GENOTYPE-PHENOTYPE ASSOCIATION ANALYSIS OF DILATED CARDIOMYOPATHY IN BECKER MUSCULAR DYSTROPHY

DISSERTATION

Presented in Partial Fulfillment of the Requirements for

the Degree Doctor of Philosophy in the Graduate

School of The Ohio State University

By

Rita Wen Kaspar, BSN, RN

*****

The Ohio State University 2009

Dissertation Committee: Approved by Professor Donna McCarthy, Advisor

Professor Federica Montanaro ______Professor Wendy Blakely Advisor Nursing Graduate Program

Copyright by

Rita Wen Kaspar

2009 ABSTRACT

Background: Becker muscular dystrophy (BMD) is caused by dystrophin gene

mutations resulting in skeletal and cardiac muscle degeneration. When cardiac

manifestations are disproportionate to skeletal muscle involvement, the disorder is

diagnosed as X-linked dilated cardiomyopathy (XLDCM). Dystrophin mutations predictive of dilated cardiomyopathy (DCM) have been suggested, but representative examples from the literature are insufficient in number to establish with certainty.

Defining the cardiac presentations of specific mutations is crucial for risk classification,

early detection and treatment of DCM to prolong survival. From the patient’s perspective, knowing how his individual genotype affects cardiac outcome may be beneficial for health management and life planning.

Purpose: To determine age of DCM manifestation in relation to dystrophin genotype and its resulting dystrophin protein modifications in BMD and XLDCM

patients.

Methods: BMD and XLDCM patients with dystrophin exon deletion mutations and with cardiac histories were collected from multiple sources. The correlations between dystrophin genotypes and cardiac phenotypes were established. Protein

modeling was conducted to evaluate structural consequences of genetic mutations.

ii Results: Data of 110 patients were included in this study, of which 76 were cardiomyopathic. Exon deletions in the amino-terminal domain of dystrophin resulted in the earliest mean age of cardiac involvement (22.5 years). Mutations affecting the spectrin repeats in the rod domain preserving Hinge 3 lead to the intermediate age of cardiomyopathy onset (29.5 years), followed the latest age of onset (46.5 years) from patients with rod-domain mutations removing Hinge 3 (p < 0.05). Mutations affecting the spectrin repeats preserving Hinge 3 were subdivided based on whether they disrupted

(out-of-phase) or preserved (in-phase) the spectrin repeats structure. Out-of-phase mutations predisposed to earlier age of cardiomyopathy onset (25.5 years) compared to in-phase mutations (36.5 years) (p < 0.05). Protein modeling analyses indicated that dystrophin protein structural modification as a result of genetic mutations may underlie the observed earlier onset of DCM in patients with out-of-phase mutations.

Conclusions: An association between dystrophin genotype and cardiac phenotype was identified in patients with BMD and XLDCM. The results enable nursing and medical practitioners to provide timely interventions for at-risk patients to optimize cardiac outcome.

iii DEDICATION

To my family

to my father and mother, Wen Wen-Shi (Mike) and Hsieh Yeh-Jung

(Anne), for giving me abundant love, hope and opportunities,

to my sister and brother, Wen Hsing-Hua (Liz) and Wen Po-Hua (Brian),

for showing me worlds beyond mine,

and to the love of my life, Brian Kevin Kaspar, for the life-changing

laughter, support and true friendship. You are my role model and my

motivation.

To my patients

for trusting me as your nurse.

iv ACKNOWLEDGMENTS

I offer my deepest appreciation to my dissertation advisor, Professor Donna

McCarthy, PhD, RN, who taught me the essence of scholarly rigor and ignited my life- long passion for nursing research.

I am indebted to Professor Federica Montanaro, PhD, for her incredible patience in mentoring me in the laboratory and her generosity in sharing her scientific expertise in muscular dystrophy.

A very special thank you to Professor Wendy P. Blakely, PhD, RN, for serving on my candidacy and dissertation committees and for opening my eyes to laboratory-based nursing research.

I thank Professor Elizabeth J. Corwin, PhD, RN, for serving as my advisor during the first year of my graduate education, and as a member on my Candidacy Committee.

In addition, I express my gratitude to Professor Jerry R. Mendell, MD, for offering significant guidance on this dissertation work and opening many doors for me to future interdisciplinary studies.

I thank Professor Hugh D. Allen, MD, for his unparalleled passion in teaching and generosity in sharing his incredible knowledge. He made this dissertation journey a humbling experience.

v I thank Professor John T. Kissel, MD, for referring patients from The Ohio State

University Medical Center, as well as Professor Kevin M. Flanigan from The University

of Utah for providing access to patients enrolled in the United Dystrophinopathy Project.

I am grateful for the collaborations with Professor William C. Ray, PhD,

Professor Carlos Alvarez, PhD, Professor Christopher H. Holloman, PhD, and Dr.

Xiomara Q. Rosales, MD, for their contributions to my dissertation study in protein structural modeling, conservation analyses, statistical consultation and graphic design of dystrophin, respectively.

Finally, I would like to acknowledge tuition and stipend supports from The Ohio

State College of Nursing for two years of Graduate Traineeship and one year of Graduate

Research Associateship, and funding from the National Institutes of Health Roadmap

Training Program in Clinical Research (T32RR023260-03).

vi VITA

September 14, 1977...... Born – Tainan, Taiwan, Republic of China

2000 – 2004...... Staff Nurse, Operating Room Alhambra Hospital Medical Center, Alhambra, CA

2002 – 2004...... Staff Nurse, Operating Room East Valley Hospital Medical Center, Glendora, CA

2004 – 2005...... Staff Nurse, Cardiothoracic Surgical Floor Duke University Medical Center, Durham, NC

2005...... B.S. Nursing, California State University, Fullerton.

2005 – 2007...... Staff Nurse, Cardiovascular Surgical Floor The Ohio State University Medical Center Ross Heart Hospital, Columbus, OH

2006 – 2008...... Graduate Trainee, The Ohio State University College of Nursing

2008 – 2009...... Graduate Research Associate, The Ohio State University College of Nursing

vii PUBLICATIONS

1. Kaspar, R. W., Wills, C. E., & Kaspar, B. K. (2009). Gene Therapy and

Informed Consent Decision-Making: Nursing Research Directions. Biological Research

for Nursing, doi:10.1177/1099800409333169.

2. Kaspar, R.W., Allen, H.D. & Montanaro, F. (2009). Current understanding and

management for dilated cardiomyopathy in Duchenne and Becker muscular dystrophy.

Journal of the American Academy of Nurse Practitioners, 21(5), 241-9.

3. Dodge, J.C., Haidet, A.M., Yang, W., Passini, M.A., Hester, M., Clarke, J.,

Roskelley, E.M., Treleaven, C.M., Rizo, L., Martin, H., Kim, S.H., Kaspar, R. W.,

Taksir, T.V., Griffiths, D.A., Cheng, S.H., Shihabuddin, L.S., & Kaspar, B.K. (2008).

Delivery of AAV-IGF-1 to the CNS Extends Survival in ALS Mice Through

Modification of Aberrant Glial Cell Activity. Molecular Therapy, 16(6), 1056-64.

FIELDS OF STUDY

Major Field: Nursing

viii TABLE OF CONTENTS

Page Abstract ...... ii Dedication ...... iv Acknowledgements...... v Vita ...... vii List of Tables ...... xii List of Figures ...... xiii List of Abbreviations ...... xv

Chapters:

1. Introduction ...... 1

2. Background ...... 4 Part I: Phenotypes and Clinical Manifestations of Dystrophin Mutation...... 4 Duchenne muscular dystrophy...... 4 Dilated cardiomyopathy pathology in Duchenne muscular dystrophy...... 7 Becker muscular dystrophy...... 8 Dilated cardiomyopathy pathology in Becker muscular dystrophy...... 10 X-linked dilated cardiomyopathy ...... 11 Other phenotypes of dystrophin mutation...... 11 Part II: Mechanisms Underlying the Phenotypes...... 12 The dystrophin gene...... 12 Dystrophin gene mutations ...... 12 The dystrophin protein...... 14 Pathophysiology in dystrophinopathies ...... 15 Interactions of dystrophin...... 18 Functional biology of dystrophin...... 21 Structure-function relationship of dystrophin...... 23 Phasing of dystrophin spectrin-like repeats ...... 25 Genotype-phenotype correlations...... 27 Part III: Dissertation Research...... 31 Synthesis of literature ...... 31 ix Study purpose...... 31 Specific aims...... 32 Theoretical Framework...... 32

3. Research design and methods ...... 36

General procedure...... 36 Grouping of patients ...... 38 Dystrophin protein modeling ...... 39 Echocardiography ...... 40 Age ...... 41 Samples...... 42 Data collection ...... 43 Data analysis ...... 44

4. Findings ...... 45

General description of samples...... 45 Mutation Group 1...... 47 Mutation Group 2...... 47 Mutation Group 3...... 48 Results for research aim 1...... 49 Results for research aim 2...... 50 Additional findings ...... 52 Rare deletion mutations ...... 52 Point mutations ...... 53

5. Discussion and conclusion...... 55

Study summary ...... 55 Discussion...... 56 Exon deletions in the amino-terminal domain...... 56 Exon deletions in the rod domain ...... 58 Study limitations ...... 61 Nursing implications...... 62 Future research directions...... 64 Conclusion ...... 65

x

Bibliography ...... 142

Appendix A: Clinical description of patients in this study affected with DCM...... 66

Appendix B: Clinical description of patients in this study unaffected with DCM...... 99

xi LIST OF TABLES

Table Page

1 Clinical phenotypes resulting from mutations in the dystrophin gene ...... 113

2 Constructs and concepts in the theoretical model...... 114

3 Cardiac function categories...... 116

4 Patient distribution according to diagnosis and source, with median ages based on cardiomyopathy categorization...... 117

5 Phasing consequences of different exon deletion mutations ...... 118

6 Pair-wise comparison of cardiomyopathic patient groups based on the location of the deletion mutation and the effects on spectrin-like repeat phasing...... 119

xii LIST OF FIGURES

Figure Page

1 Gower’s maneuver...... 120

2 Anatomy of DCM...... 121

3 The architecture of dystrophin together with other proteins on the sarcolemma ...... 122

4 Graphic representation of the dystrophin gene and protein...... 123

5 Known Pathways through which abnormal dystrophin leads to cardiomyocyte death...... 124

6 Progression from cardiomyocyte death to DCM ...... 125

7 Modeling of the rod region of spectrin ...... 126

8 Theoretical model guiding this dissertation study ...... 127

9 Graphic representation of in-phase versus out-of-phase dystrophin constructs ...... 128

10 Patient grouping by dystrophin protein domain...... 129

11 Schematic representations of the normal structure of the spectrin-like repeats in dystrophin ...... 130

12 Mutation distribution for the cardiomyopathic, grouped by diagnoses and sources...... 131

13 Distribution of mutations for cardiomyopathic patients by group...... 132

14 Age distribution of each mutation group based on cardiac involvement...... 133

xiii 15 Prediction of spectrin-like repeats re-arrangement for frequent exon deletions...... 134

16 Median ages for Group 2 mutations based on spectrin-like repeats phasing...... 135

17 Predicted models of dystrophin spectrin-like repeat structure based on in- phase and out-of-phase deletions...... 136

18 Distribution of dystrophin point mutations with XLDCM clinical diagnosis ...... 137

19 Human dystrophin 18Lysine>asparagine mutation ...... 138

20 Human dystrophin 279threonine>alanine mutation ...... 139

21 Human dystrophin 1672 asparagine>lysine mutation...... 140

22 Human dystrophin 3228 phenylalanine>leucine mutation ...... 141

xiv LIST OF ABBREVIATIONS

BMD Becker muscular dystrophy

CK creatine kinase

DAPC dystrophin-associated protein complex

DCM dilated cardiomyopathy

DMD Duchenne muscular dystrophy

DNA deoxyribonucleic acid

ECG electrocardiogram

EPSS E point-to-septal separation

IRB Institutional Review Board

MRI magnetic resonance imaging mRNA messenger ribonucleic acid

NCH Nationwide Children’s Hospital nNOS neuronal nitric oxide synthase

ORF open reading frame

OSU The Ohio State University

OSUMC The Ohio State University Medical Center

RNA ribonucleotides

UDP United Dystrophinopathy Project

xv XLDCM X-linked dilated cardiomyopathy

xvi

CHAPTER 1

INTRODUCTION

Knowledge of genetics is essential to the thorough understanding of human health and disease. The earliest genetic study on was done in the 1860’s by Gregor Mendel, who showed that traits of pea plants are passed from parents to offspring in predictable ways that are controlled by some discrete mechanisms. It is now known that those

“mechanisms” are genes. Mendel’s work established the foundation of the science of genetics, which is a branch of biology with the emphasis on heredity and variation. Less than a century took place from Mendel’s observation of heredity to the discovery of deoxyribonucleic acid (DNA) as the genetic material in 1944 by Avery, MacLeod and

McCarty (Avery, MacLeod, & McCarty, 1944). Scientific advances in genetics research have greatly enhanced our understanding of disease mechanisms and innovative means to develop new therapy and promote health.

In the field of neuromuscular disorders, Kunkel and colleagues reported the important genetic aspect of Duchenne muscular dystrophy (DMD) in 1987. They discovered that mutations in the dystrophin gene cause to abnormal dystrophin protein expression within the myofibril, resulting in the characteristic progressive muscle degeneration seen in boys with DMD (Koenig et al., 1987). Subsequent discoveries revealed that mutations in the dystrophin gene also caused other allelic disorders,

1 including BMD and dystrophin-related XLDCM. The development of DCM as part of the disease natural history in these patients has also received much recent attention due to its debilitating impact on the patient’s length and quality of life.

Understanding the genetic pathogenesis in dystrophinopathy has impacted nursing science as nursing research studies take different perspectives to address relevant issues such as newborn screening (Parsons, Clarke, Hood, Lycett, & Bradley, 2002), timing of genetic diagnosis (Parsons, Clarke, & Bradley, 2004) and family function (Chen & Clark,

2007). As an extension, this dissertation research investigated the effect of genetics on health and disease in the context of patients with dystrophin gene mutations.

Specifically, this study was a genotype-phenotype correlation analysis in BMD and

XLDCM patients. The study purpose was to elucidate whether there were particular dystrophin genetic mutations that predisposed patients to early- versus late-onset

DCM. This research pursuit is of special relevance to clinical practice, since at the present time practitioners cannot define the BMD and XLDCM patient’s risk of developing DCM in advance due to lack of reliable predictive factors. Consequently, diagnosis and treatment of cardiomyopathy in these populations does not begin until cardiac symptoms are present, leading to poorer prognosis and premature death. From the patient’s perspective, knowing how his individual genotype affects cardiac outcome may be beneficial for health management and life planning.

This dissertation research will be sequentially presented in the following 4 chapters. Chapter 2 is a review of literature. Part I introduces the clinical pictures of

DMD, BMD, XLDCM in depth, followed by a discussion of the current understanding of the pathophysiology of DCM in these patients. The focus of Part II is on the molecular

2 and genetic aspects of the dystrophin gene and its protein, and how these mechanisms relate to the clinical phenotypes. Previous genotype-phenotype correlation studies will be reviewed and the rationale of only including BMD and XLDCM patients in this study will be explained. Finally, Part III of this chapter provides the synthesis of literature and states the purpose, the research aims and the theoretical framework underlying this research.

In Chapter 3, the design and methodology of this study are described. This chapter details the procedures of subject collection, inclusion and exclusion criteria, definition and measurement of DCM, grouping of patients, methods used for dystrophin protein modeling and statistical analyses. Chapter 4 reports the results. The findings for each patient group will be discussed, and data addressing each research aim are presented. The final chapter, Chapter 5, discusses the overall meaning and significance of this study. Results of this study will be integrated with current knowledge and implications to clinical practice and future nursing research are concluded.

3

CHAPTER 2

BACKGROUND

Mutations in the dystrophin genes have been found to be responsible for three

distinct clinical presentations described below. Despite the clinical characterization of

these three diseases more than a century ago, the discovery of dystrophin and the

realization that these three diseases were genetically related have only been recent. For

this reason, this chapter will begin with the clinical description of these diseases, followed by a presentation of the genetic mutations that underlie the clinical diversity.

The current understanding of the functions of the dystrophin protein in muscle tissues will be discussed, with a particular interest on how genotype influences dystrophin function at a cellular and molecular level leading to phenotypic differences.

Part I: Phenotypes and Clinical Manifestations of Dystrophin Mutation

Duchenne Muscular Dystrophy

DMD is an X-linked neuromuscular disorder with an incidence of one in 3500 live newborn males (A. E. Emery, 1991). It is characterized by progressive, generalized weakness and muscle wasting. The disease was first described at a meeting of the Royal

Medical and Chirurgical Society in 1851 by Meryon. He described a familial pattern with a male predilection that ultimately resulted in early death (Meryon, 1851). Twenty

4 years later, Duchenne, a French neurologist, published a complete histological

description of muscle biopsies from 13 patients. He gave the name “paralysie musculaire

pseudohypertrophique” (pseudohypertrophic muscle paralysis), which accurately described the muscle paralysis and muscle hypertrophy characteristics of DMD patients

(Duchenne, 1872). The term muscular dystrophy was adopted in 1884 by Erb, who defined the disease as a primary degeneration of the muscle (Erb, 1884).

Clinically, DMD patients are diagnosed between 3 to 6 years of age with delayed

motor development and skeletal muscle weakness. Due to the X-linked inheritance

pattern, all affected patients are male. Beginning from very early childhood,

approximately half of the affected boys are unable to walk until 18 months after birth, the

age by which normal children have been walking for 6 months. As the boy becomes

older, parents may notice bilateral calf muscle enlargement, frequent falls, inability to run

well or to get up from the floor normally. Mental retardation and/or psychological

disturbances are seen in addition to muscle weakness in 30% of DMD boys (Anderson,

Head, Rae, & Morley, 2002). However, this deficit is not progressive and is not related

to severity of muscle weakness (Giliberto, Ferreiro, Dalamon, & Szijan, 2004). The boy

is usually brought to medical attention when the parents realize that something is

seriously wrong with his motor development. At school age, atypical walking patterns,

including toe-walking, pushing the abdomen forward (lordosis), waddling gait, pelvic tilt,

and difficulty climbing stairs, clearly demonstrate his difficulty in maintaining normal

standing and walking positions. These early hallmarks of DMD develop due to the

combination of the weakness of gluteus maximus and the tightness of hip flexors

(D’Angelo et al., 2009). Progressive pelvic muscle weakness in DMD boys eventually

5 leads to difficulty in rising from the floor, and their specific way of using other muscles

to get up is described as the Gower’s maneuver (Figure 1, Modified from Muscular

Dystrophy Association, 2006). In order to get up from the floor, DMD patients face the floor, plant their feet widely apart, raise their buttocks first, and use their hands to “walk”

up the legs.

The progressive muscle weakness in DMD boys eventually results in wheelchair

dependency at average age 12. Muscle wasting continues to rapidly generalize to other

parts of the body, including voluntary and involuntary muscles in the digestive tract, the

intercostals muscles and the diaphragm. Interestingly, eye muscles are spared from the

degenerative process (A. E. H. Emery, 2008). Common gastrointestinal symptoms

include delayed gastric emptying, constipation and impaction (Barohn, Levine, Olson, &

Mendell, 1988). The confinement to a wheelchair, in addition to muscle weakness,

quickly leads to joint contractures and severe scoliosis. Without appropriate postural

support or surgical correction of the scoliosis to prevent aspiration, respiratory

insufficiency develops easily particularly for patients who have become bed-ridden.

These boys need help with repositioning through the night, often leading to a parent

sleeping in the same room. The repositioning request is usually due to respiratory

compromise.

Further complicating the clinical picture of DMD is the concurrent development

of DCM, a type of cardiomyopathy characterized by enlarged chambers, thinner walls

and decreased cardiac pumping ability (Figure 2). Electrocardiographic abnormalities

include presence of short PR interval, right ventricular hypertrophy, and inferior-lateral Q

waves. These signs are seen in about half the boys and bear no relationship to the

6 presence of DCM. Many adolescent boys with DMD have increased automaticity characterized by a high average heart rate. Older boys can have supraventricular tachycardia, multiform premature ventricular contractions, ventricular tachycardia or ventricular fibrillation (Thrush, Allen, Viollet, & Mendell, 2009). Pathology followed by corroborative echocardiographic studies on DMD patients with DCM show that the left ventricle myocardial fibrosis and dilation first appear in the left ventricular wall behind the posterior mitral valve leaflet, which progress inferiorly over time toward the apex and into the septum, ultimately affecting the entire left ventricle (Allen, Mendell, & Hoffman,

2008). Despite improvements in respiratory care, most DMD patients die from cardiac failure by 25-30 years of age (English & Gibbs, 2006; Jefferies et al., 2005).

Dilated Cardiomyopathy Pathology in Duchenne Muscular Dystrophy

Many DMD patients develop sinus tachycardia by 5 years of age and conduction changes by 10 years of age. Characteristic irregular conduction patterns seen in a 12-lead electrocardiogram (ECG) non-specific to XLDCM include short PR interval, right ventricular hypertrophy, prolonged QTc interval, and prominent Q waves in leads I, aVL,

V5, and V6 or in leads II, III, aVF, V5, and V6 (Thrush, Allen, Viollet, & Mendell,

2009). Shortened PR intervals are seen in about 50% of patients, and QT prolongation is rare. From Holter ECG monitoring, resting sinus tachycardia, loss of circadian rhythm and reduced heart rate variability caused by increased sympathetic activity can be observed (Kirchmann et al., 2005). With advanced fibrosis, more serious arrhythmias may be seen, including atrial fibrillation, atrioventricular block, ventricular tachycardia and ventricular fibrillation (Corrado et al., 2002).

