Principles and Practice of the Muscular Dystrophies (Current Clinical Neurology) [1st ed. 2023] 3031440080, 9783031440083

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Current Clinical Neurology Series Editor: Daniel Tarsy

Pushpa Narayanaswami Teerin Liewluck   Editors

Principles and Practice of the Muscular Dystrophies

Current Clinical Neurology Series Editor Daniel Tarsy, Beth Israel Deaconness Medical Center Department of Neurology Boston, MA, USA

Current Clinical Neurology offers a wide range of practical resources for clinical neurologists. Providing evidence-based titles covering the full range of neurologic disorders commonly presented in the clinical setting, the Current Clinical Neurology series covers such topics as multiple sclerosis, Parkinson's Disease and nonmotor dysfunction, seizures, Alzheimer's Disease, vascular dementia, sleep disorders, and many others.

Pushpa Narayanaswami  •  Teerin Liewluck Editors

Principles and Practice of the Muscular Dystrophies

Editors Pushpa Narayanaswami Neurology Beth Israel Deaconess Medical Center/ Harvard Medical School Boston, MA, USA

Teerin Liewluck Neurology Mayo Clinic Rochester, MN, USA

ISSN 1559-0585     ISSN 2524-4043 (electronic) Current Clinical Neurology ISBN 978-3-031-44008-3    ISBN 978-3-031-44009-0 (eBook) https://doi.org/10.1007/978-3-031-44009-0 © The Editor(s) (if applicable) and The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 This work is subject to copyright. All rights are solely and exclusively licensed 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 Humana imprint is published by the registered company Springer Nature Switzerland AG The registered company address is: Gewerbestrasse 11, 6330 Cham, Switzerland Paper in this product is recyclable.

In loving memory of Dr. V. Narayanaswami, Mrs. Janaki Narayanaswami, and Ms. Fumiko Hamada. We dedicate this book humbly and reverently to all who have suffered, are suffering, or will suffer from muscular dystrophy. We hope that the advances we describe in this book will result in cures for these disorders in the near future.

Foreword

The diagnostic evaluation and management of patients with muscular dystrophies can be quite daunting. I have always felt the most important and key first step is identifying the pattern of weakness on clinical examination. Increasingly, complementary testing such as imaging of muscle by magnetic resonance imaging and ultrasound have further advanced our understanding of patterns of muscle weakness and progression of muscle degeneration in these various dystrophies. The explosion of advances in genetics has led to the identification of many various types of muscular dystrophy that share similar clinical phenotypes and, likewise, the discovery of different clinical phenotypes associated with alterations in specific genes. With genetic testing becoming more commercially available, the use of invasive muscle biopsies has diminished. However, biopsies remain invaluable, particularly when, as in the majority of cases still at this time the diagnosis remains unclear even with extensive genetic testing. I would like to congratulate the editors, Drs. Teerin Liewluck and Pushpa Narayanaswami, on this amazing new textbook, Principles and Practice of the Muscular Dystrophies. All the major forms of muscular dystrophy are covered by the leading experts in the field and are quite up to date. The need for a multidisciplinary team approach to care is emphasized. There is an important chapter developed to rehabilitation, often neglected in neuromuscular textbooks, which discusses the use of orthotics and assistive technologies to reduce disability and improve quality of life. The complexity and specific drawbacks of different forms of genetic testing are carefully explained, the utility of imaging and muscle biopsies to enhance diagnosis, and the importance of genetic counselling are described. With these advances in genetics has come a better understanding of the pathogenic bases of these different diseases which are well covered. This in turn has also led to new forms of gene therapy, including antisense oligonucleotides (ASO) that induce exon skipping, oligonucleotides that knock down mRNA expression through RNA interference (RNAi), gene replacement utilizing viral vectors, and more in development. The book even has a chapter devoted to clinical trial design. The editors and all the authors should be commended on this excellent work, which will facilitate diagnosis and improve the care of patients with muscular dystrophy. It addresses the need for a comprehensive reference incorporating the advances in radiology, genetic testing, and genetic therapies. I believe that it will become an important resource and will be on the bookshelves of all clinicians who help manage these patients. Anthony A. Amato Neuromuscular Division Brigham and Women’s Hospital Boston, MA, USA Neurology, Harvard Medical School Boston, MA, USA

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Preface

“Technology has advanced more in the last thirty years than in the previous two thousand. The exponential increase in advancement will only continue,” said Niels Bohr, the Danish physicist (1885–1962). This sentiment has never been truer than in the understanding of muscular dystrophies. From an initial descriptive period in the nineteenth century, to subsequent attempts at nosography of the disease, we arrived in the genetic era in 1987 when the gene for Duchenne muscular dystrophy was cloned and its protein product, dystrophin, identified by Hoffman, Kunkel, and colleagues. The rest, as they say, is indeed history. The classification of muscular dystrophies is now based on their genetic identity, and ongoing identification of the underlying abnormality associated with each genetic defect has provided a deeper understanding of not only the mechanisms underpinning abnormal muscle structure and function in these disorders, but also of normal muscle structure and function. The Holy Grail is an effective cure, and the last two decades have seen the beginnings of this glorious achievement. In this book, we have attempted to provide a twenty-first century update on these disorders. The phenotypic approach to clinical diagnosis remains the basis of diagnosis, but the availability of next-generation sequencing techniques has revolutionized the diagnostic algorithm of the disease. Genetic testing has superseded muscle biopsy in the algorithm. Nevertheless, Duchenne’s histologic harpoon is not ready to be laid to rest. The muscle biopsy remains relevant to confirm the effect of a genetic variant on myopathological changes and protein expression in muscle and to identify the pathological findings associated with novel genes. The phenotypic and genotypic heterogeneity of many muscular dystrophies is becoming increasingly apparent. Knowledge of the spectrum of extramuscular manifestations, particularly cardiovascular, informs judicious screening to improve outcomes. Muscle imaging has come of age as a diagnostic tool and is being investigated as a biomarker. Biomarkers in blood and tissue are being identified. Advances in rehabilitation interventions improve the quality of life at all stages of the disease. Finally, DNA- and RNA-based therapies and gene replacements have arrived. There is much to do yet, and we hope that this book will serve as a basic reference in this molecular era of muscular dystrophies. This book would not have seen the light of day without Dr. Daniel Tarsy’s encouragement. The authors who have so graciously provided their scientific, clinical, and literary expertise to the completion of this book are an absolutely stellar congregation of scientists and clinicians, and we cannot thank them enough. We extend our appreciation to Swathiga Karthikeyan, Gregory Sutorius, and Springer Nature Publishing for keeping us on task and making this book a reality. Finally, our grateful thanks to our families—Padma, Tom, Alamelu, Shruti, and Varun and to Eriko, Saya, Sota, Nikorn, and Supawadee for their unending support without which this work would not have been possible. Boston, MA, USA Rochester, MN, USA 

Pushpa Narayanaswami Teerin Liewluck

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Contents

1 An  Introduction to the Muscular Dystrophies���������������������������������������������������������   1 Teerin Liewluck and Pushpa Narayanaswami 2 Dystrophinopathies�����������������������������������������������������������������������������������������������������  11 Partha S. Ghosh and Basil T. Darras 3 Myotonic Dystrophies�������������������������������������������������������������������������������������������������  37 Gabriella Silvestri and Anna Modoni 4 Facioscapulohumeral Muscular Dystrophy�������������������������������������������������������������  63 Johanna Hamel and Rabi Tawil 5 Autosomal  Dominant Limb-Girdle Muscular Dystrophies�������������������������������������  73 Stefan Nicolau and Teerin Liewluck 6 Autosomal  Recessive Limb-Girdle Muscular Dystrophies�������������������������������������  93 Jantima Tanboon and Ichizo Nishino 7 Oculopharyngeal Muscular Dystrophy��������������������������������������������������������������������� 123 Bernard Brais 8 Distal Muscular Dystrophies������������������������������������������������������������������������������������� 131 Bjarne Udd 9 GNE Myopathy����������������������������������������������������������������������������������������������������������� 147 Zohar Argov and Stella Mitrani-Rosenbaum 10 Emery-Dreifuss Muscular Dystrophies��������������������������������������������������������������������� 159 Yukiko K. Hayashi 11 Congenital Muscular Dystrophies����������������������������������������������������������������������������� 175 Hugh J. McMillan and Maryam Oskoui 12 Myopathies  with Myofibrillar Pathology����������������������������������������������������������������� 193 Pitcha Chompoopong and Margherita Milone 13 Oculopharyngodistal Myopathy ������������������������������������������������������������������������������� 213 Masashi Ogasawara and Ichizo Nishino 14 Genetic  Diagnosis and Counseling in Muscular Dystrophies��������������������������������� 221 Kaitlin Smith and Matthew Wicklund 15 Muscle  Imaging in Muscular Dystrophies ��������������������������������������������������������������� 233 Doris G. Leung 16 The  Role of the Muscle Biopsy in the Era of Genetic Diagnosis����������������������������� 255 Edoardo Malfatti 17 Systemic  Complications of Muscular Dystrophies��������������������������������������������������� 269 Charles Kassardjian and Teerin Liewluck xi

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18 Molecular  Genetic Therapies in the Muscular Dystrophies����������������������������������� 281 Stefan Nicolau and Kevin M. Flanigan 19 Physical  Therapy, Bracing and Surgical Treatment in Muscular Dystrophies ����������������������������������������������������������������������������������������������������������������� 303 Andrew Skalsky and Phoebe Scott-Wyard 20 Trial  Design and Outcome Measurement in Muscular Dystrophies ��������������������� 331 Pushpa Narayanaswami Index������������������������������������������������������������������������������������������������������������������������������������� 341

Contents

Contributors

Zohar Argov  Hadassah Medical Center, The Faculty of Medicine, The Hebrew University of Jerusalem, Jerusalem, Israel Bernard  Brais Departments of Neurology and Neurosurgery and Human Genetics, Rare Neurological Disease group, Faculty of Medicine, McGill University, Montreal Neurological Institute-Hospital, Montreal, QC, Canada Pitcha Chompoopong  Department of Neurology, Mayo Clinic, Rochester, MN, USA Basil T. Darras  Department of Neurology, Boston Children’s Hospital, Boston, USA Kevin  M.  Flanigan  Center for Gene Therapy, Nationwide Children’s Hospital, Columbus, OH, USA Departments of Pediatrics and Neurology, The Ohio State University, Columbus, OH, USA Partha S. Ghosh  Department of Neurology, Boston Children’s Hospital, Boston, USA Johanna  Hamel  Neurology, Pathology and Laboratory, Medicine, Neuromuscular Disease Unit, University of Rochester Medical Center, Rochester, NY, USA Yukiko  K.  Hayashi Department of Pathophysiology, Tokyo Medical University, Tokyo, Japan Charles Kassardjian  St. Michael’s Hospital, University of Toronto and Institute for Health Policy, Management and Evaluation, Li Ka Shing Knowledge Institute, Toronto, ON, Canada Doris G. Leung  Kennedy Krieger Institute, Johns Hopkins University School of Medicine, Baltimore, MD, USA Teerin Liewluck  Division of Neuromuscular Medicine and Muscle Laboratory, Department of Neurology, Mayo Clinic, Rochester, MN, USA Edoardo Malfatti  APHP, Centre de Référence de Pathologie Neuromusculaire Nord-Est-Ilede-France, Henri Mondor Hospital, Créteil, France Univ Paris Est Créteil, INSERM, IMRB, Créteil, France Hugh  J.  McMillan Department of Pediatrics, Children’s Hospital of Eastern Ontario, University of Ottawa, Ottawa, ON, Canada Department of Pediatrics and Department of Neurology and Neurosurgery, McGill University, Montreal, QC, Canada Division of Pediatric Neurology, Montreal Children’s Hospital, McGill University Health Centre, Montreal, QC, Canada Margherita Milone  Department of Neurology, Mayo Clinic, Rochester, MN, USA Stella Mitrani-Rosenbaum  Hadassah Medical Center, The Faculty of Medicine, The Hebrew University of Jerusalem, Jerusalem, Israel

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Anna  Modoni Neurology Unit, Fondazione Policlinico Universitario A Gemelli, IRCCS, Rome, Italy Pushpa Narayanaswami  Department of Neurology, Beth Israel Deaconess Medical Center/ Harvard Medical School, Boston, MA, USA Stefan Nicolau  Center for Gene Therapy, Nationwide Children’s Hospital, Columbus, OH, USA Ichizo Nishino  Department of Neuromuscular Research, National Institute of Neuroscience, National Center of Neurology and Psychiatry (NCNP), Tokyo, Japan Department of Genome Medicine Development, Medical Genome Center, National Center of Neurology and Psychiatry (NCNP), Tokyo, Japan Ichizo Nishino  Department of Neuromuscular Research, National Institute of Neuroscience, National Center of Neurology and Psychiatry (NCNP), Tokyo, Japan Masashi  Ogasawara Department of Neuromuscular Research, National Institute of Neuroscience, National Center of Neurology and Psychiatry (NCNP), Tokyo, Japan Department of Pediatrics, Showa General Hospital, Tokyo, Japan Maryam Oskoui  Department of Pediatrics and Department of Neurology and Neurosurgery, McGill University, Montreal, QC, Canada Division of Pediatric Neurology, Montreal Children’s Hospital, McGill University Health Centre, Montreal, QC, Canada Phoebe  Scott-Wyard Division of Pediatric Rehabilitation Medicine, Rady Children’s Hospital San Diego, San Diego, CA, USA Department of Orthopedics, University of California San Diego, La Jolla, CA, USA Gabriella Silvestri  Department of Neuroscience, School of Medicine and Surgery, Università Cattolica del Sacro Cuore, Rome, Italy Neurology Unit, Fondazione Policlinico Universitario A Gemelli, IRCCS, Rome, Italy Andrew  Skalsky  Division of Pediatric Rehabilitation Medicine, Rady Children’s Hospital San Diego, San Diego, CA, USA Department of Orthopedics, University of California San Diego, La Jolla, CA, USA Kaitlin Smith  University of Colorado, Aurora, CO, USA Jantima  Tanboon Department of Neuromuscular Research, National Institute of Neuroscience, National Center of Neurology and Psychiatry (NCNP), Tokyo, Japan Department of Genome Medicine Development, Medical Genome Center, National Center of Neurology and Psychiatry (NCNP), Tokyo, Japan Department of Pathology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand Rabi Tawil  Neurology, Pathology and Laboratory, Medicine, Neuromuscular Disease Unit, University of Rochester Medical Center, Rochester, NY, USA Bjarne Udd  Folkhalsan Research Center, Helsinki, Finland Matthew Wicklund  University of Colorado, Aurora, CO, USA Department of Neurology, UT Health San Antonio, San Antonio, TX, USA

Contributors

1

An Introduction to the Muscular Dystrophies Teerin Liewluck and Pushpa Narayanaswami

Introduction The term muscular dystrophy is derived from the Latin, musculus (muscle), and the Greek, dys (bad, ill or difficult) and troph (nourishment). Muscular dystrophies encompass a large, clinically and genetically heterogenous group of primary progressive diseases of skeletal muscle leading to muscle wasting and weakness, with variable age of onset, ranging from in utero to late adulthood. Necrotic and regenerating myofibers and an increase in endomysial and perimysial fibrous and fatty connective tissue are the pathological hallmarks of muscular dystrophies, and these features are often referred to as “dystrophic changes” (Fig. 1.1) [1]. These disorders are often associated with extramuscular manifestations, most commonly cardiac and respiratory, but also with ophthalmological, dermatological, cognitive, and other manifestations. These extramuscular features can narrow the differential diagnosis of the type of dystrophy, and influence management. This chapter provides a broad overview of muscular dystrophies and their classification, pathogenesis, diagnosis, and management. Specific muscular dystrophies are discussed in their dedicated chapters. In the pre-genetic era, the classification of muscular dystrophies was based on the pattern of weakness, age of onset, and mode of inheritance, if known. (Table  1.1). The first description of muscular dystrophies perhaps goes as far back as 1830, when Sir Charles Bell, famous for his description of facial paralysis, may have described a case of muscular dystrophy [2]. However, it was not until 1852, when Edward Meryon, an English neurologist,  provided the first detailed T. Liewluck (*) Division of Neuromuscular Medicine and Muscle Laboratory, Department of Neurology, Mayo Clinic, Rochester, MN, USA e-mail: [email protected] P. Narayanaswami (*) Department of Neurology, Beth Israel Deaconess Medical Center/ Harvard Medical School, Boston, MA, USA e-mail: [email protected]

clinicopathological description of a disorder of progressive muscle weakness affecting young boys. The first eponym associated with a muscular dystrophy, Duchenne muscular dystrophy (DMD), was that of Guillaume-Benjamin-Amand Duchenne, a French neurologist, who, in 1868, described all the cardinal clinical features of the disease, except for the hereditary component, calling it “progressive muscular atrophy with degeneration” [3]. By the late nineteenth century, another clinically distinct muscular dystrophy, currently known as facioscapulohumeral muscular dystrophy (FSHD), was recognized [4]. Patients with myotonic dystrophy and oculopharyngeal muscular dystrophy (OPMD) were first reported in 1909 and 1915, respectively [5, 6]. The term limbgirdle muscular dystrophy (LGMD) was coined in 1953 to describe a distinct type of autosomally inherited, proximal muscular dystrophy, which was clinically distinguishable from the hitherto recognized muscular dystrophies of that time [7]. In 1955, Dr. Peter Emil Becker, a German neurologist, described a new X-linked muscular dystrophy with later age of onset and milder phenotype compared to DMD, which was subsequently named Becker muscular dystrophy (BMD) [8]. Approximately a decade later, the first cases of EmeryDreifuss muscular dystrophy (EDMD) were reported [9]. In 1977, Satoyoshi and Kinoshita described an autosomal dominant myopathy with preferential involvement of ocular, facial, bulbar and distal limb muscles, which is now known as oculopharyngodistal myopathy (OPDM) [10]. Owing to advances in molecular genetics, we now know that there are 2 genetically distinct subtypes of myotonic dystrophies [type 1 (DM1) and type 2 (DM2)] and FSHD [type 1 (FSHD1) and type 2 (FSHD2)]. LGMD and EDMD are not merely single entities, but, in fact, there are at least 30 genetically distinct subtypes of LGMD, and 6 genetic subtypes of EDMD. These further highlight the genetic heterogeneity of muscular dystrophies. Moreover, there is evidence that a mutation in a single gene can give rise to more than one clinical or histopathological phenotype (phenotypic heterogeneity), expanding the disease spectrum of individual

© The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 P. Narayanaswami, T. Liewluck (eds.), Principles and Practice of the Muscular Dystrophies, Current Clinical Neurology, https://doi.org/10.1007/978-3-031-44009-0_1

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T. Liewluck and P. Narayanaswami

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a

b

c

d

e

f

Fig. 1.1  Histopathology of muscular dystrophy. (a, hematoxylin and eosin) Muscle biopsy of a patient with early stage Duchenne muscular dystrophy shows a marked variation in fiber size, a mild increase of fibers harboring internal nuclei (asterisk), scattered fiber splitting (arrow), occasional necrotic fibers (arrowhead) and increased endomysial connective tissue. Dystrophin C-terminal immunoreactivity is absent (b) compared to control (c). (d, hematoxylin and eosin) Muscle

biopsy of a patient with advanced stage limb-girdle muscular dystrophy (LGMD) type R9 (LGMD-R9) displays marked increase of perimysial and endomysial fibrous and fatty connective tissue, consistent with near-endstage muscle. (e, Congo red) Muscle biopsy of an LGMD type R2 (LGMD-R2) patient reveals congophilic deposit in the blood vessel (arrowhead). (f, hematoxylin and eosin) There is a small perivascular collection of mononuclear cells in the perimysium (arrow)

Table 1.1  Classification of muscular dystrophies Diseases Congenital muscular dystrophies Duchenne muscular dystrophy Becker muscular dystrophy

Gene(s) or underlying genetic defects Several genes

XR

DMD

XR

DMD

Late childhood-­ adulthood

CTG expansion in 3’ UTR of DMPK

In Distal predominant and facial utero-adulthood weakness Adulthood Proximal predominant with or without mild facial weakness

Myotonic dystrophies (DM) DM1 AD DM2

AD

CCTG expansion in intron 1 of CNBP

Facioscapulohumeral muscular dystrophies (FSHD) FSHD1 AD Hypomethylation of contracted D4Z4 repeats on chromosome 4q35 and 4qA haplotype FSHD2 Digenic Hypomethylation of normal sized D4Z4 repeats and 4qA haplotype secondary to SMCHD1, DNMT3B, or LRIF1 mutations Limb-girdle muscular dystrophies (LGMD) LGMD-D AD Several genes LGMD-R

Typical age at onset Typical pattern of weakness In Proximal predominant utero-infantile Early childhood Proximal predominant

Inheritance AR or AD

AR

Emery-Dreifuss muscular XR, AD or dystrophies AR

Several genes Several genes

Proximal predominant

Infantile to adulthood Infantile to adulthood

Facial and scapuloperoneal weakness Facial and scapuloperoneal weakness

Childhood-­ adulthood Childhood-­ adulthood Childhood-­ adulthood

Proximal predominant Proximal predominant Scapuloperoneal weakness

1  An Introduction to the Muscular Dystrophies

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Table 1.1 (continued) Diseases Oculopharyngeal muscular dystrophy

Inheritance AD > AR

Oculopharyngodistal myopathy Myofibrillar myopathies (MFM) Distal myopathiesa

AD AD, AR or XR AD, AR or XR

Gene(s) or underlying genetic defects GCN repeat expansion in exon 1 > point mutations in PABPN1; N represents any A, T, C or G nucleotide CGG expansion in 5’UTR of LRP12, GIPC1, NOTCH2NLC and RILPL1 Several genes Several genes

Typical age at onset Adulthood

Adulthood Childhood-­ adulthood Childhood-­ adulthood

Typical pattern of weakness Ocular, pharyngeal and proximal weakness Ocular, pharyngeal, facial and distal weakness Various patterns of weakness Distal predominant weakness

AD autosomal dominant, AR autosomal recessive, UTR untranslated region, XR X-linked recessive Not all subtypes of distal myopathies are considered muscular dystrophies

a

Fig. 1.2 Venn diagram displays overlapping clinical phenotypes between muscular dystrophy subtypes. In red font are the genes wherein mutations can cause a limb-girdle muscular dystrophy (LGMD)-like phenotype but are not classified as LGMD. It is important to note that

Becker muscular dystrophy (BMD) can mimic LGMD.  BMD is not classified as LGMD because of its X-linked inheritance. A few congenital myasthenic syndrome (CMS) genes are also shown

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gene defects and blurring the boundaries of each muscular dystrophy subtype (Fig. 1.2). For example, mutations in the lamin A/C-encoding gene (LMNA) can cause EDMD, LGMD and congenital muscular dystrophy (CMD) [11], and defects in the dysferlin (DYSF)- and anoctamin 5 (ANO5)-encoding genes give rise to both autosomal recessive LGMD (LGMD-R2 and R12, respectively) and to the Miyoshi distal myopathy phenotype [12]. In fact, members of the same family bearing a mutation in these genes may present with either a LGMD or Miyoshi phenotype; these phenotypes tend to merge over time with disease progression. Mutations in the genes coding for merosin (LAMA2) and collagen VI (COL6A1, COL6A2 and COL6A3) were first described in CMD patients and later in LGMD patients [1]. Some inherited diseases of skeletal muscle [e.g. myofibrillar myopathies (MFM) and certain subtypes of distal myopathies] are mislabeled as myopathies despite their progressive nature and the histopathological feature of a dystrophy [13, 14].

T. Liewluck and P. Narayanaswami

some muscular dystrophies such as OPMD or OPDM, the pathomechanisms of the underlying genetic defect remains largely unknown.

Diagnosis of Muscular Dystrophies Patients suspected to have muscular dystrophies require a comprehensive evaluation, combining clinical, serological, and electrophysiological studies to select an appropriate genetic test in order to achieve a definitive diagnosis. The place of the muscle biopsy in this algorithm has evolved in the era of next generation sequencing (NGS), and tends to be later in the diagnostic pathway, often after NGS testing.

Clinical Approach

An insidious onset of slowly progressive muscle weakness is characteristic of muscular dystrophies. History should focus Pathogenesis on the age of onset and family history of similar illnesses. The age of onset may be difficult to identify in these insidiDefects in several components of muscle fibers, ranging ous disorders. Information regarding the pregnancy, quickfrom the extracellular matrix, basement membrane, sarco- ening, labor and delivery and neonatal abnormalities such as lemma, sarcomere, sarcoplasmic proteins, nuclear envelope congenital hip dislocation should be obtained. Motor and and nuclear matrix can cause muscular dystrophies. The sar- mental milestones, childhood history of participation in colemma is subject to constant shear-stress due to contractile sports and a history of learning disabilities should be evaluforces transmitted to it from the sarcomere. The resultant ated. A family history of a muscular disorder may not be damage is repaired by proteins such as dysferlin and anocta- apparent, and indirect evidence of family members being min-­5. Mutations in genes encoding sarcolemmal proteins unable to ambulate, requiring assistive devices or being typically lead to destabilization of the sarcolemma and sub- wheelchair-bound should be sought. A family history of sequent myofiber degeneration [15]. However, defects in extramuscular manifestations is also important. A careful some sarcolemmal proteins (dysferlin and anoctamin-5) pri- pedigree chart of the family should be constructed when a marily interfere with the repair machinery rather than the family history is present, to evaluate the probable mode of integrity of the plasma membrane [16]. inheritance. Absence of family history does not preclude the Generally, mutations in the extracellular matrix and base- diagnosis of muscular dystrophy. ment membrane proteins (e.g., merosin and collagen VI) A detailed neuromuscular examination to identify specause CMD, while mutations in sarcolemmal proteins cause cific patterns of muscle weakness is an integral part of the DMD, BMD and LGMD. Defects in nuclear envelope pro- evaluation of these patients. Weakness in the muscular dysteins (nuclear envelopathies) typically give rise to EDMD trophies, like in most muscle diseases, is generally symmet[12, 17]. Defects in Z-disc-related proteins or chaperone-­ ric, but asymmetric weakness can be seen in some muscular assisted selective autophagy (CASA) underlie MFM [13, dystrophies, e.g., FSHD and LGMD-R12 [4, 22]. Muscle 18]. However, the emerging phenotypic heterogeneity of pseudohypertrophy or atrophy may accompany the weakmutant genes resists this over-simplification. For example, ness. Scapular winging can be seen in FSHD and in other mutations in collagen VI- and merosin-encoding genes can muscular dystrophies such as EDMD and LGMD-R1. also give rise to the LGMD phenotype [1]. Both CMD and Clinical myotonia (action or percussion-induced) is a key LGMD phenotypes can also occur with mutations of several feature of both DM1 and DM2; these patients may report proteins involved in the glycosylation of alpha-dystroglycan, muscle stiffness or impaired relaxation of muscles in addia heavily glycosylated sarcolemmal protein connecting the tion to weakness. Clinical myotonia tends to be less promisarcolemma to the basement membrane [19]. nent in DM2 than in DM1[23]. Contractures are common in In DM1, DM2 and FSHD, the primary genetic defects advanced stages of all muscular dystrophies when mobility cause aberrant expression of toxic proteins or RNA, leading is severely impaired; however, contractures occur in the early to myofiber degeneration and weakness [20, 21]. Finally, in stages of certain muscular dystrophies (e.g., EDMD and col-

1  An Introduction to the Muscular Dystrophies

lagen VI-related muscular dystrophies) when weakness is not prominent and is a diagnostic feature of these disorders. These contractures often involve the elbow flexors and the Achilles tendon. In addition to weakness, muscular dystrophy patients may develop myalgia and/or recurrent rhabdomyolysis. Myalgia may be persistent or episodic, precipitated by exercise or other factors such as infection. A history of episodes of myalgia associated with dark colored urine suggests rhabdomyolysis. Some DM2 patients may present with profound myalgia without significant muscle weakness [20]. Myalgias and recurrent rhabdomyolysis are classically considered indicative of metabolic myopathies, but may also be an initial presentation in certain subtypes of muscular dystrophies, e.g. dystrophinopathies and some subtypes of LGMD-R (e.g. R1, R2, R9 and R12) [24]. In these muscular dystrophies with recurrent rhabdomyolysis, the “pseudo-metabolic” phenotype, muscle weakness may not be evident between episodes of rhabdomyolysis, but serum creatine kinase (CK) levels generally remain elevated between episodes. Although muscular dystrophies are primary diseases of skeletal muscle, extramuscular manifestations can occur in several muscular dystrophies. The most common are cardiac and respiratory involvement, which may vary from mild to severe and in some disorders, contribute significantly to quality of life and mortality. Other extramuscular systems involved include the central nervous system, eyes and skin. History should probe into extramuscular symptoms (dyspnea, chest pain, palpitations, developmental disabilities, cataracts, skeletal abnormalities, etc.). While there is no cure for muscular dystrophies, early recognition and prompt treatment of underlying cardiac and respiratory complications improves quality of life and prolongs life-expectancy. A history of early onset cataracts in the family raises the possibility of myotonic dystrophy. A positive family history of Paget disease of bone or frontotemporal dementia suggests multisystem proteinopathies. Extramuscular phenotypes of each muscular dystrophy are discussed with the individual disorders and are summarized in Chap. 17.

Laboratory Evaluation Elevated serum CK levels are a well-known feature of primary disorders of muscle, including muscular dystrophies. However, there is no consensus regarding the degree of elevation. CK levels can be normal or mildly to markedly elevated, depending on the subtype of muscular dystrophy, and generally correlate with a number of necrotic fibers. Muscular dystrophies due to sarcolemmal defects (e.g. DMD, BMD and LGMD) typically have greater numbers of necrotic fibers and higher CK levels compared to muscular dystrophies due to defects of nuclear envelope (EDMD), myotonic

5

dystrophies, FSHD, OPMD or collagen VI-related muscular dystrophies [15, 25]. There is wide overlap in the range of serum CK levels and they are usually not diagnostic of a specific subtype of muscular dystrophy. As the disease progresses, serum CK levels often fall and can be lower than normal, reflecting loss of muscle fibers and fibrofatty replacement (“end-stage muscle”). Some patients with muscular dystrophies (Calpainopathies [CAPN3], ANO5, Sarcoglycanopathies, and others) may present with asymptomatic/ pre-symptomatic or paucisymptomatic hyperCKemia [26, 27]. HyperCKemia can also occur in non-dystrophic myopathies, neuromuscular junction disorders and neurogenic disorders [e.g. spinal muscular atrophy (SMA), spinobulbar muscular atrophy (SBMA), and amyotrophic lateral sclerosis (ALS)] [28]. In motor neuron diseases, serum CK levels can be markedly elevated, similar to that observed in patients with muscular dystrophies featuring sarcolemmal defects [28]. Elevation of serum aspartase transaminase (AST) and alanine transaminase (ALT) is considered a diagnostic hallmark of liver disease. However, both enzymes are also expressed in skeletal muscle. Therefore, muscular dystrophy patients can have elevation of serum AST and ALT (“transaminitis” or “hypertransaminasemia”) without underlying liver disease. It is not uncommon for patients to be detected to have transaminitis on routine laboratory testing, and then undergo extensive evaluation for underlying liver disease before being referred to a neurologist for consideration of a neuromuscular etiology. Gamma glutamyl transferase (GGT) is more specific to hepatocytes compared to AST and ALT.  Hypertransaminasemia with normal GGT should prompt clinicians to measure serum CK levels [29].

Electrodiagnostic Evaluation Nerve conduction studies are generally normal in muscular dystrophies, except for those disorders with a concomitant peripheral neuropathy (Chap. 17) or in the presence of severe distal weakness (Chap. 8). Coexistent disorders such as diabetes mellitus may cause an underling neuropathy. Low-­ frequency repetitive stimulation of motor nerves may elicit a decremental response in some muscular dystrophies that are associated with a defect of neuromuscular transmission, e.g. CMD or LGMD due to mutations in genes encoding GDP-­ mannose pyrophosphorylase B (GMPPB) and plectin (PLEC) (Chap. 17) [30]. Needle electromyography (EMG) generally shows an “irritable myopathy”, characterized by increased insertional activity, fibrillation potentials or positive sharp waves, and short-duration, low-amplitude and complex motor unit potentials with early recruitment. The density of fibrillation potentials and positive sharp waves

6

correlates with the extent of necrotic fibers and fiber splitting in individual patients [31]. These abnormalities are usually seen in early disease when there is active muscle fiber necrosis. Decreased insertional activity and a mixed population of short-duration, low-amplitude and long-duration, large-­ amplitude motor unit potentials indicate chronicity and can be seen in advanced disease [32]. Myotonic discharges (electrical myotonia) are characteristic of DM1 and DM2, but these can also occur in non-­ dystrophic myotonias, some muscular dystrophies (e.g. LGMD-R12 and caveolin-3-associated muscular dystrophies) and other myopathies, especially acid-alpha glucosidase deficiency (Pompe disease), immune mediated necrotizing myopathy (IMNM) and MFM [32]. The presence of associated clinical myotonia should point to myotonic dystrophies, although clinical myotonia can be minimal or absent in DM2. Myotonic discharges in DM1 have a typical waxing and waning characteristic, while in DM2 they may appear as waning discharges or could be very subtle and hard to appreciate on the needle EMG [23, 33]. Rippling muscle diseases (RMD) refer to a group of muscle hyperexcitability disorders, clinically characterized by ripples that travel across the muscle and are typically electrically silent. RMD can be hereditary or immune-mediated [34]. Hereditary RMD is associated with mutations in genes coding for caveolin-3 and cavin-1.  Antibodies to cavin-4 have been identified in patients with immune-mediated RMD [12, 34].

Genetic Diagnosis In patients with a classical phenotype of repeat expansion disorders (DM1, DM2, and OPMD) and repeat contraction diseases (FSHD1 and FSHD2), genetic tests specific to these diseases should be performed as part of the initial evaluation; muscle biopsy is not necessary if the genetic test confirms the diagnosis [35]. OPDM is a repeat expansion disorder (Chap. 13), but a genetic test is not commercially available at this time. Muscle biopsy could serve as a diagnostic test for OPDM, although the findings are not entirely specific [36]. For other muscular dystrophies, previous diagnostic algorithms included clinical evaluation to identify distinguishing features such as ethnicity, clinical features, extramuscular manifestations etc. that may provide clues to narrow the differential diagnosis, followed by muscle biopsy, to identify histopathological and/or proteomic clues [37]. In the absence of specific distinguishing features, muscle biopsy would follow clinical evaluation; targeted genetic testing, often one candidate gene at a time, or small panels of genes, would be performed based on histopathologic features e.g., rimmed vacuoles, myofibrillar pathology, etc. [37]. The advent of NGS has revolutionized the diagnostic approach to inherited

T. Liewluck and P. Narayanaswami

myopathies as it allows analysis of several genes simultaneously in a much shorter time and lower cost compared to Sanger sequencing. Therefore, NGS has become the diagnostic test of choice and bypasses the muscle biopsy in diagnosis of hereditary muscle diseases [35]. Chap. 14 outlines the genetic diagnosis of each type of muscular dystrophies and discusses pre-and post-test genetic counseling. When a molecular diagnosis cannot be confirmed by NGS, muscle biopsy is the next step. Histopathological findings guide further evaluation or assist in interpretation of variants of uncertain significance (VUS) (see Section “Myopathological Diagnosis”) [35]. VUS refers to a variation in a genetic sequence for which the association with disease is uncertain because  although the variant has not yet been reported to be associated with a disease, it is also not reported in normal genetic libraries. Muscle imaging [Computerized Tomography (CT scan), Magnetic Resonance Imaging (MRI) and muscle ultrasound] (Chap. 15) is increasingly used in research and more recently, in clinical practice. Imaging provides specific patterns of muscle involvement in some hereditary myopathies, such as the Collagen VI disorders, which can be of diagnostic value. Additionally, imaging modalities provide both qualitative and quantitative estimates of adipose tissue deposition, which can be used as a biomarker of disease progression [38]. With emerging disease specific radiological patterns of muscle involvement, muscle imaging could be also useful in validating the pathogenicity of VUS [38]. If a diagnosis remains in doubt, further genetic tests, e.g. whole exome sequencing (WES), whole genome sequencing (WGS) or RNA sequencing, may provide the answer [35].

Myopathological Diagnosis Muscular dystrophies result in a fairly uniform histopathological appearance, as described above, known collectively as dystrophic changes or dystrophic features. These non-­specific dystrophic changes do not offer diagnostic clues to the underlying genetic defect or type of dystrophy. The severity of dystrophic findings varies with disease stage and type of dystrophy. Inflammatory infiltrates can occur in muscular dystrophies. In some types of muscular dystrophies (e.g., FSHD, LGMD-R1, LGMD-R2 and LMNA-CMD), the inflammatory reaction can be as prominent as that seen in inflammatory myopathies, and some patients may be misdiagnosed as having refractory myositis. Sarcolemmal expression of major histocompatibility complex-1 (MHC-1) is considered a pathological hallmark of inflammatory myopathies, but it has also been reported in the aforementioned muscular dystrophies featuring inflammatory infiltrates [39–42]. A new role of muscle biopsy in the genomic era is to validate the pathogenicity of VUS identified by NGS.  This is

1  An Introduction to the Muscular Dystrophies

done by demonstrating the presence or absence of the expected functional consequences of the variant. For example, interstitial congophilic deposits without systemic amyloidosis have been reported in LGMD-R2 and LGMD-R12 [43]. Therefore, its presence could support the pathogenicity of a VUS in DYSF or ANO5. Pleomorphic hyaline materials observed on modified Gomori trichrome stained section and abnormal accumulation of Z-disc related proteins are the pathological hallmarks of MFM [13]. Its presence suggests that VUS in MFM-related genes could be pathogenic. Immunohistochemical or western blot studies of targeted proteins could also aid in validating the pathogenicity of VUS. For example, in patients with a VUS in fukutin-related protein-encoding gene (FKRP), abnormal alpha-­dystroglycan immunoreactivity supports a diagnosis of LGMD-R9 due to FKRP mutations [19]. Chap. 16 elaborates the role of muscle biopsy in the genomic era.

