The natural history of a genetic subtype of arrhythmogenic right ventricular cardiomyopathy caused by a p.s358l mutation in TMEM43

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1 Clin Genet 2013: 83: Printed in Singapore. All rights reserved Original Article 2012 John Wiley & Sons A/S. Published by Blackwell Publishing Ltd CLINICAL GENETICS doi: /j x The natural history of a genetic subtype of arrhythmogenic right ventricular cardiomyopathy caused by a p.s358l mutation in TMEM43 Hodgkinson KA, Connors SP, Merner N, Haywood A, Young T-L, McKenna WJ, Gallagher B, Curtis F, Bassett AS, Parfrey PS. The natural history of a genetic subtype of arrhythmogenic right ventricular cardiomyopathy caused by a p.s358l mutation in TMEM43. Clin Genet 2013: 83: John Wiley & Sons A/S. Published by Blackwell Publishing Ltd, 2012 To determine the phenotype and natural history of a founder genetic subtype of autosomal dominant arrhythmogenic right ventricular cardiomyopathy (ARVC) caused by a p.s358l mutation in TMEM43. The age of onset of cardiac symptoms, clinical events and test abnormalities were studied in 412 subjects (258 affected and 154 unaffected), all of which occurred in affected males significantly earlier and more often than unaffected males. Affected males were hospitalized four times more often than affected females (p ) and died younger (p 0.001). The temporal sequence from symptoms onset to death was prolonged in affected females by 1 2 decades. The most prevalent electrocardiogram (ECG) manifestation was poor R wave progression (PRWP), with affected males twice as likely to develop PRWP as affected females (p 0.05). Left ventricular enlargement (LVE) occurred in 43% of affected subjects, with 11% fulfilling criteria for dilated cardiomyopathy. Ventricular ectopy on Holter monitor was common and occurred early: the most diagnostically useful clinical test. No symptom or test could rule out diagnosis. This ARVC subtype is a sex-influenced lethal arrhythmogenic cardiomyopathy, with a unique ECG finding, LV dilatation, heart failure and early death, where molecular pre-symptomatic diagnosis has the greatest clinical utility. Conflictofinterest The authors have declared that there is no conflict of interest. KA Hodgkinson a,b, SP Connors c, N Merner b, A Haywood b, T-L Young b, WJ McKenna d, B Gallagher e, FCurtis b, AS Bassett f and PS Parfrey a a Clinical Epidemiology Unit, Memorial University, Health Sciences Centre, St. John s, Newfoundland, Canada, b Discipline of Genetics, Memorial University, Health Sciences Centre, St. John s, Newfoundland, Canada, c Division of Cardiology, Memorial University, St. John s, Newfoundland, Canada, d Institute of Cardiovascular Science, University College London and The Heart Hospital, University College London Hospitals Trust, London, UK, e Division of Pathology, James Paton Memorial Hospital, Gander, Newfoundland, Canada, and f Clinical Genetics Research Program, Centre for Addiction & Mental Health, University of Toronto, Toronto, Ontario, Canada Key words: ARVC autosomal dominant cohort DCM founder SCD TMEM43 Corresponding author: Kathy Hodgkinson, Clinical Epidemiology and Genetics, Room 5339, Faculty of Medicine, Memorial University of Newfoundland, St. John s, Newfoundland and Labrador, Canada AIB 3 V6. Tel.: (709) ; fax: (709) ; khodgkin@mun.ca Received 7 February 2012, revised and accepted for publication 19 June

2 Hodgkinson et al. Arrhythmogenic right ventricular cardiomyopathy (ARVC), genetically and clinically heterogeneous cause of sudden cardiac death (SCD) in young people, is difficult to clinically diagnose (1). Current diagnostic criteria are descriptive and require a number of major and minor criteria to be achieved following detailed visualization techniques (2, 3). Newfoundland is a founder population of 510,000 people derived from 20 30,000 settlers resulting in a high prevalence of some genetic diseases. In the 1970s, one Newfoundland family with an autosomal dominant history of early SCD was ascertained. One family member was diagnosed with ARVC (4), a newly emerging diagnosis in the literature (5, 6). Currently, this pedigree has 1200 subjects across 10 generations and can be traced back to a single founder pair born in 1799 and Subsequently, 14 other large families with a similar presentation were ascertained, in which ARVC was primarily diagnosed using the 1994 criteria (7, 8) in at least one member of each family. This genetic subtype was linked to chromosome 3p25 in 1998 (9) (OMIM #604400: ARVD5 ), and subsequently all 15 families were shown to share a disease-associated DNA haplotype (10). These families became the genetically homogeneous founder population in which a missense mutation within TMEM43 (p.s358l) was