7 With DMD, myocardial fibrosis and dilation first appear in the left ventricular

wall behind the posterior mitral valve leaflet, which over time progress inferiorly toward

the apex and into the septum, ultimately affecting the entire left ventricle. Visualization

of posterior epicardial thinning and areas of abnormal and/or absent wall movement (e.g.

dyskinesis & akinesis), as well as direct measurement of systolic and diastolic

dysfunction are possible with echocardiography or other imaging methods such as

magnetic resonance imaging (MRI). The present echocardiographic standard established

to predict cardiac dysfunction is the ejection fraction; below 55% is considered abnormal

in the DMD population (Jefferies et al., 2005; Duboc et al., 2007). Other signs of dilation include increased left ventricular diameter and volume, decreased shortening fraction, and development of mitral valve regurgitation. With echocardiography, the E point-to- septal separation (EPSS) measures the minimal separation between the mitral valve anterior leaflet and the ventricular septum during early diastole. An EPSS of greater than

5-5.5mm suggests that the left ventricle has become more enlarged and can provide a

clue for cardiac anatomical changes (Anderson, 2007). Likewise, the sphericity index,

calculated by dividing the length of the left ventricle by the width of the left ventricle, is

very useful in evaluating the shape of the left ventricular chamber. A sphericity index of

greater than 0.66 suggests the presence of DCM, in which the elongated shape of the

normal heart becomes spherical (Tani, Minich, Williams, & Shaddy, 2005).

Becker Muscular Dystrophy

The allelic variant disorder of DMD is BMD, with symptom manifestations

similar to DMD but they appear later and are less severe. The reported incidence for

8 BMD is at least 1 in 18,450 male live births (Bushby, Thambyayah, & Gardner-Medwin,

1991). The spectrum of clinical presentation and severity of BMD is broader than that of

DMD. In general, the BMD patient begins to feel muscle weakness and cramping from

his lower limbs at any age during adolescence or adulthood. Toe-walking, waddling gait

and lordosis as seen in DMD patients can also be observed in the older BMD patient.

There is a wide variation of the severity of muscular degeneration among BMD patients.

Some patients become wheelchair-dependent, yet some can function well with light-duty

ambulatory aids, such as a cane. Once the BMD patient has become wheelchair-

dependent, his risk of developing immobility-related complications is similar to that of

the DMD patient. Mental and psychological deficits, while noted in some BMD patients, are present much less commonly than in DMD (A. E. H. Emery, 2008).

Despite the milder skeletal muscle degeneration in BMD, almost all BMD patients still develop DCM (Nigro et al., 2006). Similar to DMD, the severity and onset of cardiac symptoms in BMD patients are unrelated to the degree of skeletal myopathy

(Kirchmann, Kececioglu, Korinthenberg, & Dittrich, 2005; Nigro et al., 1995). In fact,

DCM is the predominant clinical feature in some BMD cases. Different from DMD, resting tachycardia due to increased automaticity of sympathetic activation is not characteristic in BMD patients (Kirchmann et al., 2005; Vita et al., 2001). Further, decreased ventricular function resulting from myocardial fibrosis in BMD patients can first be seen in the right ventricle. As left ventricular dysfunction progresses, many BMD patients may have symptoms of congestive heart failure since they are ambulatory, in contrast to DMD patients who are largely immobile and asymptomatic. Because of the milder symptoms, most BMD patients live through adulthood and are able to have

9 children. Their sons will be unaffected, yet their daughters are all carriers who may pass the disease-causing mutation on the X-chromosome to their children. Therefore, families of BMD patients require appropriate genetic counseling for reproductive planning.

Dilated Cardiomyopathy Pathology in Becker Muscular Dystrophy

BMD patients have ECG presentations similar to those seen in DMD, including deep Q waves in leads I, aVL (or II, III aVF) and V6, tall R waves in V1 and shortened

PR intervals. Bundle-branch block, non-specific ST changes and negative T waves have also been reported (Melacini et al., 1996), as have atrial and/or ventricular arrhythmias such as tachycardia, fibrillation and flutter (Suselbeck, Haghi, Neff, Borggrefe, &

Papavassiliu, 2005). However, different from DMD, the Holter ECG in Becker patients

does not show the increased automaticity of sympathetic activation (Kirchmann et al.,

2005; Vita et al., 2001). The fibrosis in BMD has a similar pattern of progression;

however, it often begins in the right ventricular wall. The echocardiographic

measurement standards are the same as in DMD. In addition, measurement of the peak systolic and early diastolic strain rates (the rate of deformation of a myocardial segment)

may detect abnormal myocardial function not revealed by the measurement of ejection

fraction (Meune et al., 2004).

In summary, damage to muscle tissues, particularly respiratory and cardiac muscles, significantly shortens the life expectancy of DMD and BMD patients. With advances in technology, respiratory care for these patients has greatly improved.

However, DCM has become the leading cause of death among DMD and BMD patients

(Eagle et al, 2002).

10 X-Linked Dilated Cardiomyopathy

In rare cases, mutations in the dystrophin gene are also the cause for some familial XLDCM in which the phenotype is cardiac-specific, generally sparing skeletal muscle. Particularly, study of a group of dystrophin-related XLDCM patients with no detectable skeletal muscle weakness revealed no dystrophin protein expression in the cardiac biopsy but present in the skeletal muscle biopsy (Milasin et al., 1996; Ferlini et al., 1998; Muntoni et al., 1993; Muntoni et al., 1995; Bies et al., 1997). The strong cardiac involvement and mild skeletal muscle phenotype in these patients could be attributed to differences in protein expression between these two types of muscle tissues.

However, this explanation cannot be extrapolated to BMD and XLDCM patients with disease-causing mutations in other regions of the dystrophin gene and with proven expression of the mutant dystrophin protein in cardiac muscle (Arbustini et al, 2000;

Muntoni et al., 1997; Towbin et al, 1993; Anan et al., 1992; Fanin, Melacini, Angelini, &

Danieli, 1999; Ortiz-Lopez, Li, Su, Goytia, & Towbin, 1997; Politano et al., 1999).

These cases suggest that some mutations might affect domains of the dystrophin protein critical to its function in cardiac tissue.

Other Phenotypes of Dystrophin Mutation

In addition to affecting the function of cardiac and skeletal muscles, dystrophin gene mutations can also be accompanied by the clinical findings in Table 1. The involvement of non-muscle organs agrees with the expression of tissue-specific forms of dystrophin in these tissues, particularly the brain (Davies & Nowak, 2006). Of note, while muscle degeneration is progressive, mental deficits do not worsen over time.

11

Part II: Mechanisms Underlying the Phenotypes

The Dystrophin Gene

The dystrophin gene, positioned at Xp21.2, is the largest gene known in the

human genome, encompassing 2.3 million base pairs of the genomic sequence, which is

about 1.5% of the entire X chromosome. The dystrophin gene is comprised of 79 exons

and its structure is complex. It contains multiple promoters and regulatory elements in its

non-coding regions that drive tissue-specific expression of at least 9 distinct dystrophin

protein isoforms of varying size and function (Leiden Muscular Dystrophy pages, 2006).

In muscle, dystrophin includes 79 exons that encode a 13.9 kilobases messenger

ribonucleic acid (mRNA) transcript (Kunkel, Beggs, & Hoffman, 1989; Mandel, 1989;

Manole, 1995).

Dystrophin Gene Mutations

This large size of dystrophin increases the likelihood of the gene to develop

sporadic mutations through rearrangement and recombination events. Mutations on dystrophin gene lead to errors when encoding for dystrophin protein, leading to various

phenotypic consequences, including DMD, BMD and XLDCM. As described by the

“reading frame theory” (Monaco et al., 1988), in most cases, patients whose dystrophin

mutations disrupt the translational reading frame have more severe skeletal muscle

symptoms (e.g. DMD), and patients whose dystrophin mutations do not disrupt the

translational reading frame have milder phenotypes (e.g. BMD). Early studies

investigating the pattern of genetic mutations among both DMD and BMD patients have

12 shown that exon deletion is the most common form of dystrophin mutations (Darras et

al., 1988; Forrest et al., 1987). This observation is consistent with later findings indicating that exon deletions are distributed throughout the dystrophin gene in DMD and

BMD, large deletions (>10 exons) occur more frequently between exons10-45, and smaller deletions (<10 exons) around exons 45-55. Deletion mutations at the 5’ and 3’ ends are rarer compared to their occurrence at the center region of the gene (80%) (Prior,

2006). Another type of mutation, exon duplication, is seen among both DMD and BMD

patients. Exon duplication mutations in the dystrophin gene are much rarer than exon

deletion mutations (~6% of all cases) (Hu, Ray, Murphy, Thompson, & Worton, 1990).

In DMD, most exon duplication mutations occur at the 5’ end of the dystrophin gene,

versus the central region for BMD cases (Prior, 2006).

Point mutations of the dystrophin gene that evolve as missense or nonsense

mutations are also present among both DMD and BMD patients (Lenk et al., 1996; Prior

et al., 1996). Point mutations at regions where dystrophin interacts with its neighboring

proteins, such as γ-actin, lead to more the more severe DMD phenotype (Prior et al.,

1995). Interestingly, among BMD patients collected in this dissertation study, some also

have point mutations at dystrophin’s protein-binding regions, providing a hint that there

must be other mechanisms involved in determining the BMD patients’ milder symptoms.

Indeed, a fascinating group of dystrophin gene mutations has been classified as “splicing

mutation” (Roest et al., 1996). Since conventional mutation detection techniques focus

on the exons of dystrophin, mutations in some patients cannot be detected. Using the

ribonucleotides (RNA)-based approach, small DNA point mutations within the introns

(non-coding region) can be detected. DNA sequence change within the intron can change

13 the normal splicing pattern in transcription, explaining variations in phenotype (Roest et

al., 1996). Finally, insertion mutations in the dystrophin gene account for another type of

rare mutation, which has been noted in both DMD and BMD (Gurvich et al., 2008;

Musova et al., 2006).

Some mutations of the dystrophin gene occur in the non-coding region. However,

they still affect the final product of the dystrophin protein, since they may affect the

promoters or tissue specific regulatory elements, such as enhancers. In these

circumstances, the phenotypic consequence is tissue-specific. Examples of these have

primarily been shown in patients harboring genetic mutations in dystrophin’s muscle

promoter or first exon regions (Milasin et al., 1996; Muntoni et al., 1996; Muntoni et al.,

1993; Muntoni et al., 1995). The heart is the preferentially affected muscle in these patients because the compensatory up-regulation of dystrophin expression from alternative dystrophin promoters that use a different first exon only occurred in the skeletal muscle but not in the cardiac muscle (Bastianutto et al., 2001).

The Dystrophin Protein

Mutations in the dystrophin gene can lead to lack of protein expression or to expression of an altered dystrophin protein, leading to the varied clinical presentations described above. It is therefore important to understand the structure of the dystrophin protein and the functions carried out by its domains. The dystrophin protein in the muscle is a long rod-shaped protein localized under the muscle fiber membrane, or the sarcolemma. As illustrated in Figure 3, the unique position of the dystrophin protein in the muscle fiber makes itself an important link between the intracellular cytoskeleton and

14 the extracellular matrix during muscle contraction and stretch. Figure 4 is a

representation of the dystrophin gene and protein. As shown, there are four major

domains of the dystrophin protein: (1) the amino terminal domain (N-terminus,

approximately exons 1-8) anchoring to the intracellular cytoskeleton; (2) the rod domain

(approximately exons 9-64 and primarily comprised of spectrin-like repeats) that offers flexibility to absorb the impact of muscle contraction; (3) the cysteine-rich domain

(approximately exons 65-69) that links the intracellular cytoskeleton to the extracellular matrix; and (4) the carboxyl terminal (C-terminus, approximately exons 70-79), which is less critical for preserving muscle function. These domains are connected by four hinges, which can be thought as joints in a body that allow bending and movements.

In addition to its localization at the muscle fiber membrane, immunostaining

studies have indicated that dystrophin is particularly concentrated in myotendinous and myomuscular junctions, which are implicated in the transmission of contractile force from myofibrils to extracellular elements of muscle tissue (Bassett et al., 2003; Samitt &

Bonilla, 1990). Myotendinous and myomuscular junctions are attachment sites between the ends of muscle fibers and tendons or serially arranged muscle fibers, respectively, which ultimately transmit the force of muscle fiber contraction to bone.

Pathophysiology in Dystrophinopathies

When the function of the dystrophin protein is compromised due to gene mutations, the sarcolemma is more fragile and susceptible to damage from muscle contractions leading to increased permeability in the membrane. Extracellular calcium enters the muscle fiber more easily, which in turn lead to protein degradation from

15 calcium-activated proteases (Constantin, Sebille, & Cognard, 2006). Evidence from

DMD skeletal muscle biopsies has shown that the level of dystrophin protein in muscle is

≤ 5%, much less than the amount of dystrophin found in BMD patients. The increased

permeability of the sarcolemma also leads to the release of intracellular muscle proteins

from the myofiber, such as creatine kinase (CK). Increased serum level of CK (>35-174

U/L) is a hallmark of muscle damage and is a diagnostic tool of these two types of

muscular dystrophy. Serum CK levels vary with the age and clinical progression in

DMD and BMD patients, and it can reach values 200 to 300 times higher than normal

(Zatz et al., 1991).

Defects in or absence of dystrophin protein in the cardiomyocyte results in DCM

through a pathway similar to that described above. Specifically, recent studies have suggested that absence or mutation of dystrophin disrupts the function of membrane ion channels, particularly the sarcolemmal stretch-activated channels, which respond to mechanical stress (Woolf et al., 2006). When cardiomyocytes with deficient or mutant dystrophin stretch during ventricular filling, the stretch-activated channels do not open appropriately, leading to increased influx of calcium (Williams & Allen, 2007). The high intracellular calcium activates calcium-induced calpains, a group of proteases, which degrade troponin I and compromise cardiomyocyte contraction (Feng, Schaus,

Fallavollita, Lee, & Canty, 2001; Gao et al., 1997). Calpain-mediated destruction of membrane protein allows more calcium entry. Eventually, the chronic calcium overload leads to the cardiomyocyte death (Raymackers et al., 2003). Figure 5 is a graphic representation of the pathophysiology event in DCM caused by abnormal dystrophin.

16 Unlike skeletal muscle, cardiac muscle repeatedly contracts at least 86,400 times

per day in the adult and more in the child. The calcium fluxes associated with each excitation contraction cycle are thought to accelerate the deterioration process within cardiomyocytes as compared to skeletal myofibers. In addition, calcium buffering and handling are different between cardiac and skeletal muscle. Cardiomyocyte death initiates an inflammatory response during which macrophages migrate to clear the damaged cells and debris. Following macrophage recruitment, fibroblasts invade the damaged area and form scar tissue (fibrosis) in the heart. Fibrotic tissue is very inflexible compared to the normal cardiac tissue and thus renders myocardial contraction inefficient. The fibrosis begins from the left ventricular wall in DMD and from the right and left ventricular walls in BMD, starting in the epicardium and advancing into the

endocardium (Frankel & Rosser, 1976). It progressively spreads throughout most of the

outer half of the ventricular wall. This pattern of fibrosis is unique to dystrophinopathy.

The fibrotic region will gradually stretch, become thinner, lose contractility, and result in

DCM. The dilation of the heart increases left ventricular volume, decreases systolic function, and can lead to mitral valve regurgitation, all of which result in decreased cardiac output and hemodynamic decompensation. The cardiac phenotype in each DMD or BMD patient results from the patient’s particular type of dystrophin gene mutation; however, the relationship between genotype and phenotype remains elusive. Figure 6 depicts the continuing pathologic progression of DCM following cardiomyocyte death.

17 Interactions of Dystrophin

The pathophysiological findings described above relate to the structural and

intracellular signaling functions mediated by the direct and indirect interactions of

dystrophin with a large number of proteins. Dystrophin is an essential member of the

dystrophin-associated protein complex (DAPC) at the sarcolemma (Figure 3) (Yoshida et al., 2000). Dystrophin binds to the intracellular cytoskeleton through γ-actin filaments at its N-terminus and at the central rod domain around spectrin-like repeats 11-17

(Sutherland-Smith et al., 2003; Winder, Gibson, & Kendrick-Jones, 1995). Although dystrophin binds actin, it does not interact with the actin filaments mediating muscle contraction at the sarcomere. At the distal end, dystrophin is connected to the extracellular matrix through the transmembrane protein β-dystroglycan, whose carboxy- terminal cytoplasmic domain binds directly to dystrophin and provides a physical link to other proteins in the DAPC, including α-dystroglycan, the transmembrane sarcoglycans

(α, β, γ, δ, є and ζ subunits), sarcospan, and laminin.

Furthermore, recent data proposed that dystrophin also binds to the anchoring proteins ankyrin-B and ankyrin-G. In particular, ankyrin-G binds to both dystrophin and

β-dystroglycan and plays a role in restricting them at costameres (sub-sarcolemmal protein assemblies circumferentially aligned in register with the Z-disk of myofibrils)

(Ayalon, Davis, Scotland, & Bennett, 2008). The physical arrangement of dystrophin and DAPC creates a transmembrane micronetwork composed of the intracellular actin, the cell membrane and the basal lamina. This structure can be thought as a bolt (e.g. dystrophin-bolt), which functions to protect the sarcolemma from mechanical stress during repeated cycles of muscle contraction and relaxation. Loss of its integrity

18 produces muscle fiber fragility, susceptibility to myofibril necrosis and eventually

degeneration of the muscle (Davies & Nowak, 2006).

The extreme C-terminus of dystrophin directly interacts with syntrophins (5

known isoforms) and dystrobrevins (at least 3 isoforms and mainly α in skeletal muscle).

Syntrophins and α-dystrobrevins have independent interactions with dystrophin and they

also directly bind to each other. Through the syntrophins, the DAPC anchors to signaling

molecules such as neuronal nitric oxide synthase (nNOS) and ion channels (Davies &

Nowak, 2006; Ervasti & Sonnemann, 2008). The crucial role of dystrophin in a number

of sarcolemmal signaling pathways is evidenced by its involvement and interactions with

many signaling proteins, including calmodulin (Anderson, Rogers, & Jarrett, 1996),

calmodulin-dependent kinase (Madhavan & Jarrett, 1999), extracellular signal-regulated

protein kinases (Shemanko, Sanghera, Milner, Pelech, & Michalak, 1995), casein kinase,

p34cdc2, calcineurin (Michalak, Fu, Milner, Busaan, & Hance,1996), protein kinase A

(Senter, Ceoldo, Petrusa, & Salviati, 1995), and protein kinases C and G (Luise et al.,

1993).

Given that there is no clear relationship between the severity of the DMD and/or

BMD patient’s skeletal muscle wasting and the severity of his DCM, questions have been raised regarding whether the functional differences between cardiac and skeletal muscle dystrophin are due to different protein associations. Investigations aimed to address this issue have utilized animal models. A widely utilized animal model to study dystrophinopathy is the mdx mouse, which expresses no dystrophin protein.

Approximately two to three weeks after birth, the mdx mouse begins to develop rapid, progressive myopathy until about seven weeks of age. The myopathy and interstitial

19 fibrosis slows down but continues throughout the mouse’s 2-year life span. Cardiac phenotype of the mdx mouse is milder than that seen in human patients; however, it does develop ECG abnormality, ventricular fibrosis and DCM during mid-to-late adulthood

(Chu et al., 2002; Quinlan et al., 2004).

Past studies have found that mdx animals over-expressing altered dystrophin transgenes which consisted of no to minimal rod domain (e.g. micro- or mini-dystrophin) demonstrated phenotypic rescue only in the skeletal muscle, but not in the cardiac muscle

(Harper et al., 2002). Therefore, it seems that the rod domain of dystrophin may not be entirely essential for normal skeletal muscle function, yet it is necessary for normal cardiac function. This result suggests structural and functional differences exist between skeletal muscle and cardiac dystrophin. Indeed, the cardiac dystrophin protein has been shown to have unique binding partners. For example, the cardiac sodium channel Nav1.5 associates with cardiac dystrophin through the mediation of α- and β- syntrophins.

Observation in the mdx5cv animals showed that normal cardiac dystrophin is required for proper functioning of Nav1.5, which maintains physiologic sodium current and regular conduction (Gavillet et al., 2006). Deficiency of dystrophin in the cardiomyocyte has been reported to lead to enhanced calcium influx and delayed calcium channel inactivation due to deregulation to the properties of the dihydropyridine receptors, the L- type calcium channel and the sarco(endo)plasmic reticulum Ca2-ATPase (Leonoudakis et al., 2004; Sadeghi, Doyle, & Johnson, 2002; Woolf et al., 2006). Anatomically, dystrophin has been found to only be present in the T-tubule and microfilaments of the cardiac, but not skeletal muscle, and there is a subset of cardiac dystrophin that does not establish interaction with DAPC (Kaprielian, Stevenson, Rothery, Cullen, & Severs,

20 2000; Meng, Leddy, Frank, Holland, & Tuana, 1996; Peri, Ajdukovic, Holland, & Tuana,

1994).

Functional Biology of Dystrophin

The functional role of dystrophin can be best understood by observing consequences of dystrophin deficiency. The impact of the absence of dystrophin is evidenced by DMD patients’ severe phenotype, even though in the DMD muscle, dystrophin’s functional analog protein, utrophin, has been reported to be up-regulated and forms an utrophin-bolt at the sarcolemma. The micronetwork connecting the basal lamina and intracellular actin held together by the utrophin-bolt is structurally weaker because only a small amount of β-dystroglycan is bound to utrophin. Moreover, there are not as many utrophin-bolts present on the DMD muscle fiber as compared to the number of dystrophin-bolts in the normal muscle (Ishikawa-Sakurai, Yoshida, Imamura, Davies,

& Ozawa, 2004; Tanaka, Ishiguro, Eguchi, Saito, & Ozawa, 1991). Consequently, some

DAPC proteins are decreased in the DMD muscle. For example, expression of β- dystroglycan and the sarcoglycans is greatly reduced (Mizuno, Yoshida, Nonaka, Hirai,

& Ozawa, 1994; Ozawa, Noguchi, Mizuno, Hagiwara, & Yoshida, 1998; Ozawa,

Mizuno, Hagiwara, Sasaoka, & Yoshida, 2005), and the syntrophins, nNOS, sarcospan and dystrobrevin are nearly absent (Brenman, Chao, Xia, Aldape, & Bredt, 1995;

Metzinger et al., 1997; Ohlendieck et al., 1993).

