������������������������������   47 4.4.2 Induction of Fetal Form of Hemoglobin (HbF)������������������   48 4.4.3 Hematopoietic Stem Cell Transplantation��������������������������   48 4.4.4 Splenectomy ����������������������������������������������������������������������   48 4.4.5 Iron Chelation Therapy������������������������������������������������������   49 4.5 Factors Affecting Global Distribution of Thalassemia ������������������   49 4.5.1 Consanguinity ��������������������������������������������������������������������   49 4.5.2 Nutrition and Infections������������������������������������������������������   49 4.5.3 Migration����������������������������������������������������������������������������   49 4.5.4 Prevention ��������������������������������������������������������������������������   50 4.6 Molecular Diagnosis of Thalassemia����������������������������������������������   50 4.6.1 PCR and Sequencing Strategies������������������������������������������   50

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4.6.2 Single-Stranded Conformational Polymorphism (SSCP)��������������������������������������������������������   50 4.6.3 Allele-Specific Oligonucleotide Probes (ASOs)����������������   50 4.6.4 Amplification Refractory Mutation System (ARMS)��������   50 4.6.5 GAP-PCR and MLPA��������������������������������������������������������   50 4.6.6 Method Using Melting Curve Analysis������������������������������   51 4.6.7 Hemoglobin Electrophoresis����������������������������������������������   51 4.7 Conclusion��������������������������������������������������������������������������������������   51 References��������������������������������������������������������������������������������������������������   51 5 Duffy Blood Group Locus ����������������������������������������������������������������������   55 5.1 Introduction������������������������������������������������������������������������������������   55 5.2 Polymorphisms and Epidemiology of Duffy Gene������������������������   56 5.3 Duffy Antigen and Malaria ������������������������������������������������������������   58 5.3.1 Duffy Antigen as Balancing Selection in Malaria��������������   58 5.3.2 Circulation of Duffy Gene Allelic Variants in Humans������   59 5.4 Invasion of P. vivax in Duffy-Negative Individuals (Duffy-Independent Invasion Pathways)����������������������������������������   60 5.5 Chemokines Act as Ligand for DARC��������������������������������������������   61 5.6 Duffy Negative Phenotype and Cancer Susceptibility��������������������   61 5.7 Discussion ��������������������������������������������������������������������������������������   63 5.8 Conclusion��������������������������������������������������������������������������������������   64 References��������������������������������������������������������������������������������������������������   64 Part II Metabolic Enzymes 6 Pyruvate Kinase Deficiency��������������������������������������������������������������������   69 6.1 Introduction������������������������������������������������������������������������������������   69 6.2 Biochemical Function and Deficiency of Enzyme��������������������������   70 6.3 Molecular Biology of Pyruvate Kinase Gene ��������������������������������   71 6.4 Epidemiology of Pyruvate Kinase Deficiency��������������������������������   72 6.5 Analysis of Genetic Variants in pklr Gene��������������������������������������   72 6.5.1 Pyruvate Kinase L272V Mutation��������������������������������������   72 6.5.2 Pyruvate Kinase Arginine/Glutamine (R41Q) Mutation����������������������������������������������������������������   72 6.5.3 Pyruvate Kinase Glutamate/Lysine (Glu277Lys) Mutation��������������������������������������������������������   73 6.6 Pyruvate Kinase Deficiency as Protective Trait Against Malaria������������������������������������������������������������������������������   73 6.6.1 Copy Number Variation in pklr Gene ��������������������������������   74 6.7 Mechanism of Protection Against P. falciparum Caused Malaria ������������������������������������������������������������������������������   75 6.8 Conclusion��������������������������������������������������������������������������������������   77 References��������������������������������������������������������������������������������������������������   77

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7 Glucose 6-Phosphate Dehydrogenase Deficiency����������������������������������   79 7.1 Introduction������������������������������������������������������������������������������������   80 7.2 Biochemical Function and Deficiency of G6PD Enzyme��������������   80 7.3 Epidemiology of G6PD Deficiency������������������������������������������������   81 7.4 Molecular Biology��������������������������������������������������������������������������   82 7.5 Glucose 6-Phosphate Dehydrogenase Polymorphisms������������������   83 7.6 G6PD Deficiency a Balancing Trait Against P. falciparum Caused Malaria ������������������������������������������������������������������������������   84 7.7 Detection of G6PD Deficiency ������������������������������������������������������   85 7.7.1 Fluorescent Spot Test����������������������������������������������������������   85 7.7.2 Spectrophotometric Assay��������������������������������������������������   86 7.7.3 Cytochemical Assay������������������������������������������������������������   86 7.8 Conclusion��������������������������������������������������������������������������������������   86 References��������������������������������������������������������������������������������������������������   86 Part III Host Immune Response 8 TNF Genetic Polymorphisms������������������������������������������������������������������   91 8.1 Introduction������������������������������������������������������������������������������������   91 8.2 Molecular Biology��������������������������������������������������������������������������   93 8.3 TNF-Alpha Mediates Action Through Binding with TNFR1 and TNFR2��������������������������������������������������������������������������������������   93 8.4 Pathologic Changes in Cerebral Malaria (CM)������������������������������   94 8.5 TNF Promoter Polymorphisms and P. falciparum Malaria������������   95 8.6 Conclusion��������������������������������������������������������������������������������������   97 References��������������������������������������������������������������������������������������������������   98 9 iNOS Genetic Polymorphisms����������������������������������������������������������������  101 9.1 Introduction������������������������������������������������������������������������������������  101 9.2 Types and Physiological Functions of Nitric Oxide Synthase��������  103 9.2.1 Nitric Oxide Synthase 1 (Neuronal Nitric Oxide Synthase)������������������������������������������������������������������  103 9.2.2 Nitric Oxide Synthase 2 (Inducible Nitric Oxide Synthase)����������������������������������������������������������������������������  103 9.2.3 Nitric Oxide Synthase 3 (Endothelial Nitric Oxide Synthase)����������������������������������������������������������������������������  104 9.3 Structure of Nitric Oxide Synthase Gene����������������������������������������  104 9.4 Inducible Nitric Oxide Synthase and Malaria��������������������������������  106 9.5 Inducible Nitric Oxide Synthase (iNOS) Polymorphisms and Malaria ������������������������������������������������������������������������������������  107 9.6 Discussion ��������������������������������������������������������������������������������������  109 9.7 Conclusion��������������������������������������������������������������������������������������  109 References��������������������������������������������������������������������������������������������������  111

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10 Human Complement Receptor 1 Polymorphisms��������������������������������  113 10.1 Introduction������������������������������������������������������������������������������������  113 10.2 Molecular Genetics of CR1������������������������������������������������������������  114 10.2.1 Knops Blood Group Antigens ������������������������������������������  115 10.3 Role of CR1 in P. falciparum Malaria Pathogenesis����������������������  115 10.3.1 Rosetting ��������������������������������������������������������������������������  117 10.3.2 Malaria Induced Severe Anemia ��������������������������������������  117 10.4 Human Complement Receptor 1 (CR1) Gene Polymorphisms������  117 10.4.1 Human Complement Receptor 1 Polymorphisms and Malaria�����������������������������������������������������������������������  119 10.5 Conclusion��������������������������������������������������������������������������������������  121 References��������������������������������������������������������������������������������������������������  121 11 Interferon-Alpha Receptor-1 (IFNAR1) Polymorphisms��������������������  123 11.1 Introduction������������������������������������������������������������������������������������  124 11.2 Interaction of IFNs with IFNAR1 and Production of Effects ��������  125 11.3 Interferon-Alpha Receptor-1 (IFNAR1) Gene and Protein������������  126 11.3.1 Promoter Polymorphisms��������������������������������������������������  126 11.3.2 Coding Polymorphism������������������������������������������������������  126 11.3.3 IFNAR1 Protein����������������������������������������������������������������  127 11.4 Interferon-Alpha Receptor-1 (IFNAR1) Polymorphisms Impact Malaria��������������������������������������������������������������������������������  129 11.5 Conclusions������������������������������������������������������������������������������������  129 References��������������������������������������������������������������������������������������������������  131 Part IV Cytoadherence 12 Intercellular Adhesion Molecule-1 Polymorphisms������������������������������  135 12.1 Introduction������������������������������������������������������������������������������������  136 12.2 Structure and Function of ICAM1 Gene ����������������������������������������  137 12.2.1 ICAM-1 Protein����������������������������������������������������������������  137 12.3 Mechanism of Cytoadherence in P. falciparum Malaria����������������  138 12.4 ICAM-1 Gene Polymorphisms ������������������������������������������������������  140 12.5 ICAM1 Gene Polymorphisms and Malaria ������������������������������������  141 12.6 Soluble ICAM-1������������������������������������������������������������������������������  143 12.7 Conclusion��������������������������������������������������������������������������������������  143 References��������������������������������������������������������������������������������������������������  144 13 Platelet Endothelial Cell Adhesion Molecule-1 (PECAM-1) Polymorphisms ����������������������������������������������������������������������������������������  147 13.1 Introduction������������������������������������������������������������������������������������  148 13.2 Structure and Function of PECAM-1 ��������������������������������������������  149 13.3 Mechanisms of Malaria Pathogenesis��������������������������������������������  150 13.4 Genetic Polymorphisms in PECAM-1 Gene and P. falciparum Malaria��������������������������������������������������������������������������������������������  152 13.4.1 Promoter Polymorphisms��������������������������������������������������  154

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13.5 Discussion ��������������������������������������������������������������������������������������  154 13.5.1 Antiadhesion Therapies����������������������������������������������������  155 13.6 Conclusions������������������������������������������������������������������������������������  156 References��������������������������������������������������������������������������������������������������  156 14 Vascular Cell Adhesion Molecule-1 (VCAM-1) Polymorphisms ��������  159 14.1 Introduction������������������������������������������������������������������������������������  160 14.2 Structure of VCAM-1 ��������������������������������������������������������������������  163 14.3 Inflammatory Role Played by VCAM-1 ����������������������������������������  163 14.4 Pathology in Cerebral Malaria��������������������������������������������������������  164 14.5 Polymorphisms in VCAM-1 Gene and P. falciparum Caused Malaria��������������������������������������������������������������������������������������������  165 14.6 Other Human Host Receptors for Infected Erythrocytes����������������  166 14.6.1 Hemoglobin C and Hemoglobin S������������������������������������  168 14.7 Compounds Targeting Cytoadherence of Infected Red Blood Cells������������������������������������������������������������������������������������������������  168 14.8 Conclusion��������������������������������������������������������������������������������������  169 References��������������������������������������������������������������������������������������������������  169

About the Author

Tabish Qidwai is an Assistant Professor at SRM University, Uttar Pradesh. India. He previously served as an Assistant Professor (contract) in the Department of Biotechnology at Babasaheb Bhimrao Ambedkar University, Lucknow (A Central University) from 2015 to 2018; Assistant Professor in the Department of Biotechnology at Raja Balwant Singh College, Agra, UP. He received his M.Tech in Biotechnology from Institute of Engineering and Technology (IET), Lucknow and his Ph.D in Biotechnology from Dr. A.P.J.  Abdul Kalam Technical University, Lucknow. His research interests are focused on the functional genomics and immunology especially polymorphisms, immunogenetics, and computational biology. He has received various prestigious awards including the Rashtriya Gaurav Award for remarkable contributions to Science and Technology, Chancellor Gold Medal for first rank in University. He has more than 8 years of teaching experience in genomics, immunology, and computational biology. He has published more than 30 research and review articles in peer-reviewed international journals and authored numerous book chapters. He is a member of several national and international scientific societies and organizations including the Indian Science Congress Association, Indian Biophysical Society, Society of Biological Chemists India, Member of International Association of Engineers (IAENG), China, and European Federation of Biotechnology.  

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1

Human Genetics and Infectious Disease

Abstract

Infectious disease has profound impact on the human genome. DNA sequence variation present in human genome affects susceptibility to infectious disease and disease outcome. Infections of virus, bacteria, parasite and other diseases cause asymptomatic to lethal conditions which may be determined by DNA sequence variations in individuals. DNA sequence variation such as single nucleotide polymorphisms (SNPs), microsatellite repeats, and copy number variations (CNVs) have demonstrated their role in susceptibility/resistance to diseases. Out of these variations, SNP, the most common type of genetic variation influences susceptibility/resistance to disease and drug response. Malaria is a parasitic infectious disease which exerts the strongest selective force on human genome and shapes many genomic regions. Due to driving force created by the malaria, many genetic variations in the human genome have been selected and shown protection against the disease and most of them related to the structure and function of erythrocytes. Human genome develops genetic control mechanisms through natural selection to provide resistance against the disease. Development of drug resistance in parasite, genetic complexity of parasite and lack of effective treatments, created the need of exploration of host genetic factors in pathogenesis, outcome, and risk of disease. Demonstrating in what way, host genes confer their effects would be helpful in identification of novel therapeutic approaches to combat malaria. Moreover, it could determine genetic risk of an individual human being to disease. The aim of present chapter is to cover information related to human genetics, polymorphisms in human genome and their role in susceptibility/resistance to P. falciparum caused malaria.

© Springer Nature Singapore Pte Ltd. 2021 T. Qidwai, Exploration of Host Genetic Factors associated with Malaria, https://doi.org/10.1007/978-981-33-4761-8_1

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Keywords

DNA sequence variation · SNP · Malaria · Selective force · Susceptibility/resistance · Endemic region

1.1

Introduction

Infectious disease like malaria has contributed huge disease burden in several countries. According to the World Health Organization (WHO), it has been estimated that human mortality due to malaria in sub-Saharan Africa would reach 769,000 in the year 2020. This number is two times the number of deaths happened in 2018  in this area (WHO 23 April 2020). This would denote reappearance of malaria mortality. SubSaharan Africa is responsible for around 93% of all malaria cases and 94% of deaths in 2018. South-East Asia region and Eastern Mediterranean region accounted for 3.4% and 2.1% cases respectively (World Malaria Report 2019). Furthermore, Tuberculosis and HIV are also prevalent infectious diseases. WHO report has identified worldwide around 10.0 million cases of tuberculosis as well as 1.2 million deaths due to tuberculosis among HIV negative people in the year 2018 (Global Tuberculosis Report 2019). The advent of genomics has been found a major breakthrough in the field of molecular biology and medical genetics. Genetic makeup of an individual determines the susceptibility/resistance to infectious, noninfectious, and other diseases (Qidwai 2016; Qidwai and Khan 2016). Single nucleotide polymorphisms (SNPs), microsatellite repeats, and copy number variations (CNVs) are important DNA sequence variations in human genome influencing disease susceptibility/resistance (Qidwai 2016). Genetic predisposition is the outcome of specific genetic variations that are often inherited from a parent. Such genetic changes may play role in disease development but do not cause it directly. Infectious disease shapes the genomic regions of human genome. Clinical consequence of infectious disease is identified by complex interactions among the microorganism, host genetic factors and environment. Epidemiological studies demonstrated differences in terms of prevalence, pathogenesis and severity of disease within and among populations exposed to same infectious agent. This fact is underlying significance of genetic background of an individual. Genetic susceptibility of host to infectious disease has been demonstrated (Clementi and Di Gianantonio 2006). Remarkable attribute of most infections in human population globally and overall history is their considerable interindividual phenotypic variability, varying from asymptomatic to fatal infections (Casanova and Abel 2018). Genetic basis for interindividual variation in susceptibility to human infectious diseases has been found informative in field of medical genetics. This has led to identification of association between genetic variations and common infectious diseases such as malaria, human immunodeficiency virus-1 (HIV-1)  infection and other infectious diseases (Hill 2001), for example, absence of Duffy antigen in human erythrocyte decreases susceptibility to Plasmodium vivax parasite (Qidwai and Khan 2016). Similarly, deletion of 32 base pairs in human chemokine receptor 5 (CCR5) gene alters progression of HIV-1 (Vannberg et al. 2011).

1.3 Predisposition to Disease

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Malaria, a widespread infectious disease shapes the human genome and shows balanced polymorphisms (de Mendonça et  al. 2012). Biological win of malaria parasite depends on their capacity to invade, grow, and survive in human erythrocytes. Genetic diseases including thalassemia, glucose-6-phosphate dehydrogenase (G6PD) deficiency, sickle cell hemoglobin, and other diseases related to erythrocyte structure and function had been selected to utmost frequencies due to their malaria-­ protective effects (Kariuki and Williams 2020). Due to increase in incidence of malaria, genetic complexity of parasite and development of drug resistance in parasite, there is a need to explore host genetic background for reduction of malaria. It would be helpful in identification of therapeutic approaches to combat malaria. The present chapter covers information of human genome and infectious disease with special emphasis on P. falciparum caused malaria. Moreover, genetic variations in human genome and disease predisposition of infectious diseases and Mendelian resistance have been highlighted.

1.2

Hypothesis

Infectious disease including malaria acts as driving force for selection of genetic variation in genome. Human genome develops genetic control mechanisms to provide resistance against the disease pressure. For example, disadvantage of sickle cell hemoglobin is associated with advantage of protection against P. falciparum malaria. Genetic makeup of population and disease endemicity influence the selection of DNA sequence variation in populations. Malaria shows balanced polymorphisms where deleterious effect of disease is associated with protection to malaria. Mostly, genetic variations in genes related to erythrocyte structure and function have been found for protection against malaria in endemic regions of the disease. Exploration of such balanced polymorphisms is important in the identification of genetic risk of a population to malaria. Due to genetic complexity and multiple stages in life cycle of malaria parasite, it is difficult to treat malaria. Increase in incidence of malaria has created the need of extensive effort to develop effective treatment options. The high mortality rate associated with P. falciparum infection makes it strong selective force. In that way outcome of severe disease, balance negative phenotype linked to  the mutation. Therefore, it is valuable to highlight significance of the study of natural mechanisms of resistance as the basis for numerous efforts to develop novel therapeutic approaches. It could either check expansion of malaria or reduce its major complications. This chapter is focused on the role played by the host genetic variation in susceptibility to infectious disease and disease outcome.

1.3

Predisposition to Disease

Genetic predisposition is the increased probability of developing a specific disease on the basis of genetic makeup of an individual human being. Genetic variation may produce huge or minor effects on developing a specific disease. Mutations in BRCA1 or BRCA2 genes increase an individual’s risk of developing breast cancer and

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Carrier Father

Unaffected child female

Carrier child male

Carrier Mother

Carrier child female

Affected child male

Fig. 1.1  Genetic variation in CFTR and disease predisposition

ovarian cancer to a great extent. Cystic fibrosis transmembrane conductance regulator (CFTR) gene mutation leads to a disease state named as cystic fibrosis (CF) which is a recessive disease. CF disease condition arises when a person have mutation in both copies of the CFTR gene (Fig. 1.1). Individuals with CF develop endocrine, gastrointestinal, pancreatic, liver, and reproductive disorders (Elborn 2016). More recently, it has been identified that CF carriers are at increased risk for a broad CF-related disease states (Miller et al. 2020). Role of many rare mutations in susceptibility to particular phenotypes of infectious disease have been studied (Chapman and Hill 2012). Mendelian susceptibility to mycobacterial disease (MSMD) arises due to inborn errors of interferon-gamma (IFN-γ) immunity (Bustamante et al. 2014; Rosain et al. 2019).

1.3.1 G  enetic Predisposition to Infections Due to Mendelian Traits Mutation in many genes influence predisposition to specific infections in healthy individuals. Complement deficiencies are responsible for predisposition to bacterial infection (Neisseria species). Autosomal recessive mutations in components of complement system, C5, C6, C7, C8A, C8B, C8G and C9 ensures disease predisposition. Properdin, a glycoprotein acts as a positive regulator of alternative pathway of complement system, its deficiency has played role in predisposition to bacterial infection (Neisseria species) (Picard et al. 2006).

1.3.1.1 Mycobacterial Disease Predisposition Mendelian susceptibility to mycobacterial disease (MSMD) is a rare inherited form including multiple genes. It has been identified that, MSMD comprising seven autosomal (IFNGR1, IFNGR2, STAT1, IL12B, IL12RB1, ISG15, and IRF8) and two X-linked (NEMO, CYBB) gene (Bustamante et al. 2014). All the nine genes products are associated with IFN-γ-dependent immunity (Bustamante et  al. 2014). Genetic variations in genes involved in host immune response such as IFNGR1,

1.4 Mendelian Resistance

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IFNGR2, STAT1, IL12B, and IL12RB have shown their role in many disorders (Dorman et al. 2004). Mutations in STAT1 (signal transducer and activator of transcription 1) affect its DNA-binding domain and showed partial dominant STAT-1 deficiency (Picard et al. 2003).

1.3.1.2 IRAK-4 Deficiency and Predisposition to Streptococcus pneumoniae Infection Interlukin-1receptor (IL-1R) associated kinase-4 (IRAK-4) deficiency is an autosomal recessive disorder causes a life-threatening disease. In this disease, patient is not able to produce pro-inflammatory cytokines. Predisposition to pneumococci was determined in patients having inherited IRAK-4 deficiency. 1.3.1.3 X-Linked Lymphoproliferative (XLP) and Predisposition to Epstein-Barr Virus Infection The X-linked recessive lymphoproliferative disease (XLP) has shown association with susceptibility to B cell-tropic Epstein-Barr virus (EBV). The  SH2D1A gene encodes a protein which acts as a signalling lymphocytic activation molecule (SLAM)-associated protein (SAP). Hemizygous mutations in SH2D1A gene give rise to a disease state known as XLP (Sayos et al. 1998). Because of impaired signaling pathways, XLP patients are no longer able to provide resistance against viral infection. Hence, such patients have predisposition to virus infection.

1.4

Mendelian Resistance

Mendelian traits are transferred from one generation to other by dominant and recessive alleles of one gene while Non-Mendelian traits are not controlled by dominant or recessive alleles, and they might involve more than one gene. Polygenic traits are considered as Non-Mendelian traits. Few Mendelian traits conferred resistance to infections because they are responsible for absence of receptors used by invading microorganisms. Genetic variations in genes may confer resistance against disease without any reduction in fitness of individuals. Therefore, individuals having wild-type alleles are susceptible to pathogens while individuals with mutant alleles show approximately complete protection against pathogens. For example, protection against Plasmodium vivax caused malaria is confirmed by absence of DARC (Duffy antigen receptor for chemokines). Duffy antigen act as coreceptor for P. vivax invasion in erythrocytes (Fig.  1.2) (Miller et al. 1976). SNP in promoter of DARC gene affects binding of transcription factor, GATA-1 to its promoter thereby, stopping gene transcription in erythroid cells (Alcais et al. 2009). This is a type of recessive trait affecting malaria frequency. Mutation in DARC gene has not been found in Europe however, its high frequency (nearly 80%) has been reported in malaria endemic regions such as African countries. It has also been reported that parasite infected some populations even with the absence of DARC on their erythrocytes. This leads to exploration of an alternative receptor named as transferrin receptor 1 (TfR1) on erythrocytes for P. vivax

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Fig. 1.2  Invasion of P. vivax in human erythrocytes using Duffy receptor. Lack of Duffy antigen on human erythrocytes decreases risk of P. vivax infection. High frequency of Duffy negative individuals provides resistance to malaria in African countries

recognition and invasion (Gruszczyk et al. 2018). Hence, DARC is not only a coreceptor for binding of P. vivax. Deletion of 32 base pairs (Δ32) in CCR5 shows strong protection against human HIV-1 infection (Alcais et  al. 2009). CCR5 modifies HIV-1 disease progression (Vannberg et al. 2011). CCR5 along with CD4 acts as coreceptor for CCR5 tropic HIV-1 invasion in CD4+ T cells. Principal resistance allele, CCR5Δ32 derived from an ancestor of European origin. Erythrocyte P antigen acts as cellular receptor for parvo-virus B19 which causes erythema infectiosum. It has been found that individuals with erythrocytes lacking receptor are resistant to infection with this virus (Brown et  al. 1994). Norovirus causes gastroenteritis and it has shown association with fucosyltransferase 2 (FUT2) gene mutations. This gene expresses α (1, 2)-fucosyltransferase protein, which controls expression of ABH histo-blood group antigens present on the surface of epithelial cells and in mucosal secretions (Marionneau et al. 2002).

1.5

 enome Variability, Natural Selection, G and Infectious Diseases

The human genome comprises information about human physiology, development, diseases, evolutionary past and ongoing change. Inherited variations in DNA sequences contribute to phenotypic variations, affecting a population’s characteristics, risk to diseases and response to environment and drugs. DNA sequence variations in genome are crucial for understanding population substructure, natural selection as a result of disease pressure and genetic drift due to population migration. DNA sequence variations such as STRs (Short Tandem Repeats or microsatellites), VNTRs (Variable Number of Tandem Repeats), deletion/insertion mutations, single nucleotide polymorphisms (SNPs), and copy number variation (CNVs) have been studied in context of miscellaneous diseases and evolutionary studies.

1.5 Genome Variability, Natural Selection, and Infectious Diseases

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Infectious agents shape genetic variation in modern populations and are being attracted attention of researchers very much in the field of medical genomics. During developmental process, human beings migrated from one region to other region throughout the world. Due to such migration, populations faced different pathogens. Natural selection increases frequency of those alleles which are beneficial in new environments in both host and pathogenic invader (Karlsson et al. 2014). This ancient history now impacts susceptibility to infectious diseases in humans and contributes to common diseases that demonstrate geographical variations.

1.5.1 Single Nucleotide Polymorphisms (SNPs) Single nucleotide polymorphism is the most common type of genetic variation in the human genome. Variation at a single base pair position in DNA is called as SNP with frequency of more than 1% in a population. The human DNA sequence carries around one SNP every 1000–2000 nucleotides (Li and Sadler 1991). SNPs have been found enormously informative in disease gene mapping and tracing evolutionary past of populations. Many studies are available covering SNP in susceptibility to disease and response to drugs (Fig. 1.3). Many studies have been carried out to explore the association of SNPs with various infectious and noninfectious diseases in different populations of the world.

1.5.1.1 Coding Polymorphisms The SNP in coding region of gene may cause non-synonymous change resulting in substitution of an amino acid in protein which in turn affects structure of protein and hence the function of protein. Change in structure and function of protein may be associated with susceptibility/resistance to disease. Moreover change in structure due to genetic alterations may have a role in evolution (Fig. 1.4). 1.5.1.2 Regulatory/Promoter Polymorphisms SNPs in regulatory or promoter region of gene may influence the expression of gene which in turn affects the level of protein. Change in level of protein may be associated with susceptibility/ resistance to disease (Fig. 1.4). Moreover, change in level of protein may have a role in evolution.

1.5.2 Copy Number Variations (CNV) Copy number variation (CNV) is a type of polymorphisms, commonly found in eukaryotic genomes varying from yeast (Liti et al. 2009) to human beings (Mills et al. 2011). Many CNVs in human have been identified that confer susceptibility/ resistance to diseases using numerous mechanisms for example, gene dosage, gene disruption, and gene fusion etc. (Figs. 1.5 and 1.6). CNVs may give rise Mendelian/ sporadic traits, associated with complex diseases. CNVs, particularly gene

1  Human Genetics and Infectious Disease

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Single nucleotide polymorphisms (SNPs) paly role

SNPs in susceptibility/ resistance to disease and disease outcome

SNPs in evolution SNPs in drug response

SNP determines genetic risk of an individual to disease and consequent disease manifestation

SNPs in drug resistance in pathogenic invader (parasite, bacteria)

Fig. 1.3  This flowchart represents the role played by SNPs. SNPs determine genetic risk of an individual to disease and outcome of disease. Moreover, it has shown its role in drug response in humans, drug resistance in bacteria and parasite, and evolution

Single nucleotide Polymorphisms (SNPs) in human genes

SNPs in promoter region

SNPs in coding region of gene can produce non-synonymous

Alteration in protein level

Affects susceptibility /resistance to disease

Amino acid substitution in protein

Play role in drug response

Affects susceptibility /resistance to disease

Fig. 1.4  Proposed model represents the role of SNPs in disease predisposition/resistance, response to drug and evolution

1.5 Genome Variability, Natural Selection, and Infectious Diseases

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Copy number variation (CNV) polymorphisms in human genome

Affects chromatin structure

Dosage effect and over expression of protein

Gene disruption

Gene fusion

Alteration in protein expression

Disease susceptibility or resistance

Change in drug response (resistance/sensitivity)

Fig. 1.5  Illustration is representing the role played by copy number variation (CNV) in disease predisposition and drug response

Gene1

CNV Gene1

Gene1

(a)

Gene1

Gene2

Gene3

Gene4

CNV Gene1

Gene4

(b) Fig. 1.6 (a) Duplication of gene 1 and (b) deletion of gene 2 and gene 3 in human genome

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duplication and exon shuffling could be predominant mechanism driving evolution of gene and genome (Zhang et al. 2009). The selective forces controlling distribution of CNVs within populations is identified but inadequately (Cheeseman et al. 2016). The beneficial nature of CNVs have been reported, such as, amplification of CCL3L1 gene decreases risk of HIV progression (Gonzalez et al. 2005) and amylase copy number is correlated with dietary starch levels in human beings (Perry et al. 2007).

1.6

 alaria, an Infectious Parasitic Disease Shapes M Human Genome

Malaria is caused by many Plasmodium species such as Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, Plasmodium ovale, and Plasmodium knowlesi. Among these P. falciparum is the most prevalent and responsible for majority of deaths (WHO 2019). According to the data of World Health Organization, malaria has produced 405,000 deaths in 2018, out of which 90% of these deaths happened in sub-Saharan Africa (WHO 2019). Malaria acts as selective pressure and has been predicted as driving force for sickle cell disease, thalassemia, glucose-­ 6-phosphate dehydrogenase deficiency (Kwiatkowski 2005), and other defects of erythrocytes. Most of genetic alterations are related to erythrocyte structure and functions and include the most common Mendelian diseases. Malaria exerts selective pressure on human genome, this evidence came from the fact that sickle cell (HbS) allele has been found in high frequencies in malaria-exposed populations in spite of lethal repercussion resulting from homozygotes (Feng et  al. 2004; Hedrick 2004). Diverse populations have evolved autonomous responses to malaria locally as well as globally (Kwiatkowski 2005). One of the most common examples is HBB gene, in which three different SNPs in coding region protects against malaria. In HbS, glutamate substituted to valine, in HbC, glutamate substituted to lysine at six positions and in HbE, glutamate substituted to lysine at 26 position. HbS allele is the most commonly found in Africa but rare in Southeast Asia whereas reverse is found for the HbE allele. Polymorphisms in erythrocyte and immune response genes and their role in malaria outcome have been illustrated (Fig. 1.7). The genetic changes that affect malaria outcome have been demonstrated (de Mendonça et al. 2012; Kariuki and Williams 2020). Major calcium transporter protein, PMCA4 (plasma membrane calcium-­ transporting ATPase4) in erythrocytes is encoded by the gene ATP2B4. Genetic variations in promoter region of ATP2B4 cause diminished expression of PMCA4 protein. This is due to alteration in binding of transcription factors that regulate its expression (Zambo et al. 2017; Malaria GEN 2019). Studies suggested that PMCA4 confers a 40% protective effect (Timmann et  al. 2012; Band et  al. 2015). This decreased PMCA4 expression changes intracellular calcium homeostasis and influence growth of parasite throughout its intra-erythrocytic stage (Tifert et al. 2005). Glycophorins act as invasion ligands for Duffy-Binding-Like (DBL) domains of a variety of P. falciparum merozoite proteins. Malaria resistance genes consist of a

1.7 Conclusion

11 Membrane Ovalocytosis Spherocytosis Elliptocytosis

Hemoglobin HbS, HbC, HbE, αthalassemia, βthalassemia

ATP2B4 CR1

Surface antigen Fy GYP A

Enzymes G6PD deficiency PK deficiency

GYPB GYP A/B

Blood groups (ABO) Fig. 1.7  The illustration demonstrates the genetic changes that affect malaria outcome. Hybrid of Glycophorin A and B is Dantu blood group antigen which provides protection against the disease

complex structural rearrangement in glycophorin gene cluster that give rise gain of two glycophorin (GYPB-A) hybrid genes to encode Dantu blood group antigen (Lefer et al. 2017). Dantu blood group antigen in homozygous state, showed strong protective effect (nearly 74%) against severe P. falciparum malaria (Malaria GEN 2019).

1.7

Conclusion

After infection, all individuals do not develop clinical disease. Human genetic variations show key role in shaping susceptibility/resistance to diseases. Susceptibility/ resistance to infectious disease probably follows a simple or complex array of inheritance and genetic alterations in many genes confer predisposition/resistance to infection. Malaria is considered as the strongest known selective pressure, so far. This infectious disease acts as driving force for selection of many traits. Host and parasite displayed complex interactions. Human genetic factors have been shown to play a role in pathogenesis, severe outcome and risk of infectious disease. In malaria endemic regions, certain genetic variations have been selected to provide resistance against disease. Most of the selected genetic variations are associated with structure

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and function of erythrocytes. Genetic polymorphisms may provide resistance to a particular pathogenic invader with no measurable reduction in fitness. Development of drug resistance in parasite, presence of inadequately effective vaccines, prompted to analyze the role of host genetic background and malaria. Exploration of host genetic factors could be important in identification of therapeutic approaches against malaria.

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Kariuki SN, Williams TN. Human genetics and malaria resistance. Hum Genet. 2020; https://doi. org/10.1007/s00439-­020-­02142-­6. Karlsson EK, Kwiatkowski DP, Sabeti PC. Natural selection and infectious disease in human populations. Nat Rev Genet. 2014; 15(6):379-93. Kwiatkowski. How Malaria Has Affected the Human Genome and What Human Genetics Can Teach Us about Malaria. Am J Hum Genet. 2005; 77(2): 171–192. Lefer EM, Band G, Busby GBJ, Kivinen K, Le QS, Clarke GM, Bojang KA, Conway DJ, Jallow M, Sisay-Joof F, Bougouma EC, Mangano VD, Modiano D, Sirima SB, Achidi E, Apinjoh TO, Marsh K, Ndila CM, Peshu N, Williams TN, Drakeley C, Manjurano A, Reyburn H, Riley E, Kachala D, Molyneux M, Nyirongo V, Taylor T, Thornton N, Tilley L, Grimsley S, Drury E, Stalker J, Cornelius V, Hubbart C, Jefreys AE, Rowlands K, Rockett KA, Spencer CCA, Kwiatkowski DP, Network Malaria Genomic Epidemiology. Resistance to malaria through structural variation of red blood cell invasion receptors. Science 2017, https://doi.org/10.1126/ science.aam6393. Li WH and Sadler LA. Low nucleotide diversity in man. Genetics, 1991, 129: 513-523. Liti G, Carter DM, Moses AM, Warringer J, Parts L, James SA, Davey RP, Roberts IN, Burt A, Koufopanou V, et  al.. Population genomics of domestic and wild yeasts. Nature, 2009, 458:337-341. Malaria GEN MG, Network E.  Insights into malaria susceptibility using genome-wide data on 17,000 individuals from Africa, Asia and Oceania. Nat Commun, 2019 10(1):5732. https://doi. org/10.1038/s41467-­019-­13480-­z Marionneau S, Ruvoën N, Le Moullac-Vaidye B, Clement M, Cailleau-Thomas A, Ruiz-Palacois G, Huang P, Jiang X, Le Pendu J. Norwalk virus binds to histo-blood group antigens present on gastroduodenal epithelial cells of secretor individuals. Gastroenterology. 2002; 122(7):1967-77. Miller AC, Comellas AP, Hornick DB, et al. Cystic fibrosis carriers are at increased risk for a wide range of cystic fibrosis-related conditions. Proc Natl Acad Sci U S A. 2020; 117(3):1621-1627. https://doi.org/10.1073/pnas.1914912117. Miller LH, Mason SJ, Clyde DF, Mc Ginniss MH. The resistance factor to Plasmodium vivax in blacks. The Duffy-blood-group genotype, FyFy. N Engl J Med. 1976 Aug 5; 295(6):302-4. Mills RE, Walter K, Stewart C, Handsaker RE, Chen K, Alkan C, Abyzov A, Yoon SC, Ye K, Cheetham RK, et al. Mapping copy number variation by population-scale genome sequencing. Nature, 2011, 470:59–65. Perry GH, Dominy NJ, Claw KG, Lee AS, Fiegler H, Redon R, Werner J, Villanea FA, Mountain JL, Misra R, et al.. Diet and the evolution of human amylase gene copy number variation. Nat Genet. 2007, 39:1256–1260. Picard C, Puel A, Ku CL, Casanova JL. Primary immunodeficiencies associated with pneumococcal disease. Curr Opin Allergy Clin Immunol, 2003, 3: 451–459. Picard C, Casanova JL, Abel L. Mendelian traits that confer predisposition or resistance to specific infections in humans. Curr Opin Immunol, 2006, 18: 383–390. Qidwai T, Khan MY.  Impact of genetic variations in C-C chemokine receptors and ligands on infectious diseases. Hum Immunol. 2016; 77 (10):961-971. Qidwai T. Chemokine genetic polymorphism in human health and disease. Immunol Lett. 2016; 176:128–138. https://doi.org/10.1016/j.imlet.2016.05.018. Rosain J, Kong XF, Martinez-Barricarte R, et al. Mendelian susceptibility to mycobacterial disease: 2014–2018 update. Immunol Cell Biol. 2019; 97(4):360-367. https://doi.org/10.1111/ imcb.12210. Sayos J, Wu C, Morra M, Wang N, Zhang X, Allen D, van Schaik S, Notarangelo L, Geha R, Roncarolo MG et al.: The X-linked lymphoproliferative-disease gene product SAP regulates signals induced through the co-receptor SLAM. Nature 1998, 395:462-469. Tifert T, Lew VL, Ginsburg H, Krugliak M, Croisille L, Mohandas N.The hydration state of human red blood cells and their susceptibility to invasion by Plasmodium falciparum. Blood, 2005, 105(12):4853–4860. Timmann C, Thye T, Vens M, Evans J, May J, Ehmen C, Sievertsen J,Muntau B, Ruge G, Loag W, Ansong D, Antwi S, Asafo-Adjei E, Nguah SB, Kwakye KO, Akoto AO, Sylverken J,

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Brendel M, Schuldt K, Loley C, Franke A, Meyer CG, Agbenyega T, Ziegler A, Horstmann RD.  Genome-wide association study indicates two novel resistance loci for severe malaria. Nature, 2012, 489(7416):443–446. https://doi.org/10.1038/nature11334 Vannberg FO, Chapman SJ, Hill AV.  Human genetic susceptibility to intracellular pathogens. Immunol Rev. 2011; 240(1):105-16. World Health Organization. (2019). Global tuberculosis report 2019. World Health Organization. https://apps.who.int/iris/handle/10665/329368 WHO (2019). World malaria report 2019. Geneva: World Health Organization. WHO (2020). The potential impact of health service disruptions on the burden of malaria. World Health Organization. Zambo B, Varady G, Padanyi R, Szabo E, Nemeth A, Lango T, Enyedi A, Sarkadi B. Decreased calcium pump expression in human erythrocytes is connected to a minor haplotype in the ATP2B4 gene. Cell Calcium. 2017;65:73–79.