identified in 2008 (11). The TMEM43 protein was simultaneously defined as LUMA, a trans-nuclear membrane protein widely distributed among tissues and species (11, 12). To assess the phenotype and natural history of disease caused by this mutation, clinical events and test results were compared in affected vs unaffected family members (family controls) born at an a priori 50% risk, an ascertainment strategy unrelated to clinical presentation. Previous ARVC studies assessing natural history and phenotype involved genetically and clinically heterogeneous groups with variable clinical presentations, family histories, causes and rates of death, and no control group (13 15). In this study, retrospective cardiac data from affected and unaffected subjects (familial controls) were obtained from medical records and clinical interviews. Prospective serial 12-lead electrocardiograms (ECGs), echocardiograms and Holter monitors were available from 1998 onward (10). This multigenerational genetically homogeneous population is the largest single well-characterized genetic subtype of ARVC worldwide. This affords a unique opportunity to provide invaluable data (including prevalence and incidence of symptoms and clinical events, and their diagnostic utility) for cardiologists, geneticists and genetic counselors. Methods Study population and mutation testing Fifteen families referred to a genetic cardiomyopathy clinic had a history of SCD and were found to have a disease-associated haplotype at the ARVD5 locus on 3p25 (16), subsequently shown to have a p.s358l mutation in TMEM43 (11). These families were Fig. 1. Transverse section of heart [55-year-old male p.s358l (TMEM43) positive]. Note biventricular dilatation and severe atrophy of right ventricle with fibrofatty replacement. characterized by autosomal dominant (AD) inheritance of early death and heart failure, a single causative mutation, and at least one subject in each family diagnosed with ARVC using original criteria (Fig. 1, Table S1, Supporting Information) (7). All subjects born at a 50% a priori risk of disease by pedigree analysis (n = 639) from 1800 onward were identified from these 15 families. Subjects were considered affected (n = 285) if they had a minimum of one of the following: (i) they had the TMEM43 p.s358l mutation, (ii) they were an obligate carrier, (iii) SCD occurred under 50 years or (iv) they were resuscitated from ventricular tachycardia (VT) or fibrillation (VF) under 50 years. Subjects were unaffected (n = 164) if they did not have the TMEM43 p.s358l mutation (Table S2). Remaining subjects were unknown (n = 190). To minimize the bias present in recognizing cases with serious clinical disease, and missing cases with minimal disease, the analysis was limited to 496 subjects from sibships where the disease status (affected or unaffected) of 50% of siblings was known (Fig. 2). Subjects in the unknown group (n = 84) were not studied further. Thus, 412 (226 males and 186 females) subjects were included in the study: 258 affected (149 males and 109 females) and 154 unaffected (77 males and 77 females). Clinical data collection All possible subjects born at an a priori 50% risk of inheriting ARVC (including subjects with only birth and death dates) were counted. Following genetic counseling and informed consent (Human Research Ethics Board of the Eastern Health Care Corporation of St. John s study # ), all past and current medical records were reviewed for all subjects. As with most genetic family studies, subjects were ascertained at different stages of their disease process. Prior to 1998, only subjects with a clinical problem were ascertained 322

3 Natural history of a genetic subtype of ARVC Fig. 2. Schematic pedigree illustrating ascertainment strategy for reducing severe case bias. (VT, heart failure or death). Post 1998, subjects at 50% risk of ARVC were evaluated through a genetics cardiac clinical program (10). Assessments included a detailed medical history by a cardiac nurse (blind to clinical or DNA test results), physical examination, 12-lead ECG, 2D echocardiogram and 24-h Holter monitor. All clinical assessments were repeated at 1- to 3-year intervals. All extant medical charts were obtained, although early SCD and a lack of rural physicians historically influenced their availability, thus raising a potential bias. As the median age to death (from birth) or last follow-up, however, for those without charts was not significantly different to those with charts [data not shown (dns)], it was acceptable. Medical charts were available for the majority of unaffected subjects. Clinical signs and symptoms in affected subjects Age at onset of palpitations, syncope, pre-syncope, chest pain, symptomatic left heart