Due to the fragile structure of the cell membrane, the DMD muscle cell is more prone to rupture at its delicate loci during muscle contractions. The “leaky” muscle cell membrane explains the finding that some cytosolic proteins, such as CK, are found in the

21 extracelluar milieu in patients with dystrophinopathy. The consequence of the leaky

membrane is the long-term loss of cytosolic proteins which eventually disturbs nutrition

homeostasis and metabolic processes within the cell, while allowing entrance of

substances from extracelluar fluids, including calcium, which plays a role in activating

calpain, a protease known to damage myofibrils in dystrophinopathy (Bodensteiner &

Engel, 1978; Bullard, Sainsbury, & Miller, 1990).

The absence of dystrophin in the myofibril also affects the related signaling

pathways. Each of the proteins in the signaling pathways described previously regulates

cellular defense mechanisms such as anti-apoptotic pathways, stress-response pathways,

antioxidant defense pathways and anti-inflammatory pathways, all of which affect muscle

death or survival and have been implicated in the pathogenesis of DMD and BMD

(Petrof, 2002). For instance, in the normal muscle the signaling protein nNOS physically

binds to the DAPC via α-syntrophin and caveolin-3, preferentially localizes to the

sarcolemma of fast fibers and functions to catalyze the production of nitric oxide. In the

mdx mouse, nNOS expression in the muscle is significantly lower and is mislocalized inside the cytosol. It is speculated that one cause for DMD pathology originates from the

misplacement of nNOS, which consequently increases free radical production and

susceptibility to inflammation (Brenman, Chao, Xia, Aldape, & Bredt, 1995; Chang, et

al., 1996).

In the cardiac muscle, the lack of the dystrophin protein leads to significant

functional compromise. Milasin (1996) reported a patient whose skeletal muscle

dystrophin was shown immunocytochemically to be reduced in quantity but was

normally distributed. However, his cardiac dystrophin was undetectable. This patient

22 was only 24 years old when he was admitted to the hospital for severe heart failure that

had developed within the preceding 6 months. Prior to this event, the patient did not have

any skeletal muscle symptoms (Milasin et al., 1996). In another similar case, a young man with no significant skeletal muscle weakness was found to be in severe heart failure when he was 20 years old. Further examinations confirmed the moderately reduced amount of skeletal muscle dystrophin and the complete absence of cardiac dystrophin

(Bies et al., 1997). Both of these patients have confirmed dystrophin genetic mutations

which resulted in no cardiac dystrophin protein expression (former: a G to T transversion

at the first exon-intron boundary that led to a splice-site mutation; latter: duplication of

exons 2-7).

The severe cardiac phenotype is not only due to the lack of cardiac dystrophin

expression but also to the altered composition of other proteins in the cardiomyocyte as a result of dystrophin’s absence. In the case of the second patient mentioned above, his α- dystroglycan was lost in the heart, thus the linkage between the DAPC and the extracellular matrix is disrupted (Bies et al., 1997). This finding does not suggest that α- dystroglycan is the only protein that is affected by alterations to dystrophin. Rather, it provides a glimpse to the complicated dynamics existing among cardiac dystrophin and other proteins which are uniquely different from skeletal muscle dystrophin.

Structure-Function Relationship of Dystrophin

Transgenic animal studies have shown that deletion of either the N-terminus or the rod-domain actin-binding site resulted in a mild muscular dystrophy phenotype, but deletion of both actin-binding domains created a severe phenotype despite restoration of

23 DAPC proteins (Corrado et al., 1996; Cox, Sunada, Campbell, & Chamberlain, 1994;

Greenberg, Sunada, Campbell, Yaffe, & Nudel, 1994; 760 Warner et al., 2002). These

findings suggest that there may be redundancy with two actin-binding domains, and

binding to actin from at least one domain is crucial for normal function. At the central

rod domain, the unique flexibility and elastic properties has been well recognized as an

important biological function of the protein. Interestingly, there have been mild cases of

BMD whose dystrophin mutations eliminated most of the rod domain (England et al.,

1990; Matsumura et al., 1994). This phenomenon raised the idea of designing a

minimally-composed dystrophin construct for gene therapy, such as mini- or micro-

dystrophin, as another approach to elucidate the structure-function relationship of the

dystrophin protein. Indeed, functional mini- and micro- dystrophin constructs with very

little rod domain but with the amino-terminal actin-binding domain have shown that the

flexibility of rod domain does not seem absolutely essential for normal functioning of

dystrophin in the skeletal muscle; rather, having the actin-binding properties is more

important (Harper et al., 2002).

The cysteine-rich domain and the C-terminus are two regions where dystrophin has interactions with many proteins. At the N-terminus end of the cysteine-rich domain

is the WW domain, where binding with β-dystroglycan is present. At the C-terminus, the coiled-coil motif forms the binding site for dystrobrevin and syntrophins (Ishikawa-

Sakurai, Yoshida, Imamura, Davies, & Ozawa, 2004; Blake, Weir, Newey, & Davies,

2002). The cysteine-rich domain seems to play an essential role for normal dystrophin

functioning, since there has been no report of BMD patients with deletions in this region.

Even missense mutations predicted in-frame deletions in this region all lead to the severe

24 DMD phenotype (Beggs et al., 1991; Ishikawa-Sakurai, Yoshida, Imamura, Davies, &

Ozawa, 2004; Koenig et al., 1989; Lenk et al., 1996; 763 Goldberg et al., 1998).

Different from the cysteine-rich domain, the C-terminus does not seem to be as crucial, as

evidenced by mild BMD phenotype resulting from C-terminus mutations (Kimura et al.,

2009; Suminaga, Takeshima, Wada, Yagi, & Matsuo, 2004; Patria, Alimsardjono, Nishio,

Takeshima, Nakamura, & Matsuo, 1996). Summarizing dystrophin structure-function

studies to date, note that most studies have defined the essential components of

dystrophin based on exhaustive testing in the skeletal muscle. However, as mentioned

earlier, mini- and micro- dystrophin constructs optimized for skeletal muscle do not fully

restore cardiac function implying that they lack protein domains that are functional in the

myocardium. The two major domains lacking in mini- and micro dystrophins are the rod

domain and the carboxyl terminus.

Phasing of Dystrophin Spectrin-Like Repeats

The structural arrangement, also termed phasing, of the dystrophin rod domain

also plays a role in maintaining its function. The dystrophin rod domain is composed of

25 degenerate copies of an amino acid sequence with significant homology to the protein

spectrin, forming 24 canonical spectrin-like repeats and one truncated repeat which comprises Hinge 3. The structure of dystrophin’s spectrin-like repeats are found in several cytoskeletal proteins and are hypothesized to consist of a series of three α-helix

coiled-coil “beads”. For spectrin, it was suggested that each of the three α-helices is

similar in length and is connected by a flexible linker sequence, or loop, to form a

flexible rod-like domain (for graphic illustration see Figure 7) (Parry, Dixon, & Cohen,

25 1992;783 Speicher & Marchesi, 1984). However, for dystrophin, more recent modeling demonstrated that the dystrophin sequence consists of a long contiguous α-helical bridge joining the beads, thereby resulting in a nested repeat structure and a relatively rigid backbone (Calvert, Kahana, & Gratzer, 1996; Cross, Stewart, & Kendrick-Jones, 1990).

A shorter α-helix folds back to stabilize the interaction of adjacent helix segments (Fig.

11A) (Cross et al., 1990; Koenig & Kunkel, 1990). The exon boundaries do not correlate with the physical boundaries of individual spectrin repeats at the protein level.

Therefore, deletion mutations that remove different combinations of exons can disrupt the folding pattern of spectrin repeats to different degrees. In mdx mice, which lack dystrophin, over-expression of transgenic dystrophin constructs that preserve the correct nested repeating structure or phasing (in-phase) of spectrin repeats rescued the skeletal muscle dystrophic phenotype better than a dystrophin construct with a disrupted repeat pattern (out-of-phase) (Harper et al., 2002). It has not yet been determined whether the phasing of spectrin-like repeats is similarly important for the function of dystrophin in human hearts.

In summary, it is clear that the role of dystrophin is structurally and functionally complex in the muscle fiber. For a less severe muscular phenotype in animal models and humans due to dystrophin mutations, it seems that keeping at least one binding site with actin, preserving the cysteine-rich domain and the C-terminus, as well as maintaining the phasing pattern of the rod domain are important.

26 Genotype-Phenotype Correlations

The size of dystrophin gene deletion mutation does not have absolute correlation

with the severity of the resulting phenotype, since there are DMD patients with small

deletions and BMD patients with large deletions. Rather, the amount of dystrophin

protein produced appears to play a more important role. Our current understanding of

this paradox is heavily founded upon the “reading-frame” rule. The genetic and

phenotypic differences between DMD and BMD were first delineated by Monaco and colleagues (1988). Immunohisochemical studies of muscle biopsies from more severe patients, most of whom had DMD, had shown the high prevalence of dystrophin protein deficiency or truncation, whereas at least partially functional dystrophin protein was found in patients with less severe symptoms (e.g. BMD). Further examinations of the dystrophin genotypes linked dystrophin protein deficiency/truncation to mutations that disrupted the translational open reading frame (ORF), and partial dystrophin protein expression/function to mutations that preserved the ORF. Therefore, the “reading-frame theory” was proposed (Monaco, Bertelson, Liechti-Gallati, Moser, & Kunkel, 1988). The reading frame theory contends that due to an eventual stop codon, the frame-shifted mRNA would prematurely terminate amino acid synthesis during translation, producing a fragmented dystrophin protein that is likely to be non-functional or degraded. On the other hand, deletions that still maintain the mRNA ORF for translation of amino acids would produce shorter, lower molecular weight dystrophin molecules that are presumably semi-functional.

This theory held true in 92% of the cases in a later study involving 258 patients with dystrophin exon deletion mutations (Koenig et al., 1989). In the Leiden Open

27 Variation Database, established by the Leiden University in Netherlands as a registry of

dystrophin mutations found worldwide (Fokkema, den Dunnen, & Taschner, 2005), the

reading-frame theory held true 91% of the time in 4700 cases of DMD and BMD

(Aartsma-Rus, Van Deutekom, Fokkema, Van Ommen, & Den Dunnen, 2006). The 8-

9% of the cases for which the reading-frame theory does not explain phenotype has been

expanded to 30% in a recent study (Kesari et al., 2008). Of 56 BMD patients with

deletion or duplication mutations in this study, 17 demonstrated frame-shift mutations,

challenging the well-believed reading-frame logic. Exceptions to the reading-frame

theory may be due to alternative splicing of exons or other epigenetic mechanisms. In

other words, genetic factors can explain the clinical course of a DMD or BMD patient to

a great extent according to established research. However, it is important to note that

exceptions do exist. Thus, in the clinical setting, each patient should be assessed and

counseled on an individual bases.

The correlation of DCM to specific dystrophin genotypes is a challenging one.

The fact that protein is the biological basis for cellular function precludes DMD patients as appropriate subjects for this type of study. Despite the wide spectrum of dystrophin

mutations among DMD patients, the common feature among them is the near complete or

complete lack of dystrophin protein in the muscle fiber, leading to the relatively

homogeneous phenotypes which define this disease. It is difficult for genotype-

phenotype analyses conducted in the DMD population to be informative, since in general

all genotypes result in the same phenotype. On the other hand, most, but not all, patients

with BMD or dystrophin-related XLDCM represent a group of individuals who have

various dystrophin gene mutations, who do produce dystrophin proteins that reflect their

28 unique dystrophin genotypes, and who likely present different clinical pictures of the

disease as a consequence of their genotypes. This group of patients is more suitable for

the research of dystrophin genotypes and resulting cardiac phenotypes since their

phenotypes can be explained by both their dystrophin protein make-up and genotypes.

Given the low prevalence of BMD patients and the even rarer cases of dystrophin-

linked XLDCM, past genotype-phenotype correlation studies have typically suffered

from low numbers of patients that display a large range of genetic mutations. For

example, a few genotype-phenotype studies have been performed in BMD patients in an

attempt to correlate sites of dystrophin mutations with the occurrence and/or severity of

cardiomyopathy. From these studies a possible correlation between DCM development

and deletions of exons 48 and 49 of the dystrophin gene was suggested (Maeda et al.,

1995; Melacini et al., 1993; Nigro, Politano, Nigro, Petretta, & Comi, 1994). In addition

to small numbers of subjects (e.g. 31 subjects in Melacini et al. (1993) study), most patients enrolled in these studies have mutations involving exons 48 and 49, thus mutations elsewhere along the dystrophin gene were significantly under-represented. No correlation of cardiomyopathy was found with mutations affecting the 5’ coding region of the dystrophin gene (Melacini et al., 1993; Nigro et al., 1994). However, this is a region found to strongly associate with dystrophin-related XLDCM in other studies (Bies et al.,

1997; Feng, Yan, Buzin, Towbin, & Sommer, 2002; Gold et al., 1992; Milasin et al.,

1996; Muntoni et al., 1993; Muntoni et al., 1995; Novakovic et al., 2005; Saotome,

Yoshitomi, Kojima, & Kuramochi, 2001). Further, upon re-analysis of the Melacini

(1993) study as part of the preliminary research for this dissertation study, it was found that some patients classified as having cardiac involvement by the study’s investigators

29 showed no abnormalities in echocardiogram measurements such as ejection fraction, but

primarily had electrocardiogram abnormalities. For this dissertation study, cardiac

involvement was defined based upon abnormalities in echocardiogram rather than

electrocardiogram, which in dystrophinopathy reflects cardiac function poorly (Corrado

et al., 2002; Thrush et al., 2009).

Of relevance, findings from a recent study associated more severe skeletal muscle

symptoms with deletion mutations that include exons 45-49 but exclude the adjacent exons 51-52 (Carsana et al., 2005). Such a correlation had not been observed in the two previous studies since patients with both large and small deletions were inappropriately pooled together. The pooling strategy was unfortunately dictated by the limited number

of available patients; consequently, less common deletions affecting other regions of

dystrophin have been left unstudied. Another recent genotype-phenotype correlation

study included 7 BMD and 62 DMD patients (Jefferies et al., 2005). They found a

significant association of cardiomyopathy with mutations affecting exons 12 and 14 to

17, as well as a possible protection conferred by mutations in exons 51 to 52.

Unfortunately, this study appears to have pooled DMD and BMD patients in order to

achieve statistical significance at these loci. This method is arguable since as stated

before, DMD patients do not usually express dystrophin and therefore while their

genotype may vary, any correlation to a phenotype in the absence of a produced protein is

difficult to interpret.

30 Part III: Dissertation Research

Synthesis of Literature

Dystrophin is an important protein present in muscle tissue. Mutations in the

dystrophin gene give rise to DMD, BMD and XLDCM. Evidence suggests that mild

phenotypes of these disorders are associated with the mutant dystrophin protein binding

with actin, maintaining the cysteine-rich domain and preserving proper phasing of the

spectrin-like repeats of the rod domain. DCM is the primary cause of death among most

BMD patients. One of the aforementioned studies (Nigro et al., 1994) followed 68 BMD

patients over a 17-year period and reported that 100% of patients had either preclinical or

clinical cardiac involvement by 30 years of age, regardless of dystrophin genotype. This

report suggests that perhaps the most important question to ask in research is not whether each patient’s dystrophin genotype would give rise to DCM. Rather, the most relevant question for patients is how soon they will develop DCM based on their own dystrophin mutation, so that early cardiac surveillance and preventive measures can begin in a timely

manner to potentially delay DCM and improve quality of life. The investigation of how

dystrophin genotype affects each patient’s age of DCM development from the

perspective of understanding how genotype modifies the protein’s molecular

conformation and cellular function has not been pursued.

Study Purpose

To determine age of DCM manifestation in relation to dystrophin genotype and its resulting dystrophin protein modifications in BMD and XLDCM patients.

31 Specific Aims

1. To determine the association between DCM age of onset and dystrophin deletion

mutations when patients are categorized based on the affected dystrophin protein

domain boundaries. The hypothesis is that the loss or alteration of specific functional

domain(s) of the dystrophin protein will be associated with earlier age of DCM

manifestation.

2. To determine if structural arrangements of the dystrophin rod-domain spectrin-like

repeats affect age of DCM manifestation among BMD patients. The hypothesis is

that severe disruption of dystrophin protein structure will lead to younger age of

DCM manifestation.

Theoretical Framework

The theoretical foundation (Figure 8) that guides the logic of the design and analysis of this dissertation study was extracted from two seminal human and animal studies (Harper et al., 2002; Monaco et al., 1988). The three constructs, genotype, protein structure and phenotype are the main stance of the model, and they are color- coordinated with the concepts (Figure 8). Concepts in green background originate from the reading-frame theory proposed by Monaco et al. (1988) and guide the process of subject selection. Concepts in orange background are established based on findings of

Harper et al. (2002). They guide the data analysis approach to establish a genotype- phenotype correlation. Table 2 is a detailed definition and description of each construct and concept in the theoretical model.

32 The basis of the theory stems from the understanding that DNA encodes RNA, which then encodes protein through the transcription and translation processes.

Acknowledging epigenetic mechanisms, the correct DNA sequence is a primary determinant of the correct coding for the protein, which is the functional unit of normal physiology. Monaco and colleagues (1988) first observed that intragenic deletions that result in shifting of the subsequent triplet codon reading frames (frame-shift) were primarily carried by DMD patients, whereas deletions that result in in-frame mutations were mostly found in BMD. As a result, a “reading frame theory” was proposed by

Monaco (1988), contending that severe deficiency or truncation of dystrophin protein found in DMD is due to the loss of the translational reading frame, whereas the dysfunctional or partial dystrophin protein found in BMD is due to in-frame mutations.

Later, Koenig et al (1989) conducted a genotype-phenotype analysis on 258 DMD and

BMD patients to test Monaco’s (1988) reading frame theory. The genotype-phenotype linkage found in 92% of the subject in this study was supportive of the theory.

In the animal study by Harper and colleagues (2002), different dystrophin constructs carried by the transgenic animals were composed based on varying assemblies of dystrophin’s spectrin-like repeats. Functional and molecular analyses of these transgenic animals demonstrated that the phasing pattern of spectrin-like repeats affected dystrophin function. In this experiment, one transgenic mouse expressed dystrophin exon deletions resulting in the coding of 8 full spectrin-like repeats, forming an “in-phase” composition of the central rod domain. This group of animals was compared with another group from an earlier study (Phelps et al., 1995), which had dystrophin exons 17-

48 deletion, forming 8 full and 1 partial (out-of-phase) spectrin-like repeats in the rod

33 domain. As shown in Figure 9, animals with dystrophin deletion of exons 17-48 partially

expressed spectrin-like repeat 19, whereas another group did not express spectrin-like

repeat 19 at all. Even though dystrophin from the second group of animals had lower

molecular weight than the first group (222kD vs. 228kD), their functional and molecular

outcomes were close to their wild-type counterparts. These findings indicated that in-

phase composition of dystrophin rod domain led to similar quadriceps and diaphragm

muscle morphology. They also had similar specific contractile force levels of diaphragm

and extensor digitorum longus muscles, compared to wild type animals (Harper et al.,

2002; Phelps et al., 1995).

While Monaco et al. (1988) and Harper et al. (2002) tested the reading frame

theory and the impact of spectrin-like repeat phasing on the skeletal muscle outcome in

human and mouse, no previous effort has explore how the human patient’s cardiac

phenotype may be affected by these mechanisms. The reading-frame hypothesis

(Monaco et al., 1988) supported that BMD patients are the best population for studying

functional roles of specific dystrophin gene regions, due to the fact that they have

dystrophin protein in their muscle. By correlating the BMD patient’s clinical muscle performance deficit with their genotype, one can speculate the function of the mutated

region of dystrophin. These findings of Harper et al. (2002) established the theoretical

hypothesis that for those BMD patients whose dystrophin deletion mutations affected the

central rod domain of dystrophin, their clinical outcomes would be worse (e.g. younger

age of DCM manifestation) when the mutation is disruptive to the spectrin-like repeats

structure (out-of-phase). Likewise, when the BMD patient’s mutation resulted in an in-

phase structure of the spectrin-like repeats, they may present with a better outcome as

34 measured by older age of DCM manifestation. Extending the skeletal muscle findings

from Harper et al. (2002) to cardiac studies in BMD patients, a hypothesis is formed based on the impact of the phasing pattern of spectrin-like repeats and is used to guide the

analysis of DCM clinical phenotype variations in the BMD population.

35

CHAPTER 3

RESEARCH DESIGN AND METHODS

General Procedure

Research protocol was approved by the Institutional Review Board (IRB) at

Nationwide Children’s Hospital (NCH), which waived additional IRB review by The

Ohio State University (OSU) due to contractual agreement between the two institutions.

Patients were enrolled from the Muscular Dystrophy Clinics at NCH and the OSU

Medical Center (OSUMC). Medical records of BMD patients from NCH and OSUMC were reviewed. For patients who had been clinically diagnosed with BMD but had not been genotyped, the clinical research staff at both clinics invited the patients to consider participation in the national United Dystrophinopathy Project (UDP), which is based in

the University of Utah, Salt Lake City, Utah. By agreeing to be a UDP participant, the

patient provides a blood sample that is sent to the UDP Center at University of Utah to be genotyped free of charge.

