Original Articles. Analysis of Dystrophin Deletion Mutations Predicts Age of Cardiomyopathy Onset in Becker Muscular Dystrophy

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1 Original Articles Analysis of Dystrophin Deletion Mutations Predicts Age of Cardiomyopathy Onset in Becker Muscular Dystrophy Rita Wen Kaspar, PhD, RN; Hugh D. Allen, MD; Will C. Ray, PhD; Carlos E. Alvarez, PhD; John T. Kissel, MD; Alan Pestronk, MD; Robert B. Weiss, PhD; Kevin M. Flanigan, MD; Jerry R. Mendell, MD; Federica Montanaro, PhD Downloaded from by guest on June 30, 2018 Background Becker muscular dystrophy (BMD) and X-linked dilated cardiomyopathy often result from deletion mutations in the dystrophin gene that may lead to expression of an altered dystrophin protein in cardiac muscle. Cardiac involvement is present in 70% of BMD and all X-linked dilated cardiomyopathy cases. To date, the timing of cardiomyopathy development remains unpredictable. We analyzed 78 BMD and X-linked dilated cardiomyopathy patients with common deletion mutations predicted to alter the dystrophin protein and correlated their mutations to cardiomyopathy age of onset. This approach was chosen to connect dystrophin structure with function in the heart. Methods and Results Detailed cardiac information was collected for BMD and X-linked dilated cardiomyopathy patients with defined dystrophin gene deletion mutations. Patients were grouped based on the dystrophin protein domain affected by the deletion. Deletions affecting the amino-terminal domain are associated with early-onset dilated cardiomyopathy (DCM; mid-20s), whereas deletions removing part of the rod domain and hinge 3 have a later-onset DCM (mid-40s). Further, we modeled the effects of the most common mutations occurring in the rod domain on the overall structure of the dystrophin protein. By combining genetic and protein information, this analysis revealed a strong correlation between specific protein structural modifications and DCM age of onset. Conclusions We identified specific regions of the dystrophin gene that when mutated predispose BMD patients to early-onset DCM. In addition, we propose that some mutations lead to early-onset DCM by specific alterations in protein folding. These findings have potential implications for early intervention in the cardiac care of BMD patients and for therapeutic approaches that target the heart in dystrophinopathies. (Circ Cardiovasc Genet. 2009;2: ) Key Words: cardiomyopathy genetics risk factors muscular dystrophy dystrophin The dystrophin gene, located on the X-chromosome, is the largest known human gene (2.4 Mb, 79 exons), resulting in a high rate of spontaneous disease-causing mutations (30% of cases) with deletions forming the majority ( 60%). Dystrophin plays an essential structural role in both cardiac and skeletal muscle, protecting the sarcolemma from mechanical stresses of muscle contraction. Complete loss of dystrophin leads to Duchenne muscular dystrophy (DMD), the most common severe form of childhood muscular dystrophy, complicated by skeletal muscle degeneration and dilated cardiomyopathy (DCM). Clinical Perspective on p 551 In contrast to the well-defined clinical course of DMD, mutations that do not disrupt the reading frame can result in expression of an altered dystrophin protein, leading to a more variable clinical presentation. This includes Becker muscular dystrophy (BMD) that presents primarily with progressive skeletal muscle degeneration with variable age of onset and severity, and X-linked DCM (XLDCM) that typically has no detectable skeletal muscle signs accompanying the cardiac involvement. Although BMD is historically diagnosed based on skeletal muscle manifestations, the primary cause of death is heart failure. 1 Indeed, 70% of BMD patients develop DCM, 1 4 and a recent longitudinal study demonstrated an onset of cardiac involvement in the early teens in some BMD patients. 5 Similar to DMD and XLDCM, the severity and age of onset of cardiac involvement in BMD show no correlation to skeletal muscle involvement. 2,3 Furthermore, DCM is often Received March 25, 2009; accepted September 21, From the Center for Gene Therapy (R.W.K., J.R.M., F.M.), The Research Institute at Nationwide Children s Hospital; College of Nursing (R.W.K.), The Ohio State University; Division of Pediatric Cardiology (H.D.A.), The Ohio State University College of Medicine, Nationwide Children s Hospital, Heart Center; Battelle Center for Mathematical Medicine (W.C.R.), The Research Institute at Nationwide Children s Hospital; Biophysics Graduate Program (W.C.R.), The Ohio State University; Center for Molecular and Human Genetics (C.E.A.), The Research Institute at Nationwide Children s Hospital; Departments of Pediatrics (C.E.A., J.R.M., F.M.) and Neurology (J.T.K., J.R.M.), The Ohio State University College of Medicine, Columbus, Ohio; Department of Neurology (A.P.), Washington University, St. Louis, Mo; and Departments of Genetics (R.B.W.) and Pediatrics (K.M.F.), University of Utah School of Medicine, Salt Lake City, Utah. The online-only Data Supplement is available at Correspondence to Federica Montanaro, PhD, The Research Institute at Nationwide Children s Hospital, Center for Gene Therapy, 700 Children s Drive, WA3020, Columbus, OH montanaf@pediatrics.ohio-state.edu 2009 American Heart Association, Inc. Circ Cardiovasc Genet is available at DOI: /CIRCGENETICS

2 Kaspar et al Dystrophin Mutations Predict Cardiomyopathy Onset 545 Downloaded from by guest on June 30, 2018 diagnosed after cardiac symptoms manifest, diminishing the efficacy of cardioprotective drugs. Therefore, the identification of parameters for cardiac risk assessment before symptom manifestation bears undeniable relevance for the clinical care of BMD patients and would assist in patient stratification in clinical trials testing the efficacy of cardiac treatments. It is with this in mind that we extensively examined a large cohort of BMD and XLDCM patients with deletion mutations to explore the hypothesis that loss of specific domains of the dystrophin protein predispose to early-onset cardiomyopathy by potentially affecting protein expression levels, function, and/or structure. This hypothesis was born from previous studies exploring correlation between genotype and the presence of DCM in BMD patients 2 4 as well as reports on the association of deletions in specific dystrophin domains with severity of skeletal muscle symptoms. 6 8 Materials and Methods Patient Sources and Data Collection This is a cross-sectional retrospective study of patient information obtained from 3 major sources: (1) a database from the Muscular Dystrophy Association clinics at Nationwide Children s Hospital and The Ohio State University Medical Center; (2) the United Dystrophinopathy Project database (PI, Kevin Flanigan, Co-PIs Jerry R Mendell and Alan Pestronk); and (3) published studies. Information was obtained related to age of onset and severity of skeletal and cardiac muscle manifestations, description of cardiac evaluation and skeletal muscle biopsy, the gene mutation, family history, and serum creatine kinase. Echocardiograms at Nationwide Children s Hospital were interpreted by the same cardiologist (H.D.A.). The Institutional Review Board at Nationwide Children s Hospital and The Ohio State University approved all protocols. Inclusion and Exclusion Criteria Inclusion criteria included (1) a diagnosis of BMD or XLDCM or both, (2) cardiac evaluation, and (3) a confirmed exon deletion mutation spanning up to 11 exons. The 11 exon limit was set to exclude large deletions potentially affecting multiple functional domains of dystrophin, while including most patients who were found to have deletions affecting 1 to 8 exons. Patients with exon 45 to 55 deletions known to be associated with mild, late-onset skeletal muscle involvement 9,10 were also included. More stringent criteria potentially compromised statistical power. Exclusion criteria included (1) reported or suspected cardiac viral infections, (2) cardiac biopsy without dystrophin expression, (3) deletions restricted to noncoding regions of the dystrophin gene potentially containing ill-defined regulatory elements, (4) subjects younger than 12 years without proven family history of BMD, and (5) wheelchair-dependent patients by age 12 years carrying a diagnosis of BMD (preferably considered a severe form of dystrophinopathy 11 ). Deletions selectively affecting myocardial expression of dystrophin have been excluded from this study primarily because of low patient numbers, precluding meaningful statistical analysis. Supplemental Table I lists subjects excluded based on these criteria. Of 320 subjects initially screened for inclusion in this study, 118 satisfied the selection criteria. Definition of Cardiomyopathy and Disease Onset Cardiomyopathy was defined as follows: ejection fraction 55% or shortening fraction 32% or both. The ejection fraction cutoff agrees with previous studies based on the natural history of the disease 5,12 and with timing of cardiac drug intervention common in clinical practice for BMD patients. When available, additional parameters were considered to support cardiac dilation: E-point septal separation above 5 mm, left ventricular (LV) end-diastolic diameter above 58 mm or above 2 z scores when indexed to body surface area, or cardiomegaly consistent with cardiomyopathy by chest radiograph. Electrocardiograms were not used to define cardiomyopathy. 13 In this cross-sectional study, the time at which abnormal cardiac findings were first reported defines the onset of cardiomyopathy. The age of onset of DCM represents the youngest reported age at which cardiac parameters met the definition of cardiomyopathy. For analyses involving noncardiomyopathic patients, age corresponds to the oldest reported age at which cardiac findings were normal. Echocardiography For United Dystrophinopathy Project and Muscular Dystrophy Association clinic patients, images from 2D and Doppler ultrasound studies were evaluated by standard techniques. Measurements included LV diameter, shortening fraction (LV diastolic diameter minus LV systolic diameter divided by LV diastolic diameter), and ejection fraction (Simpsons formula applied to planimeterized diastolic and systolic LV cavity images derived from the apex view). For published cases, deviations from this methodology can be found in the original articles (supplemental Tables II and III). Patient ing Patients were categorized into 3 groups based on the affected functional domain of the dystrophin protein. 1: subjects with deletions affecting any portion of the actin-binding amino-terminal domain of dystrophin (exons 2 to 9). Hinge 3 (a specific protein sequence joining 2 segments of dystrophin that allows flexible movement accounting for intrinsic protein folding) has been implicated in skeletal muscle involvement 6 and thus served to divide BMD subjects into 2 additional groups. 2: subjects with deletions preserving hinge 3 and affecting exons 45 to 49 (spectrin repeats 17 up to 19). 3: subjects with deletions affecting exon 50 or 51 or both, removing or disrupting hinge 3. Dystrophin Protein Modeling Rod-region spectrin repeats were modeled based on the published structure of repeats 15 and 16 of chicken brain -spectrin (PDB 1U5P). 14 The structure was manually extended by replication and RMS alignment of corresponding terminal residues, using PyMol ( The structure was briefly minimized using VMD/NAMD ( to remove significant bad contacts. 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 realignment of the resulting fragments. A brief minimization using VMD/NAMD was used to correct any significant misplacement of fragment ends. Statistical Analysis Nonparametric Kruskal-Wallis test was performed for cross-group age comparisons (a priori P 0.05), followed by Mann Whitney U test post hoc comparisons among groups (Bonferroni adjustment was used to achieve overall significance of P 0.05). Mann Whitney U test was used to compare age of cardiomyopathy onset between in-phase and out-of-phase mutations in group 2 patients. For blood relatives, only 1 patient was randomly selected for inclusion in statistical analyses. Three sibling pairs were identified within cardiomyopathic patients: 224 and 225, 251 and 3, AH11 and MJ13 (supplemental Table II). Concordance in the age of cardiomyopathy onset was observed among siblings. All data were analyzed in SPSS version 15 (SPSS, Chicago).