7

Management of Muscular Dystrophies

Currently, the management of muscular dystrophies remains symptomatic and supportive. It requires a multidisciplinary team, consisting of neurologists, physiatrists, physical therapists, occupational therapists, speech therapists, respiratory therapists, nutritionists, geneticists or genetic counselors, cardiologists, pulmonologists, orthopedists, psychologists, and perhaps psychiatrists. Patients should be followed closely by a physical medicine and rehabilitation team (Chap. 19) because disabilities emerge and evolve as diseases progress. Early detection and prompt treatment of extramuscular manifestations (Chap. 17), especially cardiorespiratory complications, can improve quality of life and prolong life expectancy. Although there is as yet no cure for muscular dystrophies, there are disease modifying therapies available for DMD, the most common type of muscular dystrophy. Glucocorticoids (prednisone, prednisolone, deflazacort and recently vamorolone) have been the cornerstone of pharmacotherapy for Differential Diagnosis of Muscular DMD for several years. In 2016, the United States Food and Dystrophies Drug Administration (FDA) approved the first drug, Eteplirsen, in a new class of genetic therapies, antisense oliPhenotypic overlap between mutations in different genes is gonucleotides (ASOs), for a subset of DMD patients. (Chap. increasingly observed as more patients undergo genetic test- 18) [51]. Three more exon-­skipping ASOs have been since ing. It is important to consider other hereditary myopathies approved. These agents restore the reading frame of dystroand congenital myasthenic syndromes (CMS) in the differ- phin gene (DMD), converting the out of frame mutation to an ential diagnosis. In cohorts of genetically uncharacterized in-frame one, and allowing the expression of a truncated dysLGMD, comprehensive genetic studies have identified trophin protein. This essentially converts the severe DMD pathogenic mutations in genes underlying hereditary non-­ phenotype to a milder phenotype, resembling BMD [51]. dystrophic myopathies or CMS in a proportion of patients Very recently, the US FDA approved a recombinant gene [44–46]. Therefore, a broader NGS panel, including not only therapy for DMD boys aged 4-5 years. This is designed to muscular dystrophy-related genes, but also non-dystrophic deliver a gene encoding micro-dystrophin, which is a trunhereditary myopathy and CMS-related genes, has a higher cated form of thedystrophin gene containing selected yield of achieving molecular diagnosis compared to an NGS domains of the functional dystrophin protein. Other genetic panel limited to genes coding for the muscular dystrophies therapies or pharmacotherapies have provided promising [35]. In patients without definite genetic diagnosis after results in pre-clinical models of muscular dystrophies, but undergoing a comprehensive genetic testing, one should con- they failed to provide the same impact in human trials [52, sider the possibility of IMNM. Patients withIMNM typically 53]. A lack of knowledge of the underlying pathomechapresent with subacute and rapidly progressive proximal nisms, the natural history of each disorder or the appropriate weakness associated with marked elevation of CK levels; outcome measures may, at least in part, be responsible for however, a rare chronic and slowly progressive form of these failures. Clinical trial design and outcome measurement IMNM, mimicking muscular dystrophies, has been recently are discussed in Chap. 20. recognized [47–49]. Atypical pathological findings e.g., myofibrillar pathology or mitochondrial abnormalities, have also been reported in IMNM [49]. Distal predominant weak- Future Prospects ness, resembling hereditary distal myopathies, can be a rare manifestation of IMNM [50]. Serologic testing for IMNM-­ The treatment of muscular dystrophies continues to be an associated antibodies [3-hydroxy-3-methylglutaryl CoA area of active research. DMD remains the major focus of this reductase (HMGCR) and signal recognition particle (SRP)] research. Improved exon-skipping strategies, microdystroshould be considered in these genetically uncharacterized phin gene replacement strategies and Cas-9/ CRISPR are muscular dystrophy or distal myopathy patients. The impor- some approaches being tested. Adeno- Associated Virus tance of recognizing IMNM cannot be overemphasized, (AAV) based vector gene therapies are being investigated for because it is a treatable disorder. some of the recessive LGMDs. Methods to block the forma-

8

tion of, or to effect degradation of, toxic mRNAs in FSHD and DM1 are being tested in pre-clinical studies. One challenge in the treatment of these disorders is the delivery of the drug effectively to target tissues. Nanomedicine is a fast-­ advancing field that develops and studies compounds that are between 1–100 nm (nanoparticles) to optimize drug delivery to target tissues. Nanoparticles have been used to deliver the Cas-9/CRISPR complex in mdx-mice which are deficient in dystrophin [54]. As potential therapeutic agents for these slowly progressive disorders are tested in clinical trials, the need for valid, reliable clinical outcome measures that are responsive to small changes becomes paramount. Refinement of surrogate outcomes such as imaging and identification of other biomarkers is critical. The rarity of these disorders will necessitate large multicenter collaborations, and registries will provide valuable observational information.

Conclusion Since the first description of muscular dystrophies nearly 2 centuries ago, the advances of molecular genetics have clarified the heterogeneity of the muscular dystrophies, aided in their classification and transformed the diagnostic algorithm. The role of diagnostic muscle pathology has evolved and now plays a key role in demonstrating the functional consequences of VUS disclosed by NGS. Multidisciplinary care is a crucial part of patient management as the diseases are incurable at the present time. The advent of genetic therapies for a subset of DMD patients has spurred research into development of disease modifying therapies for other muscular dystrophies.

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T. Liewluck and P. Narayanaswami 8. Zeidman LA, Kondziella D.  Peter Becker and his Nazi past: the man behind Becker muscular dystrophy and Becker myotonia. J Child Neurol. 2014;29(4):514–9. 9. Heller SA, Shih R, Kalra R, Kang PB. Emery-Dreifuss muscular dystrophy. Muscle Nerve. 2020;61(4):436–48. 10. Satoyoshi E, Kinoshita M.  Oculopharyngodistal myopathy. Arch Neurol. 1977;34(2):89–92. 11. Maggi L, Carboni N, Bernasconi P. Skeletal muscle Laminopathies: a review of clinical and molecular features. Cell. 2016;5(3) 12. Liewluck T, Milone M. Untangling the complexity of limb-girdle muscular dystrophies. Muscle Nerve. 2018;58(2):167–77. 13. Selcen D.  Myofibrillar myopathies. Neuromuscul Disord. 2011;21(3):161–71. 14. Milone M, Liewluck T. The unfolding spectrum of inherited distal myopathies. Muscle Nerve. 2019;59(3):283–94. 15. Ozawa E, Nishino I, Nonaka I. Sarcolemmopathy: muscular dystrophies with cell membrane defects. Brain Pathol. 2001;11(2):218–30. 16. Croissant C, Carmeille R, Brevart C, Bouter A. Annexins and membrane repair dysfunctions in muscular dystrophies. Int J Mol Sci. 2021;22(10) 17. Somech R, Shaklai S, Amariglio N, Rechavi G, Simon AJ. Nuclear envelopathies--raising the nuclear veil. Pediatr Res. 2005;57(5 Pt 2):8R–15R. 18. Kley RA, Olive M, Schroder R. New aspects of myofibrillar myopathies. Curr Opin Neurol. 2016;29(5):628–34. 19. Taniguchi-Ikeda M, Morioka I, Iijima K, Toda T.  Mechanistic aspects of the formation of alpha-dystroglycan and therapeutic research for the treatment of alpha-dystroglycanopathy: a review. Mol Asp Med. 2016;51:115–24. 20. Meola G, Cardani R.  Myotonic dystrophies: an update on clinical aspects, genetic, pathology, and molecular pathomechanisms. Biochim Biophys Acta. 2015;1852(4):594–606. 21. Hamel J, Tawil R.  Facioscapulohumeral muscular dystrophy: update on pathogenesis and future treatments. Neurotherapeutics. 2018;15(4):863–71. 22. Liewluck T, Winder TL, Dimberg EL, Crum BA, Heppelmann CJ, Wang Y, et  al. ANO5-muscular dystrophy: clinical, pathological and molecular findings. Eur J Neurol. 2013;20(10):1383–9. 23. Young NP, Daube JR, Sorenson EJ, Milone M.  Absent, unrecognized, and minimal myotonic discharges in myotonic dystrophy type 2. Muscle Nerve. 2010;41(6):758–62. 24. Lahoria R, Milone M. Rhabdomyolysis featuring muscular dystrophies. J Neurol Sci. 2016;361:29–33. 25. Yonekawa T, Nishino I. Ullrich congenital muscular dystrophy: clinicopathological features, natural history and pathomechanism(s). J Neurol Neurosurg Psychiatry. 2015;86(3):280–7. 26. Rubegni A, Malandrini A, Dosi C, Astrea G, Baldacci J, Battisti C, et al. Next-generation sequencing approach to hyperCKemia: a 2-year cohort study. Neurol Genet. 2019;5(5):e352. 27. Soontrapa P, Liewluck T. Anoctamin 5 (ANO5) muscle disorders: a narrative review. Genes (Basel). 2022;13(10) 28. Chahin N, Sorenson EJ. Serum creatine kinase levels in spinobulbar muscular atrophy and amyotrophic lateral sclerosis. Muscle Nerve. 2009;40(1):126–9. 29. Wright MA, Yang ML, Parsons JA, Westfall JM, Yee AS. Consider muscle disease in children with elevated transaminase. J Am Board Fam Med. 2012;25(4):536–40. 30. Nicolau S, Kao JC, Liewluck T. Trouble at the junction: when myopathy and myasthenia overlap. Muscle Nerve. 2019;60(6):648–57. 31. Sener U, Martinez-Thompson J, Laughlin RS, Dimberg EL, Rubin DI.  Needle electromyography and histopathologic correlation in myopathies. Muscle Nerve. 2019;59(3):315–20. 32. Liewluck T. In: Rubin DI, editor. Electrodiagnostic assessment of myopathies. 5th ed. New York, NY: Oxford University Press; 2021. 33. Logigian EL, Ciafaloni E, Quinn LC, Dilek N, Pandya S, Moxley RT 3rd, et  al. Severity, type, and distribution of myotonic dis-

1  An Introduction to the Muscular Dystrophies charges are different in type 1 and type 2 myotonic dystrophy. Muscle Nerve. 2007;35(4):479–85. 34. Dubey D, Beecher G, Hammami MB, Knight AM, Liewluck T, Triplett J, et  al. Identification of Caveolae-associated protein 4 autoantibodies as a biomarker of immune-mediated rippling muscle disease in adults. JAMA Neurol. 2022;79(8):808–16. 35. Nicolau S, Milone M, Liewluck T.  Guidelines for genetic testing of muscle and neuromuscular junction disorders. Muscle Nerve. 2021;64(3):255–69. 36. Kumutpongpanich T, Liewluck T.  Oculopharyngodistal myopathy: the recent discovery of an old disease. Muscle Nerve. 2022;66(6):650–2. 37. Narayanaswami P, Weiss M, Selcen D, David W, Raynor E, Carter G, et al. Evidence-based guideline summary: diagnosis and treatment of limb-girdle and distal dystrophies: report of the guideline development subcommittee of the American Academy of Neurology and the practice issues review panel of the American Association of Neuromuscular & Electrodiagnostic medicine. Neurology. 2014;83(16):1453–63. 38. Leung DG.  Magnetic resonance imaging patterns of muscle involvement in genetic muscle diseases: a systematic review. J Neurol. 2017;264(7):1320–33. 39. Confalonieri P, Oliva L, Andreetta F, Lorenzoni R, Dassi P, Mariani E, et  al. Muscle inflammation and MHC class I up-regulation in muscular dystrophy with lack of dysferlin: an immunopathological study. J Neuroimmunol. 2003;142(1–2):130–6. 40. Arahata K, Ishihara T, Fukunaga H, Orimo S, Lee JH, Goto K, et al. Inflammatory response in facioscapulohumeral muscular dystrophy (FSHD): immunocytochemical and genetic analyses. Muscle Nerve Suppl. 1995;2:S56–66. 41. Komaki H, Hayashi YK, Tsuburaya R, Sugie K, Kato M, Nagai T, et al. Inflammatory changes in infantile-onset LMNA-associated myopathy. Neuromuscul Disord. 2011;21(8):563–8. 42. Darin N, Kroksmark AK, Ahlander AC, Moslemi AR, Oldfors A, Tulinius M.  Inflammation and response to steroid treatment in limb-girdle muscular dystrophy 2I.  Eur J Paediatr Neurol. 2007;11(6):353–7. 43. Liewluck T, Milone M. Characterization of isolated amyloid myopathy. Eur J Neurol. 2017;24(12):1437–45.

9 44. Kuhn M, Glaser D, Joshi PR, Zierz S, Wenninger S, Schoser B, et  al. Utility of a next-generation sequencing-based gene panel investigation in German patients with genetically unclassified limb-­ girdle muscular dystrophy. J Neurol. 2016;263(4):743–50. 45. Yu M, Zheng Y, Jin S, Gang Q, Wang Q, Yu P, et  al. Mutational spectrum of Chinese LGMD patients by targeted next-generation sequencing. PLoS One. 2017;12(4):e0175343. 46. Savarese M, Di Fruscio G, Torella A, Fiorillo C, Magri F, Fanin M, et al. The genetic basis of undiagnosed muscular dystrophies and myopathies: results from 504 patients. Neurology. 2016;87(1):71–6. 47. Ikeda K, Mori-Yoshimura M, Yamamoto T, Sonoo M, Suzuki S, Kondo Y, et  al. Chronic myopathy associated with anti-­ signal recognition particle antibodies can be misdiagnosed as Facioscapulohumeral muscular dystrophy. J Clin Neuromuscul Dis. 2016;17(4):197–206. 48. Mohassel P, Landon-Cardinal O, Foley AR, Donkervoort S, Pak KS, Wahl C, et  al. Anti-HMGCR myopathy may resemble limb-­ girdle muscular dystrophy. Neurol Neuroimmunol Neuroinflamm. 2019;6(1):e523. 49. Nicolau S, Milone M, Tracy JA, Mills JR, Triplett JD, Liewluck T. Immune-mediated necrotizing myopathy: unusual presentations of a treatable disease. Muscle Nerve. 2021;64(6):734–9. 50. Moshe-Lilie O, Ghetie D, Banks G, Hansford BG, Chahin N.  Unusual cases of anti-SRP necrotizing myopathy with predominant distal leg weakness and atrophy. Neuromuscul Disord. 2022;32(2):170–5. 51. Mackenzie SJ, Nicolau S, Connolly AM, Mendell JR. Therapeutic approaches for Duchenne muscular dystrophy: old and new. Semin Pediatr Neurol. 2021;37:100877. 52. Merlini L, Sabatelli P. Improving clinical trial design for Duchenne muscular dystrophy. BMC Neurol. 2015;15:153. 53. Rybalka E, Timpani CA, Debruin DA, Bagaric RM, Campelj DG, Hayes A. The failed clinical story of Myostatin inhibitors against Duchenne muscular dystrophy: exploring the biology behind the Battle. Cell. 2020;9(12) 54. Ahmed Z, Qaisar R. Nanomedicine for treating muscle dystrophies: opportunities, challenges, and future perspectives. Int J Mol Sci. 2022;23(19)

2

Dystrophinopathies Partha S. Ghosh and Basil T. Darras

Introduction The dystrophinopathies are X-linked recessive disorders caused by mutations in the DMD gene leading to reduced or absent dystrophin, the protein product of the gene. Males are clinically affected, while females may be asymptomatic or manifesting carriers. There is a wide spectrum of clinical manifestations of dystrophinopathies: Duchenne muscular dystrophy (DMD), the most common form of muscular dystrophy due to absent or severely reduced amounts of dystrophin protein with a relentlessly progressive and fatal course; Becker muscular dystrophy (BMD), a milder phenotype due to reduced amounts of partially functional dystrophin protein; an intermediate phenotype and X-linked dilated cardiomyopathy (DCM). In this chapter, our primary focus will be on DMD and BMD. With advances in symptomatic and supportive management in the last 2 decades, the life expectancy of DMD patients has increased substantially. There have been several advancements in clinical and translational research that have paved the way for the development of new treatments to address the genetic defect.

[5, 6]. Before this description by Duchenne, isolated cases were reported in the first half of the nineteenth century by other European physicians [8, 9]. In 1955, Becker, Kiener and Walton first proposed the milder form of X-linked muscular dystrophy which was subsequently named as Becker muscular dystrophy (BMD) [10]. However, at that time, it was not clear that DMD and BMD were allelic disorders. The mapping of the gene responsible for DMD at chromosome Xp21 was made possible with advances in genetics in the early 1980s [11, 12]. In 1987–1988, Kunkel and colleagues cloned and sequenced the complete complementary DNA (cDNA) of the DMD gene and the protein product was named dystrophin [13–15]. Dystrophin was localized within the sarcolemma and was noted to be absent in DMD and decreased in BMD [16–18]. The journey of DMD thus evolved from the description of the clinical entity in the nineteenth century to understanding the genetic basis of the disease in 1980s and to the first US Food and Drug Administration (FDA) approved dystrophin restorative therapy in the form of exon skipping using antisense oligonucleotide technology in 2016.

History

Epidemiology

DMD is named after the French neurologist Duchenne [1, 2]. He first described this entity in 1861 under the term “hypertrophic paraplegia of infancy of cerebral origin” [3]. In 1865, he devised an instrument (“histologic harpoon”) for muscle biopsy and provided a detailed analysis of 13 of his own cases [4–7]. In 1868, Duchenne revised the term to “pseudo-­ hypertrophic muscular paralysis” to emphasize the fact that the weakness was of muscular rather than of cerebral origin

DMD is the most common form of muscular dystrophy with an estimated incidence of about 1 in 5000 live male births [19]. The incidence of BMD is about one-third of DMD and varies from 1 in 18,000 to 1 in 31,000 male births [20–22]. Population studies in northern England report an incidence of 1 in 5618 live male births for DMD [22], whereas in Nova Scotia, Canada, the incidence of DMD was 1  in 4500 live male births from 1968–2008 [23].

P. S. Ghosh (*) · B. T. Darras Department of Neurology, Boston Children’s Hospital, Boston, USA e-mail: [email protected]; [email protected] © The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 P. Narayanaswami, T. Liewluck (eds.), Principles and Practice of the Muscular Dystrophies, Current Clinical Neurology, https://doi.org/10.1007/978-3-031-44009-0_2

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P. S. Ghosh and B. T. Darras

12

Etiopathogenesis DMD is the largest known human gene with a 14 kilobase (Kb) transcript and 79 consecutive exons spanning 2.2 megabases (Mb) on the short arm of the X chromosome [14–16, 24].

Dystrophin Isoforms There are several tissue specific isoforms of dystrophin, driven by a specific promoter which facilitates transcription from their first exon [25–28]. (Fig. 2.1). The three main promoters of the DMD are the Brain (B), the Muscle (M) and the Purkinje (P), which drive the production of the full-length dystrophin protein of 427 kilo Dalton (KDa) designated as B/Dp427, M/Dp427, and P/Dp427, respectively [28]. The muscle isoform is expressed in skeletal, smooth and cardiac muscles. It is first detected at 9 weeks of gestation and its expression increases as myoblasts continue to mature [12]. The brain isoform is highly expressed in the neocortex and hippocampus while the Purkinje isoform is expressed in the cerebellum [12]. There are several short isoforms of dystrophin which are transcribed by at least four first exons situated adjacent to the promoters and localized within introns 29 in the DMD gene (Retinal isoform or Dp260, R), 44 (Brain specific isoform or Dp140, B3), 55 (Schwann cell isoform or Dp116, S) and 62 (General isoform or Dp71, G) [24–28].

Dystrophin Protein (Fig. 2.2a, b) Dystrophin has four functional domains: amino (N)-terminal, rod, cysteine-rich and carboxy (C)-terminal. The N-terminal

domain is encoded by exons 1–8 and binds to actin through three high-affinity actin-binding sites. This domain shares homology with other actin-binding proteins (e.g. α-actinin and β-spectrin) and interacts with the cytoskeleton [28]. Studies have shown that deletion of these actin binding sites do not cause a significant reduction of in-vitro actin-binding affinity [29]. In addition, deletions of these regions are seen in some BMD patients [30, 31]. These observations suggest that dystrophin may contain other actin-binding domains or is able to associate with additional cytoskeletal proteins [28]. The large central rod domain is encoded by exons 9–63, comprising of 24 homologous “spectrin-like” repeats forming an α-helical structure [32]. Each repeat is encoded by two exons and repeats are interrupted by two non-helical regions known as “hinges” which confer flexibility to the rod domain during muscle contraction [32]. The cysteinerich domain is encoded by exons 64–69, located near the C-terminal region and stabilizes the binding between β-dystroglycan and dystrophin on the sarcolemmal membrane [33]. The C-terminal domain encoded by exons 70–79 plays an important role in binding to various adjacent proteins. The C-terminal and cysteine-rich domains act as a bridge that link the cytoskeleton with sarcolemmal proteins that in turn bind with extracellular matrix proteins. These membrane proteins are collectively called dystrophin associated protein complex (DAP), comprising of 3 main groups: dystroglycans (α- and β-dystroglycan); sarcoglycans (α, β, γ, and δ –sarcoglycan); syntrophin/dystrobrevin group (α-syntrophin, β1-syntrophin, β2-syntrophin, α-dystrobrevin, and β-dystrobrevin) [12, 28]. Specifically, the region encoded by exons 71–74 binds to α – and β syntrophin in-vitro and modulates the functional interaction between dystrophin and syntrophin [34–36]. This region also links with nitric oxide synthase (nNOS) via dystro-

ROD LIKE DOMAIN EXONS 9-63 ACTIN BINDING DOMAIN EXONS 1-8 1

2a/b 4

Dp427B Dp427M

9

SPECTRIN LIKE REPEATS EXONS 64-69 19

29

Dp260

44

Dp140

55

Dp116

62

Dp71

68

70

7879

C TERMINAL DOMAIN EXONS 70-79

Dp427P

Fig. 2.1  Dystrophin gene structure and protein domains. Schematic representation of 79 exons of dystrophin gene with isoforms and protein domains. Lines in red represent the 5′ full length promoters and their first exon (isoforms Dp427B-M-P). Lines in blue represent the 3′ promoters and their first exons of isoforms: Dp260 (retinal), Dp140 (brain 3), Dp116 (Schwann cells), Dp71 (general). In green are repre-

sented exon alternatively spliced or skipped. Boxes’ different blue/violet colors explain the protein domains corresponding to the different exonic regions. Reproduced with permission from Elsevier from Ferlini A. et al. The medical genetics of dystrophinopathies: Molecular genetic diagnosis and its impact on clinical practice. Neuromuscular Disorders 2013;23 (1):4–14 (Fig. 1)

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13

a

b

Fig. 2.2 (a) The dystrophin-associated protein complex. Dystrophin acts as an important link between the internal cytoskeleton and the extracellular matrix. Neuronal nitric oxide synthase (nNOS) binds to α-syntrophin but also has a binding site in repeat 17 of the rod domain of dystrophin. αDG, α-dystroglycan; βDG, β-dystroglycan. Reproduced with permission from Springer Nature from Fairclough RJ et  al. Therapy for Duchenne muscular dystrophy: renewed optimism from genetic approaches. Nature Reviews Genetics 2013;14:373–378

(Fig. 1). (b): Wild-type dystrophin. Full-length dystrophin comprises an aminoterminal actin-binding domain, four hinge domains (H1–H4) and a rod domain consisting of 24 spectrin-like repeats (R1–R24), within which lie a second actin-binding domain, a cysteine-rich domain (CRD) and a carboxy-terminal domain (CTD). Reproduced with permission from Springer Nature from Fairclough RJ et al. Therapy for Duchenne muscular dystrophy: renewed optimism from genetic approaches. Nature Reviews Genetics 2013;14:373–378 (Figure 2a)

brevin. Absence of dystrophin in DMD downregulates nNOS which plays a critical role in reduced tissue perfusion and muscle damage [37].

tion of proteolytic enzymes like calpains due to an influx of extracellular calcium [40]. There is progressive degeneration of larger muscle fibers while smaller fibers like those in extraocular muscles are relatively spared because the mechanical stress per unit of the muscle membrane surface is much less in the smaller fibers [41].

Pathogenesis The primary role of dystrophin in skeletal muscle is mechanical stabilization of the sarcolemma, as evidenced by increased susceptibility to contraction-induced sarcolemmal rupture in the mdx mouse model [38]. Secondary loss of DAP due to dystrophin deficiency contributes to further destabilization of the muscle cell membrane from contractile forces, resulting in focal tears during contractile activity [39]. This in turn leads to muscle fiber necrosis from activa-

Mutations in the Dystrophin Gene (Table 2.1) The mutation rate is relatively high in the dystrophin gene. One third of the cases are due to de novo mutations; this presents a challenge to reduce population disease burden as new dystrophinopathy cases cannot be prevented even with good prenatal genetic counseling [42]. This high mutation

P. S. Ghosh and B. T. Darras

14 Table 2.1  Type and Frequency of Mutations Held within the TREAT-­ NMD DMD Global Database Total Large mutations Large deletions (≥ 1 exon) Large duplications (≥ 1 exon) Small mutations Small deletions (< 1 exon) Small insertions (< 1 exon) Splice sites (20% by Western blot

XR X-linked recessive, I.Q. intelligence quotient. Reproduced with permission from Elsevier from Darras BT, Menache-Starobinski CC, Hinton V, Kunkel LM. Dystrophinopathies. Chap. 30. In: Neuromuscular Disorders of Infancy, Childhood, and Adolescence: A Clinician’s Approach. 2nd edition. San Diego: Academic Press, 2015. pp. 551–92 (Table 30.1) a Uses monoclonal antibodies to the carboxy-terminus, amino-terminus, and mid-rod domain (6–10 antibody) of dystrophin b The quantity of dystrophin is expressed as a percentage of control values (standardized versus myosin post transfer with Coommasie stain) c Normal molecular weight is 427 kDa

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a

P. S. Ghosh and B. T. Darras

b

Fig. 2.3  A boy with Duchenne muscular dystrophy, at the ages of 8 years (a) and 11.5 years (b). Note enlargement of gastrocnemii muscles bilaterally, known as “pseudohypertrophy.” Also note the progression in foot position from plantigrade (a) to mild equinovarus (b). Reproduced with permission from Elsevier from Darras BT, Menache-Starobinski

CC, Hinton V, Kunkel LM. Dystrophinopathies. Chap. 30. In: In: Darras BT, Jones HR Jr., Ryan MM, De Vivo DC (editors). Neuromuscular Disorders of Infancy, Childhood, and Adolescence: A Clinician’s Approach. second edition. San Diego: Academic Press, 2015. pp. 551– 92 (Fig. 30.5)

lordosis), knee extensors more than flexors, elbow flexors and extensors more than deltoids. Gowers’ sign which is a manifestation of proximal lower limb muscle weakness is a useful bedside test where affected patients turn their face to the floor when arising from a supine position, then spread their legs and use their hands to climb up their thighs to an upright position [12]. Early involvement of the neck flexors as evidenced by the inability to lift the head against gravity in the supine position is common in DMD [12]. Hypertrophy of the calf muscles (Fig. 2.3) is a useful sign. In the early phase, there is true hypertrophy of the muscle fibers which are then replaced by fibrous and fatty tissue in the late stages of the disease (pseudohypertrophy) [12]. However, several other muscles can be hypertrophied, such as quadriceps, gluteal muscles, deltoid, infraspinatus, tongue and rarely masseter muscles [12]. DMD is a relentlessly progressive disease with gradually evolving weakness of the lower limb muscles from 7 years onwards to non-ambulatory status by 12–13 years historically in steroid naïve patients. This is followed by weakness of the upper limb muscles and development of scoliosis from paraspinal muscle weakness and atrophy. However, the rate of progression of weakness varies substantially among DMD patients and even within siblings of the same family, suggesting the presence of genetic modifiers in disease severity [42]. Baseline 6-minute walk distance (6MWD) which is an important functional measure in neuromuscular disorders and age (≥7 years) are strong predictors of loss of ambulation (LOA) in DMD patients; baseline 6MWD 60% of the rod domain (deletion of exons 17–48) in one BMD patient resulted in a very mild disease and forms the basis of microdystrophin constructs for DMD gene transfer therapy [102]. Survival is typically beyond the

18

third decade, and patients usually die from respiratory failure or cardiomyopathy in their fourth to sixth decades of life [60, 99].

Extra-Muscular Involvement On occasion, cardiac involvement in BMD may be more severe than the skeletal muscle involvement and can precede muscle weakness by several years [103–106]. Patients with deletions affecting N-terminal domain are more likely to experience early-onset cardiomyopathy [107]. Similarly, cognitive and behavioral problems are less severe in BMD patients, although mean IQ scores are slightly lower than the general population [12].

Other Dystrophinopathy Phenotypes Intermediate Phenotype These patients are so called “mild DMD or severe BMD” as they are in between the two classic phenotypes. In a natural history study, these patients usually remain ambulatory before age 13 but become wheelchair dependent before age 16 [12]. An important clinical clue is preservation of their ability to flex their neck against gravity which differentiates them from the classic DMD phenotype [12]. This phenotypic variability can be partly explained by the genetic modifiers that influence ambulatory status, steroid responsiveness, and cardiomyopathy [65]. Some of these genetic modifiers have negative or positive effect on the phenotype. Osteopontin, known as secreted phosphoprotein 1 (SPP1), is an acidic glycoprotein that plays important role in bone-remodeling, immune function, and muscle repair; it’s promoter is activated by transforming growth factor β (TGFβ) family members [65]. A single nucleotide polymorphism (SNP) in the promoter of SPP1 is associated with early LOA in DMD patients [108]. It is also noted that patients with certain SPP1 variants respond poorly to steroids [109]. Latent TGFβ binding protein 4 (LTBP4) is a member of the fibrillin superfamily that binds to TGFβ in the extracellular matrix and regulates TGFβ activity [110]. Certain LTBP4 genotypes have a protective effect with delayed LOA, glucocorticoid responsiveness as well as late onset of cardiomyopathy [110–112]. In two sets of brothers with DMD who were discordant for their LTBP4 haplotypes, the brothers with the protective allele had delayed LOA compared to the brothers without that allele [110].  MD-Associated Dilated Cardiomyopathy D Several members of a large multi-generation family were described in 1987 to have dilated cardiomyopathy without skeletal myopathy and linkage analysis identified the locus to Xp21 of DMD [113, 114]. DMD-CM typically presents in

P. S. Ghosh and B. T. Darras

males in the second or third decade with rapidly progressive course; associated ventricular arrhythmias are common [114, 115]. Female carriers develop mild cardiomyopathy in the fourth or fifth decade and exhibit slow progression [12]. Elevated CK is an important finding in this condition [12] and should alert the cardiologist to suspect DMD-CM. Patients with severe cardiomyopathy do not produce dystrophin in their cardiac muscle while their skeletal muscle is unaffected [116].

Female Carriers As dystrophinopathies are X-linked recessive disorders, women carry and transmit the affected gene on one X chromosome but usually do not manifest the disease due to the presence of a normal X chromosome. Women carriers can infrequently develop clinical manifestations, the so called “manifesting carriers” (MC). Several mechanisms have been proposed to explain MC [117–121]. The most frequently described mechanism is non-random or skewed X-chromosome inactivation (XCI) wherein expression of the X chromosome with the mutated allele is favored [120]. It is generally thought that more severe skewing of XCI (ratio > 90:10) is associated with more severe symptoms in MC, however, this association is not definitive [121]. The phenotype in monozygotic female twins with DMD gene mutations are often discordant due to differential XCI in the early embryonic stage [122, 123]. Other mechanisms for MC status are balanced X-autosome translocations with breakpoints at Xp21 (most common) [124], Turner syndrome [125], X chromosome uniparental disomy [126], and male pseudo-­ hermaphroditism due to a mutation in the androgen receptor gene [127]. With regards to the clinical features of MC, one study found that 5% of the carriers had myalgias/cramps without muscle weakness, 17% experienced mild-to-moderate muscle weakness, and 8% had DCM [128]. Another descriptive study of clinical and genetic features of 15 MC (excluding those with only myalgias/cramps) among 860 patients in the United Dystrophinopathy Project (UPD) found that symptom onset ranged from 2 to 47 years. The phenotype varied from DMD-like progressive disease to very mild-BMD like presentation. Eight patients had male relatives with DMD [129]. Manifesting carriers can pose diagnostic challenges in the absence of a family history of dystrophinopathy, as 7 out of 15 MC in this study had negative family history [129]. About 10% of women with elevated CK (typically >1000 U/L) and myopathic histology were found to be MC [130]. CK was elevated (2–10 times the upper limit of normal, mean 306 U/L) in 30–50% of dystrophinopathy carriers in one study; 22% were MC in this study [128]. In this study there was no significant difference of CK level between asymptomatic and MC [128].

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 ifferential Diagnosis of Muscular D phenotype of Dystrophinopathies

19

muscle with fibrous tissue [133, 134]. As a general rule, CK levels are much higher in DMD compared to BMD; by age 5 CK levels are about 50–200 times the upper limit of normal Limb-girdle muscular dystrophy (LGMD) are a diverse in DMD and 20–200 times the upper limit in BMD [132, group of disorders that can be of autosomal dominant or 134]. However, it is not always possible to reliably differentirecessive inheritance. Among LGMD types, sarcoglycanop- ate DMD from BMD based on CK levels alone because of athies (LGMD-R3–5) and LGMD-R9 resemble dystrophi- the overlap in the range of levels. One study found that CK nopathies (proximal weakness, high CK and calf hypertrophy) levels were 2–10 times the upper limit of normal in 30–50% and may have cardiomyopathy (see Chap. 6) [11, 12]. of the female carriers of DMD or BMD; 22% of carriers Clinically, these conditions are difficult to differentiate from were MC.  Mean CK level was not different in MC and dystrophinopathies in boys without a family history. Emery-­ asymptomatic carriers [128]. Another study found that Dreifuss muscular dystrophy (EDMD) is characterized by daughters of obligate carriers have a disproportionate decline the clinical triad of early onset proximal joint contractures, in CK and pyruvate kinase (PK) with age as compared to progressive muscle weakness typically starting in a scapulo-­ non-carrier females, suggesting that the rate of carrier detecperoneal distribution, and cardiac involvement (arrhythmias tion will be higher in the first two decades [135]. and cardiomyopathy) (see Chap. 10). Proximal joint contractures and scapulo-peroneal pattern of weakness help to differentiate EDMD from dystrophinopathies. EDMD can be X Electromyography linked [Emerin (EMD) and four and a half LIM domain 1 (FHL1)], autosomal dominant [Lamin A/C (LMNA), Electromyography (EMG) in DMD reveals increased inserNesprin-1 (SYNE1), Nesprin-2 (SYNE2), Transmembrane tional activity, abnormal spontaneous activity (fibrillation Protein 43 (TMEM43)] and autosomal recessive [LMNA, potentials and positive sharp waves) and brief, small amplitude motor unit potentials with early recruitment. The irritaSUN domain containing protein-1 (SUN1), Titin (TTN]. Congenital muscular dystrophies are a phenotypically bility is attributed to muscle fiber necrosis and is less apparent and genotypically diverse group of disorders (see Chap. 11). in BMD.  In end stage disease, when muscle fibers are These patients present with early onset weakness, hypotonia, replaced by fibro-fatty tissue, insertional activity is reduced, high CK and sometimes central nervous system manifesta- spontaneous activity is no longer seen and both short and tions (seizures, cortical malformations, and white matter long duration polyphasic motor units are seen, reflecting changes) [11, 12]. Most congenital muscular dystrophies chronic disease. However, because the findings are non-­ present with muscle weakness before 2 years of age, which specific [12], and the procedure is associated with some discomfort, EMG is of limited utility in the diagnosis of DMD is uncommon in DMD patients. Spinal muscular atrophy (SMA) is an autosomal recessive especially when a family history of the disorder is present. In disorder due to homozygous deletions of SMN1 (5q-SMA) sporadic cases and in BMD, because the differential diagnoin >95% of cases. SMA type 3 can present with progressive sis is broader, EMG may be useful in confirming a myopathic proximal limb weakness after 18 months of age and rarely process and directing further testing. can have calf hypertrophy. Early loss of tendon reflexes, normal or mild elevation of CK, and neurogenic changes on electromyography (EMG) help to differentiate SMA from Magnetic Resonance Imaging (MRI) muscular dystrophies [11, 12]. Muscle MRI (mMRI) is a noninvasive imaging modality to asses morphologic dystrophic abnormalities in DMD [136]. (see Chap. 14). Qualitative measures (signal intensity Investigations changes on T1 and T2 W images) assess muscle edema, fat infiltration and muscle volume; quantitative techniques Creatine Kinase (T1map, T2map, diffusion-­weighted imaging [DWI], and Among several serum muscle enzymes used to detect myop- Dixon) can precisely measure “fat fraction” (FF) of the musathies, CK is the most sensitive and cost-effective screening cle. During the early phase of DMD, muscle edema is test in clinical neuromuscular practice [131]. CK levels are observed (suggesting inflammation, seen as hyperintense invariably elevated in patients with dystrophinopathy and signals on short tau inversion recovery, water T2-Dixon, or continue to increase with age, reaching a peak by 2–3 years fat-suppressed T2-weighted sequences) while fatty infiltraof age [132]. CK levels then progressively decline with age tion and atrophy of the muscles (seen as hyperintense signals at a rate of about 20% per year due to replacement of the on T1-weighted images) occur later [137–141]. There is a

20

differential pattern of muscle involvement in DMD with severe fibro-fatty changes in the gluteus medius, gluteus minimus and adductor magnus, moderate to severe changes in gluteus maximus and quadriceps muscles with sparing of the gracilis, sartorius and semimembranosus muscles [142]. Superficial posterior (soleus and lateral head of gastrocnemius) and lateral leg muscles (peronei) are preferentially affected with relative sparing of deep posterior (tibialis posterior and popliteus muscles) and anterior leg muscles (tibialis anterior) [142]. This differential pattern of muscle involvement in mMRI may be useful to distinguish DMD from other LGMDs with similar phenotype such as LGMD-R3–5 (Sarcoglycanopathies) and LGMD-R9 (FKRP) [142]. Magnetic resonance spectroscopy (MRS) including 1H and 31P spectroscopy provide further insights into the biochemical composition and metabolic processes within tissue samples [143].1H-MRS which is independent of lipid infiltration can identify muscle damage through inflammation and edema within muscle [143]. 31P-MRS assesses energy metabolism through quantification of the phosphocreatine, inorganic phosphate, and ATP. With disease progression in DMD, fat replaces functional muscle tissue leading to reduction of oxidative capacity of the dystrophic muscle which in turn results in quantifiable metabolic changes measured via 31 P-MRS [143]. mMRI is an objective, reproducible technique, although it may be limited by patient co-operation in younger children, and can be performed during the ­ambulatory and nonambulatory phases of the disease [144]. mMRI is useful in documenting disease progression over time by precisely quantifying the fat fraction (FF) of the muscle [145–151]. A recent systematic review found a moderate to strong correlation between mMRI measurements (in particular fatty infiltration using 3-point Dixon technique) and motor function tests in ambulatory DMD patients, making it useful as a biomarker in clinical trials [144].

Muscle Biopsy The pathological evaluation of dystrophinopathy includes identification of dystrophic muscle pathology and the immunohistochemical evaluation of dystrophin epitopes. Muscle biopsies are less frequently used if genetic testing is easily available and affordable [152]. As discussed above, it can be helpful when there is a discordance between the clinical phenotype and “reading frame rule” and in patients with undetectable deep intronic mutations [28, 32]. Muscle biopsy also has a role in sporadic cases when the differential diagnosis is wider and in female carriers (asymptomatic or MC) with elevated CK without family history of dystrophinopathy. Muscle biopsy is increasingly used in clinical trials as a sur-

P. S. Ghosh and B. T. Darras

rogate biomarker of efficacy to quantify dystrophin protein expression in trials of gene restoration treatments [65].