failure, cardiac hospitalization and death were documented. A bias would occur if there was a significant difference between subjects with both a medical chart and a clinical assessment when compared with subjects with only a chart. However, there was no significant difference in time to any cardiac manifestation in male subjects by data source (dns). This was true for female subjects, except for palpitations, which were more likely in the74 (87%) females with both a clinical assessment and chart compared with 11 (13%) with chart alone [p = 0.01, relative risk (RR) 3.1, 95% confidence interval (95% CI) ]. Thus, palpitation data in females are presented only from the former group (n = 74). Cardiac testing Abnormalities on the first ECG, Holter monitor and 2D echocardiogram provided prevalence data. Incidence data (age of onset) were calculated on serial testing after a normal first test. One physician, blind to clinical status, read ECGs. Consensus regarding abnormalities was obtained following discussion with a second physician. Poor R wave progression (PRWP) was defined as an R wave <3mminV3(17)andright precordial QRS prolongation as >110 ms in leads V1, V2 and V3. The incidence analysis included only ECG features with a prevalence of >5%, which were PRWP and premature ventricular contractions (PVCs). T wave inversion and epsilon waves (7) were thus excluded (<5% prevalence). Thus, de novo PRWP and PVCs were noted in affected subjects with serial ECGs after excluding subjects with these features at baseline. Echocardiographic LVE was defined as LV end diastolic diameter (LVEDD) >112% corresponding to 2 standard deviations (SD) above the predicted mean (Henry s formula) (18). Also, LVE > 117% was assessed (>3 SD above the predicted mean). In affected subjects with serial echocardiograms, the age at onset of de novo LVE was calculated after excluding those with LVE at baseline. 323

4 Hodgkinson et al. Ventricular arrhythmia and PVC morphology were assessed from full disclosure Holter data (lead 2 when available). In affected subjects with serial Holter monitors, the age of onset of de novo 200 PVCs or 1000 PVCs was determined after excluding subjects with these features at baseline. Genetic analysis Screening for the TMEM43 p.358l mutation DNA was extracted from peripheral lymphocytes using standard methods (19). Subjects (n = 295) initially had DNA haplotype analysis at the 3p25 ARVD5 locus (10, 16) and subsequent direct mutation analysis for the p.s358l mutation in TMEM43 (11). Presence (n = 144) or absence (n = 151) of this mutation was determined (11). Statistical analysis This is a sex-influenced disease, so data for males and females were stratified (11, 16, 20). The Kaplan Meier product limit method was used for time-to-event analysis. Subjects were censored (an observation with incomplete information: removed prior to reaching the end point of the analysis) at last clinic visit, death, and at implantable cardioverter-defibrillator (ICD) surgery or heart transplant. RR was calculated using Cox s proportional regression. The SPSS statistics package (v18) was used for analyses (SPSS, Chicago, IL). The diagnostic utility of symptoms and test results from the first assessment was determined. The likelihood ratio (LR) of having and not having inherited ARVC was calculated by sensitivity/1-specificity and 1- sensitivity/specificity, respectively. Generally, LRs over 10 (diagnosis) or under 0.2 (no diagnosis) indicate good utility. Missing data This is an epidemiological study of a familial disorder with subjects born at an a priori 50% risk from several generations. Clinical data are from routine testing, reflecting the reality of general medicine. This reflects the paucity of RV data on every subject that may not be present on routine echocardiograms. All clinical data sources were used (historic and current) from Newfoundland, mainland Canada, and the United States. Missing clinical test results reflect early deaths in the affected group (85% of the absent data). Results Incident symptoms and clinical outcomes in ARVD5 Affected males were significantly more likely than unaffected males to develop all cardiac manifestations (Table 1). The incidence data (Fig. 3) illustrate the cumulative percentage of subjects who developed the anomaly by age. Only palpitations and pre-syncope occurred at a younger average age than SCD (with potential for clinical diagnosis); 53% developing both these features by 40 years (Fig. 3a) compared with 17% and 27% of unaffected males, respectively (dns). Heart failure was absent in unaffected males but developed in 16% of affected males by 40 years and in 46% by 60 years (Fig. 3a). Median survival in affected males was 45 years (95% CI: 38 52), with one death (accidental) in unaffected males (p = 0.001). Only syncope and palpitations were significantly more likely in affected compared with unaffected females (Fig. 3b). Hospitalization for cardiac problems, heart failure and death were significantly higher in affected men than affected women (Table 1). The temporal sequence in affected subjects from cardiac symptoms to death is prolonged in females compared with males (Fig. 3a,b). Prevalent events on clinical testing 12-Lead ECG At least one 12-lead ECG was available for 300 subjects (167 affected and 133 unaffected). Of the 167 affected, 117 (70%) were mutation carriers and 23 (14%) were obligate carriers. The most prevalent features in the affected group were PRWP and PVCs (Table 2). Echocardiography At least one 2D echocardiogram was available for 248 subjects [144 (68 males and 76 females) affected and 104 (50 males and 54 females) unaffected]. Five echocardiograms with no available measurements were excluded from the analysis, of which 3 males and 1 female (all deceased) were reported to have a massively dilated heart. The most prevalent feature in affected subjects was LVEDD > 112% (21) (Table 2). Twelve (18%) affected males (median age 33 years) developed a dilated LV with global systolic dysfunction and stage 3 to 4 New York Heart Association (NYHA) clinical criteria (22), fulfilling criteria for dilated cardiomyopathy (DCM). Two males presented with features fulfilling DCM diagnostic criteria (aged 23 years). Four females (5%) developed stage 2 NYHA and fulfilled criteria for DCM at a median age of 62 years. In affected males older than 30 years who presented with or developed DCM, 20% received heart transplant compared with 1 female. Holter monitor At least one Holter monitor was available for 239 subjects (146 affected and 93 unaffected). Ventricular ectopy occurred frequently in the affected group with 200 PVCs observed in 64% of subjects assessed (Table 2). Of 51 full disclosure Holter monitors from affected subjects, 63% of PVCs were of left bundle branch block morphology (RV origin). Incident events on clinical testing The incidence data (Fig. 4) illustrate the cumulative percentage of subjects who developed the anomaly 324

5 Natural history of a genetic subtype of ARVC Table 1. Median age to clinical cardiac manifestations in those with medical records data Cardiac feature Palpitations Pre-syncope Chest pain Syncope Heart failure Hosp Death Affected males, n = 87 n event % Med age (years) to event 39 (35 44) 39 (33 45) 45 (38 52) 61 (35 86) 63 (46 80) 43 (38 47) 45 (38 52) (95% CI) Unaffected males, n = 65 n event % Med age (years) to event 67 (50 84) 64 (48 79) 66 (47 85) (95% CI) c Affected males vs unaffected males p b RR (95% CI) c 3.4 ( ) 2.2 ( ) 2.0 ( ) 2.3 ( ) 10.2 ( ) 31.5 ( ) Affected females, n = 85 n event 49 a % Median age (years) to 41 (36 46) 49 (41 57) 59 (44 74) 66 (52 79) 71 ( ) 66 (55 76) event (95% CI) c Unaffected females, n = 68 n event % Med age (years) to event 62 (42 82) 66 (37 95) (95% CI) c Affected females vs unaffected females p b RR (95% CI) c 1.8 ( ) ns ns 3.8 ( ) ns ns ns Affected males vs affected females p RR (95% CI) c ns 1.9 ( ) 1.8 ( ) Q 5.2 ( ) 4.4 ( ) 7.4 ( ) RR, relative risk. a The denominators for this analysis, therefore, were 74 mutation affected vs 63 mutation unaffected. b This p value is derived from the log-rank assessment of the Kaplan Meier analysis. c Where median ages are not provided, 50% of the subjects did not reach the end point in the Kaplan Meier analysis. 325

6 Hodgkinson et al. Table 2. Prevalence of abnormalities on initial 12-lead ECG, echocardiogram and Holter monitor in ARVD5 Affected group Unaffected group Analyses 12-Lead ECG (n = 300) a Male Female Total Male Female Total χ 2 χ 2 N Affected males vs unaffected males Affected females vs unaffected females Mean age at test (SD) 30.0 (12.0) 37.8 (15.8) 33.3 (16.1) 38.2 (12.9) n % n % N (%) n % n % N (%) PRWP (29) (1) p p PVCs (28) (<1) p p QRS > 110 ms (19.8) (4.6) p p = (ns) Septal Q waves (14) (0) p p Echocardiogram (n = 248) N Mean age at test (SD) 31.6 (13.0) 39.6 (15.1) 32.6 (15) 38.4 (12.1) LVE > 2 SD (43) (14) p p LVE > 3SD (30) (6) p p Dyskinesia global/focal (24) p p LAE (17) (9) ns ns RVD (12) (6) p 0.05 ns FS < 25% (12) (3) p 0.02 ns LVH (10) (6) ns ns Holter monitor N Mean age at test (SD) 31.1 (13.3) 37.7 (15.5) 33.1 (15.9) 38.2 (12.8) PVCs 200/24 h (64) (1) p p PVCs 1000/24 h (56) (1) p p couplet (56) (2) p p runbigeminy (39) (1) p p run non-sustained VT (37) p p ECG, electrocardiogram; FS, fractional shortening; LAE, left atrial enlargement; LVE, left ventricular enlargement indexed to height and weight; LVH, left ventricular hypertrophy; PRWP, poor R wave progression; PVC, premature ventricular complex; RVD, right ventricular dilatation; VT, ventricular tachycardia. a Features analyzed, but data not shown due to low prevalence in the affected group (<5%) included: ventricular tachycardia, atrial fibrillation, L and R axis deviation, T wave inversion in V1, V2, V3, first degree AV block, long and short QT interval, short PR, epsilon wave, tall R in V1, dilated L or R atrium, L or R ventricular hypertrophy, L or R bundle branch block, L anterior or posterior hemiblock, inferior lateral and anterior Q waves, inferior, anterior and lateral ST depression, inferior, anterior and lateral ST elevation, flat, inverted or lateral T waves. 326