3 546 Circ Cardiovasc Genet December 2009 Table 1. Patient Distribution According to Diagnosis, Source of Information, and Categorization as Affected or Not With DCM Cardiomyopathic Noncardiomyopathic Diagnosis Source n Median Age* N Median Age n XLDCM Publication BMD Publication BMD UDP The median age in years of the patients in each category is indicated as well as the No. of patients (n). *Youngest age when fulfilling criteria for a diagnosis of cardiomyopathy. Oldest age at which cardiac function was found to be within normal parameters. Downloaded from by guest on June 30, 2018 Results Patient Selection and Description A total of 118 BMD and XLDCM patients (supplemental Tables II and III) were enrolled. Table 1 shows the breakdown of patients based on diagnosis, source, and whether they were categorized as cardiomyopathic or noncardiomyopathic. Only subjects with cardiomyopathy (n 78) were required to test our hypothesis that the age of DCM manifestation is associated with deletion of specific dystrophin protein domains. However, we did analyze noncardiomyopathic patients for evidence of a cardioprotective effect of some deletion mutations. We found that the noncardiomyopathic BMD patients were significantly younger than cardiomyopathic BMD patients (P 0.001) and that their deletion mutations overlap with those of cardiomyopathic patients (supplemental Figure 1). This suggests that noncardiomyopathic patients were too young to manifest cardiac involvement and will require follow-up studies to further test the hypothesis under consideration. Distribution of Mutations Relative to Dystrophin Protein Structure and Diagnosis To determine whether all cardiomyopathic patients can be combined for maximum statistical power, we first tested whether the source of patient information (published versus United Dystrophinopathy Project) or the diagnosis (BMD Figure 1. Mapping of deletion mutations in cardiomyopathic patients. Dystrophin protein domains and corresponding exons are schematically represented at the top. regions are indicated below by a line. The number of patients with any given deletion mutation is indicated in parenthesis. Patients are grouped based on diagnosis and source. Dystrophin domains: N indicates amino terminus (diagonal stripes); R, spectrin repeats 1 through 24 (gray); H, hinges 1 through 4 (black); CR, cysteine-rich domain (white); CT, carboxyl terminus (vertical stripes).

4 Kaspar et al Dystrophin Mutations Predict Cardiomyopathy Onset 547 Downloaded from by guest on June 30, 2018 Figure 2. ing of deletion mutations based on the affected protein domain. A, Top: schematic representation of dystrophin protein domains and corresponding exons. Three patient groups are indicated in relation to dystrophin. Bottom: mapping of deletion mutations for cardiomyopathic patients falling within each group. Number of patients is shown in parenthesis. B, Dot plot of the age distribution of cardiomyopathic patients in each group. Bars indicate median ages. Median age of DCM onset is significantly different between each group (P 0.016, Mann Whitney U test), except group 1 and 2. versus XLDCM) influences the age of cardiomyopathy onset. No significant effect was found (P 0.9). Therefore, the United Dystrophinopathy Project patient population is comparable with published case reports with respect to age, and XLDCM patients did not differ from cardiomyopathic BMD patients in their median age of cardiac involvement. Next, we mapped the location of deletion mutations of cardiomyopathic patients to determine whether BMD and XLDCM patients differ in the affected dystrophin protein domains. The deletion mutations found in these patients clustered around 2 dystrophin protein regions: the aminoterminal domain corresponding to exons 2 to 7, and a region in the rod domain centered around hinge 3, corresponding to exons 45 to 55 (Figure 1). This distribution is in agreement with previous reports on mutation hot spots for BMD patients. 15,16 Deletions found in XLDCM patients overlapped or in some cases were identical to those reported for BMD patients. Thus, XLDCM and BMD patients do not segregate into separate groups based on deletion mutation site or age of DCM manifestations. Taken together, these results indicate that patients can be combined for statistical analyses regardless of diagnosis or source of information. Description of Patient s 1 (Figure 2A) includes 11 patients with deletions affecting exons 2 to 9 coding for the actin-binding aminoterminal domain of dystrophin. No information on dystrophin expression in the myocardium of these patients is available. 2 represents the majority of patients (67%) and involves deletions affecting exons 45 to 49 (spectrin repeats 17 to 19) that preserve hinge 3 of the dystrophin protein (Figure 2A). This group comprises 56 cardiomyopathic patients and shows the broadest age range of all 3 groups, with most patients falling between 15 and 55 years. A single outlier (161; supplemental Table II) was diagnosed at the age of 70 years with an ejection fraction of 27% suggestive of advanced disease. Cardiac biopsy information was available for 8 patients from published case reports (supplemental Table II). Dystrophin staining could be detected in the myocardium but was often fainter than in control tissues and was discontinuous along the cardiomyocyte membrane. 3 includes 11 patients with deletions between exons 45 and 55 that remove or disrupt hinge 3 (Figure 2A). Of note, none of the patients was younger than 30 years. Cardiac biopsies were available for 4 patients (supplemental Table II) and showed reduced levels of dystrophin expression with a discontinuous pattern along the cardiomyocyte membrane. Association of Deletion Mutations With DCM 1 patients had the earliest age of DCM manifestations (median: 23 years) followed by group 2 patients (median: 29.5 years; Figure 2B). 3 patients developed DCM later in the course of the disease (median: 43 years; Figure 2B). A significant difference was detected among the groups (P 0.001, Kruskal-Wallis test) and for all post hoc pairwise comparisons (P 0.016) except between groups 1 and 2 (P 0.03). Thus, BMD patients with deletions that lie within exons 45 to 55 resulting in a dystrophin protein lacking hinge 3 have a significantly later-onset DCM compared with patients with overlapping deletions that preserve hinge 3 or with mutations affecting the aminoterminal region of dystrophin. By contrast, patients with deletion mutations affecting exons 2 to 9 or exons 45 to 49 are at risk of developing DCM in their second and third decades of life, respectively.

5 548 Circ Cardiovasc Genet December 2009 Downloaded from by guest on June 30, 2018 Disruption of Spectrin Repeat Phasing Results in Early DCM Since group 2 patients showed the widest age range, we further investigated whether a second factor could be responsible for this heterogeneity. Previous studies focusing on this region have suggested a potential association of deletions of exon 48 or 49 or both with a more severe cardiomyopathy. 2,4 Subdividing group 2 patients based on the presence or absence of exon 48 or 49 or both did not distinguish 2 subpopulations with significantly different ages of DCM onset (P 0.2). One mechanism by which genotype can influence the age of DCM manifestations is by causing protein structure rearrangements that are more or less compatible with the cellular functions of dystrophin. Previous evidence in mice has shown that the phasing of the dystrophin spectrin repeats affects function in skeletal muscle to 49 code for spectrin repeats 17 (partial) to 19. Because exon boundaries do not correlate with the physical boundaries of individual spectrin repeats at the protein level, different combinations of exon deletions could affect spectrin repeat phasing. For each group 2 mutation, the amino acid sequence of dystrophin was analyzed to assess whether the deleted sequence would disrupt (out of phase) or preserve (in phase) the known spectrin repeat pattern 18 (supplemental Figure II). Subdividing group 2 patients based on phasing pattern revealed that disruption of spectrin repeat phasing is associated with significantly earlier onset of DCM (26 versus 36 years, Figure 3A). We further compared the age of DCM manifestations between all patient groups taking phasing into account (Table 2). No significant difference was detected in the age of DCM manifestations between groups 1 and 2 out-of-phase mutations. Thus, out-of-phase mutations in the rod domain and deletions in the actin-binding amino-terminal domain are both associated with early age cardiomyopathy. By contrast, group 2 in-phase mutations led to a significantly later age of DCM manifestations compared with group 1 patients but did not differ from group 3 patients (Table 2). These results indicate that the effect of deletion mutations on the phasing pattern of spectrin repeats 17 to 19 is a strong determinant of DCM age of onset. Based on available information from cardiac biopsies, both in-phase and out-of-phase mutations result in expression of a mutant dystrophin protein in cardiomyocytes (supplemental Table II) suggesting that the observed difference in age of DCM is not due to obvious differences in the level of cardiac dystrophin expression. Effects of Out-of-Phase and In-Phase Deletions on Dystrophin Structure To further investigate the mechanism by which phasing affects dystrophin function, we modeled the effects of deletion mutations on the rod domain of dystrophin for group 2 mutations. Each spectrin repeat is composed of a long -helix 1 connected to a shorter -helix 2 by a flexible linker sequence (supplemental Figure 2A). Figure 3B illustrates how the -helices of the spectrin repeats interlock with each other forming a stable yet flexible rod-shaped structure. In-phase mutations remove 1 or more interlocking units and Figure 3. Disruption of spectrin repeat phasing is associated with an earlier DCM onset and is predicted to alter overall dystrophin structure. A, Dot plot distribution of cardiomyopathic patient age versus spectrin repeat phasing. Out-of-phase mutations in 2 patients lead to a significantly earlier age of cardiac manifestations (26 years) compared with in-phase mutations (36 years; P 0.003). Bar indicates median age in years. B, Modeling of a 5-repeat segment of the rod domain of dystrophin highlighting interlocking of adjacent helix 1 regions (orange-red) stabilized by a helix 2 (gray). This arrangement results in a nested repeat structure and provides the rod domain with a relatively rigid backbone. The structure is oriented with the amino terminus to the lower left, and the carboxyl terminus to the upper right. Inset shows a diagram of the entire dystrophin protein. Gray: rod domain; red: hinges; green: amino terminus; blue: cysteine rich region and carboxyl terminus. C, Model of the out-of-phase exons 45 to 47 deletion mutation. The structural alteration reverses the direction of extension of the helix 1 and defines a new axis for the latter portion of the rod. The structure is shown in the folded position that may be energetically favored. Asterisk: deleted sequence site. D, Model of the out-of-phase exon 48 deletion. This structural alteration recapitulates the sequence pattern of hinge 3, leading to an extended rod configuration. Highlighted in green (arrow) is a short stretch of amino acids that could adopt a range of structures, varying from an induced helix as shown through an unstructured loop to a structured turn, which reverses the axis of the rod. Asterisk: deleted sequence site. simply result in a shortening of the rod-domain (supplemental Figure 2B). By contrast, out-of-phase mutations join the helices 1 and 2 together, removing the intervening linker sequence (supplemental Figure 2C). This is predicted to result in potentially dramatic changes to the rod domain that would affect the overall dystrophin structure. Based on our modeling, most out-of-phase mutations are predicted to bend the rod domain and reverse the directionality of the entire carboxyl-terminal part of dystrophin (Figure 3C). One exception is deletion of exon 48, which is predicted to introduce a new hinge adjacent to hinge 3 (Figure 3D). Such major alterations to the overall protein structure are likely to affect the function of dystrophin.