Histopathological Findings Light microscopic findings include variation in fiber size (large hypertrophic fibers, fiber splitting, and atrophic fibers), increased internal nuclei, necrotic fibers, regenerating fibers, increased endomysial and perimysial connective tissue and inflammatory infiltrates of cytotoxic T cells and macrophages in the endomysium, perimysium and perivascular spaces. These changes are non-specific and constitute general features of a dystrophic process with superadded inflammatory infiltration [153–155].  ystrophin (IHC) Staining D IHC is performed using commercially available antibodies which recognize epitopes in the N-terminal, rod, and C-terminal domains of the dystrophin protein [156, 157]. It is important to perform IHC with antibodies directed against epitopes located in at least two domains of dystrophin because a mutation affecting one domain may render the immunostaining completely absent if the antibody to an epitope in that domain is used, despite retained dystrophin activity in other domains. This may result in some BMD patients being falsely labelled as DMD [158, 159]. In DMD almost all of the muscle fibers show markedly reduced or absent sarcolemmal dystrophin immunoreactivity. In most biopsies, mild dystrophin expression is detected in less than 1% of fibers- the “revertant” fibers, due to a somatic mutation affecting a portion of the gene, which restores the reading frame by suppressing the out-of-frame mutation [160, 161]. In BMD there is reduced sarcolemmal dystrophin staining, affecting all muscle fibers in a patchy distribution [162, 163]. A mosaic pattern, where individual fibers react either strongly or not at all to the immunostain (rather than partial or reduced immunostaining) is a feature of MC [164].  estern Blot Analysis W WB is used for quantification of the dystrophin protein (% of normal control) and for assessing the size of the residual dystrophin protein [12]. Similar to IHC, antibodies directed against different domains of the dystrophin protein are used. In most clinical laboratories, WB is performed by antibodies against the C- terminal and the mid-rod domain. This combination is predicted to be 99% accurate [12].However, WB analysis may be subject to technical variations across laboratories due to differences in their internal controls; most laboratories use myosin as internal control [12]. In DMD, there is a significant quantitative reduction in the amount of dystrophin (0–5% of normal controls) when C- terminal antibodies are used because most DMD mutations result in severely truncated dystrophin molecules [12]. Dystrophin levels can

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be as high as 25% of control levels when rod or amino-­ terminal antibodies are used; however, the size of the molecule is always smaller than normal [156, 165]. In BMD, WB analysis yields variable results. Using antibodies directed against three sub- domains of dystrophin protein, WB analysis can show reduced or normal quantity (20%–100%) of dystrophin of altered size, or reduced quantity (20%–50%) of dystrophin of normal size. The molecular weight of the dystrophin molecule is reduced in about 80% of BMD patients (deletions, lower molecular weight with larger deletions), increased in about 5% (duplications) and normal in about 15% [156, 163, 165]. There is an inverse correlation between the amount of dystrophin and severity of the BMD phenotype; patients with 3–10% of dystrophin protein have a severe phenotype regardless of protein size [166]. As there is a considerable overlap between the amount of dystrophin in patients with DMD, BMD and intermediate phenotypes, identification of these phenotypes solely based on dystrophin quantification may not be accurate [156]. These results should be interpreted in the context of the clinical phenotype.

 enetic Testing Strategies (Fig. 2.4) G In a boy with suspected dystrophinopathy, the first test is identification of deletion or duplication by multiplex ligation probe amplification (MLPA) or array comparative genomic hybridization (CGH) or chromosomal microarray (CMA) which have largely replaced previous methods to detect deletions/duplications using multiplex PCR, Southern blotting, and FISH [42, 167–171]. If a mutation is not identified by MLPA or CMA, then Sanger sequencing of the DMD gene is performed to detect small mutations. This sequence of testing remains the most efficient approach for a patient with a likely clinical diagnosis of dystrophinopathy. If Sanger sequencing of the dystrophin gene is uninformative and dystrophinopathy is a strong consideration, muscle biopsy with IHC and WB analysis should be performed after excluding mimics of DMD/BMD through further genetic testing. RNA can be isolated from the biopsied muscle tissue and may reveal an aberrant dystrophin mRNA transcript (deep intronic mutation) [42]. With increasing availability of next-­ generation sequencing techniques such as whole-exome sequencing it is possible that in the future MLPA, CGH, and

Suspected dystrophinopathy Check for deletions/duplications involving ≥1 exon using MLPA or array CGH

Deletion or duplication detected

Single exon deletion detected by MLPA

No deletion and/or duplication detected

Perform confirmatory study

Perform small mutation analysis

Single exon deletion confirmed

Single exon deletion not confirmed

Dystrophinopathy confirmed

Fig. 2.4  Genetic testing strategies in DMD. Diagnostic decision tree to confirm the genetic diagnosis of dystrophinopathies. Boys with typical symptoms of a dystrophinopathy and increased plasma creatine kinase (CK) levels should undergo genetic testing to confirm the diagnosis. Initially, multiplex ligation-dependent probe amplification (MLPA) or array comparative genome hybridization (CGH) should be used to identify causative DMD variants; however, if a causative variant is not iden-

Causative variant found

Causative variant not found

Consider • Muscle biopsy and protein and/or mRNA analysis • Differential diagnosis

tified (which occurs in ~25% of patients), small mutation analysis should be used. Boys without an identified causative variant after small mutation analysis should be referred for muscle biopsy and protein or mRNA analysis. Reproduced with permission from Springer Nature from Duan D et  al. Duchenne muscular dystrophy. Nature Reviews Disease Primers 2021;7 (13):1–19 (Fig. 4)

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Sanger sequencing will be replaced by this technique where a single step diagnosis for deletions, duplications and small mutations in DMD gene can be carried out [172, 173].

Genetic Counseling and Carrier Testing As dystrophinopathy is an X-linked disorder, the father of an affected male does not have the disease and is not a carrier. A mother with an affected son and one other affected male relative in the maternal line of inheritance is an obligate carrier [12]. If the mother has more than one affected son and negative family history, possible mechanisms are a germline mutation or germline mosaicism [12]. If the mother carries the same mutation as the proband then the risk of transmitting the mutant alleles is 50% in each pregnancy; boys who inherit the mutations are affected and girls who do so are carriers. If a maternal mutation is not detected, it possible that the proband has a de novo mutation. However undetected germline mosaicism in mothers (15–20%) simulates de novo disease in the proband and confers a risk of transmitting the disease to future offspring. This risk further increases if the mother possesses both somatic and germline mosaicism [174]. Prenatal testing is offered to pregnant mothers who are known carriers by either chorionic villous sampling at 10–12 weeks or amniocentesis at 15–18 weeks [12]. Preimplantation genetic screening can also be offered to at risk families undergoing in-vitro fertilization.

Management of DMD In this section, we will focus on the management strategies for DMD as there is no specific treatment for BMD yet and most of the anticipatory guidance for rehabilitation, cardiac, pulmonary, and orthopedic management are similar to those in DMD management. The last two decades have seen major breakthroughs in the field of clinical/translational medicine that has led to the approval of many treatment strategies for DMD. Despite these advances, DMD remains a relentlessly progressive and ultimately fatal disease. The comprehensive care guidelines for DMD were published in 2010 [175, 176] and updated in 2018 [177–179]. These guidelines provide a comprehensive and broad framework of DMD management and are supported by the US Centers for Disease Control and Prevention (CDC) with input from the TREAT- NMD network for neuromuscular diseases, the Muscular Dystrophy Association, and Parent Project Muscular Dystrophy [177]. It is increasingly recognized that the comprehensive management of a DMD patient is not confined to neuromuscular medicine but extends across multiple specialties. (Fig. 2.5) This multidisciplinary approach improves care and survival of DMD patients [66, 180–182]. With anticipatory diagnos-

P. S. Ghosh and B. T. Darras

tic and therapeutic strategies, it is now easier to identify and treat potentially modifiable disease complications in DMD.  With increased life expectancy of DMD patients, there is an increased emphasis on the quality of life and general well-being of DMD patients and their caregivers. The neuromuscular physician remains the key person and plays a pivotal role in the overall care of the patient throughout their lifetime [177]. Once the diagnosis is established, the neuromuscular physician broadly discusses with the parents the key principles of medical management and the role of the various team members in the care of the child and actively seeks their participation in the entire process. It is important to stress the need for continued follow-up at set timeframes with various specialists to provide optimal care. The established management guidelines may need to be individually tailored according to the needs of patients and their families.

Pharmacological Management Corticosteroids Oral glucocorticoids remain the cornerstone of pharmacologic management in DMD, although their precise mechanism of action remains unknown [182, 183]. Corticosteroids (CS) reduce muscle inflammation and increase muscle mass through the stimulation of insulin-like growth factors, decreased cytokine production, decreased lymphocyte reaction, enhanced myoblast proliferation and upregulation of synergistic molecules [182]. CS slow the rate of progression of muscle weakness and prolong ambulation in DMD [184– 186]. In the long term, CS improve upper limb function, preserve cardiorespiratory functions and reduce the need for scoliosis surgery [187–193]. Current recommendations are to initiate CS irrespective of the type of mutation between 3 and 5 years of age although there is wide variation in practice. Ideally, CS should be started when the patients achieve a plateau phase in their motor development and are not yet losing function, which is usually between 4 and 5 years of age. This approach attempts to balance the long term negative effects of CS on growth and development by starting too early in childhood with reducing effectiveness by starting after functional decline has commenced. The CS preparations commonly used are prednisone/prednisolone and deflazacort. Deflazacort was approved in February 2017 by the US FDA for DMD patients 2 years and older. Prednisone/prednisolone are widely available and inexpensive while deflazacort is expensive. The recommended daily doses are 0.75 mg/kg/day for prednisolone/prednisone and 0.9  mg/kg/day for deflazacort [177, 194]. A higher daily dose of prednisolone/prednisone (1.5 mg/kg/day) is not associated with increased benefits, while doses of 0.3 mg/kg/day are less effective [12]. There are different regimens for steroid doses: daily, 10 days on/off

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Neuromuscular management

Stage 1: At diagnosis

23 Stage 2: Early ambulatory

Stage 3: Late ambulatory

Stage 4: Early non-ambulatory

Stage 5: Late non-ambulatory

Lead the multidisciplinary clinic; advise on new therapies: provide patient and family support, education, and genetic counselling Ensure immunisation schedule is complete

Assess function, strength, and range of movement at least every 6 months to define stage of disease

Discuss use of glucocorticosteroids

Initiate and manage use of glucocorticosteroids

Refer female carriers to cardiologist

Help navigate end-of-life care

Rehabilitation management

Provide comprehensive multidisciplinary assessments, including standardised assessments, at least every 6 months Provide direct treatment by physical and occupational therapist, and speech-language pathologists, based on assessments and individualised to the patient

Assist in prevention of contracture or deformity, overexertion, and fall; promote energy conservation and appropriate exercise or activity; provide orthoses, equipment, and learning support

Continue all previous measures; provide mobility devices, seating, supported standing devices, and assistive technology; assist in pain and fracture prevention or management; advocate for funding, access, participation and self-actualisation into adulthood

Gastrointestinal and nutritional management

Endocrine management

Measure standing height every 6 months Assess non-standing growth every 6 months Assess pubertal status every 6 months starting by age 9 years Provide family education and stress dose steroid prescription if on glucocorticosteroids

Include assessment by registered dietition nutritionist at clinic visits (every 6 months); initiate obesity prevention strategies; monitor for overweight and underweight, especially during critical transition periods Provide annual assessments of serum 25-hydroxpitamin D and calcium intake Assess swallowing dysfunction, constipation, gastro-oesophageal reflux disease, and gastroparesis every 6 months Initiate annual discussion of gastrostomy tube as part of usual care

Respiratory management

Provide spirometry teaching and sleep studies as needed (low risk of problems)

Assess respiratory function at least every 6 months

Ensure immunisations are up to date: pneumococcal vaccines and yearly inactivated influenza vaccine Initiate use of lung volume recruitment Begin assisted cough and nocturnal ventilation

Consult cardiologist; assess with electrocardiogram and echocardiogram* or cardiac MRI

Assess cardiac function annually; initiate ACE inhibitors or anglotensin receptor blockers by age 10 years

Assess cardiac function at least annually, more often if symptomsor abnormal imaging are present; monitor for rhythm abnormalities Use standard heart failure interventions with deterioration of function

Bone health management

Cardiac management

Add daytime ventilation

Assess with lateral spine x-rays (patients on glucocorticosteroids: every 1-2 years; patients not on glucocorticosteroids: every 2-3 years) Refer to bone health expert at the earliest sign of fracture (Genant grade 1 or higher vertebral fracture or first long-bone fracture)

I

Transitions

Psychosocial management

Orthopaedic management

Assess range of motion at least every 6 months Monitor for scoliosis annually Refer for orthopaedic surgery if needed (rarely necessary)

Monitor for scoliosis every 6 months

Refer for surgery on foot and Achilles tendon to improve gait in selected situations

Consider intervention for foot position for wheelchair positioning; initiate intervention with posterior spinal fusion in defined situations

Assess mental health of patient and family at every clinic visit and provide ongoing support Provide neuropsychological evaluation/interventions for learning, emotional, and behavioural problems Assess educational needs and available resources (individualised education programme, 504 plan); assess vocational support needs for adults

I

Promote age-appropriate independence and social development

Engage in optimistic discussions about the future, expecting life into adulthood

Foster goal setting and future expectations for adult life; assess readiness for transition (by age 12 years)

Initiate transition planning for health care, education, employment, and adult living (by age 13-14 years); monitor progress at least annually; enlist care coordinator or social worker for guidance and monitoring Provide transition support and anticipatory guidance about health changes

Fig. 2.5  Comprehensive care of individuals with Duchenne muscular dystrophy. Care for patients with Duchenne muscular dystrophy is provided by a multidisciplinary team of health-care professionals; the neuromuscular specialist serves as the lead clinician. The figure includes assessments and interventions across all disease stages. *Echocardiogram for patients 6 years or younger. †Cardiac MRI for

patients older than 6 years. Reproduced with permission from Elsevier from Birnkrant DJ et al. Diagnosis and management of Duchenne muscular dystrophy, part 1: diagnosis, and neuromuscular, rehabilitation, endocrine, and gastrointestinal and nutritional management. Lancet Neurology 2018;17 (3):251–267 (Fig. 1)

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(0.75 mg/kg/day of prednisone), and high dose weekend (10 mg/kg of prednisone divided over 2 days over the weekend) [195, 196]. The intermittent dosing and weekend high dose regimens are more beneficial from the side effect standpoint (less Cushingoid features, behavioral problems, hypertension, effects on linear growth and bone mineral density) compared to the daily regimen [197, 198]. A 5-year, randomized, double-blind study (FOR-DMD) comparing daily deflazacort, daily prednisone, and intermittent (10 days on, 10 days off) prednisone has been completed; results are awaited [199]. In non-ambulatory patients, the decision to continue of CS is a balance between the potential benefits (preservation of cardiorespiratory function and prevention of scoliosis) against the risk of side effects (osteoporosis, fractures and obesity). Patients should be counseled regarding the adverse effect profile of steroids before initiation of treatment. The adverse effects are the main reason for discontinuation or dosage modification of CS in DMD [184]. Weight gain and behavioral problems are often responsible for treatment discontinuation [183]. Other side effects such as Cushingoid facies, hirsutism, delayed puberty, and gastrointestinal problems do not usually lead to discontinuation of treatment [184]. Parents should be provided anticipatory dietary advice and resources to manage behavioral problems. Behavioral problems may be magnified by pre-existing neurobehavioral issues related to the disease. If the adverse effects are unmanageable, the dose of CS can be reduced by 25–33% and reassessed after a month [177]. Other options include changing from a daily to an intermittent dosing regimen or switching from prednisone to deflazacort as the latter has been associated with less weight gain [200]. Parents should be counseled against abrupt discontinuation of steroids to avoid life threatening adrenal crisis. Families should be educated about symptoms of adrenal crisis and supplementation of stress doses of CS during periods of stress such as fever, infections and surgery [177]. There is increasing interest in the development of newer agents which target the inflammatory cascade similar to CS, without the systemic toxicity of steroids. Vamorolone is a steroid analog with membrane-stabilizing and anti-­ inflammatory properties [inhibition of Nuclear factor-κB (NF-κB)] [201]. Daily vamorolone treatment suggested improved muscle function at doses of 2.0 and 6.0 mg/kg/d, with less weight gain than prednisone at doses of 2.0 mg/ kg/d in an exploratory 24-week open-label study [202]. Edasalonexent is also an inhibitor of NF-κB [203]. Although well tolerated at the dose of 100 mg/kg/day, edasalonexent did not show significant improvements in primary and secondary functional endpoints in steroidnaïve patients, although a subgroup analysis suggested benefits in slowing disease progression if initiated before the age of 6 years [203].

P. S. Ghosh and B. T. Darras

Dystrophin Restoration Treatments The aim of these approaches is to restore the reading frame of the dystrophin gene transcript so that a partially functioning dystrophin protein is produced, thus changing the severe DMD phenotype into a less severe BMD phenotype. Exon Skipping Exon skipping therapy in DMD restores the reading frame using synthetic antisense oligonucleotides (ASO) targeted to the dystrophin pre-messenger RNA (pre-mRNA) to skip out-­of-­frame mutations in exon 51 [204]. Approximately 70% of mutations in DMD are located between exons 45 and 55. Exon 51 skipping restores the reading frame and is applicable to about 13% of patients [205]. The first clinical trials involving exon 51 skipping therapy used the compounds drisapersen and eteplirsen [206, 207]. Eteplirsen received accelerated conditional FDA approval in 2016 based on a clinical trial of 12 patients that demonstrated a 23% increase in dystrophin-positive muscle fibers compared to baseline at 24 weeks of treatment with 30 mg/kg/ week, an increase of dystrophin quantity by WB (0.08% to 0.93%) after 188 weeks of treatment [208] and a 151 m difference in the decline of 6MWD at 36 months compared to matched historical controls [207]. It is the first drug to get FDA approval using dystrophin quantification as a surrogate outcome measure for DMD. Subsequently, golodirsen, an ASO for patients amenable to exon 53 skipping was approved by the US FDA in December 2019 and is applicable to about 10% of DMD patients [209]. Viltolarsen, also an exon 53 skipping ASO was approved in August 2020 [210]. Patients with deletion of exon 52, comprising about 2% of all DMD patients, are amenable to either exon 51 or 53 skipping [204]. Casimersen, an ASO for patients with variants amenable to exon 45 skipping was approved by the US FDA in February 2021 [209]. Chap. 17 discusses these therapies in detail. Nonsense Suppression Nonsense DMD mutations account for about 10% of the mutations. They result in premature stop codons, producing a truncated, non-functional protein [42]. The read-through strategy results in suppression of the stop codon, thus restoring the reading frame and producing partially functional dystrophin protein [211]. The proof of concept for this therapy was demonstrated by the use of the aminoglycoside antibiotic gentamicin [211]. Ataluren, an oral small molecule has a better safety profile and is effective in promoting read-­ through of the premature stop codon [212]. It received conditional approval in Europe in 2014 based on a phase 2 study which showed increased dystrophin expression on muscle biopsy. The trial narrowly missed significance although showing a 31 m improvement in 6MWD. However, after a second review of post-hoc data of rapidly declining patients

2 Dystrophinopathies

where the drug improved 6MWD by 68 m [213]. A phase 3 study failed to demonstrate improvement in 6MWD in ambulatory DMD patients, but showed a significant improvement of 43 m in 6MWD over 48 weeks in a pre-specified subgroup of patients with baseline 6MWD ≥300 m and A, c.1327T>C, c.1333G>A, c.1661A>C, c.1706T>C and c.1715G>C) [96–98]. The clinical phenotypes of these patients were broadly similar to those with the

two aforementioned deletions: asymptomatic hyperCKemia or relatively mild muscular dystrophy, often with late onset and prominent axial involvement. All dominant missense CAPN3 variants were located either in the proteolytic domain or in the calpain-type β-sandwich domain, and at least two of the variants impaired proteolytic activity in  vitro [96, 98]. Other missense variants may interfere with calpain 3 activity by disrupting binding of calmodulin, which is a positive regulator of calpain 3 activity [97]. Lastly, there have also been two reports of dominant calpainopathies related to a small 3-nucleotide deletion in CAPN3 (c.759-761delGAA) [105, 106]. Both patients presented with camptocormia beginning in their 60s and had mild or moderate pelvic girdle weakness. One patient also had lobulated fibers on biopsy. Calpain-3 levels were normal on Western blot, but proteolytic activity was impaired. It is notable that this variant was previously reported in combination with a second pathogenic variant in many individuals with LGMD-R1 [88, 107–111]. However, both of the patients discussed above reported similar symptoms in their fathers, suggesting dominant inheritance rather than a second variant that may have gone undetected. The c.759-761delGAA vari-

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ant is present in the gnomAD database with an allele frequency of 0.00004. The identification of this variant in both dominantly and recessively inherited types raise the ­possibility that some dominant and recessive calpainopathies may exist as a spectrum, rather than entirely distinct entities. The discovery of dominant calpainopathies related to missense variants creates a challenge for interpreting the significance of single variants in CAPN3. In these cases, evaluation of pathogenicity will require careful review of family history in search of subtle signs of myopathy in apparently unaffected relatives, as well as correlation with clinical findings, muscle imaging, western blot, and proteolytic activity.

LGMD-D5, Collagen VI-Related Clinical Phenotype Variants in the collagen VI genes are associated with a spectrum of clinical phenotypes. The most severely affected patients present in infancy with Ullrich congenital muscular dystrophy (UCMD), which is characterized by hypotonia, a

d

weakness, proximal joint contractures, torticollis, and distal hyperlaxity [112–114]. Most affected children never walk or lose the ability to walk before adulthood [115]. On the milder end of the spectrum, Bethlem myopathy (BM) presents with proximal weakness and contractures. The age of onset varies from childhood to mid-adulthood, but many patients do have a history of hypotonia or delayed motor milestones [112, 113, 116]. Some affected patients have congenital contractures that resolve within the first years of life, before reappearing at an older age. Long finger flexor contractures are a typical finding of the disease. Both the weakness and contractures in BM are slowly progressive, but most patients remain ambulatory into their 40s [115, 116]. BM is classified as LGMD-D5 according to the 2017 ENMC classification of LGMD (Fig. 5.4) [5]. An intermediate phenotype also exists between UCMD and BM. The relative severity of contractures and weakness is variable. In some patients, contractures are the predominant cause of disability, while a minority have minimal or no contractures [117–119]. Skin involvement can occur in any form of collagen VI-related myopathy, including keratosis pilaris, keloid scars, and a soft or velvety texture of the palms and feet [112]. Respiratory involvement is common in UCMD, and f

b e

c

Fig. 5.4  Clinical and histological findings of LGMD-D5. (a, b). Long finger flexor contractures preventing finger extension during wrist extension. (c, e). Achilles tendon contractures preventing passive dorsi-

flexion. (d) Keloid-type scar. (f) Variation in fiber size, fiber splitting, endomysial fibrosis and perimysial fatty infiltration without necrotic or regenerating fibers. Figure courtesy of Dr. Carsten Bonnemann

5  Autosomal Dominant Limb-Girdle Muscular Dystrophies

half of patients require initiation of non-invasive ventilatory support by age 11–12 [115, 120]. Respiratory involvement is less frequent in BM, but a minority of patients require ventilatory support despite remaining ambulatory, highlighting the need for monitoring of pulmonary function [115, 120]. CK levels in collagen-VI related myopathies are normal or mildly elevated, and EMG shows myopathic findings.

Muscle Imaging A typical pattern of muscle involvement has been described in collagen VI-related myopathies, which is characterized by T1 hyperintensity affecting the periphery of the vasti and gastrocnemius, along with the central region of the rectus femoris [121–123]. The gracilis, sartorius, and adductor longus are typically spared. This characteristic pattern can be used to distinguish Collagen VI myopathies from other myopathies with similar clinical features, including LGMDs and myopathies with prominent contractures. Similar imaging findings, however, have occasionally been reported in other myopathies, including those related to LAMA2, VWA1, and CAPN3 [124–126]. More severely affected patients with UCMD and intermediate phenotypes can have more diffuse muscle involvement, which obscures the classic findings of collagen VI-related myopathies [122].

Histopathology Histological findings in collagen VI-related myopathies evolve over the course of the disease [112, 113]. Early in the disease, even patients with UCMD may show only mild non-­specific myopathic abnormalities or fiber type disproportion [127]. Overt dystrophic features emerge over time with progression of the disease. Abnormalities of collagen VI immunostaining are the key histological hallmark of collagen VI-related myopathies. Staining is only absent in the minority of patients with recessive disease [128]. In dominant collagenopathies, collagen VI remains expressed, but the normal localization to the basement membrane is lost [129]. This is best seen by co-staining with another basement membrane marker, such as collagen IV, perlecan, or laminin [118, 127, 130].

Molecular Mechanisms Collagen VI is a key structural component of the extracellular matrix of many tissues, including muscle and skin. Each collagen VI monomer is composed of three different chains (α1, α2, and α3), which are encoded by COL6A1, COL6A2, and COL6A3, respectively [131]. These three chains associ-

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ate via their central triple helical domains to form a triple helix. Collagen VI monomers are then assembled via disulfide bonds into antiparallel dimers and tetramers, which form the collagen VI filaments [131, 132]. A minority of patients have recessive collagen VI-related myopathies due to biallelic truncating variants, which usually present with severe (UCMD) phenotype [128, 133, 134]. The majority of collagen VI-related myopathies are caused by variants interfering with filament assembly. The most common of these are located in the triple helical domains and consist of either splice site variants resulting in in-frame exon skipping, or missense variants affecting glycine residues in the conserved Gly-X-Y motifs. RNA sequencing also recently identified a recurrent deep intronic variant in COL6A1 that may be missed by conventional diagnostic testing such as exome sequencing [135, 136]. Assembly of collagen VI monomers proceeds from the C- to the N-terminal end [132]. Variants located near the C terminus prevent triple helix formation and thus act in a recessive fashion. In contrast, variants in the N-terminal region allow incorporation of the defective chain into the triple helix, but the resulting monomers are unable to associate with wild-type monomers to assemble into filaments [132]. This leads to a potent dominant negative effect. The phenotypes of affected patients span the range of severity from BM to UCMD.  Glycine substitutions in triplets 10-15 are associated with more severe disease [118], while milder phenotypes are seen with splice site variants that also disrupt the cysteine residues required for disulfide bond formation and dimer assembly [132, 137].

Caveolinopathies Clinical Phenotype Variants in CAV3, which encodes caveolin-3, have been associated with a number of neuromuscular phenotypes, including rippling muscle disease (RMD), limb-girdle weakness (formerly LGMD1C), distal myopathy, asymptomatic hyperCKemia, rhabdomyolysis, cardiomyopathy, and cardiac conduction abnormalities. Individual patients can present with an overlap of these features, and patients with the same underlying variant can present with discordant phenotypes, often even within the same family [138–141]. CAV3-related dominant limb-girdle weakness (formerly LGMD1C) was initially described in two Italian families with onset of mild-to-moderate proximal weakness at approximately age 5, as well as calf hypertrophy [142]. The affected individuals were found to have markedly reduced caveolin-3 expression by immunofluorescence and western blot, leading to the discovery of heterozygous variants in CAV3. Many more individuals with proximal weakness were subsequently described, either with or without muscle

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­rippling [138, 143–146]. Onset has been reported as late as the 70s [144]. RMD is the most distinctive presentation of caveolinopathies, characterized by wave-like contractions that slowly spread over the muscle following stretching or percussion [147, 148]. Other manifestations of RMD are percussion-­ induced rapid contraction and percussion-induced muscle mounding. The muscle contractions in RMD are usually (but not always) electrically silent, which was identified very early as a feature distinguishing RMD from myotonic disorders [147, 149]. The age onset is variable, ranging from childhood to early adulthood. It should be noted that RMD may also be caused by recessive variants in PTRF, encoding cavin-1, or by an acquired immune-mediated process due to anti-cavin 4 autoantibodies [150–152] PMID: 3569196. Less frequently, caveolinopathies can present with a distal myopathy involving upper and lower limb muscles, with or without concomitant RMD [153–156]. In some patients, the muscle involvement is prominently asymmetric [157, 158]. Exercise-induced muscle stiffness and myalgias are common in all CAV3-associated phenotypes and are sometimes the only manifestation of the disease [145]. In some patients, exercise intolerance is also associated with recurrent rhabdomyolysis [159]. Other patients have mild or moderate persistent hyperCKemia [109, 160, 161]. Some individuals present with only asymptomatic hyperCKemia [162, 163]. Caveolinopathies usually demonstrate dominant inheritance, with incomplete penetrance. There have however also been a limited number of reports of recessive inheritance with either RMD or LGMD phenotypes [164–168]. Some, but not all of these individuals were reported to have more severe disease, including extraocular muscle and cardiac involvement. In other cases, however, CAV3 variants reported to cause recessive disease were subsequently identified at higher than expected frequencies in the general population, including in the homozygous state [163, 169, 170]. Lastly, it should be noted that CAV3 variants have also been associated with cardiac phenotypes, including hypertrophic cardiomyopathy, dilated cardiomyopathy, long QT syndrome, and sudden infant death syndrome [146, 171– 173]. To date, overlap between cardiac and skeletal muscle phenotypes has only rarely been reported [168, 173].

Muscle Imaging There is only very limited data on muscle MRI in caveolinopathies. One study found preferential involvement of the rectus femoris and semitendinosus in four individuals with childhood-onset RMD [174], while another reported normal MRI findings in 3 of 4 patients presenting with exercise intolerance, myalgias, and rhabdomyolysis [159].

S. Nicolau and T. Liewluck

Histopathology Muscle histopathology in patients with CAV3-related myopathies shows a variable degree of dystrophic changes. Markedly reduced or absent sarcolemmal expression of caveolin-3 is a consistent hallmark of the disease, regardless of the presenting phenotype. Reduced caveolin-3 expression can also be seen by western blot. Whereas caveolin-3 expression is uniformly reduced in caveolinopathies, the reduction is patchy in immune-mediated RMD [151, 152].

Molecular Mechanisms Caveolins are a group of integral membrane proteins that oligomerize on the internal surface of caveolae, which are flask-shaped invaginations of the plasma membrane involved in endocytosis and other cellular processes. Caveolins play an important role in signaling processes by binding a number of signaling molecules in their inactive state [175, 176]. Caveolae have also been implicated in mechanosensing and protection against stretch-induced cellular injury [175]. Caveolin-1 and caveolin-2 are ubiquitously expressed, while caveolin-3 is the muscle-specific member of the family. Caveolin-3 has been associated with a number of functions in muscle fibers. Several lines of evidence point to a role for caveolin-3  in myoblast fusion and muscle development [177–179]. Caveolin-3 also plays a role in regulation of cellular energy metabolism, and its upregulation promotes myoblast growth [180]. T-tubule formation is also dependent on caveolin-3, and caveolinopathy patients demonstrate morphological abnormalities of T-tubules [181, 182]. Caveolin-3 is necessary to maintain plasma membrane expression of dysferlin, a key mediator of membrane repair in muscle disease [183]. Caveolin-3 is also known to co-localize and interact with the dystrophin glycoprotein complex [184]. Caveolin-3 and dystrophin bind to the same site on β-dystroglycan, and transgenic overexpression of caveolin­3  in mice results in a myopathic phenotype, with significantly reduced dystrophin and dystroglycan expression [185]. There are no consistent genotype-phenotype associations in caveolinopathies. The same variant can lead to different muscle phenotypes even within the same family [109, 141].

 iagnostic Approach to the Dominant D LGMDs Dominant LGMDs often lack distinctive clinical features, with the exception of LGMD-D3 and LGMD-D5. LGMD-D3 features early cataracts and finger flexor involvement, while LGMD-D5 features cutaneous findings, distal hyperlaxity,

5  Autosomal Dominant Limb-Girdle Muscular Dystrophies

early contractures, and a distinctive MRI appearance. A specific diagnosis in other dominant LGMDs is generally only possible by genetic testing. It is also important to remember that dominant LGMDs share many features with other categories of dominant myopathies (e.g. myotonic dystrophy type 2, myofibrillar myopathies and multisystem proteinopathies), and the latter will generally form the bulk of the differential diagnosis in any given patient. For example, LGMD-D5 shares clinical features with congenital myopathies, congenital muscular dystrophies, and Emery-Dreifuss muscular dystrophy. Likewise, the histological findings of dominant LGMDs also overlap with other groups of disorders: LGMD-D1 and LGMD-D2 show findings of myofibrillar myopathy, while LGMD-D3 shares pathological features with multisystem proteinopathies and vacuolar myopathies [77, 186, 187]. Finally, de- novo variants and reduced penetrance can account for the absence of a family history in some cases of dominant LGMDs. This leads to overlap with other myopathies, including recessive LGMDs, dystrophinopathies, and even acquired myopathies. As with many other inherited myopathies, the diagnosis of dominant LGMDs is generally best made through broad genetic testing, such as a large gene panel or whole exome sequencing [188]. It is important for clinicians to ensure such gene panels include not only genes associated with dominant LGMDs, but also their possible mimics. It is now becoming increasingly common to obtain genetic testing as a first-line diagnostic test, and to reserve muscle biopsy for cases that cannot be solved through genetic testing alone (e.g. if genetic testing is unrevealing or identifies a variant of uncertain significance). Nonetheless, decisions on the choice of first-line diagnostic test will need to be weighed on an individual basis.

Management of Dominant LGMDs At present, no disease-modifying therapy is available for dominant LGMDs. Gene therapy development efforts have been undertaken for LGMD-D5, but they are complicated by the necessity to achieve efficient allele-specific knockdown of the mutant allele (Chap. 18) [189–191]. Treatment for dominant LGMDs therefore remains largely supportive, including measures aimed at preserving mobility, preventing injuries, and managing respiratory and cardiac involvement [87]. To date, varying degrees of respiratory involvement have been found in LGMD-D1, D2, D3, and D5. Respiratory involvement has not been seen in LGMD-D4, but it does occur in recessive calpainopathies. Cardiac conduction abnormalities have been occasionally reported in LGMD-D1, and there is evidence implicating DNAJB6 in cardiac function, as discussed above. Cardiac involvement has not been reported in LGMD-D2, D3, and D4. LGMD-D5 is generally

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not thought to affect the heart, and the rare cardiac abnormalities seen in affected patients are generally thought to be unrelated to the disease [112, 192].

Conclusion In summary, the current LGMD classification includes five molecularly defined dominant LGMDs, caused by variants in the DNAJB6, TNPO3, HNRNPDL, CAPN3, and collagen VI genes. LGMD-D1-D4 are rare and their molecular basis was only discovered in the last 10 years. Increasing use of next generation sequencing has, however, identified a growing number of affected families, which has allowed a better understanding of the clinical spectrum of these disorders. In most cases, the clinical picture of dominant LGMDs overlaps considerably with recessive LGMDs and other proximal myopathies, though certain subtypes do have distinctive features, such as finger flexor involvement in LGMD-D3. Dominant LGMDs also share histological and pathophysiological features with other groups of myopathies. LGMD-D1 and D2 display features of myofibrillar myopathy, including protein aggregates and autophagic vacuoles, while LGMD-D3 is pathophysiologically and histologically related to vacuolar myopathies and multisystem proteinopathies. Meanwhile, the rapidly growing range of variants associated with LGMD-D4 will force changes to the traditional interpretation of single variants in CAPN3. Taken together, these discoveries have expanded our knowledge of the clinical and genetic spectrum of dominant myopathies, a process which is certain to continue in the coming years. Acknowledgements  The authors would like to thank Professor Bjarne Udd (of the Neuromuscular Research Center, University of Tampere and Tampere University Hospital, Tampere, Finland), Professor Corrado Angelini (of the Neuromuscular Lab, Department of Neurosciences, University of Padova, Padova, Italy), and Dr. Carsten G. Bonnemann (of the Neuromuscular and Neurogenetic Disorders of Childhood Section, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD, USA) for providing clinical, histological, and MRI images of LGMD-D1, LGMD-D2, and LGMD-D5, respectively.