Permission was obtained to access clinical information of patients enrolled in

UDP, providing another source for patients for this study. In addition, a comprehensive

literature search was conducted to collect published case reports on BMD and XLDCM

patients. Search engines utilized included PubMed and Google, in addition to permitted

access to the Leiden Open Variation Database (Leiden University, the Netherlands), a

36 voluntary world registry of dystrophin mutations. Inclusion/exclusion criteria are described below under Data Collection.

All patients were de-identified and assigned a unique identification number. A separate file was kept to track their names and where they were from. A comprehensive database was generated as a Microsoft Excel file to store all patients’ clinical information prior to statistical analyses. Collection of clinical information included the following if available:

• Mutation of DNA (e.g. c.494A>T; in-frame or out-of-frame)

• Mutation of RNA (e.g. Exon 45-48 deleted)

• Mutation of amino acid (i.e. p.2740Asp>Gly)

• Affected exon(s) (e.g. exon 50~51)

• Type of mutation (e.g. deletion)

• Phasing pattern of the dystrophin spectrin-like repeats (e.g. in-phase or out-of-

phase)

• Technique used for genotyping

• Clinical diagnosis

• Source (e.g. UDP or Leiden)

• Clinical history in narrative form, including age of skeletal symptoms onset,

initial symptoms, age of diagnosis, wheelchair dependency,

immunohistochemistry results from muscle biopsies, muscle strength,

medications, pulmonary function test results, serum CK level, mental

disability/disturbance, family history, date of birth, gender and race.

37 • Cardiac history in narrative form, including age of first cardiac symptoms (if any),

age and detailed results of electrocardiogram, echocardiogram, available cardiac

MRI, chest X ray, cardiac catheterization or any other procedural/physical

examinations of the heart, history of cardiac surgeries/diseases, and cardiac

medications and dosages.

• Assigned cardiac function category (as defined in Table 3).

To ensure the independence of subjects, when siblings occurred in the database, only one from each pair was included in statistical analyses. For statistical analysis, an

SPSS (v15, SPSS Inc., Chicago, Illinois) file was generated and some of the above information, particularly age, mutations, echocardiographic results and cardiac function categories, were entered using corresponding number codes (e.g. BMD = 1; XLDCM =

2).

Grouping of Patients

To determine whether genetic mutations affecting specific functional domains of dystrophin protein correlate with an earlier age of cardiac involvement, patients were categorized into 3 groups based on the functional domain affected by the exon deletion

(Figure 10). Group 1 included patients with mutations that affected any portion of the amino-terminal actin-binding domain of dystrophin (exons 2 to 8). The region surrounding Hinge 3 was separated into two groups based on prior reports from BMD patients that correlated between skeletal muscle involvement and whether Hinge 3 was affected in the protein product (Carsana et al., 2005). Group 2 included patients with deletions contained within exons 45 to 49. These deletions affect spectrin repeats 17 up

38 to 19 but leave Hinge 3 intact. Group 3 included patients with deletions affecting exons

50 and/or 51, thus removing or disrupting Hinge 3. These patients had deletions that span from exon 45 up to exon 55 and affect spectrin repeats 17 up to 21.

Dystrophin Protein Modeling

Dr. Will Ray, a collaborating biophysicist of this study, conducted the 3- dimensional modeling of dystrophin rod domain to study how in-phase and out-of-phase deletion mutations would affect the structure and stability of dystrophin. The rod-region spectrin-like repeats were modeled based on the published structure of repeats 15 and 16 of chicken brain alpha spectrin (RCSB Protein Data Bank code 1U5P) (Kusunoki,

Minasov, Macdonald, & Mondragon, 2004). The structure was manually extended by replication and root mean square alignment of corresponding terminal amino acid residues, using PyMol (http://www.pymol.org/). The structure was briefly minimized using molecular dynamics within VMD/NAMD

(http://www.ks.uiuc.edu/Research/namd/), a biomolecular simulation and analysis program. The minimization was carried out in 4 steps by solvating each manually constructed repeat structure within NAMD, freezing the structure and minimizing the waters to the structure, releasing the waters and freezing the backbone and minimizing side chain rotamers, releasing the backbone and minimizing the entire repeat structure within the frozen water, then finally releasing the entire system and applying 5000 2fs steps of molecular dynamics at 310K (W. C. Ray, personal communication, February 20,

2009). This minimization was not intended to predict the fine-grained structural stability of the various conformations, but eliminated exceptionally bad chain conformations and 39 steric or electrostatic interferences that may have been created by the manual modeling

procedure. The 3-dimensional schematic of dystrophin spectrin-like repeats 16-20

constructed by this method is seen in Figure 11B. The hinge region and out-of-phase

deletion mutation structures were constructed by manual deletion of structurally

equivalent residues from the extended spectrin repeat, and structural re-alignment of the

resulting fragments. A similar minimization was used to finalize the structural

compaction resulting from the deletion mutations.

Echocardiography

Echocardiography evaluation is sensitive to anatomical and physiological changes of a dilated heart. Signs of DCM sensitive to direct visualization and measurements in an echocardiography study include: dilated ventricles and atria, areas of dyskinesis and akinesis, increased left ventricular volume, decreased ventricular wall thickness, and

mitral regurgitation. With additional calculations, echocardiography is also sensitive to

other measurements that can reflect cardiac geometric distortion consequential to DCM,

including (1) ejection fraction ([left ventricular diastolic volume – left ventricular systolic

volume] / left ventricular diastolic volume); (2) shortening fraction ([left ventricular end-

diastolic diameter – left ventricular end-systolic diameter] / left ventricular end-diastolic

diameter); (3) E-point septal separation (EPSS, the minimal separation between the mitral valve anterior leaflet and the ventricular septum during early diastole); (4) sphericity index (left ventricular short to long axis ratio); and (5) cardiac mass (Anderson,

2007; Solomon, 2007; Tani et al., 2005).

40 The following criteria defined presence of cardiomyopathy in this study: ejection fraction below 55%, shortening fraction below 33%, diagnosis of cardiomyopathy, or cardiomegaly. These criteria were determined based on established standards in the

DMD and BMD populations (Jefferies et al., 2005; Duboc et al., 2007). When available, additional parameters were included to confirm cardiac dilation: EPSS or left ventricular end diastolic dimension (Table 3.1). An EPSS of greater than 5-5.5mm suggests that the left ventricle has become more dilated and provides a clue for cardiac functional changes

(Anderson, 2007). In the adult, the left ventricular internal dimensions during systole and diastole in a normal heart are <37mm and <56mm, respectively (Solomon, 2007). In this study, left ventricular internal dimension >58mm described DCM.

Age

Age of cardiac manifestation was reported as the youngest age at which the patient’s cardiac parameters meet the definition of cardiomyopathy, or the oldest age at which the patient is defined as unaffected with cardiomyopathy as outlined above. The purpose of defining “age” differently for cardiomyopathic versus non-cardiomyopathic patients was to best capture the true age of DCM onset. For example, if a patient had cardiac data showing normal at age 17, and DCM at ages 25, and 28, respectively, the age of 25 will be collected as his age of DCM onset, because this is the age closest to when his DCM was noted. On the other hand, if a patient’s cardiac data showed normal, normal, and normal at ages 33, 36, and 45, respectively, the age collected for this patient would be 45, since this is the age closest to when he actually developed DCM.

41 Samples

Although DMD, BMD and dystrophin-related XLDCM patients have mutations in

their dystrophin gene, DMD patients were excluded. This is because the minimal

phenotypic variation in DMD patients caused by dystrophin mutations is primarily frame-

shifting and it uniformly gives rise to no dystrophin protein expression and severe muscle degeneration. A meaningful genotype-phenotype association cannot be established when there is a variety of genotypes yet only one phenotype (lack of dystrophin protein in the case of DMD). For this reason, only those patients with confirmed dystrophinopathy and whose diagnosis was not DMD were enrolled in this study. It thus included BMD and dystrophin-related XLDCM.

Data from BMD and XLDCM with dystrophin gene in-frame deletion mutations of less than 12 exons were collected from sources discussed under General Procedure.

The reason to only have included patients with exon deletion mutation was because deletion mutation is the most common among BMD patients. Thus, it was realistic to plan to conduct a statistical genotype-phenotype analysis based on a population that was

large enough to generate meaningful data. Furthermore, even if non-deletion mutations

such as duplications and insertions were included, it would not have been appropriate to

mix them with deletion mutations because each type of mutation affects the protein

differently. The deletion size limit of less than 12 exons was selected so that we could

include enough number of BMD and XLDCM patients to maintain statistical power,

while avoiding deletions that are large enough to affect more than one functional domains

of the dystrophin protein, which would lead to misinterpretation of the genotype-

phenotype correlation.

42 Power analysis for sample size estimation for single-gene genotype-phenotype correlation studies in dystrophinopathy or other rare disorders has not been proposed.

The sample sizes of past studies as well as this dissertation study were limited to the availability of eligible patients. The goal of this study was to recruit more than 100 BMD or XLDCM patients, which would be the largest number to date. New BMD patients at

NCH or OSU clinics were referred to Dr. Hugh D. Allen at NCH for cardiac evaluation and echocardiography to minimize inter-rater variability.

Data Collection

To be included in this study, patients must have: (1) a diagnosis of BMD and/or

XLDCM; (2) confirmed exon deletion mutations encompassing less than 12 exons; and

(3) echocardiographic measurements or compelling clinical evidence for DCM, which combines echocardiographic abnormalities, heart failure, cardiomegaly or cardiac transplantation. Patients were excluded if their dystrophin gene mutations affected the first exon or the 3’ untranslated region, or if their cardiac biopsies showed no dystrophin expression. BMD patients were excluded if they were wheelchair-dependent by 12 years of age, were younger than 12 years of age without family history, had out-of-frame mutations and if no muscle biopsy was available to confirm dystrophin protein expression. Finally, patients with reported or suspected cardiac viral infection were excluded.

43 Data Analysis

The non-parametric Kruskal-Wallis test (one-way ANOVA by ranks) was performed for cross-group comparisons of age (a priori p < 0.05). Mann-Whitney U tests were utilized for post hoc comparisons among groups (a priori p < 0.016 to achieve overall significance of p < 0.05 using the Bonferroni adjustment). For those with mutations in dystrophin central rod domain, the difference in age of DCM manifestation was used to indicate the consequences of spectrin-like repeat structural changes. This age difference between patients with in-phase and out-of-phase mutations was analyzed with a two-sample t-test (a priori p < 0.05). All data were analyzed in SPSS (v15, SPSS Inc.,

Chicago, Illinois).

44

CHAPTER 4

FINDINGS

General Description of Samples

Data from 309 patients were initially collected based on their medical diagnoses of BMD or dystrophin-related XLDCM. After applying inclusion and exclusion criteria, data from 110 patients were included in this study. The age range for patients with DCM was 12 to 76 years, while the age range for patients without DCM was 3 to 50 years.

Detailed clinical information, including genotype, musculoskeletal and cardiac involvement information on each patient is presented in Appendices A and B as supplemental data.

In this study, “age” was defined as follows: for those affected with DCM, it was the youngest age at which the patient’s cardiac parameters met the definition for DCM according to criteria listed in Table 3; for those unaffected with DCM, it was the oldest age at which the patient’s cardiac status was considered normal according to the same criteria. Table 4 shows number of patients according to diagnosis and source, as well as median ages based on cardiomyopathy categorization. As shown, the non- cardiomyopathic patients were significantly younger than the cardiomyopathic ones (p <

0.001), regardless of the source. This suggested that the non-cardiomyopathic patients

45 may be unaffected because they were not old enough to develop heart disease, rather than

having specific genotypes protecting them from developing DCM.

To determine whether all cardiomyopathic patients can be combined for

maximum statistical power in genotype analysis, we tested whether the clinical diagnosis

(e.g. BMD vs. XLDCM) or the source of patient information (e.g. from publications vs.

not from publications) would influence age of DCM onset. When comparing the ages of

DCM onset between cardiomyopathic patients with either XLDCM or BMD diagnosis,

no statistical differences were found. Further, when comparing the median ages for

cardiomyopathic patients across data sources, we found no statistically significant

differences between patients collected from published literature versus those from NCH

and OSUMC. These results demonstrated that it is appropriate to combine all BMD and

XLDCM patients for genotype-phenotype analyses even though they may have different

clinical diagnoses and were from different sources.

Next, deletion mutations for cardiomyopathic patients (n = 76) were mapped out

graphically to determine whether different dystrophin protein domains were affected in

XLDCM versus BMD patients (Figure 12). The deletion mutations found in BMD and

XLDCM patients clustered around the same two regions of the dystrophin protein: the

amino-terminal domain corresponding to exons 2 to 13, and a region in the rod domain

centered around Hinge 3, corresponding to exons 45 to 55. This distribution was in

agreement with previous reports on mutation hotspots for BMD patients (Worton &

Thompson, 1988; Worton, Molnar, Brais, & Karpati, 2001). In addition to the similar

mutation distribution, some particular mutations, such as exons 45-48 or 45-55 deletions,

were shared by patients with both diagnoses.

46

Mutation Group 1

Mutations of all cardiomyopathic patients are illustrated in Figure 13 by mutation group. As defined in Chapter 3, patients in Group 1 were those with mutations that affected any portion of the actin-binding N-terminus domain of dystrophin (exons 2 to 8).

Ten patients in Group 1 were cardiomyopathic and 4 were non-cardiomyopathic, with median ages 22.5 years and 19.5 years, respectively (Figure 14). Lack of age related statistical significance between these two groups was most likely due to the small sample size. From the age information of Group 1 cardiomyopathic patients, there were 2 sub- groups, one in the mid-teens, and the other in the late 20’s (Appendix A). Further investigation on additional parameters that might provide a hint that there were indeed 2 sub-groups showed that neither the exon deletion pattern nor the onset and severity of skeletal muscle symptoms redistributed Group 1 patients into two distinct sub-groups.

Among all 3 groups, Group 1 patients showed the widest spectrum of skeletal muscle involvement, ranging from clinically undetectable muscle weakness to loss of ambulation in the late teens (Appendices A & B).

Mutation Group 2

Group 2 included patients with deletions contained within exons 45 to 49. These deletions affect spectrin repeats 17 up to 19 but leave Hinge 3 intact. The majority (67%) of patients in this study had Group 2 mutations. This group included 54 cardiomyopathic and 20 non-cardiomyopathic patients. The median age for DCM was 29.5 years, significantly older than the non-cardiomyopathic patients (18.5 years) (p < 0.001) (Figure

47 14). The age range for DCM was from 15 to 55 years, with the exception of one outlier

(ID: 161), who was diagnosed at age 70 with a low ejection fraction of 27%, suggesting advanced disease that had not been detected earlier (Appendix A). Most patients in this group, whether affected with cardiomyopathy or not, showed mild skeletal muscle involvement, with only a few patients who became wheelchair-dependent in their mid

20’s to late 40’s (Appendices A & B). Eight cardiomyopathic patients in Group 2 had cardiac biopsy data which showed detectable cardiac dystrophin, yet it was fainter than control and discontinuous along cardiomyocyte membrane.

Mutation Group 3

Group 3 included patients with deletions affecting exons 50 and/or 51, thus

disrupting or removing Hinge 3. These patients had deletions that spanned from exon 45

to exon 55, affecting spectrin repeats 17 up to 21. Twelve had DCM and 9 did not. The median age for the cardiomyopathic patients in Group 3 was 46.5 years, the oldest among all three groups. The cardiomyopathic patients were significantly older than their non- cardiomyopathic counterparts, whose median age was 9 years (p < 0.001) (Figure 14).

Among cardiomyopathic patients, two older patients with deletion mutations spanning exons 45 to 55 showed only asymptomatic borderline cardiac involvement at ages 69 (ID:

162) and 76 (ID: 248). Even though their ejection fraction values were above 55%, patient 162 showed cardiomegaly by chest roentgenogram and patient 248 had a reduced shortening fraction of 28.9%. Cardiac biopsies with dystrophin immunostaining from 4 cardiomyopathic patients in this group showed detectable dystrophin staining with a discontinuous pattern at reduced levels. It is interesting to note that all patients in Group

48 3 were older than 30 years of age. In addition, all patients whose skeletal muscle information was available had mild skeletal muscle symptoms (Appendices A & B), with the exception of subject MJ01, who became wheelchair-dependent at age 40.

Results for Research Aim 1

The first research aim was to determine the association between DCM age of onset and dystrophin deletion mutations when patients are categorized based on the affected dystrophin protein domain boundaries. The median ages of DCM manifestation for patients in each mutation group were calculated as described above. They were 22.5,

29.5 and 46.5 years for Groups 1, 2 and 3 mutations, respectively (Figure 14). The non- parametric Kruskal-Wallis test showed significant differences in age among the groups (p

< 0.0005). Significant differences were detected for all post-hoc Mann-Whitney U pair- wise comparisons: p = 0.010 for Group 1 versus Group 2; p < 0.001 for Group 1 versus

Group 3; and p = 0.001 for Group 2 versus Group 3. Therefore, deletion mutations affecting these three separate domains of the dystrophin protein were associated with significant differences in the age of cardiac manifestations. More specifically, patients with deletion mutations affecting exons 2 to 9 or exons 45 to 49 were at risk of developing DCM in their second and third decade of life, respectively. In contrast, BMD patients with deletions that were within exons 45 to 55 resulting in a dystrophin protein lacking Hinge 3 were protected from early-onset DCM.

49 Results for Research Aim 2

The second research aim was to determine if structural re-arrangements of the dystrophin rod-domain spectrin-like repeats affect age of DCM manifestation among

BMD patients. At the rod domain of dystrophin, 50 exons (exons 10-60) encode for 24 spectrin-like repeats as well as Hinges 1 and 2. The exon boundaries at the gene level do not correspond to the boundaries of each spectrin-like repeat at the protein level (Figure

4). Different exon deletions result in different re-arrangements of spectrin-like repeats

(e.g. either in-phase or out-of-phase). As illustrated in Figure 15, several possible exon deletion patterns, such as deletions of exons 46-49, 48-49, 45-46, 45-48 and 47-48, will all predict the in-phase composition of the spectrin-like repeats. Likewise, a number of exon deletions, such as deletion of exons 45-47, will disrupt the structure of the spectrin- like repeats and thus make them out-of-phase.

Transgenic animals over-expressing dystrophin with an in-phase rod domain demonstrate a better phenotype as compared to those over-expressing an out-of-phase rod domain (Harper et al., 2002). With a large number of DCM-affected patients in mutation

Group 2 (n = 54), we were able to examine this research aim statistically. For Group 2 mutations, there were 10 unique types of exon deletion mutations. Each of these mutations and its resulting spectrin-like repeats phasing are listed in Table 5. Evaluation of the age of patients in Group 2 based on their spectrin-like repeat phasing showed that patients with in-phase mutations had a significantly later age of DCM manifestation (36.5 years) compared to those with out-of-phase mutations, (25.5 years) (p = 0.002). These data are shown in Figure 16.

50 We then compared the age of DCM manifestation of Group 2 mutations (both in-

phase and out-of-phase) individually with Group 1 and Group 3 mutations (Table 6).

The age of DCM manifestation for Group 2 in-phase mutations was significantly older

than Group 1 mutations (36.5 vs. 22.5 years, p < 0.001) but did not differ from that of

Group 3 mutations. The age of DCM manifestation for Group 2 out-of-phase mutations did not differ from that of Group 1 mutations but was significantly younger than Group 3 mutations (25.5 vs. 46.5 years, p < 0.001). Thus, out-of-phase mutations in the rod

domain and deletions in the amino-terminal actin-binding domain are both associated

with early age cardiomyopathy. These results indicated that the effect of deletion

mutations on the phasing pattern of spectrin repeats 17 to 19 strongly correlates with the

age of onset of cardiomyopathy.

We explored whether the earlier onset of DCM observed in out-of-phase

mutations was due to absence of cardiac dystrophin protein expression. Based on

available cardiac biopsy information (Appendix A), the expression of a mutant cardiac

dystrophin protein was present in both in-phase and out-of-phase mutations (Arbustini et

al., 2000; Fanin, Melacini, Angelini, & Danieli, 1999; Maeda et al., 1995; Muntoni et al.,

1997; Politano et al., 1999). This suggests that the significant difference in the age of

DCM onset between in-phase and out-of-phase mutations is not due to obvious differences in the level of cardiac dystrophin expression.

Collaborating with Dr. Will Ray, the predicted models of dystrophin rod domain as a result of either in-phase or out-of-phase mutations were established using methods described in Chapter 3. In-phase mutations were predicted to have the effect of shortening the rod domain (Figure 17A), whereas out-of-phase mutations were predicted

51 to graft non-homologous sub-regions together (e.g. Helix 1 onto Helix 2 and vice versa),

leading to much more detrimental changes to the rod domain structure (Figure 17B and

C). Such major alterations to the overall protein structure are likely to affect the function

of dystrophin. Utilizing the mutation of exons 45-47 deletion as the example for the out-

of-phase structure, we showed that this particular mutation caused an attachment of a

Helix 2 onto the adjacent Helix 1 (asterisk, Figure 17B), losing a flexible linker sequence.

This structural alteration reverses the direction of extension of the Helix 1 and defines a

new axis for the latter portion of the rod. Most other out-of-phase mutations were

predicted to bend the rod and reverse its direction, with the exception of exon 48 deletion,

which was predicted to give rise to the creation of a new hinge, geographically close to

Hinge 3 (Figure 17C).

