Alessandra Lorenzon

Mutations in Desmoglein-2 Gene Are Associated With Arrhythmogenic Right Ventricular Cardiomyopathy

Background— Arrhythmogenic right ventricular cardiomyopathy (ARVC) is an inherited cardiomyopathy characterized by progressive myocardial atrophy with fibrofatty replacement. The recent identification of causative mutations in plakoglobin, desmoplakin (DSP), and plakophilin-2 (PKP2) genes led to the hypothesis that ARVC is due to desmosomal defects. Therefore, desmoglein-2 (DSG2), the only desmoglein isoform expressed in cardiac myocytes, was screened in subjects with ARVC. Methods and Results— In a series of 80 unrelated ARVC probands, 26 carried a mutation in DSP (16%), PKP2 (14%), and transforming growth factor-&bgr;3 (2.5%) genes; the remaining 54 were screened for DSG2 mutations by denaturing high-performance liquid chromatography and direct sequencing. Nine heterozygous DSG2 mutations (5 missense, 2 insertion-deletions, 1 nonsense, and 1 splice site mutation) were detected in 8 probands (10%). All probands fulfilled task force criteria for ARVC. An endomyocardial biopsy was obtained in 5, showing extensive loss of myocytes with fibrofatty tissue replacement. In 3 patients, electron microscopy investigation was performed, showing intercalated disc paleness, decreased desmosome number, and intercellular gap widening. Conclusions— This is the first investigation demonstrating DSG2 gene mutations in a significant number of ARVC-unrelated probands. Cardiac phenotype is characterized clinically by typical ARVC features with frequent left ventricular involvement and morphologically by fibrofatty myocardial replacement and desmosomal remodeling. The presence of mutations in desmosomal encoding genes in 40% of cases confirms that many forms of ARVC are due to alterations in the desmosome complex.

Compound and Digenic Heterozygosity Contributes to Arrhythmogenic Right Ventricular Cardiomyopathy

OBJECTIVES The aim of this study was to define the genetic basis of arrhythmogenic right ventricular cardiomyopathy (ARVC). BACKGROUND Arrhythmogenic right ventricular cardiomyopathy, characterized by right ventricular fibrofatty replacement and arrhythmias, causes sudden death. Autosomal dominant inheritance, reduced penetrance, and 7 desmosome-encoding causative genes are known. The basis of low penetrance is poorly understood. METHODS Arrhythmogenic right ventricular cardiomyopathy probands and family members were enrolled, blood was obtained, lymphoblastoid cell lines were immortalized, deoxyribonucleic acid was extracted, polymerase chain reaction (PCR) amplification of desmosome-encoding genes was performed, PCR products were sequenced, and diseased tissue samples were studied for intercellular junction protein distribution with confocal immunofluorescence microscopy and antibodies against key proteins. RESULTS We identified 21 variants in plakophilin-2 (PKP2) in 38 of 198 probands (19%), including missense, nonsense, splice site, and deletion/insertion mutations. Pedigrees showed wide intra-familial variability (severe early-onset disease to asymptomatic individuals). In 9 of 38 probands, PKP2 variants were identified that were encoded in trans (compound heterozygosity). The 38 probands hosting PKP2 variants were screened for other desmosomal genes mutations; second variants (digenic heterozygosity) were identified in 16 of 38 subjects with PKP2 variants (42%), including desmoplakin (DSP) (n = 6), desmoglein-2 (DSG2) (n = 5), plakophilin-4 (PKP4) (n = 1), and desmocollin-2 (DSC2) (n = 1). Heterozygous mutations in non-PKP 2 desmosomal genes occurred in 14 of 198 subjects (7%), including DSP (n = 4), DSG2 (n = 5), DSC2 (n = 3), and junctional plakoglobin (JUP) (n = 2). All variants occurred in conserved regions; none was identified in 700 ethnic-matched control subjects. Immunohistochemical analysis demonstrated abnormalities of protein architecture. CONCLUSIONS These data suggest that the genetic basis of ARVC includes reduced penetrance with compound and digenic heterozygosity. Disturbed junctional cytoarchitecture in subjects with desmosomal mutations confirms that ARVC is a disease of the desmosome and cell junction.