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6

Autosomal Recessive Limb-Girdle Muscular Dystrophies Jantima Tanboon and Ichizo Nishino

Introduction Classification and Nomenclature of LGMD Walton and Nattrass classified limb-girdle muscular dystrophy (LGMD) in 1954 as a distinctive heterogenous group of neuromuscular diseases defined by early childhood to adulthood onset pelvic and shoulder girdle weakness [1]. LGMD was considered a subtype of muscular dystrophy, distinct from other described muscular dystrophies, including Duchene muscular dystrophy (DMD), facioscapulohumeral humeral muscular dystrophy (FSHD), myotonic dystrophy, distal myopathy, and ocular myopathy [1]. Walton and Nattrass observed a non-uniform but more benign clinical progression of LGMD compared to DMD [1]. In 1995, the European Neuromuscular Centre (ENMC) workshop study group (1995 ENMC-LGMD) introduced the alphanumeric system for LGMD classification and nomenclature, using LGMD1 and LGMD2 to indicate autosomal dominant (AD) and autosomal recessive (AR) mode of inheritance, respectively [2]. English alphabets were sequentially assigned to

J. Tanboon Department of Neuromuscular Research, National Institute of Neuroscience, National Center of Neurology and Psychiatry (NCNP), Tokyo, Japan Department of Genome Medicine Development, Medical Genome Center, National Center of Neurology and Psychiatry (NCNP), Tokyo, Japan Department of Pathology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand I. Nishino (*) Department of Neuromuscular Research, National Institute of Neuroscience, National Center of Neurology and Psychiatry (NCNP), Tokyo, Japan Department of Genome Medicine Development, Medical Genome Center, National Center of Neurology and Psychiatry (NCNP), Tokyo, Japan e-mail: [email protected]

the causative genes by the timeline of the linkage discovery [2]. Because of the evolutions of gene sequencing/molecular genetic technologies, the rapidly growing lists of LGMD2 associated genes exhausted all 26 alphabets in 2016 [3, 4]. In 2017, the ENMC LGMD workshop study group (2017 ENMC-LGMD) introduced the new LGMD nomenclature system using “LGMD-D” for dominant inheritance and “LGMD-R” for recessive inheritance; numbers were assigned for causative genes in order of their discovery, followed by the name of the affected protein (Table  6.1) [4]. The affected protein is also included in the nomenclature [4]. The 2017 ENMC-LGMD defines LGMD as a genetic condition primarily affecting skeletal muscle associated with progressive, predominantly proximal muscle weakness at presentation, increased serum creatine kinase (CK) level, degenerative changes (i.e., fatty replacement of skeletal muscle) over the disease course on muscle imaging, and dystrophic changes (i.e., presence of necrosis and regeneration of muscle fibers, fibrosis, and fatty infiltration) on muscle biopsy [4]. These criteria must be reported in at least two unrelated families to qualify a condition as a form of LGMD [4]. The patient must achieve independent walking before the disease onset to differentiate LGMD from congenital muscular dystrophy (CMD) [4]. In the 2017 ENMC-LGMD classification, nine disorders formerly recognized as LGMD subtypes (5 LGMD1 and 4 LGMD2) under the 1995 ENMC-LGMD classification were excluded because they did not satisfy the criteria (e.g., distal weakness, reported in only one family, histology not consistent with dystrophy) or false linkages (Table  6.2) and sarcoglycan-­related LGMDs were intentionally re-ordered by the series of Greek alphabets instead of their discovery timeline [4, 5]. Thus, some of the running numbers assigned for the causative genes in the 2017 ENMC-LGMD classification do not correspond to the alphabetical order in the 1995 ENMC-LGMD classification [5]. From our perspective, there are some inconsistencies in the 2017 ENMC-­ LGMD classification. For instance, unlike sarcoglycan-related

© The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 P. Narayanaswami, T. Liewluck (eds.), Principles and Practice of the Muscular Dystrophies, Current Clinical Neurology, https://doi.org/10.1007/978-3-031-44009-0_6

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94 Table 6.1  Autosomal recessive limb-girdle muscular dystrophy: the 2017 ENMC-LGMD classification Disease progressiona Slow

Extramuscular involvement Heart Lung Other feature(s) Rare +/−

Slow DMD-like DMD-like DMD-like DMD-like Slow

− + + + + Rare

+/− Distal weaknessb + + + + Rare Distal weaknessb

Slow Slow

+/− +

+/− +

Slow

+/−

+/−

Variable Slow

+/− Rare

? MRb Rare Asymmetrical involvementb ? + MRb, +/− brain abnormality −

AR-LGMD LGMD-R1

Old name Chromosome Gene LGMD2A 15q15.1 CAPN3

LGMD-R2 LGMD-R3c LGMD-R4c LGMD-R5c LGMD-R6c LGMD-R7

LGMD2B LGMD2D LGMD2E LGMD2C LGMD2F LGMD2G

LGMD-R8 LGMD-R9d

LGMD2H 9q33.1 LGMD2I 19q13.32

TRIM32 FKRP

LGMD-R10

LGMD2J

TTN

LGMD-R11d LGMD-R12

LGMD2K 9q34.13 LGMD2L 11p14.3

POMT1 ANO5

Age of onseta Childhoodadulthood Young adulthood Childhood Childhood Childhood Childhood Adolescent-Young adulthood Young adulthood Adolescent-Young adulthood Childhood-Young adulthood Childhood Adulthood

LGMD-R13d LGMD-R14d

LGMD2M 9q31.2 LGMD2N 14q24.3

FKTN POMT2

Childhood Childhood

? Variable

+/− +/−

LGMD-R15d

LGMD2O 1p34.1

POMGNT1

Rapid

−

LGMD-R16d

LGMD2P 3p21.31

DAG1

LGMD-R17 LGMD-R18d

LGMD2Q 8q24.3 LGMD2S 4q35.1

PLEC TRAPPC11

Infantile-­ childhood Childhood-­ adulthood Childhood Childhood

LGMD-R19d

LGMD2T 3p21.31

GMPPB

LGMD-R20d

LGMD2U 7p21.2

LGMD-R21d

LGMD2Z 3q13.33

CRPPA/ ISPD POGLUT1

LGMD-R22

LGMD-R23

− − − −

21q22.3 21q22.3 2q37.3 6q22.33

COL6A1 COL6A2 COL6A3 LAMA2

LGMD-R24d

−

3p22.1

POMGNT2

LGMD-R25

LGMD2X 6q21

LGMD-R26

−

2q31.2

DYSF SGCA SGCB SGCG SGCD TCAP

6q21

BVES/ POPDC1 POPDC3

14q32.33

JAG2

−

8p11.21

POMK

−

12p12.1

PYROXD1

LGMD-R27 POMK-­ relatedd PYROXD1-­ related

2p13.2 17q21.33 4q12 13q12.12 5q33.2-q33.3 17q12

Childhood-­ adulthood ChildhoodYoung adulthood Infantileadulthood Childhoodadulthood Childhoodadulthood Infantile-­ childhood Childhoodadulthood AdolescentAdulthood InfantileYoung adulthood Infantile-­ childhood Childhoodadulthood

Moderate-slow − Slow - > rapid Moderate

− −

−

+/−MR

Variable

+

Slow

−

+/−myasthenic features +/−MR, +/− liver, brain, eye abnormality Rare +/−MR, +/−myasthenic features + +/−MR, +/− brain abnormality +/−

Slow

−

+/−

Skin lesionsb,e

Slow

+/−

−

?

−

−

+/− epilepsy, +/−MR, +/− WM abnormality +/−MR

Slow

+

−

?

−

−

Slow

+/−

+

?

−

−

Slow

−

+

Moderate-slow +/−

+/− +/−

Axial weakness esp. neck flexor +/−MR, +/− brain abnormality

Note AR-LGMD autosomal recessive limb girdle muscular dystrophy, CK creatine kinase, + usually present, − usually absent/not reported, +/− variable, ? not enough information, MR mental retardation, WM white matter a Typical presentation b Common involvement c Sarcoglycanopathy-related LGMD d Alpha-dystroglycanopathy-related LGMD e Keloids, hypertrophic scars, hyperkeratosis pilaris

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6  Autosomal Recessive Limb-Girdle Muscular Dystrophies Table 6.2  Conditions previously classified as autosomal recessive limb-girdle muscular dystrophy Old name LGMD2R LGMD2V LGMD2W LGMD2Y

Chromosome 2q35 17q25.3 2q14.3 1q25.2

Gene DES GAA LIMS2/PINCH2 TOR1AIP1

New nomenclature Myofibrillar myopathy Pompe disease LIMS2/PINCH2-related myopathy TOR1AIP1-related myopathy

Reason for exclusion Distal weakness, muscle pathology of myofibrillar myopathy Known entity, muscle pathology of Pompe disease Reported in one family Reported in one family

LGMDs that are sequentially grouped by their discovery timeline despite the reordering based on the Greek alphabet, alpha-dystroglycan (α-DG)-related LGMDs when ordered by their discovery timeline are not sequential. In our opinion, α-DG-associated LGMDs should either be grouped by the associated-enzyme/protein affected or be arranged by the order of enzymes/proteins involved in glycosylation. This chapter summarizes underlying mechanism and clinicopathological features of LGMD-R. Collagen VI-, sarcoglycan- and α-DG-related LGMDs are grouped into separate sections. Except for α-DG-related LGMDs, all LGMDs are arranged in the numerical order of the new classification. The recently reported AR-LGMDs without officially assigned number (i.e., POMK- and PYROXD1-related LGMDs) are also included.

LGMD-R1: CAPN3-Related LGMD CAPN3 encodes calpain 3 (CAPN3), a cytoplasmic calcium-­ dependent cysteine protease preferentially present in skeletal muscle [6]. The interaction between CAPN3 and the N2A region of titin suppresses the rapid autodegradation property of CAPN3 [6]. Translocalization of CAPN3 from M-line to N2A region during sarcomere stretch facilitates recruitment of muscle ankyrin repeat protein 2 (MARP2), a mechanosensing protein involved in skeletal muscle differentiation, growth, and remodeling pathways [6–8]. Additionally, the interaction between CAPN3 and calcium-handling proteins is essential for calcium homeostasis [9, 10]. LGMD-R1, CAPN3-related LGMD, is the most common LGMD accounts for 20% -45% of genetically defined LGMD in large studies [11–14] with an estimated prevalence of 8.4 per million based on the public databases [15]. LGMD-R1 is the most common AR-LGMD in Western countries [11–14, 16]; it is the second most common AR-LGMD after LGMD-R2 (DYSF-related LGMD) in some countries in Latin America and Asia including Japan [17–19] (Fig. 6.1). LGMD-R1 is characterized by mild to moderate hyperCKemia with trunk muscle weakness and symmetrical and simultaneous (within 2  years) proximal muscle weakness of both the pelvic and shoulder girdles [20–25]. The age of onset varies from childhood to adulthood [20–25]. At presentation, pelvic girdle weakness is more common than shoulder girdle weakness [21–24]; rare patients who develop

Fig. 6.1  Frequency of autosomal recessive limb girdle muscular dystrophy subtypes in Japan. LGMD-R2 (dysferlin-related LGMD) and LGMD-R1 (calpain 3-related LGMD) are the most common LGMD subtypes in Japan. The data are based upon 192 Japanese AR-LGMD patients whose genetic diagnosis was made at the national center of neurology and neuropsychiatry (NCNP), Tokyo, Japan from September 2014 to December 2020. Abbreviations: DYSF dysferlin, CAPN3 calpain 3, SGCA alpha-sarcoglycan, POMGNT2 protein O-mannose β1,4-­N-­ acetylglucosaminyltransferase 2, ANO5 anoctamin 5, GMPPB guanosine diphosphate mannose pyrophosphorylase B, SGCG gamma-­sarcoglycan, FKRP fukutin-related protein, FKTN fukutin, POMT2 protein O-mannosyl-transferase 2, SGCB beta-sarcoglycan. “Others” includes CRPPA, cytidine diphosphate -ribitol pyrophosphorylase (previously known as ISPD, isoprenoid synthase domain-containing protein), LAMA2 laminin-α2, POMGNT1 protein O-mannose β1,2-Nacetylglucosaminyltransferase 1, TRAPPC11 transport protein particle complex 11 and MICU1, mitochondrial calcium uptake 1, 0.5% each. Courtesy of Dr. Mariko Okubo, MD, PhD. Department of Neuromuscular Research, National Center of Neurology and Psychiatry, Tokyo, Japan

shoulder girdle weakness before pelvic girdle weakness have older age of onset [22]. The most prominently affected muscles are hip adductors, hamstrings, and trunk extensors [23, 26] (Fig. 6.2a–c). The presence of contractures (usually mild at presentation), scapular winging, and typical pattern of muscle involvement suggest the diagnosis of LGMD-R1. Contractures most commonly affect the Archilles tendon, but can also affect finger, wrist, and elbow flexors [21, 23]. Loss of independent ambulation is observed approximately

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Fig. 6.2  Muscle imaging and pathology in LGMD-R1 (CAPN3-related LGMD). Muscle CT (a–c) of a 24-year-old man with LGMD-R1. At the pelvic level, fatty infiltration and atrophy of tensor fascia lata (green arrowhead), gluteus minimus (yellow arrowhead), gluteus medius (orange arrowhead), and gluteus maximus (pink arrowhead) are observed (a). Posterior thigh muscles, including biceps femoris (green arrowhead), semitendinosus (yellow arrowhead), semimembranosus (orange arrowhead), and adductor magnus (pink arrowhead), are severely affected. Mild fatty infiltration is observed in vastus intermedius (blue arrowhead) (b). At the calf level, soleus (green arrowhead)

and gastrocnemius (yellow arrowheads) are moderately and mildly affected, respectively (c). Muscle biopsy (d–e) shows fiber size variation, necrotic (black asterisks) and regenerating fibers (white asterisks), and increased endomysial connective tissue on hematoxylin and eosin stain (d). At higher magnification, scattered eosinophilic infiltration is observed (yellow arrowheads) (e). NADH-TR stain highlights lobulated fibers (yellow asterisks) (f). Note: c-f Bar = 20 microns CT, computed tomography; NADH-TR, nicotinamide adenine dinucleotide tetrazolium reductase. Muscle CT: Courtesy of Dr. Ohnmar, MD, Yangon General Hospital, Yangon, Myanmar

20  years after the onset [20–25]. Respiratory involvement can be present at late stage but is not prominent; cardiac involvement is rare [21, 23–25]. The “pseudocollagen sign“, characterized by striped appearance on T1 weighted MRI and other findings that mimic MRI appearance in COL6-related myopathy are commonly present in moderately affected muscles of lower limb e.g. “sandwich sign” in the vastus lateralis and “target sign” in the rectus femoris [26–28]. The “sandwich sign” is defined by the presence of high signal intensity on T1 weighted MRI at the periphery of the muscle, indicating more severe involvement with relative sparing of the central area in the vastus lateralis [27, 28]. The “target sign” shows the opposite pattern of involvement in rectus femoris; this pattern is also known as “central shadow” described on muscle ultrasound of COL6-related myopathy) [27–29]. The finding is associated with longer disease duration and more severe disease course [26]. Common muscle pathology findings include dystrophic features and lobulated fibers (Fig. 6.2d–f); lobulated fibers are seen on nicotinamide-adenine dinucleotide reductase (NADH-TR stain) and are likely caused by

increased expression of actin filament proteins (Fig.  6.2f) [30, 31]. Eosinophil infiltration in muscle tissue, which was previously erroneously termed “eosinophilic myositis”, may be observed in the early stage of the disease [32]. (Fig. 6.2e). The pathomechanism of eosinophil infiltration in LGMD-R1 has not been elucidated. This phenomenon is uncommon in our Japanese cohort (unpublished data). Protein analysis either by immunohistochemical study (IHC) or Western blotting (WB) in LGMD-R1 can be challenging. While severe reduction or complete loss of full-length CAPN3 detected by protein analysis is suggestive of CAPN3 deficiency, the result could be secondary to its rapid autodegradation property or secondary to a defect of other associated proteins; secondary CAPN3 reduction has been reported in patients with primary genetic defect in DYSF, FKRP ANO5, and TTN genes [24, 33]. In contrast, a normal amount of CAPN3 can be associated with mutations inactivating enzymatic function while preserving the amount of the protein. Decreased or absence of sarcolemmal dysferlin expression due to ­secondary deficiency, commonly observed in LGMD-R1, adds to the complexity of the analysis [21, 24, 33].

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LGMD-R2: DYSF-Related LGMD DYSF encodes dysferlin (DYSF), a plasma membrane and endosomal vesicle protein of the ferlin family ubiquitously present in human tissue [34, 35]. In skeletal muscle, dysferlin is present at T-tubule and sarcolemma. It regulates sarcolemmal repair, T-tubule formation and maintenance, calcium homeostasis, vesicle trafficking, and macrophage signaling [36–41]. Thrombospondin-1 is upregulated in dysferlin-­ deficient muscle, which in turn induces macrophage infiltration, leading to the sustained inflammatory process and myositis-mimic pathology [42]. Dysferlinopathy-related muscle weakness is classified based on the distribution of muscle weakness at onset into two subtypes: LGMD-R2 with predominantly proximal limb-girdle muscle weakness and Miyoshi myopathy (MM) with predominantly distal muscle weakness [43]. LGMD-R2 accounts for 18%–28% of reported LGMD [11–14] with an estimated prevalence of 7.5 per million based in public databases [15]. A growing body of evidence suggests that LGMD-R2 and MM are the same disease, as they show no significant difference in genotype or in the progression of the disease clinically and on imaging. Members of a family with the same mutation can present with either phenotype, and the phenotypes merge over time [43–46]. Thus, in our opinion, both conditions should be combined into a single entity, LGMD-R2/MM.  Most LGMD-R2/MM patients develop symptoms in early adulthood, but the onset can vary from early childhood to late adulthood [43, 45]. Inability to stand on tiptoes is a common feature of both conditions, due to weakness of the gastrocnemius [43]. The upper limb is less affected; distal upper limb weakness is not different in both conditions [43]. At presentation, the CK level is mildly to markedly elevated [45]. Selective muscle atrophy in the early stage creates unusual muscle bulges including “diamond on quadriceps sign”, “calf-head on a trophy sign”, and “boule du biceps” [47]. The “diamond on quadriceps sign” is caused by quadriceps atrophy, especially the upper and lower parts of vastus lateralis and rectus femoris, with relatively preserved central portion [48]. The “calf-head on a trophy sign” on the upper back and shoulders is caused by thinning of the trapezius muscle and a pattern of varying atrophy and hypertrophy of the deltoid, supraspinatus, infraspinatus and subscapularis; the most upper and lower borders of trapezius are relatively preserved or enlarged [49]. A biceps lump, “boule du biceps”, is caused by relatively spared proximal part of biceps short head with distal atrophy (Fig. 6.3) [50, 51]. On imaging, the gastrocnemius and the soleus are commonly affected, early in the disease course. A combination of early fatty infiltration of posterior calf, posterior thigh, pelvic, paraspinal, and scapular muscles supports the diagnosis of LGMD-R2/MM [46] (Fig.  6.4a–c). Loss of independent ambulation is usually observed approximately 20 years after

Fig. 6.3 Boule du biceps in LGMD-R2 (DYSF-related LGMD). Bulging of the proximal portion of short head biceps brachii (Boule du biceps, yellow arrowhead) at flexion compared to the distal portion (green arrowhead). Courtesy of Dr. Rasha El Sherif, MD, PhD, Myo-­ Care National Foundation, Cairo, Egypt

disease onset [22, 25]. Decreased forced vital capacity (FVC) to less than 80% predicted value is observed in 24–30% of patients with only rare requirement for non-invasive ventilation [45, 52]. Atrial electrical abnormality, abnormal P wave, is observed in 58% [52]; the finding may prone LGMD-R2 patients to develop atrial fibrillation. Other cardiac abnormalities are less likely to be associated with LGMD-R2 [52]. Muscle pathology can be variable, revealing a spectrum of abnormalities from mild myopathic changes to severe dystrophic changes [47]. Amyloid deposition in the walls of the intramuscular blood vessels and in the perimysial connective tissue is estimated to be present in 30% of LGMD-R2/MM, due to misfolded dysferlin protein ­ (Fig. 6.4d–f) [53, 54]. Inflammatory cell infiltration in the perivascular region and around necrotic fibers, consisting of macrophages and helper T-cells and expression of major histocompatibility complex I (MHC class I) on non-necrotic fibers, mimicking changes in inflammatory myopathy, is reported in 60–70% of patients and is a relatively early feature [47, 55, 56]; these findings are uncommon in our

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Fig. 6.4  Muscle imaging and pathology in LGMD-R2 (DYSF-related LGMD). Muscle CT (a–c) of a 43-year-old man with LGMD-R2. Pelvic muscles are less affected than thigh and calf muscles. At the pelvic level, there is mild fatty infiltration in the glutei (yellow arrowhead  =  gluteus minimus; orange arrowhead  =  gluteus medius; pink arrowhead = gluteus maximus) (a). At the thigh level, adductors (adductor magnus = pink arrowhead), semimembranosus (orange arrowhead), biceps femoris (green arrowhead), and the vasti are moderately to severely affected (vastus medialis = deep blue arrowhead; vastus intermedius  =  blue arrowhead; vastus lateralis  =  purple arrowhead). Sartorius (yellow arrow) and gracilis (green arrow) are mildly involved. Semitendinosus (yellow arrowhead) is relatively spared (b). At the calf level, soleus (green arrowhead) and gastrocnemius (yellow arrowheads)

are severely affected (c). Muscle biopsy, hematoxylin and eosin stain, shows fiber size variation, necrotic (black asterisk) and regenerating fibers (white asterisk), and endomysial fibrosis (d). Negative immunohistochemical staining for dysferlin is diagnostic for LGMD-R2 (e). Normal sarcolemmal staining for dysferlin (f). Amyloid myopathy in LGMD-R2 (g–i). Vessels with amyloid deposition are highlighted by Congo red stain (g). Amyloid deposits show typical apple green birefringent under polarized light (h). To detect Congo red-positive material (amyloid), fluorescence microscope with rhodamine filter is more sensitive than light microscope (i) Note: d-i bar = 20 microns. Muscle CT: Courtesy of Dr. Madoka Mori-Yoshimura, MD, PhD, Department of Neurology, National Center of Neurology and Psychiatry, Tokyo, Japan

Japanese series. Absent or severely reduced dysferlin expression by IHC or immunoblot in muscle tissue or by immunoblot in CD14+ peripheral blood mononuclear cells (PBMC) help confirm the diagnosis [47]. Very low protein levels are more likely to be detected by IHC than WB, although partial protein defects may be missed. Secondary deficiency of dysferlin on IHC can be seen in mutations in dysferlin associated proteins such as CAPN3 and caveolin 3, dystrophin, sarcoglycans, and others, resulting in false positives [47].

LGMD-R3-R6: Sarcoglycan-Related LGMDs The sarcoglycan complex (SGC), composed of α-, β-, γ-, and δ- sarcoglycans, is a transmembrane tetrameric subcomplex in the dystrophin-glycoprotein complex (DGC). SGC plays an important role in DGC and sarcolemmal stabilization [57, 58]. Mutations in sarcoglycan genes (SGCA, SGCB, SGCG, and SGCD encoding α-, β-, γ-, and δ- sarcoglycans, respectively) cause LGMD-R3 (α-sarcoglycan-related), LGMD-R4 (β-sarcoglycan-related), LGMD-R5 (γ-sarcoglycan-related),

6  Autosomal Recessive Limb-Girdle Muscular Dystrophies

and LGMD-R6 (δ-sarcoglycan-related); the conditions are collectively called “sarcoglycanopathies” [58–60]. The other sarcoglycans, ε- and ζ-sarcoglycans, are not associated with muscular dystrophy [57, 58]. ε-sarcoglycan (encoded by SGCE), consisting of the ubiquitously present isoform (ε-SG1) and the brain specific isoform (ε-SG2), is associated with myoclonus/ dystonia [61, 62]. ζ-sarcoglycan (encoded by SGCZ) is a functional γ-sarcoglycan homologue present in various tissue but abundantly present in the brain [63]; ζ-sarcoglycan has yet to be associated with human disease [58]. The estimated prevalence of LGMD-R3, LGMD-R4, LGMD-R5, and LGMD-R6 are 3.4, 0.8, 0.17, and 0.07 per million, respectively [15]. Sarcoglycanopathies are ­characterized by rapidly progressive predominantly proximal lower limb weakness, a DMD-like phenotype [59, 60, 64]. Axial muscle weakness, scoliosis, joint contractures, calf hypertrophy, and scapular winging are frequent [59, 60, 64]. The age of onset varies from early childhood to adulthood [59, 60, 64]. LGMD-R3 (α-sarcoglycan-related) associates with the later age of onset and slower disease progression [59, 64]. Respiratory insufficiency and dilated cardiomyopathy are common and usually develop approximately 20  years after disease onset, although the timeline can be variable [59, 60, 64]. Overall cardiac involvement (including cardiomyopathy and heart rhythm abnormalities) is more common in LGMD-R4 (β-sarcoglycan-related) than the other subtypes [59, 64]. On muscle imaging, the most severely and earliest affected muscles are glutei (especially gluteus maximus), thigh adductors (especially adductor magnus) and posterior thigh groups [65, 66]. A proximodistal gradient of involvement is commonly observed in vastus lateralis and other severely affected muscles [65]. A pattern of concentric fatty infiltration around the distal femoral diaphysis, affecting the vastus intermedius and vastus medialis with sparing of the rectus femoris, vastus lateralis and short head of the biceps femoris is described in 14/17 (83%) of patients in one study [66]. The lower leg muscles are relatively spared [65, 66]. Muscle biopsy shows myopathic or dystrophic changes. Reduction of one or more sarcoglycans on IHC is suggestive of sarcoglycanopathy. However, the result is not genotype specific because the expression of all four sarcoglycans on IHC can be variable across the sarcoglycanopathies [67]. On WB analysis, expression of α- and γ-sarcoglycan can be reduced in all sarcoglycanopathies [67]. The interpretation of IHC and WB in sarcoglycanopathies is further complicated by alterations of expression of the DGC proteins and vice versa [67]. In one study, only 19% of specimens were correctly genotyped based on their IHC and WB expression patterns [67]. In genetically confirmed cases, the level of protein expression tends to inversely correlate with disease severity and the rate of progression of weakness [59, 64].

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LGMD-R7: TCAP-Related LGMD TCAP encodes titin cap (TCAP) also known as telethonin, a multifunctional sarcomeric protein of skeletal and cardiac muscle [68, 69]. Telethonin forms a titin-telethonin complex anchoring titin to the Z-disc and interacts with other proteins such as myostatin, FATZ (filamin-, actin- and telethonin-­ binding protein of the Z-disc), and Ankrd2 (Ankyrin repeat domain-containing protein 2); it is suggested that titin regulates the development and maintenance of the sarcomere, maintains muscle mass, and is involved in cell signaling [70–76]. The estimated prevalence of LGMD-R7, TCAP-­ related LGMD [77, 78], is 0.04 per million [15]. The usual age of onset is in adolescence or young adulthood; childhood onset is less frequently observed. Typical clinical presentations include proximal muscle weakness, early distal muscle involvement, and calf hypertrophy [79, 80]. The CK level is mildly to moderately elevated [79–81]. Asymmetric involvement [80–84], early joint contractions [79, 80, 82, 84], and scapular winging [79, 80] are described. Common muscle imaging findings include involvement of the posterior compartment of the pelvic muscles and involvement of all compartments of the thigh muscles; sartorius and gracilis are relatively spared [80]. The disease is slowly progressive; loss of ambulation develops in the fourth decade [79–81]. Cardiopulmonary involvement is rare and mild [79, 80]. Common pathological findings in muscle include internalized nuclei and lobulated fibers; rimmed vacuoles and nemaline bodies are occasionally reported [77, 79–84]. Absence of telethonin protein on IHC or WB is diagnostic [78, 79].

LGMD-R8: TRIM32-Related LGMD TRIM32 encodes tripartite motif-containing 32 (TRIM32), a ubiquitous multifunctional E3 ubiquitin ligase involved in muscle and nerve homeostasis, glucose metabolism, and tumorigenesis [85, 86]. In skeletal muscle, TRIM32 regulates myoblast proliferation and differentiation, senescence of satellite cells, p62-related autophagy, and interacts with various proteins [86–88]. TRIM32 contains a tripartite motif (a RING finger domain, two B-box domains, and a coiled-­ coil region) in the N-terminal and six NHL repeats on the C-terminal [85, 86]. Mutations in B-box domains are associated with Bardet-Biedl syndrome (BBS) characterized by obesity, pigmentary retinopathy, polydactyly, hypogonadism, renal and cardiac abnormalities, and cognitive impairment [89]. Most cases of LGMD-R8 are due to mutations in NHL repeats [85–87, 90–94]. RING domain mutations are associated with mixed LGMD-R8 and BBS phenotypes [87]. A few patients develop concomitant neurogenic changes or cognitive impairment, possibly due to alteration of protein

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interactions [91–94]. Initially described in Hutterites, the disorder has been subsequently reported in other ethnic groups [90, 95, 96]. Most LGMD-R8 patients present with predominantly proximal muscle weakness with normal CK or mild to moderate hyperCKemia in early adulthood. However, the age of onset can range from the first to the fifth decade of life [90–96]. Facial, axial, and distal muscle weakness, and scapular winging are variable [87, 90]. Cardiac and respiratory involvement is observed at late stages [91, 92]. The disease is slowly progressive and loss of independent ambulation occurs approximately 20 years after the disease onset [90]. On imaging, the posterior muscles of thigh and calf are preferentially affected. Anterior thigh muscle involvement is observed at late stages [90]. More diffuse and severe involvement is observed in non-NHL mutations [87, 94]. Muscle biopsy may contain rimmed vacuoles or segmental multiple small vacuoles, that qualify for a diagnosis of sarcotubular myopathy (STM), or nonspecific dystrophic changes [87, 97]. Absence or reduction of TRIM32 on WB is diagnostic [87, 92].

J. Tanboon and I. Nishino

guanosine triphosphate (GTP) [102]. GDP-Man and dolicholphosphate (Dol-P) are required for Dol-P-Man synthesis in the endoplasmic reticulum (ER) membrane by dolichol-phosphate-mannose (DPM) synthase complex (DPM1, DPM2, and DPM3) [103]; dolichol kinase (DOLK) is required for the formation of Dol-P [104]. In the first step of O-mannosylation, protein O-mannosyl-transferase 1 (POMT1)/POMT2 complex transfers O-mannosyl residue from Dol-P-Man to serine/ threonine residues of α-DG [101]. For core M1 and core M2, protein O-mannose β1,2-N-­acetylglucosaminyltransferase 1 (POMGNT1) catalyzes the transfer of β1,2-linked GlcNAc to O-mannose of α-DG in the Golgi apparatus [105]. N-acetylglucosaminyltransferase-IX (GnT-IX also known as GnT-Vb) is responsible for β1,6-­linked GlcNAc branching in core M2. Notably, O-mannosyl glycans with core M2 are only present in the brain because GnT-IX/Vb is specifically expressed in the brain [106, 107]. Core M3 is synthesized in ER by sequential action of POMT1/POMT2 complex, protein O-mannose β1,4-N-­acetylglucosaminyltransferase (POMGNT2) and β1,3-N-­acetylgalactosaminyltransferase (B3GALNT2). POMGNT2 adds β1,4-linked GlcNAc to the O-mannosyl residue [108]. Subsequentially, B3GALNT2 Alpha-Dystroglycan-Related LGMD adds β1,3-linked GalNAc [108]. Protein O-mannose kinase (POMK) phosphorylates the C6 hydroxyl of mannose after LGMD-R9, LGMD-R11, LGMD-R13–R16, synthesis of core M3 [108]. The phosphorylated core M3 is LGMD-R18–R21, and LGMD-R24 further modified in the Golgi apparatus by fukutin (FKTN), fukutin-related protein (FKRP), ribitol-5-phosphate xylosylDystroglycan (DG), encoded by dystrophin-associated glyco- transferase (RXYLT1 previously known as transmembrane protein 1 (DAG1) gene, is the major non-integrin adhesion protein 5,TMEM5), β1,4-glucuronyltransferase (B4GAT1 also complex in many developing and mature mammalian tissues known as β1,3-N-­acetylgucosaminyltransferase 1, B3GNT1), including skeletal muscle and brain. DG is composed of a and like-­acetylglucosaminyltransferase (LARGE). In cytosol, highly glycosylated extracellular alpha-subunit cytidine diphosphate -ribitol pyrophosphorylase (CRPPA pre(α-dystroglycan, α-DG) anchored to a transmembrane beta-­ viously known as isoprenoid synthase domain-containing prosubunit (β-dystroglycan, β-DG), forming a molecular link tein, ISPD) synthesize cytidine diphosphate -ribitol between the extracellular matrix (ECM) and the intracellular (CDP-Rbo), the donor substrate for FKTN and FKRP [109, cytoskeleton [98]. α-DG binds to the laminin-globular 110]. FKTN transfers the first ribitol phosphate (RboP) from domains of various ECM binding partners including laminin, CDP-Rbo to GalNAc in CoreM3. Then, FKRP sequentially agrin and neurexin; this binding is calcium-dependent, and transfers the second RboP from CDP-Rbol to the first RboP the binding affinity is likely dependent on the level of forming tandem RboP on core M3 [111]. Xylose and glucO-mannosyl glycosylation (O-mannosylation) of α-DG [99, uronic acid are added at the end of tandem RboP by RXYLT1 100]. O-mannosyl glycans are classified into three core types and B4GAT1, respectively [112–114]. LARGE extends the based on the linkage of N-acetylgluocosamine (GlcNAC) to xylose and glucuronic acid disaccharide repeating unit formthe core mannose (Man): core M1 (GlcNAc-β1,2-Man), core ing full length matriglycan providing binding site for matrix M2 [GlcNAc-β1,2-(GlcNAc-β1,6)-Man], and core M3 ligands [115, 116]. [N-acetylgalactosamine (GalNAc)-β1,3-GlcNAc-β1,4Disorders related to DAG1 mutations directly affected (phosphate-6)-Man] [98](Fig. 6.5). The core M1, M2, and DG function and structure, and are regarded as “primary M3 of α-DG use dolichol-phosphate-mannose (Dol-P-Man) α-dystroglycanopathy”. On the other hand, conditions as the substrate in the first step of O-mannosylation [101]. In related to mutations in genes encoding essential enzymes for cytosol, guanosine diphosphate-mannose pyrophosphorylase O-mannosylation and associated trafficking proteins are (GMPPB) catalyzes guanosine diphosphate-mannose (GDP- regarded as “secondary α-dystroglycanopathy”. To date, Man) synthesis from mannose-1-phosphate (Man-­1-­P) and α-dystroglycanopathy associated with the LGMD phenotype

6  Autosomal Recessive Limb-Girdle Muscular Dystrophies

Fig. 6.5 Protein O-mannosylation of alpha-dystroglycan (α-DG): Dystroglycan (DG) is encoded by a single gene (DAG1) and cleaved into α-DG and β-DG (not shown). After posttranslational O-mannosylation, α-DG can be classified into three types of O-mannosylglycans based on the linkage of N-acetylgluocosamine (GlcNAC) to the core mannose: core M1, core M2 and core M3. The synthesis of O-mannosylglycans starts in endoplasmic reticulum (ER) by protein O-mannosyltransferase 1 (POMT1) and POMT2 catalyzing the transfer of mannose residue from dolichol-phosphate-mannose to serine/threonine residue of α-DG (step 1). In core M1 and core M2, the addition of GlcNAc to the O-mannosyl residue (step 2) and the following steps occur in Golgi apparatus. In core M1, protein O-mannose β1,2-N-acetylglucosaminyltransferase 1 (POMGNT1) catalyzes the β1,2-linkage of GlcNAc to O-mannose. The branching β1,6-linkage of GlcNAc to O-mannose in core M2 is catalyzed by N-acetylglucosaminyltransferase-IX (GnT-IX). The further synthesis of core M1 and core M2 peripheral structures involves various glycosylation enzymes (not shown). In core M3, protein O-mannose β1,4-N-­ acetylglucosaminyltransferase 2 (POMGNT2) catalyzes β1,4-linkage of GlcNAc to the O-mannosyl residue (step 2) and β1,3-N-­ acetylgalactosaminyltransferase (B3GALNT2) sequentially adds β1,3-­ linked N-acetylgalactosamine (GalNAc) to the POMGNT2 product (step 3). Protein O-mannose kinase (POMK) phosphorylates the O-mannose that is previously modified by both POMGNT2 and B3GALNT2 (step 4). The core M3 is further modified in the Golgi apparatus. Two ribitol phosphate molecules are sequentially added to GalNAc by fukutin (FKTN) and fukutin related protein (FKRP) forming tandem ribitol phosphate (step 5). Ribitol-5-phosphate xylosyl-

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transferase (RXYLT1) and β1,4-glucuronyltransferase (B4GAT1) sequentially adds xylose (step 6) and glucuronic acid (step 7) at the end of tandem ribitol phosphate. Extension of the xylose and glucuronic acid disaccharide repeating unit forming the full length matriglycan (step 8) is catalyzed by like-acetylglucosaminyltransferase (LARGE). Notably, the synthesis of dolichol-phosphate-mannose requires the activities of the following enzymes: guanosine diphosphate-mannose pyrophosphorylase (GMPPB), dolichol kinase (DOLK) and dolichol-­ phosphate-­ mannose (DPM) synthase complex (DPM1, DPM2, and DPM3). The synthesis of cytidine diphosphate -ribitol, the donor substrate for FKTN and FKRP, requires cytidine diphosphate -ribitol pyrophosphorylase (CRPPA) activity. Transport protein particle complex 11 (TRAPPC11) involves in ER to Golgi apparatus trafficking. Protein O-glucosyltransferase 1 (POGLUT1) involves in O-glucosylation and O-xylosylation to modify and regulate epidermal growth factor (EGF)like repeats in several proteins including Notch and its ligands. POGLUT1 recessive mutations associate with defects in Notch pathway and secondary alpha-dystroglycan hypoglycosylation. Jagged Canonical Notch Ligand 2 (JAG2) involves in Notch pathway and limb girdle muscular dystrophy (LGMD) R27 (JAG2-related LGMD) but not α-DG-related LGMD. Note: The numbers in circle indicate steps in O-mannosylation. Enzymes/proteins in red color are associated with α-DG-related AR-LGMD (see text). B3GNT1, β1,3-N-­ acetylgucosaminyltransferase 1; CDP, cytidine diphosphate; CTP, cytidine triphosphate; Dol, dolichol; GDP, guanosine diphosphate; GTP, guanosine triphosphate; ISPD, isoprenoid synthase domain-containing protein; TMEM5; transmembrane protein 5

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is caused by mutations in DAG1, GMPPB, POMT1, POMT2, POMGNT1, POMGNT2, POMK, CRPPA (ISPD), FKTN, FKRP, TRAPPC11, and POGLUT1, (see below). Alpha-dystroglycanopathy is a collective term for a wide range of clinically distinct disorders: CMD with brain and eye abnormalities including Walker-Warburg Syndrome (WWS), muscle-eye-brain disease (MEB), Fukuyama ­congenital muscular dystrophy (FCMD); CMD with intellectual disability without structural abnormality (CMD-ID; CMD without intellectual disability (CMD-no ID); LGMD with intellectual disability (LGMD-ID); LGMD without intellectual disability (LGMD-no ID) and rarely, asymptomatic hyperCKemia [117, 118]. WWS has onset prenatally or at birth, and children have severe brain anomalies including agyria, lissencephaly, hydrocephalus, cerebellar involvement and agenesis of the corpus callosum. Eye abnormalities include congenital cataracts, microphthalmia and buphthalmos. Motor development is absent and death usually occurs before 1  year of age. MEB disease is associated with less severe brain anomalies than WWS. Brain MRI shows polymicrogyria, pachygyria, cerebellar dysplasia and brainstem anomalies. Eye abnormalities are similar to WWS.  Rare patients acquire walking and learn to speak a few words. Muscle biopsy findings in these disorders include myopathic or dystrophic changes. IHC and WB show decreased or absent α-DG core protein and glycosylated epitopes; the latter can be detected by VIA4–1 and IIH6 which detect the laminin-binding glycan on α-DG. However, when glycosylation is incomplete, the interpretation of VIA4–1 and IIH6 immunoreactivity can be difficult. Laminin overlay assay shows reduced laminin binding activity (Fig. 6.6). Notably, clinical severity, histopathology changes, and the levels of glycosylated α-DG levels are not consistently correlated [119].

LGMD-R9: FKRP-Related LGMD FKRP encodes fukutin-related protein (FKRP), which is localized in the Golgi apparatus [120]. FKRP and FKTN, both RboP transferases, sequentially transfer RboP to the core M3 [110, 111]. FKRP also regulates fibronectin sialylation within the Golgi; alteration of this property and fibronectin-collagen function may provide explanation for the broad phenotypic spectrum of FKRP-related mutations [121]. Notably, LGMD-R9 is the most common phenotype of FKRP-related mutations with an estimated prevalence of 4.5 per million [15, 122]. Most patients carry a homozygous c.826C > A (p. Leu276Ile) FKRP mutation and present with a mild form of LGMD [122–124]. Other presentations

J. Tanboon and I. Nishino

include exertional fatigue and exercise-induced rhabdomyolysis/myoglobinuria [125]. The disease onset is usually in the second decade of life and is sometimes precipitated by an antecedent febrile illness [126]. Moderate to marked hyperCKemia, calf hypertrophy, and cardiopulmonary involvement are common [122–125]. In patients with homozygous p. Leu276Ile, loss of ambulation occurs approximately 30 years after the onset. Patients with compound heterozygous p.Leu276Ile mutations are more severely affected with earlier age of onset and age of loss ambulation [122]. On muscle imaging, the concentric fatty infiltration of muscles around the distal femoral diaphysis is observed with progressive postero-anterior muscle involvement [127]. Peripheral sparing of vastus lateralis reversing the “sandwich” pattern COL6-related LGMD is observed [127].