7 Natural history of a genetic subtype of ARVC (a) (b) Fig. 3. (a) Cumulative incidence of cardiac manifestations in affected males. (b) Cumulative incidence of cardiac manifestations in affected females. by age. These data show that >200 PVCs on Holter monitor are the earliest manifestation in both sexes, occurring at a younger age in males (median 25 years) than females (median 48 years) (Table 3), thus the only test able to clinically diagnose males prior to death (Fig. 4a). Dilatation on echocardiogram (LVE > 2 SD) arises frequently in middle age in both sexes (Fig. 4a,b). Ventricular ectopics on ECG are noted at a later age than PVCs identified on a Holter monitor. Using only subjects with the TMEM43 mutation assessing survival did not significantly change the results (Fig. S2, Supporting Information). Diagnostic utility The only LR >10 was observed in males with heart failure (LR 17), and this is a late manifestation so of no diagnostic value. For clinical test results, the presence of PRWP and PVCs on 12-lead ECG in males and PVCs >200 and >1000 on Holter monitor for both males and females had a LR >10. The sensitivity of these tests increased with age. As LRs <0.2 were not achieved, clinical manifestations and test results are inadequate to rule out having inherited the mutation. Discussion This is a unique population in which to assess in an unbiased manner the natural history and phenotype of this ARVC genetic subtype. Clinical diagnosis The diagnostic guidelines for ARVC comprise major and minor criteria from which a diagnosis can be made if a pre-set threshold is met (7). A recent revision of these includes mutation status as a major criterion and redefine levels of diagnosis (definite, borderline and possible) (2). These descriptive criteria are inherently biased due to the skewed ascertainment of those presenting with severe disease and because many require tertiary-level cardiac testing. Subjects in Newfoundland often presented with death, in rural areas, with no previous medical record. A diagnosis of ARVC could not be made as they would meet only one major criterion (autopsy determining evidence of the disease), and, if the pathology was at a small unit, not even then as deaths were often recorded as likely arrhythmia. In these families however, the disease was AD and thus obligate carrier status could be assigned. The use of modified diagnostic criteria (obligate carrier status, SCD under 50 years and serious arrhythmias requiring cardioversion) was essential for the epidemiological studies with this population as the diagnosis had to be effective, reproducible, easily defined, useful retrospectively and consistent. Natural history This phenotype is malignant in males with an early age of onset of cardiac symptoms followed closely by death. A more benign phenotype was observed in females with later onset of symptoms, less frequent hospitalizations, 327

8 Hodgkinson et al. Table 3. Incident events on 12 lead ECG, Holter monitoring, and echocardiogram Test Feature Sex Disease status # without clinical feature on first test with a second clinical test Incident cases Median age to onset of feature Log-rank p value( ) RR Cox p value 95% CI Affected males vs affected females ECG PRWP M No events No events No events p 0.05, RR= 2.3, 95% 41 0 CI ( ) F PVCs M p 0.004, RR = 3.7, 95% 40 1 CI ( ) F Holter >200 PVCs M p 0.02, RR= 3.6, 95% CI ( ) F >1000 PVCs M No events No events No events p 0.003, RR = 5.4, 95% CI ( ) F Echo LVE > 2 SD M p F LVE > 3 SD M No events No events No events F ECG, electrocardiogram; LVE, left ventricular enlargement; PRWP, poor R wave progression; PVC, premature ventricular contraction. 328