6 Kaspar et al Dystrophin Mutations Predict Cardiomyopathy Onset 549 Downloaded from by guest on June 30, 2018 Table 2. Pairwise Comparison of Cardiomyopathic Patient s Based on the Location of the Deletion Mutation and the Effects on Spectrin Repeat Phasing s n Median Age, y P 2 (in phase) vs: (out of phase) (out of phase) vs: vs: The Mann Whitney U test was used for post hoc statistical comparisons among all 4 groups. Significance was set a priori at P (Bonferroni adjustment for overall significance of P 0.05). Significant P values are italicized. The median age of cardiac manifestations is indicated for each group. Discussion An association of genotype with DCM has long been suspected but has not been clearly established. 2 4,24 In this study, we demonstrated that genotype is a determinant of the age of DCM manifestations in BMD and XLDCM patients. The large sample size (78 patients) and stringent selection criteria including only patients with small deletions (11 exons) enabled us to capture informative dystrophin protein domains. This allowed patient grouping based on dystrophin mutations affecting a single rather than multiple adjacent functional protein domains, thus increasing the statistical power of the study. We also provided novel evidence of a strong association of cardiomyopathy with specific structural alterations of the dystrophin rod domain. Previous studies reported contradictory findings on the potential link of deletions including exon 48 or 49 or both with severity and occurrence of DCM. 2 4,24 Analysis of our 56 patients in group 2 showed that DCM in this region is more sensitive to altered phasing of the spectrin repeats rather than absence or presence of any individual exon. Our analysis of the effects of specific deletions on the 3D structure of dystrophin and their correlation with cardiac phenotype highlights the importance of integrating protein structure information in genotype-phenotype studies. Our results also indicate that the rod domain of dystrophin may not be as permissive to alterations as previously thought. This study suggests that preservation of phasing delays the onset of DCM by about a decade. This is likely not due to a difference in expression of cardiac dystrophin protein because Arbustini et al 20 reported similar amount and distribution of dystrophin in cardiac biopsy samples from BMD patients with either in-phase or out-of-phase mutations. Rather, our modeling suggests that the alterations caused by out-of-phase mutations extend beyond the spectrin repeat unit and may lead to a severely altered configuration of the rod domain, ultimately affecting the entire dystrophin protein. This major structural change is likely a main determinant of early-onset DCM. Interestingly, most group 2 BMD patients have late-onset skeletal muscle symptoms and mild disease progression, irrespective of the effects of their mutation on phasing. This is in agreement with studies in dystrophin-null mdx mice expressing a minidystrophin construct that lacks the exons 45 to 49 region but has an intact hinge 3 domain. In these mice, only a partial restoration of cardiac function was achieved despite a complete rescue of the skeletal muscle pathology. 25 Thus, cardiac dystrophin may be particularly sensitive to structural disruptions of the exons 45 to 49 region compared with skeletal muscle dystrophin. The reasons for this disparity are currently unknown but highlight the importance of mapping domains of dystrophin essential for cardiac function to improve on current treatment approaches relying on exon skipping or gene replacement with mini-/microdystrophin constructs. This study provides expected median ages of onset of DCM associated with 3 distinct regions of the dystrophin protein and with specific rearrangements of its rod domain. The deletion mutations studied here are among the most frequent, rendering our findings relevant to most BMD and XLDCM patients. Of interest, within groups, XLDCM patients did not have an earlier age of cardiomyopathy compared with BMD patients. Instead, the earliest age of DCM is associated with mutations affecting the amino-terminal domain of the protein (early 20s), and out-of-phase mutations in the exons 45 to 49 region of the dystrophin rod domain (mid-20s). Although cardiac expression of dystrophin has been confirmed in several out-of-phase group 2 patients, such information is not available for group 1 patients. The best studied mutations affecting the 5 region of the dystrophin gene (including the muscle promoter, exon 1, or intronic regions that alter exon splicing ) lead to a selective lack of cardiac dystrophin. Although none of our group 1 patients had mutations affecting noncoding regions or exon 1, we cannot exclude the possibility that the early DCM onset in group 1 patients reflects a selective absence of cardiac dystrophin. Of note, the median age of XLDCM patients with lack of cardiac dystrophin (24 years for 6 independent families, supplemental Table I) is very similar to that of group 1 patients (23 years). Further studies are needed to determine the mechanism(s) by which deletion mutations in the aminoterminal region lead to earlier onset of cardiomyopathy compared with mutations affecting other regions. A greater awareness of the value of cardiac tissue sampling at the time of cardiac transplantation and the design of transgenic mdx mice mimicking human mutations could yield important information on cardiac-specific mechanisms regulating this region of dystrophin at a transcriptional and protein level. Significantly later DCM onset is associated with group 2 in-phase (mid-1930s) and group 3 mutations (mid-1940s). The cardio-protective effect of hinge 3 deletion seen in group 3 patients mirrors findings reported for skeletal muscle in both mice and humans. 6,17 However, although loss of hinge 3 delays onset of cardiomyopathy, it correlated with slower disease progression in skeletal muscle but had no effect on age of onset. 6 Because of the small number of group 3 patients with identical deletions, we could not determine whether this partially protective effect is associated with a specific structural alteration of the dystrophin protein back-

7 550 Circ Cardiovasc Genet December 2009 Downloaded from by guest on June 30, 2018 bone. Further studies are needed to explain the significant cardio-protective effect conferred by the loss of hinge 3. The median age of cardiac involvement for each patient group reported here is currently the best approximation available for this patient population. This information is valuable because cardiac involvement in BMD patients is often asymptomatic in its initial stages and can therefore be underestimated. Because genotyping has become a more common practice, the median ages reported here may prove valuable for individualized risk assessment and for timely cardiac evaluation and intervention. An important next step is to conduct a large-scale longitudinal study to further refine the age of DCM onset associated with the dystrophin domains identified here. This information underscores the importance of genotype information in the cardiac care of BMD patients and bears relevance to the design of therapies aimed at the myocardium in BMD, XLDCM, and DMD patients. Acknowledgments We acknowledge the input of the United Dystrophinopathy Project Consortium including the following individuals: Brenda Wong at Cincinnati Children s Hospital Medical Center, Richard Finkel, Carsten Bonnemann, and Livje Medne at Children s Hospital of Philadelphia, Julaine Florence and Anne Connolly, Washington University, Katherine Mathews, University of Iowa, Jacinda Sampson, Mark Bromberg, and Kathryn J. Swoboda, University of Utah, and John W. Day, University of Minnesota. We thank Dr Xiomara Rosales for her diagrams of the alignment of dystrophin exons with protein domains and Brent Yetter for assistance in the identification of patients seen at Muscular Dystrophy Association clinics who were suitable for this study. We also thank Drs Carlos Miranda, Jennifer Thomas-Ahner, and Christopher Pierson for editing assistance and for mentorship and support to R.W.K. from Dr Donna McCarthy, Professor of Nursing at The Ohio State University. We are indebted to Dr Christopher Holloman from the College of Mathematical and Physical Sciences at The Ohio State University for assistance with statistical analysis. Sources of Funding Supported by a grant from the NIH Roadmap Training Program in Clinical Research (T32-RR , to R.W.K.). The United Dystrophinopathy Project is supported by grants from the National Institute of Neurological Diseases and Stroke (R01 NS043264) and the National Center for Research Resources (M01-RR00064, to the University of Utah, Dr L. Betz, P.I.). None. Disclosures References 1. Bushby K, Muntoni F, Bourke JP. 107th ENMC international workshop: the management of cardiac involvement in muscular dystrophy and myotonic dystrophy. 7th-9th June 2002, Naarden, the Netherlands. 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Eur Neurol. 1999;42: Nakamura A, Yoshida K, Fukushima K, Ueda H, Urasawa N, Koyama J, Yazaki Y, Yazaki M, Sakai T, Haruta S, Takeda S, Ikeda S. Follow-up of three patients with a large in-frame deletion of exons in the Duchenne muscular dystrophy (DMD) gene. J Clin Neurosci. 2008;15: Brooke MH, Fenichel GM, Griggs RC, Mendell JR, Moxley R, Miller JP, Province MA. Clinical investigation in Duchenne dystrophy. II. Determination of the power of therapeutic trials based on the natural history. Muscle Nerve. 1983;6: Duboc D, Meune C, Lerebours G, Devaux JY, Vaksmann G, Becane HM. Effect of perindopril on the onset and progression of left ventricular dysfunction in Duchenne muscular dystrophy. J Am Coll Cardiol. 2005; 45: Thrush PT, Allen HD, Viollet L, Mendell JR. Re-examination of the electrocardiogram in boys with Duchenne muscular dystrophy and correlation with its dilated cardiomyopathy. Am J Cardiol. 2009;103: Kusunoki H, Minasov G, Macdonald RI, Mondragon A. 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Cardiac dystrophin abnormalities in Becker muscular dystrophy assessed by endomyocardial biopsy. Am Heart J. 1995;129: Arbustini E, Diegoli M, Morbini P, Dal Bello B, Banchieri N, Pilotto A, Magani F, Grasso M, Narula J, Gavazzi A, Vigano M, Tavazzi L. Prevalence and characteristics of dystrophin defects in adult male patients with dilated cardiomyopathy. J Am Coll Cardiol. 2000;35: Politano L, Passamano L, Petretta VR, Nigro V, Papparella S, Nigro G, Santangelo L, Esposito MG, Come LI, Nigro G. Familial dilated cardiomyopathy associated with the typical dystrophin BMD mutation: report on two additional cases. Acta Myol. 1999;3: Muntoni F, Di Lenarda A, Porcu M, Sinagra G, Mateddu A, Marrosu G, Ferlini A, Cau M, Milasin J, Melis MA, Marrosu MG, Cianchetti C, Sanna A, Falaschi A, Camerini F, Giacca M, Mestroni L. Dystrophin gene abnormalities in two patients with idiopathic dilated cardiomyopathy. Heart. 1997;78: Fanin M, Melacini P, Angelini C, Danieli GA. Could utrophin rescue the myocardium of patients with dystrophin gene mutations? J Mol Cell Cardiol. 1999;31:

8 Kaspar et al Dystrophin Mutations Predict Cardiomyopathy Onset Politano L, Colonna-Romano S, Esposito MG, Nigro V, Comi LI, Passamano L, Nigro G. Genotype-phenotype correlation in patients with deletions of Duchenne/Becker gene. Acta Cardiomyologica. 1991;3: Bostick B, Yue Y, Long C, Marschalk N, Fine DM, Chen J, Duan D. Cardiac Expression of a mini-dystrophin that normalizes skeletal muscle force only partially restores heart function in aged Mdx mice. Mol Ther. 2009;17: Milasin J, Muntoni F, Severini GM, Bartoloni L, Vatta M, Krajinovic M, Mateddu A, Angelini C, Camerini F, Falaschi A, Mestroni L, Giacca M. A point mutation in the 5 splice site of the dystrophin gene first intron responsible for X-linked dilated cardiomyopathy. Hum Mol Genet. 1996;5: Muntoni F, Cau M, Ganau A, Congiu R, Arvedi G, Mateddu A, Marrosu MG, Cianchetti C, Realdi G, Cao A, Melis MA. Brief report: deletion of the dystrophin muscle-promoter region associated with X-linked dilated cardiomyopathy. N Engl J Med. 1993;329: Muntoni F, Wilson L, Marrosu G, Marrosu MG, Cianchetti C, Mestroni L, Ganau A, Dubowitz V, Sewry C. A mutation in the dystrophin gene selectively affecting dystrophin expression in the heart. J Clin Invest. 1995;96: Downloaded from by guest on June 30, 2018 CLINICAL PERSPECTIVE Exon deletions of the dystrophin gene lead to Becker muscular dystrophy and X-linked dilated cardiomyopathy. Both conditions are associated with cardiomyopathy with variable onset between the second and sixth decade of life. Better understanding of the predictive pathogenic factors influencing time of onset and severity of cardiac involvement would enable clinicians to begin early intervention, and potentially prevent premature death. In this study, insight into the evolution of cardiomyopathy was gained from analyzing a large patient population with the most prevalent exon deletions affecting discrete dystrophin protein domains. Four patient groups emerged from our study. Their expected ages of cardiomyopathy onset seem to be associated with the location of the exon deletion mutation and the effects on dystrophin protein structure. The complexity of our findings illustrates that dystrophin exon deletions must be correlated with protein structural alterations to predict outcomes. Prospective testing of these relationships potentially will empower clinicians to use genotype information to intervene more effectively in the treatment of patients with Becker muscular dystrophy or X-linked dilated cardiomyopathy. In addition, the findings pave the way for improvements on current therapeutic approaches targeting the heart in dystrophinopathies and may be valuable for patient stratification in clinical trials.

9 Analysis of Dystrophin Deletion Mutations Predicts Age of Cardiomyopathy Onset in Becker Muscular Dystrophy Rita Wen Kaspar, Hugh D. Allen, Will C. Ray, Carlos E. Alvarez, John T. Kissel, Alan Pestronk, Robert B. Weiss, Kevin M. Flanigan, Jerry R. Mendell and Federica Montanaro Downloaded from by guest on June 30, 2018 Circ Cardiovasc Genet. 2009;2: ; originally published online September 30, 2009; doi: /CIRCGENETICS Circulation: Cardiovascular Genetics is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX Copyright 2009 American Heart Association, Inc. All rights reserved. Print ISSN: X. Online ISSN: The online version of this article, along with updated information and services, is located on the World Wide Web at: Data Supplement (unedited) at: Permissions: Requests for permissions to reproduce figures, tables, or portions of articles originally published in Circulation: Cardiovascular Genetics can be obtained via RightsLink, a service of the Copyright Clearance Center, not the Editorial Office. Once the online version of the published article for which permission is being requested is located, click Request Permissions in the middle column of the Web page under Services. Further information about this process is available in the Permissions and Rights Question and Answer document. Reprints: Information about reprints can be found online at: Subscriptions: Information about subscribing to Circulation: Cardiovascular Genetics is online at:

10 SUPPLEMENTAL MATERIAL Supplemental Table 1: Patients excluded from analysis Reason for exclusion # of patients % of total Mutation other than deletion Deletion > 12 exons Insufficient cardiac information a Possible DMD b Suspected Viral Myocarditis No cardiac dystrophin expression c Other d Total a. b. c. d. Information on the patient age at the time of cardiac evaluation was missing, or, for published case reports, a qualitative description without specific echocardiography measurements was provided. These patients were diagnosed as BMD but were either wheelchair dependent in their early teens, or younger than 12 years of age, had no family history of the disease, had out-of-frame deletion mutations, and a severe skeletal muscle involvement. Among these patients, 17 had mutations affecting non-coding regions some of which were not deletions 1-7. Only one patient in this category has a deletion mutation affecting the coding region. These patients represent 8 independent families. These patients had deletions of 12 or less exons within the rod domain affecting exons 12 to 44. Because of their heterogeneity and low number they could not be included in the current analysis. Page 1 of 30

11 References: 1. Ferlini A, Galie N, Merlini L, Sewry C, Branzi A, Muntoni F. A novel Alu-like element rearranged in the dystrophin gene causes a splicing mutation in a family with X-linked dilated cardiomyopathy. Am J Hum Genet. 1998;63(2): Bies RD, Maeda M, Roberds SL, Holder E, Bohlmeyer T, Young JB, Campbell KP. A 5' dystrophin duplication mutation causes membrane deficiency of alpha-dystroglycan in a family with X-linked cardiomyopathy. J Mol Cell Cardiol. 1997;29(12): Saotome M, Yoshitomi Y, Kojima S, Kuramochi M. Dilated cardiomyopathy of Becker-type muscular dystrophy with exon 4 deletion--a case report. Angiology. 2001;52(5): Yoshida K, Ikeda S, Nakamura A, Kagoshima M, Takeda S, Shoji S, Yanagisawa N. Molecular analysis of the Duchenne muscular dystrophy gene in patients with Becker muscular dystrophy presenting with dilated cardiomyopathy. Muscle Nerve. 1993;16(11): Muntoni F, Wilson L, Marrosu G, Marrosu MG, Cianchetti C, Mestroni L, Ganau A, Dubowitz V, Sewry C. A mutation in the dystrophin gene selectively affecting dystrophin expression in the heart. J Clin Invest. 1995;96(2): Muntoni F, Cau M, Ganau A, Congiu R, Arvedi G, Mateddu A, Marrosu MG, Cianchetti C, Realdi G, Cao A, Melis MA. Brief report: deletion of the dystrophin muscle-promoter region associated with X-linked dilated cardiomyopathy. N Engl J Med. 1993;329(13): Milasin J, Muntoni F, Severini GM, Bartoloni L, Vatta M, Krajinovic M, Mateddu A, Angelini C, Camerini F, Falaschi A, Mestroni L, Giacca M. A point mutation in the 5' splice site of the dystrophin gene first intron responsible for X-linked dilated cardiomyopathy. Hum Mol Genet. 1996;5(1): Page 2 of 30

12 Supplemental Table 2: Affected patients XLDCM, BMD XLDCM, BMD [1] 1) [1] 2) Age at consultation: 33 years. Mild 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. Age at consultation: 28 years. Mild 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. SL BMD UDP Onset of skeletal muscle symptoms: 2 years. Age at diagnosis: 5.5 years. Toe walking. Wheelchair dependent: 30 years. Vignos Scale (34 years): upper = 3 / lower = 9. MJ03 3 BMD MDAc Age at diagnosis: 8 years. Calf hypertrophy. CK = 18,000 U/L. Ambulant at age 14. Negative family history. Page 3 of 30 Severe congestive cardiomyopathy. EF= 25%. Severe congestive cardiomyopathy. EF= 45%. EF = 35%. Palpitations. Peripheral edema. Clinically significant arrhythmia. Heart transplant at 34 years. Cardiac data at 13 years of age: EF = 50%. Cardiac data at 16 years of age: SF = 28.39%, LV diastolic septal thickness: 0.80cm, LVd = 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: LV = 1.2sd, EPSS = 14mm, SF = 23%, EF = 45%

13 SL BMD UDP Onset of skeletal muscle symptoms: 1 year. Elevated CK. Vignos Scale (24 years): upper = 1 / lower = 3. Positive family history. MJ BMD MDAc Age at consultation and diagnosis: 25 years. Diffuse muscle stiffness, cramping, weakness. CK = 1200~2000 U/L. Biopsy showed mild muscle fiber atrophy with mild focal chronic inflammation. SL BMD MDAc Onset of skeletal muscle symptoms: 3 years. Diagnosis: 10years. Wheelchair dependent: 25.5 years. Negative family history. SL BMD UDP Symptom of weakness. Wheelchair dependent: 17 years. Negative family history XLDCM [2] DCM 10) BMD, XLDCM XLDCM, BMD [3] 4) Age at consultation: 12 years. Presented to the emergency room with dyspnoea, diaphoresis and vomiting for one week. Neurological exam showed no skeletal myopathy or abnormalities. CK = 270 U/L. Negative family history. Onset of skeletal muscle symptoms: 17 years. Age at consultation: 33 years. Wheelchair dependent. [4] Age at consultation: 14 years. Neurological exam: mild generalized muscle weakness, slight muscular atrophy with a limb-girdle distribution. CK =5-15ukat/l (normal <3.3ukat/l). Normal EMG. Biopsy showed mild myopathy. Negative family history. DCM diagnosis. EF = 25%, SF = 8%. Heart transplant at 22 years. Normal ECG. All valves normal, LV global systolic dysfunction and hypokinesis, EF = 35% EF <20%; SF = 22% EF = 17%, SF = 8% DCM diagnosis. LVEDd = 58mm (Z score 5.2), SF = 16%, EF = 30%. Moderate mitral regurgitation. Severe DCM, polymorphic PVCs. Symptoms of heart failure. Congestive heart failure symptoms. Cardiomegaly by chest X-ray. ECG: tachycardia, left atrial and ventricular hypertrophy, and strain. LVEDd = 82 mm, SF = 11%, considerable left atrial dilatation. No coronary artery abnormalities per angiography. Patient died 1 month later. Cardiac biopsy: nonspecific changes with interstitial fibrosis compatible with DCM Page 4 of 30

14 BMD [5] 2) BMD [6] 6) BMD [7] 1) Age at consultation: 31 years. Mild progressive limb-girdle weakness, calf pseudohypertrophy, elevated CK. Biopsy showed discontinuous dystrophin staining. Age at consultation: 26 years. Major impairment of motor function. Wheelchair dependent. FVC = 42%. CK=265 U/L. Negative family history BMD [8] Age at consultation: 37 years. Muscle weakness present BMD [9] 20) BMD [10] B27) BMD [6] 1) BMD [6] 12) BMD [6] 14) Age at consultation: 20 years. Clinical Severity: Mild. Dystrophin staining showed small number of negative fibers. Western blot: reduced amount (40%) and size (360 kda). Positive family history. Age at consultation: 35 years. Cramps, myalgia, myoglobinuria, calf hypertrophy. CK = 378 U/L. Western blot: Normal dystrophin amount but reduced size (380 kda). Age at consultation: 17 years. Minor impairment of motor function. FVC = 96%. Age at consultation: 22 years. Minor impairment of motor function. FVC = 98%. Age at consultation: 26 years. Minor impairment of motor function. FVC = 98%. LVEDd = 58mm, LVEDs = 47mm, EF = 47%, LV posterior wall = 10mm, Interventricular septum = 12mm. Cardiac Biopsy: No interstitial fibrosis. Dystrophin staining: discontinuous (partial or intermittent surface membrane staining) ECG showed tall R wave in V1, lateral Q 26 2 and T abnormal. QTc: 441ms. QT/PQ = 8.6. LVEDd = 66mm. SF = 13%. Onset of congestive heart failure 22 2 symptoms. EF = 25%. Cardiac Biopsy: dystrophin staining showed reduced with rod domain antibody, irregular with aminoand carboxyl-terminal antibodies. LVEDd = 58mm, EF = 42% Normal ECG. Normal Holter ECG. Normal LA volume. LV EDV = 64 ml/m 2. EF = 53%. LV wall motion: normal. RV EDV = 57 ml/m 2. RVEF = 70%. Normal RV wall motion. ECG showed LBBB. Holter ECG showed polymorphic ventricular arrhythmias (Lown grade 3). LVEDV = 85 ml/m 2. EF = 39%. LV wall motion: anterior septum akinesia. ECG showed incomplete RBBB, QTc: 399ms. QT/PQ = LVEDd = 51mm. SF = 24%. Normal ECG. QTc: 391ms. QT/PQ = LVEDd = 55mm. SF = 29%. Normal ECG. QTc: 402ms. QT/PQ = LVEDd = 55mm. SF = 16% Page 5 of 30