LGMD-R11: POMT1-Related LGMD POMT1 encodes protein O-mannosyltransferase 1 (POMT1), the critical enzyme in the first step of O-mannosylation pathway in α-DG [98]. Co-expression of POMT1 and its homolog POMT2 in the ER is required to transfer the O-mannosyl residue from Dol-P-Man to serine/threonine residues [101]. Interestingly, murine POMT1 (mPOMT1) is highly expressed in brain, muscle, and eyes of mice embryos and in skeletal and cardiac muscle of the adult mouse. This corresponds to the organ involvement in human POMT1- mutations [128]. Notably POMT1 is the most common causative gene for WWS [129]. POMT1-related LGMD is characterized by childhood onset LGMD-ID (typically without structural brain anomalies) with moderate to marked hyperCKemia [130–132]. Variable contractures, calf hypertrophy, and cardiomyopathy are reported [130–132]. Information on muscle imaging is limited; a pattern reported in two siblings suggests progressive postero-anterior thigh muscle involvement [133]. Loss of ambulation occurs at a variable age [132].

LGMD-R13: FKTN-Related LGMD FKTN encodes fukutin (FKTN), a RboP transferase localized in the Golgi apparatus. FKTN transfers RboP from CDP-Rbo to the core M3 protein of α-DG; FKRP then transfers the second RboP to the first one [110, 111]. FKTN mutations are commonly associated with FCMD, the second most common muscular dystrophy in Japan after DMD [134–136]. FCMD is characterized by CMD with cortical dysgenesis and eye abnormalities; the overall features can be categorized as a spectrum of MEB [117, 134]. Other FKTN-­

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a

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b

c

Fig. 6.6  Pathologic diagnosis of alpha-dystroglycanopathy. Muscle biopsy, hematoxylin and eosin stain shows moderate to marked fiber size variation with clusters of regenerating fibers (yellow arrowhead), increased number of fibers with internalized nuclei and endomysial fibrosis (a). Decreased or absent immunohistochemical stain for glycosylated alpha-dystroglycan (IIH-6) compared to the control (inset) (b). Western blotting (c) shows normal beta-dystroglycan in both patient and control. The patient’s specimen shows decreased molecular weight

of alpha-dystroglycan core protein, with loss of activity against glycosylated aDG antibody (VIA4–1) and decreased laminin binding in the laminin overlay assay. Note: a–b Bar  =  50 microns; aDG, alpha-­ dystroglycan; bDG, beta-dystroglycan; C, control; P, patient. Western blotting and laminin overlay assay: Courtesy of Ms. Megumu Ogawa and Dr. Yoshihiko Saito MD, PhD, Department of Neuromuscular Research, National Center of Neurology and Psychiatry, Tokyo, Japan

associated conditions outside Japan are rare and mostly characterized by mild LGMD phenotype with early childhood to juvenile onset proximal muscle weakness, calf hypertrophy, normal intelligence and brain structure (135–

137). Cardiac involvement is variable [135, 137]. Information on muscle imaging is limited although a pattern suggests progressive postero-anterior thigh muscle involvement [137].

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LGMD-R14: POMT2-Related LGMD

LGMD-R18: TRAPPC11-Related LGMD

POMT2 encodes protein O-mannosyltransferase 2 (POMT2), the ER-located protein O-mannosyl transferase homologous to POMT1. There are two murine POMT2 transcripts, somatic specific (sPOMT2) and testicular specific (tPOMT2) [138]. Notably, sPOMT2 is prominently expressed in developing muscle, eye, and brain [138]. POMT2-related LGMD is characterized by early childhood onset (or infantile onset in the case of CMD) weakness commonly associated with moderately to highly elevated CK and intellectual disability (LGMD-ID) [139, 140]. Brain structural abnormality is ­variable [139, 140]. Prominent involvement of the gluteus, paraspinal and hamstring muscles is noted [139]. Scapular winging, scoliosis, calf hypertrophy, and cardiopulmonary involvement are present in some cases [139, 140]. Loss of ambulation occurs approximately 30  years after the onset [139].

Transport protein particle (TRAPP) complex is essential for transportation of newly secreted protein via membrane-­ bound vesicles from the ER to the Golgi apparatus [150]. TRAPPC11 encodes transport protein particle complex 11 (TRAPPC11) which is necessary for the integrity and stability of the TRAPP complex and for the synthesis of lipid-­ linked oligosaccharides [150, 151]. TRAPPC11 mutations are associated with secretory cargo retention, hypoglycosylation, and unfolded protein response [151]. Although TRAPPC11 mutations were originally thought to be associated with hypoglycosylation of N-linked oligosaccharides [151], defects in both N-linked and O-linked glycosylation with a delayed vesicular transport in Golgi apparatus were reported in one case [152]. TRAPPC11-related conditions include congenital disorder of glycosylation (CDG); CMD-ID with/without structural brain anomaly; CMD with fatty liver and infantile-onset cataract; myopathy with cerebral atrophy, intellectual disability, scoliosis, achalasia and alacrimia; myopathy with intellectual disability and movement disorder (ataxia, chorea); and LGMD [152–158]. TRAPPC11-related LGMD is rare and characterized by childhood onset with mild to marked hyperCKemia [153, 157]. Restrictive respiratory dysfunction, variable intellectual disability, and eye abnormalities (cataract, esotropia, myopia, amblyopia) are noted [153]. Hip dysplasia and scoliosis were reported in siblings from one family [153]. Other features include skeletal deformities such as microcephaly, retrognathia, camptodactyly and pes cavus. Hepatic cholestasis, thrombocytopenia, nephropathy and osteopenia were reported in one case [152]. Rare features include abnormal electroencephalogram (EEG) or history of seizures, and cerebral atrophy on MRI [153]. Severely limited mobility is observed approximately 20 years after disease onset [153]. Muscle biopsy shows non-specific myopathic changes. Immunoblot of cultured fibroblasts shows TRAPPC11 reduction [153]. Because abnormal α-DG and glycosylation is demonstrated in muscle tissue, but not in cultured fibroblasts of TRAPPC11-related CMD [159], similar findings are expected in LGMD-R18.

LGMD-R15: POMGNT1-Related LGMD POMGNT1 encodes protein O-mannose β1, 2-N-acetylglucosaminyltransferase 1 (POMGNT1), a glycosyltransferase located in the Golgi apparatus. POMGNT1 participates in the formation and clustering of α-DG core M1. The POMGNT1-FKTN complex is also required for the modification of core M3 [105]. Among the α-DG-related disorders, POMGNT1-mutations are commonly associated with MEB and rarely with LGMD [117, 141–143]. To our knowledge, only three POMGNT1-related LGMD patients have been reported, characterized by infantile to childhood onset weakness with normal intelligence, mildly to markedly elevated CK level, and lordosis [143–145]. Severe myopia and calf hypertrophy were observed in one patient [144]. Profound weakness was noted after 5–7  years of disease onset [144, 145].

LGMD-R16: DAG1-Related LGMD DAG1 mutations are rare and associated with a wide range of phenotypes including WWS, MEB, LGMD-ID without structural anomaly, LGMD, and asymptomatic hyperCKemia [118, 143, 146, 147]. Most reported patients have brain abnormalities or cognitive impairment. To our knowledge, four patients with DAG1-related LGMD have been reported: one patient presented with childhood-onset LGMD-ID without structural brain anomaly, hyperCKemia, calf hypertrophy, and ankle joint contractures [148, 149]; two siblings presented with onset in their 30 s of mild LGMD [147]; and one had LGMD without information on the age of onset [143].

LGMD-R19: GMPPB-Related LGMD GMPPB encodes guanosine diphosphate mannose pyrophosphorylase B (GMPPB), located in ER, which catalyzes the formation of GDP-Man from GTP and Man-1-P [102]. GMPPB-related LGMD is associated with childhood or adulthood onset proximal muscle weakness and moderate hyperCKemia with and without intellectual disability. On imaging, the most affected muscles are erector spinae, hamstrings, and gastrocnemius [102, 160–163]. Calf hypertro-

6  Autosomal Recessive Limb-Girdle Muscular Dystrophies

phy and cardiac involvement are variable. Respiratory involvement is rare [143, 160–167]. Loss of ambulation is reported approximately 15 and 40 years after disease onset in a childhood-onset and an adult-onset patient, respectively [165, 166]. In addition to the clinical spectrum usually described in α-dystroglycanopathy, GMPPB mutations are also associated with overlapping myasthenic and myopathic features including congenital myasthenic syndrome (CMS) with myopathic features and LGMD/CMS [143, 160, 163– 167]. Abnormal glycosylation of acetylcholine receptor ­subunits is the likely cause for the myasthenic features [164]. In the setting of early childhood to adulthood onset proximal muscle weakness without facial/ocular involvement and moderately elevated CK, evidence of decremental response on repetitive nerve stimulation and evidence for abnormal glycosylation detected by IHC or WB is highly suggestive of GMPPB mutations and should prompt genetic testing for this disorder [160, 164]. Not all GMPPB-related LGMD are associated with myasthenic features and GMPPB-related myasthenic symptoms show variable response to symptomatic treatments with acetylcholine esterase inhibitors [160, 164].

LGMD-R20: CRPPA/ISPD-Related LGMD CRPPA (previously known as ISPD, isoprenoid synthase domain-containing protein) encodes CDP-L-ribitol pyrophosphorylase A (CRPPA) in the Golgi apparatus. This enzyme synthesizes CDP-Rbo from cytidine triphosphate (CTP) and RboP [110]; CDP-Rbo is used by FKTN and FKRP in the next steps of O-mannosylation [110]. CRPPA/ISPD-related LGMD is associated with childhood to early adulthood onset proximal muscle weakness with a DMD-like pattern of muscle involvement. The CK level ranges from mildly to markedly elevated. Cardiopulmonary involvement is present in late disease [168, 169]. Intellectual disability with or without structural brain abnormality, cerebellar involvement (cysts, hypoplasia), brainstem hypoplasia and fourth ventricular dilatation are occasionally present [168]. The clinical progression is variable. Childhood onset is reported to be associated with earlier loss of ambulation [168, 169]. On imaging, the thigh and calf muscles are diffusely affected but sartorius, gracilis, tibialis anterior, and tibialis posterior are relatively spared even in severely affected patients [168, 169].

LGMD-R21: POGLUT1-Related LGMD POGLUT1 encodes protein O-glucosyltransferase 1 (POGLUT1), located in the ER.  POGLUT1 catalyzes O-glucosylation and O-xylosylation to modify and regulate

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epidermal growth factor (EGF)-like repeats in several proteins including Notch and its ligands [170, 171]. O-glucosylation is essential for NOTCH1 receptor trafficking to the cell surface where it binds Notch ligands. In skeletal muscle, Notch signaling promotes quiescence (a non-proliferative state) in muscle stem cells (satellite cells) [172]. POGLUT1 mutations impair Notch signaling and reduce the number of satellite cells [3]. LGMD-R21, POGLUT1-related LGMD, is associated with slowly progressive proximal muscle weakness, scapular winging, and normally to mildly elevated CK level. The weakness predominantly affects the lower extremities [3, 173]. Onset ranges from infantile to the fifth decade. Patients with adolescent onset develop loss of ambulation during the third to fifth decades. Variable respiratory involvement is observed after the mid-40 s. Cardiac involvement is not seen. A patient with hypotonic presentation at birth (floppy infant) later developed mild limb girdle atrophy and weakness with contractures of the upper limbs, mild facial weakness, ptosis, and nasal voice [3, 173]. A typical imaging pattern in the early stage of LGMD-R21 is the “inside-to-outside” pattern of fatty degeneration; the inner region of the muscle is more severely affected than the outer region. This pattern, however, will eventually be lost as the disease progresses [3, 173]. Muscle biopsy shows myopathic/dystrophic changes with lobulated fibers. Decreased number of satellite cells can be demonstrated by IHC against PAX7 (Paired Box Protein 7), a transcriptional factor normally present in nucleus of satellite cells. Hypoglycosylation in muscle tissue is demonstrated by decreased expression of glycosylated α-DG (VIA4–1 and IIH6 on IHC and WB) and abnormal laminin overlay (on WB). Normal glycosylation in cultured fibroblasts supports the hypothesis that hypoglycosylation in LGMD-R21 is a secondary phenomenon. Reduction of NOTCH1 intracellular domain (NICD) is demonstrated on WB [3, 173].

LGMD-R24: POMGNT2-Related LGMD POMGNT2 encodes protein O-linked mannose β-1,4-N-­ acetylglucosaminyltransferase 2 (POMGNT2) also known as glycosyltransferase-like domain containing 2 (GTDC2), located in the ER. POMGNT2 forms β-1,4-GlcNAc by transferring GlcNAc from UP-GlcNAc to the initial O-mannose residue; this is the first step of core M3 formation. Conditions related to POMGNT2 mutations are WWS, LGMD, and an intermediate phenotype [174–177]. POMGNT2-related LGMD is rare and characterized by infantile or childhood onset with or without intellectual disability. Structural brain abnormalities are not observed [175]. The muscle weakness is minimal to mild. The CK level is mildly to moderately elevated. On imaging, diffuse muscle atrophy was observed

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in one patient with infantile onset. Muscle biopsy IHC and WB are notable for hypoglycosylation observed in secondary α-dystroglycanopathy [175].

LGMD-R10: TTN-Related LGMD TTN encodes titin, the largest protein in the body spanning from Z-disc (N-terminus) to M-band (C-terminus) of the sarcomere [178]. Titin binds to myosin, actin and multiple other proteins. Titin acts as a sarcomeric scaffold and is involved in sarcomere assembly, stability, protein quality-control pathways, and mechanosignaling pathways for fiber trophicity and remodeling [178–181]. LGMD-R10 was originally described as a childhood-onset LGMD with moderately elevated CK level, associated with homozygous FINmaj TTN mutation, a Finnish founder mutation resulting in a 11  bp insertion-deletion (in-del) mutation exchanging 4 amino acids in exon 364 (M-band exon 6, Mex6) [182–185]. Loss of ambulation occurs around 20  years after disease onset [185]. Contractures, mild scapular winging, late respiratory failure, and late, mild cardiac involvement can be present [186]. Compound heterozygous FINmaj mutations and frameshift mutations involving exons encoding for the M-band and PEVK-region are associated with infantile/ childhood onset LGMD [187]. Compound heterozygous mutations comprising an essential splice site c.107377  +  1G  >  A in intron 362 (previously reported in dilated cardiomyopathy) along with a second truncating mutation, are associated with childhood/early adult-onset LGMD with variable contractures and early cardiac involvement [188]. At the early stage of disease, muscle imaging may not show any abnormality [186, 188]. The distribution of muscle weakness varies between mutations [186, 188]. Muscle biopsy in homozygous FINmaj mutations shows dystrophic changes while internalized nuclei and variable rimmed vacuoles can be present in other variants [188, 189]. WB analysis can be challenging as candidate missense variants may show normal titin C-terminal pattern [189]. Additionally, mutations affecting C-terminal proteins where titin interacts with CAPN3 may cause secondary CAPN3 deficiency [183, 187, 188].

LGMD-R12: ANO5-Related LGMD ANO5 encodes Anoctamin 5 (ANO5), a calcium-activated chloride channel preferentially expressed on the ER of skeletal muscle [190]. ANO5 mediates chloride uptake into the ER to maintain electroneutrality and facilitate calcium sequestration during plasma membrane repair [190]. ANO5

J. Tanboon and I. Nishino

also appears at the sarcolemma after injury and mediates phospholipid scrambles; ANO5-mediated phospholipid scrambling facilitates cell–cell fusion of mononucleated muscle progenitor cells, which is required for muscle repair. ANO5  mutation is associated with abnormal trafficking of annexin proteins, which mediate membrane repair, to the site of cellular injury. It does not appear however, that ANO5 phospholipid scrambles are essential for repair, and it is unclear if defective phospholipid scrambles underlie the pathophysiology of ANO5-related disorders [191]. LGMD-R12 (ANO5-related LGMD) is common in European countries because the presence of the European founder mutation, c.191dupA, and the Finnish founder mutation, c.2272C  >  T [192–194]. The estimated prevalence in European countries is 28.5 per million [15]. LGMD-R12 is characterized by adult-onset (in the third and fourth decade), male predominance, proximal muscle weakness of pelvic girdle and lower extremities with moderate to marked hyperCKemia [194–196]. On imaging, the most affected muscles are the posterior compartment muscles of the thighs and calves. Paraspinal muscles are mildly affected [194, 197, 198]. Asymmetrical muscle involvement is common [192, 194, 196, 197]. Calf hypertrophy could be present at the early stage. Most patients are still ambulant at older ages [194– 196]. Cardiopulmonary involvement was initially thought to be infrequent but there are reports of cardiac involvement in 10–30% of patients and include arrhythmias, left ventricular dysfunction and dilated cardiomyopathy [194–196]. ANO5 is also associated with other phenotypes including a distal myopathy, Miyoshi Myopathy Type 3 (MM-3), asymptomatic hyperCKemia and pseudometabolic myopathy with exercise intolerance with or without rhabdomyolysis. Muscle biopsy may be normal, or show non-­specific myopathic or dystrophic changes. Inflammatory infiltrates have been described. Amyloid deposition is noted in intramuscular blood vessels and the interstitium (perimysium, endomysium). These deposits consist of amyloid chaperone proteins, but not ANO5 [194, 199]. Given the overlapping clinical and histological features, the major differential diagnosis for LGMD-R12 is LGMD-R2. Assessment of dysferlin protein expression by IHC and WB can help to differentiate these entities. Evaluation of ANO5 protein expression by WB is possible, but an antibody suitable for ANO5 detection by IHC is not yet commercially available [200].

LGMD-R17: PLEC-Related LGMD PLEC encodes plectin, a multimodular cytolinker ubiquitously present in many cell lines e.g., muscle cells, epithelial cells, endothelial cells, fibroblasts, Schwann cells, and

6  Autosomal Recessive Limb-Girdle Muscular Dystrophies

astrocytes [201, 202]. Plectin primarily interacts with and crosslinks intermediate filaments and anchors them; it also binds to actin-based filaments, microtubules, and many other membrane and cytoskeletal proteins [201, 202]. In skeletal muscle, subcellular localization of four major plectin isoforms (P1d, P1f, P1b and P1) creates an intermediate filament network linking DGC, cytoplasmic organelles, sarcolemma, and extracellular compartment, which is essential for cellular integrity and stress response/adaptation. P1d links and anchors desmin to the Z disks. P1f binds dystrophin and β-dystroglycan in the DGC. P1b links mitochondria to the desmin network and P1 binds to intermediate filaments in the nuclear/ER membrane and forms a recruitment ­platform for other intermediate filaments [201, 202]. PLEC-­related mutations are associated with the following phenotypes either separately or in combination: epidermolysis bullosa simplex, muscular dystrophy, and myasthenic syndrome [203]. LGMD-R17, associated with disruption of P1f (homozygous c.1_9del in PLEC exon1f), is characterized by early childhood onset LGMD without skin involvement [204, 205]; adult onset is also reported; calf pseudohypertrophy, paraspinal muscle involvement and myasthenia (fatigable ptosis) can be present [205] LGMD-R17 is slowly progressive or stable until the late teen years but may show rapid progression at the later stages of disease; loss of ambulation in 20 s–30 s is observed [204, 205]. Muscle biopsy shows dystrophic changes with increased internalized nuclei and some angular fibers [204, 205]. Absence of plectin immunoexpression is diagnostic [204].

LGMD-R22: COL6A1, COL6A2 and COL6A3-­ Related LGMD Collagen VI is a heterotrimeric protein ubiquitously present in the ECM. Collagen VI isoform is composed of three distinct polypeptide chains, α1(VI), α2(VI) , and α3(VI) encoded by COL6A1, COL6A2, and COL6A3, respectively, forming a heteromeric triple helix monomer. There are also three other chains, α4, α5 and α6, which may replace the α3 chain. Intracellularly, the monomers assemble into dimers, which subsequently form tetramers. The secreted tetramers form an extracellular beaded microfibril network by end-to-­end overlapping association of the tetramers. The beaded microfibrils of Collagen VI anchor tissues to basement membranes and interact with ECM proteins [206, 207]. In skeletal muscle, defective collagen VI impairs autophagy causing mitochondrial dysfunction,

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spontaneous apoptosis, and myofiber degeneration [208]. Satellite cell renewal and muscle regeneration are also affected [209]. Mutations in COL6A1, COL6A2, and COL6A3 are associated with several collagen VI-related myopathies including Ullrich congenital muscular dystrophy (UCMD) on the severe end of the spectrum, Bethlem myopathy (BM) on the mild end, and intermediate phenotypes [210]. Both AD and AR mode of inheritances are reported in all spectrums [206]. AD-related BM (LGMD-D5) is more common than AR-related BM (LGMD-R22). Both conditions show similar phenotypes and are characterized by early childhood to adult onset of gradually progressive proximal muscle weakness and contractures. The presence of distal joint hyperlaxity and posteriorly prominent calcanei are typical for UCMD [211]. The contractures typically affect the Achilles tendons and elbows. CK level is normal or slightly elevated. Contractures of long finger flexors create typical “Bethlem sign” on wrist dorsiflexion- a gesture resembling the Thai “Wai” or the Indian “Namaste” greetings without complete finger extension. Loss of independent ambulation usually occurs in the fifth decade of life. Skin features including keloids, hypertrophic scars, and hyperkeratosis pilaris are frequent [210, 211] (Fig.  6.7). The boundary between the intermediate phenotype and BM is not clearly delineated, although severe muscle involvement and declining pulmonary function differentiate intermediate phenotypes from BM [212]. On imaging, the most frequent and the most affected muscles are the vasti. In the vastus lateralis, the more affected peripheral portion but relatively preserved central portion creates the “sandwich sign” [27, 28]. This pattern can be also present in other muscles in UCMD and BM [213]. An inverse pattern with a more affected central area and relatively preserved peripheral portion of the rectus femoris creates the “target sign” [27, 28] (Fig. 6.8). Muscle biopsy shows dystrophic changes with disproportionate endomysial fibrosis in relation to the rarity of necrotic and regenerating fibers. In the relevant clinical context, abnormal collagen VI IHC expression patterns, including complete collagen VI deficiency and “sarcolemmal-specific collagen VI deficiency (SSCD)” defined by preserved interstitial collagen VI expression but decreased expression in the sarcolemma, should raise the diagnosis of collagen VI-related conditions [214] (Fig.  6.9). However, relatively normal collagen VI IHC staining on muscle biopsy cannot completely rule out the diagnosis of LGMD-R22 [215]. Collagen VI IHC and WB on cultured skin fibroblasts is a useful diagnostic procedure [215].

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Fig. 6.7  Characteristic clinical features of Bethlem myopathy and Ullrich congenital muscular dystrophy Bethlem myopathy patients (a– e) typically show flexion contractures of the elbows (a), ankles (b), wrists and fingers (c, d). Bethlem sign is shown in c. In Ullrich congenital muscular dystrophy (f–k), proximal joint contractures e.g. elbows (h) and hyperlaxity of the distal joints e.g. ankles (i), toes (j) wrists and

fingers (k) and posteriorly protruding calcanei are noted (g). Keloids are common in both disorders (e, f). (Source: Fig. 12.1 from Bushby, K.M.D., Collins, J., Hicks, D. (2014). Collagen Type VI Myopathies. In: Halper, J. (Eds) Progress in Heritable Soft Connective Tissue Diseases. Advances in Experimental Medicine and Biology, vol 802. Springer, Dordrecht. Reproduced with permission)

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Fig. 6.8  Transverse T1 weighted muscle MRI at the thigh in collagen VI-related muscular dystrophy. Fatty infiltration at the periphery of vastus lateralis (purple arrowhead) with relative sparing of the central part creating “sandwich” or “tigroid” sign. The opposite pattern is observed in rectus femoris (blue arrowhead) creating “target sign” which also

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known as “central shadow”. In this patient, adductor longus (yellow arrowhead) and gracilis (green arrowhead) are relatively spared. Courtesy of Dr. Keiko Ishigaki, MD, PhD Department of Pediatrics, Tokyo Women’s Medical University, Tokyo, Japan

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Fig. 6.9  Collagen VI immunohistochemistry in collagen VI-related muscular dystrophy. Two patterns of collagen VI distribution: (1) sarcolemma-­specific collagen VI deficiency (SSCD) showing decreased

sarcolemmal expression but presence of interstitial deposits (a) and (2) complete deficiency (b). Normal collagen VI staining on sarcolemma (c). Note: a-c bar = 10 microns

LGMD-R23: LAMA2-Related LGMD

Cardiomyopathy has been reported in one patient [220]. Respiratory involvement is not reported. The oldest reported patient remained ambulant at the age of 69 [218]. Muscle biopsy shows myopathic/dystrophic changes. Variably decreased laminin-α2 expression is noted on IHC and WB with overexpression of laminin-α5 due to molecular compensation [217]. Decreased laminin-α2 IHC expression is also present in skin biopsy [220]. The clinical phenotype also includes a severe CMD with many patients showing white matter abnormalities and neuronal migration defects on brain MRI. Extramuscular features include seizures and intellectual disability in some patients. Mild sensorimotor demyelinating neuropathy is reported. Respiratory dysfunction and swallowing difficulty are frequent. LAMA2-CMD is one of the most common CMD worldwide [221].

LAMA2 encodes laminin-α2 (merosin), a subunit of the trimeric laminin-211 protein, present in ECM of skeletal muscle, Schwann cells, astrocytes and pericytes of cerebral capillaries [188]. Laminins provide tissue stability by creating a primary scaffold attaching the ECM to the cell surface through laminin receptors (e.g.,α-DG) and serve as a platform for other ECM proteins to attach [216]. LAMA2-related LGMD is characterized by childhood to adult onset, slowly progressive muscle weakness with various degrees of central nervous system involvement including epilepsy, mental retardation, and cerebral white matter abnormalities [217– 220]. CK level is moderately increased. Axial and neck muscle involvement and calf hypertrophy are variably present.

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LGMD-R25: BVES/POPDC1-Related LGMD BVES encodes Blood Vessel Epicardial Substance (BVES), a member of Popeye domain containing (POPDC) protein family, also known as POPDC1. POPDC proteins are transmembrane proteins involved in the cyclic adenosine monophosphate (cAMP) signaling pathway and regulation of transmembrane trafficking of interacting proteins [222, 223]. Co-expression of BVES/POPDC1 and POPDC2 is required for proper membrane localization [224]. Interaction of BVES/POPDC1 and ANO5 is involved in myoblast differentiation [225]. BVES/POPDC1 is present in striated muscle, smooth muscle, brain, liver, gastrointestinal tract, and epithelial cells. The other POPDC isoforms, POPDC2 and POPDC3, are expressed in cardiac and skeletal muscle. POPDC2 expression is more prominent in cardiac muscle while POPDC3 expression is more prominent in skeletal muscle [222, 223]. BVES/POPDC1-related LGMD is characterized by childhood to adult onset proximal muscle weakness with mild to marked elevation in CK levels [224, 226–229]. Cardiac involvement in the form of atrioventricular block is common [226–229]. This is likely due to mutations affecting BVES/POPDC1-POPDC2 complex formation [224]. Cardiac involvement is not observed in mutations that leave the protein interaction intact [224]. Respiratory involvement is not reported. Asymmetric muscle involvement, atrophy of paraspinal, humeral, pectoral, and anterior and posteromedial thigh muscles and scapular winging are present in some patients. Loss of independent ambulation is observed beyond age 40 years [224, 226–229]. A patient presenting with syncope, high CK and cardiac conduction defects without muscle weakness, despite the presence of a myopathic EMG and mild dystrophic changes on biopsy has been reported [226]. Muscle biopsy shows myopathic/dystrophic changes. Decreased sarcolemmal staining [224, 226– 228] but increased perinuclear localization of IHC for BVES/ POPDC1 and POPDC2 [227] indicates abnormal membrane trafficking properties of the mutant gene and its associated protein (POPDC2). The total level of BVES/POPDC1 expression in WB can be normal or near normal if the mutation only affects membrane trafficking properties of the gene, and in turn, of the expressed protein [227].

LGMD-R26: POPDC3-Related LGMD POPDC3 encodes Popeye domain containing 3 (POPDC3), a transmembrane protein involved in cAMP signaling. POPDC3-related LGMD is characterized by adolescent to adult-onset proximal muscle weakness with moderately to markedly elevated CK levels. Cardiopulmonary involvement is not reported [230–232]. Calf hypertrophy, pectoral atro-

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phy, and biceps atrophy are variably reported. Asymmetric muscle involvement is not uncommon [230]. Two patients have been reported to have broad-based kyphosis of the thoracolumbar spine [231, 232]. Muscle MRI shows fat infiltration in paraspinal muscles, thigh muscles, and medial gastrocnemius [230]. Muscle biopsy shows myopathic/dystrophic changes [232]. WB of the reported cases show reduction of POPDC3 [230, 232] along with POPDC1 and POPDC2 proteins [230].

LGMD-R27: JAG2-Related LGMD JAG2 encodes Jagged Canonical Notch Ligand 2 (Jagged2, JAG2), a transmembrane protein that interacts with the receptor signaling Notch pathway. Jagged2 structures share some similarities to the other three canonical Notch ligands (i.e. delta-like ligand 1(DLL1), DLL4 and Jagged1). The extracellular architecture of the ligands consists of an N-terminal C2 domain, a Delta/Serrate/Lag-2 (DSL) domain, and EGF repeats [233]. Notch receptor-ligand interaction initiates a proteolytic cascade which releases and translocates the intracellular domain of the Notch receptor (NICD) to the nucleus to promote gene transcription [234]. Biallelic JAG2 variants were recently reported in association with LGMD phenotype in 23 patients from 13 families [235]. In silico analysis predicted misfolding of the C2 domain and EGF repeats in missense variants and mRNA decay in nonsense and frameshift variants. Interestingly, while heterozygous loss-of-function mutation in DLL1, DLL4, and JAG1 are described in various severe congenital conditions, heterozygous loss-of-function JAG2 variants are present in asymptomatic individuals. The observation indicates that JAG2 is less sensitive to gene dosage variation than the other canonical Notch ligand-associated genes although the mechanism is yet to be elucidated. JAG2-related LGMD is characterized by infancy to young adulthood onset with predominantly proximal muscle weakness in the lower extremities and prominent axial muscle weakness, especially of the neck flexors. Scoliosis and joint contractures are present in approximately half the patients. Muscle atrophy may be generalized or affect the proximal upper extremities, distal lower extremities or the neck muscles, and is reported in approximately one-third of the patients. Rigid spine, mild facial muscle weakness, and ptosis are less commonly reported. Ophthalmoplegia is not seen. Impaired pulmonary function is observed in half the patients but none require respiratory support. Cardiac involvement is rare. Loss of ambulation occurs during childhood or adolescence [235]. CK is normal or slightly elevated. EMG shows a myopathic pattern. Nerve conduction studies s are normal; in one

6  Autosomal Recessive Limb-Girdle Muscular Dystrophies

patient with borderline low amplitude responses in the superficial peroneal and sural nerve has been reported. Muscle MRI shows more severe fatty infiltration in the proximal muscles than distal muscles. In contrast to “inside-out” pattern of fatty degeneration in another Notch pathway-related LGMD, LGMD-R21 (POGLUT1-related LGMD), LGMD-R27 shows progressive “outside-in” degeneration mainly observed in the vastus lateralis, vastus intermedius, vastus medialis, and less frequently in the long head of the biceps femoris and the gluteus medius. Central fatty transformation is observed in rectus femoris. Although the fatty transformation in the vasti and rectus femoris may resemble the “sandwich” and “target” signs reported in LGMD-R22 (collagen VI-related LGMD) [27–29] and LGMD-R1 (CALPN3-related LGMD) [26], presence of the pattern in pelvic and calf muscles may help distinguish LGMD-R27 from these entities (Chap. 15). Muscle biopsy shows either a myopathic or a dystrophic pattern. Oxidative stains show myofibrillar architecture distortion including lobulated, whorled, moth-eaten fibers and fibers containing core-like areas. The diagnosis requires clinical and imaging correlation and genetic confirmation [235].

Recently Described AR-LGMDs POMK-Related LGMD POMK encodes protein O-mannose kinase (POMK), located in ER. At the last step of core M3 formation in ER, POMK transfers a phosphate group from adenosine 5′-triphosphate (ATP) to the C6 position of the O-mannose residue and forms the phospho-core M3 structure of α-DG [108]. Matriglycan is a scaffold for ECM proteins containing Lam-G domains such as laminin, agrin and perlecan [115, 116]. Like-acetyl-glucosaminyl transferase (LARGE, also known as LARGE 1) synthesizes and extends matriglycan on to α-DG. POMK is required for (LARGE-mediated generation of full-length matriglycan on α-DG [236]. POMK-­ related LGMD is rare and characterized by infantile or childhood onset weakness, moderately to markedly increased CK levels, and calf hypertrophy [237, 238]. Learning disability, arachnoid cysts, and mega cisterna magna are reported in infantile onset patients [237]. Mild left ventricular enlargement and borderline weakened function was reported in one patient [238]. Pulmonary involvement is not reported. The patients remain ambulant into young adulthood. Muscle biopsy shows either dystrophic change or chronic myopathic change with sparse inflammation. Based on functional studies [237], we expect typical α-DG hypoglycosylation to be demonstrated on muscle biopsy. In a report of two patients with CMD- ID with onset at birth, born of consanguineous parentage, congenital hypomyelination

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was noted on brain MRI. POMK was absent from skin fibroblasts and skeletal muscle, and a lack of α-DG expression was noted on muscle biopsy [239].

PYROXD1-Related LGMD PYROXD1 encodes pyridine nucleotide-disulphide oxidoreductase domain 1 (PYROXD1), found in the nucleus and striated sarcomeric components of skeletal muscle. The tRNA-ligase complex catalyzes pre-tRNA and mRNA splicing. The catalytic subunit of tRNA ligase is RTCB, which is highly susceptible to oxidative damage. PYROXD1 binds to RTCB in the pre-tRNA-ligase complex and converts NADPH to NADP. This prevents oxidative inactivation of the tRNA-­ ligase complex [240]. Homozygous or compound heterozygous PYROXD1 missense mutations are generally associated with childhood- or adulthood-onset LGMD; c.464A > G is the most common mutation [241–246]. Splice site mutations are usually associated with younger age of onset and with other features such as joint hypermobility, contractures, and rigid spine [241, 243, 244, 246]. CK levels are normal or slightly elevated. Axial and facial muscle weakness have been reported [241, 244, 246]. The condition is slowly progressive, such that the patients with childhood-onset disease lose ambulation in the fourth decade while the patients with adulthood-onset remain ambulant even over 60 years [241– 246]. Restrictive lung disease is present in some patients [241, 243, 244]. Cardiac involvement is not reported. Muscle imaging shows generalized fat marbling [241, 243, 245]. Muscle biopsy may be distinctive, combining features of central core, minicore, centronuclear, nemaline and myofibrillar pathology or show non-specific dystrophic features [241–246].

 iagnostic Approach and Future Treatments D for AR-LGMD Establishing a clinical diagnosis of AR-LGMD can be challenging because the of the typical limb-girdle pattern of weakness overlaps with various combinations of weakness in other muscle groups. Additionally, extramuscular involvement varies, affecting several systems. The recessive mode of inheritance adds another challenge to establishing a diagnosis. AR-LGMD are rare and can skip generations or present as an isolated case in a family. Thus, it can be difficult to differentiate AR-LGMD from acquired myopathies such as inflammatory myopathy in an individual without family history of a similar disorder. Myopathic and dystrophic changes in muscle biopsy, although suggestive of muscular dystrophy in the relevant clinical context, are not specific for disease subtyping without evidence for specific protein alterations.

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The interpretation of loss of protein on IHC can be difficult due to secondary protein abnormalities that the primary disorder causes. Some of these muscle biopsy findings can also be present in inflammatory myopathy. An accurate genetic diagnosis is important to determine prognosis and appropriate management and treatment strategies. Genetic information is also essential for genetic counselling and for disease screening and detection and further monitoring in current and future family members. Enrollment in clinical trials requires a confirmed genetic diagnosis. A guideline for diagnosis and management of LGMD provides an algorithmic approach to the diagnosis, but does not include the place of next-generation sequencing in the algorithm [247]. Generally, AR-LGMD should be considered in the differential diagnosis of limb-girdle type weakness and family history with possible recessive mode of inheritance. Noninvasive modalities such as obtaining a detailed history including family pedigree chart, a careful examination with attention to the pattern of weakness and evaluation for extramuscular manifestations, muscle imaging and laboratory investigations (e.g. CK, serology for myositis specific antibodies, thyroid function tests etc.), EKG, ECHO and pulmonary function tests should be performed early in the evaluation to exclude acquired conditions that are treatable, and to detect cardiorespiratory complications that can be treated to improve quality of life and survival. Additional clinical information

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such as age of onset, ethnicity, other extramuscular manifestations, and pattern of muscle involvement on imaging (Chap. 15) may help narrow the list of potential genetic conditions to be tested. Unless the disease has a characteristic clinical picture and the genetic test is only for confirmation of a highly likely clinical diagnosis, single gene tests are no longer used. Targeted whole exome sequencing (WES), “neuromuscular panels”, are often used as the initial genetic test, and it should be ascertained that the panels incorporate the conditions that are suggested by the clinical differential diagnosis. (Chap. 14) If the results of targeted WES reveal a pathogenic variant that has previously been associated with a clinical phenotype of LGMD, the diagnosis is confirmed. If targeted WES reports novel variants in known causative genes for neuromuscular disease (variants of uncertain significance, VUS), further investigations are required, such as muscle biopsy to evaluate the functional consequences of the variant. IHC or WB on muscle biopsy, evaluating the protein that is predicted be affected by the VUS may help to confirm the pathogenicity of the novel variants if the protein product of the gene is reduced, absent or of abnormal size on WB. For patients without any detected variant on targeted WES, whole exome or whole genome sequencing may be required. Further transcriptome, proteome, and functional studies on muscle biopsy relevant to candidate gene alterations should be considered (Fig.  6.10) [247]. (Chap. 14).

