The natural history of a genetic subtype of arrhythmogenic right ventricular cardiomyopathy caused by a p.S358L mutation in TMEM43

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 ≤ 0.0001) 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.     

Role of genetic testing in arrhythmogenic right ventricular cardiomyopathy/dysplasia

Barahona-Dussault C, Benito B, Campuzano O, Iglesias A, Leung TL, Robb L, Talajic M, Brugada R. Role of genetic testing in arrhythmogenic right ventricular cardiomyopathy/dysplasia.
In a cohort of patients with confirmed or suspected arrhythmogenic right ventricular cardiomyopathy/dysplasia (ARVC/D), genetic testing is useful in confirming the diagnosis, particularly in individuals who do not completely fulfil Task Force criteria for the disease, thereby also enabling the adoption of preventive measures in family members. Due to the high percentage of novel mutations that are expected to be identified in ARVC/D, the use of genetic screening technology based on the identification of known mutations seems to have very restricted value. Our results support that the presence of certain genetic variations could play a role in the final phenotype of patients with ARVC/D, where single and compound mutation carriers would have more symptomatic forms of the disease and the polymorphism P366L could be associated to a more benign phenotype.     

‘It had to be done’: genetic testing decisions for arrhythmogenic right ventricular cardiomyopathy

Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a heritable disease of the heart muscle, causing life‐threatening ventricular arrhythmias, sudden cardiac death and/or biventricular heart failure. Little research examines ARVC genetic test decisions, despite the gravity of the condition. This qualitative study used semi‐structured interviews to explore the testing decisions of 21 individuals across 15 families segregating a well‐studied, particularly lethal form of ARVC caused by a p.S358L TMEM43 mutation. Genetic testing decisions were rarely described as ‘decisions’ per se, but rather ‘something that had to be done’. This perception was attributed to personality type or personal suspicion of carrying the TMEM43 mutation, but most often was described in the context of testing for other family members, usually children. Participants related a strong need to rule out risk, more for children than for themselves, but lingering doubts remained about personal and children's risk for ARVC, even when gene test results were negative. Study findings highlight the interdependent nature of genetic test decisions and suggest that an individualistic conception of autonomy in genetic services may not meet the needs of affected families. Findings also suggest the need for follow‐up support of families affected by ARVC, including for those individuals testing negative for the family mutation.     

Detection of genomic deletions of PKP2 in arrhythmogenic right ventricular cardiomyopathy

Arrhythmogenic right ventricular cardiomyopathy (ARVC) is an inherited myocardial disease that predominantly affects the right ventricle and is associated with ventricular arrhythmias that may lead to sudden cardiac death. Mutations within at least seven separate genes have been identified to cause ARVC, however a genetic culprit remains elusive in approximately 50% of cases. Although negative genetic testing may be secondary to pathogenic mutations within undiscovered genes, an alternative explanation may be the presence of large deletions or duplications involving known genes. These large copy number variants may not be detected with standard clinical genetic testing which is presently limited to direct DNA sequencing. We describe two cases of ARVC possessing large deletions involving plakophilin‐2 (PKP2) identified with microarray analysis and/or multiplex ligation‐dependent probe amplification (MLPA) that would have been classified as genotype negative with standard clinical genetic testing. A deletion of the entire coding region of PKP2 excluding exon 1 was identified in patient 1 and his son. In patient 2, MLPA analysis of PKP2 revealed deletion of the entire gene with subsequent microarray analysis demonstrating a de novo 7.9 Mb deletion of chromosome 12p12.1p11.1. These findings support screening for large copy number variants in clinically suspected ARVC cases without clear disease causing mutations following initial sequencing analysis.     