Part I RBC Disorders

2

Sickle Cell Gene

Abstract

Sickle cell gene is the signature of distribution of Plasmodium falciparum caused malaria throughout the world. Malaria parasite has imposed strong selective force on human genome in malaria endemic regions. Populations residing in these regions have developed protective mechanisms through natural selection against the disease pressure. Sickle cell trait, a common hemoglobin mutation worldwide, has protective effects against severe malaria in endemic regions. High incidence of malaria and insufficiency of effective treatments prompted to explore natural genetic control mechanisms against the disease. Treatment options of malaria are limited as parasite has development resistance to the most of available antimalarial drugs. This chapter covers information linked to incidence of sickle cell disease in malaria endemic regions and its impact on P. falciparum malaria. Keywords

Plasmodium falciparum malaria · Host genetic factor · Sickle cell disease · Selection · Endemic region

2.1

Introduction

Plasmodium falciparum malaria is a foremost reason of mortality and morbidity worldwide. Nearly, 219 million malaria cases and 435,000 deaths happened due to malaria in 2017 (WHO 2019). Completion of human genome project and advent of high throughput molecular biology techniques have eased the detection of role played by human host genetic factors and parasite variability in the susceptibility or resistance of disease. In evolutionary history, many natural genetic control strategies have been developed for resistance against infectious diseases. Hemoglobinopathies defend from life-threatening manifestation of malaria, © Springer Nature Singapore Pte Ltd. 2021 T. Qidwai, Exploration of Host Genetic Factors associated with Malaria, https://doi.org/10.1007/978-981-33-4761-8_2

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18

particularly in endemic regions of disease. Malaria is considered as the strong selective force for several erythrocytic defects, for instance sickle cell disease, thalassemia, and glucose-6-phosphate dehydrogenase (G6PD) deficiency. The most important of which is sickle cell disease (SCD) which caused nearly, 90% decline in risk of severe P. falciparum malaria in sub-Saharan Africa (Taylor et al. 2012). Heterozygotes for the sickle gene (AS) provide protection against malaria while homozygous for sickle gene give rise sickle cell anemia (SCA) which has lethal effects. Experimental model suggested that AS heterozygotes P. falciparum-infected red cells are preferentially eliminated by macrophages (Luzzatto 2012). Sickle cell trait is prevalent throughout world, making SCD one of the most common monogenetic diseases throughout the world (Pecker and Naik 2018). Presence of high frequency of sickle cell hemoglobin (HbS) in malaria endemic regions provides the evidence of natural selection of sickle cell allele under the pressure of malaria. Available antimalarial drugs are not so effective as a consequence of development of drug resistance in parasite. Increase in incidence of malaria and inadequate therapeutic approaches encouraged us to understand the human host genetic factors associated with malaria. This chapter is aimed to explore recent findings related to sickle cell disease as a genetic control mechanism to provide survival advantage in populations residing in malaria endemic regions.

2.2

Hypothesis

Genetic makeup of an individual contains information of human physiology, development, disease, and evolution. Different person may differ in their response to an infectious disease. High frequency of malaria is reported from malaria endemic regions, which is encountered by selection of genetic variations in human genome. Many red cell defects confer resistance to malaria are traditional examples of genetic footprints chosen in response to malaria disease pressure. Haldane hypothesized, that due to variations in genetic makeup people might have different risk of death caused by a parasite infection. If a human gene confers resistance its frequency could increase upon exposer of population to the parasite in spite of deleterious effect of that particular gene (Luzzatto 2012). Heterozygote of sickle cell disease has been found in high frequency in malaria endemic regions as protective trait.

2.3

Balanced Polymorphisms

Malaria shows balanced polymorphisms in which frequency of disease causing allele increases which is found helpful in protection against disease. For example, in response to malaria pressure high frequency of sickle cell disease is detected in malaria endemic regions. In a single population, presence of larger number of heterozygotes provides advantage of being, “malaria-resistant.” Advantage of heterozygotes coexists with

2.5 Epidemiology of Sickle Cell Hemoglobin

19

disadvantage of homozygotes. Hence, it is named a balanced polymorphism. Many association studies have been done to inspect the role of host genetic factors and susceptibility/resistance to malaria in different populations (Qidwai and Khan 2016; Sinha et  al. 2009). High prevalence of host erythrocyte polymorphisms such as alpha-thalassemia, hemoglobin variants (HbS, HbC and HbE), complement receptor-­1 (CR1) deficiency, G6PD deficiency and south-east Asian ovalocytosis (SAO) have been reported in malaria endemic regions (Pasvol et al. 1978). Studies have established that these polymorphisms decrease risk of severe P. falciparum malaria and known to contribute a survival advantage in humans against malaria (Weatherall 1987; Adewoyin 2015). Most of the studies related to these genetic variations have been done in malaria endemic areas of African countries.

2.4

Sickle Cell Disease

Normal adult hemoglobin (HbAA) has alpha globins expressed by HBA1 and HBA2 genes and beta globins expressed by HBB gene. HBB gene has three distinct coding polymorphisms, each has provided resistance to malaria in sub-Saharan Africa, Middle East and central India. Single base substitution in gene encoding beta globin chain of hemoglobin causes substitution of amino acid valine for glutamate (Glu → Val) on sixth amino acid position. This variant form of normal adult hemoglobin is called sickle cell hemoglobin (HbS) resulting in sickle cell disease (SCD). Homozygous states of HbS with two affected beta chains (HbSS) is called as sickle cell anemia (SCA), in which individuals usually die before adulthood, while heterozygous carrier state (HbAS) has sickle cell trait and are generally asymptomatic with protective effect from P. falciparum. This disadvantage of homozygous state and advantage of heterozygous are coexisting, hence called as balanced polymorphism. Glutamate is negatively charged, polar, and hydrophilic which is substituted by valine, a neutral, less polar, and hydrophobic amino acid (Fig. 2.1). This substitution induces polymerization of hemoglobin in the erythrocytes. Abnormal valine residue produces intra-erythrocytic hydrophobic interaction of sickle cell hemoglobin under deoxy conditions which lead to their precipitation and polymer formation resulting sickle shape of erythrocytes. The distinctive feature of sickle cell hemoglobin is that it is normal in oxygenated form and becomes abnormal in deoxygenated form (Wishner et al. 1996; Modell and Darlison 2008).

2.5

Epidemiology of Sickle Cell Hemoglobin

In numerous countries, 10–40% of the population comprises sickle cell gene resulting in sickle cell disease prevalence of nearly 2% (Regional Committee for Africa 2011). It has been reported that around 5–7% population has an abnormal hemoglobin gene (WHO 2019) and more than 312,000 infants has been born with homozygous HbS every year with enormous births arising in developing world. Approximately, 230,000 such births annually reported in sub-Saharan Africa

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2  Sickle Cell Gene

+

βAβA

βAβS

βSβS

HbA Normal

HbS Sickle cell

-

Normal beta chain of Hb

NH3

Val His Leu Thr Pro Glu

Glu

Sickle cell beta chain of Hb

NH3

Val His Leu Thr Pro Val

Glu

1. HbA = aa-bb = 1 glutamate in each b chain. 2. HbS = aa-b*b* = both glutamate replaced by valine 3. HbAS=aa-b*b=only one glutamate replaced. Glutamate still present on wild polypeptide Fig. 2.1  Illustration represents normal hemoglobin, sickle cell trait, and sickle cell anemia

(Serjeant and Serjeant 2001). Sickle cell trait frequency varies 10–45% in various regions of sub-Saharan Africa (Okwi et al. 2010). Sickle cell hemoglobinopathy and G6PD deficiency are the main health problems in tribal communities in India. The first documentation of sickle hemoglobin in India was in 1952 in tribal populations of Nilgiri hills in South India (Colah et al. 2015). In the same year, occurrence of sickle hemoglobin was reported in tea garden workers of Assam who migrated from tribal groups in Bihar and Odisha. Since then, in India many population groups have been screened. HbS is frequently found in central India, Andhra Pradesh, Maharashtra, Odisha, and Rajasthan. Rourkela of Odisha state has high frequency of sickle cell disease (0–22.4%). More than half (60.64%) cases of sickle cell found in four states: Gujarat (n = 29), Maharashtra (n = 32), Odisha (n = 37), and Chhattisgarh (n  =  53) (Hockham et  al. 2018). Heterozygotes of sickle cell hemoglobin, the HbAS is seemingly healthy and show protection against malaria and hence sickle cell defects are kept in high frequency as balanced polymorphism in several malaria endemic populations of Africa and India (Kreuels et al. 2010; Piel et al. 2013).

21

2.7 Malaria and Sickle Cell Hemoglobinopathy (HbS) Carrier Father Sickle cell trait (HbAS)

Unaffected child female HbAA

Carrier child male HbAS

Carrier Mother Sickle cell trait (HbAS)

Carrier child female HbAS

Affected child male HbSS

Fig. 2.2  Genetic variation in beta gene of hemoglobin and its inheritance

2.6

Genetic Origin of Sickle Cell Disease

It is believed that the source of SCD is Africa and Asia. SCD is the consequence of natural mutation affecting gametes and moved to successive generations. Prevalence of SCD is present in other regions of the world including the United States of America (USA), Europe, London and Paris (Modell et  al. 2007; Eridani 2011). Migration and interracial marriages are proposed to be the reasons that have led to an increase in frequency and genetic heterogeneity of hemoglobinopathies across the world (Eridani 2011). Genetic variation in beta gene of hemoglobin and its inheritance has been shown (Fig.  2.2). Haplotype study of the beta globin chain genes has identified African haplotypes (Senegal, Benin, Bantu, and Cameroon), and Asian haplotype (Arab-Indian) (Aidoo et al. 2002). Differential association of these haplotypes with disease has been found. The Bantu and Arab-Indian haplotypes have shown association with the most severe disease phenotype and mild phenotype respectively. It has been suggested that there is probable correlation between advent of agriculture and malaria. Agriculture was originated from Middle East but developed in Middle East, China, and Mexico. Population explosion forced migration of populations to new uncultivated land resulted, spread of agriculture from its origin to different parts like Africa, Europe, and eastwards to India (Allison 1964).

2.7

Malaria and Sickle Cell Hemoglobinopathy (HbS)

Studies have provided that there is strong correlation between malaria and sickle cell hemoglobin (HbS) in diverse populations of the world. Global distribution of sickle cell trait is varying with malaria endemicity. The highest allele frequency of this trait and high malaria endemicity was reported (Menzel and Thein 2009). Slow increase in HbS allele frequency in endemic areas in Africa and India showed that

22

2  Sickle Cell Gene

malaria protection through HbS includes heightening of innate as well as acquired immunity to P. falciparum. Determinants of hemoglobin F (HbF) levels and co-­ inheritance of alpha-thalassemia are the best characterized genetic modifiers of sickle cell trait. In Mali village, the highest exposure to malaria infection documented, survival benefit due to sickle cell trait could occur but no significant reduction in parasite load has been found. Malaria episode has been appeared significantly late in children with heterozygous sickle cell trait (Menzel and Thein 2009). The malaria occurrence in Odisha state of India need to pay special attention as the state contributes high incidence of disease in this country. Nearly 50% of demises arise as a result of malaria and 30% of P. falciparum malaria is reported from this Indian state. High frequency of SCD in the Odisha state is accompanied by exertion of selective force by malaria.

2.7.1 Hemoglobin C (HbC) HbC is another important hemoglobin variant protecting individuals against malaria in heterozygous (HbAC) as well as homozygous (HbCC) state. The impact of HbAS and HbAC genotypes on the incidence of parasitemia, uncomplicated malaria and anemia was analyzed (Jha et al. 2012). The mechanism of protection by HbAS and HbAC against malaria may be because of increasing antibody response to antigens or by means of other immune mechanisms (Agarwal et al. 2000). The variant HbC is been in various regions of West Africa but not as much of HbS, this may suggests lower frequencies of HbS allele in West Africa (Eridani 2011). Although it offers resistances against malaria in homozygous and heterozygous as well but a greater protective effect has been found in homozygous state (Williams et  al. 2005a, b). This is recognized on the basis of observation of decreased parasite cytoadherence, unusual PfEMP-1 expression, clustering of erythrocyte band 3 protein and transformed erythrocyte membrane in appearance of HbC. HbC, thalassemias along with membrane protein defects protect from malaria. Many of these mutations cause hemolysis in association with accumulation of free heme. HbE is another important hemoglobin variant common in South East Asia. Chotivanich et al. (2002) suggested that erythrocytes from HbE-heterozygous individuals are comparatively resistant to invasion by P. falciparum. Homozygotes generally has symptom less anemia.

2.7.2 Mechanism of Protection by Sickle Cell Hemoglobin Although several studies have been done to explore the protection caused by sickle cell hemoglobin but the mechanism by which sickle cell hemoglobin protects the individuals from malaria is still under investigation. Few genetic traits are most commonly expressed in erythrocytes to provide protection, for example, Duffy

2.7 Malaria and Sickle Cell Hemoglobinopathy (HbS)

23

Increased splenic phagocytosis (Luzzatto et al., 1970) Premature hemolysis, parasite death (Rothet al. 1978) HbS provides protection through

Impaired hemoglobin digestion (Pasvol 1980) Weakened cytoadherence (Cyrklaff et al. 2011) Acquired host immunity (Williamset al. 2005)

Fig. 2.3  The flow chart is illustrating mechanism through which sickle cell heterozygous individuals (HbAS) are comparatively protected from severe P. falciparum malaria

negative red cell causes failure of P. vivax invasion, hemoglobin variant CC causes impaired intra-erythrocytic growth and heterozygous sickle cell trait (HbAS) causes enhanced removal of parasitized red cells (Fig.  2.3). Luzzatto et  al. (1970) suggested that sickling may provide a mechanism for rapid clearance of infected erythrocytes by the spleen. Heightened phagocytosis of parasite infected mutant erythrocytes may denote a common strategy for protection against malaria disease in nonimmune persons with sickle cell trait and beta-thalassemia (May et al. 2007). One report elucidated that sickle cell hemoglobin is considerably more unstable than normal hemoglobin and through clustering of erythrocytes membrane proteins, it could hasten their elimination by phagocytic cells of immune system. Other in  vitro study demonstrated parasite growth inhibition and reduced invasion into sickle cell erythrocytes (Shear et  al. 1993). Study suggested that this is to some extent, due to a decrease of hemoglobin solubility under low oxygen tension and augmented sickling of parasitized erythrocytes with raised removal by reticuloendothelial system of the human body (Hebbel et al. 1988). Eridani (2011) suggested that protection against malaria is caused by a comparatively high rate of free heme production from sickle cell hemoglobin than normal hemoglobin. Such faster heme loss from oxygenated sickle cell hemoglobin is due to accelerated autoxidation (Higgs et al. 1982). Too much formation of superoxide by sickle erythrocytes and abnormal deposition of heme and heme proteins on membranes of sickle erythrocytes have been reported (Cholera et al. 2008). Other hypothesis proposed that P. falciparum Erythrocyte Membrane Protein 1 (PfEMP1) expression is reduced on erythrocytes with sickle cell hemoglobin (Baruch et al. 1997) (Fig. 2.4). PfEMP1 interacts with host adhesion molecules and involved in cytoadherence resulting decreased interaction of infected cells to the endothelium (Cholera et al. 2008; Archer et al. 2018). Nearly one half of cytoadherence has been seen in sickle cell erythrocytes infected with parasite.

24

2  Sickle Cell Gene

Fig. 2.4 Alternative hypothesis proposed that PfEMP1expression is reduced on erythrocytes with sickle cell hemoglobin (Cholera et al. 2008; Archer et al. 2018)

Reduced

expression

of

Pf EMP1

on

erythrocytes having sickle cell hemoglobin

Sickle cell erythrocyte leads to the reduced binding of infected cells to the endothelium

Reduced cytoadherence of infected erythrocytes

2.7.3 Hemoglobin Degradation by Plasmepsins The exact mechanism by which sickle cell trait give rise resistance to malaria has to be analyzed by considering the degradation of normal hemoglobin and sickle cell hemoglobin in infected erythrocyte by parasite proteolytic enzymes. It is likely that this protection is mediated by altered susceptibility of mutant hemoglobin to hydrolysis by parasite proteases, including falcipains. But other study has demonstrated that, sickle cell hemoglobin is cleaved in an identical mode to normal hemoglobin (Ezebuo et al. 2012). Membrane lipid oxidation of the parasite happens due to deoxygenation of hemoglobin and formation of methemoglobin. They may be responsible for malaria resistance in individuals with hemoglobin variants (HbAS).

2.8

Other Natural Protective Mechanisms

Besides hemoglobin polymorphisms, varying frequencies of many of other genetic variants have been found in malaria endemic regions. Cockburn et al. (2004) suggested that, low CR1 expression allele is linked with alpha-thalassemia in malaria endemic areas of Papua New Guinea that gave protection from severe falciparum malaria. Population in endemic region of India has very low erythrocyte cell surface CR1 levels. Higher frequencies of low expression alleles (exon 22 and intron 27 mutant L) has been found (Opi et al. 2016; Sinha et al. 2009; Kreuels et al. 2009). Therefore, it has been suggested that high frequencies of mutant alleles are associated with low CR1 expression on the erythrocytes which in turn is associated with protection from falciparum malaria in endemic region of India.

2.9 Conclusions

25

2.8.1 Knops Blood Group System CR1 genetic variants also produce Knops blood group system of antigens. Knops blood group comprises allelic pairs Swain-Langley 1 and 2 (S/1 and S/2), McCoy a and b (McCa and McCb) and Knops a and b (Kna and Knb). The S/2 and McCb are present in higher frequencies in African than non-Africans. CR1 Knops blood group alleles S/2 and McCb and homozygous HbSS have shown a positive association with CR1 level on erythrocytes, whereas alpha-thalassemia is associated with reduced erythrocyte CR1 level (Opi et al. 2016; Sinha et al. 2009).

2.8.2 Adhesion Molecules Platelet cell adhesion molecule-1 (PECAM1) gene (rs668, exon 3, C/G) displayed differential association with disease in in endemic and non-endemic regions of India. G allele has been reported as a risk factor for malaria in endemic region of India (Sinha et al. 2008). African populations comprise remarkably high frequency of mutations in CD36. Mutation leading to CD36 deficiency showed association with susceptibility to severe malaria. It has been suggested that CD36 is a contributing factor of developing cerebral malaria as compared to other severe malaria complications (Aitman et  al. 2000). High occurrence of the Gerbich-negative blood group which is originated as a consequence of mutation in gene encoding glycophorin C, or GYPC-Dex3 has shown association with resistance to malaria in Papua New Guinea populations (Booth and McLoughlin 1972). Gerbich blood group antigens present on glycophorin C and D serve as receptors for P. falciparum whereas decreased levels of glycophorin C and D have shown association with hereditary elliptocytosis (Walker and Reid 2010).

2.9

Conclusions

Due to genetic complexity and multiple phases in life cycle of malaria parasite, it is difficult to treat malaria. Furthermore, parasite developed resistance to majority of antimalarial drugs. Due to high incidence of malaria, the researchers are being done extensive effort to develop effective treatment options. Sickle cell trait provides survival benefit to humans against P. falciparum caused malaria in endemic regions of sub-Saharan Africa and India, where high frequency of malaria has been found. Genome develops genetic control mechanism through natural selection against infectious disease. Sickle cell gene and malaria both coexist in malaria endemic regions. Disadvantage of mutation is associated with advantage of protection against the infectious disease. It could be important to focus on study of natural genetic control mechanisms as the basis for repeated efforts to generate vaccines for malaria prevention.

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Menzel S, Thein SL (2009) Genetic architecture of hemoglobin F control. Curr Opin Hematol 16(3):179-186. Modell B, Darlison M (2008) Global epidemiology of haemoglobin disorders and derived service indicators. Bulletin of the World Health Organization 86:480-487. Modell B, Darlison M, Birgens H et al (2007) Epidemiology of haemoglobin disorders in Europe: an overview. Scand J of Clinical and Labo Invest 67: 39-69. Natasha M. Archer, Nicole Petersen, Martha A. Clark, Caroline O. Buckee, Lauren M. Childs, and Manoj T. Durai Singh. Resistance to Plasmodium falciparum in sickle cell trait erythrocytes is driven by oxygen-dependent growth inhibition. PNAS, 2018 115 (28) 7350-7355. Okwi AL, Byarugaba W, Ndugwa CM, Parkes A et  al (2010) An up-date on the prevalence of sickle cell trait in Eastern and Western Uganda. BMC Blood Disorders 10: 5. Opi DH, Uyoga S, Orori EN, Williams TN, Rowe JA (2016). Red blood cell complement receptor one level varies with Knops blood group, α+thalassaemia and age among Kenyan children. Genes and Immunity 17:171–178. Pasvol G, Weatherall DJ, Wilson RJ (1978) Cellular mechanism for the protective effect of hemoglobin S against P. falciparum malaria. Nat 274(5672):701-703. Pecker LH, Naik RP. The current state of sickle cell trait: implications for reproductive and genetic counseling. Blood. 2018; 132(22):2331–2338. Piel FB, Patil AP, Howes RE et al (2013) Global epidemiology of Sickle haemoglobin in neonates: a contemporary geostatistical model-based map and population estimates. The Lancet 381: 142-151. Qidwai T, Khan MY (2016) Impact of genetic variations in C-C chemokine receptors and ligands on infectious diseases. Hum Immun 77:961-971. Regional Committee for Africa (2011) Sickle-Cell Disease: a strategy for the WHO African Region. 60. https://apps.who.int/iris/handle/10665/1682 Serjeant GR. Serjeant BE (2001) The epidemiology of sickle cell disorder: a challenge for Africa. Archives of Ibadan Medicine 2: 46-52. Shear HL, Roth EF, Fabry ME, et al (1993) Transgenic mice expressing human sickle hemoglobin are partially resistant to rodent malaria. Blood 81(1):222-226. Sinha S, Qidwai T, Kanchan K, Anand P, Jha GN, Pati SS, Mohanty S, Mishra SK, Tyagi PK, Sharma SK. Indian Genome Variation Consortium, Venkatesh V, Habib S. Variations in host genes encoding adhesion molecules and susceptibility to falciparum malaria in India. Malar J. 2008 Dec 4;7:250. https://doi.org/10.1186/1475-­2875-­7-­250. PMID: 19055786; PMCID: PMC2612678. Sinha S, Jha GN, Anand P, Qidwai T et al (2009) CR1 levels and gene polymorphisms exhibit differential association with falciparum malaria in regions of varying disease endemicity Human Immunology 70:244-250. Taylor SM, Parobek CM, Fairhurst RM. Haemoglobinopathies and the clinical epidemiology of malaria: a systematic review and meta-analysis. Lancet Infec Dis. 2012; 12(6):457-68. Walker PS, Reid ME (2010) The Gerbich blood group system: a review. Immunohe-matology 26:60-5. Weatherall DJ (1987) Common genetic disorders of the red cell and the malaria hypothesis. Ann Trop Med Parasitol 81(5):539- 548. WHO (2019) World Malaria Report, World Health Organization. Williams TN, et al. (2005a) An immune basis for malaria protection by the sickle cell trait. PLoS Med 2:e128. Williams TN, Mwangi TW, Wambua S, Alexander ND (2005b) Sickle Cell Trait and the Risk of Plasmodium falciparum Malaria and Other Childhood Diseases. Infect Dis 192(1): 178-186. Wishner BC, Ward KB, Lattman EE, Love WE (1996). Crystal structure of sickle-cell deoxyhemoglobin at 5 resolution. J MoleBiolo 98:179-191.

3

Alpha-Thalassemia

Abstract

Alpha-thalassemia is a type of recessive genetic disease affecting people in several parts of the world. This disease appears because of genetic defect(s) in alpha-globin chain of hemoglobin. Although alpha-thalassemia frequently exists in Africa, South East Asia, Middle East and India, but its prevalence has increased in other regions of the world for instance North European countries and Northern America as well. There is a strong relationship between alpha-thalassemia and Plasmodium falciparum caused malaria. Malaria forces the natural selection of genetic variations leading to diseases such as thalassemia, sickle cell hemoglobin and other diseases connected to the structure and function of erythrocytes. Malaria and alpha-thalassemia are coexisting in endemic regions. Because of the high occurrence of malaria in endemic regions, the human genome develops genetic control mechanisms to overcome the action of disease. This chapter is aimed to investigate the possible association of malaria with alpha-thalassaemia among different populations of the world. It would be helpful in identification of new therapeutic approach in malaria prevention and control. Keywords

Alpha-thalassemia · Globin gene · Malaria · Balancing trait · Endemic region

3.1

Introduction

Hemoglobin disorders such as thalassemia and sickle cell disease cause significant morbidity and mortality worldwide (Crighton et  al. 2016). Alpha-thalassaemia is monogenic, autosomal recessive disorder, specifically common in Mediterranean countries, South East Asia, Africa, Middle East, and India. Due to demographic alterations during last few years, frequency of alpha-thalassemia has increased in © Springer Nature Singapore Pte Ltd. 2021 T. Qidwai, Exploration of Host Genetic Factors associated with Malaria, https://doi.org/10.1007/978-981-33-4761-8_3

29

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3 Alpha-Thalassemia

North European countries and Northern America. Approximately 7% of pregnant females have beta or alpha zero thalassemia, or variant form of hemoglobins (Modell and Darlison 2008). Hemoglobin disorders including Alpha-thalassaemia were formerly distinguishing features of the tropics and subtropics but presently found globally because of migration (Weatherall and Clegg 2001a, b). Alpha-thalassemia is categorized by a shortage in making of alpha-globin chains of hemoglobin. Human hemoglobin consist of four chains: HbF known as fetal hemoglobin carries two alpha and two gamma chains (α2γ2) and HbA named as adult hemoglobin carries two alpha and two beta chains (α2β2). Globin gene production is regulated by alpha gene cluster and beta gene cluster positioned on chromosomes number 16 and 11 respectively. Hemoglobin gene variants are categorized into, structural variants arises because of change in amino acid sequence and consequent making of unusual hemoglobin and other group includes thalassaemias that arises due to lower/non production of globin chains. Diminished expression of alpha-globin chains give rise surplus beta-like globin chains which form gamma4 (γ4) tetramers, named as Hb Bart’s (in fetal life), while beta 4 (β4) tetramers, named as HbH (in adult life) (Harteveld and Higgs 2010). Individuals having mutations that influence alpha-globin genes, showed association with nominal anemia called as alpha-­ thalassemia trait. Multiple heterozygotes and some homozygotes of alpha-­ thalassaemia contain average severe anemia represented by existence of HbH in peripheral blood. Human individual with very small or no alpha-globin chain production has severe form of anemia with no production of alpha genes and is named as Hb Bart’s Hydrops Fetalis Syndrome (Farashi and Harteveld 2018; Harteveld and Higgs 2010). Malaria is one of the major problems of many countries including India. In malaria endemic regions, it acts as driving force for selection of traits like sickle cell hemoglobin and thalassemia. It is supposed that globin gene disorders have been selected to protect carriers from P. falciparum malaria. In those areas where malaria is endemic, high frequency of this trait has been reported. Usually, 3–40% of people comprise one of these variants; incidence of hemoglobin disorders varies from 0.3 to 25 for each 1000 births in endemic regions (Angastiniotis and Modell 1998). In response to high frequency of malaria, human genome has developed genetic control mechanisms to overcome the disease. This chapter is focused on alpha-­ thalassemia, its type, epidemiology and its role in P. falciparum caused malaria in various population.

3.2

Mechanism

The name thalassemia originates from the Greek name “Thalassa,” which meant the spirit of the sea in Greek mythology. Although thalassemias are reported throughout the world but first cases of thalassemia has been seen in Mediterranean Basin. Alpha-thalassemia a monogenic disorder affected 5% population of the world (Vichinsky 2010).

3.3 Epidemiology

31

Alpha-thalassemia offers protection against malaria through

Improved immune clearance

Diminished survival of malaria parasite in erythrocytes

Decreased ability of parasite for invading erythrocytes

Fig. 3.1  The flowchart represents alpha-thalassemia in protection against malaria

In spite of widespread studies, the mechanism of protection is still need to explore (Galanello and Cao 2011). However, it has been found that alpha and beta hemoglobin mutations protect against malaria through improved immune clearance, diminished survival of malaria parasite in erythrocytes and decreased ability of parasite for invading erythrocytes (Fig. 3.1). Children in Papua New Guinea (PNG) population with alpha-thalassemia have their erythrocytes abnormally small and more plentiful, caused a mild form of anemia, as compared with erythrocytes without thalassemia. The alpha-thalassemias give rise to an advantage against malaria infection. It has been found that several nations in the Mediterranean, comprising Greece, eradicated malaria nonetheless increase in the number of imported cases have seen due to increased global travel, climate changes, and spread of people from malaria endemic regions (Opi et al. 2014). Mutation in beta chain of hemoglobin confers protection against malaria, however presence of thalassemia along with sickle cell anemia (SCA), make the individual susceptible to malaria infection (Leffler et  al. 2017) as both mutations principally cancel each other (Leffler et al. 2017).

3.3

Epidemiology

Alpha-thalassaemia occurs at high frequencies in Africa, Middle East, India, Southeast Asia, southern China, and sometimes in Mediterranean area. Carrier frequency of alpha-thalassaemia is very high (80–90%) in some populations, approximately at fixation (Harteveld and Higgs 2010). HbH disease is widely distributed across the world, mainly, it has been found in South East Asia, Middle East and Mediterranean countries. Likewise, Hb Bart’s Hydrops fetalis mostly present in South East Asia. Over the years due to massive population movement, globin gene disorders spread in North European, North American, and Australian (Galanello and Cao 2011). Approximately 7% of pregnant women consists of beta or alpha

32

3 Alpha-Thalassemia

zero thalassemia (Modell and Darlison 2008). Around 15% of American black population are silent carriers of alpha-thalassemias. One study in Morocco has identified six different alpha-globin genetic disorders (Laghmich et al. 2019). In India, prevalence of alpha-thalassemias is around 12.9%. The highest prevalence of alpha-thalassemia has been found in Punjabi population derived from northern area of India (Nadkarni et al. 2008). Very high incidence of thalassemia has been reported in Maldives. Thalassemia is commonly found in areas where malaria disease is endemic as unusual erythrocyte provides adverse milieu for malarial parasite to complete its life cycle (Nadkarni et al. 2008).

3.4

Types of Alpha-Thalassemia

Alpha-thalassemia is classified according to number of nonfunctional alpha-globin chain. Four types of alpha-thalassemia have been found in human beings including alpha-thalassemia minor (alpha-thalassemia trait), Alpha-thalassemia silent carrier, Hemoglobin H (HbH) disease and Hb Bart’s hydrops fetalis (Fig. 3.2).

3.4.1 Alpha-Thalassemia Trait The alpha-thalassemia trait is expressed as αα/−− or α−/α− (Fig.  3.3a). Usually, alpha-thalassemia traits (αα/−−) do not cause health problem. In this trait both male and female parents consist of trans-form of alpha-thalassemia trait (α−/α−). Individuals having alpha-thalassemia trait consist of small erythrocytes and a low erythrocytes count.

Alpha-thalassemia

4 loci affected

3loci affected

HbH disease

2 loci affected

2 loci on same chromosome Alpha0-thalassemia

2 loci on different chromosome Alpha+-thalassemia

Hydrops fetalis (alphathalassemia)

Fig. 3.2  Classification of different form of alpha-thalassemia

1 locus affected

Silent alphathalassemia

3.4 Types of Alpha-Thalassemia

(a) Alpha-thalassemia trait

(b) Silent carrier state

33

(c) Hb H disease

(d) Hydrops fetalis (alpha-thalassemia major)

Fig. 3.3  Cartoon representation of different types of alpha-thalassemia

3.4.2 Silent Carrier State of Alpha-Thalassemia Silent carrier state of alpha-thalassemia arises due to the loss of a single alpha allele and expressed as αα/α− (Fig. 3.3b). In this type of thalassemia, deletion or nonfunctional one alpha-globin gene is found. No clinical or hematologic anomalies are linked with this type of alpha-thalassemia.

3.4.3 Hemoglobin H (Hb H) Disease Hemoglobin H disease is produced due to defect at 3loci. This trait is expressed as α−/−−. Hemoglobin H (HbH) disease arises in offspring when one parent consist of alpha-thalassemia trait in cis form represented as αα/−− and another parent has

3 Alpha-Thalassemia

34

alpha-thalassemia in silent carrier form denoted as αα/α−. After each pregnancy, there may be 25%, chance of having a child with HbH disease (Fig. 3.3c). Individuals with HbH disease have symptoms of an enlarged spleen, low erythrocyte counts, and gall stones.

3.4.4 Hb Bart’s Hydrops Fetalis Syndrome Hydrops fetalis is severe health problem develops due to non-production of alpha-­ globin that give rise death before or shortly after birth. Generally, children born with hydrops fetalis do not live, as they are not able to make enough hemoglobin. Usually, Hb Bart’s Hydrops fetalis syndrome arises in offspring because of inheritance of a deletion allele with nonfunctional alpha-globin genes from both male and female parents. It has been found in few cases that a compound heterozygosity for two diverse defects is accountable for a state which is between HbH disease and Hb Bart’s Hydrops Fetalis Syndrome. Both parents with alpha-thalassemia trait in cis form expressed as αα/−−, may contain 25%, chance of having a child with hydrops fetalis (−−/−−) (Fig. 3.3d).

3.5

Molecular Basis of Thalassemia

The alpha-globin production is controlled by four alpha-globin genes present on every copy of chromosome number 16 in normal human beings. Mostly, loss of one (−α) or both (−−) alpha genes give rise to alpha-thalassaemia. Usually, reason includes passing of nonfunctional alpha-globin genes (because of a deletion allele) from both male and female parents. One embryonic gene (ζ), two minor alpha-like genes (αD), two pseudogenes (ψα1 and ψζ) and two alpha genes (α2 and α1) constitute the alpha-globin gene cluster in telomeric area of small arm of chromosome 16 (16p13.3) (Higgs 1993; Hughes et al. 2005) (Fig. 3.4a). Genes cluster expressing beta-like globin chains on small arm of chromosome 11 (11p15.5), comprises embryonic ε-globin gene, fetal globin genes Gγ and Aγ, pseudogene ψβ and adult δ- and β-globin genes (Fritsch et al. 1980; Farashi and Harteveld 2018) (Fig. 3.4b).

3.6

Inheritance Pattern

The alpha-thalassemia inheritance follows a complex pattern. Every individual receives two alpha-globin alleles from male and two from female parent. Assuming that both parents have lost at least one alpha-globin allele, their progenies may comprise Hb Bart syndrome, HbH disease, or alpha-thalassemia trait. Risk is determined by number of missing alleles and the combination of HBA1 and HBA2 genes. Half of alpha-globin genes are lost/defective in alpha-thalassemia trait. If both abnormal alpha-globin genes present on same chromosome it will be called as cis position and when on each chromosome in the pair it will be named as trans

35

3.7 Diagnosis

Chromosome 16

16p:13.3

5’

ζ

α2

α1

3’

(a)

Chromosome 11

11p:15.4

5’

ε

γ

γ

δ

β

3’

(b) Fig. 3.4 (a) Figure represents alpha-globin gene cluster on small arm of chromosome 16. (b) Representation of genes expressing beta-like globin chains present on chromosome 11

position. When both abnormal genes are present on contrasting chromosomes in both male and female parents then their offspring will receive alpha-thalassemia trait (Fig. 3.5a). One parent with alpha-thalassemia trait in cis position (two abnormal alpha-­ globin genes on same chromosome) and another parent with silent carrier state alpha-thalassemia (a single abnormal alpha-globin gene on a chromosome) will produce 25% progeny with normal Hb, 25% HbH disease, 25% alpha-thalassemia and 25% alpha-thalassemia silent carrier (Fig. 3.5b). Once male and female parents comprise alpha-thalassemia trait in cis position will produce 25% progeny with normal Hb, 50% Alpha-thalassemia (cis) and 25% Hydrops fetalis (alpha-­ thalassemia major) (Fig. 3.5c).

3.7

Diagnosis

3.7.1 Hematological Test Usually, extent of microcytic, hypochromic anemia is determined by number of alpha genes mutated and shows connection with decrease in alpha-chain synthesis (Weatherall and Clegg 2001a, b; Higgs 1993; Wilkie 1991). HPLC and capillary electrophoresis are used to distinct abnormal hemoglobin fractions (Fig. 3.6).