Additional Findings

Rare Deletion Mutations

Five patients meeting inclusion/exclusion criteria were eventually removed from

this genotype-phenotype analysis. These patients had deletion mutations between exons

10 and 20. These patients were affected with DCM and had deletion mutations of exons

10-16 (n=1), 11-13 (n=1), 13 (n=2) and 13-19 (n=1), and their median age of DCM

manifestation was 40.6 years old. Elimination of these patients does not imply non-

significance of the mutations. Rather, the number of patients in this group was too small

to be analyzed statistically.

52 Point Mutations

In the process of data collection, we encountered several cases of dystrophin point mutations. The genotype-phenotype association in these patients was not analyzed due to the rarity of dystrophin point mutations. As shown in Figure 18, 8 patients with dystrophin point mutations had the XLDCM clinical diagnosis. These patients were identified in published literature, with the following mutations: (1) 18Lysine>asparagine

(n = 1) (Feng et al., 2002); (2) 279threonine>alanine (n = 5, all from same family) (Berko

& Swift, 1987; Ortiz-Lopez, Li, Su, Goytia, & Towbin, 1997); (3) 1672 asparagine>lysine (n = 1) (Feng, Yan, Buzin, Sommer, & Towbin, 2002); and (4) 3228 phenylalanine>leucine (n = 1) (Feng et al., 2002). The phenotypic presentation of all of these patients was cardiac-specific without any skeletal muscle symptoms. The DCM in these patients was very severe, with the average age of cardiac symptom manifestations being 17 years old. In particular, a previous study revealed that the amino acid substitution of threonine to alanine at position 279 (Hinge 1) disrupts the sequence of a evolutionarily highly conserved region, leading to a change in amino acid polarity and

consequently substituting a β-sheet for α-helix and changing the secondary and tertiary protein structure of dystrophin (Kahana, Marsh, Henry, Way, & Gratzer, 1994; Koenig &

Kunkel, 1990; Ortiz-Lopez et al., 1997).

It is interesting that all of these point mutations occurred at regions where dystrophin is known to have interactions with its neighboring proteins, such as actin and the dystroglycan complex. Further, from the conservation analyses performed by our collaborating geneticist, Dr. Carlos Alvarez, we found that all of these loci are highly evolutionary conserved. Data from conservation analyses are shown in Figures 19 to 22.

53 These two mechanisms may explain the severe and early-onset DCM seen in these patients.

In addition to XLDCM patients with dystrophin point mutations, 3 BMD patients also had point mutations. Their mutations were: (1) 165aspartic acid>valine (at N- terminus); (2) 2740aspartic acid>glycine (rod domain at exon 56); and (3) 3368aspartic acid>glycine (at C-terminus). All 3 patients were from the UDP patient pool. The patients with 165aspartic acid>valine and 3368aspartic acid>glycine mutations were affected with DCM at 17 and 46 years old, respectively. The patient with 2740aspartic acid>glycine mutation was unaffected with DCM at 34 years of age.

54

CHAPTER 5

DISCUSSION AND CONCLUSION

Study Summary

The primary strengths of this study were the large sample size (N=110) and the

stringent selection criteria that only included patients with small deletions, allowing the

grouping of patients based on mutations affecting well defined regions of the dystrophin protein without compromising statistical power. Due to the low incidence of BMD, previous genotype-phenotype correlation studies as reviewed in Chapter 2 were too

underpowered to detect the effects of mutations in regions of the dystrophin gene that are

not mutation hot-spots. The number of patients analyzed in past studies ranged from 19

to 84 and included patients with very large exon deletions (up to 43 consecutive exons).

In this dissertation, we studied patients with dystrophin exon deletion mutations

collectively without separating them by clinical diagnoses, since high genetic (e.g.

mutation distribution) and phenotypic (e.g. DCM age of onset) similarities between BMD

and XLDCM patients were found. Median ages of cardiac manifestation were identified

with respect to 3 distinct regions of dystrophin protein as well as to specific patterns of

the rod-domain spectrin-like repeat re-arrangements. The earliest age of DCM

development, 22.5 years, was associated with dystrophin deletion mutations affecting

exons 2 to 9 (Group 1). In comparison, deletion mutations in the region between exons

55 45 and 55 which resulted in removal of Hinge 3 of the dystrophin protein (Group 3) were

found to be associated with the latest onset of DCM, 46.5 years. Giving rise to DCM age

of onset in-between the youngest and the oldest ages were deletion mutations affecting

exons 45 to 49 (Group 2). The median age of DCM onset for this patient group was 29.5

years. Interestingly, when this group was divided into 2 subgroups based on the phasing

pattern of the affected spectrin-like repeats, we found that out-of-phase deletion

mutations led to an earlier age of DCM onset, 25.5 years, compared to 36.5 years for in- phase deletion mutations. Further, 3-dimensional modeling studies suggested that the earlier DCM age of onset may be explained by the predicted aberrant grafting of non- homologous sub-regions of dystrophin rod-domain spectrin-like repeats, causing severe changes to the protein’s structure and possibly impacting function.

Discussion

Exon Deletions in the Amino-Terminal Domain

Deletions affecting the 5’ region of the dystrophin gene, including the muscle promoter region, exon 1, or intronic regions that alter exon splicing, have been associated with a mild clinical course of skeletal muscle degeneration yet with severe DCM. For these patients, the preferential cardiac involvement was found to correlate with the complete absence of cardiac dystrophin. The molecular mechanisms underlying this phenomenon include tissue-specific use of alternative dystrophin promoters, disruption of tissue-specific enhancers, and differences in alternative splicing between skeletal and cardiac muscle (Muntoni et al., 1994; Kimura et al. 2007; Bies et al., 1997; Milasin et al.

1996; Muntoni et al., 1993; Muntoni et al., 1995; Neri et al., 2007). In order to minimize

56 the error of misinterpreting that the patient’s age of DCM onset was due to his dystrophin genotype rather than the absence of cardiac dystrophin protein, we purposefully excluded those known to lack cardiac dystrophin expression in this study. Because of the finding that the age of DCM development in XLDCM patients with no cardiac dystrophin reported in the literature was very similar to that of our patients in Group 1, we speculate that despite our effort to eliminate patients known to have no cardiac dystrophin, at least some of our Group 1 patients still owe their early DCM manifestation to the lack of cardiac dystrophin. Nevertheless, given the variety of different exon deletion patterns in

Group 1, the probability for the majority of Group 1 patients to have early DCM because of this particular cause is small.

An alternative hypothesis to explain the early-onset DCM and milder musculoskeletal symptoms in patients with mutations in the amino-terminal domain is that although dystrophin’s affinity for actin is lost in both cardiac and skeletal muscle resulting from the deletion, the effect may be more severe in the cardiac muscle due to its different mechanical stress, geometry and physiology. Several lines of evidence support this possibility. One study showed that approximately 35% of the cardiac dystrophin protein tightly binds to actin and is an important part of the cardiomyocyte contractile apparatus. In the presence of cardiomyopathy, this association is progressively disrupted as demonstrated in hamster models (Meng, Leddy, Frank, Holland, & Tuana, 1996).

More recent studies have also confirmed that alterations in cardiac dystrophin, specifically cleavage of the amino-terminal domain, accompany and may precede cardiac dysfunction not related to dystrophin mutations in both rodents and humans (Vatta et al.,

2002; Toyo-Oka et al., 2004). Taken together, these observations suggest that in the

57 cardiac muscle, the amino-terminal domain of dystrophin plays an essential role in

tethering and stabilizing the contractile apparatus via its binding with actin and that loss

of this link leads to cardiomyopathy.

Exon Deletions in the Rod Domain

The rod domain of dystrophin is usually considered as a spacer with elastic

properties (Ervasti & Sonnemann, 2008). Since mutations giving rise to a dystrophin

protein with either a longer or shorter rod domain are typically associated with a late

onset of skeletal muscle symptoms and a slow disease progression, dystrophin rod

domain appears to be permissive to alterations without much major or severe impact on

skeletal muscle function (England et al., 1990; Dastur, Gaitonde, Khadilkar, & Nadkarni,

2008; Mirabella et al., 1998). This phenomenon has led recent gene therapy researchers

to design miniature dystrophin constructs small enough to be packaged into appropriate

viral vectors by removing a large portion of dystrophin rod domain (Scott et al. 2002;

Draviam, Wang, Li, Xiao, & Watkins, 2006; Bostick et al., 2009).

However, as reviewed in Chapter 2, not all re-arrangements of the dystrophin rod

domain are compatible with normal or near-normal skeletal muscle function. Deletions

affecting exons 45 to 48 have been associated with the loss of dystrophin’s ability to bind

nNOS in humans and mice (Wells et al., 2003). Further, past efforts attempting to

substitute dystrophin spectrin-like repeats with homologous spectrin-like repeats from the

alpha-actinin 2 protein found that this type of substitution leads to a loss of dystrophin function as well as the development of myopathic skeletal muscle features (Harper et al.,

2002). These observations strongly suggest that the rod domain of dystrophin is not just

58 a spacer; rather, it is likely an important mechanical modulator between the distal dystrophin domains and other proteins and thus is more sensitive to structural alterations than previously thought.

Extending the discussion from skeletal muscle dystrophin to cardiac dystrophin, we questioned whether all rod-domain deletion mutations would have the same impact on the function of cardiac dystrophin. Previous reports have shown that deletions of exons

45-52 and 45-55, although both affecting the distal rod-domain, were associated with normal cardiac function (former) versus congestive heart failure (latter) at relatively similar ages (29 and 36 years, respectively) (Melacini et al., 1996; Tasaki et al., 2001).

The phenotypic difference resulting from different rod-domain deletion mutations, as defined by DCM age of onset, was also reflected in our results, particularly between patients whose deletions removed Hinge 3 with those whose deletions preserved Hinge 3.

First, removal of Hinge 3 in Group 3 patients conferred a protective effect in cardiac decline, leading to a significantly later onset of cardiomyopathy. Although prior human and animal studies have shown a correlation between the absence of Hinge 3 and milder skeletal muscle pathology (Ferreiro et al., 2009; Carsana et al., 2005; Harper et al.,

2002), this dissertation study is the first to demonstrate that removal of dystrophin Hinge

3 due to exon deletion mutations leads patients to have the oldest age of DCM manifestation (46.5 years old). This was not detected in prior studies because patients with or without Hinge 3 were not analyzed as separate groups (Melacini et al., 1993;

Nigro et al., 1994; Politano et al., 1991). Due to the paucity of information on the structure of Hinge 3 and a low number of patients with similar deletions, we could not model the effects of mutations on the dystrophin protein backbone for Group 3 mutations.

59 Therefore, further studies are needed to elucidate the significant cardio-protective effect

conferred by the loss of Hinge 3.

Second, we have shown that deletion mutations preserving proper phasing of the

spectrin-like repeats upstream to Hinge 3, specifically the region encoded by exons 45 to

49, significantly delayed DCM development by a decade when compared with out-of-

phase re-arrangements in the same region. This is likely not due to difference in cardiac

dystrophin protein expression since similar amount and distribution of dystrophin in

cardiac biopsy samples from BMD patients with either in-phase or out-of-phase

mutations has been reported (Arbustini et al., 2000). Our 3-dimensional modeling of

dystrophin rod domain conformation modeled after in-phase and out-of-phase exon

deletions suggested that the structural alterations caused by out-of-phase mutations

extend beyond the disruption within each spectrin-like repeat unit and lead to a severely

distorted rod-domain configuration, ultimately damaging the entire dystrophin protein.

This major change in the structure of dystrophin is likely the primary determinant of the early onset of DCM in patients with out-of-phase mutations.

Another interesting finding was that most patients in Group 2 had late-onset

skeletal muscle symptoms and mild disease progression, irrespective of the spectrin-like

phasing pattern, providing a hint that cardiac dystrophin may be more sensitive than

skeletal muscle dystrophin to structural disruption in the exons 45-49 region. Recent

studies have tested the ability of synthetic dystrophin constructs with a shortened rod

domain to restore cardiac function in the mdx mouse. While these constructs rescued

skeletal muscle, they were not able to reverse all cardiac parameters to normal (Bostick et

al., 2009; Gregorevic et al., 2006; Townsend et al., 2007). Since transgenic constructs

60 are not subject to alternative splicing or tissue specific differences in exon skipping, these

results indicate a genuine difference in dystrophin protein domains required for cardiac

and skeletal muscle function.

Study Limitations

The genetic, cardiac and other relevant clinical information for most patients in

this study was collected retrospectively from published case reports, OSU, NCH and

UDP. Most patients had had echocardiography performed by various cardiologists and

had been genotyped at different institutions prior to the initiation of the study. To a certain extent, some inter-observer variability was inherent in this research design since taking measurements and making interpretations of an echocardiogram can be subjective and genotyping methodologies vary. Within our ability, we referred newly-identified patients to Dr. Hugh D. Allen at NCH for echocardiographic evaluation and enrolled them in the UDP study for dystrophin genotyping to take place at the UDP headquarters in University of Utah. Although some subjects in this study had echocardiography performed incidentally (e.g. during an emergency room visit), in most cases their echocardiographic evaluations were performed as a protocol of a research program, a setting under which diagnostic procedures are presumed to be of high quality. The genotyping method for the majority of patients was performed with multiplex PCR and

Southern blotting techniques, which have low error rates (Prior & Bridgeman, 2005).

Other genotyping methods included multiplex ligation-dependent probe amplification

(Schwartz & Duno, 2004), detection of virtually all mutations- single-strand conformation polymorphism analysis (Mendell et al., 2001), and single condition

61 amplification/internal primer sequencing (Flanigan et al., 2003), which have all showed

high efficiency and accuracy in detecting small deletion mutations.

Another limitation for this study is the cross-sectional data collection. Due to the

rare incidence of BMD and XLDCM, it was not realistic for this study to set out to

longitudinally perform cardiac surveillance for a large number of patients prior to cardiac

pathology. Therefore, to capture the age closest to the true onset of DCM, “age” for

those affected with DCM was defined as the youngest age at which the patient’s cardiac

parameters met the definition for cardiomyopathy according to our criteria, and for those

unaffected with DCM, “age” was the oldest age at which the patient’s cardiac status was

considered normal according to the same standards. We believe that the median age of cardiac involvement for each of the patient groups reported in this study is useful in revealing differences among these groups and is the best approximation available for this patient population.

Nursing Implications

DCM development is part of the BMD disease course. Due to lack of evidence, clinicians cannot define the patient’s risk of developing DCM based on the genetic diagnosis. Consequently, diagnosis and treatment of DCM in these populations often does not begin until cardiac symptoms manifest, leading to poor prognosis and premature death. In this study, we performed a genotype-phenotype analysis to identify dystrophin mutations predictive of the timing for DCM development in patients with BMD or

XLDCM. The results demonstrated that genotype is a determinant of the age for DCM development in BMD and XLDCM patients and provided novel evidence to support the

62 association between age of cardiac phenotype and specific structural alterations of the dystrophin protein rod domain.

This new knowledge enables nurses in genetic counseling and advanced practice of cardiology or neurology to customize education for patients and their families on what to expect with the disease in the context of progressive skeletal muscle degeneration and cardiac involvement. During the process of data collection, we encountered several patients who refused to be genotyped because the genotype information would not change how their disease would be managed (L. Viollet, personal communication, 2008).

The findings of this study may help these patients justify genotyping. In addition, cardiac function monitoring may be incorporated into routine visits for appropriate patients as a standard of practice, so that cardiac treatment may be started during early stages of DCM.

For patients with no or mild skeletal muscle symptoms and seem suitable for heart transplantation, the screening and approval process necessary to place the patient on the transplant waiting list may be initiated in advance.

The disease natural history of BMD is different from that of DMD because of its wide spectrum of disease onset and severity that are difficult to foretell. Upon diagnosis of DMD, most patients and their families can anticipate wheelchair-dependency around age 12 and the end of life at the third decade. However, BMD patients cannot determine their disease course with as much certainty. Particularly, knowing that DCM is the leading cause of death but not when to expect cardiac deterioration in life can bring a tremendous amount of frustration and stress to the BMD patient and his family. Even though each patient’s disease course may have individual differences, the findings in this study on DCM age of onset based on dystrophin genotype can enable the practitioner to

63 offer the patient and family a general picture of the patient’s cardiac disease progression and prognosis when consulting with patients and families.

Future Research Directions

One direction for future clinical research is to begin longitudinal cardiac monitoring in a large group of BMD patients with detailed genotype data. This will help refine the true age of DCM onset associated with various dystrophin domains identified in this study. Nursing and medical interventions may consequently be designed to prevent or delay the onset and progression of DCM. Because different dystrophin genotypes result in different cardiac phenotypes, individual genotypes potentially also dictate treatment responses to common medication regimen to treat DCM, such as an angiotensin-converting enzyme inhibitor. An important clinical investigation for patients with different genotypes will be to explore factors that may optimize his treatment outcome, through clinical trials testing various dosages, treatment timings, or different ways to combine multiple classes of drugs and integrate lifestyle modifications (e.g. diet, exercise regimen) with pharmaceutical therapies.

Alongside clinical research, it is imperative for laboratory studies to delineate specific mechanisms by which exon deletion mutations in the amino-terminal region of dystrophin lead to such an early onset of DCM when compared with mutations affecting other regions of the protein. Human cardiac tissue samples from explanted hearts may be a valuable source for this research. Alternatively, creating a new animal model of dystrophin-related DCM can also provide a useful in vivo approach to study the genetics and molecular pathology of this disease. Our structural predictions of dystrophin rod-

64 domain resulting from specific exon deletions as well as how they correlated with

different ages of DCM manifestation indicate that normal cardiac function requires

structural integrity of the dystrophin protein. This is an important aspect to consider

when designing mini- or micro-dystrophin constructs for gene therapy of DCM in this

patient population.

Conclusion

The dissertation research was the first to demonstrate that dystrophin genotype is a determinant for DCM age of onset in patients with BMD and XLDCM. Our novel genotype analysis approach highlighted the importance to understand how the protein structure is affected by the genotype when studying genotype-phenotype associations.

Our results identified specific dystrophin protein regions that when lost or altered due to gene mutations predispose patients to early- versus late-onset cardiomyopathy. Using this knowledge, nurses in advanced practice and/or in genetic counseling may begin early cardiac surveillance, make timely referrals and facilitate in life-planning for at-risk patients.

65

APPENDIX A

CLINICAL DESCRIPTION OF PATIENTS IN THIS STUDY AFFECTED WITH DCM

66 Group 1 Patients. ID Deleted Dx/Age* Source Clinical Cardiac Exons information information 3 2-7 XLDCM, [1] Age at Severe BMD/28 (Patient 1) consultation: congestive 33 years. cardiomyopathy. Mild EF= 25%. myopathy with preferential cardiac involvement beginning from the second decade of life. CK= 800 U/L. Biopsy showed increased central nucleation, fiber splitting and fiber size variation, carnitine deficiency. Slightly fainter dystrophin staining intensity than normal controls. Western blot: reduced dystrophin size. Brother of #251. Negative family history. 251 2-7 XLDCM, [1] (Patient Age at Severe BMD/28 2) consultation: congestive 28 years. cardiomyopathy.

67 Mild EF= 45%. myopathy with preferential cardiac involvement beginning from the second decade of life. CK= 2000 U/L. Biopsy showed mild myopathy, increased numbers of central nuclei and fiber splitting, fiber size variation. Slightly fainter dystrophin staining intensity than normal controls. Western blot: reduced dystrophin size. Brother of #3. Negative family history. ID Deleted Dx/Age* Source Clinical Cardiac Exons information information SL1032 3 BMD/23 UDP Onset of EF = 35%. skeletal Palpitations. muscle Peripheral symptoms: 2 edema. years. Age at Clinically diagnosis: 5.5 significant years. Toe arrhythmia. walking. Heart transplant

68 Wheelchair at 34 years. bound: 30 years. Vignos Scale: upper = 3 / lower = 9. ID Deleted Dx/Age* Source Clinical Cardiac Exons information information MJ03 3 BMD/13 MDC Age at Cardiac data at diagnosis: 8 13 years of age: years. Calf EF = 50%. hypertrophy. CK = 18,000 Cardiac data at U/L. 16 years of age: Ambulant at SF = 28.39%, age 14. LV diastolic Negative septal thickness: family 0.80cm, LVIDd history. = 55.3mm (1.02 z-score), LV diastolic wall thickness: 0.80cm. No global LV dysfunction, overall LV systolic wall motion is low- normal.

Cardiac data at 18 years of age: LVID = 1.2sd, EPSS = 14mm, SF = 23%, EF = 45%. SL187 3-7 BMD/22 UDP Onset of DCM diagnosis. skeletal EF = 25%, SF = muscle 8%. Heart symptoms: 1 transplant at 22 year. years. Elevated CK. Vignos Scale: upper = 1 / lower = 3.

69 Positive family history. ID Deleted Dx/Age* Source Clinical Cardiac Exons information information MJ18 3-7 BMD/29 MDC Age at Normal ECG. consultation All valves and normal, LV diagnosis: 25 global systolic years. Diffuse dysfunction and muscle hypokinesis, EF stiffness, = 35%. cramping, weakness. CK = 1200~2000 U/L. Biopsy showed mild muscle fiber atrophy with mild focal chronic inflammation. SL1077 3-11 BMD/19 UDP Symptom of EF = 17%, SF = weakness. 8%. Wheelchair bound: 17 years. Vignos Scale: NSI/NSI. Negative family history. 20 5 XLDCM/12 [2] (Patient Age at DCM diagnosis. DCM 10) consultation: LVEDd = 12 years. 58mm (Z score Presented to 5.2), SF = 16%, the EF = 30%. emergency Moderate mitral room with regurgitation. dyspnoea, diaphoresis and vomiting for one week. Neurological

70 exam showed no skeletal myopathy or abnormalities. CK = 270 U/L. Negative family history. ID Deleted Dx/Age* Source Clinical Cardiac Exons information information 157 5-9 BMD, [3] (Patient Onset of Severe DCM, XLDCM/33 4) skeletal polymorphic muscle PVCs. symptoms: 17 Symptoms of years. Age at heart failure. consultation: 33 years. Wheelchair bound. 140 6-13 XLDCM, [4] Age at Congestive heart BMD/14 consultation: failure 14 years. symptoms, two Neurological month history of exam: mild weight loss, generalized increasing muscle fatigue, and weakness, periods of slight abdominal pain muscular and vomiting atrophy with after upper a limb-girdle respiratory tract distribution. infections. Chest CK =5- X-ray showed 15ukat/l cardiomegaly. (normal < ECG showed 3.3ukat/l). tachycardia, left Normal atrial and EMG. Biopsy ventricular showed mild hypertrophy, myopathy. and strain. Negative LVEDd = 82 family mm, SF = 11%, history. considerable left atrial dilatation.