Fig. 6.10  Diagnostic approach for AR-LGMD. Note:*Myositis specific antibodies may include but not limited to anti-TIF1-γ, NXP-2, MDA5, Mi-2, SAE, Jo-1, OJ, PL-7, PL-12, EJ, KS, Zo, Ha, Ly, SRP, and HMGCR antibodies. IHC: immunohistochemical study

6  Autosomal Recessive Limb-Girdle Muscular Dystrophies

Current management of AR-LGMD relies on multiple medical specialties for systemic and supportive treatments. The efficacy of non-specific medical therapy (i.e. immunomodulatory therapy, myostatin inhibitors) has not been proven in LGMD.  Deflazacort, a glucocorticoid was not effective for LGMD-R2 (dysferlin-related LGMD) in a phase III study (ClinicalTrials.gov NCT00527228) [248]. A phase III study of deflazacort in LGMD-R9 (FKRP-related LGMD) (clinicaltrials.gov NCT03783923) was terminated due to the COVID-19 pandemic. A phase II open label study (ClinicalTrials.gov NCT04054375) of weekly corticosteroids in muscular dystrophy (WSiMD) evaluated weekly prednisone in several subtypes of AR-LGMD and in Becker muscular dystrophy. The results are pending. A phase II study of domagrozumab, a myostatin inhibitor, showed no efficacy in improving muscle strength or function in FKRP-­ related LGMD (ClinicalTrials.gov NCT02841267) [249]. There are no LGMD-specific therapies beyond Phase II development. Gene therapy with SRP-9004 (scAAVrh74. tMCK.hSGCA, a self-complementary adeno-associated virus serotype rhesus 74 (AAVrh74) vector delivering the human SCGA) is being explored in LGMD-R3 (SCGA-­ related LGMD). A phase II study with isolated limb infusion of scAAVrh74.tMCK.hSGCA via vascular route increased knee extensor muscle strength and fiber diameter in two of six patients but the six-minute walk times decreased or remained the same (ClinicalTrials.gov NCT01976091) [250]. In addition to gene therapy (ClinicalTrials.gov NCT05230459, NCT05224505), oral administration of BBP-418 (ribitol, Rbo) is being investigated for the t­ reatment of FKRP-related LGMD (ClinicalTrials.gov NCT04800874); Rbo and CDP-Rbo were effective in mouse models of FKRPand CRPPA  (ISPD)-related LGMD [251–253]. Autologous gene-edited muscle stem Cells (GenPHSats-bASKet) to repair muscular dystrophy causing mutations are also being explored for LGMD treatment (ClinicalTrials.gov NCT05588401).

Summary The development of advanced gene sequencing technologies has accelerated the discovery of causative and candidate genes for AR-LGMD.  The older nomenclature system is now replaced by the 2017 ENMC-LGMD classification to accommodate the rapid growing list of genetically confirmed AR-LGMD.  Although this new system uses a numerical order to indicate the order of causative gene discovery, the causative genes for sarcoglycanopathy are intentionally reordered by the Greek alphabets assigned to the sarcoglycan isoforms. This classification system may be convenient, but has limitations, as discussed above. The classification of

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these disorders will continue to evolve. Although AR-LGMDs are genetically heterogenous, their clinical and pathological phenotypes can overlap, making diagnosis of the subtype challenging. Next generation sequencing has revolutionized the diagnosis of these disorders. Treatment is mainly supportive and includes management of cardiorespiratory complications, arguably the most serious extramuscular manifestations. Basic and clinical research to treat the underlying genetic defect is ongoing. The rarity of these disorders is a major challenge to clinical trials.

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7

Oculopharyngeal Muscular Dystrophy Bernard Brais

Introduction

ally more than 10 years after the onset of the first cardinal symptoms [5]. As the disease progresses, all extraocular Oculopharyngeal muscular dystrophy (OPMD) is a late muscles are affected. Limitation of eye elevation is observed onset inherited muscle disease clinically characterized by in most older patients. Incomplete horizontal gaze paresis, progressive ptosis of the eyelids and dysphagia, associated though not universal, can be observed in older patients, but is with unique tubulofilamentous intranuclear inclusions (INI) rarely associated with diplopia. In advanced stages of the on muscle biopsy [1]. OPMD is usually transmitted as an disease, the eyelids become very thin and transparent. The autosomal dominant trait, but a much rarer recessive form forehead is permanently wrinkled, the eyebrows are raised, has also been described [2]. OPMD is caused by short trinu- and the supraorbital ridges appear prominent, due to over cleotide repeat (GCN)11–18 expansions in the polyadenylate-­ activity of the frontalis muscles (Fig. 7.1a). There is usually binding protein nuclear 1 (PABPN1) gene. OPMD was first no severe facial diplegia. Dysphagia is first for solid foods described by Taylor in 1915 in four members of a French-­ and slowly progresses. Early symptoms that are suggestive Canadian family living in the Boston area [3]. In 1962, of mild dysphagia are lengthening of mealtime and avoidVictor, Hayes and Adams coined the name to underline the ance of certain dry and fibrous foods such as nuts, rice and preponderant symptoms related to the eyelid ptosis and dys- celery [13]. Patients will learn from affected family members phagia, but also noted the accompanied limb-girdle weak- and through their own experiences different coping strategies ness [4]. Many regional clusters of higher prevalence of that will render eating easier, such as swallowing liquids OPMD have been described in the Québec province of with solid food, swallowing small bits that lessen greatly the Canada [2, 5], Israeli Bukhara Jews [6], Uruguay, [7] Spanish risk of airway obstruction, avoiding foods that are harder for Americans living in New Mexico [8] and California, USA them to swallow, adding extra sauce and refraining from [9, 10]. Cases of OPMD are reported in more than 50 coun- talking while eating. Unfortunately, late in the disease many tries. The highest prevalence are 1:1000 in Quebec French-­ patients may refrain from eating in public. Aspiration pneuCanadians and 1:600 in Bukhara Jews leaving in Israel [6, monia is frequent as the diseases progresses and up to 32% 11]. The estimated prevalence in Europe based on a French will die of respiratory complications, which is far more frestudy is in the order of 1:100,000 [12]. quent than in the general population [5]. All patients develop limb-girdle weakness of variable severity. Early on, patients complain of more difficulty getting up from a chair, going up Clinical Phenotype stairs and walking uphill. Though only a minority of patients will lose their walking ability on flat surfaces, a significant OPMD usually manifests in the fifth or sixth decade with number will require walking aids such as canes and walkers, eyelid ptosis, dysphagia, or both. Limb-girdle weakness, in particularly outside their home [5, 14]. Home adaptations to particular in the lower extremities, always follows but usu- improve access and limit falls are usually needed. Late in the course, patients with more severe disease will develop distal limb weakness usually accompanied by loss of deep tendon B. Brais (*) reflexes. There is no strong evidence that OPMD is associDepartments of Neurology and Neurosurgery and Human ated with an increased risk of developing cognitive dysfuncGenetics, Rare Neurological Disease group, Faculty of Medicine, tion, but data comparing the prevalence of cognitive changes McGill University, Montreal Neurological Institute-Hospital, Montreal, Quebec, Canada in OPMD families between affected and unaffected siblings e-mail: [email protected]

© The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 P. Narayanaswami, T. Liewluck (eds.), Principles and Practice of the Muscular Dystrophies, Current Clinical Neurology, https://doi.org/10.1007/978-3-031-44009-0_7

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124

a

B. Brais

d

b

c

Fig. 7.1 (a) Image of patient that shows ptosis despite previous surgery, frontalis wrinkling and temporalis muscle atrophy. (b) Electron micrograph showing a subsarcolemmal skeletal muscle nucleus filled

with OPMD filaments, white arrow (x20,000). (c) Tubular filaments often in tangles (x100,000). (d) Cane with an integrated half step

or between OPMD patients and the general population are sparse [15, 16]. The age of onset of autosomal dominant OPMD is often difficult to ascertain [5]. A study of 72 French-Canadian symptomatic carriers of a (GCN)13 repeat expansion established a mean age of onset for ptosis of 48.1 (26–65) years and for dysphagia of 50.7 (40–63) years [13]. A more recent large retrospective study of carriers of repeat expansions of the same size reported the average of onset of ptosis and dysphagia to be around 54 years [5, 13]. In a large study of 354 French cases carrying different expansion sizes, in which the mean age of clinical diagnosis for (GCN)13 carriers was

64 years, larger repeat size inversely correlated with an earlier age of first consultation though not with declared aged of onset [17]. The other signs observed as the disease progresses are: proximal upper extremity weakness (38%), mild facial muscle weakness (43%), limitation of upgaze (61%), dysphonia (hypophonia and nasal speech) (67%), proximal lower extremity weakness (71%) and tongue atrophy and weakness (82%) [13]. The relative prevalence of these clinical characteristics varies between cohorts even in carriers of the same mutation size [5, 17]. Life expectancy of dominant OPMD was reported to be normal when compared to unaffected family members in one

7  Oculopharyngeal Muscular Dystrophy

New Mexico Latino American cohort, although reduced by about 10 years than the general USA population [8]. Deaths usually occur at an advanced age, often due to causes unrelated to OPMD, but up to 32% are reported to die of respiratory conditions including aspiration pneumonia, other pneumonias, influenza and other respiratory disorders [5]. Malnutrition and aspiration pneumonia are important contributors to the cause of death in older OPMD patients. Earlier treatment of pharyngeal dysfunction and better nutrition can improve overall quality of life and likely life expectancy.

Diagnosis and Differential Diagnosis Clinical criteria for OPMD are: A positive family history with involvement of two or more generations; the presence of ptosis (defined as either vertical separation of at least one palpebral fissure that measures less than 8 mm at rest or previous corrective surgery for ptosis) and the presence of dysphagia, defined as swallowing time greater than seven seconds when drinking 80 mL of ice-cold water [11]. A clinical diagnosis of OPMD can often be made based on these criteria. The diagnosis is more difficult when the family history is uninformative. Myotonic dystrophy type 1 and 2 are usually distinguishable clinically and excluded by genetic testing. Ptosis can be seen in some congenital myopathies, but their earlier age of onset, more extensive muscle involvement and less frequent associated dysphagia allows distinguishing them on clinical grounds. In familial ptosis, blepharophimosis, extraocular muscle fibrosis and congenital myasthenic syndrome, the onset of the ptosis occurs at a younger age and there is no accompanying dysphagia. Ocular myasthenia gravis is the most important disease to exclude in the work-up of patients with ptosis. In most myasthenic patients, the fluctuating diplopia, fatigability, and response to anticholinesterase drugs establish the diagnosis. The presence of circulating Anti-acetylcholine freceptor and Anti-­ muscle specific kinase antibodies and demonstration of a neuromuscular transmission defect by electrophysiologic testing (repetitive nerve stimulation of single fiber electromyography) can further support the diagnosis. Late-onset Kearns-Sayre syndrome (KSS), a mitochondrial disorder, should also be entertained in the differential diagnosis and may be difficult to distinguish from OPMD. Severe dysphagia and a dominant inheritance may exist in KSS, and the visceral, cardiac, or retinal changes are frequently absent in late-onset KSS [18]. A diagnosis of KSS is established upon the identification of mitochondrial DNA deletions. It should be noted that occasional ragged red fibers can be observed in OPMD muscle as in older patients with other muscle diseases.

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Dysphagia is observed in other muscle disorders, such as myotonic dystrophy, hereditary inclusion body myopathy (hIBM) and immune-mediated myopathies, especially sporadic inclusion body myositis (sIBM), but these diseases can be distinguished from OPMD by their clinical and pathological features. For example, in immune-mediated myopathies, ptosis is very rare, but dysphagia can occur and sometimes is the presenting symptom in sIBM [19–24]. Limb muscle weakness is usually prominent and there are no reports of ocular paresis in sIBM. In hIBM (Chap. 9), ptosis is not a common finding, and mutation testing can distinguish the two conditions [2, 25]. The pathological differences in the observed filaments on electron microscopy between OPMD and hIBM filaments can also help in distinguishing the two conditions. Distal muscle involvement is excessively rare in the early stages of OPMD but can be present in older patients, though the frequency has not been well studied. Early distal weakness accompanied by ptosis and dysphagia should raise the suspicion for oculopharyngodistal myopathy (OPDM) rather than OPMD [26, 27]. Expansion repeats in LRP12, GIPC1, NOTCH2NLC, NUTM2BAS1, and RILP1 have recently been found to cause OPDM (Chap. 13), but these mutations are only starting to be tested in clinical laboratories [28–34]. Facial diplegia is a shared feature of many OPDM cases, whereas frontalis overactivity with resultant wrinkling of the forehead is more prominent in OPMD. Though muscle biopsies in many OPDM cases were reported to show only nonspecific dystrophic features, some genetically confirmed cases were found to have p62-positive intranuclear inclusions in muscle and neurons. These inclusions consist of filaments of 13-18 nm in diameter, larger than the usual 8.5-nm OPMD filaments [26, 27, 32, 33, 35].

Laboratory Findings Electromyographic (EMG) studies of facial and limb muscles generally show a myopathic pattern (short duration, low amplitude, polyphasic motor unit potentials with early recruitment), though mixed population of long duration and high amplitude and short duration and low amplitude motor unit potentials in limb muscles has been reported in some patients and likely reflects the chronicity of the disorder [36]. Sensory and motor conduction velocities are either normal or, less frequently, slightly reduced [37]. The serum creatine kinase (CK) and aldolase levels are generally within normal limits or only mildly elevated. However, CK values may be 2–5 fold elevated compare to the upper limit of normal, mostly in individuals who have more limb-girdle involvement, are taking statins or are very physically active [38]. Manometry and video fluoroscopy studies of pharyngeal and esophageal motility show weak, prolonged, and repeti-

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tive pharyngeal contractions, but contraction of the upper esophageal sphincter is usually normal; however, sphincter relaxation, which depends on pharyngeal pressure, is late and incomplete [39–41]. These physiological changes explain the mechanisms of the dysphagia. Though video fluoroscopic assessment is the gold standard to evaluate dysphagia and pulmonary aspiration risk in OPMD, it should not lead to early Percutaneous Endoscopic Gastrostomy (PEG) placement on the basis of small aspirations since they may occur very early in the disease when no recurrent aspiration pneumonias have occurred. Patients develop progressively coping strategies to swallow that decrease greatly their risk of life-threatening respiratory obstruction. The adaptive swallowing behaviors explain why life expectancy is close to normal and allows, despite severe dysphagia, to continue oral feeding late in the disease. Muscle Computed Tomography (CT) studies and magnetic resonance imaging (MRI) show abnormal fatty-­ infiltration signal and atrophy in many muscles, most frequently in the tongue, the adductor magnus and soleus [42]. The use of MRI as an outcome measure of progression in OPMD is still under investigation as are patient-reported outcome measures (PROMs) [42–44]. The pathological changes seen in the muscle fibers of extraocular and other voluntary muscles vary according to the stage of the disease and the muscle biopsied. Although all skeletal muscles are likely affected, the extraocular, lingual, pharyngeal, and diaphragmatic muscles are selectively more severely involved at autopsy [45]. Muscle biopsy typically show loss of muscle fibers with fatty infiltration, variation in fiber size, increased in the number of internal nuclei, and increased interstitial fibrosis. Fibers undergoing necrosis and phagocytosis are rare. Inflammatory changes are usually not present. Histochemical studies reveal small angulated fibers more frequently in type 1 than type 2 fibers, that often react strongly for oxidative enzymes such as NADH-tetrazolium reductase or myofibrillar ATPase, and rimmed vacuoles [46]. Though the small angulated fibers may suggest an underlying denervating process, their occurrence may be mostly due to the advanced age of patients. Fibrosis of the cricopharyngeal muscle is believed to play a role in restricting sphincter opening [47]. Rimmed vacuoles are not specific for OPMD. The vacuoles consist of irregularly round or polygonal clear spaces lined by a ring of material that is basophilic with the hematoxylin and eosin stain and stains red with Gomori trichrome stain. The rimmed vacuoles are of autophagic nature and have acid phosphatase activity. The classical ultrastructural change in OPMD are the intranuclear tubular filaments with 8.5  nm outer and 3  nm inner diameters that form INI (Fig. 7.1b and c) [1]. The filaments are unbranched, often course rectilinearly, are sometimes striated with 7 to 7.5 nm of periodicity, and are up to

B. Brais

0.25 μm in length [46]. They are disposed in various directions and frequently form tangles or palisades. Large collections of filaments appear as clear zones surrounded by chromatin in the affected nuclei on semithin epoxy section by phase-contrast microscopy. Serial semithin sections suggest that the filamentous inclusions occur in all muscle fiber nuclei [48]. The percentage of INI positive nuclei per slide, 4.9% in heterozygotes and 9.4% in homozygotes has been shown to correlate with clinical severity [49]. The inclusions are present only in muscle fibers and not in the nuclei of any other cells in muscle; and described in anterior horn neurons only in one report [50]. Rarely, larger and longer tubular filaments of 16-18 nm in diameter and 1.5 μm in length, resembling sIBM, that are less organized and predominantly cytoplasmic can be found in muscle fibers [51].

Molecular Genetics and Diagnosis The OPMD locus was mapped to chromosome 14q11.2  in 1995 [11]. More than 98% of cases with a classic OPMD phenotype carry a single dominant (GCN)12–18 cryptic expansion in the PABPN1 gene [2]. The short trinucleotide repeat (GCN)11–18 are located immediately after the ATG start codon of the first exon of the polyadenylate-binding protein nuclear 1 gene (PABPN1), previously known as PABP2 [2]. The normal (GCN)10 sequence coding the 10 alanine stretch is: (GCG)6(GCA)3(GCG)1. The mutations consist of cryptic triplet expansions coding for alanine affecting the original (GCA)3, leading to mutations that are coded by. (GCG)6(GCN)4–11(GCG)1 referred to conventionally as (GCN)11–18 mutations [52]. Rare point mutations have been described, which generate a lengthened (GCN)10 stretch to (GCN)13 by causing an amino acid substitution at position c.35G > C (p.Gly12Ala) [53]. All mutations cause the lengthening of an N-terminal polyalanine domain (Ala)11–18 of the PABPN1 protein. Dominant and recessive OPMD are mitotically and meiotically stable. The severity of the dominant OPMD phenotype is variable between carriers of the same mutation even in the same family [5, 54]. A large French study also documents a wide spectrum of severity for carriers of the same size of repeat expansion as was shown in other large studies of carriers of the same size of mutations [5, 14, 17]. Of note, the disease severity can be quite different in patients from the same and carrying the same founder mutation [5]. Some of the more severe cases are compound heterozygotes for the dominant mutation and a (GCN)11 polymorphism in the other copy of the PABPN1 gene [2, 17]. This polymorphism has a 1–2% prevalence in North America, Europe and Japan. The more severe phenotypes observed in homozygotes and compound heterozygotes for a dominant and a recessive mutation suggest a clear gene dosage effect. The possibility that the

7  Oculopharyngeal Muscular Dystrophy

(GCN)11 allele could also be a dominant mutation has been raised by one group, though there is no consensus on this [54]. The relative frequencies of the different mutation sizes vary between countries, but (GCN)13 mutation is the most frequent mutation observed worldwide and together with (GCN)14, comprises 60% of cases [17]. Based on haplotype analysis it is clear that (GCN)n mutations have arisen independently many times during human history [6, 55]. Gene dosage has a clear influence on the age of onset and severity of the OPMD phenotype, with the most severe OPMD phenotype observed in carriers of two dominant (GCN)12–18/(GCN)12–18 OPMD mutations [2, 17, 49]. Recessive OPMD patients carrying two (GCN)13 mutations are more severely affected than dominant OPMD patients as shown in Jewish and French-Canadian cohorts [49]. The pathological diagnosis of OPMD has been replaced by genetic testing [2]. A single polymerase chain reaction (PCR) can establish the diagnosis. It can also be seen by next generation parallel sequencing such as Whole Exome and Genome sequencing, although some commercially available NGS platforms may not identify repeat disorders. Uncovering of an expanded (GCN)n size has a sensitivity and specificity close to 100%. The indications for DNA testing of a symptomatic individual are: (1) confirmation of the diagnosis, (2) the clinical picture presents a diagnostic dilemma, (3) evaluation the size of the mutation as a possible contributor to severity, (4) to exclude a compound heterozygote status in a patient with a severe earlier onset form of the disease, and (5) to exclude recessive OPMD. Since OPMD is inherited in both an autosomal dominant and an autosomal recessive manner, establishing the nature of the mutation(s) in a patient is essential to ensure appropriate genetic counseling. Every child of an individual heterozygous for a (GCN)12–18 mutation has a 50% chance of inheriting the disease-causing mutation. The offspring of an individual with autosomal recessive (GCN)11/(GCN)11 OPMD are obligate heterozygotes (carriers) for a mutant OPMD allele but this will most likely not lead to disease. One group has proposed that a (GCN)11 mutation alone could lead to a very late form of dysphagia [54], but this is unlikely since the relatively high carrier frequency of this mutation (as high as 1–2% in some populations), and would have resulted in many more cases being reported since genetic diagnosis became available [2]. Much care should be taken before requesting the predictive testing of an asymptomatic individual, because no preventive treatment is yet available. If presymptomatic testing is performed it should be accompanied by genetic counseling and psychological support.

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Pathogenesis The pathophysiology of OPMD is still largely unknown despite extensive work by many groups since the identification of the mutation in 1998 [2]. Various nuclear inclusion dependent and independent mechanisms have been proposed [56]. Though most hypotheses suggest that the expansion of the polyalanine stretch leads to a gain of function through aggregation of the protein, there is evidence that a relative decrease in level of normal PABPN1 may be central to the pathophysiology in a dominant negative fashion [57–59]. Since PABPN1 is an ubiquitous polyadenylation factor essential for the formation of the poly(A) tails of all human mRNA, the mutated protein could lead to a wide range of cellular dysfunction. Since it shuttles between the nucleus and the cytoplasm, both nuclear and cytoplasmic functions could be perturbed. This complexity has hampered international efforts to uncover how the mutations lead to a late-­ onset dystrophy. Complex perturbations of general processes such as mRNA expression profiles, polyadenylation site selection, abnormal myogenesis, mitochondrial dysfunction and muscle regeneration may be responsible for cell death [56]. Many groups have demonstrated that alternative polyadenylation initiation occurs in human muscle and myoblasts and in different cell and animal models, more generally leading to a shortening of polyalanine tails of mRNAs [56, 60– 62]. Some researchers have proposed an attractive argument that the altered processes are reminiscent of premature aging of the muscle [56]. However, the complex changes at the cellular level have not yet led to well defined therapeutic targets others than trying to decrease aggregation of PABPN1 protein and the expression of the mutated expanded allele. Many groups have studied cellular and animal models for OPMD. Expression of a mutated allele under the native promoter in a transgenic mice model was found not to lead to a major muscle clinical phenotype [63]. Until recently, the great majority of models have relied on over-expression of large expanded (GCN)n PABPN1. The most studied model to date has been the mice transgenic (GCN)17 mice PABPN1 model with a very high level of PABPN1 expression because of the human skeletal actin promoter [64, 65]. Despites its extensive use, it remains an imperfect model because over expressing even of wild type PABPN1 is known to lead to intranuclear aggregation [64]. Similar over-expression model in cells, C. elegans and zebrafish have been used to screen for candidate therapeutic compounds that have yet to undergo trials in humans. More recently, a mice transgenic model expression with a knock-in 17 alanine PABPN1 under a native promoter in mice with only one mutated (GCN)10 copy of PABPN1 showed a mild phenotype [59].

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Therapy

B. Brais

eye syndrome or poor orbicularis oculi function with incomplete eye closure. The evaluation of symptomatic dysphagia should be There is no disease-modifying therapy for OPMD.  A high prompted by moderate to severe dysphagia, suspected aspiprotein diet is generally recommended. Encouragement to ration pneumonia, marked weight loss, or recurrent pneumolimit social withdrawal at mealtime is important when the nias. Cricopharyngeal myotomy will alleviate symptoms in dysphagia becomes more severe. Patients and family memmost cases. Unfortunately, dysphagia will slowly reappear bers should be reassured about the low risk of fatal choking since patients learn to only swallow small bits. As aspiration over years and some patients with advanced disease have pneumonia is a frequent cause of death late in the diseases limited benefit; the 10.8% morbidity and 2.8% mortality are course, patient should be advised to obtain medical advice not negligible [67, 68]. Furthermore, if the surgical proceexpeditiously if they have a productive cough accompanied dure is not one of an extensive myotomy, the dysphagia may by fever. Strong mouthwashes may limit salivary pharyngeal not be improved and can even be worsened [39]. Severe dyspooling during the night by decreasing saliva viscosity. phonia and lower esophageal sphincter incompetence are Aerobic exercises for cardiovascular fitness in these late-­ contraindications to surgery. Though dilatation of the upper-­ onset disease should be encouraged but strenuous exercise esophageal sphincter has not been found to be efficacious for should be avoided. A large percentage of patients will use more than a few months and requires repetition of the proceeither a cane or a walker late in the course of the disease. A dure, it has become the first line treatment for dysphagia in cane with an integrated half step has been shown to help OPMD in many countries [41, 69]. Pharyngeal botulinum ambulant patients to conserve some autonomy (Fig.  7.1d) toxin injections has been used, but the clinical benefits have Walking sticks appear to be better to increase walking speed not been well documented and it may lead to increased dysand diminish risk of fall especially on uneven terrains than a phonia, worsened dysphagia and local hemorrhage [70]. cane. Rarely the more severe cases will require a wheelchair Autologous myoblast transplantation in pharyngeal and crior scooters mostly to travel longer distances. Prevention of copharyngeal muscles has been found to be technically featraumatic fractures due to falls is paramount. Adaptations to sible and lead to integration of the myoblasts into the facilitate access in the home is very important, especially pharyngeal muscles [71]. However, since all patients also since going up stairs becomes increasingly difficult as the underwent simultaneous cricopharyngeal myotomy in this diseases progresses. Patients may benefit from lift chairs. study, it is unclear if myoblast transplantation alone had therElectrically operated hospital beds to raise the head of the apeutic effect. Another limitation is the difficulty in producbed helps limit salivary choking at night and ability to adjust ing large numbers of autologous myoblasts. Now that a the height of the bed helps getting in and out of bed. Driving pharyngeal corrective gene therapy is being investigated, it is is usually not an issue until very late in the course of disease, unlikely that myoblast transfers will continue to be explored as a viable treatment for OPMD [72]. Unfortunately, despite with rare cases requiring modified manual controls. Surgical treatments are used to correct eyelid ptosis and extensive efforts to identify new therapeutic molecules using improve swallowing in moderately to severely affected indi- animal and cellular models there is no solid evidence that viduals. Two types of operations are used to correct ptosis any presently US FDA approved drug will be efficacious in with overall good results: (1) resection of the levator palpe- OPMD. Only trehalose, after having been found to modify brae aponeurosis and (2) frontal suspension of the eyelids. the phenotype in the A17 transgenic mice model, was tried in Resection of the aponeurosis is easily performed but usually a yet to be published phase 2 intravenous open-labeled needs to be repeated every 3–6 years with declining success human trial [65]. The field of OPMD research and treatment development with each surgery. Frontal suspension of the eyelids is now the gold-standard surgical treatment of ptosis in OPMD [66]. continue to evolve, although OPMD is often perceived as It consists of tying a synthetic sling to the tarsal plate of the one of the less severe muscular dystrophies with already upper eyelid and attaching it to the fascia of the frontalis available surgical treatments. However, its high prevalence muscle, which is relatively preserved in OPMD, thus allow- in certain populations, and the involvement of oculobulbar ing it to raise the eyelids. Its major advantages are that it is muscles makes it particularly attractive for studies of regional permanent and modification of over or under correction of anatomical treatment. Large natural history studies and the ptosis are done under local anesthesia in the weeks fol- research to define better outcome measures that are neceslowing surgery. Surgery is recommended when the ptosis sary to serve as the basis for future robust study designs are interferes with vision or cervical pain appears secondary to ongoing in the Netherlands and in Canada [73]. the constant dorsiflexion of the neck. Relative contraindications to blepharoplasty are marked ophthalmoplegia, a dry-­ Acknowledgements None.

7  Oculopharyngeal Muscular Dystrophy

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B. Brais 60. Jenal M, et  al. The poly(a)-binding protein nuclear 1 suppresses alternative cleavage and polyadenylation sites. Cell. 2012;149(3):538–53. 61. de Klerk E, et al. Poly(a) binding protein nuclear 1 levels affect alternative polyadenylation. Nucleic Acids Res. 2012;40(18):9089–101. 62. Chartier A, et  al. Mitochondrial dysfunction reveals the role of mRNA poly(a) tail regulation in oculopharyngeal muscular dystrophy pathogenesis. PLoS Genet. 2015;11(3):e1005092. 63. Dion P, et al. Transgenic expression of an expanded (GCG)13 repeat leads to weakness and coordination defects in mice. Neurobiol Dis. 2005;18:528–36. 64. Davies J, et al. Doxycycline attenuates and delays toxicity of the oculopharyngeal muscular dystrophy mutation in transgenic mice. Nat Med. 2005;6:672–7. 65. Davies JE, Sarkar S, Rubinsztein DC.  Trehalose reduces aggregate formation and delays pathology in a transgenic mouse model of oculopharyngeal muscular dystrophy. Hum Mol Genet. 2006;15(1):23–31. 66. Kalin-Hajdu E, et  al. Comparison of two polypropylene frontalis suspension techniques in 92 patients with Oculopharyngeal muscular dystrophy. Ophthalmic Plast Reconstr Surg. 2017;33(1):57–60. 67. Coiffier L, et  al. Long-term results of cricopharyngeal myotomy in oculopharyngeal muscular dystrophy. Otolaryngol Head Neck Surg. 2006;135(2):218–22. 68. Brigand C, et al. Risk factors in patients undergoing cricopharyngeal myotomy. Br J Surg. 2007;94(8):978–83. 69. Mathieu J, et al. A pilot study on upper esophageal sphincter dilatation for the treatment of dysphagia in patients with oculopharyngeal muscular dystrophy. Neuromuscul Disord. 1997;7(Suppl 1):S100–4. 70. Youssof S, Spafford M. Reply: to PMID 24259282. Muscle Nerve. 2014;50(5):870–1. 71. Perie S, et  al. Autologous myoblast transplantation for oculopharyngeal muscular dystrophy: a phase I/IIa clinical study. Mol Ther. 2014;22(1):219–25. 72. Malerba A, et al. PABPN1 gene therapy for oculopharyngeal muscular dystrophy. Nat Commun. 2017;8:14848. 73. Kroon R, et  al. Longitudinal assessment of strength, functional capacity, oropharyngeal function, and quality of life in oculopharyngeal muscular dystrophy. Neurology. 2021;97:e1475–83.

8

Distal Muscular Dystrophies Bjarne Udd

Introduction Distal muscular dystrophy refers to a group of genetic muscle diseases presenting at the onset with weakness of hands and fingers and/or feet and toes, combined with progressive atrophy of the corresponding distal muscles. In the literature most of these diseases carry names with the term distal myopathy because of historical reasons. The first publications in the field used distal myopathy to describe this phenotype although these diseases clearly are muscular dystrophies by the clinical definition: hereditary muscle diseases leading to progressive loss of muscle fibers, being replaced by fibrofatty connective tissue. Within this group, as with other genetic disorders, there is considerable clinical variability (Table 8.1). Proximal muscles may or may not be affected at a later stage of the disease and rarely, cardiac, respiratory, bulbar or facial muscles may also be involved. The clinical phenotype may vary from severe early onset forms that may present in the first year of life with later loss of ambulation to very mild late adult onset forms with ankle dorsiflexion or finger extension weakness starting in the eight decade.

Clinical Evaluation For the clinician, neuropathies are the first consideration when evaluating patients with distal muscle weakness and atrophy. However, if a patient with lower leg atrophy shows retained Achilles tendon reflexes, a myopathy is possible. If

anterior compartment wasting is combined with sparing of extensor digitorum brevis muscle and the patient lifts the toes when trying and failing to walk on heels, again a myogenic disease is more likely than a neurogenic one. Laboratory investigations include serum creatine kinase (CK), muscle magnetic resonance imaging (MRI) and neurophysiology. If a distinction between neuropathy and myopathy is clear based on this preliminary assessment, then molecular genetics can follow or be combined with a targeted muscle biopsy. More advanced histopathological techniques and refined cell and molecular biology studies have resulted in a better understanding of the pathophysiology of distal muscular dystrophies. Muscle imaging has proved to be an essential tool in the diagnosis and for the interpretation of novel variants of uncertain significance identified in known genes. In the last few years, massive parallel sequencing has contributed to identify disease-causing variants in over a dozen novel genes and revealed the first digenic mechanism causing a distal muscular dystrophy as well as the first X-linked distal muscular dystrophy (Table 8.1). Many of the currently known distal muscular dystrophy genes may also cause other clinical phenotypes such as limb-girdle muscular dystrophy (LGMD), contributing to the diversity of genetic myopathies [1–5]. Other genetic or acquired muscle diseases may occasionally also present with a distal phenotype and these need to be considered in the differential diagnosis (Table 8.2). In this chapter, distal muscular dystrophies are divided into 3 groups based on age of onset of weakness (early or childhood onset, juvenile to early adult onset and late adult onset).

B. Udd (*) Folkhalsan Research Center, Helsinki, Finland e-mail: [email protected] © The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 P. Narayanaswami, T. Liewluck (eds.), Principles and Practice of the Muscular Dystrophies, Current Clinical Neurology, https://doi.org/10.1007/978-3-031-44009-0_8

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132 Table 8.1  Distal muscular dystrophies and causative genes Clinical entity Adult—late onset diseases Welander distal myopathy (WDM) Digenic SQSTM1 and TIA1 mediated distal dystrophy Tibial muscular dystrophy (Udd myopathy) Vocal cord and pharyngeal distal myopathy Distal Actininopathy Oculopharyngodistal myopathy (OPDM)

Gene(s)

References

TIA1 SQSTM1 + TIA1 TTN MATR3 ACTN2 NOTCH2NLC, LRP12, GIPC1, RILPL1 PLIN4 VCP SMPX

AD DG AD AD AD>AR AD AD AD XR

Hackman et al. (2012) Lee et al. (2018) Hackman et al. (2002) Senderek et al. (2009) Savarese et al. (2019), Inoue et al. (2021) Deng et al. (2020); Ishiura et al. (2019); Saito et al. (2020); Sone et al. (2019) Ruggieri et al. (2020) Palmio et al. (2011) Johari et al. (2021)

MYOT LDB3

AD AD

Penisson-­Besnier et al. (2006) Griggs et al. (2007)

DES CRYAB

AD>AR AD

Sjoberg et al. (1999) Reichlich et al. (2010)

DYSF TTN GNE

AR AR AR

Liu et al. (1998) Evila et al. (2017) Kayashima et al. (2002)

PLIN4 mutated distal muscular dystrophy VCP distal muscular dystrophy SMPX mutated distal muscular dystrophy Myofibrillar distal muscular dystrophies Distal Myotilinopathy Late onset distal myopathy (Markesbery-­Griggs, Zaspopathy) Desminopathy Alpha-B crystallin-­mutated distal dystrophy Juvenile-early adult onset distal muscular dystrophies Miyoshi myopathy Recessive distal titinopathy Distal myopathy with rimmed vacuoles (Nonaka and GNE myopathy) Distal ABD-filaminopathy DNAJB6 distal muscular dystrophy Rimmed vacuolar distal neuromyopathy Distal ANO5 anoctaminopathy RYR1 mutated calf distal muscular dystrophy ADSSL1 distal myopathy Early-childhood onset distal muscular dystrophies Laing distal myopathy Nebulin distal muscular dystrophy

FLNC DNAJB6 HSPB8 ANO5 RYR1 ADSSL1

AD AD AD AR AD,AR AR

Duff et al. (2011) Ruggieri et al. (2015), Palmio et al. (2020) Ghaoui et al. (2016) Penttilä et al. (2012) Laughlin et al. (2017), Jokela et al. (2019) Park et al. (2016)

MYH7 NEB

AD AR > AD

KLHL9 distal muscular dystrophy

KLHL9

AD

Meredith et al. (2004) Wallgren-­Pettersson al. (2007), Kiiski et al. (2019) Cirak et al. (2010)

AD autosomal dominant; AR autosome recessive; DG digenic; XR X-chromosomal recessive Table 8.2  Other muscular dystrophies with occasional distal onset

1.  Hereditary Myopathies   −  Facioscapulohumeral muscular dystrophy (FSHD)   −  Myotonic dystrophy type1 (DM1)   −  Hereditary myopathy with early respiratory failure (HMERF)- titinopathy   −  Scapuloperoneal syndromes (FHL1 and TRIM32);   −  Other nemaline and rod-core myopathies (TPM2 and ACTA1)   −  Multisystem proteinopathy due to HNRNPA1 mutations   − Telethoninopathy   − Caveolinopathy   −  Centronuclear myopathy due to DNM2 mutations (dynaminopathy)   − Glycogenoses (e.g. glycogen branching and debranching enzyme deficiency, and polyglucosan body myopathy due to GYG1 mutations)   −  Neutral lipid storage myopathy due to PNPLA2 mutations   −  Mitochondrial distal myopathy (POLG)   −  Collagen XII-myopathy   −  Cystinosis-associated myopathy 2.  Acquired Myopathies   −  Sporadic inclusion body myositis (s-IBM)   −  Light chain (AL) amyloidosis-associated myopathy   −  Sarcoid myopathy

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 dult: Late Onset Distal Muscular A Dystrophies Welander Distal Myopathy (WDM): TIA1 WDM was first reported as an autosomal dominant late adult-onset disease with onset of muscle weakness in the finger extensors in a large Swedish cohort [6]. Later, the finger flexors, toe and ankle extensors are affected. The age of onset is 40–60 years; the disease is slowly progressive and patients remain ambulant. Sensory impairment in the distal extremities has been reported. Creatine kinase (CK) levels are usually normal. MRI shows fatty involvement and atrophy of forearm and lower leg extensors, usually also involving the calf muscles (Fig.  8.1). Muscle biopsy reveals myopathic changes and fibers with rimmed vacuoles [7]. The disorder was subsequently reported in Finnish and rare British patients. The causative founder mutation in Scandinavian patients (p.E384K) in the TIA1 gene on chromosome 2p13 was first reported in 2013 [8]. TIA1 encodes an RNA-binding protein, T cell-restricted intracellular antigen 1, a key regulator of mRNA-translation. TIA1 is a component of stress granules, which are cytoplasmic ribonucleoprotein aggregates that sequester untranslated messenger ribonucleic acid (mRNA) in response to cellular stress. It is proposed that the TIA1 mutation increases stress granule aggregation, dysregulating stress granule dynamics and leading to abnormal autophagy [8].

 igenic Cause of Distal Muscular Dystrophy: D SQSTM1 mutations Combined with Polymorphism in TIA1 Dominant mutations in sequestosome 1-encoding gene (SQSTM1) cause Paget disease of the bone with reduced penetrance [9]. If these mutations co-segregate with a common polymorphism in TIA1 (population frequency of 1%), the phenotype is not Paget disease, but a WDM-identical distal muscular dystrophy phenotype (Fig.  8.2). Thus the TIA1 polymorphism is capable of diverting the SQSTM1 affected

Fig. 8.1  Muscle MRI (axial T1 sequence) of the lower legs in a patient with Welander distal myopathy showing extensive fatty degeneration both in anterior and posterior compartment muscles of the lower legs

Fig. 8.2  Muscle MRI (axial T1 sequence) of the lower legs in a patient with digenic SQSTM1 and TIA1 mutated distal muscular dystrophy showing extensive fatty degeneration both in anterior and posterior compartment muscles of the lower legs

Fig. 8.3  Muscle MRI (axial T1 sequence) of the lower legs in a patient with Tibial muscular dystrophy, late onset dominant distal titinopathy showing extensive fatty degeneration in the anterior compartment muscles of the lower legs

tissue from bone to muscle. The basis for this co-interaction relied on the evidence that the SQSTM1 gene product, p62 protein, also interferes with the stress granule dynamics pathway [9]. As a further proof of this effect of the digenic mechanism, a control cohort of 50 Paget patients with the same SQSTM1 mutations did not have the TIA1 polymorphism [9].

 ibial Muscular Dystrophy (Udd Myopathy): T Dominant Distal Titinopathy-TTN Tibial muscular dystrophy (TMD) was described long before the gene was known [10]. The onset is after age 35–55 with weakness in ankle dorsiflexion and atrophy of the anterior lower leg muscles. Slow progression with very late proximal weakness preserves walking capacity until very advanced ages. Extensor digitorum brevis and hand muscles remain spared. Serum CK is normal or slightly elevated and MRI shows fatty degeneration in the anterior tibial muscles with later involvement of long toe extensors (Fig. 8.3), hamstrings and the gluteus minimus. Biopsy of an affected muscle shows rimmed vacuoles but may be near normal in clinically unaffected muscles, with only an increase of internal nuclei [10]. A common Finnish founder mutation (FINmaj) in the last exon of titin gene (TTN) on chromosome 2q31 was reported in 2002 [11]. The mutation is a complex 11-base pair inser-

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tion–deletion leading to an exchange of 4 amino acids in the last exon of the enormous gene, without interrupting the reading frame. Missense variants in the same exon 364 were later identified in other populations [11–13]. The subsequently identified recessive distal titinopathy with earlier onset is discussed later in this chapter. The huge titin protein is the third most abundant protein in the muscle filament system and the backbone of the sarcomere, forming a continuous filament system along the full length of the myofibril [14]. The C-terminus of titin bears binding sites for the protease calpain-3. The FINmaj mutation alters the proteolytic cleavage of C-terminal titin [15, 16]. A large number of alternative splicing events results in a similar number of different transcripts depending on developmental stage or type of muscle tissue [17, 18]. Causative mutations thus result in many different disease phenotypes affecting skeletal muscle, cardiac muscle or both [19, 20].

 ocal Cord and Pharyngeal Distal Myopathy: V MATR3 A large North American family [21] and a subsequent Bulgarian dominant pedigree [22] were reported with vocal cord and pharyngeal- distal myopathy (VCPDM) with onset of weakness in these muscle groups in late adulthood [21–23]. Many patients develop respiratory failure later in the disease [24]. CK levels are normal or mildly elevated and muscle pathology shows rimmed vacuoles. Fatty involvement of anterior and posterior compartments in lower legs (Fig.  8.4) and hamstrings is evident on MRI [25]. The disease is usually caused by a p.S85C mutation in MATR3 [22], which encodes a nuclear matrix protein involved in the regulation of RNA-metabolism [26]. Variants in MATR3 have also been associated with ALS [27], but VCPDM is clearly a myopathy based on pathology and the lack of long duration, high amplitude motor unit potentials on needle EMG, despite the frequently observed severe atrophy of intrinsic hand muscles mimicking a motor neuron disease [28].