36

3 Alpha-Thalassemia

3.7.2 Molecular Diagnosis Several molecular techniques are available for diagnosis of thalassemia. Diagnosis of hemoglobin H (Hb H) disease has been carried out using multiplex PCR and reverse dot blot hybridization. Multiplex-PCR is utilized for amplification of target fragments and then hybridization with allele-specific oligonucleotide probes which are bound on a nylon membrane (Nittayaboon, and Nopparatana 2018). Southern blotting and DNA sequence analysis are used in characterization of most alpha-­ globin rearrangements. These techniques are so laborious and hence, fast screening assays need to investigate. Technique of Gap-PCR has been established for seven

a

Alpha- thalassemia trait

Alpha- thalassemia trait

Alpha- thalassemia trait Fig. 3.5 (a) Male and female parents consists of alpha-thalassemia trait with two abnormal genes of each parent are present on contrasting chromosomes. The progeny produced from these parents will have alpha-thalassemia trait. (b) The figure represents the inheritance pattern of alpha-­ thalassemia trait and silent carrier state parents, resulting progeny will have 25% normal Hb, 25% HbH, 25% alpha-thalassemia, and 25% alpha-thalassemia silent carrier. (c) In this cartoon both parents consists of alpha-thalassemia trait. Abnormality in alpha-globin genes is present on same chromosome of each parent

3.7 Diagnosis

37

b

Alpha- thalassemia trait

25% Normal Hb

Fig. 3.5 (continued)

25% Hb H disease

Silent carrier state

25% Alpha-thalassemia

25% Alpha-thalassemia silent carrier

38

3 Alpha-Thalassemia

c

Alpha-thalassemia

25% Normal hemoglobin

Alpha-thalassemia

50% Alpha-thalassemia (Cis)

25% Hydrops fetalis (alphathalassemia major)

Fig. 3.5 (continued)

utmost common alpha-thalassaemia deletions (Chong et  al. 2000). Multiplex Ligation-dependent Probe Amplification (MLPA) is used for diagnosis (White et al. 2004). It is already known that alpha-thalassemias, are usually generated by partial or complete deletion of one or both alpha genes in haploid genome or, hardly, due to deletion of alpha-globin major regulatory element (HS-40). MLPA is used to identify changes that are accountable for alpha-thalassemia, mainly huge deletions leading to complete alpha cluster and/or deletions in HS-40 area (Mota et al. 2017).

3.9 Alpha-Thalassemia and Malaria

39

Normal erythrocyte

RBC

WBC

Microcytic

RBC

WBC

Fig. 3.6  Structure of normal erythrocyte and microcytic

3.8

Therapy

3.8.1 Blood Transfusion Blood transfusion is done to keep a pretransfusional Hb level and a post-transfusion Hb level of 9–10  g/dl and 13–14  g/dl respectively. This administration usually inhibits growth impairment, organ injury, and bone malformations.

3.8.2 Transplantation of Bone Marrow and Cord Blood Transplantation of bone marrow is only conclusive medication existing for thalassemic patients. Still, main limitations of allogenic bone marrow transplantation have been found. Cord blood transplantation may also be used.

3.8.3 Gene Therapy In thalassemia, use of gene therapy is not sufficiently effective. Usefulness of this therapy will be determined by the effectiveness of gene delivery and numerous additional factors including viral titers, non-oncogenic insertion, differential expression of globin genes and differential contributions of beta-thalassemia phenotype.

3.9

Alpha-Thalassemia and Malaria

Malaria caused by Plasmodium falciparum is an example of balancing selection. It exerts driving force for selection of thalassemia, sickle cell anemia, and glucose 6 phosphate dehydrogenase deficiency to protect from malaria particularly in endemic

40

3 Alpha-Thalassemia

regions. In balancing selection one disease is selected against the pressure of other disease for example, high frequencies of hemoglobinopathies have been found in endemic regions of malaria. Disadvantage of genetic diseases (hemoglobinopthies) are associated with advantage of protection from malaria.

3.9.1 Alpha-Thalassemia Trait and P. falciparum Malaria Protection Globally, around 3.2 billion people are at risk for malaria (The National Institute of Allergy and Infectious Disease reports). WHO declared that around half of population of the world is at risk and nearly 90 countries and regions throughout the world have cases of malaria transmission (WHO 2018). Elevated frequency of alpha-­ thalassemia reported in those regions where malaria is endemic. In non-malarial environments, alpha-thalassemia is rare. In malarial surroundings, loss of alpha-­ globin genes (loss of one/two copies) in usual diploid genotypes provides resistance (lesser death) to malaria. Increase in alpha−− (−−α) haplotype (with one deleted gene) was associated with fitness in Kenyan and Papua New Guinea populations. Double deletion frequency (−− haplotypes) is greater in few Asian populations as compared to single deletions and heterozygotes with normal alpha++ (++α) haplotypes are likely to keep highest fitness. The genetic investigation has provided evolutionary framework for detection of global frequency of alpha-thalassemia in both malaria endemic and non-endemic environments (Hedrick 2011). Mechanism of malaria protection by alpha-thalassemia has been proposed. Parasite infected alpha-thalassemic erythrocytes showed abnormality in presentation of P. falciparum erythrocyte membrane protein 1 (PfEMP1) on surface. It may be hypothesized that other form of hemoglobins such as HbE and unpaired alpha-­ globin chains protect against malaria by an analogous mechanism (Krause et  al. 2012). It may be suggested that alpha-thalassemia damages cytoadherence of infected erythrocytes to microvascular endothelial cells and monocytes. P. falciparum exports PfEMP1 proteins and centralize them in knob-like protrusions on surface of their host erythrocytes. Such host cell alteration allows a big mass of parasitized erythrocytes to sequester in venous micro vessels and evade elimination from bloodstream by spleen. Nevertheless, adherence of parasitized erythrocytes to microvascular endothelial cells and monocytes too contributes to malaria disease by causing systemic microvascular inflammation (Krause et  al. 2012). Alpha-­ thalassaemia trait shows association with severe malaria protection. Heterozygous children are linked with decreased risk of parasitemia compared to heterozygous adults and pregnant women. Kids having heterozygous alpha-thalassaemia trait demonstrated decreased risk of asexual parasite carriage. Though, no link between alpha-thalassaemia trait and risk of gametocyte carriage has been found. The study investigated malaria association with alpha-thalassaemia among Ghanaian population (Lamptey et al. 2019). In Tanzania, a malaria endemic region, alpha (+)-thalassemia is commonly found and linked with P. falciparum transmission intensity (Enevold et  al. 2007). Alpha+-thalassemia prevalence has been connected with

References

41

malaria endemicity in sub-Saharan Africa (Enevold et al. 2007). This study is supported by previous finding conducted in Sardinia (Siniscalco et  al. 1961) and Melanesia (Flint et al. 1986). One report suggested a negative interaction between sickle cell hemoglobin and alpha+-thalassemia (Williams et al. 2005). When the two traits are inherited together with, then malaria-defensive outcome of either trait is vanished. High frequencies of either trait are accumulated in remarkably high malaria transmission areas.

3.10 Conclusion Alpha-thalassemia, a recessive, monogenic disorder is affecting people all over the world. It has shown a protective association with P. falciparum malaria, both are coexisting in malaria endemic regions. Development of drug resistance in malaria parasite, lack of effective treatment options and high mortality rate caused by malaria prompted to explore the role of genetic control mechanisms against malaria. Natural selection of alpha-thalassemia has been found a good strategy against malaria. Detection of the possible association of malaria with alpha-thalassaemia among different populations of the world would be helpful in identification of new therapeutic approach in malaria prevention and control.

References Angastiniotis M, Modell B.  Global epidemiology of hemoglobin disorders. Ann N Y Acad Sci 1998; 850: 251-69. https://doi.org/10.1111/j.1749-­6632.1998.tb10482.x pmid: 9668547. Chong SS, Boehm CD, Higgs DR, Cutting GR: Single-tube multiplex-PCR screen for common deletional determinants of alpha-thalassemia. Blood. 2000, 95: 360-362. Crighton G, Wood E, Scarborough R, Ho PJ, Bowden D.  Haemoglobin disorders in Australia: where are we now and where will we be in the future? Intern Med J. 2016; 46 (7): 770-779. https://doi.org/10.1111/imj.13084. Edward F Fritsch, ∗Richard M Lawn, †Tom Maniatis. Molecular cloning and characterization of the human β-like globin gene cluster. Cell. 1980; 19 (4): 959-972. https://doi. org/10.1016/0092-­8674(80)90087-­2. Enevold A, Alifrangis M, Sanchez JJ, Carneiro I, Roper C, Børsting C, Lusingu J, Vestergaard LS, Lemnge MM, Morling N, Riley E, Drakeley CJ.  Associations between alpha+-thalassemia and Plasmodium falciparum malarial infection innortheastern Tanzania. J Infect Dis. 2007; 196(3):451-9. Farashi S, Harteveld CL. Molecular basis of α-thalassemia. Blood Cells Mol Dis. 2018; 70:43-53. https://doi.org/10.1016/j.bcmd.2017.09.004. Flint J, Hill AVS, Bowden DK, et  al. High-frequencies of alpha-thalassemia are the result of natural-­selection by malaria. Nature 1986; 321:744–50. Galanello, R., Cao, A. Alpha-thalassemia. Genet Med, 2011, 13, 83-88. https://doi.org/10.1097/ GIM.0b013e3181fcb468. Harteveld CL, Higgs DR. Alpha-thalassaemia. Orphanet J Rare Dis. 2010 28; 5:13. https://doi. org/10.1186/1750-­1172-­5-­13. Hedrick PW. Selection and mutation for α Thalassemia in nonmalarial and malarial environments. Ann Hum Genet. 2011; 75(4):468-74. https://doi.org/10.1111/j.1469-­1809.2011.00653.

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Higgs DR: Alpha-thalassaemia. Baillieres Clin Haematol. 1993, 6: 117-150. https://doi. org/10.1016/S0950-­3536(05)80068-­X. Hughes JR, Cheng JF, Ventress N, Prabhakar S, Clark K, Anguita E, De Gobbi M, de Jong P, Rubin E, Higgs DR. Annotation of cis-regulatory elements by identification, subclassification, and functional assessment of multispecies conserved sequences. Proc Natl Acad Sci U S A. 2005 Jul 12;102(28):9830-5. https://doi.org/10.1073/pnas.0503401102. Epub 2005 Jul 5. PMID: 15998734; PMCID: PMC1174996. Krause MA, Diakite SA, Lopera-Mesa TM, Amaratunga C, Arie T, Traore K, Doumbia S, Konate D, Keefer JR, Diakite M, Fairhurst RM. α-Thalassemia impairs the cytoadherence of Plasmodium falciparum-infected erythrocytes. PLoS One. 2012; 7(5):e37214. https://doi. org/10.1371/journal.pone.0037214. Laghmich A, Alaoui Ismaili FZ, Barakat A, Ghailani Nourouti N, Khattab M, Bennani Mechita M. Alpha-Thalassemia in North Morocco: Prevalence and Molecular Spectrum. Biomed Res Int. 2019; 2019: 2080352. https://doi.org/10.1155/2019/2080352. Lamptey H, Ofori MF, Adu B, Kusi KA, Dickson EK, Quakyi I, Alifrangis M. Association between alpha-thalassaemia trait, Plasmodium falciparum asexual parasites and gametocyte carriage in a malaria endemic area in Southern Ghana. BMC Res Notes. 2019;12(1):134. https://doi. org/10.1186/s13104-­019-­4181-­8. Leffler EM, Band G, Busby GBJ, et al. Resistance to malaria through structural variation of red blood cell invasion receptors. Science. 2017; 356:eaam6393. Modell B, Darlison M. Global epidemiology of haemoglobin disorders and derived service indicators. Bull World Health Organ. 2008; 86(6):480-487. https://doi.org/10.2471/blt.06.036673. Mota NO, Kimura EM, Ferreira RD, et al. Rare α0-thalassemia deletions detected by MLPA in five unrelated Brazilian patients. Genet Mol Biol. 2017; 40(4):768-773. Nadkarni A, Phanasgaonkar S, Colah R, Mohanty D, Ghosh K. Prevalence and molecular characterization of alpha-thalassemia syndromes among Indians. Genet Test. 2008; 12(2):177-80. Nittayaboon K, Nopparatana C. Molecular characterization of Hb H disease in southern Thailand. Int J Hematol. 2018; 108(4):384-389. https://doi.org/10.1007/s12185-­018-­2494-­3. Opi DH, Ochola LB, Tendwa M, et  al. Mechanistic studies of the negative epistatic malaria-­ protective interaction between sickle cell trait and α+thalassemia. EBioMedicine. 2014; 1: 29-36. Siniscalco M, Bernini L, Motulsky AG, Latte B. Favism and thalassaemia in Sardinia and their relationship to malaria. Nature 1961; 190:1179. Vichinsky E. Complexity of alpha thalassemia: growing health problem with new approaches to screening, diagnosis and therapy. Ann N Y Acad Sci 2010; 1202:180-7. Weatherall DJ, Clegg JB. Inherited haemoglobin disorders: an increasing global health problem. Bull World Health Organ. 2001a; 79(8):704-12. Weatherall DJ, Clegg JB: The Thalassaemia Syndromes. 2001b. White SJ, Vink GR, Kriek M, Wuyts W, Schouten J, Bakker B, Breuning MH, den Dunnen JT: Two-color multiplex ligation-dependent probe amplification: detecting genomic rearrangements in hereditary multiple exostoses. Hum Mutat. 2004, 24: 86-92. https://doi.org/10.1002/ humu.20054. Wilkie AO: The alpha-thalassaemia/mental retardation syndromes: model systems for studying the genetic contribution to mental handicap. University of Oxford; 1991. Williams TN, Mwangi TW, Wambua S, et al. Negative epistasis between the malaria-protective effects of alpha+-thalassemia and the sickle cell trait. Nat Genet 2005; 37:1253–7. World HealthOrganization. “Malaria.” Accessed December 11, 2018.

4

Beta-Thalassemia

Abstract

Mutation in gene encoding beta protein chain consequences in either decreased or no production of beta chains which results a disease state named as beta-­ thalassemia. The reduction or lack of beta protein chains leading to the production of defective erythrocytes. The prevalence of disorder is reported in Mediterranean, Middle East, Africa, and Indian subcontinent. Symptomatic cases of the disease are appearing about 1  in 100,000 individuals in general population. Limited therapies with effective results are available, so far. This prompted to study the disease prevalence and existing treatment options of the disease. Currently, erythrocytes transfusions, iron chelation treatment are being used against disease. Induction of fetal hemoglobin with potential pharmacological compounds, bone marrow transplantation, and stem cell therapy are being investigated. Therefore, the present chapter is focused to explore molecular epidemiology of disease and recent advances in the treatment strategies of the disease. Keywords

Beta-thalassemia · Hemoglobin · Molecular epidemiology, Therapeutic strategies

4.1

Introduction

Beta-thalassemia is a type of autosomal recessive blood disorder, affecting nearly 40,000 people worldwide annually (Cappellini et al. 2014). Beta-thalassemia causes reduction of hemoglobin and deficiency of red blood cells. The hemoglobinopathies are divided in two categories; one class is of structural variants, which involve

© Springer Nature Singapore Pte Ltd. 2021 T. Qidwai, Exploration of Host Genetic Factors associated with Malaria, https://doi.org/10.1007/978-981-33-4761-8_4

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defects in both alpha and beta chains and includes different form of hemoglobin such as HbS, C, and E.  The other category includes alpha- and beta-thalassemia variants which arise due to reduced production of either alpha or beta chains of hemoglobin. Mutations in gene cause diminished RNA production, processing, or stability that result in reduced protein production or stability. Reduced biosynthesis of one globin chain resulting accumulation of surplus polypeptides expressed by normal gene. Such imbalance in chain leads to abnormal red blood cell (RBC) maturation (Fig. 4.1) and microcytosis (Sabath 2017). On the basis of severity thalassemia may be classified as thalassemia major, thalassemia intermedia, and thalassemia minor. Thalassemia major is more severe as compared to thalassemia intermedia. The symptoms of thalassemia major disease may appear within 2 years of life. Beta-thalassemia is largely caused by single base substitution, small deletions or insertions mutations within gene encoding beta-­ globin chain or its immediate flanking DNA sequence and rarely by gross deletions. Approximately, greater than 350 beta-thalassaemia mutations are characterized so far (Kountouris et  al. 2014; De Sanctis et  al. 2017). Beta-thalassaemia resulting from partial reduction in synthesis of beta-globin chains are represented as beta++ (mild form) and beta+ (severe form) while total reduction in beta-globin chain is expressed as beta0. The hemoglobinopathies have been found as balanced polymorphism in many parts of the world and appears to provide resistance against malaria disease. It has been reported that people suffering from thalassemia and sickle cell anemia are resistant to malaria (Sinha et al. 2008; Qidwai et al. 2014; Weatherall 2010). The present chapter is focused to cover molecular epidemiology of thalassemia and treatment strategies of disease. Moreover, existing diagnostics approaches of disease are analyzed.

Platelets

Platelets

WBC

WBC

RBCs

Abnormal RBCs (a)

(b)

Fig. 4.1 (a) Normal RBCs and (b) abnormal RBCs produced in beta-thalassemia

4.2 Molecular Biology and Epidemiology of Beta-Thalassemia

4.2

45

 olecular Biology and Epidemiology M of Beta-Thalassemia

4.2.1 Molecular Biology of Disease There are four polypeptide chains in human hemoglobin, two alpha plus two gamma chains in fetal hemoglobin (HbF) and two alpha plus two beta chains in adult hemoglobin (Hb A). In alpha-thalassemia there is defective production of alpha chain of globin protein while defective production or no production of beta chain of globin protein leads to beta-thalassemia. No production of beta-globin protein due to mutated allele causes beta0-thalassemia while decreased amounts of protein biosynthesis cause beta+-thalassemia. Heterozygous condition of each kind of allele is represented as beta-thalassemia minor. Compound heterozygous of two beta+-thalassemia alleles or single beta+ plus single beta0 allele produce more severe form named beta-thalassemia intermedia. In beta-thalassemia intermedia disease condition, anemia, hemolysis, iron loading and occasional requirement for transfusion are common. Human subjects having two beta0 thalassemia alleles encompass the most severe type of disease named beta-thalassemia major. This form of disease causes transfusion dependent anemia, severe iron loading due to transfusion and reduced life (Sabath 2017). Approximately 95% of beta-thalassemias arise because of point mutations. Such mutation causes anomalous gene transcription, RNA processing or stability, or nonsense mutations. As result abnormal protein is formed leading to different form of thalassemia (Fig. 4.2). Unlike alpha-thalassemia, in which deletions in alpha-globin gene cluster mostly liable for mutations in majority of cases, beta-thalassemias arises due to mutations involving within beta gene or its immediate flanking regions (Giardine et al. 2011). The different phenotypic variations formed by deletions in beta-globin gene cluster are categorized as beta-thalassemia, delta- beta-­thalassemia, Epsilon-gamma-delta-beta-thalassemia and hereditary persistence of fetal hemoglobin (HPFH) (Başak 2007).

4.2.2 Epidemiology of Beta-Thalassemia High burden of thalassemias have been reported in Mediterranean countries, Africa, Middle East, Southeast Asia, Melanesia and India (Kountouris et al. 2014; Ladis et al. 2013), Central Asia, Southern China and nations along north coast of Africa and in South America (Flint et al. 1998). Nearly 5% of world’s populations have been predicted as carriers of this disease. It is expected that 60,000 kids with alpha-­ thalassemia major and 43,917 symptomatic beta-thalassemia are born yearly. Majority of cases are being reported from developing countries (Lin et  al. 2014; Weatherall and Clegg 2001; Galanello and Origa 2010). Very recently in Southeastern China one study showed high prevalence of thalassemia with complicated gene mutations (Huang et al. 2019). Characterization of thalassemia and hemoglobinopathies in prenatal diagnosis was carried out in northern part of Thailand and findings

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Point mutation in beta-globin gene or its immediate flanking regions

Altered stability of RNA

Abnormal RNA transcription

Abnormal RNA processing

Non-sense mutations

Abnormal protein production

Beta-thalassemia Fig. 4.2  Development of beta-thalassemia due to point mutation in beta-globin gene or its immediate flanking regions

suggested high prevalence of beta0-thalassemia in these parts as compared to other regions of Thailand (Mankhemthong et al. 2019). Frequency of beta-thalassemia trait in Turkey has been found 2.1%. In Greece, beta-thalassaemia carriers have been found 7.4% (De Sanctis et al. 2017). Higher incidence of alpha/beta-thalassemia has been found in Hakka population of southern Jiangxi province (9.49%) in China (Lin et al. 2014). In India overall prevalence of beta-thalassemia trait has been reported 2.78% (Mohanty et  al. 2013). But in different Indian states its frequency varied from 1.48% to 3.64%. Frequency of beta-thalassemia also varied among different ethnic populations in India. Nearly, 4693 endogamous communities and 427 tribal groups have been present in India. Though hemoglobinopathies are being reported in all Indian states but prevalence is fairly variable. Prevalence of beta-thalassemia varied in different states and populations. One study in Maharashtra state on Sindhis population has reported the prevalence of beta-thalassemia trait 16.81% (Mulchandani et  al. 2008). Further, investigation of beta-thalassemia in subcastes of Sindhis identified that prevalence was enormously variable and highest prevalence in Larkhana Sindhis (17%) whereas lowest prevalence (8%) of beta-thalassemia found in Dadu Sindhis (Jawahirani et  al. 2007). Prevalence of beta-thalassemia has been demonstrated in different states. The beta-thalassemia detected 1–6% in Maharashtra and 0–9.5% in Gujarat (Colah et al. 2010). On the basis of religion, frequency of the beta-thalassemia trait varied. Majority of the people were affected from Hindu religion in India. The 78.2% people from Hindu, 9% Muslims, 6.4% Sikhs, 3.2% Christians, 2.1% Jains,

4.4 Current Treatment Options

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and 1% Buddhists were suffering from beta-thalassemia in studied region of India (Mohanty et al. 2013).

4.3

 emoglobinopathies Selected as Balancing Trait H Against Malaria

Plasmodium falciparum malaria is very common in developing countries. Falciparum malaria shows the phenomenon of balancing selection. It has been suggested that people suffering from thalassemia offer resistance against falciparum malaria. The heterozygous condition in beta-thalassaemia provided a selective advantage against malaria (Haldane 1949). Studies have revealed that there is strong correlation between malaria and sickle cell hemoglobin (HbS) in different populations of world. Distribution of sickle cell trait varies with malaria endemicity throughout the world. The highest allele frequency of this trait with high malaria endemicity has been well documented (Eridani 2011). Determinants of hemoglobin F (HbF) levels (Menzel and Thein 2009) and co-inheritance of alpha-thalassemia are the best recognized genetic transformers of sickle cell trait. In a Mali village, highest exposure to malaria infection established that the survival advantage conferred by sickle cell trait against the disease in humans could happen without a significant reduction of parasite load. A significant delayed time to first malaria episode in children with heterozygous sickle cell trait has been shown (Eridani 2011). One report identified that complement receptor1 level varies with Knops blood group and alpha+-thalassaemia in Kenyan children (Opi et al. 2016).

4.4

Current Treatment Options

Although several therapies are available against beta-thalassemia (Fig. 4.3) but none has found efficient. Therefore, researchers are trying to explore effective therapies against the disease.

4.4.1 Transfusion Transfusion strategy is one of the common treatments in patients suffering from beta-thalassemia major for their survival, but many complications arise because of transfusion. Iron overload and related complications, such as cardiac, liver and endocrine problems are common in transfusion treatment. Moreover, alloimmunization could happen, in which recipient generates an immune response to donor antigens. Alloimmunization may produce several complications. Hence, other alternative approaches are being adopted for disease treatment.

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Therapy against beta-thalassemia

Transfusion

Iron chelation therapy by chemical compounds

Stem cell transplantation

HbF induction by pharmacological agents

Splenectomy

Fig. 4.3  Treatment options of beta-thalassemia

4.4.2 Induction of Fetal Form of Hemoglobin (HbF) Fetal hemoglobin induction approach can be employed by using suitable inducing agents, for instance hydroxyurea is used in patients with beta-thalassemia to increase production of gamma-globin, which binds to alpha-chains to produce HbF, dealing imbalance in globin chains. As a consequent, ineffective erythropoiesis and hemolysis decreases and total hemoglobin increases.

4.4.3 Hematopoietic Stem Cell Transplantation In the current situation, stem cell therapy is being explored against several life threating diseases. Presently, hematopoietic stem cell transplantation (HSCT) treatment is only one approach to cure beta-thalassemia (Cappellini et al. 2014). In this method donor and patients ideally should have identical human leukocyte antigen (HLA), however approximately 60% of patients do not contain such family donor. Under such circumstances, transplant from matched unrelated donors could be expected (Lucarelli et al. 2012).

4.4.4 Splenectomy In patients of beta-thalassemia major, huge amount of cells are pooled in spleen leading to spleen enlargement (hypersplenism). Splenectomy can be performed in specific, defined clinical circumstances including splenic enlargement. Though,

4.5 Factors Affecting Global Distribution of Thalassemia

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good clinical management might delay or stop hypersplenism, dropping requirement for splenectomy.

4.4.5 Iron Chelation Therapy In beta-thalassemia patients, regular transfusion causes accumulation of iron. Iron overload may cause several complications. Administration of regular transfusion and chelation regimens has improved survival of patients in beta-thalassemia major disease. Several iron chelating agents are available (Cappellini et  al. 2014). Deferoxamine (DFO) is the first existing chelator and has been used since1980s regularly.

4.5

Factors Affecting Global Distribution of Thalassemia

4.5.1 Consanguinity Consanguinity is one of the most important factors affecting prevalence of thalassemia. Consanguineous marriages are particularly common in several parts of the world. Approximately, 25–70% of male and females are from related families. Various influences like religious, cultural and economic issues are usually apparent explanations for these marriages (Rudra et  al. 2016). Close marriages (marriage between family members) are frequent in some communities.

4.5.2 Nutrition and Infections It is already mentioned that majority of thalassemia cases are reported from developing countries. People residing in developing countries do not have sufficiently enough nutrition and social status. Therefore, they are suffering from nutritional and infectious diseases. Malaria is an infectious disease caused by different species of Plasmodium. Plasmodium falciparum malaria exerts selective force on human genome to select those traits which act against disease in endemic regions. High frequencies of hemoglobinopathies are reported in endemic regions of malaria.

4.5.3 Migration In last decades, migration of people with multiple ethnicities has changed the population demography. Such changes affect the incidence of hemoglobinopathies. Currently, healthy carriers of this trait are residing in many non-endemic parts of the world, and severely affected babies are being produced in those areas where these diseases were earlier rare or unknown. It appears that distribution of thalassemia

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gene affects whole ethnic groups worldwide except those originating from western, central and north Europe.

4.5.4 Prevention Presently, many countries have developed extensive national prevention programs, including awareness in public, carrier screening and information on prenatal diagnosis and preimplantation diagnosis (Cao and Kan 2013). As a result of prevention programs, burden of thalassemia major has decreased substantially in last two decades in many countries like Italy, Cyprus, and Greece.

4.6

Molecular Diagnosis of Thalassemia

4.6.1 PCR and Sequencing Strategies PCR and sequencing strategies are used for diagnosis of beta-thalassemia. In majority of cases beta-thalassemia arises due to point mutation, hence sequencing techniques are important in disease diagnosis.

4.6.2 Single-Stranded Conformational Polymorphism (SSCP) SSCP is used for detection of single base substitution in a given DNA sample. Thalassemia can be diagnosed by using SSCP.

4.6.3 Allele-Specific Oligonucleotide Probes (ASOs) Allele-specific oligonucleotide probes (ASOs) are used to hybridize amplified DNA fragments bound to nylon membrane by dot blotting (Ristaldi et al. 1989).

4.6.4 Amplification Refractory Mutation System (ARMS) Amplification refractory mutation system (ARMS) is used to detect mutation in a given DNA sample. This technique is widely used to detect thalassemia.

4.6.5 GAP-PCR and MLPA The larger beta0-thalassaemia deletion alleles (290  bp to 67  kb) are detected by means of gap PCR and/or multiplex ligation-dependent probe amplification (MLPA) analysis (Waye et al. 1994; Harteveld et al. 2005).

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4.6.6 Method Using Melting Curve Analysis The method is helpful in detection of multiple mutations simultaneously in a single tube. This method was used to identify 24 beta-thalassemia mutations commonly found in the southern Chinese population (Xiong et al. 2011).

4.6.7 Hemoglobin Electrophoresis The isoelectric focusing has largely replaced cellulose acetate alkaline electrophoresis. This technique is used to separate hemoglobin A, hemoglobin F in addition to this common variants hemoglobins S and C. The raised hemoglobin A2 associated with beta-thalassemia can also be separated.

4.7

Conclusion

Thalassemia is a global health problem affecting several people. Although presence of thalassemia is found throughout the world but comparatively more diseased people are being reported from developing countries. A lot of promising diagnostic methods are existing and being used for disease diagnosis. Currently available treatment options in thalassemia are not quite well. Therefore, researchers are being explored novel therapeutic approaches against thalassemia. Presently, stem cell therapy is being used and it has been found interesting against disease. Induction of erythropoiesis using molecules is also an attractive approach. Moreover, management of iron load in both transfusion dependent and nondependent people are important. We have to look at those factors which have contribution in thalassemia distribution, for example, population migration, close marriages. We should conduct awareness programs and make aware the people about the disease. Acknowledgement  The author is thankful to Prof. B.N. Mishra for giving inspiring suggestions during writing of this book chapter.

References Cappellini MD, Viprakasit V, Taher AT.  An overview of current treatment strategies for b-­thalassemia. Expert Opinion on Orphan Drugs, 2014, 2(7):665-679. Sabath DE. Molecular Diagnosis of Thalassemias and Hemoglobinopathies: An ACLPS Critical Review. Am J Clin Pathol. 2017; 148(1):6-15. Kountouris P, Lederer CW, Fanis P, Feleki X, Old J, Kleanthous M.  Itha Genes: an interactive database for haemoglobin variations and epidemiology. PLoS One. 2014 24; 9(7):e103020. De Sanctis V, Kattamis C, Canatan D, Soliman A, Elsedfy H, Karimi M, Daar S, Wali Y, Yassin M, Soliman N, Sobti P, Al Jaouni S, El Kholy M, Fiscina B, Angastiniotis M. β-Thalassemia

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Distribution in the Old World: an Ancient Disease Seen from a Historical Standpoint. Mediterr J Hematol Infect Dis. 2017; 9(1): e2017018. Sinha S, Qidwai T, Kanchan K, Anand P, Jha GN, Pati SS, Mohanty S, Mishra SK, Tyagi PK, Sharma SK; Indian Genome Variation Consortium, Venkatesh V, Habib S. Variations in host genes encoding adhesion molecules and susceptibility to falciparum malaria in India. Malar J. 2008;7: 250. https://doi.org/10.1186/1475-­2875-­7-­250. Qidwai T, Jamal F, Singh S. Exploring putative molecular mechanisms of human pyruvate kinase enzyme deficiency and its role in resistance against Plasmodium falciparum malaria. Interdiscip Sci. 2014;6(2):158-66. https://doi.org/10.1007/s12539-­013-­0025-­8. Weatherall DJ. The inherited diseases of hemoglobin are an emerging global health burden. Blood. 2010; 115: 4331-4336. Giardine B, Borg J, Higgs DR, Peterson KR, Philipsen S, Maglott D, Singleton BK, Anstee DJ, Basak AN, Clark B, et  al. 2011. Systematic documentation and analysis of human genetic variation in hemoglobinopathies using the microattribution approach. Nat Genet 43: 295–301. Başak AN. The molecular pathology of β-thalassemia in Turkey: the Bogaziçi University experience. Hemoglobin. 2007, 31:233–241. Ladis V, Kaagiorga-Langana M, Chouliaras Tsiarta I. Thirty-year experience in preventing hemoglobinopathies in Greece: achievements and potentials for optimisations. Eur J Haematol. 2013, 90:313–322. Flint J, Harding RM, Boyce AJ, Clegg JB. The population genetics of the haemoglobinopathies. Baillieres Clin Haematol 1998, 11:1-51. Lin M, Zhong TY, Chen YG, Wang JZ, Wu JR, Lin F, Tong X, Yang HT, Hu XM, Hu R, Zhan XF, Yang H, Luo ZY, Li WY, Yang LY. Molecular Epidemiological Characterization and Health Burden of Thalassemia in Jiangxi Province, P. R. China. PLoS ONE, 2014, 9(7): e101505. Weatherall DJ, Clegg JB. Inherited haemoglobin disorders: an increasing global health problem. Bull World Health Organ, 2001, 79: 704–712. Galanello R, Origa R. Beta-thalassemia. Orphanet J Rare Dis, 2010, 5: 11. Huang H, Xu L, Chen M, Lin N, Xue H, Chen L, Wang Y, He D, Zhang M, Lin Y. Molecular characterization of thalassemia and hemoglobinopathy in Southeastern China. Sci Rep. 2019; 9(1):3493. Mankhemthong K, Phusua A, Suanta S, Srisittipoj P, Charoenkwan P, Sanguansermsri T. Molecular characteristics of thalassemia and hemoglobin variants in prenatal diagnosis program in northern Thailand. Int J Hematol. 2019 Jun 25. https://doi.org/10.1007/s12185-­019-­02694-­y Mohanty D, Colah RB, Gorakshakar AC, Patel RZ, Master DC, Mahanta J, et al. Prevalence of β-thalassemia and other haemoglobinopathies in six cities in India: A multicentre study. J Community Genet 2013, 4:33-42. Mulchandani DV, Fulare MB, Zodpey SP, Vasudeo ND Prevalence and some epidemiological factors of beta-thalassaemia trait in Sindhi community of Nagpur City, India. Indian J Public Health. 2008, 52(1):11-5. Jawahirani A, Mamtani M, Das K, Rughwani V, Kulkarni H Prevalence of beta-thalassaemia in subcastes of Indian Sindhis: Results from a two-phase survey. Public Health. 2007,121 (3):193-8. Colah R, Gorakshakar A, Phanasgaonkar S, D’Souza E, Nadkarni A, Surve R, Sawant P, Master D, Patel R, Ghosh K, Mohanty D Epidemiology of beta-thalassaemia in Western India: mapping the frequencies and mutations in sub-regions of Maharashtra and Gujarat. Br J Haematol. 2010,149(5):739-47. Haldane JBS. Disease and evolution. La Ricerca Scientifica. 1949; 19:68–76. Eridani S.Sickle cell protection from malaria. Hematology Reports, 2011, 3:e24. Menzel S, Thein SL.  Genetic architecture of hemoglobin F control. Curr Opin Hematol 2009, 16(3):179-186. Opi DH, Uyoga S, Orori EN, Williams TN, Rowe JA. Red blood cell complement receptor one level varies with Knops blood group, α+thalassaemia and age among Kenyan children. Genes and Immunity 2016, 17:171-178. Lucarelli G, Isgrò A, Sodani P, Gaziev J. Hematopoietic stem cell transplantation in thalassemia and sickle cell anemia. Cold Spring Harb Perspect Med 2012;2(5):a011825.

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5

Duffy Blood Group Locus

Abstract

Duffy protein is expressed on the human erythrocytes acts as receptor for chemokines and malaria parasite, Plasmodium vivax. Duffy receptor protein of human erythrocytes binds with Duffy binding protein of P. vivax (PvDBP). Most commonly, three genetic variants (FY*A, FY*B, and FY*O) of Duffy gene have been found in different population of world. In FY*O variant, Duffy protein is not expressed. Presence/absence of Duffy protein depends on the endemicity of malaria. In response to disease pressure in endemic regions, positive natural selection of FY*O allele provides resistance against infection by P. vivax. However, P. vivax infects Duffy-negative individuals using other receptors. Investigation of genetic control mechanisms to minimize the problem of malaria especially in endemic regions needs to be done. This chapter is aimed to analyze the genetic variation and natural selection of Duffy antigen and its importance in protection against malaria. Besides, mechanism of parasite invasion in Duffy-­ negative individuals is highlighted. Keywords

Duffy protein · Natural selection · Malaria · Genetic variations · Duffy negative

5.1

Introduction

Malaria caused by Plasmodium vivax produced significant morbidity and mortality in populations of endemic regions (Barber et  al. 2015; Popovici et  al. 2020). Distribution of P. vivax is very wide and nearly more than three billion people living within P. vivax transmission boundaries (Battle et al. 2019; Popovici et al. 2020). Therapeutic approaches against malaria are not very well however, some genetic control mechanisms have been developed against the pressure of disease. Example © Springer Nature Singapore Pte Ltd. 2021 T. Qidwai, Exploration of Host Genetic Factors associated with Malaria, https://doi.org/10.1007/978-981-33-4761-8_5

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of genetic control mechanism includes selection of Duffy negative phenotype. Therefore, investigation of natural control mechanism against malaria is important. Malaria parasite remains present in human erythrocyte during its blood stage. During inflammation, a wide variety of chemicals are released from blood cells that bind with Duffy glycoprotein receptor on erythrocytes. The Duffy protein serves as receptor for malaria parasite Plasmodium vivax during invasion of parasite in erythrocytes. Duffy protein named as Duffy antigen receptor for chemokines (DARC), alternatively called ACKR1 (atypical chemokine receptor 1) as it serves as receptor for chemokines. DARC may act as scanvenger on the surface of erythrocytes to remove surplus of toxic chemokines produced under pathological circumstances (Langhi and Bordin 2006). The Duffy protein is expressed by FY gene. Duffy gene is situated on q arm of chromosome1, showing several polymorphisms. Three allelic variants of Duffy gene are very common in different populations (Miller et  al. 1976). The genetic variants that lead to Duffy negative have been found the target of natural positive selection in African countries. Human individuals lacking Duffy antigen is comparatively resistant to invasion by P. vivax. Duffy protein on erythrocyte surface acts as receptor of P. vivax this evidence came from fact that individual of African origin lacking Duffy antigens are protected from P. vivax infections (Miller et al. 1976). This doctrine was challenged by the findings that Duffy-negative people infected by P. vivax (Menard et al. 2010). It has been reported that P. vivax infects Duffy negative individuals in African countries (Ryan et  al. 2006; Popovici et  al. 2020). Invasion of parasite in Duffy negative individuals has been shown (Lo et al. 2019). The aim of this chapter is to analyze the Duffy antigen and its role in resistant to malaria. Moreover, Duffy antigen polymorphisms in different populations have been covered.