A genetic variants database for arrhythmogenic right ventricular dysplasia/cardiomyopathy

Arrhythmogenic right ventricular dysplasia/cardiomyopathy (ARVD/C) is a hereditary cardiomyopathy characterized by fibrofatty replacement of cardiomyocytes, ventricular tachyarrhythmias and sudden death. ARVD/C is mainly caused by mutations in genes encoding desmosomal proteins. However, the pathogenicity of variants is not always clear. Therefore, we created an online database ( www.arvcdatabase.info ), providing information on variants in ARVD/C‐associated genes. We searched the literature using ARVD/C and its underlying genes as search terms. From the selected papers and our unpublished data, we collected details on the type of mutation and information provided at the protein level. A “details page” contains clinical data and references. To aid the interpretation of missense mutations, we provide data from in silico prediction methods. In May 2009 the database contained 481 variants in eight genes. A total of 144 variants are considered pathogenic, 73 are unknown/unclassified, and 264 have no known pathogenicity. The database was converted into the Leiden Open Variation Database (LOVD) format, a gene‐centered collection of DNA variations. The ARVD/C database will be useful for both researchers and clinicians. It can be searched to determine if variants have been published and whether they are considered pathogenic. External users are invited to add information to improve the quantity and quality of the data entered. Hum Mutat 30:1–6, 2009. © 2009 Wiley‐Liss, Inc.     

Etiopathogenesis of arrhythmogenic right ventricular cardiomyopathy

Arrhythmogenic right ventricular cardiomyopathy (ARVC) is characterised by progressive fibro-fatty replacement of right ventricular myocardium. Earlier studies described ARVC as non-inflammatory, non-coronary disorder associated with arrhythmias, heart failure and sudden death due to functional exclusion of the right ventricle. Molecular genetic studies have identified nine different loci associated with ARVC; accordingly each locus is implicated for each type of ARVC (ARVC1–ARVC9). So far five genes have been identified as containing pathogenic mutations for ARVC. Though mutations in each of the gene/s indicate disruption of different pathways leading to the condition, the exact pathogenesis of the condition is still obscure. This review tries to understand the pathogenesis of the condition by examining the individual proteins implicated and relate them to the pathways that could play a role in the aetiology of the condition. Cardiac ryanodine receptor (RYR-2), which regulates intra-cellular Ca²⁺ concentration by releasing Ca²⁺ reserves from the sarcoplasmic reticulum (SR), was the first gene for ARVC. The mutation in this gene is believed to disrupt coupled gating of RYR-2, causing after-depolarisation, leading to arrhythmias followed by structural changes due to altered intra-cellular Ca²⁺ levels. Three other genes implicated for ARVC, plakoglobin (Naxos disease), desmoplakin (ARVC8) and plakophilin (ARVC9) have prompted the speculation that ARVC is primarily a disease of desmosomes. But identification of TGF-3 for ARVC1 and the role of all these three genes (plakoglobin, desmoplakin and plakophilin) in cardiac morphogenesis indicate some kind of signal-transducing pathway disruption in the condition. The finding that ARVC as a milder form of Uhls anomaly indicates similar ontogeny for the condition. Further, discovery of apoptotic cells in the autopsy of the right ventricular myocardium of ARVC patients does indicate a common pathway for different types of ARVCs, which is more specific for the right ventricular myocardium involving desmosomal plaque proteins, growth factors and Ca²⁺ receptors.     

Clinical usefulness of immunohistochemistry for plakoglobin,N-cadherin,and connexin-43 in the diagnosis of arrhythmogenic right ventricular cardiomyopathy