B. Udd

Fig. 8.5  Muscle MRI (axial T1 sequence) of the lower legs in a patient distal actininopathy showing extensive fatty degeneration in most muscles of the lower legs, with long toe extensors and flexors less involved

Distal Actininopathy: ACTN2 Distal actininopathy is an adult onset dominant disease reported from Spain and Sweden. It usually presents in the fourth to fifth decades with bilateral or unilateral foot drop (Fig.  8.5) progressing to posterior leg compartment and proximal lower limb muscle weakness resulting in loss of ambulation around the eight decade. Initial asymmetric hypertrophy of the calf and quadriceps may be seen, followed in later stages of the disease by atrophy of these muscles. The upper limbs are relatively unaffected [29]. Serum CK levels are variable, ranging from mildly elevated in most patients to markedly elevated up to 10 times the upper limit of normal, especially in younger adults. Asymptomatic hyperCKemia is a rare presentation. Muscle MRI reveals fatty infiltration limited to the tibialis anterior in early disease, although long extensors and posterior leg compartment may also be involved. With later disease, the thigh and gluteal muscles also show fatty degeneration. Imaging findings are frequently asymmetric. Muscle pathology is dominated by rimmed vacuoles with some myofibrillar disarray and lobulated fibers. Electron microscopy reveals autophagic debris and some undulation of the Z-disk [29]. Missense variants in the ACTN2 (alpha actinin-2) gene were identified as fully co-segregating with the disease in the reported families; one of these is apparently a founder mutation in the Basque population [29]. ACTN2 encodes α-actinin2, a structural protein of the Z-disk interacting with titin and many other Z-disk located proteins such as myotilin [30–32]. Recently out-of-frame mutations in the last exon were identified as the cause a dominant distal myopathy with facial weakness [33]. Mutations in ACTN2 have also been shown to cause congenital myopathy [34], recessive core myopathy [35] and cardiomyopathy [36].

Distal Myopathy with Sarcoplasmic bodies: MB Fig. 8.4  Muscle MRI (axial T1 sequence) of the lower legs in a patient with Matrin3 mutated distal muscular dystrophy showing extensive fatty degeneration both in anterior and posterior compartment muscles of the lower legs

A Swedish family with autosomal dominant adult onset thenar atrophy and finger flexor weakness was reported in 1980 [37]. Subsequently the disorder has been described from

8  Distal Muscular Dystrophies

Sweden, France and the Netherlands [38]. The disease progresses more rapidly than other distal myopathies, with patients developing proximal muscle weakness, lower extremity and axial muscle weakness, requiring a wheelchair in some patients by 10–15 years of onset. Patients may also present with proximal lower extremity or axial weakness and have thenar muscle atrophy when examined [38]. CK is normal to moderately elevated. Muscle MRI reveals atrophy of involved muscles including paraspinal muscles. Dilated cardiomyopathy may occur, and respiratory failure requiring ventilatory support develops in many patients about 10 years after disease onset. The disease is named for characteristic inclusion body on muscle biopsy termed sarcoplasmic body. Sarcoplasmic bodies are round/oval, brown on hematoxylin and eosin, red on trichrome staining with a glassy appearance, easily detected even on unstained sections, sometimes occurring in longitudinal rows in both fiber types. They are perinuclear or intermyofibrillar on electron microscopy. Some are membrane bound and homogeneous, while others are not membrane bound, and contain filamentous material. They are autofluorescent with laser excitation. Myopathic changes accompany these bodies in affected muscles [37, 38]. One unique causative variant in the Myoglobin-encoding gene (MB), occurring in the primary family and in several unrelated families was recently identified [38]. The characteristic muscle pathology was a unifying finding but the clinical weakness was not distinctly distal in the later identified families, but more proximo-distal [38].

 culopharyngodistal Myopathy (OPDM): CGG O and GGC Expansions—See Chap. 13  LIN4 Mutated Distal Muscular dystrophy: PLIN4 P An autosomal dominant adult-onset distal muscular dystrophy with rimmed vacuoles first described in an Italian kindred mapped to a locus on 19p13.3 [39] and was recently shown to harbour a variant in the perilipin-4 (PLIN4) gene [40]. The disease presents between 26–73 years, with earlier onset noted in later generations, likely due to awareness of the disease. Clinical features ranged from no symptoms to bilateral foot drop, weakness of neck flexors and milder involvement of the finger and wrist extensors and shoulder girdle. Bulbar and cardiac involvement were not noted. Muscle biopsy revealed a myopathy with uniquitin positive rimmed vacuoles [40]. The gene normally contains a repeat tract, 31 repeats of 99 nucleotides, which in the affected members of the family was expanded to 40 repeats. Perilipins coat the phospholipid layer around lipid droplets and regulate lipid droplets. The identified repeat expansion is trans-

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Fig. 8.6  Muscle MRI (axial T1 sequence) of the lower legs in a patient with VCP mutated distal muscular dystrophy showing early fatty degenerative changes in the anterior compartment muscles of the lower legs

lated into a longer protein and causes misfolding with subsequent accumulation of the PLIN4 protein [40].

VCP Distal Muscular Dystrophy: VCP The common phenotype of mutated valosin containing protein (VCP) is a combination of hereditary inclusion body myopathy (hIBM) with proximal weakness andscapular winging, Paget disease of the bone and frontotemporal dementia (IBMPFD) [41–43]. However, a distal phenotype without scapular winging also exists, as reported in a large dominant Finnish family, with onset in adulthood (4th–sixth decades) of weakness in the anterior leg and intrinsic hand muscles [44]. Three of 9 patients in the original report developed dementia in the sixth to seventh decades. Paget disease did not occur in this family. Myopathic changes with rimmed vacuoles in the muscle biopsy, normal to mildly elevated CK levels and fatty replacement of anterior lower leg muscles on MRI were the characteristic clinical findings (Fig.  8.6). Proximal muscle weakness or respiratory involvement did not develop on follow-up. A VCP mutation P137L in exon 4 was detected in these patients [44].

 MPX Mutated Distal Muscular Dystrophy: S SMPX Through a large European collaboration, the first X-linked distal muscular dystrophy was identified in 2021, and found to be caused by several missense mutations in the small muscle protein X gene (SMPX) on Xp22.12 [45]. The age of onset is in the third-fourth decades. Patients develop moderate to severe distal weakness in the finger extensors or ankle dorsiflexors with late progression to proximal lower limb involvement. Progression to moderate to severe extensor predominant distal upper extremity weakness and mild proximal upper extremity weakness results in a pattern of severe

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distal >proximal upper and lower extremity weakness over decades; scapular winging may be present. Facial, bulbar, respiratory and cardiac muscles are spared [45]. CK is mildly elevated. MRI reveals early fatty changes in the anterior compartment of the lower leg (Fig. 8.7) spreading to the posterior compartment and later to the thigh muscles. The various mutant proteins have aggregating propensities and thus the muscle pathology is dominated by rimmed vacuoles and cytoplasmic inclusions some of which contain amyloid [45]. The vacuoles stain positive for several proteins including SMPX and ubiquitin. It is suggested that the missense mutations cause a gain-of-function muscle disease due to aggregation of the mutant protein [45].

 yofibrillar Distal Muscular Dystrophies (See M Chap. 12) Myotilinopathy: MYOT Mutations in the myotilin gene (MYOT) cause very late onset ankle weakness (5th–eighth decade) affecting both dorsal and plantar flexors [46–48]. Despite the late onset, the progression of the disease can be significant, and may cause loss of ambulation about 10  years after onset. Lower extremity pain and cramps and inability to walk on the toes are common early symptoms. Ankle jerks are absent, without sensory loss, even at initial examination. The muscle histopathology is dominated by myofibrillar disorganization

B. Udd

with myotilin accumulations and both rimmed and non-­ rimmed vacuoles. Amyloid deposits are not seen. The soleus muscle is usually first affected followed by tibialis anterior and gastrocnemius medialis muscles based on MRI findings (Fig. 8.8) in presymptomatic and symptomatic patients [49]. Some myotilinopathy patients have a limb-girdle phenotype (previously LGMD1A) [50], but the LGMD nomenclature/classification was replaced by the term myofibrillar myopathy (see Chaps. 5 and 12). Considerable proximal involvement may also result from homozygosity of milder dominant variants [51]. In some patients the protein accumulations containing myotilin may appear as spheroid bodies [52], although most aggregates form filamentous bundles on electron microscopy.

 arkesberry-Griggs Distal Muscular Dystrophy M (Zaspopathy): LDB3 A dominant LDB3 mutation is the cause of one of the canonical distal muscular dystrophy families reported in the 1970’s [53, 54]. LDB3 encodes Z-band alternatively spliced PDZ motif-containing protein (ZASP), which, as the name implies, is a Z-line protein. Along with myotilin and α-actinin, ZASP contributes to the stability of the Z line in anchoring the contractile filaments and titin. Patients develop ankle weakness after the age of 40 years (Fig. 8.9) with mild, subsequent involvement of the proximal muscles [53, 54]. Very late cardiomyopathy, either dilated or hypertrophic, has been reported in some patients [55, 56]. The muscle pathology is very similar to the myotilinopathies with heavily disorganized myofibrillar structures and protein accumulation and with both rimmed and non-rimmed vacuoles [54]. On electron microscopy the protein aggregates form characteristic filamentous bundles similar to myotilinopathy [57].

Desminopathy: DES Fig. 8.7  Muscle MRI (axial T1 sequence) of the lower legs in a male patient with X-linked SMPX mutated distal muscular dystrophy showing extensive fatty degeneration in the anterior compartment muscles of the lower legs and less in the soleus muscle

Fig. 8.8  Muscle MRI (axial T1 sequence) of the lower legs in a patient with distal myotilinopathy showing extensive fatty degeneration in the posterior compartment muscles of the lower legs, most of all in the soleus muscles and early changes in the anterior and lateral compartments

The first family later identified as desminopathy was reported in 1943 [58, 59]. Muscle weakness and atrophy starts in early adulthood in the hands and the lower legs, progressing

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acting with desmin in the assembly of intermediary filaments [63, 64]. This protein accumulates with desmin and myotilin in the myofibrillar aggregates on muscle pathology. The ultrastructural findings of granulofilamentous inclusions are similar to those in desminopathy.

Fig. 8.9  Muscle MRI (axial T1 sequence) of the lower legs in a patient with LDB3 mutated zaspopathy showing extensive fatty degeneration both in anterior and posterior compartment muscles of the lower legs with less involvement of the the peroneal muscles and toe flexors

Fig. 8.10  Muscle MRI (axial T1 sequence) of the lower legs in a patient with desminopathy at an early stage of the disease showing most fatty degeneration the left peroneal muscles and milder changes in the anterior compartment muscles and left peroneus of the lower legs

later to proximal muscles. Cardiomyopathy and cardiac arrhythmia are usually present. Cardiac involvement and respiratory failure may occur before limb muscle weakness. MRI typically reveals early involvement of peroneal muscles followed by tibialis anterior, gastrocnemius and soleus muscles (Fig. 8.10) along with the semitendinosus [49]. Serum CK is mildly elevated. The muscle pathology shows desmin and Z-disk protein accumulations, which can be less prominent, and therefore not detected, compared to myotilinopathy and zaspopathy. Electron microscopy reveals granulofilamentous accumulations [57]. Rarely a more severe clinical phenotype is the result of recessive biallelic desminopathy [60].

J uvenile to Early Adult-Onset Distal Muscular Dystrophies Miyoshi myopathy: DYSF Miyoshi et al., in the 1960s, reported patients with early adultonset weakness, myalgia and atrophy of the calf muscles [65]. Very high serum CK is a major hallmark of the disease, even in presymptomatic patients. Some patients may also have ankle dorsiflexion weakness. Correspondingly, MRI shows severe fatty replacement of the calf muscles (Fig.  8.11) and muscle biopsy reveals necrotic fibers with occasional inflammation. Biallelic recessive mutations in dysferlin gene (DYSF) are the cause of the disease [66]. Dysferlin protein has a function in sarcolemmal fusion events and resealing after damage due to the shearing forces of repeated contractions, which likely explains the CK leakage and fiber necrosis [67]. Dysferlin expression by immunostaining in muscle tissue or immunoblotting in muscle tissue or peripheral blood monocytes aids the diagnosis, and can also be used to verify the pathogenicity of variants of uncertain significance (VUS) identified in DYSF during genetic testing. Miyoshi myopathy and LGMD-R2 (previously LGMD2B), one of the common LGMDs, show overlapping features [68], and after years of disease progression the two phenotypes align and may be indistinguishable [69] (See Chap. 6).

Recessive Distal Titinopathy: TTN Truncating mutations in the last two exons of TTN, either in homozygosity or combined with a second recessive mutation in

Alpha-B Crystallinopathy: CRYAB Dominant mutations in α-B crystallin gene (CRYAB) can give rise to muscular dystrophy resembling desminopathy both clinically and pathologically [61, 62]. The major differences from desminopathy are the presence of cataracts; dysphagia or dysphonia may also be present. Muscle MRI shows fatty degenerative changes in tibialis anterior, gastrocnemius medialis and vastus muscles [49]. α-B-crystallin is a small heat-shock protein (HSPB5), a molecular chaperone inter-

Fig. 8.11  Muscle MRI (axial T1 sequence) of the lower legs in a yang patient with Miyoshi distal dysferlinopathy showing extensive fatty degeneration of the medial gastrocnemius muscles of the lower legs

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Fig. 8.12  Muscle MRI (axial T1 sequence) of the lower legs in a patient with juvenile onset recessive distal titinopathy showing the typical pattern of extensive fatty degeneration both in the soleus, anterior and lateral compartment muscles of the lower legs with asymmetry especially in the medial gastrocnemius

B. Udd

(Fig. 8.13) (see Chap. 9). In the thigh, involvement of hamstring muscles with sparing of the quadriceps is characteristic. The disease is progressive and half of the patients lose ambulation 10 years after symptom onset. CK is mildly elevated and muscle pathology findings are dominated by rimmed vacuoles [72, 73]. After disease causing founder mutations in GNE were identified [74] in patients from Japan and the Middle East, the disease has been identified worldwide. GNE encodes the enzyme epimerase-kinase, which is the rate-limiting enzyme in the sialic acid biosynthesis pathway [75]. Hyposialylation of muscle proteins was proposed as a major pathomechanistic hypothesis, but a recent study could not confirm consistent hyposialylation, suggesting that the pathophysiology is still unsettled [76]. Despite many years of clinical trials, no definitive therapeutic success has so far been reported from therapeutic substitution attempts with sialic acid.

Distal filaminopathy: FLNC Fig. 8.13  Muscle MRI (axial T1 sequence) of the lower legs in a patient with GNE mutated recessive distal muscular dystrophy showing extensive fatty degeneration in anterior compartment muscles of the lower legs

trans, cause early/juvenile onset recessive distal titinopathy affecting the anterior tibial muscles, with difficulty in heel walking [70]. Serum CK is slightly to markedly elevated; muscle biopsy reveals myopathic changes and on muscle MRI, fatty changes in lower leg anterior compartment muscles are combined with severe involvement of the soleus muscle (Fig. 8.12). Founder nonsense mutations in the second last exon 363  in southeastern and eastern Europe as well as a frameshift founder mutation in the Iberian population cause higher prevalence of recessive distal titinopathy in these regions. In some families different second causative variants segregating in the family may cause ‘pseudodominant’ pedigrees complicating the diagnostics and genetic counselling [70]. Moreover, elusive second mutations in TTN may not be easily identified by conventional pipelines used to analyse sequencing data and finding the final diagnosis may need application of copy number variation (CNV) analytics and RNA-sequencing [71].

After the initial description in a large Australian family, this dominant disease has been identified in many populations [77, 78]. Weakness of handgrip may be the first sign in young adults followed by calf weakness. The progression is slow, with later involvement of proximal muscles, but patients remain ambulant. CK is mildly elevated and muscle MRI reveals fatty replacement in the calves (Fig. 8.14) Muscle pathology usually shows non-specific myopathic changes without vacuoles or myofibrillar abnormalities unlike in the other forms of filaminopathies. FLNC mutations underlying this distal phenotype are located in the N-terminal actin-­binding domain, and result in increased actin-binding or located in the Ig-like domain 15. These mutations are different from those associated with the predominantly limb-­girdle form with myofibrillar changes on biopsy [79, 80].

 istal Myopathy with Rimmed vacuoles D (Nonaka Distal Myopathy, GNE Myopathy): GNE This recessively inherited disease causes ankle dorsiflexion weakness and foot drop with atrophy of the anterior compartment muscles of the lower legs in early adulthood

Fig. 8.14  Muscle MRI (axial T1 sequence) of the lower legs in a patient with early adult onset distal ABD-filaminopathy showing extensive fatty degeneration in the posterior compartment muscles of the lower legs with just minor changes in the peroneal muscles

8  Distal Muscular Dystrophies

Fig. 8.15  Muscle MRI (axial T1 sequence) of the lower legs in a patient with DNAJB6 mutated distal muscular dystrophy showing extensive and selective fatty replacement in posterior compartment muscles of the lower legs

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Fig. 8.16  Muscle MRI (axial T1 sequence) of the lower legs in a patient with HSPB8 mutated distal neuromyopathy showing fatty degenerative changes in the anterior compartment muscles of the lower legs

DNAJB6 Distal Muscular Dystrophy: DNAJB6 While mutations in DNAJB6, specifically in the G/F domain, were first identified in patients with LGMD (LGMD-D1; formerly LGMD1D) [81], some of the G/F domain mutations are also associated with a myopathy with distal onset. However, a very distinct distal muscular dystrophy phenotype is typically a result of mutations in the N-terminal J-domain [82]. A dominant mutation, p.A50V has been identified in several populations. These patients have adult-onset ankle plantar flexion weakness and loss of toe walking with severe dystrophic fatty replacement in the calf muscles (Fig. 8.15). Later hand weakness and very late involvement of proximal muscles is a common pattern of disease evolution. Serum CK levels are elevated and the muscle biopsies show both accumulations of myofibrillar proteins and rimmed vacuoles. The DNAJB6 encoded protein is a ubiquitous co-­chaperone and member of the DNAJ/HSP40 family [83], normally functioning as a partner in larger chaperone protein complexes. The dominant negative effect of the mutants causes malfunction of these complexes leading to accumulation of misfolded proteins and secondary decompensated autophagic re-cycling paralleled by the rimmed vacuolar pathology [63].

Fig. 8.17  Muscle MRI (axial T1 sequence) of the lower legs in a patient with ANO5 distal anoctaminopathy showing extensive fatty degeneration of the medial gastrocnemius muscles and asymmetrically of the soleus muscle in the lower legs

HSPB8 acts as stress protein with a chaperone-like activity and is a part of the Z-disc associated chaperone-assisted selective autophagy (CASA) complex [86]. The myopathological findings of myofibrillar myopathy with aggregates and rimmed vacuoles mimic those seen in myopathies caused by defects in BAG3 and DNAJB6, other proteins of the CASA complex. Recently, a novel HSPB8 variant has been found in a patient with limb-girdle myopathy without associated neuropathy [87].

Recessive Distal Anoctaminopathy: ANO5 Rimmed Vacuolar Neuromyopathy: HSPB8 Mutations in HSPB8 (small heat-shock protein-beta 8) were initially described in patients with motor neuropathy [84], but Ghaoui et al. reported patients with early adult neurogenic leg weakness evolving into distal and subsequent proximal myofibrillar and rimmed vacuolar myopathy later in the disease course [85]. EMG in these patients shows neurogenic findings in distal muscles with myopathic changes in proximal muscles. In the early stages of the disease, MRI shows either diffuse neurogenic changes (atrophy and edema without fatty replacement) in the lower legs or fatty involvement of the anterior compartment muscles (Fig. 8.16) depending on the mutation type, but with disease evolution dystrophic fatty replacement occurs both in proximal thigh and lower legs muscles.

The usual phenotype with ANO5 mutations is adult or late onset LGMD (LGMD-R12). For unknown reasons, some patients have earlier onset (15–40 years) [88, 89], with calf weakness leading to difficulties in sport activities and walking on tiptoes. The initially hypertrophic calf muscles turn into frequently asymmetric muscle atrophy (Fig. 8.17) [89]. Proximal muscle weakness and wasting is a late feature, 10–20 years after the calf weakness. Cardiomyopathy is not part of the anoctaminopathy phenotype. Because of the preferential involvement of the calf muscles, highly elevated CK levels (over 10 times the upper limit of normal) and muscle biopsy revealing scattered necrotic fibers, this disorder arises in the differential diagnosis of Miyoshi myopathy. Bi-allelic mutations in ANO5 form the genetic basis of this disorder. The protein anoctamin-5 is a cytoplasmic

140

calcium-­activated chloride channel [90, 91]. Mutations in this gene lead to reduced anoctamin-5 protein levels in the muscle either by loss of function mutants or by missense mutations destabilizing the protein and subsequent degradation [92]. Interestingly, there is considerable clinical variability with a much milder disease presentation in female patients [89].

B. Udd

J uvenile Recessive ADSSL1 Distal Muscular dystrophy: ADSSL1

Two unrelated Korean families with an autosomal recessive adolescent-onset distal (predominantly anterior leg compartment) and facial muscle weakness, mild CK elevations and myopathic features with or without rimmed vacuoles on muscle biopsy have been reported [96]. ADSSL1 encodes a muscle specific enzyme, adenylosuccinate synthase, that RYR1—Calf Distal Dystrophy: RYR1 catalyses the initial reaction in the conversion of inosine monophosphate (IMP) to adenosine monophosphate (AMP) A very unusual mild form of preferential fatty degeneration [97]. After the first report, more patients and families have of the medial gastrocnemius muscle can be the consequence been described, which largely confirm the phenotype, with of some ryanodine receptor 1 (RYR1) mutations, as recently the additional feature of quadriceps weakness developing by reported in a few families [93]. Some of the patients were toe early adulthood [98]. A sporadic case of Turkish origin and walkers in childhood, but this resolved spontaneously in one of Indian origin have been reported, with the Indian their teens. Patients report exercise-induced calf pain and patient having proximal myopathy with contractures [99]. A have very high CK levels (5–10 fold elevated). The disease is large cohort from Japan with 7 distinct biallelic mutations mild, without limitations in ambulation even in older patients. was reported to show more variable muscle involvement Some patients can actively participate in sports even in late including proximal and/or distal leg muscles, tongue, masadulthood despite fatty degeneration of the medial gastroc- seter, diaphragm, and paraspinal muscles, Dysphagia and nemius (Fig.  8.18). However, as with other RYR1-related chewing dysfunction developed in 26/ 63 patients; hypertromyopathies, central cores can be detected on muscle biopsy phic cardiomyopathy and restrictive ventilatory failure were with the typical pattern of focal irregular RYR1 staining on noted in some patients in the later stages. Muscle MRI immunohistochemistry [93]. revealed fat infiltration in the periphery of the vastus lateraThe mutant protein, RYR1, is the major calcium release lis, gastrocnemius and soleus muscles. On muscle biopsy, channel of the sarcoplasmic reticulum which in co-activation nemaline bodies, increased lipid droplets and myofibrillar with the sarcolemmal L-type voltage-gated calcium channels myopathy were commonly observed in all patients, suggestor dihydropyridine receptors, are responsible for the massive ing that the disease may be classified as nemaline myopathy cytoplasmic calcium influx activating the excitation-­ [100]. contraction coupling [94]. Dominant and recessive mutations in RYR1 present with a multitude of phenotypes [94]. Among others, a recessive Early-Childhood Onset Distal Muscular childhood-onset distal myopathy presenting with hand stiff- Dystrophies ness and facial weakness has been associated to bi-allelic RYR1 variants [95]. Laing Distal Myopathy: MYH7

Fig. 8.18  Muscle MRI (axial T1 sequence) of the lower legs in a patient with RYR1-mutated dominant distal myopathy showing specific fatty degeneration both in the medial gastrocnemius muscles of the lower legs

This disease was the first of the distal muscular dystrophies with established genetic linkage [101]. Weakness of ankle dorsiflexors and toe extensors (hanging big toe sign) occurs in early childhood, with inability to walk on heels and frequently with heel cord tightness. There is usually very slow progression, but a severe form can occur, with scoliosis and weakness of neck flexors, finger extensors and later proximal extremity and facial weakness. CK levels remain normal or very mildly elevated. On muscle biopsy the characteristic finding is type 1 fiber smallness, as in congenital fiber type disproportion. This is often combined with less distinct core or minicore lesions. Muscle MRI shows the selective involvement of tibialis anterior and long toe

8  Distal Muscular Dystrophies

141

Fig. 8.19  Muscle MRI (axial T1 sequence) of the lower legs in a patient with Laing distal myopathy showing selective and extensive fatty degeneration in the anterior compartment muscles of the lower legs

Fig. 8.20  Muscle MRI (axial T1 sequence) of the lower legs in a patient with distal nebulin myopathy showing selective fatty degeneration both the tibialis anterior muscles of the lower legs

extensor muscles (Fig. 8.19). In the more severe phenotype, calf muscles and thigh muscles can be affected although the lateral gastrocnemius and rectus femoris muscles are typically spared [102]. The disease is due to mutations in the MYH7 gene, which encodes the beta heavy chain of myosin, expressed in type 1 muscle fibers and the heart [103]. After the first mutation was identified in 2004, a large number of mutations from all over the world have been identified and most of them are concentrated in the tail region of the protein. An unusual high proportion, some 30%, of all mutations, are ‘de novo’, which means there is no family history of the disorder. Many of these mutations are re-occurring, without a founder background [104]. Mutations in the head and neck domains of the protein are associated with cardiomyopathy [105, 106], and mutations in the ultimate C-terminal region may cause ­myosin storage (hyaline body) myopathy with or without cardiac involvement [107].

A different, dominant early onset distal muscular dystrophy is associated with large in-frame NEB deletions, as described in a three-generation family with nemaline rod/cap pathology due to a deletion of 68 exons of the 183-exon NEB [112]. The deletion results in a 35% smaller sized protein causing a dominant-negative effect. The clinical picture is similar to recessive distal nebulinopathy with foot drop in childhood and finger and wrist extensor weakness in adolescence, largely sparing facial, bulbar and proximal muscles. CK can be slightly elevated. EMG is myopathic and muscle imaging shows fatty degeneration of the anterior compartment lower leg muscles [112]. Similarly, a large heterozygous de novo deletion in a sporadic young patient with asymmetric distal and facial weakness has been described, confirming the dominant effect of an abnormally truncated protein [113]. These examples show that copy number variant (CNV) analysis and RNA sequencing are essential to identify possible elusive pathogenic NEB variants [114, 115].

Distal Dystrophy with Nebulin Defects: NEB

 arly Onset Distal Dystrophy with KLHL9 E mutations: KLHL9

Bi-allelic recessive mutations in the nebulin gene (NEB) usually cause congenital nemaline myopathy with generalized muscle weakness [108, 109]. However, some recessive missense mutations lead to a much milder early-onset distal weakness predominantly affecting the anterior compartment muscles of the lower legs and later the finger and wrist extensors [110, 111]. Facial muscles are largely spared. The progression is very slow, with no major disability even in late-stage disease and patients usually remain ambulant without aids other than ankle- foot orthoses. Selective fatty degeneration in the anterior tibial muscles is the typical finding on MRI (Fig. 8.20) with more diffuse changes in late disease. EMG shows myopathic changes and CK is normal or mildly elevated. Muscle biopsy shows groups of atrophic fibers (but not angulated). Unlike other nebulinopathies, nemaline rods are not observed on light microscopy [111]. On electron microscopy, however, small rods adjacent to Z-disks may be seen [111].

A German family with early onset autosomal dominant distal weakness was reported to segregate with a heterozygous variant in the kelch-like homolog 9 gene (KLHL9) [116]. However, no other family has been identified despite extensive testing of this gene by gene panels and exome sequencing.

Management Overall, the degree of disability in distal muscular dystrophies is less severe than with most other hereditary muscle diseases and the progression is very slow in most, except for GNE-myopathy. However, most patients will need medical interventions or supporting rehabilitative measures for symptomatic management. For the everyday clinical diagnostics and evaluation of variants of uncertain significance, the algorithm below is of practical use (Fig. 8.21).

142

B. Udd

CLINICAL CRITERIA

Age of onset

Childhood to early adulthood

Teens to age 40 yrs

Late

hands AD/spor

AR/spor

Muscle imaging findings in the lower legs Ant > post

post

MYH7 DNM2 KLHL9 NEBdel

Ant > post NEB ADSSL TTN

Ant> post

mixed

AD/spor

AR/spor

AD

post

Muscle imaging findings in the lower legs Ant> post

mixed

DYSF ADB-FLNC GNE D DES HNRNPA1 A D DNAJB6 CRYAB ACTN2 R RYR1 HSPB8 OPDM PLIN4

Ant> post

post

DYSF ANO5

hands

Post >ant MYOT ZASP

YES

NO

TIA1 MATR3 VCP SQSTM1 +TIA1snp

TTN VCP SMPX

Fig. 8.21  Approach to the diagnosis of distal muscular dystrophies based on age at onset, inheritance pattern and muscle involvement patterns on imaging

The most important aspect of their care is timely recognition and management of systemic complications such as the cardiopathy in the desminopathies. Fortunately, respiratory failure is rare in the distal dystrophies as a group. For ankle dorsiflexion weakness, the use of dynamic anklefoot orthoses (DAFOs) makes walking easier. Various orthotic adaptations can be used for finger weakness. Modifications in everyday life tools assist hand function. Surgical correction of contractures has been used in severe cases.

Conclusions Distal muscular dystrophies are rare disorders. Many developments in molecular genetic techniques over the last 20 years have helped to uncover the genetic cause of several

distal muscular dystrophies. However, the molecular diagnosis in many families and patients remains unsolved. Much remains to be improved in the bioinformatic tools to uncover causative abnormalities from genomic and RNA sequencing data. Optimal clinical care and the development of treatment strategies are directly depending on a correct understanding of the underlying primary defect and pathomechanism. Constitution of large international consortia such as the Solve-RD in Europe will increase the diagnostic rate. Why some genetic defects preferentially affect the distal limb muscles in the pathologic process leading to progressive loss of muscle tissue is unclear. Clarifying this ­preference should reveal novel insights into the molecular basis of the downstream molecular pathomechanisms after the gene defect, and also provide better insights for therapeutic options.

8  Distal Muscular Dystrophies

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146 101. Laing NG, Laing BA, Meredith C, et  al. Autosomal dominant distal myopathy: linkage to chromosome 14. Am J Hum Genet. 1995;56(2):422–7. 102. Dubourg O, Maisonobe T, Behin A, Suominen T, Raheem O, Penttilä S, Parton M, Eymard B, Dahl A, Udd B. A novel MYH7 mutation occurring independently in French and Norwegian Laing distal myopathy families and de novo in one Finnish patient. J Neurol. 2011;258:1157–63. 103. Meredith C, Herrmann R, Parry C, et  al. Mutations in the slow skeletal muscle fiber myosin heavy chain gene (MYH7) cause Laing early-onset distal myopathy (MPD1). Am J Hum Genet. 2004;75(4):703–8. https://doi.org/10.1086/424760. 104. Lamont P, Wallefeld W, Hilton-Jones D, Udd B, Argov Z, Barboi A, Bonneman C, Boycott K, Bushby K, Connolly A, Davies N, Beggs A, Cox G, DeChene E, Jungbluth H, Muelas N, Palmio J, Penttilä S, Schmedding E, Suominen T, Straub V, Staples C, Van den Bergh P, Vilchez J, Wagner K, Wheeler P, Wraige E, Laing N. New mutations widen the phenotypic spectrum of slow skeletal/ β-cardiac myosin (MYH7) distal myopathy. Hum Mutation. 2014;35(7):868–79. 105. Morales A, Kinnamon DD, Jordan E, et  al. Variant interpretation for dilated cardiomyopathy: refinement of the American College of Medical Genetics and Genomics/ClinGen guidelines for the DCM precision medicine study. Circ Genom Precis Med. 2020;13(2):e002480. https://doi.org/10.1161/ CIRCGEN.119.002480. 106. Das KJ, Ingles J, Bagnall RD, Semsarian C. Determining pathogenicity of genetic variants in hypertrophic cardiomyopathy: importance of periodic reassessment. Genet Med. 2014;16(4):286–93. https://doi.org/10.1038/gim.2013.138. 107. Tajsharghi H, Oldfors A, Macleod DP, Swash M.  Homozygous mutation in MYH7  in myosin storage myopathy and cardiomyopathy. Neurology. 2007;68(12):962. https://doi.org/10.1212/01. wnl.0000257131.13438.2c. 108. Lehtokari VL, Kiiski K, Sandaradura SA, et al. Mutation update: the spectra of nebulin variants and associated myopathies.

B. Udd Hum Mutat. 2014;35(12):1418–26. https://doi.org/10.1002/ humu.22693. 109. Feingold-Zadok M, Chitayat D, Chong K, et  al. Mutations in the NEB gene cause fetal akinesia/arthrogryposis multiplex ­ congenita. Prenat Diagn. 2017;37(2):144–50. https://doi. org/10.1002/pd.4977. 110. Lehtokari VL, Pelin K, Herczegfalvi A, et al. Nemaline myopathy caused by mutations in the nebulin gene may present as a distal myopathy. Neuromuscul Disord. 2011;21(8):556–62. https://doi. org/10.1016/j.nmd.2011.05.012. 111. Wallgren-Pettersson C, Lehtokari VL, Kalimo H, et  al. Distal myopathy caused by homozygous missense mutations in the nebulin gene. Brain. 2007;130(Pt 6):1465–76. https://doi.org/10.1093/ brain/awm094. 112. Kiiski KJ, Lehtokari VL, Vihola AK, et al. Dominantly inherited distal nemaline/cap myopathy caused by a large deletion in the nebulin gene. Neuromuscul Disord. 2018;29:97–107. https://doi. org/10.1016/j.nmd.2018.12.007. 113. Sagath L, Lehtokari VL, Välipakka S, Vihola A, Gardberg M, Hackman P, Pelin K, Jokela M, Kiiski K, Udd B, Wallgren-­ Pettersson C.  Congenital asymmetric distal myopathy with hemifacial weakness caused by a heterozygous large de novo mosaic deletion in nebulin. Neuromuscul Disord. 2021;31(6):539–45. 114. Kiiski K, Lehtokari VL, Loytynoja A, et  al. A recurrent copy number variation of the NEB triplicate region: only revealed by the targeted nemaline myopathy CGH array. Eur J Hum Genet. 2016;24(4):574–80. https://doi.org/10.1038/ejhg.2015.166. 115. Hamanaka K, Miyatake S, Koshimizu E, et al. RNA sequencing solved the most common but unrecognized NEB pathogenic variant in Japanese nemaline myopathy. Genet Med. 2019;21(7):1629– 38. https://doi.org/10.1038/s41436-­018-­0360-­6. 116. Cirak S, Von Deimling F, Sachdev S, et al. Kelch-like homologue 9 mutation is associated with an early onset autosomal dominant distal myopathy. Brain. 2010;133(7):2123–35. https://doi. org/10.1093/brain/awq108.