5.2

Polymorphisms and Epidemiology of Duffy Gene

Gene for Duffy protein (FY) is polymorphic, characterized by three different allelic variants such as FY*A, FY*B and FY*O.  FY*A and FY*B alleles express Fy (a+b−), Fy (a−b+), and Fy (a+b+) serological phenotypes. FY*B is ancestral allelic form of Duffy gene. Single non-synonymous change at 125 nucleotide position causes amino acid change at 42 position in Duffy receptor protein (Aspartate FY*B to Glycine FY*A) (Fig. 5.1). This change results FY*A variant. Single base substitution at promoter region (T-33C) in the binding site for transcription factor GATA1 blocks expression of FY gene. The FY*O allele gives rise to Fy (a−b−) phenotype (Chittoria et al. 2012). Absence of Duffy receptor on erythrocyte gene blocks invasion of P. vivax in erythrocytes (Chittoria et al. 2012). FY*O allele has been found close to fixation in sub-Saharan Africa populations and hardly ever found in populations outside of Africa. This finding has suggested a selection for FY*O allele in African population (Howes et al. 2011). FY*O allele has not been found in Indians (Chittoria et al. 2012), almost absent from Asia and Europe (Howes et al. 2011) whereas FY*A and FY*B alleles were

5.2 Polymorphisms and Epidemiology of Duffy Gene

+1 Exon1

57

Exon2

GATA-1 Gene

G A 125 3 AAAn

mRNA

Protein

+

NH3

COO-

42 Glycine FY*A Aspartate FY*B Fig. 5.1  Illustration of Duffy gene FY, mRNA, and protein. Single base substitution at nucleotide position 125 results change of an amino acid at 42 position in protein. Glycine is present in FY*A polymorphic form and Aspartate is present in FY*B polymorphic form at position 42

widely distributed among Indians. Genotypes FY*A/FY*A, FY*A/FY*B, and FY*B/FY*B are formed by two alleles, FY*A and FY*B, which are distributed among Indians (Chittoria et  al. 2012). Erythrocytes with FY*A and FY*B allele make functional proteins on its surface whereas FY*O makes erythrocyte surfaces Duffy protein deficient. Erythrocytes harboring FY*O do not express Duffy protein because of mutation in the gene promoter, which suppress the erythroid-specific gene expression (Miller et al. 1976; Tournamille et al. 1995). Studies have identified that absence of DARC in erythrocytes prevent P. vivax infection (Miller et al. 1976; Tournamille et al. 1995). FY*B has been distributed in Europe and Asia. FY*A is the predominant form, existing in modern human populations. Greatest frequency of this allele has been reported in Asia (nearly >80%) and in Europe (30–50%) (Howes et al. 2011). FY*A is reported in southern Africa and absent in western and central Africa (Howes et al. 2011). FY*A allele shows near fixation frequency in parts of eastern Asia and the Pacific. Furthermore, heterozygous individuals have reduced expression of DARC and showed partial protection against P. vivax. Infection of P. vivax is uncommon in Africa this is because of near fixation of FY*O, in Africa. It is commonly found among black individuals but rare among other races. Study by Cavasini et al. 2007 has shown that a low genotype frequency of the FYO is exhibited in P. vivax-endemic areas of Brazilian Amazon region. A study on tribes of Andaman and Nicobar islands, India, showed very high frequency of the FYO genotype and almost complete absence of P. vivax infection in that area (Das et al. 2005). Similar findings have been reported in malaria endemic regions of PNG, where emergence of FY*O allele suggests that infection burden by P. vivax is associated with selection of this

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erythrocyte polymorphism (Zimmerman et al. 1999) where FY*O allele is rare in populations outside of Africa.

5.3

Duffy Antigen and Malaria

Malaria caused by Plasmodium vivax is chronic form and has been distributed outside of Africa (Gething et al. 2012; Howes et al. 2011). World Malaria report 2019 reported that P. vivax malaria is accountable for 64% of malaria cases in endemic areas of America, more than 30% in South East Asia and 40% in Eastern Mediterranean regions (World Malaria Report 2019). Infectious diseases shape human demography and genetics. Malaria is known as selective pressures in current human history (McManus et al. 2017). The Duffy antigen, is utilized by P. vivax to infect erythrocytes (Fig. 5.2).

5.3.1 Duffy Antigen as Balancing Selection in Malaria Human host and parasite both are under influence of strong selective pressure. The balancing selection at few loci might give a more uniform circulation of allele frequencies comparative to that at neutral loci. The FY*O allele, is non-expression, fixed in sub-Saharan Africa areas, is a target of positive natural selection because it provides resistance to P. vivax caused malaria (Tournamille et al. 1995). FY*B allele is identified as ancestral. It is missing from majority of human population and limited to western Asia, Europe, and Americas. The study estimated age of FY*O allele is 42,000 years, probably older than most other mutations related to malaria resistance (McManus et al. 2017). It seems this ancient allele has been fixed in African population in a situation in which all three DARC alleles segregated in this region. Therefore, presence of FY*O allele has been identified as a marker for African

P. Vivax

DBP

Duffy

Erythrocyte

Fig. 5.2  This figure represents the invasion of P. vivax in human erythrocytes using Duffy receptor on erythrocytes

5.3 Duffy Antigen and Malaria

59

lineage. Duffy blood groups are also linked to differential susceptibilities to P. vivax infection (King et al. 2011; Michon et al. 2001). Although it has been found that Duffy negative phenotype is generally present in African population but Indian study has identified, Duffy negative individuals in tribal populations of Madhya Pradesh and Andaman and Nicobar. Although malaria is frequently found in Odisha state of India but non-occurrence of Duffy negative individuals in Juango, Bonda, Kutia Kandha tribes of Odisha indicated that Duffy-negative Indians may be limited to specific tribes and places in India (Chittoria et al. 2012).

5.3.2 Circulation of Duffy Gene Allelic Variants in Humans Ancient human genomes have been screened to detect the presence of DARC alleles. FY*O mutation, has not been present in sub-Saharan Africa. The archaic hominin genomes of Denisovan and Altai Neandertal had ancestral FY*B allele, whereas an ancient Ethiopian genome carried FY*A/FY*B heterozygote. Moreover, Ust’Ishim, from Siberia, a 45,000  years old individual also has been found with FY*A/FY*B heterozygote (McManus et al. 2017). Duffy negative antigen (FA*O) is present in Africa, but almost absent in Asia and Europe (Fig. 5.3). Distribution of Duffy protein on erythrocytes in different population of world varies according to Fig. 5.3  Illustration of FY*A, FY*B and FY*O allele prevalence and P. vivax malaria. FY*B prevalent in Europe and Asia, FY*A in Asia. FY*O, in Africa, but almost absent in Asia and Europe. Nonfunctional Duffy protein is prevalent in African countries

Gene for Duffy receptor antigen protein

Three allelic variants, FY*A, FY*B and FY*O in Duffy receptor gene

FY*A and FY*B form functional proteins, whereas FY*O is not expressing protein

FY*B prevalent in Europe and Asia, FY*A in Asia. FY*O, in Africa, but almost absent in Asia and Europe

FY*B and FY*A make individual susceptible to P. vivax infection

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5  Duffy Blood Group Locus

disease endemicity. In endemic regions, due to high frequency of disease, there is selection of Duffy negative antigen (FA*O). FY*B is prevalent in Europe and Asia and FY*A in Asia. Occurrence/non-occurrence of Duffy negative antigen (FA*O) depends on disease endemicity. Therefore, high frequency of Duffy negative (FY*O) is found in malaria endemic regions (Africa). Whereas this protein is nearly absent in India. Although, Duffy negative erythrocytes provides resistance against P. vivax but it has also been suggested that P. vivax infects Duffy negative erythrocytes (Chan et al. 2020).

5.4

Invasion of P. vivax in Duffy-Negative Individuals (Duffy-Independent Invasion Pathways)

Malaria parasite, P. vivax infects Duffy-negative persons also. Evidence that P. vivax infection occurs in Duffy negative, came from travelers infected with P. vivax after returning from Africa, a region where Duffy negative is at near fixation (Twohig et al. 2019). Probably P. vivax infects Duffy negative individuals using other mechanisms. P. vivax Duffy binding protein (PvDBP) does not bind to Duffy-negative red blood cells. Very high frequency of pvdbp duplication was identified in Madagascar as compared to other parts. It has been suggested that this duplication in Madagascar may be because of presence of Duffy negative phenotype. Increased expression of PvDBP on surface of merozoite helps to interact to an unidentified low-affinity receptor. Study from Ethiopian P. vivax isolates identified a higher pvdbp amplification in human erythrocytes with FY*A allele as compared to those with FY*B (Lo et al. 2019). A lower interaction of PvDBP to FY*A is found as compared to FY*B. The pvdbp amplification selected to enhance binding affinity to erythrocytes with FY*A allele via increasing PvDBP protein on merozoites. P. vivax reticulocyte binding protein (PvRBP) a parasite ligands is involved in binding with Duffy negative erythrocytes. Amplification of pvdbp is linked with immune evasion of PvRBP. PvRBP1a and PvRBP2c have been known to interact with Duffy-negative reticulocytes, showing they might be connected with Duffy-independent invasion pathways (Chan et al. 2020). Currently, transferrin receptor 1 (TfR1) has been recognized as receptor for the ligand PvRBP2b for reticulocyte recognition and invasion (Gruszczyk et al. 2018) (Figs. 5.4 and 5.5). TfR1 receptor is located on Duffy-positive and Duffy-negative reticulocytes as well. Nevertheless, it is supposed that PvRBP2b and TfR1 binding takes place upstream of PvDBP-Duffy. Duffy-negative individuals express Duffy antigens in other cell types as well. Presence of Duffy protein on endothelial cells, demonstrates that Duffy expression is not intrinsically stopped by T to C change at position (−67) in DNA (Dechavanne et  al. 2018). Invasion of parasite in Duffy negative individuals has been shown (Lo et al. 2019). Report suggested that Duffy protein is measurable in erythroid precursor cells characteristically being present in

5.6 Duffy Negative Phenotype and Cancer Susceptibility

Plasmodium vivax

RBP

TFR1

61

Erythrocyte

Fig. 5.4  This figure demonstrates the invasion of P. vivax in human erythrocytes using a receptor other than Duffy receptor (transferrin receptor1). This receptor helps the parasite in invasion of Duffy-negative erythrocytes

bone marrow where P. vivax invasion is supposed to takes place at few level (Popovici et al. 2020).

5.5

Chemokines Act as Ligand for DARC

Cytokines and chemokines have been shown to play role in host defence, they bind with their cognate receptors and mediate the action. DARC acts as receptor for numerous pro-inflammatory cytokines secreted by immune cells named as chemokines. This receptor protein has shown association with many diseases including cancer (Ntumngia et  al. 2016). RANTES (regulated upon activation normal T-expressed and secreted), MCP-1 (monocyte chemotactic protein-1), neutrophil activating protein 2 and 3, growth-related gene alpha, and CXCL5 are important chemokines which are involved in interaction with DARC (Ntumngia et al. 2016; McMorran et al. 2012).

5.6

Duffy Negative Phenotype and Cancer Susceptibility

It is proposed that absence of DARC expression results in failure to eliminate accumulation of angiogenic chemokines, which may contribute to cancer development (Shen et  al. 2006). Consequently, high frequency of prostate cancer has been detected in male people of African ancestry which is attributed to prevalence of DARC negative in these individuals (Shen et  al. 2006; Lentsch 2002). But other studies showed slight or no association of DARC and risk/development of prostate cancer in African men. Outcome of other cancers such as breast cancer may also be associated with Duffy antigen.

Duffy negative

TFR1

Amplification of PvDBP protein on merozoites might be selected to increase affinity between FY*A erythrocytes and PvDBP protein

Amplification of Pvdbp is greater in erythrocytes with FY*A allele

Pvdbp amplification is lower in erythrocytes with FY*B one

Interaction of PvDBP to FY*A is lesser as compared to FY*B

Fig. 5.5  Duffy-negative and Duffy-positive reticulocyte. T to C mutation at (−67) position decreases binding of transcription factor GATA-1 and hence decreases gene expression but RNA formation still happening at a very low levels

negative Individuals

Invasion of parasite in Duffy

PvRBP binds with Transferrin receptor1

Duffy positive

Duffy protein

P. vivax RBP is involved in binding with Duffy negative erythrocytes

Currently, parasite can invade both Duffy negative and positive erythrocytes

62 5  Duffy Blood Group Locus

5.7 Discussion

5.7

63

Discussion

Plasmodium vivax malaria produced significant morbidity and mortality in endemic regions and its distribution is very extensive (Popovici et al. 2020). Genetic control mechanisms have been demonstrated to play vital role against the malaria disease pressure. Selection of Duffy negative phenotype has been found in malaria endemic region sub-Saharan Africa. P. vivax uses Duffy antigen for its invasion in red blood cells. Due to absence of Duffy protein parasite is no longer be able to interact with human host. However, parasite uses alternative receptors for its invasion. Reticulocyte binding protein interacts with transferrin receptor1 (TFR1) and thereby infects Duffy protein lacking individuals. Natural selection of Duffy negative (FY*O) will be important in malaria endemic regions (Fig. 5.6). Consequently, high frequency of Duffy negative (FY*O) is reported from malaria endemic regions while this phenotype is nearly absent from India. Fig. 5.6  Flow chart represents the mutation and selection in Duffy gene allele. Selection of FY*O allele causes absence of Duffy antigen thereby discourage entry of P. vivax in human erythrocyte

Ancestral form of Duffy antigen FY*B

Presence/absence of Duffy protein depends on disease endemicity

High frequency of malaria (disease pressure) exerts force to select genetic variations. Selection of FY*O in endemic region occurs

FY*O does not express Duffy protein and thereby discourage invasion of parasite in erythrocyte

Therefore, high frequency of Duffy negative (FY*O) is found in malaria endemic regions (Africa). Whereas this protein is nearly absent in India

64

5.8

5  Duffy Blood Group Locus

Conclusion

In African countries malaria burden is due to more sever parasite Plasmodium falciparum. In those countries and somewhere else, to get the eradication of all human malaria, it will be essential to adopt approaches targeting P. vivax. It has been evidenced since a long time that people having Duffy negative erythrocytes are resistant to invasion of parasite P. vivax. Natural selection of Duffy negative phenotype has been found in African countries against malaria. Usually, the genetic control mechanisms have been developed in human beings against the pressure of malaria, selection of non-expression allele FA*O is one of the important genetic control mechanisms against P. vivax. However, P. vivax also infects Duffy-negative individuals using other receptor. Understanding of important genetic control mechanism could be important in malaria prevention and treatment.

References Barber BE, William T, Grigg MJ, Parameswaran U, Piera KA, Price RN, et al. Parasite Biomass Related Inflammation, Endothelial Activation, Microvascular Dysfunction and Disease Severity in Vivax Malaria. PLoS Pathog. 2015; 11(1):e1004558. Battle KE, Lucas TCD, Nguyen M, Howes RE, Nandi AK, Twohig KA, et al. Mapping the global endemicity and clinical burden of Plasmodium vivax, 2000–17: a spatial and temporal modelling study. The Lancet. 2019. Cavasini, C.E., Mattos, L.C., D’Almeida, C.A.A., D’Almeida, C.V.S., Gollino, Y., Moretti, L.J. et al. (2007) Duffy blood group gene polymorphisms among malaria vivax patients in four areas of the Brazilian Amazon region. Malar. J. 19:167. Chan LJ, Dietrich MH, Nguitragool W, Tham WH.  Plasmodium vivax Reticulocyte Binding Proteins for invasion into reticulocytes.Cell Microbiol. 2020 Jan; 22(1):e13110. Chittoria A, Mohanty S, Jaiswal YK, Das A (2012) Natural Selection Mediated Association of the Duffy (FY) Gene Polymorphisms with Plasmodium vivax Malaria in India. PLoS ONE 7(9): e45219. https://doi.org/10.1371/journal.pone.0045219 Das, M.K., Joshi, H., Verma, A., Singh, S.S. and Adak, T. (2005) Malaria among the Jarawas, a primitive and isolated tribe on the Andaman islands. India. Ann. Trop. Med. Parasitol. 99:545-552. Dechavanne C, Dechavanne S, Metral S, Roeper B, Krishnan S, Fong R, et  al. Duffy Antigen Expression in Erythroid Bone Marrow Precursor Cells of Genotypically Duffy Negative Individuals. bioRxiv. 2018:508481. https://doi.org/10.1101/508481. Gething PW, Elyazar IRF, Moyes CL, Smith DL, Battle KE, Guerra CA, et al. A long neglected world malaria map: Plasmodium vivax endemicity in 2010. PLoS Negl Trop Dis. 2012; 6(9):e1814. PMID: 22970336 4. Gruszczyk J, Kanjee U, Chan LJ, et al. Transferrin receptor 1 is a reticulocyte-specific receptor for Plasmodium vivax. Science 359, 48 (2018). pmid:29302006. Howes RE, Patil AP, Piel FB, Nyangiri OA, Kabaria CW, Gething PW, et al. The global distribution of the Duffy blood group. Nat Commun. 2011; 2:266. https://doi.org/10.1038/ncomms1265 PMID: 21468018 King, C. L. et al. Fy(a)/Fy(b) antigen polymorphism in human erythrocyte Duffy antigen affects susceptibility to Plasmodium vivax malaria. Proc. Natl. Acad. Sci. USA 108, 20113–20118 (2011). Langhi DM Jr, Bordin JO. Duffy blood group and malaria. Hematology. 2006;11(5):389-98. Lentsch AB. The Duffy antigen/receptor for chemokines (DARC) and prostate cancer. A role as clear as black and white? FASEB J. 2002; 16(9):1093–1095.

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Lo E, Hostetler JB, Yewhalaw D, Pearson RD, Hamid MMA, Gunalan K, Kepple D, Ford A, Janies DA, Rayner JC, Miller LH, Yan G Frequent expansion of Plasmodium vivax Duffy Binding Protein in Ethiopia and its epidemiological significance. PLoS Negl Trop Dis. 2019; 13(9):e0007222. McManus KF, Taravella AM, Henn BM, Bustamante CD, Sikora M, Cornejo OE (2017) Population genetic analysis of the DARC locus (Duffy) reveals adaptation from standing variation associated with malaria resistance in humans. PLoS Genet 13(3): e1006560. https://doi.org/10.1371/ journal.pgen.1006560 McMorran BJ, Wieczorski L, Drysdale KE, et al. Platelet factor 4 and Duffy antigen required for platelet killing of Plasmodium falciparum. Science. 2012; 338(6112):1348–1351. Menard D, Barnadas C, Bouchier C, Henry-Halldin C, Gray LR, Ratsimbasoa A, et al. Plasmodium vivax clinical malaria is commonly observed in Duffy-negative Malagasy people. Proceedings of the National Academy of Sciences. 2010; 107(13):5967–71. Michon, P. et al. Duffy-null promoter heterozygosity reduces DARC expression and abrogates adhesion of the P. vivax ligand required for blood-stage infection. FEBS Lett. 495, 111–114 (2001). Miller LH, Mason SJ, Clyde DF, Mc Ginniss MH. The resistance factor to Plasmodium vivax in blacks: the Duffy-blood-group genotype, FyFy. N Engl J Med. 1976; 295(6):302–304. https:// doi.org/10.1056/NEJM197608052950602. PMID: 778616 Ntumngia FB, Thomson-Luque R, Pires CV, Adams JH.  The role of the human Duffy antigen receptor for chemokines in malaria susceptibility: currentopinions and future treatment prospects. J Receptor Ligand Channel Res. 2016; 9: 1-11. Popovici J, Roesch C, Rougeron V.  The enigmatic mechanisms by which Plasmodium vivax infects Duffy-negative individuals. PLoS Pathog. 2020; 16(2):e1008258. Ryan JR, Stoute JA, Amon J, Dunton RF, Mtalib R, Koros J, Owour B, Luckhart S, Wirtz RA, Barnwell JW, Rosenberg R. Evidence for transmission of Plasmodium vivax among a duffy antigen negative population in Western Kenya.Am J Trop Med Hyg. 2006 Oct; 75(4):575-81. Shen H, Schuster R, Stringer KF, Waltz SE, Lentsch AB. The Duffy antigen/receptor for chemokines (DARC) regulates prostate tumor growth. FASEB J. 2006; 20(1):59–64. 33. Tournamille C, Colin Y, Cartron JP, Le Van Kim C. Disruption of a GATA motif in the Duffy gene promoter abolishes erythroid gene expression in Duffy–negative individuals. Nature Genet. 1995; 10 (2):224–228. https://doi.org/10.1038/ng0695-­224 Twohig KA, Pfeffer DA, Baird JK, Price RN, Zimmerman PA, Hay SI, et  al. Growing evidence of Plasmodium vivax across malaria-endemic Africa. PLoS Negl Trop Dis. 2019; 13(1):e0007140–e. WHO (2019). World malaria report 2019. Geneva: World Health Organization. Zimmerman PA, Woolley I, Masinde GL, Miller SM, McNamara DT, Hazlett F, Mgone CS, Alpers MP, Genton B, Boatin BA, Kazura JW.  Emergence of FY*A(null) in a Plasmodium vivax-­ endemic region of Papua New Guinea. Proc Natl Acad Sci U S A. 1999 Nov 23; 96(24):13973–7.

Part II Metabolic Enzymes

6

Pyruvate Kinase Deficiency

Abstract

Pyruvate kinase is an important enzyme of glycolytic pathway that catalyzes the last step of this pathway. In human erythrocytes, there is no mitochondrion hence glycolytic pathway is the most important source of energy generation. In vitro, in vivo, and case control studies suggested that pyruvate kinase deficiency provides resistance against P. falciparum caused malaria. Erythrocytes having pyruvate kinase deficiency are less permissive for P. falciparum infection. The gene that encodes pyruvate kinase exhibits excessive diversity in malaria endemic region such as sub-Saharan Africa. Pyruvate kinase variants possibly affect frequency and intensity of malaria episodes in population exist in in malaria endemic regions. Malaria, a strong selective force shapes the genome region of pyruvate kinase gene. Co-distribution of malaria and pyruvate kinase deficiency has been reported in malaria endemic regions. High incidence of pyruvate kinase deficiency appears to maintain in malaria endemic regions as a protective mechanism against malaria. Keywords

Pyruvate kinase deficiency · P. falciparum malaria · Genetic variation · Protective mechanism · Endemic regions

6.1

Introduction

Malaria is one of the most devastating infectious disease imposed a lot of socioeconomic burden. Malaria is the source of strongest selective force, especially in endemic regions of the disease. Human genome is under the influence of selective force and host genetic factors contribute to susceptibility/resistance to parasite infections, disease pathogenesis, and disease outcome (Kwiatkowski 2005). Blood © Springer Nature Singapore Pte Ltd. 2021 T. Qidwai, Exploration of Host Genetic Factors associated with Malaria, https://doi.org/10.1007/978-981-33-4761-8_6

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6  Pyruvate Kinase Deficiency

parasitemia level, number of clinical episodes of malaria and severity of disease all demonstrate a strong heritable constituent (Bongfen et al. 2009). Population residing in malaria endemic regions have developed genetic control mechanisms against the pressure of malaria (Kwiatkowski 2005). Glucose 6-phosphate dehydrogenase (G6PD) deficiency and hemoglobinopathies are the most common examples of genetic control mechanisms found in malaria endemic regions against disease. Malaria forces the natural selection of genetic variations in human genome as the part of genetic control mechanism. Genetic factors controlling these traits studied using candidate gene and few family-based genome wide linkage association studies (Bongfen et al. 2009). Studies suggested that pyruvate kinase (PK) deficiency provides resistance against P. falciparum caused malaria. Erythrocytes having pyruvate kinase deficiency are less permissive for P. falciparum infection. In vitro study demonstrated that PK-deficiency is linked with protection against invasion and maturation of P. falciparum. The pklr gene has been found to display high diversity in malaria endemic region, sub-Saharan Africa (Ayi et al. 2008; Durand and Coetzer 2008a, b). PK enzyme catalyzes last rate-limiting step of glycolysis. Mature erythrocytes in human being is lacking mitochondria hence, PKLR enzyme becomes vital for energy production (van Bruggen et  al. 2015; van Wijk and van Solinge 2005). Coding/noncoding variations of pklr gene demonstrated differential response to P. falciparum (van Bruggen et al. 2015). Approximately, 192 pklr mutations associated with pyruvate kinase deficiency but only a small number of variants identified in sub-Saharan Africa (Ayi et al. 2008; Durand and Coetzer 2008a, b). Copy number variation polymorphism in pklr gene has also been reported (Freeman et al. 2006; Faik et al. 2017). Population-based studies demonstrated that malaria, a selective force shapes pklr genomic region in population particularly residing in malaria endemic regions such as African countries (Cape Verde, Angola and Mozambique) (Alves et al. 2010; Machado et al. 2012). Dissemination of both malaria and pyruvate kinase deficiency appears to found in the Middle East and sub-Saharan Africa. Co-distribution of PK-deficiency and malaria in endemic regions suggested emergence of natural genetic control mechanism against disease. Limited studies are available that focused on pyruvate kinase deficiency in malaria. Therefore, present chapter is aimed to analyze the pyruvate kinase deficiency as genetic control mechanism to protect malaria in endemic regions. Moreover, genetic variations of pklr gene in different population across the world have been covered.

6.2

Biochemical Function and Deficiency of Enzyme

Pyruvate kinase (PK) enzyme catalyzes the formation of pyruvate from phosphoenolpyruvate in last step of glycolysis. In humans, a pyruvate kinase protein exists in two forms named as liver/erythrocyte-specific (PKLR) and muscle specific (PKM1/2). Several mutations have been found in pklr gene that affects PK enzyme function. Enzyme with decreased pyruvate kinase function, make happen a scarcity of ATP in erythrocytes and augmented levels of other molecules produced early

6.3 Molecular Biology of Pyruvate Kinase Gene

71

Function

ATP production in erythrocytes

Deficiency

Shortage of ATP, erythrocytes destroyed and anemia

PK enzyme

Fig. 6.1  Biochemical function and deficiency of enzyme pyruvate kinase 1q22

Exon

1

2

34 5 6 7

8 9

10

11

Fig. 6.2  Chromosomal location of pyruvate kinase (pklr) gene and its exons

steps of glycolysis (Fig. 6.1). Anomalous red blood cells are collected by the spleen and destroyed. This condition leads to anemia, fatigue, and breathing problem.

6.3

Molecular Biology of Pyruvate Kinase Gene

The gene for PKLR enzyme is located on chromosome 1q21 (OMIM 609712) (Machado et al. 2012). Different isoforms are encoded by two genes, pkm on chromosome 15 and pklr on chromosome 1. The erythrocyte isoform of the gene has 11 exons (Fig. 6.2). Pyruvate kinase deficiency is common enzyme defect in glycolytic pathway found in erythrocyte. This enzyme defect causes hereditary chronic non-­ spherocytic hemolytic anemia. It is passing on as autosomal recessive trait and clinical symptoms generally arise in homozygotes and in compound heterozygotes for two mutant alleles (Secrest et al. 2020). Clinical symptoms are varying from a mild chronic hemolytic anemia to a severe anemia (Zanella et al. 2007). It has been found that those genes which provide defense against malaria are the most variable genes in human genome, for example, pklr gene presents many mutation and polymorphic sites (Machado et al. 2012).

72

6.4

6  Pyruvate Kinase Deficiency

Epidemiology of Pyruvate Kinase Deficiency

PK-deficiency is inherited in the form of autosomal recessive trait, caused due to loss-of-function mutations in PKLR (Ayi et al. 2008). Studies suggested that 192 pklr mutations associated with pyruvate kinase deficiency but only a small number of variants identified in sub-Saharan Africa (Ayi et al. 2008; Durand and Coetzer 2008a, b). G829A (Glu277Lys) mutation was found 41.7% in Mozambican PK-deficient isolates are strongly associated with reduced enzyme activity (Machado et al. 2012). Very recently, it has been identified that PK-deficiency is produced by compound heterozygosity or homozygosity for more than 300 mutations in the pklr gene (Secrest et al. 2020). Distribution of PK-deficiency varied among different ethnic population. PK-deficiency is prevalent in the Middle East and sub-Saharan Africa. Prevalence of PK-deficiency has been found 0.24% in Spain and 1.1% in Turkey. Among Asians, Hong Kong Chinese population has 0.1% whereas south Iranian population has 1.9% PK-deficiency (Yavarian et  al. 2008). A prevalence of PK-deficiency was found 3.12% in newborns from Saudi Arabia (Machado et al. 2012). PK-deficiency has shown its role in malaria protection and co-dissemination of malaria and PK-deficiency has been found (Durand and Coetzer 2008b; Machado et al. 2012).

6.5

Analysis of Genetic Variants in pklr Gene

Single nucleotide polymorphisms in pklr gene are linked with PK-deficiency and hence have shown association with disease. SNPs in coding region of gene lead to L272V, R41Q and Glu277Lys change in protein. In addition to SNPs, pklr gene showed variable copy numbers (Freeman et al. 2006; Faik et al. 2017).

6.5.1 Pyruvate Kinase L272V Mutation Genetic variants in pklr gene and malaria protection have been studied in endemic regions of Senegal and Thailand (van Bruggen et  al. 2015). Leucine to valine (L272V) substitution (rs147659527) at 272 positions has been identified in people of Mossi and Fulani ethnicity. The L272V variant appears a rare variant distributed in different African populations. The L272V mutation in homozygous was detected in an individual from Senegalese of Ndiop (van Bruggen et al. 2015).

6.5.2 Pyruvate Kinase Arginine/Glutamine (R41Q) Mutation Amino acid, arginine at 41 position (R41) is extremely conserved in pyruvate kinase family. Replacement from arginine to glutamine (R41Q) influences stability of the protein. Individuals having R41Q heterozygous showed association with a

6.6 Pyruvate Kinase Deficiency as Protective Trait Against Malaria

73

significant decrease in number of attacks with P. falciparum whereas this change is correlated with an increased Plasmodium vivax infection. R41Q polymorphism has been reported with frequency of 1.5% in Cambodians, 1.8% in Laos, 1% in Thais, 3.2% in Myanmarese, and 2.9% in Mon. Frequency of R41Q is low in Thai population (approximately1%), whereas it is higher in Thais of Karen ethnicity (approximately 5%) who reside in malaria endemic regions. Likewise, R41Q allele frequency is higher in Myanmarese population (approximately 3.2%) (Summerer et al. 2014; van Bruggen et al. 2015).

6.5.3 Pyruvate Kinase Glutamate/Lysine (Glu277Lys) Mutation The missense mutations G829A in pyruvate kinase gene results substitutions of Glutamate to lysine at 277 position, such substitution affects the structure and hence function of pyruvate kinase enzyme. This mutation was found in 41.7% of Mozambican pyruvate kinase deficient isolates having strong linkage with decreased activity. It was first study related to incidence of PK-deficiency in Africans. Global, co-distribution of malaria and PK-deficiency probably acts as selective force for high frequency of 277 Lys variant (Machado et al. 2012).

6.6

 yruvate Kinase Deficiency as Protective Trait P Against Malaria

Malaria imposed strongest known selective force on human genome which has been reflected in several molecular variants against infection and disease. The most studied are sickle cell anemia, thalassemias, and G6PD-deficiency. High frequencies of these aforementioned variants have been reported in malaria endemic regions as the part of genetic control mechanism. Selective pressure from malaria infection leads to genetic retention of few allelic variants that confer limited protection against disease in malaria endemic areas. The pklr gene displays greatest sequence diversity in malaria endemic regions. For example, malaria has been found to increase the frequency of 277 Lys variant in pyruvate kinase enzyme in endemic regions (Machado et al. 2012). Protective effect of variant was detected in murine models (Min-Oo et  al. 2003) as well as in  vitro in P. falciparum using human pyruvate kinase deficient blood (Ayi et al. 2008; Durand and Coetzer 2008b). Co-existing of malaria and PK-deficiency was found in Middle East and sub-Saharan Africa. High frequency of malaria may be acts as driving force for natural selection of PK-deficiency in these areas. On the other hand, incidence of PK-deficiency was very little in overall white populations. Furthermore, genes conferring resistance to malaria showed high variability in the human genome (Machado et  al. 2012). Prevalence of PK-deficiency in North America, Europe and Asia identified that heterozygote allele frequencies varied 1–3.6% (Beutler and Gelbart 2000) in these areas. One study in African American population identified that frequency of heterozygote allele was 2.4-fold greater in Africans than in Caucasians (Mohrenweiser

74

6  Pyruvate Kinase Deficiency

Fig. 6.3 Illustration is representing the role of genetic variations of pyruvate kinase gene and malaria protection in malaria endemic regions

Genetic variations in Pyruvate kinase gene

Loss of function or reduced activity of Pyruvate kinase

Pyruvate kinase deficiency

High frequencies of PKdeficiency in malaria endemic regions found to offer protection

1987). Studies performed in endemic areas of Senegal and Thailand found that allelic variants of pyruvate kinase gene modify susceptibility to Plasmodium infection with retention of protective alleles in populations of endemic regions (Fig. 6.3). R41Q variant of PKLR appeared in Southeast Asia, showed relationship with diminished number of malaria attacks in females from a big Thai group of Karen ethnicity. Existence of highest genetic diversity within pklr from Sub-Saharan African population and lowest pklr genetic diversity in European population (Berghout et  al. 2012) suggesting that pklr gene is being under the influence of selective pressure, maybe as a result of malaria (van Bruggen et al. 2015). Thus, along with sickle cell hemoglobin, G6PD deficiency, and several others, PKLR may denote one more example in which heterozygosity for incomplete or whole loss of function variants confer few quantity of defence against malaria.

6.6.1 Copy Number Variation in pklr Gene It has been suggested that pklr gene exhibits copy number variation (CNV) (Freeman et al. 2006). Study was conducted to investigate pyruvate kinase gene copy number in Gabonese and European individuals. Pklr gene copy has been associated with malaria severity in Gabonese people (Faik et al. 2017). Europeans has significantly higher gene copy number compared to that in Gabonese individuals. It is clearly mentioned that deficiency of pyruvate kinase provides protection against malaria (Qidwai et al. 2014). The pklr gene dosages demonstrated significant association with malaria outcome. Excessive gene copies of the gene appear to be a susceptibility factor for malaria. More gene copies express more enzymes and hence contribute

6.7 Mechanism of Protection Against P. falciparum Caused Malaria

75

Copy number variation (CNV) in Pyruvate kinase

High copy number in Pyruvate kinase

Low copy number in Pyruvate kinase

High expression of Pyruvate kinase

Low expression of Pyruvate kinase

Susceptibility factor for malaria

Pyruvate kinase deficiency

In Europeans

Protection against malaria in endemic regions such as Africa

Fig. 6.4  Proposed  illustration is  representing the role of copy number variations in pyruvate kinase gene in malaria protection in malaria endemic regions

to malaria susceptibility (Fig.  6.4). Contrary, fewer copies express less enzyme hence contributes to protection against severe malaria, especially in malaria endemic regions.

6.7

 echanism of Protection Against P. falciparum M Caused Malaria

Investigation of malaria protection mediated by PK-deficiency is important in context of malaria prevention. Researchers are being explored the mechanisms of protection confirmed by PK-deficiency. Protection against malaria is established either by causing a decrease in parasite burden or by falling number of infected erythrocytes in trophozoite and schizont stages (Ayi et al. 2008). It has been demonstrated that PK-deficient erythrocytes are less permissive to P. falciparum infection than erythrocytes with normal pyruvate kinase enzyme activity (Fig.  6.5). In addition, P. falciparum infected erythrocytes from

erythrocytes found in case subjects (in both homozygotes and heterozygotes)

found in case subjects (in

homozygotes)

Fig. 6.5  Pyruvate kinase (PK)-deficiency protects against malaria by reduced invasion of parasite in PK-deficient erythrocytes and enhanced phagocytosis of infected erythrocytes

variation leading to reduced enzyme activity

Malaria protection due to selection of genetic

2. Enhanced phagocytosis of infected erythrocytes

1. Reduced invasion of parasite

Macrophage clearance of infected

Invasion defect of erythrocytes

malaria using dual mechanism

PK-deficiency protects against

76 6  Pyruvate Kinase Deficiency

References

77

heterozygous PK-deficient patients are phagocytized at significantly higher than control erythrocytes.