Diagnosing arrhythmogenic right ventricular cardiomyopathy (ARVC) is often challenging because no single diagnostic tool is available to detect the disease. We evaluated whether analysis of plakoglobin, N-cadherin, and connexin-43 immunoreactivity can be used as a significant test in diagnosis of ARVC. We selected subjects with suspicion of ARVC (n=22) in patients who underwent endomyocardial biopsy (EMB) in Kyungpook National University Hospital (n=1326). The patients (n=22) were classified into definite ARVC patients (n=17) and borderline ARVC (n=5). We selected control subjects (n=20) who were autopsied and died of non-cardiac disease. Hematoxylin-eosin, Masson’s trichrome, and immunohistochemical stains for plakoglobin, N-cadherin, and connexin-43 were used for all specimens. Reduced immunoreactivity of plakoglobin was observed in 13 (76%) of the 17 patients with a definite ARVC and in 4 (80%) of the 5 patients with a borderline ARVC. All subjects displayed no significant reduction of the immunoreactivity for connexin-43 as well as for N-cadherin. Our investigation revealed that the immunohistochemical analysis for plakoglobin had an accuracy of 81%, 76% sensitivity, and 84% specificity in diagnosis of ARVC. Results of our study showed that the immunohistochemical analysis of plakoglobin had a relatively high sensitivity and specificity in ARVC, but immunohistochemistry for plakoglobin alone could not be relied upon as a diagnostic test for ARVC. We confirmed that N-cadherin and connexin-43 had no diagnostic value in ARVC.     

Reevaluation of genetic variants previously associated with arrhythmogenic right ventricular cardiomyopathy integrating population-based cohorts and proteomics data

Arrhythmogenic right ventricular cardiomyopathy (ARVC) is one of the most common causes of sudden cardiac death in young people. Patients diagnosed with ARVC may experience increased likelihood of development of anxiety and depression, emphasizing the need for accurate diagnosis. To assist future genetic diagnosis and avoidance of misdiagnosis, we evaluated the reported monogenic disease-causing variants in ARVD/C Genetic Variants Database, Human Gene Mutation Database, and ClinVar. Within the aforementioned databases, 630 monogenic disease-causing variants from 18 genes were identified. In the genome Aggregation Database, 226 of these were identified; 68 of which were found at greater than expected prevalence. Furthermore, 37/226 genetic variants were identified amongst the 409 000 UK biobank participants, 23 were not associated with ARVC. Among the 14 remaining variants, 13 were previously found with greater than expected prevalence for a monogenic variant. Nevertheless, they were associated with serious cardiac phenotypes, suggesting that these 13 variants may be disease-modifiers of ARVC, rather than monogenic disease-causing. In summary, more than 10% of variants previously reported to cause ARVC were found unlikely to be associated with highly penetrant monogenic forms of ARVC. Notably, all variants in OBSCN and MYBPC3 were found, making these unlikely to be monogenic causes of ARVC.     

The p.A897KfsX4 frameshift variation in desmocollin-2 is not a causative mutation in arrhythmogenic right ventricular cardiomyopathy

Mutations in genes encoding desmosomal proteins have been reported to cause arrhythmogenic right ventricular cardiomyopathy/dysplasia (ARVC/D), an autosomal-dominant disease characterised by progressive myocardial atrophy with fibro-fatty replacement. We screened 112 ARVC/D probands for mutations in desmocollin-2 (DSC2) gene and detected two different amino-acid substitutions (p.E102K, p.I345T) and a frameshift variation (p.A897KfsX4) in 7 (6.2%) patients. DSC2a variant p.A897KfsX4, previously reported as a p.E896fsX900 mutation, was identified in five unrelated probands. Four of them were found to carry one or two mutations in different ARVC/D genes. Unexpectedly, p.A897KfsX4 variation was also found in 6 (1.5%) out of 400 control chromosomes. In vitro functional studies showed that, unlike wild-type DSC2a, this C-terminal mutated protein was localised in the cytoplasm. p.A897KfsX4 variation affects the last five amino acids of the DSC2a isoform but not of DSC2b. In contrast with what we found in other human tissues, in the heart DSC2b is more expressed than DSC2a, suggesting that relative deficiency of DSC2a might be compensated by isoform b. In conclusion, DSC2 gene mutations are not frequently involved in ARVC/D. The p.A897KfsX4 variation, identified in several Italian healthy control subjects, which affects only one of the two DSC2 isoforms, may be considered a rare variant, though possibly affecting phenotypic expression of concomitant ARVC/D mutations.     