15 BMD, XLDCM [11] Onset of skeletal muscle symptoms: 19 years. Wheelchair dependent at 34 years. Positive family history. Cardiomegaly by chest X ray. ECG showed complete AV block, varying heart rate between beats/minute. EF = 30%. Doppler showed mild-to-moderate mitral regurgitation SL BMD MDAc Onset of skeletal muscle symptoms: 13 years. Diagnosed: 16 years. Symptoms: weakness, myalgias, cramping. Vignos Scale (28 years): upper = 1 / lower = 1. SL BMD MDAc Onset of skeletal muscle symptoms: 11 years. Diagnosed: 22 years. Symptoms: weakness, myalgias, cramping. Vignos Scale ( BMD, XLDCM BMD, XLDCM [7] 5) years): upper = 1 / lower = 1. CK= U/L. Negative family history. [12] Age at consultation: 32 years. CK = 2780U/L. Neurological examination: normal except for slight calf hypertrophy. Biopsy showed fiber size variation with scattered atrophic fibers and fiber hypertrophy, internalized nuclei, some necrotic fibers and fiber splitting, moderate increase of connective tissue. Western Blot: slight reduction in dystrophin size. Positive family history. Family: Brother diagnosed with BMD and DCM. Echo (18 years): abnormal, low EF. Echo (24 years): EF= 20% 18 2 EF = 30% 29 2 Onset of congestive heart failure symptoms. EF = 30%. Cardiac Biopsy: dystrophin staining showed reduced with rod domain antibody, irregular with aminoand carboxyl-terminal antibodies. DCM diagnosis. ECG showed LBBB. Marked dilation of LV. LVEDd = 44mm, LVEDV = 201ml, EF = 17%, moderate regurgitation, thrombus in the apical region of the LV chamber. Cardiac Biopsy: hypertrophic myocytes with huge and bizarre nuclei, focal interstitial fibrosis, mild endocardial fibrous thickening MJ BMD MDAc EMG at 7 years was normal but calf hypertrophy present. Onset of skeletal muscle symptoms: 22 years. Diagnosed at 32 years. Calf hypertrophy, progressive weakness and heaviness when climbing stairs. CK=1200U/L. Positive family history. Page 6 of 30 Heart transplantation at age 31. EF = 30%. Family: Grandfather died of DCM. 34 2

16 MJ BMD MDAc Onset of skeletal muscle symptoms: 10years. Diagnosed at 11years. Symptom of weakness. CK = 1245 U/L. Started cane use at age 19 and wheelchair at 23. Partially ambulatory. Biopsy showed marked variability in muscle fiber size, μm. Positive family history. AH BMD MDAc Onset of skeletal muscle symptoms: 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 ( micrometers in diameter), marked connective tissue proliferation. Wheelchair dependent by Cardiac data at 19 years of age: DCM diagnosis. Cardiac medications started. Cardiac data at 35 years of age: EF = 55% Normal ECG. EF = 51%. SF = 32% years. Negative family history BMD [13] Unavailable. EF = 42%, LVEDd = 58mm. Diagnosis of DCM based on WHO criteria. Negative for cardiac insufficiency based on European Society of Cardiology criteria XLDCM [7] 6) XLDCM [14] 2) CK = 501U/L. Negative family history. Onset of cardiac symptoms. EF = 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. Age at consultation: 33 years. No skeletal muscle symptoms. CK = 754U/L. Positive family history. DCM diagnosis pre-dating cardiac data below. Cardiac data at 33 years of age: ECG showed LBBB. Interventricular septal thickness = 8mm; Posterior wall thickness = 8mm; LVEDd = 67mm; LVEDs = 52mm; SF = 22% Page 7 of 30

17 XLDCM [14] 4) Age at consultation: 57 years. No skeletal muscle symptoms. CK = 438U/L. Positive family history BMD [15] Onset of skeletal muscle symptoms: 3 years. Age at consultation: 33 years. DCM diagnosis pre-dating cardiac data below. Cardiac data at 57 years of age: ECG showed RBBB. Interventricular septal thickness = 9mm; Posterior wall thickness = 9mm; LVEDd = 65mm; LVEDs = 58mm; SF = 11%. SF = 11%; LVEDd = 82; Interventricular septal thickness = 18mm; Posterior wall thickness: BMD [8] Age at consultation: 54 years. LVEDd = 67mm, EF = 30%, met DCM criteria of WHO. Symptoms: dyspnoea, thoracic pain BMD [9] 13) BMD [9] 25) BMD [9] 30) Age at consultation: 15 years. Clinical Severity: mild. Biopsy showed few dystrophin negative fibers. Western blot: reduced dystrophin expression (40%) and size (370 kda). Negative family history. Age at consultation: 30 years. Clinical Severity: Moderate. Biopsy showed small number of dystrophin negative fibers. Western Blot: reduced amount (40%) and size (370 kda). Positive family history. Age at consultation: 36 years. Clinical Severity: Mild. Western Blot: normal amount but reduced size (380 kda). Positive family history. ECG showed incomplete RBBB. Normal Holter ECG. LA volume = 40 ml/m 2. LV EDV = 69 ml/m 2. EF = 51%. RVEDV = 84 ml/m 2. RVEF = 63%. RV wall motion apical hypokinesia. ECG showed incomplete RBBB. Holter ECG showed isolated monomorphic PVCs (Lown grade 1). LA volume = 29 ml/m 2. LV EDV = 78 ml/m 2. EF = 48%. LV wall motion: diffuse hypokinesia. RV EDV = 64 ml/m 2. RVEF = 39%. Symptomatic. ECG showed LBBB. Holter ECG showed polymorphic ventricular arrhythmias (Lown grade 3). Positive infra-hisian block (His-ventricular interval 80ms). Pacemaker implanted. LA volume = 45 ml/m 2. LV EDV = 85 ml/m 2. EF = 39%. LV wall motion: anterior septal akinesia. RV EDV = 69 ml/m 2. RVEF = 65%. RV wall motion: septal akinesia Page 8 of 30

18 BMD [10] A14) Age at consultation: 20 years. Cramps, myalgia, myoglobinuria, calf hypertrophy. CK = 28 U/L. Western blot: reduced dystrophin amount (40%) and reduced size (370 kda). ECG showed incomplete RBBB. Normal Holter ECG. LVEDV = 69 ml/m 2. EF = 51% BMD [13] Unavailable. EF = 30%, LVEDd = 67mm. Diagnosed DCM based on WHO criteria. Diagnosed cardiac insufficiency based on European Society of Cardiology criteria BMD, XLDCM [7] 3) BMD [6] 5) CK=2203U/L. Negative family history. Age at consultation: 25 years. Major impairment of motor function but ambulant. FVC = 77%. SL BMD UDP Onset of skeletal muscle symptoms: 6.5 years. Diagnosed at 17 years. Myalgia, cramping. Vignos Scale (52 years): upper = 1 / lower = 2. Positive family history. SL BMD UDP Onset of skeletal muscle symptoms: 29 years. Diagnosis: 35years. Symptom of weakness. Wheelchair dependent at 53 years. Vignos Scale (60 years): upper = 5 / lower = BMD, XLDCM [16] 1) Onset of skeletal muscle symptoms: 60 years. Difficulties in tilling the soil with a hoe and climbing hills and stairs. Age at consultation: 73 years. Neurological exam: moderate proximal muscular atrophy and weakness, waddling gait, mild calf hypertrophy, positive Gower's sign. Walks independently. Computed tomography scan: low-density areas in the proximal muscles, especially in the thighs. CK = 681U/L. Positive family history. Onset of congestive heart failure symptoms. EF = 20%. Cardiac Biopsy: dystrophin staining reduced with rod domain antibody, irregular with aminoand carboxyl-terminal antibodies. Normal ECG. QTc: 404ms. QT/PQ = 8.8. LVEDd = 47mm. SF = 27%. Cardiac data at 47 years of age: SF = 28% Cardiac data at 52 years of age: EF = 50%. EF = 47% Diagnosis of congestive heart failure caused by DCM. Symptoms of paroxysmal chest discomfort and dyspnoea. Chest roentgenogram showed cardiomegaly and pulmonary congestion (cardiothoracic ratio = 61%). ECG showed prominent R wave in V1. EF = 27%, SF = 13.5%, positive LV hypokinesis, LVEDd = 66mm, LVEDs = 57mm Page 9 of 30

19 BMD, XLDCM [16] 4) Onset of skeletal muscle symptoms: early 30's. Awkward gait. Started use of cane in his early 40's. Wheelchair dependent in his late 40s. Age at consultation: 53 years. Marked proximal muscular atrophy and weakness, no obvious hypertrophy in the calves. CK = 217U/L. Negative family history. SL BMD UDP Onset of skeletal muscle symptoms: 10 years. Diagnosis: 22 years. Symptom of weakness. Wheelchair dependent at 48 years. Vignos Scale (48 years): upper = 2 / lower = 9. SL BMD UDP Onset of skeletal muscle symptoms: 2 years. Diagnosis: 23 years. Weakness, hypertrophy, myalgia/cramping, myoglobinuria, cognitive dysfunction. Vignos Scale (40 years): upper = 1 / lower = 5. Negative family history. SL BMD UDP Onset of skeletal muscle symptoms: 6 years. Diagnosis: 7 years. Symptom of weakness. Wheelchair dependent at 23 years of age. Vignos Scale (32 years): upper = 2 / lower = 9. Negative family history XLDCM, BMD [17] Age at consultation: 23 years. No trouble in walking. Normal muscle mass except for pseudohypertrophy of calf and quadriceps muscles; normal muscle strength. Biopsy showed internal nuclei, mild variability in the fiber size with hypotrophic and hypertrophic fibers, rare splitting fibers in an otherwise 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. DCM diagnosis. Chest roentgenogram showed cardiomegaly, bilateral pleural effusion and pulmonary congestion. ECG showed prominent Q wave in I, avl, V5-6. LV hypokinesis, LVEDd = 64mm, LVEDs = 60mm. EF = 16%, SF = 6.3% EF = 50% EF = 29% EF = 25%, SF = 14% DCM diagnosis. Symptom of palpitations. Echocardiogram showed enlarged left ventricle. Cardiac biopsy on RV: mosaic pattern of negative and positive dystrophin cardiomyocytes. Faint staining with amino- and carboxyl-terminal antibodies Page 10 of 30