6.8

Conclusion

Human host genetic factors contributed greatly in the protection against malaria. Sickle cell hemoglobin, Glucose 6 phosphate deficiency, and thalassemia and several other alleles are reported in higher frequencies in malaria endemic regions to offer resistance against malaria. Allelic variants at pyruvate kinase gene modify susceptibility to Plasmodium parasite infection in humans. It may be concluded that protective alleles may be retained in populations living in malaria endemic regions. Those genetic variants that lead to pyruvate kinase deficiency (loss of function or reduced activity) selected in malaria endemic regions. Hence, pyruvate kinase deficiency is found in malaria endemic regions as protective mechanism against malaria.

References Alves J, Machado P, Silva J, Gonçalves N, Ribeiro L, et al. (2010) Analysis of malaria associated genetic traits in Cabo Verde, a melting pot of European and sub Saharan settlers. Blood Cells Mol Dis 44: 62-68. Ayi K, Min-Oo G, Serghides L, Crockett M, Kirby-Allen M, Quirt I, Gros P, Kain KC. Pyruvate kinase deficiency and malaria. N Engl J Med. 2008; 358(17):1805-10. https://doi.org/10.1056/ NEJMoa072464. Berghout J, Higgins S, Loucoubar C, Sakuntabhai A, Kain KC, Gros P. Genetic diversity in human erythrocyte pyruvate kinase. Genes Immun. 2012; 13(1):98-102. https://doi.org/10.1038/ gene.2011.54. Beutler E, Gelbart T. Estimating the prevalence of pyruvate kinase deficiency from the gene frequency in the general white population. Blood 2000; 95: 3585-8. Bongfen SE, Laroque A, Berghout J, Gros P.  Genetic and genomic analyses of host-pathogen interactions in malaria. Trends Parasitol. 2009; 25(9):417-22. Durand PM, Coetzer TL. Pyruvate kinase deficiency in a South African kindred caused by a 1529A mutation in the PK-LR gene. S Afr Med J 2008a; 98:456–7. Durand PM, Coetzer TL.  Pyruvate kinase deficiency protects against malaria in humans. Haematologica 2008b; 93:939-40. Freeman JL, Perry GH, Feuk L, et al. Copy number variation: new insights in genome diversity. Genome Res 2006; 16:949–61. Imad Faik, Hoang van Tong, Bertrand Lell, Christian G.  Meyer, Peter G.  Kremsner, and Thirumalaisamy P. Velavan Pyruvate Kinase and Fcγ Receptor Gene Copy Numbers Associated with Malaria Phenotypes. The Journal of Infectious Diseases 2017;216:276–82 Kwiatkowski DP. How malaria has affected the human genome and what human genetics can teach us about malaria. Am J Hum Genet. 2005; 77(2):171-92. Machado P, Manco L, Gomes C, Mendes C, Fernandes N, et al. (2012) Pyruvate Kinase Deficiency in Sub-Saharan Africa: Identification of a Highly Frequent Missense Mutation (G829A;Glu277Lys) and Association with Malaria. PLoS ONE 7(10):e47071. https://doi. org/10.1371/journal.pone.0047071. Min-Oo G, Fortin A, Tam MF, Nantel A, Stevenson MM, et al. (2003) Pyruvate kinase deficiency in mice protects against malaria. Nat Genet 35: 357–362.

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Mohrenweiser HW. Functional hemizygosity in the human genome: direct estimate from twelve erythrocyte enzyme loci. Human Genet 1987; 77: 241-5. Qidwai T, Jamal F, Singh S. Exploring putative molecular mechanisms of human pyruvate kinase enzyme deficiency and its role in resistance against Plasmodium falciparum malaria. Interdiscip Sci 2014; 6:158–66. Secrest MH, Storm M, Carrington C, Casso D, Gilroy K, Pladson L, Boscoe AN. Prevalence of Pyruvate Kinase Deficiency: A Systematic Literature Review. Eur J Haematol. 2020 Apr 12. https://doi.org/10.1111/ejh.13424. Summerer M, Horst J, Erhart G, Weissensteiner H, Schonherr S, Pacher D, et al. Large-scale mitochondrial DNA analysis in Southeast Asia reveals evolutionary effects of cultural isolation in the multi-ethnic population of Myanmar. BMC Evol Biol. 2014; 14:17. https://doi.org/10.118 6/1471-­2148-­14-­17 van Bruggen R, Gualtieri C, Iliescu A, Louicharoen Cheepsunthorn C, Mungkalasut P, Trape J-F, et al. (2015) Modulation of Malaria Phenotypes by Pyruvate Kinase (PKLR) Variants in a Thai Population. PLoS ONE 10(12): e0144555. https://doi.org/10.1371/journal.pone.0144555 van Wijk R, van Solinge WW. The energy-less red blood cell is lost: erythrocyte enzyme abnormalities of glycolysis. Blood. 2005; 106(13):4034–42. Yavarian M, Karimi M, Shahriary M, Afrasiabi AR (2008) Prevalence of pyruvate kinase deficiency among the south Iranian population: quantitative assay and molecular analysis. Blood Cells Mol Dis 40: 308-311. Zanella A, Fermo E, Bianchi P, Chiarelli LR, Valentini G (2007) Pyruvate kinase deficiency: the genotype-phenotype association. Blood Rev 21: 217-231.

7

Glucose 6-Phosphate Dehydrogenase Deficiency

Abstract

Pentose phosphate pathway is sole source of NADPH in mammalian system. Glucose 6-phosphate dehydrogenase (G6PD) is one of the most important enzymes of this pathway. NADPH is utilized to reduce glutathione which is oxidized by free radicals in the human erythrocytes. It has been reported that deficiency or reduced activity of G6PD enzyme causes lack of NADPH resulting oxidative stress and hemolysis of human erythrocytes. Human host genetic factors play very important role in P. falciparum infection, pathogenesis, and disease outcome. G6PD deficiency in human is an important genetic factor showing a very strong correlation with malaria. G6PD deficiency is the natural genetic control mechanism developed against malaria. It is maintained in high frequency in malaria endemic regions as the part of balanced polymorphisms. Malaria is the strongest known selective force which causes natural selection of DNA sequence variations in human genome. Many genetic variations in G6PD have been reported in various ethnic populations and accountable for deficiency/decreased activity of enzyme. Therefore, study of the host genetic control mechanisms such as G6PD deficiency and its role in malaria resistance in different ethnic population is important. The aim of this present chapter is to explore G6PD deficiency and its importance in resistance to P. falciparum caused malaria. Moreover, investigation of G6PD deficiency in various populations is important as this information could be valuable while using malaria intervention that produce hemolytic anemia in deficient individuals. Keywords

Malaria · G6PD deficiency · Balanced polymorphisms · Ethnic population · Endemic regions · SNPs

© Springer Nature Singapore Pte Ltd. 2021 T. Qidwai, Exploration of Host Genetic Factors associated with Malaria, https://doi.org/10.1007/978-981-33-4761-8_7

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7.1

7  Glucose 6-Phosphate Dehydrogenase Deficiency

Introduction

Malaria is the leading cause of health problem, produced 228 million malaria cases and 405,000 death in the year 2018 worldwide. Malaria affects pregnant women and young children. Around 11 million pregnant women have been infected with malaria in sub-Saharan Africa (World Malaria Report 2019). Population living in endemic regions have developed genetic control mechanisms against malaria. For example, genetic variations leading to Glucose 6-phosphate dehydrogenase (G6PD) enzyme deficiency have been selected to offer malaria resistance in endemic regions (Qidwai et al. 2013). G6PD is a key house keeping enzyme which catalyzing first step of oxidative branch of pentose phosphate pathway. In red blood cells, this pathway is a single source for generation of NADPH, which is involved in removal of reactive oxygen species from red blood cells. Though G6PD deficiency has been connected with many clinical disorders such as neonatal jaundice, hemolytic anemia, and cardiovascular disorders (Beutler 1994), it is selectively keep going in populations worldwide (Manjurano et al. 2015). Association of G6PD deficiency with uncomplicated malaria and cerebral malaria provided conflicting results in different populations. Study identified that G6PD deficiency protects against uncomplicated malaria in African countries but not severe malaria (Mbanefo et  al. 2017). Many studies suggested that G6PD deficiency provide protection against cerebral malaria (Guindo et al. 2007; Manjurano et al. 2015; Clarke et al. 2017; Uyoga et al. 2015) and parasitemia (Louicharoen et al. 2009) as well. G6PD deficiency is not associated with severe malaria (Clark et al. 2009). P. falciparum showed deferred growth in vitro in G6PD-deficient red blood cells (Roth et al. 1983). Numerous genetic variations in G6PD gene have been detected in populations of the world resulting deficiency/ decreased enzymatic activity (Luzzatto et al. 2001). More than 130 different G6PD variants have been known to influence G6PD enzyme activity (Luzzatto et al. 2001). G6PD mutations give rise reduced enzyme activity has been associated with malarial resistance and demonstrated selection in human genome (Verrelli et  al. 2002; Verrelli et al. 2006). Therefore, investigation of genetic variations in G6PD gene that lead to reduced activity or level may cause enzyme deficiency/reduced activity is important in malaria endemic regions. In this chapter, G6PD deficiency as genetic control mechanisms against malaria has been analyzed. Moreover, genetic variations in G6PD gene in different population have been covered.

7.2

Biochemical Function and Deficiency of G6PD Enzyme

Glucose 6-phosphate dehydrogenase (G6PD) enzyme is prevailing in red blood cells and catalyzes rate limiting step of pentose phosphate pathway (PPP). Free radicals produced during oxidative stress are neutralized by glutathione which in turn reduced by NADPH generated by pentose phosphate pathway (Figs. 7.1 and 7.2). H2O2 causes oxidative damage, detoxify using glutathione and thioredoxin (Peters and Van Noorden 2009). Reduction of both glutathione and thioredoxin is carried out by NADPH (Fig.  7.2). G6PD enzyme protects erythrocytes from

7.3 Epidemiology of G6PD Deficiency NADP+

81 NADPH+H+

Glucose 6Phosphate

6-phosphogluconolactone Glucose6P- Dehydrogenase Gluconolactonase

6- phosphogluconate NADP+ 6-phospogluconate dehydrogenase

NADPH+H

CO2

Ribulose-5-phosphate Fig. 7.1  Flowchart represents oxidative branch of pentose phosphate pathway. This branch makes NADPH

oxidative stress. Deficiency of this enzyme causes hemolysis in the existence of Factors including infection, few medications and certain foods (Harcke et al. 2019). G6PD deficient erythrocytes are no longer being able to produce NADPH and reduced glutathione. Absence of reduced glutathione impairs the ability of cell to neutralize free radicals eventually leading hemolysis. During erythrocytic cycle of P. falciparum infection, a lot of free radicals are produced, which may cause hemolysis in absence of G6PD and NADPH consequently parasite killing. Probably, because of this reason the G6PD deficiency is retained in the form of balanced polymorphism in regions where malaria is endemic.

7.3

Epidemiology of G6PD Deficiency

Millions of populations residing in tropical and subtropical regions have deficiency of G6PD enzyme. Due to malaria, approximately 900,000 children have been born with a low birth weight in malaria endemic regions. Nearly, 70% deaths of children have been related to malaria World Health organization (WHO) 2019. Many genetic variations of G6PD have been found in different ethnic population of world. G6PD deficiency is commonly found in males compared to females (Ruwende and Hill 1998) and occurs most frequently in Africa, Asia, Mediterranean, and Middle East. G6PD deficiency mainly affects individuals of African and Mediterranean ethnic origins, with a prevalence of approximately 10% (Nkhoma et al. 2009). Malaria disease prevalence differs in various Indian states. About 30%

82

7  Glucose 6-Phosphate Dehydrogenase Deficiency

NADP+

Thioredoxin reductase

Reduced thioredoxin

H2O2

Peroxiredoxin Oxidized thioredoxin

H2O

NADPH

Reduced Glutathione Glutathione reductase

H2O2

Glutathione peroxidase Oxidized glutathione

H2O

NADP+ Fig. 7.2  NADPH-consuming steps (glutathione reductase and thioredoxin reductase) vital for reduction of glutathione and thioredoxin required for detoxification of H2O2

of P. falciparum caused malaria has been reported in Orissa state of India. G6PD deficiency varies among different tribes in India. Some tribes have high frequency of G6PD deficiency was found above 10%, namely Paraja (17.4%), Bhuyan (16.7%), Munda (15.9%), Kharia (12.5%), Bhumiz (12.2%), Kolha (10.7%) while Saora (8.9%), Oraon (8.6%), Gond (8.5%), Bathudi (8.2%), Santal (7.7%) and Kondh (6.2%) tribes have frequency less than 10% (Balgir 2006). Study by Sukumar et al. (2003) reported one mutation in Indian population at position 989  in exon 9 of G6PD gene but this mutation does not influence stability or activity of enzyme (Sukumar et al. 2003). Studies reported that G6PD deficiency varies 9–22% in central African states (Nguetse et al. 2016; Brito et al. 2014).

7.4

Molecular Biology

The G6PD locus is present on long arm of X chromosome (Xq28) in telomere region. This gene is flanked on either side by the genes encoding factor VIII and red/ green color pigment. Gene for the G6PD protein is 18 kb long and comprises 13 exons and 12 introns (Fig. 7.3) (Martini et al. 1986). Based on electrophoretic and biochemical properties, approximately 400 G6PD variants were detected. G6PD B variant has normal activity and is present worldwide, but those variants which give

7.5 Glucose 6-Phosphate Dehydrogenase Polymorphisms

83

X chromosome

1

2

3

4

5

6

7

8

9

10

11

12

13

Glucose6p dehydrogenase gene

Mature mRNA

1

2

3

4

5

6

7

8

9

10

11

12

13

Fig. 7.3  Illustration of X chromosome and G6PD gene coding sequence. G6PD gene consists of 13 exon and 12 introns

rise to enzyme deficiency, are limited to particular regions of the world for example, G6PD A and A− variants are found in sub-Saharan Africa and G6PD Med variant is found in Southern Europe, Middle East, and India.

7.5

Glucose 6-Phosphate Dehydrogenase Polymorphisms

Polymorphisms are existing in gene encoding G6PD, as a result different forms including G6PD B (wild type), G6PD A (non-deficient type), and G6PD A− (deficient type), are reported. The wild type variant (G6PD B) is different from both G6PD A and G6PD A− by a single base substitution at position 376 (A to G) (Hirono and Beutler 1988). G6PD A− contain an extra mutation at nucleotide 202 (G to A) giving a Valine to Methionine amino acid change (Fig. 7.4) (Tishkoff et al. 2001). A mutation in DNA sequence at position 563 substitutes serine to phenylalanine amino acid in protein sequence. Such amino acid substitution gives rise G6PD Med variant. G to A change at position 989 in exon 9 of the G6PD gene causes an Arginine to Histidine substitution in Indians (Sukumar et al. 2003). G6PD genetic variants and associated severity of hemolysis (Grace and Glader 2018) has been shown (Table 7.1).

7  Glucose 6-Phosphate Dehydrogenase Deficiency

84

G6PD gene (B) wild type

376 G6PD (A)

202

376 G6PD (A-)

A/G

G/A

A /G

Valine/Methionine

Fig. 7.4  Illustration represents the variants of G6PD gene Table 7.1  G6PD genetic variants and associated G6PD activity

7.6

Sr No. 1. 2. 3. 4. 5. 6.

G6PD subtypes G6PD A− G6PD A+ G6PD Kaiping G6PD Mahidol G6PD Mediterranean G6PD Canton

G6PD activity 10–60% 60–150% 10–60% 10–60% Less than 10% Less than 10%

 6PD Deficiency a Balancing Trait Against P. falciparum G Caused Malaria

G6PD deficiency is primarily found in areas where malaria is endemic such as Africa, Asia, and Mediterranean Europe (Peters and Van Noorden 2009). G6PD deficiency is strongly correlated with malaria endemicity. G6PD deficiency decreases risk of malaria infection. It has been observed that G6PD deficient individuals have lesser P. falciparum parasite loads than controls. In vitro study in G6PD-deficient cells demonstrated inhibited growth of parasite in first few cycles of infection (Tishkoff et al. 2001). In Africa, three variant form of G6PD have been reported, out of which the G6PD B has frequency of 60–80%, and this variant retains normal enzyme activity. G6PD A variant has frequency 15– 40% with 85% normal enzyme activity. Frequency of G6PD A− has been predicted 0–25% and it retains 12% normal enzymatic activity (Luzzatto et  al. 2001; Ruwende and Hill 1998). Out of three variant, only A− is believed to offer defence against malaria in Africa (Ruwende and Hill 1998). The G6PD Med variant retains 3% of standard enzymatic activity and generally found 2–20%, however its frequency has been reported nearly 70% among Kurdish Jews (Beutler 1994). One African case-control study showed that G6PD deficiency caused by G6PD A− variant is linked with a 46–58% decrease in risk of severe malaria. One meta-analysis identified that in

7.7 Detection of G6PD Deficiency Fig. 7.5 Glucose 6-phosphate dehydrogenase deficiency protects individuals from malaria. Based on electrophoretic and biochemical properties, several variant form of G6PD has been found. G6PD A− variant is connected with protection from malaria in Africa

85

G6PD B, G6PDA, G6PDA-, G6PD Med are important variants of G6PD

High frequency of G6PDA- variant is found in malaria endemic regions

High frequency of G6PD deficiency is linked with malaria defense

African populations, G6PD deficiency protects against uncomplicated malaria, but not severe malaria. Association of G6PD deficiency with uncomplicated malaria and cerebral malaria provided conflicting results in different populations. Study identified that G6PD deficiency protects against uncomplicated malaria in African countries however not from severe malaria (Mbanefo et al. 2017). G6PD deficiency showed no association with severe malaria (Clark et  al. 2009). Many studies suggested that G6PD deficiency provide protection against cerebral malaria (Guindo et al. 2007; Manjurano et  al. 2015; Clarke et  al. 2017; Uyoga et  al. 2015) and parasitemia (Louicharoen et al. 2009) as well. G6PD gene is X-linked therefore connected to gender (Mbanefo et al. 2017). Female heterozygotes (one normal and one mutant copy of the gene) have demonstrated protection from malaria (Manjurano et  al. 2015). G6PD deficiency in Kenya (Uyoga et  al. 2015) and Tanzania (Manjurano et al. 2015) demonstrated protection against severe malaria only in heterozygous females, however in Mali G6PD deficiency has provided defense in hemizygous males against severe malaria (Guindo et al. 2007). Homozygous female genotypes are very rare (National Organization for Rare Disorders 2019; Gómez-Manzo et al. 2016; van den Heuvel et al. 2017) because whole G6PD inactivation has not compatible with natural life. Study by Nguetse et  al. presented that G6PD deficient males and heterozygous females have lesser susceptible to severe malaria (Fig. 7.5).

7.7

Detection of G6PD Deficiency

7.7.1 Fluorescent Spot Test Normal erythrocytes have sufficient functional G6PD enzyme which convert, NADP+ into NADPH. Addition of glucose 6-phosphate and NADP+, in the presence of light of wavelength 340  nm, spot of blood fluoresces. Fluorescent spot test is

7  Glucose 6-Phosphate Dehydrogenase Deficiency

86

authentic for identification of hemizygous males and homozygous females, but not authentic for heterozygous female identification.

7.7.2 Spectrophotometric Assay Spectrophotometric test is too authentic for identification of hemizygous males and homozygous females. Identification of heterozygous genotype in female increases the problems. Quantitative spectrophotometry is gold standard for diagnosis of G6PD deficiency (Henriques et al. 2018).

7.7.3 Cytochemical Assay In cytochemical test, G6PD activity give rise to staining of individual erythrocyte via conversion of exogenous Glucose 6phosphate and NADP+. Unstained erythrocyte has slight or no G6PD enzyme activity. Light microscope can be used for identification of percentage of stained cells and unstained cells.

7.8

Conclusion

P. falciparum caused malaria is major problem throughout the world and high incidence of disease is reported in several parts of the world. Under such circumstances, human genome tried to select those variations which offer resistance to malaria. G6PD deficiency is an example of balancing selection and its high frequency is reported in those areas where high frequency of malaria has been found. The G6PD B variant has normal activity exists worldwide, but variants associated with enzyme deficiency, are limited to malaria endemic areas for instance G6PD A and A− in sub-­ Saharan Africa and G6PD Med in regions of Southern Europe, Middle East and India have been seen. Natural selection of genetic variations in G6PD gene is found as the part of genetic control mechanism in malaria endemic regions to offer resistance against P. falciparum caused malaria. Exploration of G6PD deficiency and its distribution across the world population would be suggested to notify use of malaria intervention(s) that makes acute hemolytic anemia in G6PD deficient people.

References Balgir, RS. Do tribal communities show an inverse relationship between sickle cell disorders and glucose-6-phosphate dehydrogenase deficiency in malaria endemic areas of Central-Eastern India? Homo 2006, 57:163-176. Beutler E. G6PD deficiency. Blood 1994, 84:3613–3636. Brito M, Tchonhi CL, Santos B, Veiga L.  Glucose-6-phosphate dehydrogenase deficiency in children from 0 to 14 years hospitalized at the Pediatric Hospital David Bernardino, Luanda, Angola. J Pharmacogenomics Pharmacoproteomics. 2014;5:125.

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Clark TG, Fry AE, Auburn S, Campino S, Diakite M, Green A, et al. Allelic heterogeneity of G6PD deficiency in West Africa and severe malaria susceptibility. Eur J Hum Genet. 2009;17: 1080–5. Clarke GM, Rockett K, Kivinen K, Hubbart C, Jeffreys AE, Rowlands K, et al. Characterisation of the opposing effects of G6PD deficiency on cerebral malaria and severe malarial anaemia. Elife. 2017;6:e15085. https://doi.org/10.7554/eLife.15085. Gisela Henriques, Koukeo Phommasone, Rupam Tripura, Thomas J.  Peto, Shristi Raut, et  al. Comparison of glucose-6 phosphate dehydrogenase status by fluorescent spot test and rapid diagnostic test in Lao PDR and Cambodia. Malar J. 2018; 17: 243. https://doi.org/10.1186/ s12936-­018-­2390-­6. Gómez-Manzo S, Marcial-Quino J, Vanoye-Carlo A, et al. Glucose-6-phosphate dehydrogenase: update and analysis of new mutations around the world. Int J Mol Sci. 2016; 17(12):E2069. Grace RF, Glader B.  Red Blood Cell Enzyme Disorders. Pediatr Clin North Am. 2018; 65(3):579–595. https://doi.org/10.1016/j.pcl.2018.02.005 Guindo A, Fairhurst RM, Doumbo OK, Wellems TE, Diallo DA. X-linked G6PD deficiency protects hemizygous males but not heterozygous females against severe malaria. PLoS Med. 2007;4:e66. https://doi.org/10.1371/journal.pmed.0040066. Harcke, Susan J., Rizzolo, Denise, Harcke, H.  Theodore MD G6PD deficiency, Journal of the American Academy of Physician Assistants: November 2019,32,11:21-26. https://doi. org/10.1097/01.JAA.0000586304.65429.a7 van den Heuvel EAL, Baauw A, Mensink-Dillingh SJ, Bartels M. A rare disorder or not? How a child with jaundice changed a nationwide regimen in the Netherlands. J Community Genet. 2017;8(4):335–339. Hirono A, Beutler E. Molecular cloning and nucleotide sequence of cDNA for human glucose-­6-­ phosphate dehydrogenase variant A(-). Proc Natl Acad Sci USA 1988 85: 3951–3954. Louicharoen C, Patin E, Paul R, Nuchprayoon I, Witoonpanich B, Peerapittayamongkol C, Casademont I, Sura T, Laird NM, Singhasivanon P, Quintana-Murci L, Sakuntabhai A Positively selected G6PD-Mahidol mutation reduces Plasmodium vivax density in Southeast Asians. Science. 2009 Dec 11; 326(5959):1546-9. Luzzatto L, Mehta A, Vulliamy TJ. In: Scriver CR, Beaudet AL, Sly WS, Valle D. The metabolic and molecular bases of inherited disease. McGraw-Hill, New York, 2001, 4517–4553. Manjurano A, Sepulveda N, Nadjm B, et al. African glucose-6-phosphate dehydrogenase alleles associated with protection from severe malaria in heterozygous females in Tanzania. PLoS Genet. 2015; 11(2):e1004960. https://doi.org/10.1371/journal.pgen.1004960. Martini G., Toniolo D., Vulliamy T., Luzzatto L., Dono R., Viglietto G., Paonessa G., D’Urso M., Persico M.G. Structural analysis of the X-linked gene encoding human glucose 6-phosphate dehydrogenase. EMBO J. 1986;5:1849–1855. Mbanefo EC, Ahmed AM, Titouna A, Elmaraezy A, Trang NT, Phuoc Long N, Hoang Anh N, Diem Nghi T, The Hung B, Van Hieu M, Ky Anh N, Huy NT, Hirayama K. Association of glucose-6-phosphate dehydrogenase deficiency and malaria: a systematic review and meta-­ analysis. Sci Rep. 2017 6;7:45963. https://doi.org/10.1038/srep45963. National Organization for Rare Disorders. https://rarediseases.org/rare-­diseases/glucose-­6-­ phosphate-­dehydrogenase-­deficiency. Accessed July 24, 2019. Nguetse CN, Meyer CG, Adegnika AA, Agbenyega T, Ogutu BR, Kremsner PG, et al. Glucose-6-­ phosphate dehydrogenase deficiency and reduced haemoglobin levels in African children with severe malaria. Malar J. 2016; 15:346. Nkhoma ET, Poole C, Vannappagari V, Hall SA, Beutler E. The global prevalence of glucose-­6-­ phosphate dehydrogenase deficiency: a systematic review and meta-analysis. Blood Cells Mol Dis. 2009; 42(3):267-78. Peters AL, Van Noorden CJ. Glucose-6-phosphate dehydrogenase deficiency and malaria: cytochemical detection of heterozygous G6PD deficiency in women. J Histochem Cytochem. 2009; 57(11):1003-11. Qidwai T, Khan F, Sharma B and Jamal F. Assay of Glucose 6-phosphate Dehydrogenase Enzyme and its Correlation with Disease Prevalence in Patients with Plasmodium falciparum Malaria.

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American Journal of Biochemistry and Molecular Biology. 2013, 3, 1, 135-142. https://doi. org/10.3923/ajbmb.2013.135.142 Roth EF, Raventos-Suarez C, Rinaldi A, Nagel RL. Glucose-6-phosphate dehydrogenase deficiency inhibits in  vitro growth of Plasmodium falciparum. Proc Natl Acad Sci USA 1983, 80:298–299. https://doi.org/10.1073/pnas.80.1.298. PMID: 6337374 Ruwende C, Hill A.  Glucose-6-phosphate dehydrogenase deficiency and malaria. J Mol Med (Berl). 1998; 76(8):581-8. Sukumar S, Mukherjee M.B., Colah RB, Mohanty D.  Molecular characterization of G6PD Insuli—a novel 989 CGC 3 CAC (330 Arg 3 His) mutation in the Indian population. Blood Cells, Molecules, and Diseases 2003, 30, 246–247. Tishkoff SA, Varkonyi R, Cahinhinan N, Abbes S, et  al., Haplotype Diversity and Linkage Disequilibrium at Human G6PD: Recent Origin of Alleles That Confer Malarial Resistance. SCIENCE 2001, 293. Uyoga S, Ndila CM, Macharia AW, Nyutu G, Shah S, Peshu N, et  al. Glucose-6-phosphate dehydrogenase deficiency and the risk of malaria and other diseases in children in Kenya: a case–control and a cohort study. Lancet Haematol. 2015;2:437–444. https://doi.org/10.1016/ S2352-­3026(15)00152-­0. Verrelli BC, McDonald  JH et  al., Evidence for Balancing Selection from Nucleotide Sequence Analyses of Human G6PD. Am. J. Hum. Genet. 2002, 71:1112–1128. Verrelli BC, Tishkoff SA, Stone AC, Touchman JW. Contrasting Histories of G6PD Molecular Evolution and Malarial Resistance in Humans and Chimpanzees Molecular biology and evolution. 2006; 23(8):1592–1601. World Health organization (WHO) 2019. World Malaria Report 2019, World Health Organization.

Part III Host Immune Response

8

TNF Genetic Polymorphisms

Abstract

Although a lot of development has been done for prevention of malaria, but the problem of severe form of disease and death is still the prime concern of the people living in endemic regions. Many genetic and environmental factors have been shown to influence observed variation in P. falciparum infection its progression and outcome of disease. Inflammation is one of the important events in malaria pathogenesis. Tumor necrosis factor is a potent pro-inflammatory cytokine has been shown its role in malaria pathogenesis. Elevated level of TNF has been found in malaria patients. TNF-alpha genetic variant have shown association with higher TNF-alpha in plasma and hence variants are connected with elevated plasma TNF-alpha level in malaria endemic areas. Therefore, TNF-­ alpha variants may have a role in efficiency of antiparasitic response. This chapter is aimed to explore the roles of TNF genetic polymorphisms and their role in malaria pathogenesis. Keywords

Malaria · Inflammation · TNF · Genetic variations · Disease outcome · Endemic region

8.1

Introduction

Tumor necrosis factor is an important pro-inflammatory cytokine of the immune system. Tumor necrosis factor (TNF) has very important function in host defence during infections, showing a dual role in Plasmodium falciparum infection in humans. TNF is produced to reduce parasitemia and intra-erythrocytic parasite killing and its level is elevated in serum of infected people (Carlos Penha-Gonçalves 2019). Although high TNF level is correlated with quicker parasite clearance and © Springer Nature Singapore Pte Ltd. 2021 T. Qidwai, Exploration of Host Genetic Factors associated with Malaria, https://doi.org/10.1007/978-981-33-4761-8_8

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8  TNF Genetic Polymorphisms

with resolution of malaria attacks, however, serum TNF levels continually has been increased in children with severe malaria suggesting complicated role in malaria pathogenesis (Randall and Engwerda 2010). Hence, both useful and detrimental roles of TNF on outcome of P. falciparum malaria infection have been suggested (Nguyen et al. 2017). High TNF level upregulates the expression of adhesion molecules which interact with parasite factor such as P. falciparum erythrocyte membrane protein-1 (Pf EMP-1). As a result cytoadherence, a pathological condition in cerebral malaria, increases (Sinha et al. 2008b). Single nucleotide polymorphisms (SNPs) in the promoter/enhancer region of TNF-alpha gene have been known to play a role in TNF-alpha expression and are linked with control of parasitemia and with better anti-P. falciparum IgG levels (Afridi et  al. 2012). It may be suggested that variants of TNF-alpha modulate response against P. falciparum malaria. SNPs in TNF enhancer/promoter region have been shown to modulate expression of TNF protein and hence circulating levels of TNF. TNF-alpha polymorphisms G-238A (rs361525), G-308A (rs1800629), T-857C (rs1799724), and T-1031C (rs1799964) (Fig. 8.1) have shown their role in TNF expression (Yang et al. 2017). These TNF SNPs have shown their role in different diseases (Yang et  al. 2017; Mahto et al. 2019, Qidwai and Khan 2011). Malaria infection is an example of evolutionary selection and it has been demonstrated that those genotypes in human individuals which provide survival advantage are present in higher frequency in malaria endemic regions (Kwiatkowski 2005). Generally, in malaria endemic areas, high frequencies of protective alleles have been present. In absence of perfect therapy against P. falciparum caused malaria, it is important to explore host genetic factors as it will be helpful in identification of genetic risk of a particular population and exploration of disease outcome and drug response. This chapter is aimed to investigate the role of TNF genetic polymorphisms and their role in malaria pathogenesis and disease outcome. T-1031C T-857C G-308A G-238A

TNF gene

Fig. 8.1  Tumor necrosis factor promoter genetic polymorphism

8.3 TNF-Alpha Mediates Action Through Binding with TNFR1 and TNFR2

93

Chromosome number 6 TNF LTA

LTB

Fig. 8.2  Gene for TNF is surrounded by lymphotoxin A (LTA) and lymphotoxin B (LTB) on chromosome number 6

8.2

Molecular Biology

Gene for tumor necrosis factor is present in short arm of the chromosome 6, covering around 12 kb lengths. Nearly, 43 SNPs at promoter region of TNF-alpha gene have been reported so far (Mahto et al. 2019; Yang et al. 2017). TNF protein is a member of the tumor necrosis factor family. The protein is extremely inducible, secreted, making heterotrimers with lymphotoxin-beta which anchor lymphotoxin-­ alpha to cell surface. TNF gene is surrounded by lymphotoxin A (LTA) and lymphotoxin B (LTB) on chromosome number 6 (Fig. 8.2).

8.3

 NF-Alpha Mediates Action Through Binding T with TNFR1 and TNFR2

TNF-alpha directs signals to target cells using two homologous homodimeric receptors named as tumor-necrosis factor receptor-1 or 2 (TNFR1 and TNFR2) (Derouich-­ Guergour et  al. 2001). Both TNFR1 and TNFR2 are glycoproteins present on membrane of nearly all categories of cells except erythrocytes. TNFR1 has a size of 55–60 kDa while TNFR2 has 75–80 kDa. TNFR1 exhibits constitutive expression in the most tissues but, TNFR2 is powerfully controlled during inflammatory response. TNF binds with cell surface receptors TNFR1 and TNFR2, mediating biological responses in mammalian cells. Binding of TNF with TNFR1 leads to cytotoxic effects whereas TNF binding toTNFR2 produces proliferative effects. Both receptors are co-expressed in most of the cells. It has been identified that mostly TNFR1 enables actions of soluble TNF-alpha whereas TNFR2 mediates mostly those of transmembrane TNF-alpha (Fig. 8.3). Binding of TNF with its receptors stimulates intracellular signal transduction events. TNF triggers an upstream activation of IƘƘB by phosphorylation, ubiquitination, and degradation of IƘB alpha. Subsequent phosphorylation, NF-ƘB is released and moved to the nucleus, where it interacts to those sequences in DNA that modulate expression of genes (Perkins 2007). Human TNF genotypes are linked to malaria clinical outcomes by alteration of cytokine levels in plasma. Apoptosis in CD4 displaying T cells is mediated by the TNFR1 death receptor through TNF pathway in the course of Plasmodium vivax infection (Hojo-Souza et al. 2015).

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8  TNF Genetic Polymorphisms

TNFR1 and TNFR2 both are co-expressed

Binding of TNF with TNFR1 leads to cytotoxic effects

Mostly TNFR1 mediates action of soluble TNF-alpha

TNF binding toTNFR2 produces proliferative effects

TNFR2 mediates action of transmembrane TNF-alpha

Fig. 8.3  Representation of TNF-TNF receptors interaction and production of effects

High plasma levels of both soluble receptors (sTNFR1 and sTNFR2) have been associated with numerous pathological conditions (Megnekou et al. 2010) including severe malaria. Soluble receptors, resulting from cleavage of membrane receptors and circulate in body fluids such as serum and urine under normal and pathological conditions. Expression of TNF receptors on the cell surface is regulated by many factors, for example, cytokines, protein kinases and phosphatases.

8.4

Pathologic Changes in Cerebral Malaria (CM)

TNF a pro-inflammatory cytokine is one of the most important cytokine of host defense system elects a variety of cellular responses varying from proliferation to activation of apoptosis. Elevated TNF-alpha level produces pro-inflammatory response, and is linked to brain damage in pathological conditions, for instance, cerebral malaria (CM), bacterial meningitis, multiple sclerosis and Alzheimer’s disease (Gimenez et al. 2003). Several pathological changes are reported in CM including (1) sequestration of parasitized red blood cells in cerebral vessels, endothelial cell destruction, hemorrhage, thrombosis, and inflammation (2) rupture of schizont, releases toxins that activate monocytes and macrophages to yield TNF-alpha and other cytokines. TNF-­ alpha increases expression of adhesion molecules. Parasitized erythrocytes start to express P. falciparum erythrocyte membrane protein-1 (PfEMP-1) on its surface that interacts with unparasitized erythrocytes to form “rosettes.” Pro-inflammatory cytokine such as TNF-alpha is upregulated, which in turn upregulates expression of

8.5 TNF Promoter Polymorphisms and P. falciparum Malaria

95

ICAM-1 on endothelial cells. PfEMP-1 on infected erythrocytes interacts with adhesion molecules leading to cytoadherence (Fig. 8.4).

8.5

TNF Promoter Polymorphisms and P. falciparum Malaria

During P. falciparum infection, mortality is connected with very high plasma levels of TNF-alpha (Perera et al. 2013). Many studies have been done in various populations to establish a plausible association between TNF-alpha polymorphisms and susceptibility/ resistance to P. falciparum infection/disease severity (Basu et  al. 2010; Nguyen et al. 2017; Ojurongbe et al. 2018). TNF enhancer polymorphisms (SNPs at positions −1031, −857, −376, −308 and −238) exhibit differential associations to malaria and TNF production in different populations across the world (Hananantachai et al. 2007; McGuire et al. 1994; Sinha et  al. 2008a) recommending that individual TNF responses are genetically determined (Fig. 8.5). TNF-alpha promoter polymorphisms are supposed to influence mRNA production and change plasma levels of protein. TNF-alpha (G-308A) mutant showed association with susceptibility to P. falciparum infection (Ojurongbe et al. 2018), higher levels of parasitemia (Nguyen et al. 2017) and severe malaria (Dunstan et  al. 2012). Nevertheless, in a South-West Nigerian study, infected patients have demonstrated no such association with disease (Olaniyan et al. 2016). One more common TNF-alpha promoter (G-238A) variant is also associated to increased parasitemia (Nguyen et  al. 2017) and severe P. falciparum malaria (Olaniyan et al. 2016). Gambian study showed that TNF–238 A allele is linked with susceptibility to severe malaria anemia, but not to cerebral malaria (McGuire et  al. 1999) while nearby TNF-308 A allele is linked with CM, but not with severe malaria anemia (McGuire et al. 1994; Harishankar Mahto et al. 2019). TNF-308 A allele demonstrated significant association with high P. falciparum parasitemia in children from Kenya (Aidoo et al. 2001). Although TNF promoter polymorphisms appear to have a role in the severity of malaria, but conflicting association of two major TNF alleles (TNF-308 A and TNF-238 A) and severe anemia and cerebral malaria has been found. High heterozygosity at TNF-alpha locus appears to be associated with intermediate levels of TNF-alpha expression in Western Africa (Santovito et al. 2012). It has been suggested that heterozygous genome is selected to give protective advantage against P. falciparum infection. Selection of protective alleles has been found in those populations which have been exposed to P. falciparum caused malaria in endemic regions (Fig. 8.6).