Pathologic evidence of extensive left ventricular involvement in arrhythmogenic right ventricular cardiomyopathy.

Arrhythmogenic right ventricular cardiomyopathy (also known as arrhythmogenic right ventricular dysplasia) is characterized by adipose or fibroadipose tissue replacement of the right ventricular myocardium, whereas the left ventricle is substantively spared. Two cases of the disease with evidence of extensive left ventricular involvement at pathologic examination are described. Hearts from two patients who died suddenly showed full-thickness right ventricular fatty infiltration associated with extensive left ventricular involvement (greater than 50% of myocardial thickness). These findings might explain the reported clinical features of left ventricle dysfunction in a subset of patients with arrhythmogenic right ventricular cardiomyopathy. In view of the biventricular involvement of the disease, it should simply be termed "arrhythmogenic cardiomyopathy."     

In vivo evidence of apoptosis in arrhythmogenic right ventricular cardiomyopathy.

Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a primary heart muscle disease characterized by progressive atrophy of the right ventricular myocardium with fibro-fatty replacement and the risk of electrical instability and sudden death. The disease is often familial and the aetiopathogenesis is still unknown. Recently apoptosis (genetically determined cell death) was postulated to account for progressive loss of myocardium. To establish whether apoptosis is present in ARVC, right ventricular endomyocardial biopsies from 20 patients with clinical and histological diagnosis of ARVC were examined by electron microscopy and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling method (TUNEL). Apoptotic index was calculated as the percentage of positive nuclei in sections stained by TUNEL. Cell proliferation activity was also assessed by argyrophilic staining of the nucleolar organizer region (AgNOR) and MIB-1 antibody analysis. Twenty biopsies taken from patients during monitoring of cardiac transplantation (grade 0 rejection) served as control. Occurrence of apoptosis was correlated with clinical history duration and the presence of acute symptoms and signs like angina, pyrexia, erythrocyte sedimentation rate and creatine phosphokinase elevation, as well as ST segment elevation on basal electrocardiogram. Electron microscopy and TUNEL revealed presence of apoptotic myocytes in seven cases (35%) with a mean apoptotic index of 24.4+/-9.8. The remaining 13 patients and all of the 20 controls were negative both at the electron microscopy and TUNEL. Presence of apoptosis appeared to be significantly related to clinical history duration of less than 6 months (P < 0.001) and presence of acute symptoms and signs (P = 0.007). AgNOR staining and MIB-1 antibody analysis ruled out cell proliferation activity. In conclusion, apoptosis is present in endomyocardial biopsies of patients with ARVC, especially in the early symptomatic phase of disease. Myocardial destruction with replacement by fat may be episodic rather than gradual and continuous.     

致心律失常性右室心肌病心力衰竭期的病理特点分析

目的 通过分析致心律失常性右室心肌病(ARVC)心力衰竭期的病理改变,以进一步了解其临床分期与病理表型的关系.方法 从2004-2007 年在阜外心血管病医院接受心脏移植的心力衰竭病例中,收集病理诊断为ARVC的受体心脏8例,测量心脏重量,评价左右心室心腔扩张、心肌细胞肥大、脂肪浸润、纤维化、附壁血栓和伴发心肌炎等指标,注意左心室受累情况,并进行病理分型.结果 8例中的7例为经典型(即右心室改变为主),1例为左优势型(左心室改变为主),未见双室型病例.组织学均为纤维脂肪型,未见单纯脂肪型病例.经典型病例的右心室中、重度扩张,少数有室壁瘤形成,其中6例伴左心室受累,受累左心室轻、中度扩张,心肌广泛间质纤维化,部分病例伴替代性疤痕,而脂肪浸润量小,多局限于心外膜下.左心室心肌细胞肥大普遍.而左优势型的左心室重度扩张,弥漫间质纤维化和局部透壁性脂肪浸润.8例中3例左心室明显肥厚,3例查见双室附壁血栓,1例伴局灶性心肌炎.结论 ARVC心力衰竭期的左心室受累多见而严重,左心室间质纤维化突出,心肌细胞肥大明显,但脂肪替代少见和局限.左、右心室多扩张,可见附壁血栓,应注意与扩张型心肌病等鉴别.     