20 XLDCM, BMD [17] Age: 6 years. Calf hypertrophy and elevated CK, no signs of muscle involvement. Brother of #224. DCM diagnosis at 23 years. Symptoms of dyspnea even with mild physical activity. EF = 28%, LVEDd = 72 mm BMD [9] 16) Age at consultation: 16 years. Clinical Severity: mild. FVC = 109%. Western blot: reduced dystrophin expression (50%) and size (390 kda). Negative family history. MJ BMD MDAc Motor and cognitive delay. Classified as retarded at 33 years of age. Onset of skeletal muscle symptoms: 33 years. Frequent falling. Very mild muscle involvement. CK = 1700 U/L. Dystrophin staining shows correct localization at membrane in all fibers. Western Blot: reduced levels and size. Positive family history. SL BMD UDP Onset of skeletal muscle symptoms: 6.5 years. Diagnosis: 6 years. Calf hypertrophy, myalgia, cramping. Vignos Scale (31 years): BMD [6] 11) BMD, XLDCM [18] 4) upper = 1 / lower = 2. Age at consultation: 26 years. Minor impairment of motor function. FVC = 80%. Onset of skeletal muscle symptoms: 2 years. Awkward gait, could not run as fast as his peers. Diagnosed with limb-girdle muscular dystrophy (incorrectly) and DCM at age 23 years. Age at consultation: 34 years. Clinical status: marked proximal muscular atrophy and weakness, decreased deep tendon reflexes, and waddling gait. Died of DCM at 29 years. ECG showed incomplete RBBB. Holter ECG showed isolated monomorphic PVCs documented >30/hr (Lown grade 1). LA volume normal. LV EDV= 65 ml/m 2. EF = 50%. RV EDV = 45 ml/m 2. RVEF = 51%. EF = 22%, dilated LV, no mitral regurgitation. Cardiac data at 37 years of age: Congestive heart failure. Chest X ray showed cardiomegaly EF = 40% ECG showed LV hypertrophy. QTc: 430ms. QT/PQ = LVEDd = 51mm. SF = 27%. Cardiac data at 23 years of age: DCM diagnosis. Cardiac data at 30 years of age: Occasional nocturnal orthopnea, exertional dyspnea, and hemoptysis due to left-sided heart failure Page 11 of 30

21 69 47 BMD, XLDCM [18] 3); [19] 1) BMD [20] 16) BMD [9] 19) BMD [6] 19) XLDCM [7] 2) BMD [10] B28) XLDCM [21] 1) Onset of skeletal muscle symptoms: 9 years. Positive family history. Onset of skeletal muscle symptoms: 15years. Biopsy showed positive dystrophic change in skeletal muscle. Negative family history. Age at consultation: 20 years. Clinical Severity: Mild. CK= 2700 U/L. Western blot: normal dystrophin expression but reduced size (370 kda). Positive family history. Age at consultation: 16 years. Minor impairment of motor function. FVC = 82%. CK = 63U/L. Negative family history. Age at consultation: 48 years. Cramps, myalgia, myoglobinuria, calf hypertrophy. CK = 4700 U/L. Western blot: reduced dystrophin amount (50%) and reduced size (390 kda). Age at consultation: 24 years. Dyspnea with mild physical activity, no cramps or myalgia associated with exercise. Neurological exam: no weakness or muscle wasting or hypertrophy. CK = U/L. Biopsy showed variable intensity of immunoreactivity among fibers, overall fainter than control. Positive family history. Page 12 of 30 DCM diagnosis. ECG showed infarct pattern of LV posterolateral wall. Family: Affected uncle died of congestive heart failure at age 47 years. Cardiac failure diagnosis. Death in same year. Normal ECG. Holter ECG showed isolated monomorphic PVCs (Lown grade 1). Normal LA volume. LV EDV = 65 ml/m 2. EF = 53%. RV EDV normal. RVEF normal. ECG showed RBBB, left ventricular hypertrophy, lateral Q wave. QTc: 403ms. QT/PQ = LVEDd = 52mm. SF = 29%. Onset of congestive heart failure 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 carboxylterminal domains. ECG showed LBBB. Holter ECG showed Lown grade 4b. LVEDV = 229 ml/m 2. EF = 34%. LV wall motion: diffuse hypokinesia. DCM diagnosis. ECG showed Q waves in the inferior and posterior leads, incomplete RBBB. Holter ECG showed sustained ventricular arrhythmias monitoring. LVEDd = 73 mm, EF = 27%. Angiography showed no coronary artery disease. Cardiac biopsy: no active myocarditis

22 BMD, XLDCM [22] 2) Age at consultation: 29 years. Elevated CK. Biopsy showed mild variability of fiber size and moderate interstitial fibrosis; dystrophin staining normal with carboxyl-terminal antibodies. Positive family history. Cardiac data at 29 years of age: Cardiac symptoms of dyspnea. NYHA II. Marked LV dilation; LVEDV = 176 ml/m 2, EF = 26%. The patient was treated with high doses of captopril BMD, XLDCM [23] Onset of skeletal muscle symptoms: 32 years. Biopsy showed increased variability of muscle fiber diameter, rounded and abnormally 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. AH BMD MDAc Onset of skeletal muscle symptoms: 30 years. Age at consultation: 42 years. Muscle pain and weakness. No calf hypertrophy. Normal reflexes. Related to #MJ13. Positive family history. MJ BMD MDAc Onset of skeletal muscle symptoms and diagnosis: 25 years. Chest pain, muscle weakness, fatigue climbing upstairs, calf hypertrophy. CK = 658 U/L. Related to # AH11. Positive family history BMD, XLDCM [7] 7) CK=1442U/L. Positive family history. Page 13 of 30 Cardiac data at 30 years of age: Severe LV dilation; LVEDV = 166 ml/m 2, EF = 28%. Cardiac Biopsy: Positive but faint dystrophin immunofluorescence in all cardiomyocytes. Diffuse but faint overexpression of utrophin. Cardiac data at 26 years of age: DCM diagnosis. Cardiac data at 27 years of age: Cardiac transplant for end stage DCM. DCM diagnosis. EF = 15%. Family: Brother had heart attack at age of 47 years. Severe chest pain, without vital sign and rhythm changes. Negative stress test. EF = 54%. LV free wall thickness: 0.9cm. Onset of congestive heart failure symptoms. ECG: Q waves in V4-6. EF = 35%. Cardiac Biopsy: dystrophin staining reduced with rod domain antibody, irregular with amino- and carboxylterminal antibodies

23 BMD [20] Onset of skeletal muscle symptoms: 15years. Heart failure diagnosed ) Skeletal muscle fibrosis. Positive family history XLDCM [24] Age at consultation: 36 years. Normal neurological exam. CK = 1,347U/L. DCM diagnosis. LV dilatation (LVEDd = 72mm), diffuse 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 progression in leads V,-V3 and flat T wave in leads I, avl, V5 and V6. Cardiac biopsy: myocyte degeneration, irregularly shaped nuclei BMD [25] 3) Onset of skeletal muscle symptoms: 59 years. Mild limb muscle weakness. Age of BMD diagnosis: 69 years. Moderate proximal muscular atrophy, weakness, waddling gait, no calf pseudo hypertrophy. Clinical severity: moderate. Age at consultation: 76 years. Neurological examination: walking requires effort, muscular atrophy had slightly progressed. Clinical severity: moderate. CK = 669 IU/L. Negative family history. and interstitial fibrosis. First cardiac consultation at 69 years of age: ECG showed prominent R waves in V1. No LV hypokinesis. EF = 60%, SF = 32.1%, LVEDd = 56mm. Second cardiac consultation at 76 years of age: ECG showed prominent R waves in V1, slight LV hypokinesis. EF = 56%, SF = 28.9%, LVEDd = 44mm. MJ BMD MDAc Diagnosis: 58 years. Positive family history. Cardiac data at 63 years of age: EF = 15-20%, severe mitral regurgitation. Cardiac data at 64 years of age: ECG showed ventricular tachycardia, widened QRS complex with duration about 128msec and frequent PVCs. LV and LA enlargement Page 14 of 30

24 XLDCM [7] 4) CK = U/L. Negative family history XLDCM [26] Age at consultation: 65 years. Investigated following incidental finding of elevated CK in his 5-year-old grandson. Normal muscle strength, no trophic changes. Normal CK. Normal EMG. Biopsy was normal apart from occasional small fibers and some internal nuclei. Dystrophin immunostaining: normal in distribution, slightly reduced in amount. Western blot: reduced size of dystrophin. Alpha-sarcoglycan, beta-sarcoglycan, gamma-sarcoglycan, spectrin and merosin showed normal distribution XLDCM [14] 3) XLDCM [7] 8) Age at consultation: 43 years. No skeletal muscle symptoms. CK = 96U/L. Positive family history. CK below 200U/L. Negative family history. Onset of cardiac symptoms. 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 carboxylterminal domains. Cardiac data at 60 years of age: DCM diagnosis. Severe LV dilation with reduced EF and mitral and aortic regurgitation. Coronary arteriography showed very mild atherosclerosis without significant obstructive lesions. Died suddenly of cardiac arrest at age 68. Family: Cardiomyopathy caused the death of his mother and two brothers in their sixth decade. DCM diagnosis pre-dating cardiac data below. Cardiac data at 43 years of age: ECG showed LBBB. Interventricular septal thickness = 9mm; Posterior wall thickness = 9mm; LVEDd = 72mm; LVEDs = 69mm; SF = 4%. Onset of cardiac symptoms. 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 carboxylterminal domains Page 15 of 30

25 XLDCM [21] 2) Age at consultation: 52 years. No muscle atrophy or pseudohypertrophy, normal muscle strength. CK= 84 U/L. MJ BMD MDAc Onset of skeletal muscle symptoms: birth. Diagnosis: 5 years. Clinical findings: scoliosis since the teenage years, limited mobility at 29 years. Wheelchair dependent at 40 years of age. Cardiac data at 50 years of age: Onset of congestive heart failure symptoms. Cardiac data at 52 years of age: Dyspnoeic at rest, systolic murmur present. 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, 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 reduction in amount by Western blot. Cardiac data at 33 years of age: Congestive heart failure. Pacemaker implanted. Cardiac data at 35 years of age: Severe LV dilation with global LV 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; DCM: dilated cardiomyopathy; Dx: diagnosis; ECG: electrocardiogram; EDV: end diastole volume; EF: ejection fraction (left ventricular unless otherwise specified); EPSS: E-point septal separation; FVC: forced vital capacity; LA: left atrium; LBBB: left branch bundle block; LV: left ventricle; LVEDd: left ventricular enddiastolic diameter; LVEDs: left ventricular end systolic diameter; MDAc: muscular dystrophy clinics of Nationwide Children s Hospital and The Ohio State University Medical Center; NSI: no specific information; PVC: premature ventricular contraction; RA: right atrium; Page 16 of 30