Cytoadherence on endothelial

Over expression of VCAM-1

Activates signaling molecules

TNF-alpha interacts with TNFR1

PfEMP-1 on erythrocytes

Infected erythrocytes start to produce PfEMP-1

Fig. 8.4  Leukocytes secrete TNF-alpha that upregulates expression of VCAM-1 on surface of endothelial cells

Leukocyte secretes TNF-alpha

96 8  TNF Genetic Polymorphisms

8.6 Conclusion

97

Cerebral malaria

Severe malaria

Severe malaria anemia

T-1031C T-857C G-308A G-238A TNF SNPs

TNF promoter region Fig. 8.5  Tumor necrosis factor promoter polymorphisms and their role in malaria

8.6

Conclusion

More than one century has been passed but the exact mechanism of P. falciparum infection has not been well known, so far. Inflammation is the major event in malaria disease pathogenesis. TNF is a major cytokine which is modulated during malaria infection. Alteration in TNF cytokine level may be associated with malaria disease. Polymorphisms in promoter region of gene are associated with its expression which in turn may be associated with disease. Those alleles which maintain the intermediate level of TNF may be selected as a balancing trait in response to malaria infection in endemic regions.

98

8  TNF Genetic Polymorphisms

Elevated TNF level is associated with malaria pathogenesis

Genetic variants in enhancer/promoter in TNF may influence TNF expression, hence alters circulating level of TNF

Moderate level of TNF is beneficial during malaria pathogenesis

High heterozygosity at TNF locus recommends a possible selective advantage of the heterozygote genomes

Heterozygote genomes demonstrates association with intermediate levels of TNF expression

Advantageous in endemic region Fig. 8.6  Illustration is representing the role played by genetic variants of TNF gene promoter in malaria protection

References A Santovito, P Cervella, D Schleicherova, M Delpero (2012) Genotyping for Cytokine Polymorphisms in a Northern Ivory Coast Population Reveals a High Frequency of the Heterozygote Genotypes for the TNF-α-308G/A SNP Int J Immunogenet, 39 (4), 291-5. Afridi S, Atkinson A, Garnier S, Fumoux F, Rihet P. Malaria resistance genes are associated with the levels of IgG subclasses directed against Plasmodium falciparum blood-stage antigens in Burkina Faso. Malar J. 2012; 11:308. Aidoo M., McElroy P.  D., Kolczak M.  S., Terlouw D.  J., ter Kuile F.  O., Nahlen B. et  al. (2001) Tumor necrosis factor-alpha promoter variant 2 (TNF2) is associated with pre-term delivery,infant mortality and malaria morbidity in western Kenya: Asembo Bay Cohort Project IX. Genet. Epidemiol. 21: 201–211.

References

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Basu, M. et al. Genetic association of Toll-like-receptor 4 and tumor necrosis factor-alpha polymorphisms with Plasmodium falciparum blood infection levels. Infect Genet l. 10, 686–696, https://doi.org/10.1016/j.meegid.2010.03.008 (2010). Carlos Penha-Gonçalves. Genetics of Malaria Inflammatory Responses: A Pathogenesis Perspective. Front Immunol. 2019; 10: 1771. Derouich-Guergour D., Brenier-Pinchart M.  P., Ambroise Thomas P. and Pelloux H. (2001) Tumour necrosis factor alpha receptors: role in the physiopathology of protozoan parasite infections. Int. J. Parasitol. 31: 763–769. Dunstan, S J et al. Variation in human genes encoding adhesion and proinflammatory molecules are associated with severe malaria in the Vietnamese. Genes and immunity. 13, 503–508, https://doi.org/10.1038/gene.2012.25 (2012). Gimenez F, et al. Tumor necrosis factor alpha in the pathogenesis of cerebral malaria. Cellular and Molecular Life Sciences. 2003;60(8):1623–35. Hananantachai H, Patarapotikul J, Ohashi J, Naka I, Krudsood S, Looareesuwan S, Tokunaga K. Significant association between TNF-α (TNF) promoter allele (-1031C, -863C, and -857C) and cerebral malaria in Thailand. Tissue Antigens. 2007; 69: 277–280. https://doi. org/10.1111/j.1399-­0039.2006.00756.x. Hojo-Souza N.S., Pereira D.B., Mendes T.A., Passos L.S., Gazzinelli-Guimaraes A.C., Gazzinelli-­ Guimaraes P.H. CD4 + T cells apoptosis in Plasmodium vivax infection is mediated by activation of both intrinsic and extrinsic pathways. Malar. J. 2015;14(1):5. Kwiatkowski, D.  P. How malaria has affected the human genome and what human genetics can teach us about malaria. American journal of human genetics 77, 171–192, https://doi. org/10.1086/432519 (2005). Mahto H, Tripathy R, Meher BR, et  al. (2019). TNF-α promoter polymorphisms (G-238A and G-308A) are associated with susceptibility to Systemic Lupus Erythematosus (SLE) and P. falciparum malaria: a study in malaria endemic area. Sci Rep;9(1):11752. McGuire W., Hill A. V., Allsopp C. E., Greenwood B. M. and Kwiatkowski D. (1994) Variation in the TNF-alpha promoter region associated with susceptibility to cerebral malaria. Nature 371: 508–510 McGuire W., Knight J. C., Hill A. V., Allsopp C. E., Greenwood B. M. and Kwiatkowski D. (1999) Severe malarial anemia and cerebral malaria are associated with different tumor necrosis factor promoter alleles. J. Infect. Dis. 179: 287–290 Megnekou, Simon Ako, Rose G. F. Leke and Diane Wallace Audrey Davidson Thévenon, James A.  Zhou, Rosette. Inflammation Markers for Malaria-Associated Parasitemia in Pregnant Women: Potential and 2 Correlate with Plasmodium falciparum Elevated Levels of Soluble TNF Receptors 1. https://doi.org/10.4049/jimmunol.1002293. J Immunol 2010; 185:7115-7122. Nguyen TN, Baaklini S, Koukouikila-Koussounda F, Ndounga M, Torres M, Pradel L, Ntoumi F, Rihet P (2017). Association of a Functional TNF Variant with Plasmodium Falciparum Parasitaemia in a Congolese Population. Genes Immun, 18 (3), 152-157. Ojurongbe, O. et  al. Genetic variants of tumor necrosis factor-alpha -308G/A (rs1800629) but not Toll-interacting proteins or vitamin D receptor genes enhances susceptibility and severity of malaria infection. Immunogenetics. 70, 135–140, https://doi.org/10.1007/ s00251-­017-­1032-­4 (2018). Olaniyan, S. A. et al. Tumour necrosis factor alpha promoter polymorphism, TNF-238 is associated with severe clinical outcome of falciparum malaria in Ibadan southwest Nigeria. Acta tropica. 161, 62–67, (2016). Perera, M. K. et al. Association of high plasma TNF-alpha levels and TNF-alpha/IL-10 ratios with TNF2 allele in severe P. falciparum malaria patients in Sri Lanka. Pathogens and global health 107, 21–29, (2013). Perkins N.D. Integrating cell-signalling pathways with NF-kappaB and IKK function. Nat. Rev. Mol. Cell Biol. 2007;8(1):49–62. Qidwai T, Khan F. Tumour necrosis factor gene polymorphism and disease prevalence. Scand J Immunol. 2011 Dec;74(6):522-47. https://doi.org/10.1111/j.1365-­3083.2011.02602.x. PMID: 21790707; PMCID: PMC7169614.

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Randall LM, Engwerda CR. TNF family members and malaria: old observations, new insights and future directions. Exp Parasitol. 2010; 126(3):326-31. Sinha S, Mishra SK, Sharma S, et  al. (2008a). Polymorphisms of TNF-enhancer and gene for FcgammaRIIa correlate with the severity of falciparum malaria in the ethnically diverse Indian population. Malar J.; 7:13. https://doi.org/10.1186/1475-­2875-­7-­13. Sinha, S., Qidwai, T., Kanchan, K. et al. (2008b). Variations in host genes encoding adhesion molecules and susceptibility to falciparum malaria in India. Malar J 7, 250. https://doi.org/10.118 6/1475-­2875-­7-­250 Yang, Z. C., Xu, F., Tang, M. & Xiong, X. Association Between TNF-alpha Promoter -308 A/G Polymorphism and Systemic Lupus Erythematosus Susceptibility: A Case-Control Study and Meta-Analysis. Scandinavian journal of immunology 85, 197–210, https://doi.org/10.1111/ sji.12516 (2017).

9

iNOS Genetic Polymorphisms

Abstract

Nitric oxide synthase enzyme catalyzes the formation of nitric oxide which has been shown to play an important role in Plasmodium falciparum caused malaria. There are three form of nitric oxide synthase (NOS). Out of which inducible form of nitric oxide synthase (iNOS) is implicated in malaria. Expression of iNOS is controlled by polymorphisms in the promoter region of gene. Promoter polymorphisms affect the expression of iNOS gene and thus formation of NO.  Nitric oxide (NO) reduces parasite growth, cytoadherence of parasitized erythrocytes and TNF cytotoxicity. Studies suggested a protective role of iNOS polymorphism with malaria, however few studies suggested role of iNOS polymorphisms and NO with cerebral malaria. Therefore polymorphic regulation of iNOS and alteration of NO level have been shown to play role in malaria. The iNOS promoter polymorphism in heterozygous form is present in high frequency to provide resistance against malaria in endemic regions. Exploration of polymorphisms in iNOS gene will be important in identification of genetic risk of a population to malaria. Moreover, enzyme and its product (iNOS/NO) probably act as possible diagnostic marker in evaluating clinical malaria. Keywords

iNOS · Polymorphism · NO · Gene expression · Malaria pathogenesis · Disease outcome

9.1

Introduction

Nitric oxide is formed from arginine with the help of enzyme nitric oxide synthase. It is antimicrobial, involved in signal transduction and neurotransmission. Nitric oxide synthase exists in three forms named as nitric oxide synthase 1 (Neuronal © Springer Nature Singapore Pte Ltd. 2021 T. Qidwai, Exploration of Host Genetic Factors associated with Malaria, https://doi.org/10.1007/978-981-33-4761-8_9

101

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9  iNOS Genetic Polymorphisms

nitric oxide synthase), nitric oxide synthase 2 (inducible nitric oxide synthase), and nitric oxide synthase 3 (endothelial nitric oxide synthase). Endothelial nitric oxide synthase (eNOS), inducible nitric oxide synthase (iNOS), and neuronal nitric oxide synthase (nNOS) all three catalyze the conversion of l-arginine and molecular oxygen to nitric oxide and L citrulline (Fig. 9.1). Out of three enzymes, iNOS is inducible while both, eNOS and nNOS are constitutively expressed. The iNOS (NOS2) mediated production of NO has shown its role in host resistance to infectious agents (Timothy Planche et al. 2010). NO has dual role in malarial etiology, some studies identified that NO is associated with cerebral malaria (Burgner et al. 1998). This molecule has protective role during Plasmodium infection. High concentration of NO has been found in African children (Gyan et al. 2002), in vivo studies in rodent malaria model suggested that NO has toxic effects to malaria parasites and also mediates host protective effects (Seguin et al. 1994). In addition, NO reduces cytoadherence and TNF toxicity. The precise mechanism by which NO plays role in malaria is need to explore, probably it has dual role as some studies have suggested that NO is associated with cerebral malaria (Burgner et al. 1998; Clark et al. 1993; Rockett et al. 1992), while some studies suggested a protective role of NO (Al-Yaman et al. 1998; Taylor-Robinson 1997). Cytokines and other agents induce expression of inducible NOS. Induction of inducible NOS in macrophage is crucial for control of intracellular bacteria such as Mycobacterium tuberculosis. Endothelial NOS is important as it produces NO which is a physiological vasodilator, can also deliver vasoprotection. NO acts as potent inhibitor of platelet aggregation and adhesion to the vascular wall. This also stops discharge of platelet-derived growth factors that stimulate smooth muscle Fig. 9.1 Flowchart represents production of nitric oxide from arginine

L-Arginine NADPH + O2 Nitric oxide synthase NADP+H2O

Citrulline

Nitric oxide

9.2 Types and Physiological Functions of Nitric Oxide Synthase

103

proliferation (Forstermann and Sessa 2012). Production of NO is influenced by the availability and expression of nitric oxide synthase enzyme. It is estimated that many people are suffering from malaria, nearly 405,000 deaths happened in 2018, more than 90% deaths occurring in sub-Saharan Africa (WHO 2019). In India, approximately 569 million people are living in high transmission areas (Kumar et al. 2018). Human host genetic factors play a key role in malaria pathogenesis, polymorphisms in NOS2 gene alters susceptibility/resistance to disease. DNA sequence variations in nitric oxide synthase gene influences its expression and hence are involved in NO synthesis. Limited studies of NOS2 genetic variants and susceptibility/resistance to malaria have been available. Thus, it is important to study the role of DNA sequence variation of NOS2 enzyme in malaria pathogenesis and outcome in different ethnic population. Moreover, the role played by NO in immunity against infectious disease has been covered.

9.2

 ypes and Physiological Functions of Nitric T Oxide Synthase

9.2.1 Nitric Oxide Synthase 1 (Neuronal Nitric Oxide Synthase) Three different types of nitric oxide synthase accomplish diverse functions. Neuronal form of NOS is expressed in specific neurons and implicated in synaptic plasticity. NO produced by neuronal NOS too takes part in central regulation of blood pressure. NO derived from neuronal NOS serves as atypical neurotransmitter in peripheral nervous system (PNS), which mediates relaxing components of gut peristalsis, vasodilation, and penile erection. Enzyme nNOS has played role in pathophysiology of many diseases. Irregular NO signaling has shown its role in wide range of neurodegenerative pathologies such as excitotoxicity subsequent stroke, multiple sclerosis, Alzheimer’s, and Parkinson’s diseases.

9.2.2 Nitric Oxide Synthase 2 (Inducible Nitric Oxide Synthase) Generally, inducible NOS is not constitutively expressed in cells, its expression can be induced by cytokines, bacterial lipopolysaccharide, and other agents. Expression of inducible NOS can be stimulated in nearly any cell or tissue in presence of suitable inducing agents (Forstermann and Sessa 2012). Both immune and non-­immune cells can be induced using cytokines. Induction of inducible NOS in macrophages causes production of large quantities of NO which has cytotoxic effects. Nonimmune cells can be induced by cytokines to release amounts of NO great adequate to influence neighboring cells. Cytokine-activated endothelial cells have been demonstrated to lyse tumor cells, furthermore induced hepatocytes use NO to kill malaria sporozoites (Green et al. 1990).

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9  iNOS Genetic Polymorphisms

Synaptic plasticity Blood pressure regulation nNOS Neurotransmission, erection

penile

Non-specific defense iNOS Mediators inflammation

of

Vasodilation, vasoprotection nNOS Prevention of atherosclerosis Fig. 9.2  The flow chart represents the physiological roles played by different nitric oxide synthase enzymes

9.2.3 N  itric Oxide Synthase 3 (Endothelial Nitric Oxide Synthase) Endothelial NOS is the most commonly produced in endothelial cells. Endothelial NOS has been shown to play role in vasodilation and endothelial function. Endothelial dysfunction has been found in patients with cardiovascular risk factors such as hypertension, hyper-cholesterolemia, diabetes mellitus, cigarette smoking, etc. Moreover, patients with vascular disease demonstrated endothelial dysfunction. In this condition, they are unable to make sufficient quantities of bioactive NO. Hence, not able to make NO-mediated vasodilation (Fig. 9.2).

9.3

Structure of Nitric Oxide Synthase Gene

Various types of nitric oxide synthase genes are present on different chromosomes. Gene for NOS1 and NOS2 are located on chromosome 12 and chromosome 17 respectively while gene for NOS3 is located on chromosome 7 (Fig. 9.3).

Fig. 9.3  Representation of NOS genes on chromosome 12, chromosome 17 and chromosome 7 (https://www.genecards.org/cgi-­bin/carddisp.pl?gene=NOS)

7q36.1

Chromosome 7: Cytogenetic band: 7q36.1

3. NOS3

17q11.2

Chromosome 17: Cytogenetic band: 17q11.2

2. NOS2

12q24.22

Chromosome 12: Cytogenetic band: 12q24.22

1. NOS1

9.3 Structure of Nitric Oxide Synthase Gene 105

106

9  iNOS Genetic Polymorphisms

9.4

Inducible Nitric Oxide Synthase and Malaria

Nitric oxide is an advantageous component of the innate immune system and NO produced from iNOS plays role in both pathogenesis and control of viral, bacterial, and parasitic infections (Legorreta-Herrera et al. 2011). Production of NO is mainly controlled by pro-inflammatory cytokines through transcriptional mechanisms (Ganster et al. 2001). The augmented expression of endogenous NO during blood-­ stage malaria infection demonstrated protection against P. falciparum. In an in vitro study, it has been proposed that NO might show both cytotoxic and cytostatic to P. falciparum (Rockett et al. 1991). Study on rodent models of malaria showed protective effects of NO (Dzodzomenyo et al. 2018). Nitric oxide reduces malaria parasite growth, cytoadherence and TNF toxicity (Fig. 9.4). Moreover, NO modulates immune response through regulation of apoptosis and upregulation of cytokine expression. During severe malaria disease, level of NO decreases which in turn increases the expression of adhesion molecules such as ICAM1 resulting increased cytoadherence of parasitized erythrocytes (Fig.  9.5). One study in Papua New Guinean Showed that malaria parasitemia is not associated with NO production (Boutlis et al. 2004). Cytokines, Microbes and toxins

NOS2 catalyzes formation of NO

Reduced parasite growth

Reduced ICAM1

Reduced Cytoadherence

Reduced TNF

Reduced TNF Toxicity

Fig. 9.4  Cytokines/microbes induce expression of iNOS which produces NO, reducing cytoadherence, and TNF toxicity (Hobbs et al. 2002)

9.5 Inducible Nitric Oxide Synthase (iNOS) Polymorphisms and Malaria

107

Malaria in severe condition

Intrvascular hemolysis

High level of plasma hemoglobin

Asexual parasitemia

Expression of pro-inflammatory

and Arginase

cytokines such as TNF-alpha and

Reduced nitric oxide bioavailability

TNF toxicity takes place

Nitric increases

oxide

reduction

expression

of

adhesion molecules, VCAM1

Cytokine driven expression of adhesion molecules, VCAM1 and ICAM1

Cytoadherence of parasitized cells lead to disease severity Fig. 9.5  Flowchart represents the severe condition arises during P. falciparum malaria

9.5

I nducible Nitric Oxide Synthase (iNOS) Polymorphisms and Malaria

The reports suggested associations between polymorphisms in the promoter region of the gene encoding iNOS protein, nitric oxide level and malaria disease severity (Trovoada et al. 2014). DNA sequence variations in the iNOS and their role in various diseases have been reported in different population (Qidwai and Jamal 2010). Four polymorphisms in promoter region of iNOS gene demonstrated association with variability in disease outcome, a CCTTT microsatellite repeat positioned in

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9  iNOS Genetic Polymorphisms

2.5 kb upstream from the iNOS transcriptional start site (Burgner et al. 1998) and three SNPs (G-954C) (Kun et  al. 1998), (C-1173T) (Burgner et  al. 2003) and (A-1659G) (Burgner et  al. 1998). Studies in diverse geographical regions documented contradictory opinions of importance of these polymorphisms. Different populations have shown differential association with disease, for example, Kun et al. reported protective association of iNOS promoter polymorphism with severe malaria in a Gabonese population, while, Burgner et al. reported a different association in Gambian population. Both in vitro and in vivo studies revealed contradictory results for the pentanucleotide microsatellite repeat CCTTT polymorphism. Smaller repeats are associated with cerebral malaria in Gambian children (Hobbs et al. 2002). Severe malaria has positive correlation with longer forms of this variant in Thai adults (Ohashi et al. 2002). In contrast, repeat length has not been associated with malarial disease severity in neither Tanzania (Levesque et al. 1999) nor Gabon (Kun et al. 2001). The iNOS promoter polymorphism (G-954C) at −954 position may activate the expression of iNOS and nitric oxide production (Kun et al. 1998; Hobbs et al. 2002; Cramer et al. 2004). This mutation is associated with a lower incidence of acute malaria in Iganga, Uganda (Lwanira et al. 2017). DNA binding proteins bind with promoter during transcription of gene, heterozygote G954C allele of iNOS gene bind to DNA binding protein with higher affinity, resulting increased gene expression and enhanced production of iNOS protein which in turn increases production of NO. Therefore, this mutation provides protection against severe malaria. This SNP transforms iNOS transcription and increases NO which may be important in parasite clearance and protection against P. falciparum infection. The iNOS promoter polymorphisms may play role in host defense against malaria by enhancing anti-oxidants enzymes and encouraging infection induced biochemical pathway against plasmodium pathology. Among other functions, NO seems defensive in endothelial function comprising reduced blood cell-­ endothelial cell adhesion in both in humans and animal models. It has been reported that NOS2-G954C variant has the highest frequency in Africa and at a lower frequency in Asia (Kun et al. 2001), this mutation in heterozygous form exhibited protection against severe malaria as efficiently as the sickle cell trait in Gabon (Kun et al. 2001). The C-1173T SNP was associated with protection from two syndromes of severe malaria, cerebral malaria in Tanzanian children and severe malarial anemia in a cohort of malaria patients from Kenya (Hobbs et al. 2002). The C-1173T SNP was associated with increased systemic NO levels in healthy children. A protective role of iNOS-1173 C to T against severe malaria has been shown in Tanzania (Hobbs et al. 2002) but not in Gambia (Burgner et al. 2003).

9.7 Conclusion

9.6

109

Discussion

Nitric oxide produced by inducible form of NOS has shown its role in immunity against many infectious agents including P. falciparum caused malaria. Expression of NOS gene is controlled by the presence of promoter polymorphisms. SNPs and pentanucleotide microsatellite (CCTTT) repeats in regulatory region of iNOS gene are associated with its expression and thereby involved in NO production. Regulatory polymorphisms may influence expression of gene whereas coding polymorphisms in gene may influence the activity of gene product (Qidwai and Jamal 2010). In malaria endemic regions, high frequency of the disease is maintained throughout the year. Malaria exerts selective pressure to select genetic variations in the human genome against disease pressure (Fig. 9.6). It may be suggested that those polymorphisms in human genome which provides the resistance against the disease have been found in high frequency in malaria endemic region. Those iNOS polymorphisms are selected which increase the expression of iNOS protein and thus NO level because elevated level of NO mediates resistance in human host.

9.7

Conclusion

During malaria parasite infection, inducible NOS produces NO which is toxic to parasite. Moreover, NO decreases parasite cytoadherence and tumor necrosis factor (TNF) alpha toxicity. iNOS gene expression is controlled by its promoter polymorphisms. Presence of promoter polymorphisms in inducible NOS gene affects its expression and hence iNOS enzyme level. Alteration in iNOS level affects NO production. Differential NO level is associated with variable malarial disease outcomes. Those alleles which are associated with malaria, have been selected in malaria endemic regions. Knowledge of iNOS polymorphisms, along with understanding of their functional implications, contribute epidemiologic studies of the involvement of NO in malarial pathogenesis plus individual variation in response to antimalarial drug therapy.

(a)

NO decreases parasite, reduces cytoadherence and TNF toxicity NO mediates host resistance to malaria parasite

Change in NO level Variable outcome of disease

Regulates iNOS expression Change in iNOS level

(b)

High expression alleles in iNOS gene has been selected in endemic region

• •

• •

iNOS promoter polymorphisms

Fig. 9.6  (a) Flow chart represents NO production and its role in malaria (b) and effect of promoter polymorphisms in the iNOS and alteration in NO level

•

•

Nitric oxide (NO) production

Inducible Nitric Oxide Synthase (iNOS)

110 9  iNOS Genetic Polymorphisms

References

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References Al-Yaman FM, Genton B, Clark IA. The ratio of reactive nitrogen intermediates to tumour necrosis factor and clinical outcome of falciparum malaria disease. Trans R Soc Trop Med Hyg. 1998;92(4):417-420. Boutlis CS, Weinberg JB, Baker J, et al. Nitric oxide production and nitric oxide synthase activity in malaria-exposed Papua New Guinean children and adults show longitudinal stability and no association with parasitemia. Infect Immun. 2004; 72(12):6932-6938. Burgner D, Rockett K, Ackerman H, Hull J, Usen S, Pinder M, et  al. Haplotypic relationship between SNP and microsatellite markers at the NOS2A locus in two populations. Genes Immun. 2003; 4:506-514. https://doi.org/10.1038/sj.gene.6364022 PMID: 14551604 Burgner D, Xu WM, Rockett K, Gravenor M, Charles IG, Hill AV, et al. Inducible nitric oxide synthase polymorphism and fatal cerebral malaria. Lancet. 1998; 352:1193-1194. https://doi. org/10.1016/S0140-­6736(05)60531-­4 Clark IA, et al. Nitric oxide and cerebral malaria. Lancet 1993; 341:632-633. Cramer JP, Mockenhaupt FP, Ehrhardt S, et  al. iNOS promoter variants and severe malaria in Ghanaian children. Trop Med Int Health. 2004;9(10):1074–1080. https://doi. org/10.1111/j.1365-­3156.2004.01312.x Dzodzomenyo M, Ghansah A, Ensaw N, Dovie B, Bimi L, Quansah R, et  al. (2018) Inducible nitric oxide synthase 2 promoter polymorphism and malaria disease severity in children in Southern Ghana. PLoS ONE 13(8): e0202218. https://doi.org/10.1371/journal.pone.0202218 Forstermann U, Sessa WC. Nitric oxide synthases: regulation and function. Eur Heart J. 2012;33(7):829–37, 837a–837d. https://doi.org/10.1093/eurheartj/ehr304. Epub 2011 Sep 1. PMID: 21890489; PMCID: PMC3345541. Ganster RW, Taylor BS, Shao L, Geller DA. Complex regulation of human inducible nitric oxide synthase gene transcription by Stat 1 and NF- B. Proc Natl Acad Sci USA 98: 8638 –8643, 2001. Green SJ, Crawford RM, Hockmeyer JT, Meltzer MS, Nacy CA. Leishmania major amastigotes initiate the L-arginine-dependent killing mechanism in IFN-gammastimulated macrophages by induction of tumor necrosis factor-alpha. J Immunol. 1990;145(12):4290-7. Gyan B, Kurtzhals JA, Akanmori BD, Ofori M, Goka BQ, Hviid L, Behr C Elevated levels of nitric oxide and low levels of haptoglobin are associated with severe malarial anaemia in African children. Acta Trop. 2002; 83(2):133-40. Hobbs MR, Udhayakumar V, Levesque MC, Booth J, Roberts JM, Tkachuk AN, et  al. A new NOS2 promoter polymorphism associated with increased nitric oxide production and protection from severe malaria in Tanzanian and Kenyan children. Lancet. 2002; 360(9344): 1468-1475. https://doi.org/10.1016/S0140-­6736(02)11474-­7 PMID: 12433515 Kumar A, Singh KP, Bali P, Anwar S, Kaul A, Singh OP, Gupta BK, Kumari N, Noor Alam M, Raziuddin M, Sinha MP, Gourinath S, Sharma AK, Sohail M. iNOS polymorphism modulates iNOS/NO expression via impaired antioxidant and ROS content in P. vivax and P. falciparum infection. Redox Biol. 2018; 15:192-206. Kun JF, Mordmuller B, Lell B, Lehman LG, Luckner D, Kremsner PG, et al. Polymorphism in promoter region of inducible nitric oxide synthase gene and protection against malaria. Lancet. 1998; 351:265-266 Kun JF, Mordmuller B, Perkins DJ, May J, Mercereau-Puijalon O, Alpers M, et al. Nitric oxide synthase 2(Lambarene) (G-954C), increased nitric oxide production, and protection against malaria,J Infect Dis. 2001; 184(3): 330-336. Legorreta-Herrera M, Rivas-Contreras S, Ventura-Gallegos JL, Zentella-Dehesa A. Nitric Oxide is Involved in the Upregulation of IFN-γ and IL-10 mRNA Expression by CD8+ T Cells During the Blood Stages of P. chabaudi AS Infection in CBA/Ca Mice. Int J Biol Sci 2011; 7(9):1401-1411. Levesque MC, Hobbs MR, Anstey NM, Vaughn TN, Chancellor JA, Pole A, et al. Nitric oxide synthase type 2 promoter polymorphisms, nitric oxide production and disease severity in Tanzanian children with malaria. J Infect Dis. 1999; 180: 1994-2002.

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Lwanira CN, Kironde F, Kaddumukasa M, Swedberg G. Prevalence of polymorphisms in glucose-­6-­ phosphate dehydrogenase, sickle haemoglobin and nitric oxide synthase genes and their relationship with incidence of uncomplicated malaria in Iganga, Uganda, Malar J. 2017;16(1):322. Ohashi J, Naka I, Patarapotikul J, Hananantachai H, Looareesuwan S, Tokunaga K. Significant association of longer forms of CCTTT microsatellite repeat in the inducible nitric oxide synthase promoter with severe malaria in Thailand. J. Infect Dis. 2002; 186: 578-581. https://doi. org/10.1086/341779 PMID: 12195390 Qidwai T, Jamal F.  Inducible nitric oxide synthase (iNOS) gene polymorphism and disease prevalence. Scand J Immunol. 2010 Nov;72(5):375-87. https://doi.org/10.1111/ j.1365-­3083.2010.02458.x. PMID: 21039732. Rockett KA, Awburn MM Cowden WB, Clark IA. Killing of Plasmodium falciparum in vitro by nitric oxide derivatives. Infect Immun. 1991; 59:3280-3283. Rockett KA, Awburn MM, Aggarwal BB, Cowden WB, Clark IA. In vivo induction of nitrite and nitrate by tumor necrosis factor, lymphotoxin, and interleukin-1: possible roles in malaria. Infect Immun. 1992;60(9):3725-3730. Seguin MC, Klotz FW, Schneider I, Weir JP, Goodbary M, Slayter M, Raney JJ, Aniagolu JU, Green SJ. Induction of nitric oxide synthase protects against malaria in mice exposed to irradiated Plasmodium berghei infected mosquitoes: involvement of interferon gamma and CD8+ T cells. J Exp Med. 1994; 180(1):353-8. Taylor-Robinson AW.  Antimalarial activity of nitric oxide: cytostasis and cytotoxicity towards Plasmodium falciparum. Biochem Soc Trans. 1997;25(2):262S. https://doi.org/10.1042/ bst025262s Timothy Planche, Derek C.  Macallan, Toni Sobande, Steffen Borrmann, Jürgen F.  J. Kun, Sanjeev Krishna, and Peter G. Kremsner. Nitric oxide generation in children with malaria and the NOS2G-954C promoter polymorphism. Am J Physiol Regul Integr Comp Physiol 299: R1248–R1253, 2010 Trovoada Maria de J, Martins M, Ben Mansour R, et al. NOS2 variants reveal a dual genetic control of nitric oxide levels, susceptibility to Plasmodium infection, and cerebral malaria, Infect Immun. 2014;82(3):1287–1295. https://doi.org/10.1128/IAI.01070-­13. WHO (2019). World malaria report 2019. Geneva: World Health Organization. https://www.genecards.org/cgi-­bin/carddisp.pl?gene=NOS.

Human Complement Receptor 1 Polymorphisms

10

Abstract

Severe outcome of Plasmodium falciparum infection causes severe anemia or cerebral malaria. In endemic regions, majority of the deaths resulting from complications including malaria induced severe anemia or cerebral malaria. Available studies suggested that complement receptor 1 (CR1) has a key role in pathogenesis of severe malaria anemia and cerebral malaria. Low CR1 on erythrocytes causes higher rate of removal of erythrocytes from circulation leading to severe anemia. High CR1 level causes rosette formation, hence CR1 has dual mechanism. Expression of CR1 is genetically controlled. Polymorphisms in CR1 gene have been shown to play role in CR1 expression and hence involved in malaria pathogenesis. Natural selection of genetic variations in CR1 gene has been found to provide resistance against the disease in endemic regions. High frequency of low expression allele in CR1 gene has been found in endemic regions of India and Africa. The present chapter is aimed to explore single nucleotide polymorphisms in CR1 genes and their role in CR1 expression and severity of malaria. Keywords

Complement receptor 1 · Polymorphism · Malaria · Severe anemia · Rosetting · Disease susceptibility

10.1 Introduction Human malaria caused by Plasmodium falciparum infection has been found major threat and gave rise death of several million people in malaria endemic regions. Majority of deaths occurred in sub-Saharan Africa due to problems including severe malarial anemia (SMA) or cerebral malaria (CM). Although studies have been done to investigate the role of complement receptor 1 (CR1) in the pathogenesis of SMA © Springer Nature Singapore Pte Ltd. 2021 T. Qidwai, Exploration of Host Genetic Factors associated with Malaria, https://doi.org/10.1007/978-981-33-4761-8_10

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and CM (Sinha et al. 2009; Stoute 2011) but pathogenesis in severe malaria is not well known, the precise mechanisms need to be investigated. Complement receptor-1 is a large approximately 200 kDa glycoprotein, member of a family named as “regulators of complement activation” (RCA). CR1 attach to complement cleavage products C3b and C4b hence serving as a cofactor to inactivate them (Roozendaal and Carroll 2007). CR1 expression is found on many cell types including erythrocytes, myeloid cells and lymphocytes. Erythrocytic CR1 is required for clearance of immune complexes from circulation by transferring them to macrophages in liver and spleen (Schifferli et al. 1986). Apart from its immune clearance function, erythrocyte CR1 has been implicated in development of rosettes. In rosetting, P.falciparum infected erythrocytes start to express parasite PfEMP1 which interacts with uninfected erythrocytes (Rowe et al. 1997) which is thought to be associated with disease severity. CR1 levels on erythrocytes show variability among individuals, it ranges 50–1200 molecules per cell (Cockburn et al. 2004), low expression phenotype makes less than 200 CR1 molecules per cell (Rowe et al. 1997). CR1 high (H) and low (L) expression phenotypes are genetically determined in various ethnic populations. Single nucleotide polymorphisms (SNPs) in exon 19, exon 22, intron 27, and exon 33 associated with its expression (Cockburn et  al. 2004; Birmingham et al. 2003). The relationship between these SNPs and CR1 levels on erythrocytes differs in ethnic groups (Rowe et al. 2002). CR1 genetic polymorphisms have been associated with many diseases such as inflammation and coronary artery disease (CAD) (de Vries et  al. 2017), susceptibility to leprosy (Kretzschmar et al. 2018) and Chagas disease (Sandri et al. 2018). Numerous studies have been done to investigate role of CR1 genetic polymorphisms and its association with P.falciparum malaria in Gambian, Papua New Guinea (PNG), and Indian populations with contradictory results (Cockburn et al. 2004; Sinha et  al. 2009). CR1 polymorphisms played a critical role in malaria pathogenesis. Moreover, it has been suggested that CR1 acts as an alternate receptor for invasion of erythrocytes by P. falciparum (Stoute 2011). The present chapter is aimed to explore the role played by CR1 polymorphisms in malaria pathogenesis. Besides, CR1 polymorphisms in disease severity and outcome have been investigated.

10.2 Molecular Genetics of CR1 The gene for CR1 protein is positioned on chromosome 1 in the cytogenetic band 1q32. This gene remains present in the region of RCA gene cluster (Khera and Das 2009). The CR1 gene is consists of 30 short consensus repeats. First 28 are ordered into four long homologous repeats (LHRs) each with seven consecutive short consensus repeats (Fig. 10.1). The Kna, McCa, Sla, and Yka antigens are located on CR1. CR1 gene polymorphisms affect density of CR1 molecules on cell surface. Moreover, SNPs change CR1 protein to generate a separate blood group antigen variants, called as Knops.

10.3 Role of CR1 in P. falciparum Malaria Pathogenesis LHR A

1

2

C4b

3

4

5

6

LHR B

7

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C4b/C3b

11

12

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LHR C

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18

C4b/C3b

LHR D

19

20

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22

23

24

25

26

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28

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KN

Transmembrane region

Cytoplasmic tail

Fig. 10.1  Representation of common structural variant of CR1. Amino terminal extracellular portion has 30 short consensus repeats, they are organized into four long homologous regions (LHRs). LHR-A interacts with C4b, LHR-B, and LHR-C bind C3b and C4b, as well as PfEMP-1. LHR-D too has sites specific for Mannannose binding lectin and C1q

10.2.1 Knops Blood Group Antigens The Knops blood group (KN) is present on CR1, McCoy (McC) and Swain-Langley (Sl(a)), KN antigens, demonstrated noticeable frequency differences between Caucasians and Africans. Knops blood system comprises antithetical pairs of Knops antigens a and b (Kna and Knb), McCa and McCb, KAM+/KAM− and Swain-Langley/ Vil (Sl1 and Sl2), as well as Sl4, Sl5, Yka and Sl3 (Fontes et al. 2011). Different antigens Knb, McCb, Sl2, Sl4, and KAM+ are consequence of amino acid substitutions V1561M, K1590E, R1601G, S1610T, and I1615V, respectively (Daniels et al. 2007; Moulds et al. 2002).