Genetic testing of candidate genes in arrhythmogenic right ventricular cardiomyopathy/dysplasia

Arrhythmogenic right ventricular cardiomyopathy/dysplasia (ARVC/D) is a rare cardiac genetic disease characterized by the presence of structural alterations in the right ventricle which may cause ventricular arrhythmias and may induce sudden cardiac death. ARVC/D has been associated with mutations in genes encoding myocyte adhesion proteins. However, only 30%-50% of patients have mutations in these genes. Genetic testing is useful in obtaining a diagnosis, particularly in individuals who do not completely fulfill clinical criteria, thereby also enabling the undertaking of preventive strategies in family members. The main goal of this study was to identify mutations in candidate genes associated with intercalate disks that could be potentially involved in ARVC/D pathogenesis. We analyze a cohort of 14 Spanish unrelated patients clinically diagnosed with ARVC/D without any genetic alteration in all previously known responsible genes. Thus, a genetic screening has been performed in 7 additional potential candidate genes (ACTC1 -actin alpha cardiac muscle 1-, CDHN -cadherin 2 type 1 or N-cadherin-, CTNNA1 -catenin alpha 1-, Cx43 or GJA1 -gap junction protein alpha 1-, MVCL -Metavinculin-, MYL2 -myosin light chain 2- and MYL3 -myosin light chain 3-) by direct sequencing analysis. Our genetic analysis did not identify any disease-causing mutation. Thirty single nucleotides polymorphisms were found, six of them novel. In conclusion, our ARVC/D Spanish cohort has not shown any mutations in the analyzed candidate genes despite their involvement in formation and maintenance of the intercalated disk.     

Clinical and pathologic study of two siblings with arrhythmogenic right ventricular cardiomyopathy.

ARVC is a cardiomyopathy in which the right ventricular myocardium is replaced by fibroadipose tissue. Males are affected slightly more often than females and, in those cases which are familial, the pattern of inheritance is usually autosomal dominant with incomplete penetrance. We examined the hearts of two sisters, ages 17 and 14, with no family history of heart disease. The older sibling, who was previously considered healthy, died suddenly, while the younger sibling developed congestive heart failure and received a cardiac transplant. An autopsy of the older sibling and examination of the younger sibling's excised heart revealed severe examples of ARVC with minor differences. A thick cap of fibroadipose tissue covered most, if not all, of each right ventricle and was transmural in some areas. Microscopically, lelt ventricles contained extensive myocyte disarray and multifocal fibrosis. The coronary arteries displayed intimal hyperplasia with disruption of the internal elastic lamina, similar to fibromuscular dysplasia. These two cases comprise a unique familial grouping in a polymorphic disease. Despite the male predominance and autosomal dominant inheritance in ARVC, the only members affected in this family were female, and an autosomal dominant pattern of inheritance, even with incomplete penetrance, would be unusual. In addition, we identified changes in the coronary arteries similar to fibromuscular dysplasia and corroborated recently reported changes in the left ventricle of patients with ARVC, providing evidence that this disease, in its most severe form, involves the entire heart.     

Programmed cell death in the myocardium of arrhythmogenic right ventricular cardiomyopathy in children and adults.