71 No coronary artery abnormalities per angiography. Patient died 1 month later. Cardiac biopsy: non-specific changes with interstitial fibrosis compatible with DCM.

Group 2 Patients. ID Deleted Dx/Age* Source Clinical Cardiac Exons information information 250 45 BMD/31 [5] Age at LVEDd = 58mm, (Patient consultation: 31 LVEDs = 47mm, EF 2) years. Mild = 47%, LV posterior progressive limb- wall = 10mm, girdle weakness, Interventricular calf septum = 12mm. pseudohypertrophy, Cardiac Biopsy: No elevated CK. interstitial fibrosis. Biopsy showed Dystrophin staining: discontinuous discontinuous dystrophin staining. (partial or intermittent surface membrane staining). 45 45 BMD/26 [6] Age at ECG showed tall R (Patient consultation: 26 wave in V1, lateral 6) years. Major Q and T abnormal. impairment of QTc: 441ms. QT/PQ motor function. = 8.6. LVEDd = Wheelchair bound. 66mm. SF = 13%. FVC = 42%. 193 45-47 BMD/22 [7] CK=265 U/L. Onset of congestive (Patient Negative family heart failure 1) history. symptoms. EF = 25%. Cardiac Biopsy: dystrophin staining

72 showed reduced with rod domain antibody, irregular with amino- and carboxyl-terminal antibodies. ID Deleted Dx/Age* Source Clinical Cardiac Exons information information 253 45-47 BMD/37 [8] Age at LVEDd = 58mm, consultation: 37 EF = 42%. years. Muscle weakness present. 110 45-47 BMD/20 [9] Age at Normal ECG. (Patient consultation: 20 Normal Holter ECG. 20) years. Clinical Normal LA volume. Severity: Mild. LV EDV = 64 Dystrophin staining mL/m2. EF = 53%. showed small LV wall motion: number of negative normal. RV EDV = fibers. Western 57 mL/m2. RVEF = blot: reduced 70%. Normal RV amount (40%) and wall motion. size (360 kDa). Positive family history. 189 45-47 BMD/35 [10] Age at ECG showed (Patient consultation: 35 LBBB. Holter ECG B27) years. Cramps, showed myalgia, polymorphic myoglobinuria, calf ventricular hypertrophy. CK = arrhythmias (Lown 378 U/L. Western grade 3). LVEDV = blot: Normal 85 mL/m2. EF = dystrophin amount 39%. LV wall but reduced size motion: anterior (380 kDa). septum akinesia. 141 45-47 BMD/17 [6] Age at ECG showed (Patient consultation: 17 incomplete RBBB, 1) years. Minor QTc: 399ms. QT/PQ impairment of = 10.7. LVEDd = motor function. 51mm. SF = 24%. FVC = 96%. 148 45-47 BMD/22 [6] Age at Normal ECG. QTc: (Patient consultation: 22 391ms. QT/PQ = 12) years. Minor 11.80. LVEDd = 73 impairment of 55mm. SF = 29%. motor function. FVC = 98%. ID Deleted Dx/Age* Source Clinical Cardiac Exons information information 149 45-47 BMD/26 [6] Age at Normal ECG. QTc: (Patient consultation: 26 402ms. QT/PQ = 14) years. Minor 18.00. LVEDd = impairment of 55mm. SF = 16%. motor function. FVC = 98%. 146 45-47 BMD/16 [6] Age at ECG showed tall R (Patient consultation: 16 wave in V1, lateral 8) years. Minor Q and R loss. QTc: impairment of 432ms. QT/PQ = motor function. 7.36. LVEDd = 48mm. SF = 26%. 138 45-47 BMD, [11] Onset of skeletal Chest X ray showed XLDCM/49 muscle symptoms: cardiomegaly. ECG 19 years. showed complete Wheelchair bound AV block, varying at 34 years. heart rate between Positive family 30–36 beats/minute. history. EF = 30%. Doppler showed mild-to- moderate mitral regurgitation.

Family: Brother diagnosed with BMD and DCM. 197 45-47 BMD, [7] CK=104-837U/L. Onset of congestive XLDCM/38 (Patient Negative family heart failure 5) history. symptoms. EF = 30%. Cardiac Biopsy: dystrophin staining showed reduced with rod domain antibody, irregular with amino- and carboxyl-terminal antibodies. 192 45-47 BMD, [12] Age at DCM diagnosis. XLDCM/29 consultation: 32 ECG showed 74 years. CK = LBBB. Marked 2780U/L. dilation of LV. Neurological LVEDd = 44mm, examination: LVEDV = 201ml, normal except for EF = 17%, moderate slight calf regurgitation, hypertrophy. thrombus in the Biopsy showed apical region of the fiber size variation LV chamber. with scattered Cardiac Biopsy: atrophic fibers and hypertrophic fiber hypertrophy, myocytes with huge internalized nuclei, and bizarre nuclei, some necrotic focal interstitial fibers and fiber fibrosis, mild splitting, moderate endocardial fibrous increase of thickening. connective tissue. Western Blot: Heart slight reduction in transplantation at dystrophin size. age 31. Positive family history. ID Deleted Dx/Age* Source Clinical Cardiac Exons information information MJ02 45-47 BMD/34 MDC EMG at 7 years EF = 30%. was normal but calf hypertrophy Family: Grandfather present. Onset of died of DCM. skeletal muscle symptoms: 22 years. Diagnosed at 32 years. Calf hypertrophy, progressive weakness and heaviness when climbing stairs. CK=1200U/L. Positive family history. MJ20 45-47 BMD/19 MDC Onset of skeletal Cardiac data at 19 muscle symptoms: years of age: DCM 10years. Diagnosed diagnosis. Cardiac at 11years. medications started.

75 Symptom of weakness. CK = Cardiac data at 35 1245 U/L. Started years of age: EF = cane use at age 19 55%. and wheelchair at 23. Partially ambulatory. Biopsy showed marked variability in muscle fiber size, 10-120 μm. Positive family history. ID Deleted Dx/Age* Source Clinical Cardiac Exons information information AH05 45-47 BMD/39 MDC Onset of skeletal Normal ECG. EF = muscle symptoms: 51%. SF = 32%. 5 years. Could not keep up with peers in terms of running. Diagnosis: 18 years. Difficulty climbing stairs and getting up from the floor, calf hypertrophy. CK = 2516 U/L. Biopsy showed fiber size variation (10-125 micrometers in diameter), marked connective tissue proliferation. Wheelchair bound by 42 years. Negative family history. 228 45-47 BMD/37 [13] Unavailable. EF = 42%, LVEDd = 58mm. Diagnosis of DCM based on WHO criteria. Negative for cardiac insufficiency based on European Society of Cardiology

76 criteria. ID Deleted Dx/Age* Source Clinical Cardiac Exons information information 198 45-48 XLDCM/39 [7] CK = 501U/L. Onset of cardiac (Patient Negative family symptoms. EF = 6) history. 18%. DCM diagnosis. Cardiac Biopsy: reduced dystrophin immunoreactivity with antibodies to the rod domain and irregular staining with antibodies to the amino- and carboxyl-terminal regions. 210 45-48 XLDCM/33 [14] Age at DCM diagnosis pre- (Patient consultation: 33 dating cardiac data 2) years. No skeletal below. muscle symptoms. CK = 754U/L. Cardiac data at 33 Positive family years of age: ECG history. showed LBBB. Interventricular septal thickness = 8mm; Posterior wall thickness = 8mm; LVEDd = 67mm; LVEDs = 52mm; SF = 22%. 212 45-48 XLDCM/57 [14] Age at DCM diagnosis pre- (Patient consultation: 57 dating cardiac data 4) years. No skeletal below. muscle symptoms. CK = 438U/L. Cardiac data at 57 Positive family years of age: ECG history. showed RBBB. Interventricular septal thickness = 9mm; Posterior wall thickness = 9mm; LVEDd = 65mm; LVEDs = 58mm; SF = 11%.

77 ID Deleted Dx/Age* Source Clinical Cardiac Exons information information 201 45-48 BMD/33 [15] Onset of skeletal SF = 11%; LVEDd muscle symptoms: = 82; 3 years. Age at Interventricular consultation: 33 septal thickness = years. 18mm; Posterior wall thickness: 18. 252 45-48 BMD/54 [8] Age at LVEDd = 67mm, consultation: 54 EF = 30%, met years. DCM criteria of WHO. Symptoms: dyspnoea, thoracic pain. 109 45-48 BMD/15 [9] Age at ECG showed (Patient consultation: 15 incomplete RBBB. 13) years. Clinical Normal Holter ECG. Severity: mild. LA volume = 40 Biopsy showed few mL/m2. LV EDV = dystrophin negative 69 mL/m2. EF = fibers. Western 51%. RVEDV = 84 blot: reduced mL/m2. RVEF = dystrophin 63%. RV wall expression (40%) motion apical and size (370 kDa). hypokinesia. Negative family history. 112 45-48 BMD/30 [9] Age at ECG showed (Patient consultation: 30 incomplete RBBB. 25) years. Clinical Holter ECG showed Severity: Moderate. isolated Biopsy showed monomorphic PVCs small number of (Lown grade 1). LA dystrophin negative volume = 29 mL/m2. fibers. Western LV EDV = 78 Blot: reduced mL/m2. EF = 48%. amount (40%) and LV wall motion: size (370 kDa). diffuse hypokinesia. Positive family RV EDV = 64 history. mL/m2. RVEF = 39%. 105 45-48 BMD/36 [9] Age at Symptomatic. ECG (Patient consultation: 36 showed LBBB. 30) years. Clinical Holter ECG showed Severity: Mild. polymorphic

78 Western Blot: ventricular normal amount but arrhythmias (Lown reduced size (380 grade 3). Positive kDa). Positive infra-Hisian block family history. (His-ventricular interval 80ms). Pacemaker implanted. LA volume = 45 mL/m2. LV EDV = 85 mL/m2. EF = 39%. LV wall motion: anterior septal akinesia. RV EDV = 69 mL/m2. RVEF = 65%. RV wall motion: septal akinesia. ID Deleted Dx/Age* Source Clinical Cardiac Exons information information 182 45-48 BMD/20 [10] Age at ECG showed (Patient consultation: 20 incomplete RBBB. A14) years. Cramps, Normal Holter ECG. myalgia, LVEDV = 69 myoglobinuria, calf mL/m2. EF = 51%. hypertrophy. CK = 28 U/L. Western blot: reduced dystrophin amount (40%) and reduced size (370 kDa). 227 45-48 BMD/54 [13] Unavailable. EF = 30%, LVEDd = 67mm. Diagnosed DCM based on WHO criteria. Diagnosed cardiac insufficiency based on European Society of Cardiology criteria. 195 45-48 BMD, [7] CK=2203U/L. Onset of congestive XLDCM/29 (Patient Negative family heart failure 3) history. symptoms. EF = 20%. Cardiac Biopsy:

79 dystrophin staining reduced with rod domain antibody, irregular with amino- and carboxyl-terminal antibodies. ID Deleted Dx/Age* Source Clinical Cardiac Exons information information 144 45-48 BMD/25 [6] Age at Normal ECG. QTc: (Patient consultation: 25 404ms. QT/PQ = 5) years. Major 8.8. LVEDd = impairment of 47mm. SF = 27%. motor function but ambulant. FVC = 77%. SL578 45-48 BMD/56 UDP Onset of skeletal EF = 47%. muscle symptoms: 29 years. Diagnosis: 35years. Symptom of weakness. Wheelchair bound at 53 years. Vignos Scale: upper = 5 / lower = 8. 161 45-48 BMD, [16] Onset of skeletal Diagnosis of XLDCM/70 (Patient muscle symptoms: congestive heart 1) 60 years. failure caused by Difficulties in DCM. Symptoms of tilling the soil with paroxysmal chest a hoe and climbing discomfort and hills and stairs. Age dyspnoea. Chest at consultation: 73 roentgenogram years. Neurological showed exam: moderate cardiomegaly and proximal muscular pulmonary atrophy and congestion weakness, (cardiothoracic ratio waddling gait, mild = 61%). ECG calf hypertrophy, showed prominent R positive Gower's wave in V1. EF = sign. Walks 27%, SF = 13.5%, independently. positive LV Computed hypokinesis,

80 tomography scan: LVEDd = 66mm, low-density areas LVEDs = 57mm. in the proximal muscles, especially in the thighs. CK = 681U/L. Positive family history. ID Deleted Dx/Age* Source Clinical Cardiac Exons information information 164 45-48 BMD, [16] Onset of skeletal DCM diagnosis. XLDCM/53 (Patient muscle symptoms: Chest 4) early 30's. roentgenogram Awkward gait. showed Started use of cane cardiomegaly, in his early 40's. bilateral pleural Wheelchair bound effusion and in his late 40s. Age pulmonary at consultation: 53 congestion. ECG years. Marked showed prominent proximal muscular Q wave in I, aVL, atrophy and V5-6. LV weakness, no hypokinesis, obvious LVEDd = 64mm, hypertrophy in the LVEDs = 60mm. EF calves. CK = = 16%, SF = 6.3%. 217U/L. Negative family history. SL49 45-48 BMD/47 UDP Onset of skeletal Cardiac data at 47 muscle symptoms: years of age: SF = 6.5 years. 28%. Diagnosed at 17 years. Myalgia, Cardiac data at 52 cramping. Vignos years of age: EF = Scale: upper = 1 / 50%. lower = 2. SL699 45-48 BMD/47 UDP Onset of skeletal EF = 50%. muscle symptoms: 10 years. Diagnosis: 22 years. Symptom of weakness. Wheelchair bound at 48 years. Vignos Scale: upper = 2 / lower = 9.

81 ID Deleted Dx/Age* Source Clinical Cardiac Exons information information SL8 45-48 BMD/27 UDP Onset of skeletal EF = 29%. muscle symptoms: 2 years. Diagnosis: 23 years. Weakness, hypertrophy, myalgia/cramping, myoglobinuria, cognitive dysfunction. Started wheelchair at 27 years. Vignos Scale: upper = 1 / lower = 5 at 40 years of age. 225 45-49 XLDCM, [17] Age: 6 years. Calf DCM diagnosis at BMD/23 hypertrophy and 23 years. Symptoms elevated CK, no of dyspnea even signs of muscle with mild physical involvement. activity. EF = 28%, Brother of #224. LVEDd = 72 mm.

Died of DCM at 29 years. 224 45-49 XLDCM, [17] Age at DCM diagnosis. BMD/19 consultation: 23 Symptom of years. No trouble in palpitations. walking. Normal Echocardiogram muscle mass except showed enlarged left for ventricle. pseudohypertrophy Cardiac biopsy on of calf and RV: mosaic pattern quadriceps of negative and muscles; normal positive dystrophin muscle strength. cardiomyocytes. Biopsy showed Faint staining with internal nuclei, amino- and mild variability in carboxyl-terminal the fiber size with antibodies. hypotrophic and hypertrophic fibers, rare splitting fibers in an otherwise

82 well preserved muscle. Dystrophin staining: normal with rod domain antibodies, faint with N-terminal antibody, almost normal with few areas of discontinuity with C-terminal antibody. CK = 520U/L. Condition unchanged at 26 years of age. Brother of #225. ID Deleted Dx/Age* Source Clinical Cardiac Exons information information 215 45-49 BMD/16 [9] Age at ECG showed (Patient consultation: 16 incomplete RBBB. 16) years. Clinical Holter ECG showed Severity: mild. isolated FVC = 109%. monomorphic PVCs Western blot: documented >30/hr reduced dystrophin (Lown grade 1). LA expression (50%) volume normal. LV and size (390 kDa). EDV= 65 mL/m2. Negative family EF = 50%. RV EDV history. = 45 mL/m2. RVEF = 51%. SL42 45-49 BMD/32 UDP Onset of skeletal EF = 25%, SF = muscle symptoms: 14%. 6 years. Diagnosis: 7 years. Symptom of weakness. Wheelchair bound at 23 years of age. Vignos Scale: upper = 2 / lower = 9. SL48 45-49 BMD/28 UDP Onset of skeletal EF = 40%. muscle symptoms: 6.5 years. Diagnosis: 6 years.

83 Calf hypertrophy, myalgia, cramping. Started wheelchair at 22.5 years. Vignos Scale: upper = 1 / lower = 2. ID Deleted Dx/Age* Source Clinical Cardiac Exons information information MJ14 45-49 BMD/36 MDC Motor and EF = 22%, dilated cognitive delay. LV, no mitral Classified as regurgitation. retarded at 33 years of age. Onset of Cardiac data at 37 skeletal muscle years of age: symptoms: 33 Congestive heart years. Frequent failure. Chest X ray falling. Very mild showed muscle cardiomegaly. involvement. CK = 1700 U/L. Began wheelchair use at 49 years. Dystrophin staining shows correct localization at membrane in all fibers. Western Blot: reduced levels and size. Positive family history. 147 46-49 BMD/26 [6] Age at ECG showed LV (Patient consultation: 26 hypertrophy. QTc: 11) years. Minor 430ms. QT/PQ = impairment of 10.00. LVEDd = motor function. 51mm. SF = 27%. FVC = 80%. 69 47 BMD, [18] Onset of skeletal DCM diagnosis. XLDCM/25 (Patient muscle symptoms: ECG showed infarct 3); 9 years. Positive pattern of LV [19] family history. posterolateral wall. (Patient 1) Family: Affected uncle died of

84 congestive heart failure at age 47 years. ID Deleted Dx/Age* Source Clinical Cardiac Exons information information 137 47 BMD, [18] Onset of skeletal Cardiac data at 23 XLDCM/23 (Patient muscle symptoms: years of age: DCM 4) 2 years. Awkward diagnosis. gait, could not run as fast as his peers. Cardiac data at 30 Diagnosed with years of age: limb-girdle Occasional muscular dystrophy nocturnal orthopnea, (incorrectly) and exertional dyspnea, DCM at age 23 and hemoptysis due years. Age at to left-sided heart consultation: 34 failure. years. Clinical status: marked proximal muscular atrophy and weakness, decreased deep tendon reflexes, waddling gait. 97 47-48 BMD/51 [20] Onset of skeletal Cardiac failure (Patient muscle symptoms: diagnosis. Death in 16) 15years. Biopsy same year. showed positive dystrophic change in skeletal muscle. Negative family history. 104 48 BMD/20 [9] Age at Normal ECG. Holter (Patient consultation: 20 ECG showed 19) years. Clinical isolated Severity: Mild. monomorphic PVCs CK= 2700 U/L. (Lown grade 1). Western blot: Normal LA volume. normal dystrophin LV EDV = 65 expression but mL/m2. EF = 53%. reduced size (370 RV EDV normal. kDa). Positive RVEF normal. family history. 152 48 BMD/16 [6] Age at ECG showed

85 (Patient consultation: 16 RBBB, left 19) years. Minor ventricular impairment of hypertrophy, lateral motor function. Q wave. QTc: FVC = 82%. 403ms. QT/PQ = 9.30. LVEDd = 52mm. SF = 29%. ID Deleted Dx/Age* Source Clinical Cardiac Exons information information 194 48 XLDCM/24 [7] CK = 63U/L. Onset of congestive (Patient Negative family heart failure 2) history. symptoms and DCM diagnosis. EF = 14%. Cardiac Biopsy: reduced dystrophin immunoreactivity with an antibody to the rod domain, and irregular staining with antibodies to the amino- and carboxyl- terminal domains. 190 48-49 BMD/48 [10] Age at ECG showed (Patient consultation: 48 LBBB. Holter ECG B28) years. Cramps, showed Lown grade myalgia, 4b. LVEDV = 229 myoglobinuria, calf mL/m2. EF = 34%. hypertrophy. CK = LV wall motion: 4700 U/L. Western diffuse hypokinesia. blot: reduced dystrophin amount (50%) and reduced size (390 kDa). 71 48-49 XLDCM/24 [21] Age at DCM diagnosis. (Patient consultation: 24 ECG showed Q 1) years. Dyspnea waves in the inferior with mild physical and posterior leads, activity, no cramps incomplete RBBB. or myalgia Holter ECG showed associated with sustained ventricular exercise. arrhythmias Neurological exam: monitoring. LVEDd no weakness or = 73 mm, EF =

86 muscle wasting or 27%. Angiography hypertrophy. CK = showed no coronary 540 -867 U/L. artery disease. Biopsy showed Cardiac biopsy: no variable intensity active myocarditis. of immunoreactivity among fibers, overall fainter than control. Positive family history. ID Deleted Dx/Age* Source Clinical Cardiac Exons information information 226 48-49 BMD, [22] Age at Cardiac data at 29 XLDCM/29 (Patient consultation: 29 years of age: 2) years. Elevated Cardiac symptoms CK. Biopsy of dyspnea. NYHA showed mild II. Marked LV variability of fiber dilation; LVEDV = size and moderate 176 mL/m2, EF = interstitial fibrosis; 26%. The patient dystrophin staining was treated with normal with high doses of carboxyl-terminal captopril. antibodies. Positive family history. Cardiac data at 30 years of age: Severe LV dilation; LVEDV = 166 mL/m2, EF = 28%. Cardiac Biopsy: Positive but faint dystrophin immunofluorescence in all cardiomyocytes. Diffuse but faint over-expression of utrophin. 153 48-49 BMD, [23] Onset of skeletal Cardiac data at 26 XLDCM/26 muscle symptoms: years of age: DCM 32 years. Biopsy diagnosis. showed increased variability of Cardiac data at 27 muscle fiber years of age:

87 diameter, rounded Cardiac transplant and abnormally for end stage DCM. shaped fibers, internalized myonuclei, slightly proliferated endomysial connective and fat tissue. Dystrophin staining patchy and irregular; reduced size by Western blot (380 kDa). Sarcoglycans and dystroglycan normally expressed. Utrophin detected in some muscle fibers. ID Deleted Dx/Age* Source Clinical Cardiac Exons information information AH11 48-49 BMD/42 MDC Onset of skeletal DCM diagnosis. EF muscle symptoms: = 15%. 30 years. Age at consultation: 42 Family: Brother had years. Muscle pain heart attack at age of and weakness. No 47 years. calf hypertrophy. Normal reflexes. Related to #MJ13. Positive family history. MJ13 48-49 BMD/37 MDC Onset of skeletal Severe chest pain, muscle symptoms without vital sign and diagnosis: 25 and rhythm changes. years. Chest pain, Negative stress test. muscle weakness, EF = 54%. LV free fatigue climbing wall thickness: upstairs, calf 0.9cm. hypertrophy. Started wheelchair use at 33 years. CK = 658 U/L. Related to # AH11. Positive

88 family history.