9

GNE Myopathy Zohar Argov and Stella Mitrani-Rosenbaum

Introduction Over the last four decades a large group of adult-onset hereditary myopathies with distal onset has been identified (Chap. 8). A subgroup of these neuromuscular disorders shares some of the major histological and ultrastructural features of sporadic inclusion body myositis (sIBM): presence of cytoplasmic autophagic (‘rimmed’) vacuoles and of cytoplasmic inclusions composed of clusters of tubular filaments. Thus, two terms have been used in the past to define these conditions: hereditary inclusion body myopathies (HIBM) [1] and distal myopathy with rimmed vacuoles (DMRV) [2]. A ‘prototypic’ form of HIBM, the Persian Jewish quadriceps sparing myopathy (QSM), was reported in 1984 [3]. Following our description of the genetic defect in QSM, a homozygous mutation in the gene called GNE (short term for UDP N-acetylglucosamine 2- epimerase/N-acetylmannosamine kinase) [4], it became clear that DMRV is exactly the same condition as QSM (clinically and genetically) [5]. To avoid further confusion it was decided to call this unique condition GNE myopathy [6] and this chapter focuses on this hereditary disorder.

Clinical Phenotype The age of onset is commonly in the third- fourth decade of life. Teenage onset is considered rare (less than 3% approached a physician before age 20 in our series of Middle

Z. Argov (*) (Emeritus) of Neurology, Hadassah Medical Center, The Faculty of Medicine, The Hebrew University of Jerusalem, Jerusalem, Israel e-mail: [email protected] S. Mitrani-Rosenbaum (Emerita) of Molecular Biology, Hadassah Medical Center, The Faculty of Medicine, The Hebrew University of Jerusalem, Jerusalem, Israel

Eastern patients), but onset as early as 10 years of age has been reported [7, 8]. Late onset (age 40–60 years) has also been recorded [7–10]. The initial symptom is typically a change in gait, sometimes first noticed by the patient’s family or friends. Frequent tripping and a sense of ‘instability’ are sometimes reported by the patient. Weakness at onset is usually in the anterior compartment of the lower leg leading to bilateral foot drop (which maybe slightly asymmetric in the beginning). Rarely, one may encounter patients with onset of weakness in the hip muscles [11, 12]. There have been reports of abdominal muscle weakness with protruded belly as a first or very early sign [13, 14]. Distal upper limb weakness and atrophy can also be an early feature [8, 15]. Early paraspinal musculature involvement has been described, with very few patients reaching medical attention because of it [12, 16]. With all the above-mentioned unusual features at onset one should remember that in the vast majority of patients foot drop is the first symptom and can be the sole sign for several years, thus it is not surprising that GNE myopathy is still regarded as a ‘distal myopathy‘. As the disease progresses, the calf muscles become weak, at times only in the more advanced stages. The proximal leg musculature becomes affected typically as the first region of disease spread. Usually, the iliopsoas is the first and the most affected of the hip musculature with a similar degree of weakness appearing in the glutei, hamstrings and adductors. The quadriceps muscle remains strong or minimally affected even in older patients who are wheelchair-bound or bedridden. It should be emphasized that this unique pattern of quadriceps sparing, reported first in Iranian Jews [3], is shared by most patients worldwide and is a clinical hallmark sign, even in proximal-onset GNE myopathy patients. In less than 5% of patients the quadriceps becomes weak, even to the same degree as the other hip muscles [9, 17, 18]. Such patients will lose their ambulation at an early stage of the disease. In the upper limbs, involvement of the scapular and very proximal muscles is initially found followed by weakening

© The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 P. Narayanaswami, T. Liewluck (eds.), Principles and Practice of the Muscular Dystrophies, Current Clinical Neurology, https://doi.org/10.1007/978-3-031-44009-0_9

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of the distal muscles of the arm and hand at later stages. Neck flexors become affected only in advanced stages and mild facial muscles weakness was also reported [19]. Bulbar and ocular muscles are not affected. Clinically-relevant respiratory impairment has not been observed in the Middle Eastern patient cluster [11, 20], Bulgarian Roma [8] or UK patients [21]. In contrast, it was recorded in advanced phases of the disease in the Japanese cohort [22]. These were usually wheelchair bound patients, who had low FVC values (5x ULN

Myopathic units Some (1+) FP or PSWs (functional denervation)

Lissencephaly, pachygyria, polymicrogyria, ventricular enlargement with or without hydrocephalus; brainstem & cerebellar hypoplasia

Dystrophic changes Reduced staining α-dystroglycan and merosin

High >5x ULN

Dystrophic changes Absent merosin staining

Distal hyperextensible joints (Ullrich) Contractures (esp elbow, knee, ankles) Finger flexor contractures (Bethlem) Keratosis pilaris Keloids Hypertrophic scars Cognitively normal Severe cognitive impairment Epilepsy

Normal to mildly elevated

Myopathic units Subcortical white matter Some (1+) FP or abnormalities PSWs (functional denervation) Myopathic units Unrevealing

High

Myopathic units

Risk of structural brain anomalies

Dystrophic changes

Facial weakness Ophthalmoparesis Ptosis

Normal

Myopathic units

Unrevealing

Recurrent apneas (spontaneous or with infection) Ptosis Ophthalmoparesis (25% of RAPSYN) Stridor, vocal cord paresis (DOK7) Ptosis Anticipation seen evaluate mother or father for mild symptoms / myotonia

Normal Mild CK elevation in some GFPT1 CMS patients [135] Normal

Decrement on Unrevealing RNS Repetitive CMAPs (COLQ, slow channel CMS)

Specific myopathic findings (nemaline rods, fibre type disproportion, central cores, etc) Unrevealing in most cases except for tubular aggregates in GFPT1, DPAGT1 and ALG2

Myotonic Unrevealing discharges later in life (not seen in first few years of life)

autosomal recessive ryanodine receptor 1 (RYR1)-myopathy (Fig. 11.2) [16]. Facial weakness is also a prominent feature of infants with congenital myotonic dystrophy and some CMS [17]. Among CMD, children with  merosin-negative CMD [18] may show more prominent  facial weakness,

Dystrophic or myopathic changes Absent to reduced-to-normal collagen VI staining

Unrevealing

although this can be differentiated from the other congenital disorders of muscle listed above by its elevated serum CK, typically well above 5x upper limit of normal (ULN). Ophthalmoplegia is also a feature of some  CM, most notably centronuclear myopathy, autosomal recessive RYR1-­

H. J. McMillan and M. Oskoui

178 Isolated hyperCKemia?

Cardiomyopathy?

Consider GOSR2 (myoclonic epilepsy onset later)

Normal cognition?

Structural brain malformation? Serum CK >5x ULN

Structural eye anomalies?

Consider TTN. Consider Pompe disease

White matter hyperintensities?

Consider α-dystroglycanopathies

Consider merosinopathy

INFANT < 2 yo: WEAKNESS + HYPOTONIA + HYPOREFLEXIA

Distal hyperlaxity

Serum CK normal to 1000 U/L in more than 80% of infants and children with LAMA2-related CMD [57] which can assist with its differentiation from many other congenital muscle disorders. MRI of the brain is particularly revealing in patients with LAMA2 variants, showing diffuse T2 hyperintensity in the subcortical white matter (Fig.  11.6) noted as early as

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6  months of age [58]. White matter changes characteristically spare the corpus callosum, internal capsule as well as the deep grey nuclei and cerebellum [59, 60]. As such, an MRI brain is recommended for children who are clinically suspected to have merosin-deficient CMD to aid in diagnosis [37]. Merosin deficiency CMD has been documented to be associated with a broad range of cognitive potential; however, initial reports were based upon clinical phenotype, biopsy and brain MR imaging without genetic confirmation [61]. Lower IQ scores were seen among children with findings other than white matter changes on brain MRI such as cerebellar hypoplasia [61]. Despite the presence of widespread white matter abnormalities patients do not demonstrate any consistent central nervous system dysfunction such as progressive cognitive impairment or dementia and no corticospinal tract involvement such as spasticity or hyperreflexia. Disorders of neuronal migration are uncommon (200) CGG repeats, suggesting that haploinsufficiency is not the disease mechanism and the expression of CGG-expanded RNA may be toxic in NOTCH2NLC-OPDM [13]. DNA hypermethylation has not been demonstrated yet in other OPDM subtypes. The CGG repeat expansions in NOTCH2NLC have been reported to translate into a toxic polyglycine-containing protein, uN2CpolyG, which forms intranuclear inclusions in cell and mouse models and in tissue samples from patients with NIID [44]. These mouse models show muscle weakness, neuronal cell loss with significant loss of Purkinje cells, and premature death, suggesting that the uN2CpolyG protein expression is toxic to animals [44]. Fragile X tremor ataxia syndrome (FXTAS) results from CGG repeat expansions in the 5′UTR of FMR1, which also translates into a toxic polyglycine-­containing protein, FMRpolyG, which is toxic to Drosophila and human cell lines [45]. The polyG mechanism has not been confirmed in other OPDM subtypes. However, RILPL1-OPDM shows co-localization of glycine and p62  in the intranuclear inclusions of skeletal muscle, suggesting that the pathomechanism of RILPL1-OPDM may be associated with polyglycinopathy [14]. Intranuclear inclusions in skin biopsy stain with anti-p62 antibody in LRP12-­ OPDM, GIPC1-OPDM, and NOTCH2NLC-OPDM, similar to NIID (Table  13.1), suggesting that the underlying pathomechanism of the subtypes of OPDM may be similar [41, 42]. It is therefore possible that the CGG repeat expansions in LRP12 and GIPC1 are also associated with novel polyglycine-containing proteins [23]. Further studies are necessary to unveil the pathomechanism of OPDM. Expanded CGG repeats can be pathogenic by triggering the formation of RNA foci, which sequester RNA-binding proteins that are cytotoxic in the nucleus [23]. In patients with NOTCH2NLC-OPDM, two RNA-binding proteins, Heterogeneous nuclear ribonucleoprotein A/B (hnRNP A/B) and Muscle blind like protein 1 (MBNL1), were both colocalized with p62  in intranuclear inclusions in muscle sections and cultured cells [13]. MBNL1 is also sequestered in the intranuclear inclusions, and induces RNA toxicity in myotonic dystrophy 1 and FXTAS [46, 47]. Moreover, in patients with NIID, the RNA-binding protein Src associated in mitosis, of 68  kDa (Sam68) was sequestered and

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c­ olocalized with p62 in intranuclear inclusions [48], suggesting that toxic RNA gain-of-function may also play a role in the pathogenesis of CGG repeat expansions in NOTCH2NLC [13]. Additionally, RNA Fluorescent in-situ hybridization (FISH) has demonstrated RNA foci formed by RILPL1 mRNA, and that MBNL1 and p62 were colocalized in the intranuclear inclusions in muscle biopsies, suggesting that the RNA gain-of-function mechanism is also important in RILPL1-OPDM [14]. Although there are no reports regarding RNA toxicity in LRP12-OPDM and GIPC1-OPDM, the similarity of LRP12-OPDM to RILPL1-OPDM in clinicopathological features may implicate RNA toxicity in LRP12-­ OPDM and GIPC1-OPDM. Further studies are necessary to confirm the above hypothesis.

Differential Diagnosis There are several conditions that should be considered in differential diagnoses in OPDM, such as mitochondrial myopathy with chronic progressive external ophthalmoplegia (CPEO), myasthenia gravis, congenital myasthenic syndrome, OPMD, myotonic dystrophy type 1, vocal cord and pharyngeal dysfunction with distal myopathy (VCPDM), inclusion body myositis (IBM) and facioscapulohumeral muscular dystrophy (FSHD) [15]. In particular, patients with OPMD caused by GCN repeat expansions in the PABPN1, (see Chap. 7) show clinicopathologically similar symptoms to OPDM.  Patients with OPMD typically show progressive ptosis, dysphagia, and proximal muscle weakness, whereas patients with OPDM usually show progressive ptosis, dysphagia, and predominant distal muscle weakness [12]. However, some patients with OPMD also show predominant distal muscle weakness [49] and a small proportion of OPDM patients has predominant proximal muscle involvement, [12] making it difficult to distinguish between OPMD and OPDM. Therefore, genetic analysis is necessary to differentiate OPDM from OPMD.  The onset of OPMD with GCN repeat expansions in the PABPN1 typically occurs around 60 years of age, which is older than the typical age of onset of OPDM; however, compound heterozygotes and dominant homozygotes with the expanded GCN repeats in the PABPN1 and dominant frameshift variants in HNRNPA2B1 may present with early-onset OPMD [50–53]. Intranuclear inclusions staining for p62 are a feature of LRP12-OPDM, GIPC1-OPDM, and NOTCH2NLC-OPDM, and are seen in myonuclei as well as in other tissues. Skin biopsies reveal these inclusions in the sweat glands, adipocytes and fibroblasts in all forms of OPDM but not in OPMD, IBM or GNE myopathy [41]. Anti-p62 antibody-positive intra-myonuclear inclusions are more frequent in OPMD (involving an average 11.9%  ±  1.1% of myonuclei, range

M. Ogasawara and I. Nishino

5.9–18.6%) than in LRP12-OPDM, GIPC1-OPDM, and NOTCH2NLC-OPDM (mean 0.9–1.5%, range 0–2.8%, p 150  m but 90% and also reduced expression of DUX4 target genes. Further experiments with one of the gapmers showed a partial correction of gene expression patterns and myotube atrophy, and local injection into the tibialis anterior of mice expressing DUX4 showed a 70% knockdown of DUX4 [125].

 NA Therapeutics for Other Dominant R Myopathies and LGMDs RNAi strategies similar to those employed in DM1 and FSHD can also be applied to other myopathies caused by dominant negative variants [126]. Development efforts in this regard are less advanced, in part owing to the rarity of many of these conditions. RNAi has been successfully employed in a mouse model of myotilinopathy. A miRNA targeting the MYOT gene was delivered via an AAV vector and resulted in >50% reduction in gene and protein expression. This led to a reduction in the protein aggregates that are a pathological hallmark of the disease, as well as other improvements of muscle histopathology, weight, and specific force [127]. Variants in the three collagen VI genes (COL6A1, COL6A2 and COL6A3) are among the most common causes of dominant LGMDs [128, 129]. Biallelic loss of collagen VI is deleterious and leads to recessive collagenopathies,

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whereas haploinsufficiency (loss of function of a single allele) is not associated with disease. Therapeutic strategies for dominant collagenopathies therefore require allele-­ specific knockdown, as knockdown of both alleles would be expected to worsen the phenotype. Splice site variants causing exclusion of exon 16 of COL6A3 are a common cause of the disease and present a good target for RNAi targeting the aberrant junction between exons 15 and 17. This strategy was effective in cellular models, where it silenced the disease-­causing allele without affecting the wild-type allele, resulting in increased extracellular collagen VI matrix deposition [130]. A similar strategy was employed in a recently described collagen XII myopathy caused by deletion of exon 52 of COL12A1 [131]. Variants affecting glycine residues in the triple-helical domains of the collagen proteins are also a common cause of collagenopathies and represent a more challenging therapeutic target. A set of siRNAs directed at one such missense variant resulted in knockdown of both the mutant and wild-type alleles, with only moderate specificity for the former [132].

Molecular Therapeutics for Oculopharyngeal Muscular Dystrophy Oculopharyngeal muscular dystrophy (OPMD) is an autosomal dominant muscular dystrophy caused by a polyalanine expansion in the polyadenylate binding protein nuclear 1 (PABPN1) (see Chap. 7). Knockdown of PABPN1 can be accomplished by RNAi, but in isolation, it is deleterious to muscle function, inducing muscle degeneration [133]. An effective therapeutic approach must thus also restore normal protein expression. This was initially accomplished in a murine model by co-delivery of shRNAs that silence endogenous PABPN1 expression, along with a second vector containing a codon-optimised version of PABPN1, which codes for the same amino acid sequence but has a different DNA sequence that is not recognised by the shRNAs [133]. The two therapeutic constructs were subsequently combined into a single AAV9 vector, dubbed BB-301. Intramuscular injection of BB-301 in a mouse model of OPMD resulted 80% knockdown of endogenous PABPN1 and expression of the codon-optimised version at 60% of normal levels. Treated animals showed a reduction in intranuclear inclusions, a pathological hallmark of OPMD, and restoration of muscle strength [134]. Further preclinical studies are underway in order to optimise intramuscular delivery in preparation for clinical trials. The “silence and replace” approach employed by BB-301 may also find applications in other dominant myopathies.

S. Nicolau and K. M. Flanigan

Gene Replacement Dystrophinopathies and most recessive LGMDs are caused by a loss of function of the affected gene, and these disorders could therefore be corrected by delivering a functioning copy of the gene to re-establish protein production. Different viral vectors have been tested for this purpose, including lentiviruses, retroviruses, adenovirus, herpes simplex virus, and AAV. The latter has become the preferred vector for human gene therapy [135] and 3 AAV-based gene therapy products have received regulatory approval in the United States or Europe: onasemnogene abeparvovec for spinal muscular atrophy [136], voretigene neparvovec for RPE65-related retinal dystrophy [137], and alipogene tiparvovec for lipoprotein lipase deficiency [138]. A number of features of AAV render it well-suited for gene delivery. AAV is a non-­ pathogenic virus and is incapable of independent replication. It remains episomal rather than integrating into the human genome, thus avoiding the risk of oncogenicity, and is capable of establishing lasting expression in both dividing and non-dividing cells. Several naturally-occurring and engineered AAV serotypes have been described, which differ in tissue tropism [139]. When used as a vector for gene therapy, the AAV capsid is retained, but its genome is replaced with a synthetic transgene (Fig. 18.3). The major limitation of AAV is its restricted packaging capacity of approximately 5kb, which complicates the delivery of several large genes implicated in muscle disease.

Fig. 18.3  General structure of an AAV vector used for gene therapy

18  Molecular Genetic Therapies in the Muscular Dystrophies

 ene Replacement for Duchenne Muscular G Dystrophy Among muscle disorders, DMD has been the focus of the most intense gene therapy development work. Early efforts included gene delivery by plasmids and adenoviral vectors, but these were limited by poor efficiency and strong immunogenicity, respectively [140, 141]. The dystrophin protein is composed of a number of functional domains: an N-terminal actin-binding domain, a central rod domain that includes 4 hinges and 24 spectrin-like repeats, a cysteinerich domain that includes dystroglycan-binding sites, and a C-terminal domain (see Chap. 2). The full coding sequence of dystrophin comprises 79 exons spanning 11kb, far exceeding the packaging capacity of AAV.  A number of mini- and microdystrophin constructs have been developed over the years and evaluated in preclinical studies. Three different microdystrophins are currently undergoing clinical trials: PF-06939926, SGT-001, and GNT 0004. As reviewed in detail elsewhere [142], their common features include removal of the majority of the rod domain, leaving only 4–5 spectrin-like repeats and 2–3 hinges, as well as removal of the C-terminal domain beyond the dystroglycan-binding site. They differ however in retention of the neuronal nitric oxide synthase (nNOS) binding site, as well as in the choice of promoter and AAV serotype. An early study of AAV-delivered microdystrophins described the ∆3990 construct, which is similar to PF-06939926, and was shown to restore dystrophin expression in 50–80% of muscle fibres and to correct dystrophic pathology in the mdx mouse model [143]. A closely related construct was tested in the golden retriever muscular dystrophy (GRMD) model, where it was found to restore dystrophin expression, but raised concern of an inflammatory reaction [144]. Indeed, an early study of intramuscular delivery in boys with DMD showed only sparse dystrophin expression, with evidence of an immune response against the transgene [145, 146]. Some preclinical evidence suggests that microdystrophin function is improved by having an even number of spectrin-­ like repeats and by inclusion of the second hinge [147, 148]. These features are both incorporated in the ∆R4-R23/∆71-­78 microdystrophin, on which SRP-9001 is based. This construct was used to restore dystrophin expression by local injection in the skeletal muscles and heart of mdx mice, and shown to protect treated muscles from contraction-induced injury [147, 149, 150]. Systemic treatment was also effective at restoring dystrophin expression and improving muscle pathology in young and aged mdx mice, as well as in the dystrophin-utrophin double-knockout mice, a more severe model of DMD [151–153]. More recently, an AAVrh74 vector containing a ∆R4-R23/∆71-78 microdystrophin under the control of the MHCK7 promoter showed good safety, robust restoration of dystrophin expression, and improve-

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ments in muscle force in mdx mice, providing preclinical evidence of efficacy for SRP-9001 [154]. A similar microdystrophin design was shown to be effective in two dog models of DMD [155, 156]. In muscle fibers, neuronal nitric oxide synthase (nNOS) localises to the sarcolemmal membrane by interacting with dystrophin and dystrophin-associated proteins. nNOS plays an important role in regulation of muscle blood flow and in several metabolic pathways in muscle fibers, and loss of nNOS activity has been implicated in muscle fatigue in DMD [157–159]. In addition to restoring dystrophin expression, a microdystrophin containing the nNOS binding site in spectrin-like repeats 16–17 improved muscle perfusion during exercise in mdx mice [157]. Similar constructs were found to be effective by local and systemic delivery in canine models of DMD [160–162]. SGT-001 is based on related design employing the CK8 promoter and AAV9 serotype. The efficacy of this construct was demonstrated in the more severely affected DBA/2J-mdx mouse model of DMD [163]. Published and unpublished data from early-phase trials has shown microdystrophin expression of 10–70% of normal dystrophin levels [164]. Complement activation was observed in trials of PF-06939926 and SGT-001, while rhabdomyolysis was reported with SRP-9001. Preliminary clinical data suggests functional improvements in some, but not all studies. SRP-001 was recently approved by the US FDA under an accelerated approval program for the treatment of ambulatory boys aged 4 to 5 years. It is contraindicated in patients with deletions in exon 8 or 9 due to the risk for an immune mediated myositis.

Gene Replacement for LGMDs Sarcoglycanopathies are a group of recessive limb-girdle muscular dystrophies (LGMD-R3-R6) caused by variants in one of the 4 sarcoglycan genes (SGCA, SGCB, SGCG and SGCD, encoding α-, β-, γ- and δ-sarcoglycan, respectively). α- and γ-sarcoglycanopathies are the most common subtypes in Europe and North America [129, 165]. Unlike dystrophin, sarcoglycan genes are small enough to be packaged into a single AAV vector. Mendell and colleagues developed an AAV vector carrying the SGCA gene under the control of a truncated muscle CK promoter. Local injection into the tibialis anterior of Sgca-null mice resulted in efficient myofiber transduction, overexpression of α-sarcoglycan, and restoration of the dystrophin-glycoprotein complex, without apparent toxicity [166]. A small phase 1 clinical trial involving 6 patients showed α-sarcoglycan expression up to 6 months following local injection into the extensor digitorum brevis [167, 168]. Delivery by isolated limb infusion in a further 6 individuals showed restoration of 14–25% of normal α-sarcoglycan levels and improved strength in the knee extensors, however without any improvement in 6-min walk distance [169].

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Gene therapy development efforts are also underway for the other sarcoglycanopathies. Mendell and colleagues also developed a similar vector for treatment of β-sarcoglycanopathy. Sgcb-null mice have pronounced dystrophic muscle pathology, similar to human patients. Gene transfer in these mice via intramuscular delivery reduced internal nucleation and fibrosis, increased muscle force, and enhanced resistance to contraction-induced injury. Similar benefits were seen with isolated limb perfusion. In aged mice, treatment not only prevented further fibrosis, but also partially reversed existing fibrosis [170]. A further study using systemic delivery of the SGCB gene under the control of the MHCK7 promoter showed highly efficient transduction of both heart and skeletal muscle, with expression of β-sarcoglycan up to 72% above wild-type mice. In addition to correction of dystrophic muscle pathology, treated mice also showed reduced CK levels, less kyphoscoliosis, and increased functional ability. Despite the overexpression of β-sarcoglycan, no significant toxicity was observed [171]. These encouraging results led to a phase 1/2 clinical trial evaluating systemic delivery at two different doses in 6 patients [NCT03652259]. Preliminary data presented at the 2021 Congress of the World Muscle Society shows β-sarcoglycan expression in all treated patients, reaching a mean level of 62% of normal in the high-dose cohort. Performance on functional tests at 24 months was also improved over baseline. A phase 1 dose escalation study was also completed in γ-sarcoglycanopathy, employing an AAV vector to deliver the SGCG gene under the control of the desmin promoter. The vector was administered by intramuscular injection into the extensor carpi radialis. At 30 days, γ-sarcoglycan expression was detected in 5–10% of muscle fibres in all 3 patients in the highest dose group, and to a lesser degree in 2 of the 6 patients in lower dose groups [172]. In a subsequent study, systemic delivery was evaluated in Sgcg-null mice. At the highest tested dose, this resulted in restoration of γ-sarcoglycan expression in 75–100% of muscle fibers, as well as an improvement in muscle pathology and increased performance on functional testing [173]. δ-sarcoglycanopathy is much rarer than the other 3 sarcoglycanopathies and has thus attracted fewer efforts at therapy development. SGCD gene transfer has nonetheless been evaluated in the BIO14.6 hamster [174, 175]. This long-established model of cardiomyopathy was only much later found to carry a large deletion in the δ-sarcoglycan gene [176, 177]. In these animals, gene transfer led to robust δ-sarcoglycan expression in heart and skeletal muscle, with improvements in muscle pathology, CK levels, functional testing and survival [174, 175]. Dysferlinopathies (LGMD-R2 and Miyoshi myopathy) are a clinically heterogenous group of recessive myopathies stemming from variants in the DYSF gene, which is involved in membrane repair. In contrast to sarcoglycan genes, the

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DYSF gene spans 6.2 kb and thus cannot be packaged into a single AAV vector. One solution is to design a truncated dysferlin construct analogous to microdystrophin. Llanga and colleagues engineered such a nanodysferlin lacking domains C2D-C2F, resulting in a 4.4  kb open reading frame. The nanodysferlin showed appropriate cellular localization. Systemic delivery via an AAV vector into dysferlin-deficient mice improved sarcolemmal membrane integrity and muscle function [178]. An alternative approach is to divide the full-­ length DYSF transcript between two AAV vectors. The ability to deliver fragmented genes via AAV vectors was serendipitously discovered while attempting to package oversized transgenes into AAV [179]. The process can be optimised by providing overlapping homologous regions to allow recombination in the target cell after gene transfer [180]. This strategy has demonstrated promising results in vitro and in a mouse model of dysferlinopathy [181]. In this model, treatment by intramuscular injection, regional vascular delivery, or systemic delivery resulted in significant increases in dysferlin expression [181–183]. Internal nucleation and membrane repair ability were also improved. Systemically treated animals had increased force of the diaphragm. Some of these benefits persisted for at least 13 months after gene transfer [182]. No safety concerns were identified in a non-human primate [182]. A small phase 1 study was conducted using intramuscular delivery in the extensor digitorum brevis [NCT02710500], but no results have been reported. A potential concern for gene transfer in dysferlinopathies is the progressive myopathy observed in mice over-expressing dysferlin [184].

Surrogate Gene Approaches The therapeutic strategies discussed so far aim to restore expression of the defective protein in muscular dystrophies. Other approaches however act in less direct fashion, by targeting surrogate genes that have the potential to substitute for the missing protein or to attenuate the disease phenotype through different mechanisms. Utrophin is a cytoskeletal protein that shares a high degree of homology with dystrophin and is expressed at the sarcolemmal membrane in developing muscle [185]. Postnatally, utrophin is largely replaced by dystrophin and remains expressed only at the neuromuscular junction. The ability of utrophin over-­ expression to rescue dystrophin deficiency was initially demonstrated in mdx mice [186]. The use of utrophin as a substitute for dystrophin also has the advantage of avoiding immunity against the transgene. The utrophin gene is nearly as long as dystrophin, and thus it must also be internally truncated in order to allow packaging into viral vectors. As with dystrophin, initial efforts employed adenoviral vectors [187, 188]. More recently, a micro-utrophin construct deliv-

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ered via an AAV9 vector was shown to improve histopathological, biochemical and functional deficits in mouse and canine models of DMD. In a deletional-null canine model of DMD, micro-utrophin showed robust expression and did not elicit an immune response, in contrast to microdystrophin, which elicited a strong immune response and was only sparsely expressed [189]. A different surrogate gene therapy approach for DMD centers on GALGT2, which encodes a beta-1,4-N-acetyl-­ galactosaminyltransferase. In adult muscle, this enzyme normally localizes to the neuromuscular and myotendinous junctions, where it participates in α-dystroglycan glycosylation [190]. Overexpression of the enzyme in non-synaptic regions leads to α-dystroglycan glycosylation with the cytotoxic T cell (CT) carbohydrate [191]. This ultimately results in expression of a number of synaptic proteins in extrasynaptic regions, including agrin, laminin α4, laminin α5, and utrophin [190, 192, 193]. Overexpression of Galgt2 in the mdx mouse model of DMD attenuated pathological and biochemical markers of muscular dystrophy [191, 194]. By contrast, Galgt2 knockout worsens the severity of the myopathy [195]. When delivered using the same AAV vector and promoter, GALGT2 and microdystrophin both corrected muscle specific force and resistance to eccentric contraction to ­wild-­type levels [196]. GALGT2 gene therapy also reduced cardiac pathology in these mice [197]. The mechanism of action of GALGT2 is not specific to DMD alone, and overexpression was also shown to be beneficial in three other muscular dystrophies: laminin α2-related congenital muscular dystrophy, α-sarcoglycanopathy (LGMD-R3), and FKRP-related dystroglycanopathy (LGMD-R9) [198–200]. In light of the positive results seen in mdx mice and a favourable safety profile, GALGT2 gene therapy using an AAVrh74 vector is currently being evaluated in a phase I/IIa trial in DMD [NCT03333590] [201, 202]. Myostatin is a cytokine involved in negative regulation of muscle growth. For this reason, myostatin inhibition has been proposed as a therapeutic strategy in a number of neuromuscular disorders [203, 204] and monoclonal antibodies against myostatin have been evaluated in a number of completed and still ongoing clinical trials [205–207]. Myostatin signaling is inhibited by follistatin, and intramuscular delivery of an AAV vector carrying follistatin into the hindlimbs of DMD and FSHD mouse models resulted in improvements in muscle histopathology and an increase in muscle mass and strength [208, 209]. This approach was subsequently tested in a phase I/IIa clinical trial in BMD, and 4 of 6 subjects who received intramuscular injections in the quadriceps demonstrated improved performance on the 6MWT, as well as improvements in muscle pathology [210]. The results of a trial in boys with DMD [NCT02354781] have never been reported, but the benefit in the setting of partial dystrophin expression in BMD subjects suggests that follistatin may

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prove useful as an adjunct in combination with a dystrophin-­ restoring therapy, as also suggested in animal studies [211]. Variants in LAMA2, encoding laminin α2, are one of the most common causes of congenital muscular dystrophy (see Chap. 11) [212–215]. The disease is caused by biallelic loss-­of-­function variants and would thus be amenable to correction by LAMA2 gene replacement, or by compensatory upregulation of laminin α1 [216, 217]. The large size of the laminin genes (9  kb) precludes their packaging into AAV vectors. Laminins can only function as heterotrimers, thus posing an obstacle to the creation of miniaturised constructs. Like laminin, agrin is an extracellular matrix protein that binds to α-dystroglycan. Miniagrin constructs have therefore been proposed as a substitute for laminin α2. The efficacy of this approach was first demonstrated in transgenic mice [218, 219]. Subsequently, systemic delivery of miniagrin via an AAV1 vector resulted in a correction of muscle pathology, improved mobility, and substantially improved survival [220]. Switching to the more recently described AAV9 serotype for delivery further enhanced the efficacy of miniagrin in a mouse model of LAMA2 muscular dystrophy [221]. Surrogate gene approaches have also been explored in DM1. Since MBNL1 sequestration plays a key role in pathogenesis of the disease, it is a potential target for gene therapy. Two studies employing AAV-mediated transduction or constitutive overexpression of MBNL1 in a mouse model of DM1 found that this approach corrected DM1-related splicing defects and attenuated myotonia [222, 223], but a subsequent study did not find any benefits in a different mouse model of DM1, finding instead evidence of increased muscle pathology [224]. The viability of MBNL1 overexpression as a therapeutic strategy in DM1 thus requires further study.

Genome Editing In contrast to the gene delivery approaches described above, in which the transgene remains episomal, genome editing employs the clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 system to directly modify an organism’s genomic DNA [225]. In this system, the Cas9 enzyme cleaves genomic DNA at a location to which it is directed by a guide RNA, producing a double-stranded DNA break (DSB). The break can then be repaired by non-­ homologous end joining, generally resulting in formation of an indel [226]. Alternatively, a fragment of DNA can be knocked in at the DSB site by a number of different mechanisms [227]. In vivo, genome editing machinery (i.e. the Cas9 enzyme, gRNA, and any knock-in construct) can be delivered via AAV vectors or non-viral delivery methods [228]. Genome editing has the potential to ensure durable gene expression and, in the case of the long DMD gene, to

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allow expression of longer dystrophin isoforms than could be packaged in an AAV vector. A variety of genome editing approaches have been employed to correct different pathogenic variants in the DMD gene. The mdx mouse model carries a nonsense variant in exon 23 of DMD, and excision of this exon can restore the reading frame of the gene [229–231]. In patients with deletions, a similar approach could be employed to excise an additional exon and restore the reading frame of the gene, in a manner similar to exon skipping ASOs [232]. A different approach employs only a single DSB to target the splice site of this exon. A proportion of the resulting indels will be therapeutic, either by ablating the splice site and causing exon skipping, or by changing the reading frame of the exon. This approach has been applied to correct a number of common DMD variants in cell lines and mouse models [233–238]. Alternatively, the splice site can be disrupted using base editing, which relies on an inactive Cas9 enzyme to target an adenine base editor that converts an adenine to a guanine without creating a DSB [239]. The relatively mild phenotypes of patients with large in-frame deletions in the rod domain and N-terminal mutation sites have led to the development of strategies to excise these entire regions from genomic DNA, thus potentially correcting a large number of variants with the same approach [240, 241] It should be noted that these approaches all result in expression of internally-­truncated dystrophin isoforms, rather than full-­ length dystrophin. In patients with duplications in DMD, a single gRNA targeting the duplicated region will result in two DSBs and excision of the duplicated region between them, restoring the coding sequence of the gene. This approach was recently demonstrated in a novel mouse model of DMD carrying a duplication of exons 18–30 [242]. Some nonsense variants can also be corrected by base editing to restore the DMD coding sequence [243, 244]. The majority of DMD patients however carry deletions in the gene, thus necessitating knock-in of a donor DNA fragment in order to restore full-­ length dystrophin. In mouse embryos, knock-in at the Dmd locus has been accomplished by homology-directed repair, but this mechanism is very inefficient in terminally differentiated tissues such as muscle [245]. As an alternative, homology-­ independent targeted integration employs non-­ homologous end joining, which is active throughout the cell cycle [246]. This approach was recently demonstrated in a mouse model of exon 52 deletion. In addition to replacing the deleted exon alone, it is also possible to knock into intron 51 a “superexon” containing the sequence of all downstream exons and a new polyadenylation site, thus potentially correcting all variants downstream of this point [247]. Beyond DMD, genome editing has also been employed to correct a number of other muscular dystrophies, including DM1 and FSHD. These approaches are reviewed in greater

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detail elsewhere [248–250]. In DM1, a number of groups have employed genome editing to excise the CAG repeat in DMPK [251–254]. As an alternative approach, it has been observed that single strand breaks created by Cas9 nickase induce contraction of CAG repeats during DNA repair [255]. Because the CAG repeat is located in the 3′ UTR of DMPK, it is also possible to introduce a premature PAS between the stop codon and CAG repeat, thus truncating the 3′UTR and preventing transcription of CAG repeats [254]. DMPK expression can also be inhibited by binding of a catalytically inactive Cas9 enzyme to the repeat expansion [256]. Genome editing for correction of FSHD1 is challenging due to the repetitive nature of the D4Z4 repeats and their similarity to other regions in the genome [250]. One recently demonstrated approach is to excise or disrupt the DUX4 PAS on the 4qA haplotype [257, 258]. Other approaches employ transcriptional repressors fused to a catalytically inactive Cas9 enzyme to downregulate DUX4 expression [259–261]. The ability of Cas9 to modify the genome raises the concern of potential off-target effects, and reliable characterization of off-target activity will be crucial to bring genome editing into clinical use. Because of the relatively low level of off-target genome modifications, whole genome sequencing is a relatively insensitive detection tool. Several techniques have thus been developed to identify off-target effects. These techniques include computational algorithms to predict potential off-target sites based on gRNA sequences [262, 263], as well as in vitro and in vivo assays that rely on a variety of methods to produce next-generation sequencing libraries enriched for genome editing events. The latter are based on a variety of approaches, including integration of oligonucleotide tags at DSB sites (GUIDE-seq), cleavage of circularized DNA fragments (CIRCLE-seq, CHANGE-seq), and chromatin immunoprecipitation (DISCOVER-seq) [264– 267]. Because of the imperfect sensitivity and specificity of each of these tools, the first clinical trial of systemically delivered genome editing (for hereditary transthyretin amyloidosis) relied on a combination of them to predict potential off-target sites, followed by deep sequencing of these sites in relevant human cell lines to validate the predictions [268].

Conclusion The past two decades have seen the development of a number of molecular therapeutics for muscular dystrophies and other inherited myopathies. For DMD, research on exon-­ skipping ASOs has now led to 4 FDA-approved products targeting exons 45, 51, and 53. These result in modest restoration of skeletal muscle dystrophin and mild clinical benefits. Ongoing efforts are underway to develop next-generation exon skipping strategies with greater potency, longer-lasting efficacy, and greater restoration of cardiac dystrophin. In

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parallel, three different microdystrophin gene replacement constructs are in clinical trials and have shown promising early results. A recombinant gene therapy for DMD, designed to deliver microdystrophin, has recently been approved. Gene replacement strategies are also being pursued for a number of recessive LGMDs, with promising results in preclinical and early clinical studies. For DM1 and FSHD meanwhile, RNAi or RNase H have been employed to silence or degrade toxic mRNAs, with positive results in preclinical studies. Taken together, these therapeutic developments bring the hope that effective disease-modifying treatment for common inherited myopathies and muscular dystrophies will become available in the coming years.

Appendix Since this manuscript was submitted and reviewed, the microdystrophin gene therapy discussed above as SRP-9001 received accelerated approval from the U.S. Food and Drug Administration for delivery to boys with DMD aged 4 and 5  years old, under the trade name Elevidys. Approval was based upon data from an initial first-in-human open label trial referenced in the article, as well as additional data partly described in the references below. The therapy has not yet been approved for an older patient population, and additional trials are ongoing as of publication [269, 270].

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