26 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 or equal to 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 or equal to 32%; Vignos Scale: described in JAMA (1963), 184: Published Sources: 1. Gold R, Kress W, Meurers B, Meng G, Reichmann H, Muller CR: Becker muscular dystrophy: detection of unusual disease courses by combined approach to dystrophin analysis. Muscle Nerve 1992, 15: Feng J, Yan J, Buzin CH, Towbin JA, Sommer SS: Mutations in the dystrophin gene are associated with sporadic dilated cardiomyopathy. Mol Genet Metab 2002, 77: Novakovic I, Bojic D, Todorovic S, Apostolski S, Lukovic L, Stefanovic D, Milasin J: Proximal dystrophin gene deletions and protein alterations in becker muscular dystrophy. Ann N Y Acad Sci 2005, 1048: Oldfors A, Eriksson BO, Kyllerman M, Martinsson T, Wahlstrom J: Dilated cardiomyopathy and the dystrophin gene: an illustrated review. Br Heart J 1994, 72: Maeda M, Nakao S, Miyazato H, Setoguchi M, Arima S, Higuchi I, Osame M, Taira A, Nomoto K, Toda H, et al.: Cardiac dystrophin abnormalities in Becker muscular dystrophy assessed by endomyocardial biopsy. Am Heart J 1995, 129: Steare SE, Dubowitz V, Benatar A: Subclinical cardiomyopathy in Becker muscular dystrophy. Br Heart J 1992, 68: Arbustini E, Diegoli M, Morbini P, Dal Bello B, Banchieri N, Pilotto A, Magani F, Grasso M, Narula J, Gavazzi A, et al.: Prevalence and characteristics of dystrophin defects in adult male patients with dilated cardiomyopathy. J Am Coll Cardiol 2000, 35: Martins E, Silva-Cardoso J, Silveira F, Nadais G, Goncalves FR: Left ventricular function in adults with muscular dystrophies: genotype-phenotype correlations. Rev Port Cardiol 2005, 24: Page 17 of 30

27 9. Melacini P, Fanin M, Danieli GA, Fasoli G, Villanova C, Angelini C, Vitiello L, Miorelli M, Buja GF, Mostacciuolo ML, et al.: Cardiac involvement in Becker muscular dystrophy. J Am Coll Cardiol 1993, 22: Melacini P, Fanin M, Danieli GA, Villanova C, Martinello F, Miorin M, Freda MP, Miorelli M, Mostacciuolo ML, Fasoli G, et al.: Myocardial involvement is very frequent among patients affected with subclinical Becker's muscular dystrophy. Circulation 1996, 94: Akdemir R, Ozhan H, Gunduz H, Yazici M, Erbilen E, Uyan C, Imirzalioglu N: Complete atrioventricular block in Becker muscular dystrophy. N Z Med J 2004, 117:U Piccolo G, Azan G, Tonin P, Arbustini E, Gavazzi A, Banfi P, Mora M, Morandi L, Tedeschi S: Dilated cardiomyopathy requiring cardiac transplantation as initial manifestation of Xp21 Becker type muscular dystrophy. Neuromuscul Disord 1994, 4: Sousa RC, Silva P, Pais F, Fortuna A, Relvas S, Simoes L, Miranda O, Gama V: [Dilated cardiomyopathy in a patient with Becker's muscular dystrophy. A clinical case report]. Rev Port Cardiol 1993, 12: , Shimizu M, Ino H, Yasuda T, Fujino N, Uchiyama K, Mabuchi T, Konno T, Kaneda T, Fujita T, Masuta E, et al.: Gene mutations in adult Japanese patients with dilated cardiomyopathy. Circ J 2005, 69: Finsterer J, Stollberger C, Blazek G, Kunafer M, Prager E: Cardiac involvement over 10 years in myotonic and Becker muscular dystrophy and mitochondrial disorder. Int J Cardiol 2007, 119: Yazaki M, Yoshida K, Nakamura A, Koyama J, Nanba T, Ohori N, Ikeda S: Clinical characteristics of aged Becker muscular dystrophy patients with onset after 30 years. Eur Neurol 1999, 42: Politano L, Passamano L, Petretta VR, Nigro V, Papparella S, Nigro G, Santangelo L, M.G.Esposito, Come LI, Nigro G: Familial Dilated Cardiomyopathy Associated with the Typical Dystrophin BMD Mutation: Report on Two Additional Cases. Acta Myol 1999: Yoshida K, Ikeda S, Nakamura A, Kagoshima M, Takeda S, Shoji S, Yanagisawa N: Molecular analysis of the Duchenne muscular dystrophy gene in patients with Becker muscular dystrophy presenting with dilated cardiomyopathy. Muscle Nerve 1993, 16: Yazawa M, Ikeda S, Owa M, Haruta S, Yanagisawa N, Tanaka E, Watanabe M: A family of Becker's progressive muscular dystrophy with severe cardiomyopathy. Eur Neurol 1987, 27: Page 18 of 30

28 20. Saito M, Kawai H, Akaike M, Adachi K, Nishida Y, Saito S: Cardiac dysfunction with Becker muscular dystrophy. Am Heart J 1996, 132: Muntoni F, Di Lenarda A, Porcu M, Sinagra G, Mateddu A, Marrosu G, Ferlini A, Cau M, Milasin J, Melis MA, et al.: Dystrophin gene abnormalities in two patients with idiopathic dilated cardiomyopathy. Heart 1997, 78: Fanin M, Melacini P, Angelini C, Danieli GA: Could utrophin rescue the myocardium of patients with dystrophin gene mutations? J Mol Cell Cardiol 1999, 31: Finsterer J, Bittner RE, Grimm M: Cardiac involvement in Becker's muscular dystrophy, necessitating heart transplantation, 6 years before apparent skeletal muscle involvement. Neuromuscul Disord 1999, 9: Tasaki N, Yoshida K, Haruta SI, Kouno H, Ichinose H, Fujimoto Y, Urasawa N, Kawakami T, Taniguchi M, Kurushima S, et al.: X- linked dilated cardiomyopathy with a large hot-spot deletion in the dystrophin gene. Intern Med 2001, 40: Nakamura A, Yoshida K, Fukushima K, Ueda H, Urasawa N, Koyama J, Yazaki Y, Yazaki M, Sakai T, Haruta S, et al.: Follow-up of three patients with a large in-frame deletion of exons in the Duchenne muscular dystrophy (DMD) gene. J Clin Neurosci 2008, 15: Palmucci L, Mongini T, Chiado-Piat L, Doriguzzi C, Fubini A: Dystrophinopathy expressing as either cardiomyopathy or Becker dystrophy in the same family. Neurology 2000, 54: Page 19 of 30

29 Supplemental Table 3: Non-affected patients BMD [1] 22) Age at consultation: 24 years. Clinical Severity: severe. Myoglobinuria. FVC = 92%. Positive family history. ECG showed incomplete RBBB. Holter ECG showed isolated monomorphic PVCs (Lown grade 1). Normal LA volume. LV EDV = 50 ml/m 2. EF = 67%. Normal LV wall motion. Normal RV EDV. Normal RVEF BMD [1] 29) Age at consultation: 35 years. Clinical Severity: Severe. FVC = 75% (mild restrictive respiratory insufficiency). Positive family history. Normal ECG. Holter ECG showed isolated monomorphic PVCs (Lown grade 1). Normal LA volume. LV EDV = 62 ml/ m 2. EF = 61%. Normal LV wall motion. Normal RV EDV. Normal RVEF. Normal RV motion AH BMD MDAc Ambulatory at 9 years of age. EF = 55%, SF = 34%, EPSS = 5mm. 9 1 SL BMD UDP Vignos Scale (22 years): upper = 1 / lower = 2. EF = 55% BMD [1] 1) Age at consultation: 6 years. Clinical Severity: Mild. Skeletal muscle symptoms are present. CK = 1770 U/L. Western blot: reduced dystrophin expression (60%) and size (370 kda). Negative family history. Normal ECG. Normal LA volume. LV EDV = 37 ml/ m 2. EF = 60%. Normal LV wall motion. RV EDV=41 ml/ m 2. RVEF = 68%. Normal RV wall motion. 6 1 AH14 5 BMD MDAc Onset of skeletal muscle symptoms: 7 years. Age at consultation: 13 years. Clinical Severity: Mild. Skeletal muscle symptoms are present. Elevated CK. Biopsy: dystrophic changes; Dys2 staining positive but Dys 1 and Dys 3 severely reduced; Utrophin is upregulated. Western Blot: severely reduced dystrophin. EF = 58%, SF = 31%, EPSS = 5mm, no mitral valve regurgitation, no sign of LV dilation Page 20 of 30

30 AH02 5 BMD MDAc Onset of skeletal muscle symptoms: 10 years. Frequent falling, difficulty climbing stairs, calf hypertrophy, positive Gower's sign. CK = 7651 U/L. Negative family history. SL BMD UDP Onset of skeletal muscle symptoms: 3 years. Diagnosis: 6 years. Weakness, calf hypertrophy and Gower s sign. Vignos BMD [1] 2) Scale (14 years): upper = 1 / lower = 2. Age at consultation: 6 years. Clinical Severity: Mild. CK = 5000 U/L. FVC = 56% (moderate restrictive respiratory insufficiency). LVEDd = 0.5sd, EF = 60%, SF = 40% EF = 63%, SF = 35% Normal ECG. Normal LA volume. LV EDV = 67 ml/ m 2. EF = 64%. Normal LV wall motion. RV EDV = 46 ml/ m 2. RVEF = 57%. Normal RV motion BMD [1] 24) BMD [1] 28) Age at consultation: 28 years. Clinical Severity: Moderate. Age at consultation: 33 years. Clinical Severity: Moderate. Biopsy showed small number of dystrophin negative fibers. Western Blot: reduced amount (50%) and size (380 kda). Positive family history. KJ BMD MDAc Onset of skeletal muscle symptoms: childhood. Diagnosis: 37 years. Age at consultation: 47 years. Patient is ambulant with wide-based gait with exaggerated lordosis and some waddle. SL BMD UDP Onset of skeletal muscle symptoms: 1.5 years. Diagnosis: 9 years. Toe walking, myalgia, cramping. Vignos Scale (19 years): upper = 1 / lower = 1. Negative family history. ECG showed T wave changes. Normal Holter ECG. Normal LA volume. LV EDV = 69 ml/ m 2. EF = 55%. Normal LV wall motion. RV EDV = 66 ml/ m 2. RVEF = 64%. Normal ECG. Holter ECG showed isolated monomorphic PVCs (Lown grade 1). LA volume = 29 ml/ m 2. LV EDV = 44 ml/ m 2. EF = 55%. Normal LV wall motion. RV EDV = 52 ml/ m 2. RVEF = 52%. Echocardiography and stress test negative for cardiac dilation EF=57%, SF= 32% Page 21 of 30