Group 3 Patients. ID Deleted Dx/Age* Source Clinical Cardiac Exons information information 199 45-51 BMD, [7] CK=1442U/L. Onset of XLDCM/43 (Patient Positive family congestive heart 7) history. failure symptoms. ECG: Q waves in V4-6. EF = 35%. Cardiac Biopsy: dystrophin staining reduced with rod domain antibody, irregular with amino- and carboxyl-terminal antibodies. 63 45-53 BMD/36 [20] Onset of skeletal Heart failure (Patient muscle symptoms: diagnosed. 8) 15years. Skeletal muscle fibrosis. Positive family history. 209 45-55 XLDCM/36 [24] Age at DCM diagnosis. consultation: 36 LV dilatation years. Normal (LVEDd = neurological exam. 72mm), diffuse CK = 1,347U/L. hypokinesis of the left ventricular wall motion, reduced cardiac output, EF = 16%. Chest roentgenogram showed slight cardiomegaly, bilateral pleural effusion, and pulmonary congestion. ECG showed sinus tachycardia, poor R-wave

89 progression in leads V,-V3 and flat T wave in leads I, aVL, V5 and V6. Cardiac biopsy: myocyte degeneration, irregularly shaped nuclei and interstitial fibrosis. ID Deleted Dx/Age* Source Clinical Cardiac Exons information information 248 45-55 BMD/76 [25] Onset of skeletal First cardiac (Patient muscle symptoms: consultation at 69 3) 59 years. Mild limb years of age: ECG muscle weakness. showed prominent Age of BMD R waves in V1. diagnosis: 69 years. No LV Moderate proximal hypokinesis. EF = muscular atrophy, 60%, SF = 32.1%, weakness, LVEDd = 56mm. waddling gait, no calf pseudo Second cardiac hypertrophy. consultation at 76 Clinical severity: years of age: ECG moderate. Age at showed prominent consultation: 76 R waves in V1, years. Neurological slight LV examination: hypokinesis. EF = walking requires 56%, SF = 28.9%, effort, muscular LVEDd = 44mm. atrophy had slightly progressed. Clinical severity: moderate. CK = 669 IU/L. Negative family history. 162 45-55 BMD/69 [16] Onset of skeletal No cardiac (Patient muscle symptoms: symptoms. 2) 59 years. Mild but Negative cardiac progressive failure, weakness affecting cardiomegaly on both arms and legs, chest X-ray. ECG difficulty climbing showed prominent

90 hills and stairs, R wave in V1. No difficulty lifting LV hypokinesis, luggage and LVEDd = 56mm, standing from a LVEDs = 38mm. sitting or crouching EF = 60%, SF = position. Age at 32.1%. consultation: 69 years. Neurological exam: moderate proximal muscular atrophy and weakness, especially in the lower limbs, positive Gower's sign, waddling gait, no calf hypertrophy. CK = 669 U/L. Biopsy showed fiber size variation, opaque fibers and proliferated connective tissues. Dystrophin staining: faint and discontinuous patchy pattern. Negative family history. ID Deleted Dx/Age* Source Clinical Cardiac Exons information information MJ06 45-55 BMD/63 MDC Diagnosis: 58 Cardiac data at 63 years. Positive years of age: EF = family history. 15-20%, severe mitral regurgitation.

Cardiac data at 64 years of age: ECG showed ventricular tachycardia, widened QRS complex with

91 duration about 128msec and frequent PVCs. LV and LA enlargement. ID Deleted Dx/Age* Source Clinical Cardiac Exons information information 203 48-51 XLDCM/60 [26] Age at Cardiac data at 60 consultation: 65 years of age: years. Investigated DCM diagnosis. following Severe LV dilation incidental finding with reduced EF of elevated CK in and mitral and his 5-year-old aortic grandson. Normal regurgitation. muscle strength, no Coronary trophic changes. arteriography Normal CK. showed very mild Normal EMG. atherosclerosis Biopsy was normal without significant apart from obstructive occasional small lesions. fibers and some internal nuclei. Died suddenly of Dystrophin cardiac arrest at immunostaining: age 68. normal in distribution, Family: slightly reduced in “Cardiomyopathy” amount. Western caused the death blot: reduced size of his mother and of dystrophin. two brothers in Alpha-sarcoglycan, their sixth decade. beta-sarcoglycan, gamma- sarcoglycan, spectrin and merosin showed normal distribution. 196 48-51 XLDCM/30 [7] CK = 442-515U/L. Onset of cardiac (Patient Negative family symptoms. 4) history. Congestive heart failure diagnosed. EF = 30%. Cardiac Biopsy:

92 reduced dystrophin immunoreactivity with an antibody to the rod domain, and irregular staining with antibodies to the amino- and carboxyl- terminal domains. ID Deleted Dx/Age* Source Clinical Cardiac Exons information information 211 48-52 XLDCM/43 [14] Age at DCM diagnosis (Patient consultation: 43 pre-dating cardiac 3) years. No skeletal data below. muscle symptoms. CK = 96U/L. Cardiac data at 43 Positive family years of age: ECG history. showed LBBB. Interventricular septal thickness = 9mm; Posterior wall thickness = 9mm; LVEDd = 72mm; LVEDs = 69mm; SF = 4%. 200 48-53 XLDCM/50 [7] CK below 200U/L. Onset of cardiac (Patient Negative family symptoms. 8) history. Congestive heart failure diagnosed. EF = 30%. Cardiac Biopsy: reduced dystrophin immunoreactivity with an antibody to the rod domain, and irregular staining with antibodies to the amino- and carboxyl- terminal domains.

93 ID Deleted Dx/Age* Source Clinical Cardiac Exons information information 208 49-51 XLDCM/50 [21] Age at Cardiac data at 50 (Patient consultation: 52 years of age: 2) years. No muscle Onset of atrophy or congestive heart pseudohypertrophy, failure symptoms. normal muscle strength. CK= 84 Cardiac data at 52 U/L. years of age: Dyspnoeic at rest, systolic murmur present over the mitral valve. ECG showed negative T waves in leads V5-V6. Dilated LV (LVEDd = 70mm), EF = 20%, dilated RV and both atria. Moderate mitral and tricuspidal valve regurgitation. Cardiac biopsy: significant fibrosis, separating individual cardiomyocytes in some areas, and gross variability of fiber size mainly due to hypertrophic cardiomyocytes; strong and continuous dystrophin staining with antibodies to amino-terminus, mid rod domain, and carboxyl- terminus. Slight

94 reduction in amount by Western blot. ID Deleted Dx/Age* Source Clinical Cardiac Exons information information MJ01 51-52 BMD/33 MDC Onset of skeletal Cardiac data at 33 muscle symptoms: years of age: birth. Diagnosis: 5 Congestive heart years. Clinical failure. Pacemaker findings: scoliosis implanted. since the teenage years, limited Cardiac data at 35 mobility at 29 years of age: years. Wheelchair Severe LV dilation bound at 40 years with global LV of age. dysfunction, EF = 34%.

* Age refers to the youngest age in years at which the patient fulfills our criteria for classification as affected with cardiomyopathy.

Abbreviations: AV: atrioventricular; BMD: Becker muscular dystrophy; CK: creatine kinase; Dx: diagnosis; ECG: clectrocardiogram; EDV: end diastole volume; EF: ejection fraction (left ventricle unless otherwise specified); EPSS: end-point septal separation; FVC: forced vital capacity; LA: left atrium; LBBB: left branch bundle block; LV: left ventricle; LVEDd: left ventricular end diastolic diameter; LVEDs: left ventricular end systolic diameter; MDC: muscular dystrophy clinics of Nationwide Children’s Hospital and The Ohio State University; NSI: no specific information; PVC: premature ventricular contraction; RA: right atrium; RBBB: right branch bundle block; RV: right ventricle; SF: shortening fraction; UDP: United Dystrophinopathy Project; WHO: World Health Organization.

Normal values for measured parameters: CK: 200 U/L (unless otherwise stated); EF: above 55%; EPSS: below 5 mm; FVC: 80%-120% predicted; Intraventricular Septal Thickness: below 12mm; LVEDd: below 58 mm, 2 z scores, or 2 SD; Posterior Wall Thickness: below 12mm; QTc: below 440ms; SF: above 32%; Vignos Scale: described in JAMA (1963), 184:89-96.

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96 12. Piccolo, G., Azan, G., Tonin, P., Arbustini, E., Gavazzi, A., Banfi, P., et al. (1994). Dilated cardiomyopathy requiring cardiac transplantation as initial manifestation of Xp21 becker type muscular dystrophy. Neuromuscular Disorders : NMD, 4(2), 143-146.

13. Sousa, R. C., Silva, P., Pais, F., Fortuna, A., Relvas, S., Simoes, L., et al. (1993). Dilated cardiomyopathy in a patient with becker's muscular dystrophy. A clinical case report. [Cardiomiopatia dilatada em doente com distrofia muscular de Becker. Apresentacao de um caso clinico] Revista Portuguesa De Cardiologia : Orgao Oficial Da Sociedade Portuguesa De Cardiologia = Portuguese Journal of Cardiology : An Official Journal of the Portuguese Society of Cardiology, 12(6), 563-70, 511.

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17. Politano, L., Passamano, L., Petretta, V. R., Nigro, V., Papparella, S., Nigro, G. E., et al. (1999). Familial dilated cardiomyopathy associated with the typical dystrophin BMD mutation: Report on two additional cases. Acta Myologica : Myopathies and Cardiomyopathies : Official Journal of the Mediterranean Society of Myology / Edited by the Gaetano Conte Academy for the Study of Striated Muscle Diseases, 3, 329-336.

18. Yoshida, K., Ikeda, S., Nakamura, A., Kagoshima, M., Takeda, S., Shoji, S., et al. (1993). Molecular analysis of the duchenne muscular dystrophy gene in patients with becker muscular dystrophy presenting with dilated cardiomyopathy. Muscle & Nerve, 16(11), 1161-1166.

19. Yazawa, M., Ikeda, S., Owa, M., Haruta, S., Yanagisawa, N., Tanaka, E., et al. (1987). A family of becker's progressive muscular dystrophy with severe cardiomyopathy. European Neurology, 27(1), 13-19.

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22. Fanin, M., Melacini, P., Angelini, C., & Danieli, G. A. (1999). Could utrophin rescue the myocardium of patients with dystrophin gene mutations? Journal of Molecular and Cellular Cardiology, 31(8), 1501-1508.

23. Finsterer, J., Bittner, R. E., & Grimm, M. (1999). Cardiac involvement in becker's muscular dystrophy, necessitating heart transplantation, 6 years before apparent skeletal muscle involvement. Neuromuscular Disorders : NMD, 9(8), 598-600.

24. Tasaki, N., Yoshida, K., Haruta, S. I., Kouno, H., Ichinose, H., Fujimoto, Y., et al. (2001). X-linked dilated cardiomyopathy with a large hot-spot deletion in the dystrophin gene. Internal Medicine (Tokyo, Japan), 40(12), 1215-1221.

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26. Palmucci, L., Mongini, T., Chiado-Piat, L., Doriguzzi, C., & Fubini, A. (2000). Dystrophinopathy expressing as either cardiomyopathy or becker dystrophy in the same family. Neurology, 54(2), 529-30.

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APPENDIX B

CLINICAL DESCRIPTION OF PATIENTS IN THIS STUDY UNAFFECTED WITH

DCM

99 Group 1 Patients. ID Deleted Dx/Age* Source Clinical Cardiac Exons information information 216 3-4 BMD/24 [1] Age at ECG showed (Patient 22) consultation: 24 incomplete RBBB. years. Clinical Holter ECG Severity: severe. showed isolated Myoglobinuria. monomorphic FVC = 92%. PVCs (Lown grade Positive family 1). Normal LA history. volume. LV EDV = 50 mL/m2. EF = 67%. Normal LV wall motion. Normal RV EDV. Normal RVEF. 218 3-4 BMD/35 [1] Age at Normal ECG. (Patient 29) consultation: 35 Holter ECG years. Clinical showed isolated Severity: Severe. monomorphic FVC = 75% (mild PVCs (Lown grade restrictive 1). Normal LA respiratory volume. LV EDV insufficiency). = 62 mL/ m2. EF = Positive family 61%. Normal LV history. wall motion. Normal RV EDV. Normal RVEF. Normal RV motion. 99 3-9 BMD/6 [1] Age at Normal ECG. (Patient 1) consultation: 6 Normal LA years. Clinical volume. LV EDV Severity: Mild. = 37 mL/ m2. EF = Skeletal muscle 60%. Normal LV symptoms are wall motion. RV present. CK = EDV=41 mL/ m2. 1770 U/L. RVEF = 68%. Western blot: Normal RV wall reduced motion. dystrophin expression (60%) and size (370 kDa). Negative family history.

100 AH02 5 BMD/14 MDC Onset of skeletal LVEDd = 0.5sd, muscle EF = 60%, SF = symptoms: 10 40%. years. Frequent falling, difficulty climbing stairs, calf hypertrophy, positive Gower's sign. CK = 7651 U/L. Negative family history.

Group 2 Patients. ID Deleted Dx/Age* Source Clinical Cardiac Exons information information SL188 45 BMD/15 UDP Onset of skeletal EF = 63%, SF = muscle 35%. symptoms: 3 years. Diagnosis: 6 years. Weakness, calf hypertrophy and Gower’s sign. Began wheelchair use at 12 years of age. Vignos Scale: upper = 1 / lower = 2. 217 45-47 BMD/28 [1] Age at ECG showed T (Patient 24) consultation: 28 wave changes. years. Clinical Normal Holter Severity: ECG. Normal LA Moderate. volume. LV EDV = 69 mL/ m2. EF = 55%. Normal LV wall motion. RV EDV = 66 mL/ m2. RVEF = 64%. 106 45-47 BMD/33 [1] Age at Normal ECG. (Patient 28) consultation: 33 Holter ECG years. Clinical showed isolated Severity: monomorphic Moderate. PVCs (Lown grade Biopsy showed 1). LA volume =

101 small number of 29 mL/ m2. LV dystrophin EDV = 44 mL/ m2. negative fibers. EF = 55%. Normal Western Blot: LV wall motion. reduced amount RV EDV = 52 mL/ (50%) and size m2. RVEF = 52%. (380 kDa). Positive family history. ID Deleted Dx/Age* Source Clinical Cardiac Exons information information 213 45-47 BMD/6 [1] Age at Normal ECG. (Patient 2) consultation: 6 Normal LA years. Clinical volume. LV EDV Severity: Mild. = 67 mL/ m2. EF = CK = 5000 U/L. 64%. Normal LV FVC = 56% wall motion. RV (moderate EDV = 46 mL/ m2. restrictive RVEF = 57%. respiratory Normal RV insufficiency). motion. SL382 45-47 BMD/19 UDP Onset of skeletal EF=57%, SF= muscle 32%. symptoms: 1.5 years. Diagnosis: 9 years. Toe walking, myalgia, cramping. Vignos Scale: upper = 1 / lower = 1. MJ04 45-47 BMD/17 MDC Diagnosis: 10 Echocardiography years. Elevated normal. CK. Onset of skeletal muscle symptoms: 12 years. Muscle fatigue, weakness, calf hypertrophy. MJ09 45-47 BMD/24 MDC Diagnosis: age 4 EF = 60%, SF = years. Biopsy 42.5%. showed muscle fiber size variation and necrosis. Elevated

102 CK. Negative family history.

ID Deleted Dx/Age* Source Clinical Cardiac Exons information information AH03 45-47 BMD/10 MDC Onset of skeletal EF = 55%, SF = muscle 33%. symptoms: 6 years. CK = 5650 U/L. Mild calf hypertrophy, mild lordosis, kyphosis, scoliosis, toe walking, positive Gower's sign, weakness, chronic pulmonary disease, developmental motor delay. Positive family history. AH07 45-47 BMD/9 MDC Onset of skeletal LVEDd = 0.9 sd, muscle EPSS = 2mm, SF symptoms: 5 = 40%, EF = 60%. years. Muscle fatigue, calf hypertrophy, and hip pain. CK=18137 U/L. Biopsy showed no inflammation, mild fatty replacement and mild fibrosis. Staining for dystrophin, sarcoglycans, beta-dystroglycan and laminin showed strong undisrupted sarcolemmal staining. Western Blot: several 103 dystrophin bands of reduced size. Positive family history.

ID Deleted Dx/Age* Source Clinical Cardiac Exons information information KJ03 45-47 BMD/45 MDC Onset of skeletal Echocardiography muscle normal. Stress test symptoms: 8 negative. years. Leg weakness, difficulty climbing stairs or arising from the floor. Diagnosis: 37 years. Clinical status: ambulant at age 47 years, wide-based gait with exaggerated lordosis and some waddle. Positive family history. AH10 45-48 BMD/15 MDC Diagnosis: 10 EF = 58%, SF = months of age. 32.23%. Onset of skeletal muscle symptoms: 8 years. Calf hypertrophy, muscle cramps. CK = 5 210 U/L. Positive family history. 214 45-48 BMD/10 [1] Age at ECG showed (Patient 4) consultation: 10 R/S>1. Normal years. Clinical Holter ECG. Severity: Mild. Normal LA Myoglobinuria. volume. LV EDV CK = 7300 U/L. = 50 mL/ m2. EF = FVC = 69% (mild 62%. Normal LV restrictive wall motion. RV 104 respiratory EDV = 74 mL/ m2. insufficiency). RVEF = 43%. Positive family Normal RV history. motion. ID Deleted Dx/Age* Source Clinical Cardiac Exons information information 181 45-48 BMD/20 [2] Age at ECG showed left (Patient consultation: 20 anterior fascicular A13) years. Cramps, block. Normal myalgia, Holter ECG. LV myoglobinuria, EDV = 72 mL/ m2. calf hypertrophy. EF = 63%. CK = 1774 U/L. Western blot: normal dystrophin amount but reduced size (360 kDa). 207 45-48 BMD/41 [3] Onset of skeletal ECG showed flat muscle T waves. Slight symptoms: 15 motility reduction years. Cramps. of the LV posterior Age at wall and increased consultation: 41 LV EDV. Normal years. CK = 406 LV end-diastolic U/L. No muscle pressure, EF = weakness or 58%. fatigability. Muscle hypertrophy of glutei and calf muscles. Negative family history. MJ26 45-48 BMD/29 MDC Shoulder girdle LVEDd = 0.5sd, weakness and SF = 37%, EF = difficulty running 58%, no mitral since early regurgitation. childhood. Neurological exam at 18 years: CK = 2500-3300 U/L. Biopsy: fiber size variation, atrophic 105 fibers, mild to moderate increase of internal nuclei. Western Blot: reduced dystrophin amount. ID Deleted Dx/Age* Source Clinical Cardiac Exons information information 219 45-49 BMD/50 [1] Age at ECG showed (Patient 31) consultation: 50 incomplete LBBB. years. Clinical Holter ECG Severity: Severe. showed Lown FVC = 26% grade 4b. Normal (severe restrictive LA volume. LV respiratory EDV = 74 mL/ m2. insufficiency). EF = 55%. Normal Positive family LV wall motion. history. Normal RV EDV. Normal RVEF. Normal RV motion. 103 48 BMD/13 [1] Age at ECG showed T (Patient 10) consultation: 13 wave changes and years. Clinical R/S>1. Normal Severity: Mild. Holter ECG. Skeletal muscle Normal LA symptoms are volume. LV EDV present. CK = = 49 mL/ m2. EF = 3910 U/L. 58%. RV EDV = Western blot: 55 mL/ m2. RVEF reduced = 56%. dystrophin expression (80%) and size (390 kDa). Positive family history. 184 48 BMD/24 [2] Age at ECG showed (Patient consultation: 24 R/S>1. Normal A17) years. Cramps, Holter ECG. LV myalgia, EDV = 88 mL/ m2. myoglobinuria, EF = 56%. calf hypertrophy. CK = 18 U/L. Western blot:

106 reduced dystrophin amount (60%) and reduced size (395 kDa). ID Deleted Dx/Age* Source Clinical Cardiac Exons information information 114 48 BMD/3 [4] Onset of skeletal Normal ECG. (Patients 1 muscle Normal & 2) symptoms: 3 echocardiography. years. Patients are twins. Symptoms: cramps and myalgia during routine activity. Negative Gower’s sign. EMG showed myopathic features. CK = 2655 U/L. Age at consultation: 9 years. Biopsy showed slight variability in fiber staining for dystrophin. Western blot: slightly decreased size. Negative family history. 108 48-49 BMD/14 [1] Age at ECG showed (Patient 12) consultation: 14 incomplete RBBB. years. Clinical Holter ECG Severity: Mild. showed Lown Skeletal muscle grade 1. Normal symptoms are LA volume. LV present. FVC = EDV = 77 mL/ m2. 105%. Biopsy EF = 65%. Normal showed many LV wall motion. dystrophin RV EDV =110 negative fibers. mL/ m2. RVEF = Western blot: 56%. RV wall reduced motion showed dystrophin septal akinesia.