9 Natural history of a genetic subtype of ARVC (a) (b) Fig. 4. (a) Cumulative incidence of clinical anomalies on 12-lead electrocardiogram (ECG), Holter monitor and echocardiogram in affected males following normal test results at baseline. (b) Cumulative incidence of clinical anomalies on 12-lead ECG, Holter monitor and echocardiogram in affected females following normal test results at baseline. and longer survival, and other than syncope and palpitations, there was no difference in symptoms between unaffected and affected females. Cardiac symptoms in either sex had no diagnostic utility. Ectopy occurred early in the natural history. Heart failure and chest pain were late manifestations in subjects who did not die of VF. Heart failure leading to transplant occurs in a significant minority of subjects. Despite methodological differences, the results of this study are similar to those reported in other ARVC studies, including 26 subjects with mutations in desmoplakin (23), and in a heterogeneous group of 130 affected subjects (24). Clinical features on cardiac testing This genetic subtype has similarities and differences to typical ARVC. Few subjects had T wave inversion and epsilon waves (7), but PRWP in the precordial leads was common and noted in the first subject ascertained in the 1970s prior to RV surgical therapy (4). PRWP can be caused by anterior myocardial infarction (17), but angiography in PRWP subjects was negative. Other causes include lead misplacement, obesity, LV hypertrophy and Wolff Parkinson White (WPW), and it can be a normal variant with PRWP reported in 10% of hospitalized adults with routine ECGs (25). That 2% of unaffected subjects had PRWP probably reflects one of the above. In subjects with PRWP, multiple ECGs were recorded, the subjects were not obese, LV hypertrophy was absent and WPW was excluded. The Holter monitor was the only clinical test that had diagnostic utility for both males and females with LRs >10. LV involvement This population has early ventricular ectopy with heart failure as a later manifestation in survivors. Interestingly, several males developed (or presented with) DCM (LV systolic dysfunction and heart failure). This may reflect modifying genes, environmental and/or epigenetic issues. It may also reflect the molecular pathophysiology of TMEM43 in the myocardium. TMEM43 (LUMA) is a conserved nuclear membrane protein (12) that associates with Emerin and Lamin. Mutations in LMNA (Lamin AC) are known to cause DCM (26, 27). The potential interaction of TMEM43 with Lamin and Emerin may illuminate this clinical phenotype. LVE/DCM was not restricted to one family or sibling set, reducing the likelihood of familial clustering. Survival Previous studies of genetically heterogeneous families with ARVC have reported better overall survival than observed in this study (28), although males have always had worse survival than females (6). In this study, the sex influence on mortality was reflected by the earlier 329

10 Hodgkinson et al. time to development of PRWP, LVE, and ectopy in affected men compared with affected women. Limitations of the study Multiple biases exist when describing the natural history of a difficult to diagnose inherited disease with early death. The study design includes subjects assessed retrospectively and prospectively; however, no significant difference was noted in clinical outcomes comparing groups by method of data ascertainment except for palpitations in females. Defining affected status using a major clinical event (e.g. SCD < 50 years) potentially introduces a case bias although this outcome occurs infrequently in the general population. However, survival was not different (Fig. S2) when the 57 people with a diagnosis based on a severe clinical event (Table S2) were removed from the analysis. This suggests that the decision to include those defined by clinical events is reasonable, particularly as it enhances the statistical power of the analyses pertaining to the incidence of clinical events and provides robust data regarding early death, an outcome that was not restricted to earlier generations or to isolated branches of pedigrees, and which followed a clear AD male to male transmission pattern across time. To limit ascertainment bias, only subjects from sibships where disease status in at least 50% of siblings was known were analyzed. However, more male subjects remain in the study, which probably indicates a residual severe case bias. Survivor bias is present as many affected males died with no medical chart. However, median survival of these males was not significantly different to those with a chart. Intervention with ICD significantly improves survival in TMEM43 p.s358l males (16) and females (29). Censoring at ICD implant controlled this bias. Drug treatment effects due to changes in prescribing regimes over several decades could not be assessed. In-depth assessment of RV structure during routine clinical echocardiography was not (and is not in Newfoundland) routinely performed, and this aspect of the phenotype is lacking. However, the clinical evidence supports this disease as primarily arrhythmic with a LV component (the