10.3 Role of CR1 in P. falciparum Malaria Pathogenesis Complement receptor1 performs a key role in the pathogenesis of severe P. falciparum malaria. Low CR1 level causes severe anemia whereas high CR1 level causes cerebral malaria (Fig. 10.2). Sinha et al. 2009 studied the association of erythrocyte CR1 levels with P. falciparum malaria in both endemic and non-endemic regions in India. Very low CR1 level has been detected in uninfected individuals (controls) in the malaria endemic region in India (Sinha et  al. 2009). Severe malaria patients exhibit low CR1 levels in non-endemic region, whereas severe and nonsevere malaria patients in the endemic region did not show any difference in CR1 levels. Uninfected controls had significantly lower CR1. The findings of this study are consistent with previous study including the comparison of erythrocyte CR1 levels in an endemic and non-endemic area of Kenya (Waitumbi et al. 2004). The results of the studies suggested that chronic exposure to malaria give rise lower CR1 expression on erythrocytes, however, alternative or contributing factors include differences in the genetic background or nutritional status of the populations. Polymorphisms in CR1 gene have been shown to affect CR1 levels and hence involved in severe anemia and cerebral malaria.

of

C3b

deposited

Cerebral Malaria

with non-parasitized erythrocytes

Rosetting of parasitized erythrocytes

High CR1 level on erythrocytes

Fig. 10.2  CR1 plays a dual role in malaria pathogenesis. High CR1 level causes rosetting and low level of CR1 causes severe anemia in malaria (Sinha et al. 2009)

severe anaemia

Removal of erythrocytes leading to

erythrocytes by macrophages

Phagocytosis

more susceptible to C3b deposition

Erythrocytes with low CR1 level are

CR1 on red blood cells

116 10  Human Complement Receptor 1 Polymorphisms

10.4 Human Complement Receptor 1 (CR1) Gene Polymorphisms

117

10.3.1 Rosetting During the blood stage, parasite remains present in human erythrocytes and starts to express a Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1), one of the key adhesin on parasitized erythrocytes. This parasite ligand binds with a number of receptors on endothelial cells and uninfected erythrocytes. Interaction of parasitized erythrocytes (infected erythrocytes) with uninfected erythrocytes is called as rosetting. This strategy is exploited by the parasite to remain sequestered in the microvasculature to escape from destruction in the spleen and liver. Rosetting process creates the problem of obstruction of blood flow in microcapillaries. Populations from differential malaria endemicity demonstrate variation in erythrocyte CR1 levels. CR1 levels for all control individuals from the endemic and non-­ endemic region of India have been studied and suggested that, erythrocyte CR1 levels (60–950 molecules/cell) have been found in non-endemic controls and CR1 levels ( G) variant is located in a transcription factor binding region for transcription factors, POLR2A, PHF8, ELF1, SPI1 and MAX. Polymorphism denoted as rs12626750 lies in regions that bind to ELAVL1, which stabilizes adenylate uridylate-rich elements containing mRNAs (Feintuch et al. 2018). It may be suggested that higher IFNAR1 expression drives increased interferon responsive gene expression and cytokine production to increase risk for cerebral malaria (CM). Chr21:34696785 (C > G) (a variant at position −576 relative to the transcription start site of IFNAR1) is located in a transcription factor–binding region, which can bind POLR2A, PHF8, ELF1, SPI1, and MAX and this region also contains histone marks (Feintuch et al. 2018). The association of Chr21:34696785 (C > G), also known as IFNAR1 272354C-G with CM has been found in Malawian. The association of Chr21:34696785 (C > G) with severe disease is found, which has been reported in other African and Southeast Asian countries (Feintuch et al. 2018).

11.3.2 Coding Polymorphism Polymorphism (rs2257167) that substitutes leucine to valine at 168 in IFNAR1 has been focused in several studies (Khor et al. 2007; Aucan et al. 2003). IFNAR1 polymorphisms (+17470 in intron 3 and L168V in exon 4) demonstrated an association

Chromosome 21 p13

q22.11

q22.3

Fig. 11.2  Location of IFNAR1 gene on chromosome 21.This gene is located in cytogenetic band q22.11 on chromosome 21

11.3 Interferon-Alpha Receptor-1 (IFNAR1) Gene and Protein

127

with severe malaria. Both 17470-G/G and L168V-G/G genotypes demonstrated association with protection against severe malaria and CM (Aucan et  al. 2003). IFNAR1, 267717G-T, 268710G-A, 272354C-G, 273628A-G, 279923C-T, 283708A-G polymorphisms have been studied. IFNAR1 gene polymorphisms have shown their role in susceptibility/resistance to malaria in different populations (Fig.  11.3). Genetic variants, 283708A-G and 272354C-G are identified as rs2243594 and rs2843710 respectively in dbSNP database.

11.3.3 IFNAR1 Protein IFNAR1 contains four subdomains (SD), ligand-binding region is localized to membrane-distal portion (SD1–SD3). Membrane-proximal domain (SD4) is needed for conformational change essential for signal transduction across cell membrane (Figs. 11.4 and 11.5). The type I IFN pathway is involved in host response to malaria infection, composed of two membrane spanning proteins, (IFNAR1 and IFNAR2), making a ternary complex with the ligand (Meyer 2009). IFNAR1 has paired

IFNAR2

IL-10RB

Promoter Polymorphisms

IFNAR1

IFNAR2

Intron3 17470

Exon4 L168 V

Altered expression/activity of IFNAR1 protein

Play role in susceptibility/resistance to P. falciparum malaria Fig. 11.3  IFNAR1 gene polymorphisms have shown their role in susceptibility/resistance to malaria or severity/outcome of malaria

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11  Interferon-Alpha Receptor-1 (IFNAR1) Polymorphisms

IFNAR1 protein consists of four subdomains (SD1-4)

Ligand-binding region is localized in subdomain SD1-SD3

Subdomain SD4 is required for conformational change

Fig. 11.4  IFNAR1 contains four subdomains. SD1–SD3 required for ligand binding, SD4 needed for conformational change IFNAR1

IFNAR1 IFNAR2

IFNAR2 D1

D1

JAK1

SD2 IFN

IFN D2

SD1

SD1

SD3 D2

JAK1

SD4

TYK2

IFN

SD2 SD3 SD4

TYK2

Fig. 11.5  Interaction of IFN and IFNAR1 leads to signal transduction. SD4 is involved in conformational change needed for signaling

proline residues in hinge regions between individual extracellular domains (Zhang et al. 2018). IFNAR2 consists of two subdomains named as D1 and D2. Tyrosine kinase 2 (Tyk2) is associated with IFNAR1 while Janus family kinases (Jak1) is associated with IFNAR2 (Piehler et al. 2012).

11.5 Conclusions

129

11.4 I nterferon-Alpha Receptor-1 (IFNAR1) Polymorphisms Impact Malaria Interferon-alpha receptor-1 is one of the most important protein of human host defence, its role in malaria pathogenesis is explored in many ethnic groups. Almost each cell type has IFNAR1 (Ito et al. 2006; Prinz et al. 2008). Studies identified that decreased circulating IFN-alpha levels are linked with severe P. falciparum malaria in Gabonese (Luty et al. 2000) and Kenyan (Ongecha et al. 2011) populations. Genetic variants within the IFNAR1 have been found to be associated with severe illness in African populations (Feintuch et al. 2018). Study on Indian population has provided the association of IFN-gamma, IFN-alpha, IFNGR1, and IFNAR1 polymorphisms and susceptibility/resistance to P. falciparum malaria in populations belong to differential endemicity (Kanchan et  al. 2015). IFNAR1 gene polymorphism (IFNAR1 272354C-G), identified as rs2843710 in dbSNP, which is located at −576 with respect to transcription start has been strongly associated with susceptibility to severe malaria in three populations (Gambian, Kenyan and Vietnamese) (Khor et al. 2007). Gambian case control study identified that IFNAR1-17470 G/G and IFNAR1-­ L168V G/G genotypes linked to decreased risk of severe malaria and CM. Both, IFNAR1-17470 G/G and IFNAR1-L168V G/G genotypes have been found more common in controls than in severe anemia cases and associated with protection against severe malaria and CM (Aucan et  al. 2003). Variants, rs12626750 and rs1041867 are associated with CM and causes increased IFNAR1 expression (Feintuch et al. 2018). The uncomplicated malaria is associated with genetic variant rs914142 which causes a decrease in IFNAR1 expression. Protective variant, rs914142, is associated with decreased expression of IFNAR1, while CM-associated variant, rs12626750 is associated with increased IFNAR1 expression in Malawi (Feintuch et al. 2018; Fig. 11.6). Variant alleles of rs2843710 and rs2243594 SNPs in Gambian, Vietnamese and Kenyan population showed strong association with susceptibility to severe malaria (Khor et al. 2007). The G allele of promoter SNP rs2843710 is associated with disease and A allele of intronic SNP rs2243594 demonstrated association with disease in the endemic region of India (Kanchan et al. 2015). SNPs, rs1012335 and rs2257167, showed association with protection against severe malaria, in Gambian population (Aucan et al. 2003) but no association has been found in Indian population (Kanchan et al. 2015).

11.5 Conclusions Inflammation is the major event during malaria pathogenesis. Dysregulated cytokine production or excessive expression may lead to disturbance of fine tuning of pro- and anti-inflammatory cytokines. Too much expression of pro-inflammatory cytokines such as interferon leads to a severe outcome of the disease. Interferons bind to their receptors to activate interferon responsive genes.

130

11  Interferon-Alpha Receptor-1 (IFNAR1) Polymorphisms Interferon alpha interacts with IFNAR1 and IFNAR2 to form ternary complex

Activation of signaling pathways

Induction

of

expression

of

interferon (IFN) responsive genes

Alteration in level of IFNAR1/IFN influence disease susceptibility/pathogenesis/outcome

(a)

IFNAR1

expression

is

genetically

controlled. Promoter polymorphisms in IFNAR1 affect expression of receptor

Polymorphism (rs12626750) is

Polymorphism (rs914142) is protective for cerebral malaria

associated with cerebral malaria

Polymorphism (rs12626750) demonstrated

Polymorphism (rs914142) demonstrated

increased IFNAR1 expression

lower IFNAR1 expression

Stimulation of type I IFN pathway contributes to pathogenesis of cerebral malaria

(b) Fig. 11.6 (a) Role of interferons and interferon receptors in cerebral malaria (CM) (b) Interferon receptor and ligand interaction leads to activation of signaling pathways protective variant, rs914142, demonstrated lower IFNAR1 expression whereas CM-associated rs12626750 variant is linked with its overexpression. High expression of IFNAR1 is associated with disease via activation of interferon response genes

References

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It may be suggested that induction of the type I interferon pathway might contribute to the pathogenesis of CM. This activation is carried out with the help of IFNAR1 and its over expression contributes malaria pathogenesis and outcome. Polymorphisms in IFNAR1 have been shown to affect the expression of IFNAR1 gene. Some genetic variants are associated with overexpression and hence activation of interferon responsive gene. Such activation may lead to inflammation and malaria pathogenesis.

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Part IV Cytoadherence

Intercellular Adhesion Molecule-1 Polymorphisms

12

Abstract

Cerebral malaria is the most severe condition arises from an infection of P. falciparum. Cerebral malaria is accountable for the majority of the malaria associated mortality in endemic regions. Both host and parasite factors are involving in pathogenesis of cerebral malaria. Parasitized erythrocytes sequestered through cytoadherence to vascular endothelium. The host ICAM-1 interacts with parasite erythrocyte membrane protein (PfEMP1). Cytoadherence to brain microvasculature is supposed to be involved ICAM-1. Single nucleotide polymorphisms in ICAM-1 gene have shown their role in malaria. Although many association studies have been done to investigate ICAM-1 gene polymorphisms and susceptibility to severe malarial, but results are inconclusive. Therefore, investigation of genetic polymorphisms in ICAM-1 gene and malaria would be important. The present chapter is aimed to analyze the role played by ICAM-1 in pathogenesis and outcome of malaria. Moreover, ICAM-1 polymorphisms and their role in susceptibility/resistance to malaria have been covered. Keywords

Cerebral malaria · ICAM-1 polymorphisms · Cytoadherence · Endemic regions · Susceptibility/resistance

© Springer Nature Singapore Pte Ltd. 2021 T. Qidwai, Exploration of Host Genetic Factors associated with Malaria, https://doi.org/10.1007/978-981-33-4761-8_12

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12.1 Introduction The severe outcome of Plasmodium falciparum caused malaria is characterized by the excessive expression of pro-inflammatory cytokines by reason of dysregulation of cytokine gene expression. Elevated levels of pro-inflammatory cytokines, acidosis, respiratory distress and sequestration of parasitized erythrocytes in microvasculature have been found during severe malaria (Sinha et al. 2008). Over expression of pro-inflammatory cytokines, increases the expression of host adhesion molecules on the surface of endothelial cells. P. falciparum-infected erythrocytes interact with adhesion molecules on the surface of endothelial layer called as cytoadherence which help the parasite in escaping from immune clearance. Human host adhesion molecules including intercellular adhesion molecule 1 (ICAM-1, CD54), platelet endothelial cell adhesion molecule 1 (PECAM-1, CD31), vascular cell adhesion molecule (VCAM-1), thrombospondin, Eselectin, CD36, and P-selectin serve as receptors for parasite ligands such as Plasmodium falciparum erythrocyte membrane protein-1 (PfEMP1). Parasites in infected erythrocytes express certain parasite gene such as PfEMP1 on the surface of infected erythrocytes. PfEMP1 on the infected erythrocytes mediates cytoadherence (Sherman et al. 2003). PfEMP1 protein of parasite undergoes antigenic variation. Adhesion of infected erythrocytes in brain microvasculature is supposed to involve in development of cerebral malaria (Fry et al. 2008). ICAM-1 belongs to immunoglobulin superfamily, its expression is found on endothelial cells, principally in brain, pro-inflammatory cytokines powerfully increase expression of ICAM-1. Lymphocyte function associated antigen (LFA)-1 is a leukocyte cell surface glycoprotein. Binding of LFA-1 to ICAM-1 activates natural killer cells during P. falciparum infection (Baratin et  al. 2007). ICAM-1 interacts to LFA-1 permitting leukocytes passage via blood–brain barrier (BBB). Polymorphisms in ICAM-1 gene have been correlated with many diseases such as stroke (Gao and Zhang 2019), inflammatory and neurodegenerative diseases (Amodu et al. 2005) and was detected as a risk factor for severe malaria in Nigerian children (Amodu et al. 2005). ICAM-1 polymorphisms have been shown their role in susceptibility/resistance to cerebral malaria. Amino acid change due to polymorphisms causes change in protein structure and binding affinity of ICAM-1 protein. Hence it may impact function of protein and finally affect susceptibility/resistance to disease and outcome of disease. Study of polymorphisms in ICAM-1 could be important in identification of risk of a population to develop severe malaria and cerebral malaria. Therefore, the present chapter is aimed to study the ICAM-1 gene polymorphisms in P. falciparum caused malaria. Understanding of genetic variations in the genes associated with malaria pathogenesis would be helpful in prevention and control of malaria, specifically in endemic regions.

12.2 Structure and Function of ICAM1 Gene

137

12.2 Structure and Function of ICAM1 Gene The ICAM-1 gene encodes a cell surface glycoprotein which is expressed on endothelial cells and immune cells. ICAM-1 gene is positioned on chromosome 19p13.3­p13.2 scanning 15,512 base pairs (Fig. 12.1). It consists of 7 exon; mRNA 3249 base pairs.

12.2.1 ICAM-1 Protein ICAM-1 protein has a molecular mass ranging from 75 to 115 kDa. This protein consists of 532 amino acids (Fig. 12.2). ICAM-1 interacts with integrins of type CD11a/CD18, or CD11b/CD18. ICAM-1 is upregulated on endothelial and epithelial cells at sites of inflammation, mediating vascular adhesion and paracellular migration of leukocytes with activated LFA-1 and Mac-1. It can bind many non-­ integrin ligands such as CD43/Sialophorin, fibrinogen, hyaluronan, rhinoviruses and Plasmodium falciparum-infected erythrocytes.

Chromosome19: 19p13.2 p13.3

(a)

p13.2

p13.13

q11

q11

q12

q13.49

ICAM-1

Transcription start site

ATG

K29M K56M

E283K

R397Q G241R

K469E

5’ UTR

Exon1

3’-UTR

2

3

4

5

6

7

(b)

Fig. 12.1 (a) Location of ICAM-1 gene on chromosome 19 in cytogenetic band 19p13.2 (https:// www.genecards.org/cgi-­bin/carddisp.pl?gene=ICAM1&keywords=icam1) (b) the figure demonstrates the polymorphisms in ICAM-1

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Fig. 12.2  This figure demonstrates the amino acid sequences of ICAM-1 protein P05362-1 (532 amino acids) (https://www.uniprot.org/uniprot/P05362)

12.3 Mechanism of Cytoadherence in P. falciparum Malaria Infection of P. falciparum under severe condition leads to cerebral malaria (CM) which is accountable for the most of demises in malaria endemic regions such as sub-Saharan Africa (Adukpo et al. 2013). Both, host and parasite factors are involved in CM pathogenesis causes parasite sequestration through cytoadherence to the vascular endothelium. It has been found that host endothelial receptor, ICAM-1 is likely involved in cytoadherence to brain microvasculature, whereas other receptors comprising CD36 is commonly involved in cytoadherence of parasites in other organs (Adukpo et al. 2013). ICAM-1, PECAM-1, and VCAM-1 are present on the surface of endothelial layer. Expression of ICAM-1shows both beneficial as well as deleterious effects in the infected host. ICAM-1 on monocyte takes part in the immune response to P. falciparum caused malaria. It has been demonstrated that ICAM-1 surface expression required for interferon-gamma (IFN-gamma) response of natural killer (NK) cells to malaria-infected erythrocytes. Infection of P. falciparum increases interferon which in turn upregulate expression of TNF-alpha. TNF, a pro-inflammatory cytokine, increases the expression of ICAM-1 on endothelial layer (Fig. 12.3). During blood stage, parasite remains present in human erythrocytes and start to express few its protein on the surface of infected erythrocytes such as PfEMP1. Infected

Receptor

I

Increased expression of ICAM-1 on host endothelial layer

Rupture of blood brain barrier (BBB) and severe outcome of disease and cerebral malaria

Infected erythrocytes

Expression of parasite PfEMP1 on infected erythrocytes

Pro-inflammatory cytokine, TNF up-regulation

Fig. 12.3  P. falciparum infection upregulates TNF expression. ICAM-1 and VCAM-1 expressions are inducible and their expression induced by TNF, high levels of ICAM-1 and VCAM-1 are found during acute malaria (Baruch et al. 2002). PfEMP1 on infected erythrocytes binds with ICAM-1 during P. falciparum malaria (Razakandrainibe et al. 2012). Such cytoadherence and sequestration of infected erythrocytes in brain leads to disruption of BBB and cerebral malaria

P. falciparum in human erythrocytes

Parasite infection

12.3 Mechanism of Cytoadherence in P. falciparum Malaria 139

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140

erythrocytes interact with endothelial layer via ICAM-1 resulting cytoadherence of infected erythrocytes.

12.4 ICAM-1 Gene Polymorphisms ICAM-1 polymorphisms, exon 6 (rs5498) Lysine 469 Glutamic acid (K469E), and Glycine 241Arginine (G241R) might be involved in cancer development and progression. The ICAM1 (K469E) variation found in domain 5 (D5) of ICAM-1 molecule showed association with inflammatory and neurodegenerative diseases (Amodu et al. 2005). Variant K469E has been significantly associated with susceptibility of human beings to ischemic stroke in Caucasians, but not in Asians (Table 12.1) (Gao and Zhang 2019), and suggested as a risk factor for severe malaria in Nigerian children (Amodu et  al. 2005). The ICAM-1 rs5491 (K56M or ‘ICAM-1Kilifi’) variant located in domain1 (D1) of ICAM-1, demonstrated an association to disease response in Africa. This mutation was first recognized in Kilifi region of Kenya, homozygotes for this mutation found more frequent in CM patients (Fig.  12.4). K/E mutation of ICAM-1 molecule affects dimerization of ICAM-1 (Reilly et al. 1995) plus interaction between B cells and dendritic cells. Studies suggested that glutamic acid (E) to lysine (K), modification changes protein structure and binding affinity of ICAM-1 protein (He et  al. 2014; Mohamed et  al. 2010). Consequently, most likely rs5498 polymorphism affects biological function of ICAM-1 and produce over-activated inflammatory reactions. Dimerization of ICAM-1 increases its binding to its natural ligand LFA-1. The residue key, for interaction of ICAM-1 to PfEMP1, lies in the dimer interface of domain 1, however whether this binding includes monomeric or dimeric ICAM-1 is still unidentified. G241R (glycine/arginine) is positioned within the third Immunoglobulin-like domain (D3), showing Mac-1 binding domain. Hence, it plays important role during leukocyte transmigration. ICAM-1 variant (G241R) may has association with Table 12.1  ICAM-1 genetic polymorphisms and disease Sr. no 1. 2. 3. 4. 5. 6. 7.

Polymorphisms Lys469Glu (K469E) Lys469Glu (K469E) Lys469Glu (K469E) Lys29 Met (K29M) Lys29 Met (K29M) Gly 241Arg (G241R) Gly 241Arg (G241R)

Disease P. Falciparum Malaria

References Fry et al. (2008)

Malaria, Inflammatory and neurodegenerative Ischemic stroke

Amodu et al. (2005)

Malaria (Kenya)

Gao and Zhang (2019)

Malaria (Gabon) Cancer

Fernandez-Reyes et al. (1997) Kun et al. (1999) Cheng and Liang (2015)

Fuchs uveitis

Cimino et al. (2010)

12.5  ICAM1 Gene Polymorphisms and Malaria

141

Fig. 12.4  Location of SNPs in domain1 and domain5 of ICAM-1 gene

cancer risk, in European populations (Cheng and Liang 2015). It has been hypothesized that ICAM-1 gene polymorphisms in exons lead to amino acid substitutions, such changes might alter ligand binding or stability of multimeric ICAM-1 on cell surface and hence alter signal transduction (Cimino et al. 2010).

12.5 ICAM1 Gene Polymorphisms and Malaria Malaria pathogenesis is because of adherence of parasitized red blood cells to vascular endothelium using receptors, ICAM-1, CD36, and thrombospondin (Fernandez-Reyes et al. 1997). P. falciparum infection has created selective force on the human genome and has been found driving force behind the selection of various DNA sequence variations in the human genome. ICAM-1Kilifi, mutation causes a change from lysine to methionine in a loop that binds parasitized erythrocytes, rhinoviruses, and lymphocytes. Remarkably, this mutation has been demonstrated increased susceptibility of Kenyan children to severe malaria (Kun et  al. 1999). High frequency ICAM-1Kilifi mutation has been reported within African populations, which showed increased risk of CM. ICAM-1Kilifi has decreased avidity for LFA-1 as compared to ICAM-1 reference form. Binding to soluble fibrinogen was totally

142

12  Intercellular Adhesion Molecule-1 Polymorphisms

eliminated with Kilifi variant. Studies in P. falciparum adhesion assays, identified that ITO4-A4u binding to ICAM-1Kilifi was reduced as compared to binding with reference form. These outcomes permit the chance of balancing selection between the reference and Kilifi forms of ICAM-1 via modulation of inflammatory responses and show differences within ICAM-1-binding P. falciparum isolates which may be pertinent to malaria pathogenesis (Craig et al. 2000). A high frequency ICAM-1(Kilifi) allele has been found in Tanzania, association of this allele with greater child hospitalization frequencies is independent of cytoadherence patterns of infected erythrocytes isolated from ICAM-1(Kilifi). Result of this study suggested that association between ICAM-1(Kilifi) mutation and severe malaria are not expected to be mediated via alteration in cytoadherence of infected erythrocytes (Mwanziva et al. 2010). The ICAM-1Kilifi mutant allele probably has been selected in endemic malaria. Nonetheless, unexpectedly, in a Kenyan case-control study, it was found that the ICAM-1Kilifi allele showed association to an increased susceptibility to CM. ICAM-1Kilifi has been found commonly in Kenyan and Gambian populations (approximately 30%), but not in Caucasians. It may be suggested that ICAM-1Kilifi provides a compensatory selective benefit but the mechanism is not known (Fry et al. 2008). Approximately, 55% mild malaria patients were carriers of ICAM-1Kilifi while 39% of severe malaria patients were carriers in Gabonese and Kenyan (Kun et al. 1999). In contrast, a protective effect of ICAM-1Kilifi was found in case-control study from Gabon, (Kun et al. 1999). No significant association has been found between malaria phenotypes and ICAM-1Kilifi in case-control studies in the Gambia, Thailand, Senegal, Nigeria and further studies in Kenya. Nigerian case-control study suggested a marginal association between SNP in exon 6 (rs5498) and malaria susceptibility. Very low frequencies of ICAM-1Kilifi polymorphism was detected in populations of both endemic and non-endemic regions of P. falciparum caused malaria in India, therefore it was not included in studies (Sinha et  al. 2008). Conflicting results of the studies were reported in different population across the world. Therefore, exploration of ICAM-1 genetic polymorphisms in different ethnic population would be important in detection of genetic risk of a population to malaria. Studied ICAM-1 gene polymorphisms may be responsible for altered response of an individual human being to P. falciparum infection and disease outcome (Fig. 12.5). It has been suggested that changes in levels or structure of adhesion molecules could influence response of an individual human being to P. falciparum infection and subsequent disease severity and outcome (Sinha et al. 2008). DNA sequence variations may play role in alteration of level/ structure of protein and thereby produces altered response of human host to malaria.

12.7 Conclusion Fig. 12.5 Polymorphisms in ICAM-1 gene may be responsible for altered response of an individual human being to P. falciparum infection and disease outcome

143

ICAM-1 genetic polymorphisms in coding regions

Substitution of amino acid in ICAM-1 protein

Affects structure of ICAM-1 protein

Alteration in structure of ICAM-1 protein

Altered response of an individual human being to P. falciparum infection and disease manifestation

12.6 Soluble ICAM-1 Soluble ICAM-1 circulates in the plasma, it is formed by proteolytic cleavage of cell surface ICAM-1 in response to inflammatory cytokines or endothelial damage. It has been found that soluble ICAM-1 levels were significantly higher among children with severe malaria syndromes or fatal malaria as compared to uncomplicated malaria. Presence of high soluble ICAM-1 concentrations might reflect a combination of larger inflammatory response, augmented endothelial cell stimulation with ICAM-1 peeling, or decreased soluble ICAM-1 clearance (Cserti-­ Gazdewich et al. 2010).

12.7 Conclusion ICAM-1 is present on the surface of many cells such as leukocytes, endothelial cells. ICAM-1 performs a dual role, response of host to inflammation and pathogenesis of severe malaria/ cerebral malaria as well. It serves as receptors for a number of organisms such as P. falciparum, rhinovirus, and coxsackie virus A13. ICAM-1on endothelial layer interacts with PfEMP1 expressed on infected erythrocytes. Such cytoadherence of infected erythrocytes contributes to malaria pathogenesis. Malaria is an example of evolutionary selection and balancing selection. ICAM-1 gene

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polymorphisms have shown association with disease in African as well as other countries including India. SNPs in ICAM-1 gene are expected to select for protection against malaria.

References Adukpo S, Kusi KA, Ofori MF, Tetteh JKA, Amoako-Sakyi D, et al. (2013) High Plasma Levels of Soluble Intercellular Adhesion Molecule (ICAM)-1 Are Associated with Cerebral Malaria. PLoS ONE 8(12): e84181. https://doi.org/10.1371/journal.pone.0084181. Amodu OK, Gbadegesin RA, Ralph SA, Adeyemo AA, Brenchley PE, Ayoola OO, Orimadegun AE, Akinsola AK, Olumese PE, Omotade OO: Plasmodium falciparum malaria in south-west Nigerian children: is the polymorphism of ICAM-1 and E-selectin genes contributing to the clinical severity of malaria? ActaTrop 2005, 95:248-255. Baratin M, Roetynck S, Pouvelle B, Lemmers C, Viebig NK, Johansson S et al. Dissection of the role of PfEMP1 and ICAM-1 in the sensing of Plasmodium falciparum-infected erythrocytes by natural killer cells. PLoS ONE 2007; 2: e228. Baruch DI, Rogerson SJ, Cooke BM: Asexual blood stages of malaria antigens: cytoadherence. Chem Immunol. 2002, 80: 144-162. Cheng D, Liang B.  Intercellular Adhesion Molecule-1 (ICAM-1) Polymorphisms and Cancer Risk: A Meta-Analysis. Iran J Public Health. 2015;44 (5):615-24. Cserti-Gazdewich CM, Dzik WH, Erdman L, Ssewanyana I, Dhabangi A, Musoke C, Kain KC. Combined measurement of soluble and cellular ICAM-1 among children with Plasmodium falciparum malaria in Uganda. Malar J. 2010; 9:233. https://doi.org/10.1186/1475-­2875-­9-­233 Cimino L, Boiardi L, Aldigeri R, Casali B, Nicoli D, Farnetti E, Salvarani C, Cirone D, De Martino L, Pupino A, Cappuccini L.  G/R 241 polymorphism of intercellular adhesion molecule 1 (ICAM-1) is associated with Fuchs uveitis. Invest Ophthalmol Vis Sci. 2010; 51 (9):4447-50. Craig A, Fernandez-Reyes D, Mesri M, McDowall A, Altieri DC, Hogg N, Newbold C.A functional analysis of a natural variant of intercellular adhesion molecule-1 (ICAM-1Kilifi). Hum Mol Genet. 2000;9(4):525-30. Fernandez-Reyes D, Craig AG, Kyes SA, Peshu N, Snow RW, Berendt AR, Marsh K, Newbold CI. A high frequency African coding polymorphism in the N-terminal domain of ICAM-1 predisposing to cerebral malaria in Kenya. Hum Mol Genet. 1997;6(8):1357-60. Fry, A. E., S. Auburn, M. Diakite, A. Green, A. Richardson, J. Wilson, M. Jallow, F. Sisay-Joof, M. Pinder, M. J. Griffiths, N. Peshu, T. N. Williams, K. Marsh, M. E. Molyneux, T. E. Taylor, K.A.  Rockett, and D.  P. Kwiatkowski. Variation in the ICAM1 gene is not associated with severe malaria phenotypes. Genes and immunity, 2008, 9: 462-469. Gao H, Zhang X. Associations of intercellular adhesion molecule-1 rs5498 polymorphism with ischemic stroke: A meta-analysis. Mol Genet Genomic Med. 2019; 7(6):e643. https://doi. org/10.1002/mgg3.643. He, Q., Lin, X., Wang, F., Xu, J., Ren, Z., Chen, W., & Xing, X. (2014). Associations of a polymorphism in the intercellular adhesion molecule-1 (ICAM1) gene and ICAM1 serum levels with migraine in a Chinese Han population. Journal of the Neurological Sciences, 345, 148-153. Kun JF, Klabunde J, Lell B, Luckner D, Alpers M, May J et al. Association of the ICAM-1Kilifi mutation with protection against severe malaria in Lambarene, Gabon. Am J Trop Med Hyg 1999; 61: 776-779. Mohamed, A. A., Rashed, L., Amin, H., Abu-Farha, M., El Fadl, S. A., & Pakhoum, S. (2010). K469E polymorphism of the intercellular adhesion molecule-1 gene in Egyptians with coronary heart disease. Annals of Saudi Medicine, 30, 432-436. Mwanziva C, Mpina M, Balthazary S, Mkali H, Mbugi E, Mosha F, Chilongola J. Child hospitalization due to severe malaria is associated with the ICAM-1Kilifi allele but not adherence patterns

References

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of Plasmodium falciparum infected red blood cells to ICAM-1. Acta Trop. 2010;116(1):45-50. https://doi.org/10.1016/j.actatropica.2010.05.006 Razakandrainibe R, Pelleau S, Grau GE, Jambou R.  Antigen presentation by endothelial cells: what role in the pathophysiology of malaria? Trends Parasitol. 2012; 28(4):151-60. https://doi. org/10.1016/j.pt.2012.01.004 Reilly PL, Woska JR Jr, Jeanfavre DD, McNally E, Rothlein R, Bormann BJ: The native structure of intercellular adhesion molecule-1 (ICAM-1) is a dimer. Correlation with binding to LFA-1. J Immunol 1995, 155:529-532. Sherman IW, Eda S, Winograd E: Cytoadherence and sequestration in Plasmodium falciparum: defining the ties that bind. Microbes Infect 2003, 5:897-909. Sinha S, Qidwai T, Kanchan K, Anand P, Jha GN, Pati SS, Mohanty S, Mishra SK, Tyagi PK, Sharma SK; Indian Genome Variation Consortium, Venkatesh V, Habib S. Variations in host genes encoding adhesion molecules and susceptibility to falciparum malaria in India. Malar J. 2008;7: 250. https://doi.org/10.1186/1475-­2875-­7-­250. https://www.genecards.org/cgi-­bin/carddisp.pl?gene=ICAM1&keywords=icam1. https://www.uniprot.org/uniprot/P05362

Platelet Endothelial Cell Adhesion Molecule-1 (PECAM-1) Polymorphisms

13

Abstract

During Plasmodium falciparum infection, cytoadherence, and rosetting are the main causes of pathology. Excessive cytoadherence and rosetting probably cause occlusion of the microvasculature, leading to acute pathology of severe malaria. Endothelial receptors including intercellular adhesion molecule-1 (ICAM-1), platelet endothelial cell adhesion molecule 1 (PECAM-1), vascular cell adhesion molecule (VCAM-1), CD36, thrombospondin, and E-selectin, serve as a receptors for parasite ligand PfEMP1 on infected erythrocytes. Single nucleotide polymorphisms in PECAM-1 gene have shown association with P. falciparum malaria in different populations. PECAM-1 is involved in severe outcome of the disease via cytoadherence of infected erythrocytes on endothelial layer. The presence of polymorphisms in promoter region and non-synonymous polymorphisms in PECAM-1 coding region may influence susceptibility/resistance to P. falciparum caused malaria. The genetic variations of PECAM-1 may affect the severity and outcome of P. falciparum malaria in different populations. The high incidence of malaria in endemic regions forces the human genomes to select the genetic variations of human genes involved in pathogenesis of malaria. This chapter is aimed to investigate the PECAM-1 polymorphisms and susceptibility/resistance to malaria in endemic areas. Analysis of genetic makeup of a population and its exposure to malaria and outcome of disease could be important in malaria prevention.

© Springer Nature Singapore Pte Ltd. 2021 T. Qidwai, Exploration of Host Genetic Factors associated with Malaria, https://doi.org/10.1007/978-981-33-4761-8_13

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Keywords

Adhesion molecules · P. falciparum malaria · PECAM-1 polymorphisms · Cytoadherence

13.1 Introduction Platelet endothelial cell adhesion molecule-1 (PECAM-1), also named CD31, is a transmembrane glycoprotein. PECAM-1, a member of immunoglobin superfamily, is constitutively expressed on human platelets, intracellular junctions of resting endothelial cells and on circulating monocytes, granulocytes and subgroup of T cells. PECAM-1 performs a vital role in the transendothelial movement of circulating leukocytes during inflammation and in maintenance of vascular endothelial integrity. The PECAM-1 molecule consists of six Immunoglobin (Ig)-like extracellular domains, a short transmembrane domain and a long cytoplasmic tail having multiple sites for phophorylation, lipid modifications, and other posttranslational modifications. Host and parasite factors have been known to involve in severe outcome of malaria pathogenesis. Human host molecules mediate cerebral and non-cerebral cytoadherence of parasitized erythrocytes (P. falciparum-infected erythrocytes) to endothelial layer (Serghides et al. 2003), these processes assist the parasite to escape immune clearance in the spleen. PECAM-1, CD36, intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule (VCAM-1), thrombospondin, E-selectin, and P-selectin are important adhesion molecules that serve as receptors for ligands such as Plasmodium falciparum erythrocyte membrane protein-1 (PfEMP-1), present on the surface of infected erythrocytes to mediate cytoadherence (Sinha et al. 2008). PECAM-1 on vascular endothelium interacts with PfEMP-1 leading to cytoadherence, common in both cerebral and non-cerebral malaria. Certain cytokines like TNF and IFNγ have been shown to alter the adhesion of PECAM-1 and infected erythrocytes (Treutiger et al. 1997). Due to the sequestration of infected erythrocytes, a number of alterations take place in the microcirculatory environment resulting in multiple organ dysfunction and manifestation of more severe forms of malaria, like cerebral malaria (CM). It may be suggested that PECAM-1 is a virulence-linked endothelial receptor of parasitized erythrocytes (PE). Single nucleotide polymorphisms (SNPs) in PECAM-1 gene have been shown association to many diseases. PECAM-1 gene C+373G (Leucine125Valine) in exon 3, encoding first extracellular Ig-like domain which mediates the homophilic binding of PECAM-1 (Zavrsnik et  al. 2016). The PECAM-1 gene polymorphism C+373G rs688 (Leucine125Valine) studied in Egyptian patients with coronary artery disease (El-Kishki et al. 2019). Mutant homozygotes of PECAM-1 polymorphisms (L125V and S563N) have been identified as a risk factors for CM in Thailand (Kikuchi et al. 2001).