31 SL BMD UDP Vignos Scale (10 years): upper = 1 / lower = 1. SF = 36.1% 10 2 MJ BMD MDAc Diagnosis: 10 years. Elevated CK. Onset of skeletal muscle symptoms: 12 years. Muscle fatigue, weakness, calf hypertrophy. MJ BMD MDAc Diagnosis: age 4 years. Biopsy showed muscle fiber size variation and necrosis. Elevated CK. Negative family history. AH BMD MDAc Onset of skeletal muscle symptoms: 5 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, betadystroglycan and laminin showed strong undisrupted sarcolemmal staining. Western Blot: several dystrophin bands of reduced size. Positive family history. AH BMD MDAc Onset of skeletal muscle 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. AH BMD MDAc Diagnosis: 10 months of age. Onset of skeletal muscle symptoms: 8 years. Calf hypertrophy, muscle cramps. CK = U/L. Positive family history. SL BMD UDP Onset of skeletal muscle symptoms: 6 years. Diagnosis: 6 years. Symptoms: weakness, myalgias, cramping. Negative family history. Vignos Scale (18 years): upper = 1/ lower = 1. Echocardiography normal EF = 60%, SF = 42.5% LVEDd = 0.9 sd, EPSS = 2mm, SF = 40%, EF = 60%. 9 2 EF = 55%, SF = 33% EF = 58%, SF = 32.23% EF = 55% 18 2 Page 22 of 30

32 BMD [1] 4) BMD [2] A13) Age at consultation: 10 years. Clinical Severity: Mild. Myoglobinuria. CK = 7300 U/L. FVC = 69% (mild restrictive respiratory insufficiency). Positive family history. Age at consultation: 20 years. Cramps, myalgia, myoglobinuria, calf hypertrophy. CK = 1774 U/L. Western blot: normal dystrophin amount but reduced size (360 kda) BMD [3] Onset of skeletal muscle symptoms: 15 years. Cramps. Age at consultation: 41 years. CK = 406 U/L. No muscle weakness or fatigability. Muscle hypertrophy of glutei and calf muscles. Negative family history. MJ BMD MDAc Shoulder girdle weakness and difficulty running since early childhood. Neurological exam at 18 years: CK = U/L. Biopsy: fiber size variation, atrophic fibers, mild to moderate increase of internal nuclei. Western Blot: reduced dystrophin amount BMD [1] 31) BMD [1] 10) Age at consultation: 50 years. Clinical Severity: Severe. FVC = 26% (severe restrictive respiratory insufficiency). Positive family history. Age at consultation: 13 years. Clinical Severity: Mild. Skeletal muscle symptoms are present. CK = 3910 U/L. Western blot: reduced dystrophin expression (80%) and size (390 kda). Positive family history. ECG showed R/S>1. Normal Holter ECG. Normal LA volume. LV EDV = 50 ml/ m 2. EF = 62%. Normal LV wall motion. RV EDV = 74 ml/ m 2. RVEF = 43%. Normal RV motion. ECG showed left anterior fascicular block. Normal Holter ECG. LV EDV = 72 ml/ m 2. EF = 63%. ECG showed flat T waves. Slight motility reduction of the LV posterior wall and increased LV EDV. Normal LV end-diastolic pressure, EF = 58%. LVEDd = 0.5sd, SF = 37%, EF = 58%, no mitral regurgitation. ECG showed incomplete LBBB. Holter ECG showed Lown grade 4b. Normal LA volume. LV EDV = 74 ml/ m 2. EF = 55%. Normal LV wall motion. Normal RV EDV. Normal RVEF. Normal RV motion. ECG showed T wave changes and R/S>1. Normal Holter ECG. Normal LA volume. LV EDV = 49 ml/ m 2. EF = 58%. RV EDV = 55 ml/ m 2. RVEF = 56% Page 23 of 30

33 BMD [2] A17) BMD [4] s 1 & 2) BMD [1] 12) BMD [2] A11) BMD [1] 5) Age at consultation: 24 years. Cramps, myalgia, myoglobinuria, calf hypertrophy. CK = 18 U/L. Western blot: reduced dystrophin amount (60%) and reduced size (395 kda). Onset of skeletal muscle symptoms: 3 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. Age at consultation: 14 years. Clinical Severity: Mild. Skeletal muscle symptoms are present. FVC = 105%. Biopsy showed many dystrophin negative fibers. Western blot: reduced dystrophin expression (50%) and size (390 kda). Positive family history. Age at consultation: 18 years. CK = 6427 U/L. Cramps, myalgia, myoglobinuria, calf hypertrophy. Western blot: reduced amount (50%) and size (390 kda). Age at consultation: 10 years. Clinical Severity: Mild. Skeletal muscle symptoms are present. CK = 1616 U/L. Western blot: normal dystrophin expression levels but reduced size (360 kda). Negative family history. ECG showed R/S>1. Normal Holter ECG. LV EDV = 88 ml/ m 2. EF = 56% Normal ECG. Normal echocardiography. 3 2 ECG showed incomplete RBBB. Holter ECG showed Lown grade 1. Normal LA volume. LV EDV = 77 ml/ m 2. EF = 65%. Normal LV wall motion. RV EDV =110 ml/ m 2. RVEF = 56%. RV wall motion showed septal akinesia. ECG showed incomplete RBBB. Holter ECG showed isolated monomorphic PVCs (Lown grade 1). LV EDV = 86 ml/ m 2. EF = 58%. Normal LV wall motion. ECG showed R/S>1. Holter ECG showed isolated monomorphic PVCs (Lown grade 1). Normal LA volume. LV EDV = 63 ml/ m 2. EF = 69%. Normal LV wall motion. RV EDV = 60 ml/ m 2. RVEF = 68%. Normal RV wall motion Page 24 of 30

34 SL BMD UDP Age of skeletal muscle symptom onset: 1 year. Diagnosis: 2 years. Skeletal muscle symptoms are present. Developmental delay. Vignos Scale (8 years): upper = 1/ lower = 1. SL BMD UDP Age of skeletal muscle symptom onset: 4 years. Diagnosis: 11 years. Myalgia, cramping. Vignos Scale (13 years): upper = 1 / lower = 1. Positive family history BMD [2] A18) BMD [16] 2) Age at consultation: 29 years. Cramps, myalgia, myoglobinuria, calf hypertrophy. CK = 2100 U/L. Western blot: normal dystrophin amount but reduced size (360 kda). Onset of skeletal muscle symptoms: 59 years. Mild but progressive weakness affecting both arms and legs, difficulty climbing hills and stairs, difficulty lifting luggage and standing from a sitting or crouching position. Age at 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. SF = 38.6% 8 EF = 65%, SF = 34% Normal ECG. LVEDV = 53 ml/ m 2. EF = 61% No cardiac symptoms. Negative cardiac failure, cardiomegaly on chest X-ray. ECG showed prominent R wave in V1. No LV hypokinesis, LVEDd = 56mm, LVEDs = 38mm. EF = 60%, SF = 32.1% Page 25 of 30

35 BMD [2] A5) Age at consultation: 12 years. Clinical Severity: Mild. Cramps, myalgia, myoglobinuria, calf hypertrophy. CK = 6000 U/L. FVC normal. Western blot: reduced dystrophin amount (40%) and reduced size (340 kda). Negative family history. ECG showed R/S>1 and incomplete LBBB. Holter ECG showed isolated monomorphic PVCs (Lown grade 1). Normal LA volume. LV EDV = 50 ml/ m 2. EF = 61%. Normal LV wall motion. RV EDV = 63 ml/ m 2. RVEF = 60%. Normal RV wall motion BMD [5] Onset of skeletal muscle symptoms: 5 years. Age at consultation: 9 years. Calf hypertrophy and mild proximal muscle weakness. CK = 3,000 U/L. Grandson of #203. AH BMD MDAc Onset of skeletal muscle symptoms and diagnosis: 4 years. CK = 551 U/L. Normal pulmonary function. Brother of #AH9. Positive family history. AH BMD MDAc Onset of skeletal muscle symptoms: 7 years. Weakness and muscle pain. CK = 1096 U/L. Brother of #AH8. Positive family history BMD [6] Onset of skeletal muscle 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. SL BMD UDP Onset of skeletal muscle symptoms: 5years. Diagnosis: 6.5 years. Symptom of weakness. Vignos Scale (13 years): upper = 1 / lower = 1. Positive family history. Normal echocardiography. 9 3 SF = 37%, EF = 59%, LVEDd = 1sd. 5 3 SF = 42%, EF = 62%, no mitral regurgitation. 8 3 Normal echocardiography. 4 3 SF = 39%. 4 3 Page 26 of 30

36 * 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: electrocardiogram; EDV: end diastole volume; EF: ejection fraction (left ventricular unless otherwise specified); EMG: electromyography; EPSS: E-point septal separation; FVC: forced vital capacity; LA: left atrium; LBBB: left branch bundle block; LV: left ventricle; LVEDd: left ventricular end-diastolic diameter; MDAc: muscular dystrophy clinics of Nationwide Children s Hospital and The Ohio State University Medical Center; 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 or equal to 55%; EPSS: below 5 mm; FVC: 80%-120% predicted; LVEDd: below 58 mm, 2 z scores, or 2 SD; SF: above or equal to 32%; Vignos Scale: described in JAMA (1963), 184: Published sources: 1. Melacini P, Fanin M, Danieli GA, Fasoli G, Villanova C, Angelini C, Vitiello L, Miorelli M, Buja GF, Mostacciuolo ML, et al.: Cardiac involvement in Becker muscular dystrophy. J Am Coll Cardiol 1993, 22: Melacini P, Fanin M, Danieli GA, Villanova C, Martinello F, Miorin M, Freda MP, Miorelli M, Mostacciuolo ML, Fasoli G, et al.: Myocardial involvement is very frequent among patients affected with subclinical Becker's muscular dystrophy. Circulation 1996, 94: Siciliano G, Fanin M, Angelini C, Pollina LE, Miorin M, Saad FA, Freda MP, Muratorio A: Prevalent cardiac involvement in dystrophin Becker type mutation. Neuromuscul Disord 1994, 4: Ramelli GP, Joncourt F, Luetschg J, Weis J, Tolnay M, Burgunder JM: Becker muscular dystrophy with marked divergence between clinical and molecular genetic findings: case series. Swiss Med Wkly 2006, 136: Palmucci L, Mongini T, Chiado-Piat L, Doriguzzi C, Fubini A: Dystrophinopathy expressing as either cardiomyopathy or Becker dystrophy in the same family. Neurology 2000, 54: Page 27 of 30

37 6. Lesca G, Testard H, Streichenberger N, Pelissier JF, Lestra C, Burel E, Jonveaux P, Michel-Calemard L: [Family study allows more optimistic prognosis and genetic counselling in a child with a deletion of exons of the dystrophin gene]. Arch Pediatr 2007, 14: Page 28 of 30

38 Page 29 of 30 Wen Kaspar et al.

39 Page 30 of 30 Wen Kaspar et al.