LV is more easily assessed on routine echocardiography), and the lack of understanding of the right ventricle in this population sample will alter neither the strength of the molecular diagnosis nor the correct treatment. Advantages of the study The deep genealogies in this genetically homogeneous population provided longitudinal and cross-sectional data to enable us to define phenotype and natural history. The use of unaffected family members and comprehensive chart assessment from extant and deceased subjects where disease manifestations were consistently classified was unique advantages. Regular visits to a screening clinic provided years of serial assessments in affected and unaffected subjects. The presence of the TMEM43 p.s358l mutation and/or obligate carrier status was a strategy independent of clinical manifestations. The disease caused by TMEM43 is a fully penetrant AD disorder where penetrance is defined by the onset of any clinical cardiac manifestation of disease (11), with a characteristic ECG pattern and LV involvement. This study provides accurate, clinically informative information for individuals with this mutation. It supports the concept that cardiomyopathies should be classified by a molecular genetic etiology [arrhythmogenic cardiomyopathies (30)] rather than criteria-based clinical definitions. The presence of features of ARVC (dilated RV, massive ectopy, and early death) and DCM (dilated LV and systolic dysfunction) often in siblings further underlines this concept. Is this disease ARVC or a unique cardiomyopathy with arrhythmia? It is felt that this is a subtype of ARVC but with the variability present in most AD diseases, probably due to genetic modifiers of varying penetrance. The families were ascertained because of a lethal multigenerational history of SCD (Fig. 2, Figs. S1 and S2). Some mutation carriers will develop RV or LV problems, but early death is a reality in the absence of treatment. Many individuals will never fulfill diagnostic criteria (related to the availability of relevant testing and early deaths precluding testing) but they are clearly affected. The data presented are necessary for those treating families with this mutation whether or not ARVC diagnostic criteria are met. European families recently ascertained will enable replication of these findings. Conclusions ARVC due to p.s358l in TMEM43 is a variant form of ARVC with extreme variability of expression. It is sex influenced: males are more frequently hospitalized and have heart failure and SCD at a younger age than females. It is an electrical disease with late mechanical complications (occasionally DCM) with a minority requiring transplant. Symptom onset in males is often closely followed by death. PRWP is common in the precordial ECG leads. Ventricular ectopy on Holter monitor is diagnostically useful, whereas no clinical manifestation or test could rule out a diagnosis. Supporting Information The following Supporting information is available for this article: Figure S1. Single family detailing the extensive phenotypic and genealogical data. Key: Black circles and squares are affected females and males, respectively, based on (i) SCD under 50 years, and/or (ii) cardioversion for VT or VF under 50 years, and/or (iii) obligate carrier status, and/or (iv) a p.s358l founder mutation in TMEM43. A line through a symbol indicates death. A plus sign next to a symbol indicates the presence of the TMEM43 p.s358l mutation. The arrowed individual is the original patient (4). Figure S2. Survival in affected male and affected female subjects defined by DNA analysis or obligate carrier status. Table S1. Diagnostic criteria fulfilled in probands from each family: 1994 criteria (1 major and 2 minor). 330

11 Natural history of a genetic subtype of ARVC Table S2. Method of diagnosis for 258 affected subjects defined by clinical events, mutation status and pedigree position. Additional Supporting information may be found in the online version of this article. Acknowledgements We sincerely thank the families for participating in this study, Barbara Peddle (cardiac nurse) and Elspeth Drinkell (RA). Research was supported by grants from the Canadian Institute for Health Research/ACOA (Regional Partnerships Program), the Atlantic Medical Genetics and Genomics Initiative (AMGGI) from Genome Canada and St. Jude Medical Research Grant, Canada. W. M. acknowledges the proportion of funding received by UCLH/UCL from the Department of Health s NIHR Biomedical Research Centres funding scheme. References 1. Ellinor PT, MacRae CA, Thierfelder L. Arrhythmogenic right ventricular cardiomyopathy. Heart Fail Clin 2010: 6: Marcus FI, McKenna WJ, Sherrill D et al. 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