107 expression (50%) and size (390 kDa). Positive family history.

180 48-49 BMD/18 [2] Age at ECG showed (Patient consultation: 18 incomplete RBBB. A11) years. CK = 6427 Holter ECG U/L. Cramps, showed isolated myalgia, monomorphic myoglobinuria, PVCs (Lown grade calf hypertrophy. 1). LV EDV = 86 Western blot: mL/ m2. EF = reduced amount 58%. Normal LV (50%) and size wall motion. (390 kDa).

Group 3 Patients. ID Deleted Dx/Age* Source Clinical Cardiac Exons information information 100 45-51 BMD/10 [1] Age at ECG showed (Patient 5) consultation: 10 R/S>1. Holter years. Clinical ECG showed Severity: Mild. isolated Skeletal muscle monomorphic symptoms are PVCs (Lown grade present. CK = 1). Normal LA 1616 U/L. volume. LV EDV Western blot: = 63 mL/ m2. EF = normal 69%. Normal LV dystrophin wall motion. RV expression levels EDV = 60 mL/ m2. but reduced size RVEF = 68%. (360 kDa). Normal RV wall Negative family motion. history. SL54 45-51 BMD/13 UDP Age of skeletal EF = 65%, SF = muscle symptom 34%. onset: 4 years. Diagnosis: 11 years. Myalgia, cramping. Vignos Scale: upper = 1 / lower = 1.

108 185 45-52 BMD/29 [2] Age at Normal ECG. (Patient consultation: 29 LVEDV = 53 mL/ A18) years. Cramps, m2. EF = 61%. myalgia, myoglobinuria, calf hypertrophy. CK = 2100 U/L. Western blot: normal dystrophin amount but reduced size (360 kDa). ID Deleted Dx/Age* Source Clinical Cardiac Exons information information 178 45-55 BMD/12 [2] Age at ECG showed (Patient A5) consultation: 12 R/S>1 and years. Clinical incomplete LBBB. Severity: Mild. Holter ECG Cramps, myalgia, showed isolated myoglobinuria, monomorphic calf hypertrophy. PVCs (Lown grade CK = 6000 U/L. 1). Normal LA FVC normal. volume. LV EDV Western blot: = 50 mL/ m2. EF = reduced 61%. Normal LV dystrophin wall motion. RV amount (40%) EDV = 63 mL/ m2. and reduced size RVEF = 60%. (340 kDa). Normal RV wall Negative family motion. history. 204 48-51 BMD/9 [5] Onset of skeletal Normal muscle echocardiography. symptoms: 5 years. Age at consultation: 9 years. Calf hypertrophy and mild proximal muscle weakness. CK = 3,000 U/L. Grandson of #203.

109 ID Deleted Dx/Age* Source Clinical Cardiac Exons information information AH08 49-51 BMD/5 MDC Onset of skeletal SF = 37%, EF = muscle symptoms 59%, LVEDd = and diagnosis: 4 1sd. years. CK = 551 U/L. Normal pulmonary function. Brother of #AH9. Positive family history. AH09 49-51 BMD/8 MDC Onset of skeletal SF = 42%, EF = muscle 62%, no mitral symptoms: 7 regurgitation. years. Weakness and muscle pain. CK = 1096 U/L. Brother of #AH8. Positive family history. 202 50-51 BMD/4 [6] Onset of skeletal Normal muscle echocardiography. symptoms: 2 years. Age at consultation: 4 years. CK =1300 U/L. Normal EMG. Biopsy showed rhabdomyolysis without features of muscular dystrophy. Immunolabelling for dystrophin, merosin and dysferlin were normal. Western blot: reduced size of dystrophin and slightly reduced amount of dystrophin, α and γ-sarcoglycan.

110 ID Deleted Dx/Age* Source Clinical Cardiac Exons information information SL896 50-53 BMD/4 UDP Onset of skeletal SF = 39%. muscle symptoms: 5years. Diagnosis: 6.5 years. Symptom of weakness. Vignos Scale: upper = 1 / lower = 1. Positive family history.

* Age refers to the age in years of the patient at the last reported cardiac evaluation with cardiac findings that fulfill our criteria for a classification of non-affected with cardiomyopathy.

Abbreviations: BMD: Becker muscular dystrophy; CK: creatine kinase; Dx: diagnosis; ECG: clectrocardiogram; EDV: end diastole volume; EF: ejection fraction (left ventricle unless otherwise specified); EMG: electromyography; EPSS: end-point septal separation; FVC: forced vital capacity; LA: left atrium; LBBB: left branch bundle block; LV: left ventricle; LVEDd: left ventricular end diastolic diameter; MDC: muscular dystrophy clinics of Nationwide Children’s Hospital and The Ohio State University; PVC: premature ventricular contraction; RBBB: right branch bundle block; RV: right ventricle; SF: shortening fraction; UDP: United Dystrophinopathy Project.

Normal values for measured parameters: CK: 200 U/L (unless otherwise stated); EF: above 55%; EPSS: below 5 mm; FVC: 80%-120% predicted; LVEDd: below 58 mm, 2 z scores, or 2 SD; SF: above 32%; Vignos Scale: described in JAMA (1963), 184:89-96.

Source List: 1. Melacini, P., Fanin, M., Danieli, G. A., Fasoli, G., Villanova, C., Angelini, C., et al. (1993). Cardiac involvement in becker muscular dystrophy. Journal of the American College of Cardiology, 22(7), 1927-1934.

2. Melacini, P., Fanin, M., Danieli, G. A., Villanova, C., Martinello, F., Miorin, M., et al. (1996). Myocardial involvement is very frequent among patients affected with subclinical becker's muscular dystrophy. Circulation, 94(12), 3168-3175.

3. Siciliano, G., Fanin, M., Angelini, C., Pollina, L. E., Miorin, M., Saad, F. A., et al. (1994). Prevalent cardiac involvement in dystrophin becker type mutation. Neuromuscular Disorders : NMD, 4(4), 381-386.

111

4. Ramelli, G. P., Joncourt, F., Luetschg, J., Weis, J., Tolnay, M., & Burgunder, J. M. (2006). Becker muscular dystrophy with marked divergence between clinical and molecular genetic findings: Case series. Swiss Medical Weekly : Official Journal of the Swiss Society of Infectious Diseases, the Swiss Society of Internal Medicine, the Swiss Society of Pneumology, 136(11-12), 189-193.

5. Palmucci, L., Mongini, T., Chiado-Piat, L., Doriguzzi, C., & Fubini, A. (2000). Dystrophinopathy expressing as either cardiomyopathy or becker dystrophy in the same family. Neurology, 54(2), 529-30.

6. Lesca, G., Testard, H., Streichenberger, N., Pelissier, J. F., Lestra, C., Burel, E., et al. (2007). Family study allows more optimistic prognosis and genetic counselling in a child with a deletion of exons 50-51 of the dystrophin gene. [Impact de l'etude familiale sur le pronostic et le conseil genetique chez un enfant porteur d'une deletion des exons 50-51 du gene de la dystrophine] Archives De Pediatrie : Organe Officiel De La Societe Francaise De Pediatrie, 14(3), 262-265.

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• Duchenne muscular dystrophy • Becker muscular dystrophy • X-linked DCM1, 2 • Retinal neurotransmission defect2 • Mental retardation2 • Psychiatric disturbances2 1 Can manifest without any musculoskeletal manifestation. 2 Usually present with DMD and/or BMD.

Table 1: Clinical Phenotypes Resulting From Mutations in the Dystrophin Gene

113

CONSTRUCTS CONCEPTS Genotype In-frame mutations of the dystrophin gene Theoretical Definition: result in dysfunctional or partial production of The versions of a gene for a given trait dystrophin protein. These mutations are carried by an organism (Klug, disruptions in the DNA sequence (ie. base pair Cummings & Spencer, 2006). substitution) that allow the codon reading frame to be maintained. Therefore, even though an incorrect amino acid is encoded at that mutated region, the downstream codons remain unaffected. The consequence to the protein is not as detrimental as that of frame- shift mutation.

Frame-shift mutations in the dystrophin gene result in no production of dystrophin protein in the cell. These mutations are disruptions in the DNA sequence that affect all downstream codons, in that incorrect or no amino acids can be encoded beginning from the mutation point. The end result is that a protein cannot be produced.

Exons represent the genomic DNA sequences that are retained and expressed in the final mRNA transcript (Klug, Cummings & Spencer, 2006). There are 79 exons in the dystrophin gene. Exon deletions are DNA deletion mutations that result in the removal of a section of the coding sequence. Protein Structure Dystrophin Protein Defect refers to the state Theoretical Definition: of dystrophin protein dysfunction, such as A protein is a molecule composed of reduced molecular weight or lower quantity multiple amino acids. The accurate expressed in the myofiber. conformation of its 3-dimensions relies heavily on the composing amino Dystrophin Protein Deficiency refers to the acids and is essential for normal state of dystrophin protein absence. function. The precursors of a protein are polypeptides, which are assembled Spectrin-like repeats are the structural units during the transcription process based that make up the central rod domain of the on the RNA sequence. Because RNA dystrophin protein. There are 24 of spectrin- is synthesized from a DNA template, like repeats, and their structure and the correct original DNA sequence is composition rely on the encoding exons. For crucial in the making of a protein example, exons 48 and 49 encode for the 19th (Klug, Cummings & Spencer, 2006). spectrin-like repeat, and when exon 48 deletion 114 mutation occurs, only partial portion of the 19th spectrin-like repeat can be made. The structure of spectrin-like repeats can be classified as in-phase and out-of-phase, as described earlier. Phenotype The primary clinical characteristics reflecting Theoretical Definition: dystrophin defect versus deficiency is the The observable features of an patient’s musculoskeletal symptoms. Milder organism reflecting its genetic musculoskeletal symptoms are found in BMD makeup, or the genotype (Klug, patients, presumably due to their abnormal yet Cummings & Spencer, 2006). present production of dystrophin protein. When the BMD patient’s symptoms of dystrophinopathy are predominantly cardiac pathology, the patient is classified as having X-linked DCM in this study.

Severe musculoskeletal symptoms are seen in DMD patients, 99% of whom do not express dystrophin at all in the muscle (Hoffman, 1993).

Age of DCM manifestation is the outcome phenotype for dystrophin genetic mutation. Age of DCM manifestation is not necessarily the true onset of the disease; rather, it reflects closely to the time when cardiac symptoms are noted.

Table 2: Constructs and concepts in the theoretical model.

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Non-Affected Affected Shortening ≥33% <32% Fraction Ejection Fraction ≥55% <55%

Left Vent. ≤58mm >58mm Internal Diastolic or or Dimension 2 SD/z scores 2 SD/z scores

E-Point Septal ≤5mm >5mm Separation Cardiac History None Heart block, dilated cardiomyopathy, heart failure, heart transplant

Table 3. Cardiac function categories.

116

Median Ageb Median Agea (Non- Diagnosis Source N (Cardiomyopathic) N Cardiomyopathic) N XLDCM Publication 17 30 17 - - BMD Publication 60 29.5 40 16 20 Non- BMD 33 33 19 14.5 14 publication

a Youngest age when fulfilling criteria for a diagnosis of cardiomyopathy. b Oldest age at which cardiac function was found to be within normal parameters.

Table 4: Patient numbers according to diagnosis and source, with median ages based on

cardiomyopathy categorization. For BMD patients from both sources, the median ages for the cardiomyopathic and the non-cardiomyopathic were significantly different

(p<0.001). For all cardiomyopathic patients, regardless of clinical diagnoses and sources, there was no statistically significant difference in median age. Similarly, for all non- cardiomyopathic patients, there was no statistically significant difference in median age.

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Phasing of Exon Deletion spectrin-like repeats 45 Out-of-phase 45-46 In-phase 45-47 Out-of-phase 45-48 In-phase 45-49 Out-of-phase 46-49 In-phase 47 Out-of-phase 47-48 In-phase 48 Out-of-phase 48-49 In-phase

Table 5: Phasing consequences of different exon deletion mutations.

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Median Age Median Age Group (years) Group (years) p value Vs. Group 1 22.5 0.001 Group 2 36.5 (in-phase) Vs. Group 3 46.5 0.057

Vs. Group 1 22.5 0.1 Group 2 25.5 (out-of-phase) Vs. Group 3 46.5 <0.001

Table 6: Pair-wise comparison of cardiomyopathic patient groups based on the location of the deletion mutation and the effects on spectrin-like repeat phasing. Significant p values are bolded and italicized. The Mann-Whitney U test was used for statistical comparisons. The median age of cardiac manifestations is indicated for each group.

119

1 2 3

4 5 6

Figure 1: Gower’s maneuver. DMD patients face the floor, plant their feet widely apart, raise their buttocks first, and use their hands to “walk” up the legs. Modified from Muscular Dystrophy Association (2006).

120

Figure 2: Anatomy of DCM. Left: normal heart; Right: DCM.

121

Figure 3: The architecture of dystrophin together with other proteins on the sarcolemma. Graph modified from Rodino-Klapac, Chicoine, Kaspar & Mendell (2007).

122 st s Domain

Binding ‐ : C-terminus. : C-terminus. ns as labeled (top). The fir CT gene. The colors of the exon Dystroglycan illustrates the flexibility of the the flexibility illustrates 2

#

: cysteine-rich domain; CR Domain

ein. There are three binding domai s the 79 exons of dystrophin : hinges; H Binding icted to encode. The third panel ‐ Actin : spectrin-like repeats; repeats; : spectrin-like R of the dystrophin gene and prot ure. The second panel indicate protein the exons are pred 1

#

: N-terminus; : N-terminus; N Domain

Binding ‐ Actin panel indicates the protein struct coordinate with regions of the Figure 4: Graphic representation 123 dystrophin protein.

ABNORMAL DYSTROPHIN PROTEIN

DAPC Destabilization Dysfunction of Sarcolemmal Stretch-activated Ion Channels

↑ Sarcolemmal Permeability

Release of Enzymes into Serum ↑ Entry of Extracellular Ca++

↑ Release of Ca++ from Intracellular Stores ↑ Serum CK

Ca++ Overload

Activation of Calcium-induced Proteases

Protein Degradation

Cardiomyocyte Death

Figure 5: Known Pathways through which abnormal dystrophin leads to cardiomyocyte death. DAPC: Dystrophin-associated Protein Complex; CK: creatine kinase; Ca++: calcium.

124

Figure 6: Progression from cardiomyocyte death to DCM.

125

Repeat A Repeat B

Helix 1 Helix 2 Helix 3 Helix 1 Helix 2 Helix 3 Helix 1

Folded

Helix 1 Helix 2 Helix 3 Helix 1 Helix 2 Helix 3 Helix 1 Helix 2 Helix 3 Helix 1

Repeat A Repeat B Repeat C

Figure 7: Modeling of the rod region of spectrin. Each repeat contains helices number 2, number 3 and the number 1 of the next unit. When folded into a three-dimensional structure, the linker sequence forms a flexible loop. Figure modified from Parry, Dixon and Cohen (1992).

126

Dystrophin Gene Mutations

GENOTYPE In‐frame Frame‐shift Exon mutation mutation Deletion

PROTEIN Dystrophin Dystrophin Spectrin‐like STRUCTURE Protein Defect Protein Repeats Structure Deficiency ¾In‐phase ¾Out‐of‐phase

PHENOTYPE Milder Musculoskeletal Severe Age of DCM Symptoms Musculoskeletal Manifestation ¾BMD Symptoms ¾XLDCM ¾DMD

SUBJECT SELECTION DATA ANALYSIS Main Stance (Monaco et al., 1988) (Harper et al., 2002)

Figure 8: Theoretical model guiding this dissertation study.

127

SR 19

Figure 9: Graphic representation of in-phase vs. out-of-phase dystrophin constructs. ∆17- 48 indicates deletions of exons 17-48, and ∆H2-R19 indicates deletions from Hinge 2 to spectrin-like repeat 19. Top panel is the wild type dystrophin. Numbers on the right indicate molecular weights of the coinciding dystrophin. Figure modified from Harper et al. (2002). SR: Spectrin-like repeat.

128

123

Figure 10: Patient grouping by dystrophin protein domain.

129

A B

Helix 1

Helix 2

Repeat B

Repeat A Repeat B Helix 1 Helix 2

Folded Folded

Dystrophin rod domain

Figure 11: Schematic representations of the normal structure of the spectrin-like repeats in dystrophin. A. A long α-helical segment (Helix 1) alternates with a short Helix 2 of approximately half the length of Helix 1. Variable-length linkers provide a direction-reversing turn at the end of each helix. Because the Helix 2 structures are anti-parallel to the Helix 1 structures and therefore backtrack along the rod axis, every Helix 1 overlaps its N and C terminal Helix 1 neighbors by approximately half its length. B. Representation of a five-repeat segment o f the rod domain of dystrophin based on the Cross et al. (1990) model. The structure is oriented with the N-terminal to the lower left, and the C-terminal to the upper right. The repeating motif is Helix 1 (orange-red) followed by Helix 2 (gray), with each Helix 1 protruding into the adjacent repeat motif creating an interlocking, nested structure.

130

with that specific

ources. Each horizontal (Non- publications)

s is the number of patient

, grouped by diagnoses and s

The number in parenthesi

tion for the cardiomyopathic

Figure 12: Mutation distribu bar represents an exon deletion mutation. deletion.

131

r cardiomyopathic patients by group. Figure 13: Distribution of mutations fo Figure 13: Distribution

132

46.5

29.5

18.5

22.5 19.5 9

Figure 14: Age distribution of each mutation group based on cardiac involvement.

133

Helix 1 spectrin-like repeat spectrin-like repeat Helix 2 Re-arrangement: Re-arrangement: in-phase in-phase out-of-phase out-of-phase An An B

deleted area deleted area deleted area deleted area e.g. Exons 47-48 deletion 47-48 Exons e.g. e.g. Exon 48deletion Exon e.g. e.g. Exons 45-46, 45-48 deletion 45-48 45-46, Exons e.g. A Repeat

e.g. Exons 45-47 deletion 45-47 Exons e.g. deleted area Repeat e.g. Exons 46-49, 48-49 deletion 48-49 46-49, Exons e.g. Figure 15: Distribution of mutation by group. Figure 15: Distribution

134

P = 0.002

36.5 25.5 70

60

50

40 (years)

30 Age 20

10

0 IN OUT Phasing of spectrin‐like repeats

Figure 16: Median ages for Group 2 mutation based on spectrin-like repeat phasing.

135

A

In-phase

B

Out-of-phase

C

Exon 48 deletion (Out-of-phase)

Figure 17: Predicted models of dystrophin spectrin-like repeat structure based on in- phase (A) and out-of-phase (B & C) deletions. C: A new hinge is created as a result of exon 48 deletion.

136

Actin‐Binding Domain # 1 Actin‐Binding Domain # 2 Dystroglycan‐Binding Domain

Lys18Asn 1 Thr279Ala 5(family) Asn1672Lys 1 Phe3228Leu 1

Figure 18. Distribution of dystrophin point mutations with XLDCM clinical diagnosis.

137

Figure 19. Human dystrophin 18Lysine>asparagine mutation. This mutation affects the third position (black highlighting) of a Calponin homology (CH) domain (white background). Lysine 18 is perfectly conserved in Dystrophin from fish to humans and in all known species with Utrophin (chick and mammals). Sequence flanking the CH domain is shown with gray background. Yellow highlighting shows amino acid divergence from the human sequence.

138 d on the human sequence, an evolutionarily conserved residue there is no highly similar sequence in ver, it does not appear to be a single dystrophin from fish to human shows ted below the sequence alignment using threonine 279 (brown highlighted 279 (brown threonine of two very highly conserved short of two very highly conserved , and highly charged (mappe nine mutation. This mutation is in domain is 51% conserved in zebrafish; w. We found perfect conservation of w. We found perfect conservation entirely within a supposed hinge. Howe n. The multiple sequence alignment of (secondary structure elements are predic humans. Threonine 279 is within one are serine-and proline-rich on of mutation by group. Figure x.x: Distributi positions divergent from human in yello divergent from positions in dystrophin from fish to position) Pele and Phyre programs). The full Pfam domain is any other protein. The Pfam flexible loop. Two apparent loops of the dystrophin Hinge 1 region domai sequences, both apparent beta strands top). Figure 20: Human dystrophin 279threonine>ala Figure 20: Human

139

Figure 21: Human dystrophin 1672 asparagine>lysine mutation. Asparagine 1672 (highlighted black) is conserved in all vertebrate dystrophins including the fish Tetraodon and Fugu, but not zebrafish, which has lysine. Asparagine 1672 is also widely conserved in the closely related protein Utrophin, but not in fish, which have serine at that position. The entire domain (white, with flanking sequence in gray) is very highly conserved throughout evolution. Sequence that diverges from human is highlighted yellow.

140

Figure 22: Human dystrophin 3228 phenylalanine>leucine mutation. The second EF hand domain (shown as white sequence, with flanking sequence in gray) is a dystroglycan-binding domain. It is strikingly highly conserved from fish to human (sequence divergence from human is shown in yellow). Phenylalanine 3228 is perfectly conserved in dystrophin from diverse species, including fish - except hagfish (not shown).

141

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