Arrhythmogenic right ventricular cardiomyopathy (ARVC) is characterized by fibrofatty replacement of the right ventricular myocardium. Recently, the myocardial loss in ARVC has been suggested to be related to apoptosis. However, it is still unknown whether this phenomenon is already established in the myocardium of pediatric cases with this disease. We examined the histopathologic characteristics of the ventricular myocardium in specimens obtained from 10 patients, including 3 children with ARVC, and investigated the occurrence of apoptosis in the myocardium by terminal deoxyribonucleotidyl transferase-mediated dUTP-biotin nick-end labeling (TUNEL) assay and agarose-gel electrophoresis of DNA. Endomyocardial biopsy specimens from the 10 cases and a necropsy sample from one adult case with ARVC were examined. Histopathologic examination of biopsy specimens from the pediatric cases revealed extensive fibrosis. Typical fatty infiltration was demonstrated in one of the 3 pediatric cases. These findings were similar to those in adult cases; the histopathologic index based on the severity of myocardial damage, including myocyte degeneration and fibrosis, was not significantly different from that in adult cases. TUNEL assay revealed positive reactivity of the myocardial cells. The apoptotic index was 1.4 +/- 0.4% in children and 1.6 +/- 0.5% in adults (difference not statistically significant). Agarose-gel electrophoresis of a DNA extract of the myocardial tissue of the autopsy case revealed DNA fragmentation. Cases with idiopathic ventricular tachycardia and control cases with a cardiac transplant (with no rejection) had minimal histopathologic findings and negative reactivity in the TUNEL assay. These results indicate that myocardial damage is already established in cases diagnosed as ARVC in childhood, and suggest that the myocardial damage is closely related to apoptosis in children, as well as in adults, in this disease.     

Identification of a PKP2 gene deletion in a family with arrhythmogenic right ventricular cardiomyopathy

Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a primary heart muscle disease characterized by progressive myocardial loss, with fibro-fatty replacement, and high frequency of ventricular arrhythmias that can lead to sudden cardiac death. ARVC is a genetically determined disorder, usually caused by point mutations in components of the cardiac desmosome. Conventional mutation screening of ARVC genes fails to detect causative mutations in about 50% of index cases, suggesting a further genetic heterogeneity. We performed a genome-wide linkage study and a copy number variations (CNVs) analysis, using high−density SNP arrays, in an ARVC family showing no mutations in any of the desmosomal genes. The CNVs analysis identified a heterozygous deletion of about 122 kb on chromosome 12p11.21, including the entire plakophilin-2 gene and shared by all affected family members. It was not listed on any of available public CNVs databases and was confirmed by quantitative real-time PCR. This is the first SNP array-based genome-wide study leading to the identification of a CNV segregating with the disease phenotype in an ARVC family. This result underscores the importance of performing additional analysis for possible genomic deletions/duplications in ARVC patients without point mutations in known disease genes.     

Identification of mutations in the cardiac ryanodine receptor gene in families affected with arrhythmogenic right ventricular cardiomyopathy type 2 (ARVD2)

Arrhythmogenic right ventricular dysplasia type 2 (ARVD2, OMIM 600996) is an autosomal dominant cardiomyopathy, characterized by partial degeneration of the myocardium of the right ventricle, electrical instability and sudden death. The disease locus was mapped to chromosome 1q42--q43. We report here on the physical mapping of the critical ARVD2 region, exclusion of two candidate genes (actinin 2 and nidogen), elucidation of the genomic structure of the cardiac ryanodine receptor gene (RYR2) and identification of RYR2 mutations in four independent families. In myocardial cells, the RyR2 protein, activated by Ca(2+), induces the release of calcium from the sarcoplasmic reticulum into the cytosol. RyR2 is the cardiac counterpart of RyR1, the skeletal muscle ryanodine receptor, involved in malignant hyperthermia (MH) susceptibility and in central core disease (CCD). The RyR2 mutations detected in the present study occurred in two highly conserved regions, strictly corresponding to those where mutations causing MH or CCD are clustered in the RYR1 gene. The detection of RyR2 mutations causing ARVD2, reported in this paper, opens the way to pre-symptomatic detection of carriers of the disease in childhood, thus enabling early monitoring and treatment.