13.2 Structure and Function of PECAM-1

149

Promoter SNPs in PECAM-1 gene have also shown association with malaria disease (Ohashi et  al. 2016). PECAM-1 promoter polymorphisms may affect its expression and hence the extent of cytoadherence in cerebral and non-cerebral outcome of disease. Non-synonymous coding polymorphisms in PECAM-1 may affect the activity of protein and influence cytoadherence in P. falciparum malaria. Therefore, people with different genotypes in adhesion molecules have varying severity of P. falciparum malaria. The present chapter is aimed to analyze the function of PECAM-1, its role in malaria pathogenesis. Moreover, SNPs in PECAM-1 gene and its impact on the severity and outcome of P. falciparum caused malaria in different populations have been covered.

13.2 Structure and Function of PECAM-1 The gene for PECAM-1 is located in cytogenetic band 17q23.3 on chromosome number 17 of human beings. The PECAM-1 gene is 75 kb, present near the end of the long arm of chromosome 17 (Fig. 13.1) and consists of 16 exons. PECAM-1 gene encodes a 130 kDa glycoprotein of the Ig superfamily. PECAM-1 protein is consisting of cytoplasmic domain, extracellular domain (consist of six Ig-like domains) (Fig. 13.2). PECAM-1 is predominantly found at endothelial cell intercellular junctions, where it controls leukocyte trafficking, mechanotransduction, and vascular permeability (Lertkiatmongkol et al. 2016). PECAM-1-mediated signaling is started by phosphorylation of amino acid residue serine at 702 position, which releases immunoreceptor tyrosine-based inhibitory motifs (ITIM) tyrosine 686 from its association with the plasma membrane, easing its phosphorylation by the Src-family kinase (Lertkiatmongkol et al. 2016). ITIMs in PECAM-1, when phosphorylated, recruit the protein-tyrosine phosphatase, SHP-2, resulting in formation of a PECAM-1/SHP-2 complex that functions in circulating blood cells to inhibit a plethora of tyrosine kinase initiated cellular activation events (Fig. 13.3) (Newman 1999). Endothelial PECAM-1 is able to recruit cytosolic SHP-2 to the inner face of the plasma membrane in a phospho-ITIM-­ specific manner to form a complex that functions to increase endothelial cell motility and migration (Masuda et al. 1997).

Chromosome17: 17q23.3 17p13.3

17q23.2

17q23.3

17q24.1

17q25.3

PECAM-1

Fig. 13.1  Location of adhesion molecule, PECAM-1 gene on chromosome 17 (https://www.genecards.org/cgi-­bin/carddisp.pl?gene=PECAM1&keywords=pecam1)

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13  Platelet Endothelial Cell Adhesion Molecule-1 (PECAM-1) Polymorphisms

NH2 1

Homophilic binding sites

2 3 4 5 6

Exon9 Exon10

Y 686

Exon14 Exon15

C terminal 702 7

Exon13

Exon16

Y663 Exon11 Exon12

Cytoplasmic ITIM tyrosine phosphorylated by Lyn

Fig. 13.2  PECAM-1, an important signaling molecule consists of 16 exons. PECAM-1 signaling is started by phosphorylation of amino acid residue serine at 702 position, which releases immunoreceptor tyrosine-based inhibitory motifs (ITIM) tyrosine 686 from its association with plasma membrane (Lertkiatmongkol et al. 2016)

Serine amino acid at

Release of ITIM tyrosine 686 from its

702 position undergoes

association with plasma membrane

phosphorylation

ITIMs

in

PECAM-1,

after

phosphorylation, recruit proteintyrosine phosphatase, SHP-2

PECAM-1 mediated

PECAM-1/SHP-2

Signaling

complex formation

Fig. 13.3  This flowchart represents PECAM-1 mediated signaling

13.3 Mechanisms of Malaria Pathogenesis The cytoadherence of parasitized erythrocytes to vascular endothelium mediated by PECAM-1 plays a crucial role in pathogenesis of CM. Adhesion molecules including PECAM-1 are expressed on the surface of endothelial cells. Blood flows in blood capillaries along with blood cells and infected erythrocytes (Fig. 13.4). Flow of blood with infected erythrocytes causes cytoadherence of infected erythrocytes

13.3 Mechanisms of Malaria Pathogenesis

151

Uncomplecated malaria

Cerebral malaria

Adhesion molecules (ICAM-1, PECAM-1)

Parasitized e rythrocytes

Endothelial layer Uncomplecated malaria

Cerebral malaria (a)

P. falciparum caused malaria

Uncomplecated malaria

Cerebral malaria

High TNF expression Low expression of adhesion molecules High expression of adhesion molecules

More cytoadherence No pathology and no cell death

Pathology and cell death (b)

Fig. 13.4 (a) Diagrammatic representation of adhesion of parasitized erythrocytes on the endothelial layer during uncomplicated/non-cerebral malaria and cerebral malaria. (b) Excessive cytokine production, high expression of adhesion molecules, and high level of cytoadherence on endothelial layer have been found during malaria pathogenesis

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13  Platelet Endothelial Cell Adhesion Molecule-1 (PECAM-1) Polymorphisms

(Tunon-Ortiz and Lamb 2019). Modulators of endothelial cell cross talk with pericytes and astrocytes may have a role in blood brain barrier (BBB) disruption during CM.

13.4 G  enetic Polymorphisms in PECAM-1 Gene and P. falciparum Malaria The PECAM-1 acts as an endothelial receptor for erythrocytes having P. falciparum infection. Domains 1–4 of the PECAM-1 molecule are involved in binding with parasitized erythrocytes. This is confirmed by the evidence that antibodies directed against domains 1–2 has been shown to inhibit PECAM-1-dependent adhesion of infected erythrocytes in vitro (Treutiger et  al. 1997), probably this is because of disruption of interaction with PfEMP-1 in infected erythrocytes. Several PECAM-1 genetic polymorphisms have been associated with diseases. PECAM-1 SNPs, rs668, L/V; rs12953, S/N; rs1131012, R/G; int10 novel SNP, G/A; int15, rs2070783 C/T identified in different populations (Table 13.1). The PECAM-1 exon 3 polymorphism lies in first Ig-like domain of the PECAM-1 molecule (Fig. 13.5a) which has mediated homophilic adhesion and controls leukocyte transmigration (Sinha et al. 2008; Liao et al. 1997; Novinska et al. 2006). Study by Sinha et al. (2008) on the Indian population identified that exon 8 G/A and exon 12 A/G SNPs did not show a significant association with disease (Fig. 13.5a). Exon 3 SNP demonstrated no significant association with disease in both endemic and Table 13.1 Human PECAM-1 genetic polymorphisms and diseases Sr. no 1.

Polymorphisms Exon 3 rs668 (C/G)

Change Leucine125Valine

2.

Exon 8 rs12953 (G/A)

3. 4.

Exon 12 rs1131012 (A/G) Int15, rs2070783 C/T

Serine 563Asparagine Glycine 670 Arginine –

5.

Promoter, rs9912957

–

6.

Promoter, rs1122800

–

7.

Exon 3 rs668 (C/G)

Leucine125Valine

8.

Exon 3 rs668 (C/G)

Leucine125Valine

9. 10.

Exon3 +373 C/G PECAM-1, 1688 A/G

Leucine125Valine Serine 563 Asparagine

Disease P. falciparum malaria P. falciparum malaria P. falciparum malaria P. falciparum malaria P. falciparum malaria P. falciparum malaria Type 2 diabetes mellitus Coronary artery disease Kawasaki disease Kawasaki disease

References Sinha et al. (2008) Sinha et al. (2008) Sinha et al. (2008) Sinha et al. (2008) Ohashi et al. (2016) Ohashi et al. (2016) Popovic et al. (2016) El-Kishki et al. (2019) Li et al. (2017) Li et al. (2017)

13.4 Genetic Polymorphisms in PECAM-1 Gene and P. falciparum Malaria

153

(a) rs9912957

rs1122800 Non-synonymous SNPs

Promoter SNPs

Affect the expression of PECAM-1 gene. (b) Fig. 13.5  Location of single nucleotide polymorphisms (SNPs) in PECAM-1 (a) coding SNPs and (b) promoter SNPs

non-­endemic regions whereas a significant association has been found in endemic regions. The mutant allele G demonstrated an association with susceptibility to disease in endemic areas, this mutant G allele has demonstrated, high frequency in both severe and nonsevere patients as compared to controls (Sinha et al. 2008). The exact functional effect of the exon 3 L/V mutation is not known, so far, however, if

154

13  Platelet Endothelial Cell Adhesion Molecule-1 (PECAM-1) Polymorphisms

it affects homophilic binding of PECAM-1 then it may affect leukocyte movement at the site of inflammation during disease condition. The role of PECAM-1 exon 3 SNP in infected erythrocyte sequestration has been demonstrated.

13.4.1 Promoter Polymorphisms Single nucleotide polymorphisms in promoter regions of PECAM-1 gene have also been studied (Fig. 13.5b) (Ohashi et al. 2016). Four SNPs, rs7213889, rs59573853, rs8065316, and rs12953175, were significantly associated with CM (Ohashi et al. 2016). The rs1122800-C was associated to  protection from CM in the dominant model and rs9912957-A was associated to increased risk for CM in malaria patients from Thailand population (Ohashi et al. 2016). The rs1122800-CC and rs1122800­CG genotypes showed protective effects from CM while rs1122800-GG genotype is linked to risk for CM. In vitro study in cell lines has been done to investigate the effects of PECAM-1 promoter SNPs. It has been identified that rs1122800-C allele is significantly linked to  lower expression of PECAM-1 (Ohashi et  al. 2016). A reduction in cytoadherence caused by lower expression of PECAM-1 on vascular endothelium may decrease risk for CM.

13.5 Discussion Adhesion molecules such as PECAM-1 interacts with infected erythrocytes through parasite expressed protein; this process is named as cytoadherence and appears to play role in severe/cerebral malaria. Individuals with different genotypes in PECAM-1 gene may have varying extent of cytoadherence of infected erythrocytes with PECAM-1 on vascular endothelium. SNPs in PECAM-1 gene have been implicated in susceptibility/ resistance to malaria. Coding region polymorphisms may affect activity of protein while promoter polymorphisms may affect expression of gene (Fig. 13.6). Alteration in activity or level of expression of PECAM-1 protein may affect cytoadherence of infected erythrocytes on the endothelial lining. PECAM-1 SNPs conferring protective effects have been selected in those regions having high incidence of P. falciparum malaria. The differences in cytoadherence of infected erythrocytes to PECAM-1 on vascular endothelium between different PECAM-1 genotypes may explain why some malaria patients develop CM. Genetic control mechanisms have been developed in human beings in endemic regions through natural selection, for example, high frequencies of hemoglobinopathies have been found in malaria endemic regions of Africa and India. Presence of hemoglobins C and hemoglobin S, alter the PfEMP-1 expression and thereby influence the parasite adhesion to endothelial cells, suggesting a possible mechanism for their protective effects (Taylor et al. 2012; Mahamar et al. 2017).

13.5 Discussion

155

PECAM-1 polymorphisms

Coding region nonsynonymous polymorphisms

PECAM-1 promoter polymorphisms

Influence activity of PECAM-1 protein

Influence expression of PECAM-1 protein

Alteration in structure or level of PECAM-1 protein may affect cytoadherence of infected erythrocytes on endothelial lining

Individuals with variable PECAM-1 genotypes show variability in cytoadherence of infected erythrocytes with PECAM-1 on vascular endothelium

Some people infected with P. falciparum may develops cerebral malaria Fig. 13.6  This flowchart demonstrates that PECAM-1 polymorphisms may affect its structure or expression level and thereby affect cytoadherence of infected erythrocytes to adhesion molecules. Most likely due to presence of polymorphisms some people infected with P. falciparum may develop cerebral malaria, complicated and fatal form of malaria

13.5.1 Antiadhesion Therapies Many treatment options are being used to overcome the cytoadherence of infected erythrocytes on the endothelial layer (Rowe et al. 2009). The PfEMP-1 blocking agents have been used to prevent cytoadherence of infected erythrocytes (Cooke

156

13  Platelet Endothelial Cell Adhesion Molecule-1 (PECAM-1) Polymorphisms

et al. 1998; Chen et al. 2004). Levamisole inhibits sequestration of infected erythrocytes in patients with falciparum malaria (Dondorp et  al. 2007). During malaria therapy, geographical variation needs to be considered carefully. A rosette-­disrupting drug might be of clinical benefit in sub-Saharan Africa but would not be an appropriate treatment for severe malaria in South-East Asia. On the contrary, a drug that reverses CD36 binding might be more effective in South-East Asia.

13.6 Conclusions Host adhesion molecules such as ICAM-1, PECAM-1, and VCAM-1 act as a receptor for the ligand PfEMP-1 expressed on the surface of infected erythrocytes. Adhesion of parasite infected erythrocytes on the endothelial cell cause major pathology in malaria. Polymorphisms in promoter/regulatory or nonregulatory (coding region) region may affect level/ activity of PECAM-1 which in turn influence cytoadherence of parasitized erythrocytes thereby alters severity of disease and pathology during infection. Frequency of polymorphisms in PECAM-1 gene varies according to malaria endemicity. Those variations which confer protection, would be selected by human genome in malaria endemic regions.

References Chen, Q. et al. Immunization with PfEMP1-DBL1alpha generates antibodies that disrupt rosettes and protect against the sequestration of Plasmodium falciparum-infected erythrocytes. Vaccine, 2004, 22, 2701-2712. Cooke, B.M. et  al. A recombinant peptide based on PfEMP-1 blocks and reverses adhesion of malaria-infected red blood cells to CD36 under flow. Molecular Microbiology, 1998,30, 83-90. Dondorp AM, Silamut K, Charunwatthana P, et al. Levamisole inhibits sequestration of infected red blood cells in patients with falciparum malaria. J Infect Dis. 2007;196(3):460-466. https:// doi.org/10.1086/519287 El-Kishki MM, Abdelfattah Alramly AM, Taha MO, El Din Medhat RA. Association of platelet endothelial cell adhesion molecule-1 gene polymorphism (Leu125Val) with coronary artery disease in type II diabetics and nondiabetics. Sci J Al-Azhar Med Fac Girls 2019; 3: 23-32. Rowe JA, Claessens A, Corrigan RA, Arman M.  Adhesion of Plasmodium falciparum-infected erythrocytes to human cells: molecular mechanisms and therapeutic implications. Expert Rev Mol Med. 2009;11:e16. https://doi.org/10.1017/S1462399409001082. Kikuchi M, Looareesuwan S, Ubalee R, Tasanor O, Suzuki F, Wattanagoon Y, Na-Bangchang K, Kimura A, Aikawa M, Hirayama K: Association of adhesion molecule PECAM-1/CD31 polymorphism with susceptibility to cerebral malaria in Thais. Parasitol Int. 2001, 50: 235-239. Lertkiatmongkol P, Liao D, Mei H, Hu Y, Newman PJ. Endothelial functions of platelet/endothelial cell adhesion molecule-1 (CD31). Curr Opin Hematol. 2016; 23(3):253-9. Liao F, Ali J, Greene T, Muller WA: Soluble domain 1 of platelet-endothelial cell adhesion molecule (PECAM) is sufficient to block transendothelial migration in vitro and in vivo. J Exp Med. 1997, 185: 1349-1357. Mahamar A, Attaher O, Swihart B, et  al. Host factors that modify Plasmodium falciparum adhesion to endothelial receptors. Sci Rep. 2017;7(1):13872. https://doi.org/10.1038/ s41598-­017-­14351-­7.

References

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Masuda M, Osawa M, Shigematsu H, Harada N, Fujiwara K. Platelet endothelial cell adhesion molecule-1 is a major SH-PTP2 binding protein in vascular endothelial cells. Febs Letters. 1997;408:331–336 Newman PJ. Switched at birth: a new family for PECAM-1. J Clin Invest. 1999; 103:5–9. Novinska MS, Pietz BC, Ellis TM, Newman DK, Newman PJ. The alleles of PECAM-1. Gene. 2006; 376(1):95-101. Ohashi J, Naka I, Hananantachai H, Patarapotikul J. Association of PECAM-1/CD31 polymorphisms with cerebral malaria. Int J Mol Epidemiol Genet. 2016; 7(2):87-94. E Collection 2016. Popovic D, Nikolajevic Starcevic J, Santl Letonja M, Makuc J, Cokan Vujkovac A, Reschner H, Bregar D, Petrovic D.  PECAM-1 gene polymorphism (rs668) and subclinical markers of carotid atherosclerosis in patients with type 2 diabetes mellitus. Balkan J Med Genet. 2016;19(1):63-70. Serghides L, Smith TG, Patel SN, Kain KC: CD36 and malaria: friends or foes? Trends Parasitol 2003, 19:461-469. Sinha S, Qidwai T, Kanchan K, et al. Variations in host genes encoding adhesion molecules and susceptibility to falciparum malaria in India. Malar J. 2008;7:250. Published 2008 Dec 4. https://doi.org/10.1186/1475-­2875-­7-­250. Taylor, S. M., Parobek, C. M. & Fairhurst, R. M. Haemoglobinopathies and the clinical epidemiology of malaria: a systematic review and meta-analysis. Lancet Infect Dis, 2012, 12, 457-468. Treutiger, C.J., Heddin, I A., Fernandez, V., Muller, W.A. and Wahlgren, M.  PECAM-1/CD31, an endothelial receptor for binding Plasmodium falciparum-infected erythrocytes. Nat. Med. 1997, 3:1405-1408. Tunon-Ortiz A, Lamb TJ. Blood brain barrier disruption in cerebral malaria: Beyond endothelial cell activation. PLOS Pathogens, 2019, 15(6): e1007786. Zavrsnik M, Kariz S, Makuc J, Seruga M, Cilensek I, Petrovic D. PECAM-1 Leu125Val (rs688) Polymorphism and Diabetic Nephropathy in Caucasians with Type 2 Diabetes Mellitus. Anal Cell Pathol (Amst). 2016: 3152967. https://doi.org/10.1155/2016/3152967. Epub 2016 Dec 28. Zhuoying Li, Dong Han, Jie Jiang, Jia Chen, Lang Tian, Zuocheng Yang. Association of PECAM-1 Gene Polymorphisms with Kawasaki Disease in Chinese Children. Dis Markers. 2017; 2017: 2960502. Published online 2017 Apr 23. https://doi.org/10.1155/2017/2960502 https://www.genecards.org/cgi-­bin/carddisp.pl?gene=PECAM1&keywords=pecam1

Vascular Cell Adhesion Molecule-1 (VCAM-1) Polymorphisms

14

Abstract

Host adhesion molecules play a key role in malaria pathogenesis. The Plasmodium falciparum infected erythrocytes (parasitized erythrocytes) can bind to endothelium, uninfected erythrocytes, and platelets. Adhesion of parasitized erythrocytes to endothelial cells causes their sequestration in the microvasculature of many organs including brain, heart, and lung. Interaction of parasitized erythrocytes to endothelial layer (cytoadherence) is a key in pathogenesis of severe malaria. P. falciparum infection upregulates expression of pro-inflammatory cytokines which in turn elevates expression of adhesion molecules such as vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1). VCAM-1 interacts with parasite ligand named as P. falciparum erythrocyte membrane protein1 (PfEMP-1) on the surface of infected erythrocytes which give rise cytoadherence. Role of host genetic polymorphism is evidenced in malaria, regulatory polymorphisms affect expression of gene (level of gene product) while coding polymorphisms affect protein structure and hence activity. Polymorphisms in VCAM-1 gene have been shown to influence the cytoadherence process and therefore alter the disease severity. In malaria endemic regions, high frequency of malaria exerts selective pressure on human genome to select variation which confers protection against the disease. Analysis of genetic variations of VCAM-1 and their role in resistance/susceptibility/severity of malaria have been covered in this chapter. Identification of VCAM-1 polymorphisms would be important in detection of risk factor for cerebral malaria.

© Springer Nature Singapore Pte Ltd. 2021 T. Qidwai, Exploration of Host Genetic Factors associated with Malaria, https://doi.org/10.1007/978-981-33-4761-8_14

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14  Vascular Cell Adhesion Molecule-1 (VCAM-1) Polymorphisms

Keywords

Host adhesion molecules · VCAM-1 polymorphisms · Malaria · Cytoadherence · Endemic regions · Infected erythrocytes

14.1 Introduction Vascular cell adhesion molecule-1 (VCAM-1) also named as CD106 is, a 90-kDa glycoprotein. VCAM-1 is inducible and, most commonly expressed in endothelial cells, however, other cell types such as tissue macrophages, dendritic cells, Kupffer cells, cancer cells, etc. also show expression of VCAM-1 on their surface (Sharma et al. 2017). VCAM-1 has been recognized in the year 1989, as a surface glycoprotein on endothelial cells (Kong et al. 2018). Initially, VCAM-1 was detected as a cell adhesion molecule, playing a role for regulation of inflammation-linked vascular adhesion and transendothelial movement of leukocytes, including T cells and macrophages (Kong et  al. 2018). In vitro, studies on cytoadherence of P. falciparum isolates taken from infected individuals showing various binding affinities to several human host proteins, for example, ICAM-1 (Berendt et al. 1989), CD36 (Barnwell et  al. 1989), VCAM-1 (Ockenhouse et  al. 1992), CR1 (Rowe et  al. 1997), and gC1qR/HABP1/p32 (Biswas et al. 2007) serve as receptor for binding to infected erythrocytes. VCAM-1 in human beings is involved in parasite interaction and hence cytoadherence, binding of P. falciparum parasites to VCAM-1 was demonstrated in vitro using field isolates (Ockenhouse et al. 1992). P. falciparum infected erythrocytes have ability to attach to a variety of host cells, producing different types of events. Generally three types of infected erythrocyte adhesion have been recognized, for example, binding of parasitized erythrocytes to endothelium causes cytoadherence, binding to uninfected erythrocytes produces rosetting and binding to platelets give rise platelet-mediated clumping (Fig. 14.1) (Rowe et al. 2009). Sequestration happens in the microvasculature of brain, heart, lung, and adipose tissue due to adhesion of parasitized erythrocytes to endothelial cells (Rowe et al. 2009). Expression of adhesion molecule is activated by pro-inflammatory cytokines such as tumor necrosis factor alpha (TNFα) and also by reactive oxygen species (ROS) (Kong et al. 2018). Inflammation is a key event in many diseases like malaria. TNFα is involved in pathogenesis of P. falciparum caused malaria as this molecule is associated with inflammation. It activates expression of other cytokines involved in inflammation and host cell adhesion molecules (VCAM-1 and ICAM-1). During P. falciparum infection, severe condition of the disease arises due to adhesion of infected erythrocytes on the endothelial cells. In severe malaria, pro-inflammatory cytokines are upregulated which in turn increase expression of VCAM-1 and ICAM-1 (host adhesion molecules). VACM-1 interacts with parasite derived protein on the surface of infected erythrocytes named as Plasmodium falciparum

Interaction of infected erythrocyte to uninfected erythrocytes (Rosetting)

Interaction of infected erythrocytes to platelets lead to clumping of infected erythrocytes

Fig. 14.1  P. falciparum infected erythrocytes can bind to endothelium, uninfected erythrocytes, and platelets giving different phenomenon such as cytoadherence, rosetting and clumping of infected erythrocytes

Attachment of infected erythrocytes to endothelial cells (Cytoadherence)

erythrocyte adhesion

Three major types of P. falciparum-infected

14.1 Introduction 161

162

14  Vascular Cell Adhesion Molecule-1 (VCAM-1) Polymorphisms

erythrocyte membrane protein-1 (PfEMP-1). Cytoadherence of infected erythrocytes causes intravascular mechanical obstruction. Cerebral malaria is a fatal form of malaria leading to neurological complication of infection. PfEMP-1 interacts with brain endothelial receptors, ICAM-1, VCAM-1, and cytokine-activated endothelial protein C receptor (EPCR). Blood–brain barrier (BBB) consists of a single layer of endothelial cells connected by tight junctions and underlying basal lamina. Dysregulation of BBB has been found (Brussee et al. 2015) resulting from sequestration of infected erythrocytes to the brain endothelial cells (Storm and Craig 2014). Two events are crucial in cerebral malaria (CM), excessive inflammation due to increased intracerebral pro-inflammatory cytokine response (Dunst et al. 2017) and disseminated intravascular coagulation in the brain (Moxon et al. 2013). Human host genetic polymorphism is evidenced in malaria, regulatory polymorphisms affect expression of gene while coding polymorphisms affect protein activity. It appears that polymorphisms in VCAM-1 gene influence the cytoadherence process and hence alters the disease severity and outcome. Therefore, exploration of role played by VCAM-1 and its polymorphisms in malaria is important. In malaria endemic regions, high frequency of malaria exerts selective pressure on human genome to select variation which confers protection against the disease. The present chapter is aimed to analyze the role of VCAM-1  in severe malaria. Moreover, genetic polymorphisms in VCAM-1 gene and susceptibility/outcome/resistance to P. falciparum malaria have been investigated.

Integrin on leukocytes

Associated with asthma, arthritis, cancer, transplant rejection and other diseases

Fig. 14.2  This  figure demonstrates immunoglobulin-like domains of VCAM-1. D1 and/or D4 domain of VCAM-1 present on activated endothelial cells bind to α4β1 integrin located on leukocytes, such interaction is implicated in a wide variety of diseases including cancer, arthritis and transplant rejection

14.3 Inflammatory Role Played by VCAM-1

163

14.2 Structure of VCAM-1 The human VCAM-1 gene is located on chromosome number1. This gene is located in cytogenetic band: 1p21.2. VCAM-1 protein comprises an extracellular domain made of seven homologous immunoglobulin (Ig)-like domains (Fig.  14.2), one transmembrane domain and one cytosolic domain (Polte et  al. 1991; Kim et  al. 2017, Schlesinger and Bendas 2015). It has been identified that Ig-like domain 1 and 4, 2 and 5, 3 and 6 are extremely homologous with each other (Cook-Mills et al. 2011).

14.3 Inflammatory Role Played by VCAM-1 Inflammation is the part of defence mechanism, it recruits immune cells and molecular mediators to exclude infectious agents or damaged cells. Leukocyte trafficking is an important phenomenon during inflammation, it is controlled by synchronized activities of chemokines, cell adhesion molecules and other important molecular mediators (Mitroulis et al. 2015). Macrophages, T lymphocytes, and natural killer cells secrete TNFα which starts the process of inflammation. TNFα triggers expression of ICAM-1 and VCAM-1 and other adhesion molecules to recruit leukocytes at inflamed locations using leukocyte adhesion (Fig. 14.3) (Medzhitov 2008). ICAM-1 and VCAM-1 are inducible, induced by TNFα, high expression of these molecules has been found during acute malaria infection (Baruch et al. 2002). Alteration in level or structure of these molecules may be associated with disease manifestation.

Leukocyte secretes TNFα

TNFα interacts with TNFR1

Activates signaling molecules

Over expression of VCAM-1

High expression in acute malaria infection is found Fig. 14.3  Leukocyte secretes TNFα, which upregulates expression of VCAM-1 on endothelial cells. Adhesion molecules such as ICAM-1 and VCAM-1 are inducible, induced by TNFα. High expression of these molecules reported in acute malaria infection

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14  Vascular Cell Adhesion Molecule-1 (VCAM-1) Polymorphisms

14.4 Pathology in Cerebral Malaria In inflammation, TNFα is principally secreted from leukocytes interact with TNFα receptors, leading to overexpression of VCAM-1 on endothelial cells. VCAM-1 on endothelial cells interacts with α4β1 integrin on leukocytes. Consecutively, this interaction activates VCAM-1 downstream signaling molecules. Parasitized erythrocytes adhere on endothelial cell through VCAM-1 (Fig.  14.4). Binding of PfEMP-1 to endothelial receptors initiates multiple signaling pathways in endothelial cells, as a result reorganization of tight-junction complexes happens, eventually give rise leakiness of the blood–brain barrier (Nishanth and Schluter 2019).

Leukocyte

Infected erythrocyte Cytoadherence of infected RBC P. falciparum

TNFα VCAM-1

Fig. 14.4  The figure demonstrates pathology during severe outcome of P. falciparum infection. TNFα is upregulated which in turn upregulates expression of adhesion molecules on endothelial cells and increases the adhesion of infected erythrocytes on endothelial cells

During cerebral malaria/severe malaria pathological condition arises due to

Sequestration of parasitized erythrocytes to brain endothelial cells

Too much inflammation due to augmented intracerebral pro-inflammatory cytokine response

Intravascular coagulation in brain

Dysregulation of vascular endothelial cells and loosening and leakiness of BBB

Fig. 14.5  During cerebral malaria/severe malaria pathological condition arises due to sequestration of parasitized erythrocytes and excessive inflammatory response and dysregulation of vascular endothelial cells

14.5 Polymorphisms in VCAM-1 Gene and P. falciparum Caused Malaria

165

In cerebral malaria/severe malaria pathological condition arises because of sequestration of parasitized erythrocytes to brain endothelial cells, high inflammation resulting from augmented intracerebral pro-inflammatory cytokine response and disseminated intravascular coagulation in brain leading to altered regulation of vascular endothelial cells (Nishanth and Schluter 2019) (Fig. 14.5). Pro-inflammatory cytokine such as TNFα is upregulated which in turn upregulates expression of adhesion molecules on endothelial cells (Sinha et al. 2008; Baruch et al. 2002). Increase in level of TNF cytokine has been observed during CM, even higher in fatal cases in African children (Grau et al. 1989; Kwiatkowski et al. 1990; Sahu et al. 2013), however, no correlation has been found between TNF level and CM (Esamai et al. 2003; Armah et al. 2007; Thuma et al. 2011).

14.5 Polymorphisms in VCAM-1 Gene and P. falciparum Caused Malaria Single nucleotide polymorphisms (SNPs) of VCAM-1 gene (rs3176860, rs2392221, rs3917010 and rs3176879 are connected to obesity and inflammation markers in Korean population (Yu et al. 2017). VCAM-1 gene SNPs, rs3783611, exon5 C/T; rs3783613, exon6 G/C; rs2392221, int3 C/T; rs3176860, int2 A/G (Table 14.1) have been reported. VCAM-1 gene polymorphisms, rs3917010 in myocardial infarction (Nasibullin et al. 2016) and rs1041163 in multiple sclerosis (Sanadgol et al. 2013) have been studied. In Indian populations, exonic SNPs rs3783611 and rs3783613 have been reported monomorphic, the intronic SNPs, rs2392221 and rs3176860 showed minor allele frequencies of 0.13 and 0.46, respectively (Sinha et al. 2008). Extremely low frequencies of these polymorphisms have been found in other Asian population. The exact function of adhesion molecules in mediating cytoadherence in vivo is not known very well due to minimal or no adhesion to these receptors in those Table 14.1  Demonstration of various VCAM-1 genetic polymorphisms in human and diseases Sr. no. 1. 2. 3. 4. 5. 6. 7. 8. 9.

Polymorphisms rs3783611, exon5 rs3783613, exon6 rs2392221, int3 rs3176860, int2 rs3170794, promoter rs1041163, promoter rs3176860, int2 rs2392221, int3 rs3176879

10. 11. 12.

rs3917010 rs3917010 rs1041163

Change C/T G/C C/T A/G T(−833)C T(−1592)C A/G C/T G>A Lys736Lys A>C A>C T>C

Disease Malaria Malaria Malaria Malaria Kidney transplantation Kidney transplantation obesity obesity obesity

References Sinha et al. (2008) Sinha et al. (2008) Sinha et al. (2008) Sinha et al. (2008) Kloda et al. (2013) Kloda et al. (2013) Yu et al. (2017) Yu et al. (2017) Yu et al. (2017)

obesity myocardial infarction Multiple Sclerosis

Yu et al. (2017) Nasibullin et al. (2016) Sanadgol et al. (2013)

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studies performed either in patient isolates or under flow conditions (Udomsangpetch et al. 1997; Cooke et al. 1998; Yipp et al. 2000). Binding of P. falciparum to VCAM-1 was demonstrated in vitro using field isolates (Ockenhouse et  al. 1992). Study on field isolates from Thailand has been shown that they tether and roll on VCAM-1, but static adhesion did not happen (Udomsangpetch et al. 1996). Whereas in African isolates, VCAM-1 adhesion was found very low and furthermore showed, no association with disease severity (Newbold et  al. 1997). Response of an individual human being to P. falciparum infection and following disease manifestation depend on polymorphisms in human host adhesion molecules (Fig.  14.6). Varying association of adhesion molecules with severe malaria has been found in different populations. ICAM-1-binding parasite with severe malaria has been demonstrated in different studies. Studies in Thailand, Kenya and Malawi were unsuccessful to link ICAM-1-binding parasites with severe malaria (Ockenhouse et al. 1991; Ho et al. 1991; Newbold et al. 1997; Rogerson et al. 1999) whereas Kenyan (Ochola et al. 2011) and Tanzanian (Turner et al. 2013) studies have identified a significant association of severe malaria with parasite adhesion. It has been identified that adhesion molecule such as PECAM-1 promoter/regulatory polymorphisms in gene may affect expression level of gene whereas coding region polymorphisms may affect structure and hence activity of protein (Fig. 14.6). PECAM-1 promoter SNP, rs1122800-C allele is significantly linked to lower expression of PECAM-1 (Ohashi et  al. 2016). A reduction in cytoadherence caused by lower expression of PECAM-1 on vascular endothelium may decrease risk for CM (Ohashi et al. 2016). Because of this reason people with different genotypes may have variability in outcome of severe disease. It may be hypothesized that VCAM-1 gene polymorphisms may affect expression level/activity of protein, and hence affect cytoadherence.

14.6 Other Human Host Receptors for Infected Erythrocytes The PfEMP-1 infected erythrocytes can bind with some other receptors on endothelial. PfEMP-1 interacts to endothelial protein C receptor (EPCR) (Fig.  14.7) and connected to severe malaria in Tanzania (Turner et al. 2013). Studies suggested that a 32 kDa human protein named as gC1qR/HABP1/p32 serves as a receptor for infected erythrocytes for their attachment to human brain microvascular endothelial cells. This receptor is present on platelets where it can be used for platelet-mediated clumping which is associated with severe malaria (Biswas et  al. 2007). The gC1qR/HABP1/p32 receptor connected to seizures in Mozambique (Mayor et al. 2011). Parasites isolated from children suffering from severe malaria in Kenya and Tanzania has been more likely to interact to multiple receptors than other parasites (Heddini et al. 2001).

14.6 Other Human Host Receptors for Infected Erythrocytes

167

Genetic polymorphisms in human VCAM-1 gene

VCAM-1 promoter polymorphisms

May affect gene expression

VCAM-1 coding polymorphisms

May affect structure of protein

Alteration in level or structure of protein may occur

Variability in response of an individual human being to P. falciparum infection and disease manifestation Fig. 14.6  It may be hypothesized that polymorphisms in adhesion molecules like VCAM-1 affects structure or level of protein thereby affects cytoadherence of infected erythrocytes and resulting sequestration. Hence, VCAM-1 polymorphisms may affect response of an individual human being to P. falciparum caused malaria infection

168

14  Vascular Cell Adhesion Molecule-1 (VCAM-1) Polymorphisms

P. falciparum infected erythrocytes

PfEMP-1

EPCR

Cell

membrane

Fig. 14.7  Cytoadhesion of infected erythrocyte to endothelium

14.6.1 Hemoglobin C and Hemoglobin S Hemoglobin variants C and S protect the individuals from malaria, they transform PfEMP-1 expression on infected erythrocytes and thereby parasite adhesion to endothelial cells (Mahamar et al. 2017). Because of this reason, protective effects of hemoglobinopathies have been found (Taylor et al. 2012).

14.7 C  ompounds Targeting Cytoadherence of Infected Red Blood Cells Cytoadherence and sequestration of infected erythrocytes can be prevented using drugs targeting cytoadherence of infected erythrocytes (Nishanth and Schluter 2019). Rapamycin prevents cytoadherence of infected erythrocytes through decreased ICAM-1 and VCAM-1 expression in experimental cerebral malaria. Furthermore, It has been identified that ethanolic extracts of Trichoderma stromaticum reduces inflammation and improve experimental CM by decreasing cerebral expression of ICAM-1 and VCAM-1, in that way preserving BBB integrity (Cariaco et al. 2018). Though the role of infected erythrocytes sequestration in the pathology of CM is well known but the role of infected erythrocytes sequestration in experimental CM is controversial (White et al. 2010). Since the malaria parasite is being developed resistance to the most of the drugs, therefore, new strategies are urgently needed. Exploration of specific agents targeting cytoadherence of infected erythrocytes will create new avenues for therapy of malaria and other immunological and infectious diseases. Investigation of the regulatory mechanisms of VCAM-1 expression would be a potential therapeutic target in various diseases. PfEMP-1-EPCR interaction can be used as target in malaria therapy.

References

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14.8 Conclusion VCAM-1 is an important cell adhesion molecule plays a role in regulation of inflammation-associated vascular adhesion and trafficking of leukocytes. It has been well evidenced that this molecule is associated with several inflammation-­ linked diseases, including malaria, rheumatoid arthritis, asthma and cancer. TNFα induces expression of intercellular cell adhesion molecule-1 (ICAM-1) and VCAM-1 on endothelial cells along with other adhesion molecules. P. falciparum infected erythrocytes adhere with host endothelial surfaces through adhesion molecules such as VCAM-1 and ICAM-1 leading to severe outcome of disease. It may be suggested that polymorphisms in VCAM-1 gene affect expression (level) or structure of VCAM-1 and hence influence response of an individual human being to P. falciparum infection, consequently disease manifestation. It may be hypothesized that promoter polymorphisms in VCAM-1 gene have shown association with increased expression of VCAM-1 and hence increases cytoadherence of infected erythrocytes. Further, studies are needed to confirm the exact role of VCAM-1 promoter polymorphisms in malaria pathogenesis and disease severity and outcome in endemic populations.

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