Multiple mutations in desmosomal proteins encoding genes in arrhythmogenic right ventricular cardiomyopathy/dysplasia

BACKGROUND Arrhythmogenic right ventricular cardiomyopathy/dysplasia (ARVC/D) is a progressive cardiomyopathy showing a wide clinical spectrum in terms of clinical expressions and prognoses. OBJECTIVE This study sought to estimate the occurrence of compound and double heterozygotes for mutations in desmosomal proteins encoding genes in a cohort of ARVC/D Italian index cases, and to assess the clinical phenotype of mutations carriers. METHODS Fourty-two consecutive ARVC/D index cases who fulfilled the International Task Force diagnostic criteria were screened for mutations in PKP2, DSP, DSG2, DSC2, and JUP genes by denaturing high-performance liquid chromatography (DHPLC) and direct sequencing. RESULTS Three probands (7.1%) showing a family history of sudden death carried multiple mutations. Family screening identified an additional 7 multiple-mutation carriers. Among the 7 double heterozygotes for mutations in different genes, 2 were clinically unaffected, 2 were affected, and 3 showed some clinical signs of ARVC/D even if they did not fulfill the diagnostic criteria. Two compound heterozygotes for mutations in the same gene and 1 subject carrying 3 different mutations showed a severe form of the disease with heart failure onset at a young age. Moreover, multiple-mutation carriers showed a higher prevalence of left ventricular involvement (P = .025) than single-mutation carriers. CONCLUSION Occurrence of compound and double heterozygotes in ARVC/D index cases is particularly relevant to mutation screening strategy and to genetic counseling. Even if multiple-mutation carriers show a wide variability in clinical expression, the extent of the disease is higher compared to that in single-mutation carriers.

Compound and digenic heterozygosity predicts lifetime arrhythmic outcome and sudden cardiac death in desmosomal gene-related arrhythmogenic right ventricular cardiomyopathy.

Background—Mutations in genes encoding for desmosomal proteins are the most common cause of arrhythmogenic right ventricular cardiomyopathy (ARVC). We assessed the value of genotype for prediction of lifetime major arrhythmic events and sudden cardiac death (SCD) in desmosomal gene–related ARVC. Methods and Results—The overall study population included 134 desmosomal gene mutation carriers (68 men; median age 36 years [22–52]) from 44 consecutive ARVC families undergoing comprehensive genetic screening. The probability of experiencing a first major arrhythmic event or SCD during a lifetime was determined by using date of birth as start point for the time-to-event analysis, and was stratified by sex, desmosomal genes, mutation types, and genotype complexity (single versus multiple mutations). One hundred thirteen patients (84%) carried a single desmosomal gene mutation in desmoplakin (n=44; 39%), plakophilin-2 (n=38; 34%), desmoglein-2 (n=30; 26%), and desmocollin-2 (n=1; 1%), whereas 21 patients (16%) had a complex genotype with compound heterozygosity in 7 and digenic heterozygosity in 14. Over a median observation period of 39 (22–52) years, 22 patients (16%) from 20 different families had arrhythmic events, such as SCD (n=1), aborted SCD because of ventricular fibrillation (n=6), sustained ventricular tachycardia (n=14), and appropriate defibrillator intervention (n=1). Multiple desmosomal gene mutations and male sex were independent predictors of lifetime arrhythmic events with a hazard ratio of 3.71 (95% confidence interval, 1.54–8.92; P=0.003) and 2.76 (95% confidence interval, 1.19–6.41; P=0.02), respectively. Conclusions—Compound/digenic heterozygosity was identified in 16% of ARVC-causing desmosomal gene mutation carriers and was a powerful risk factor for lifetime major arrhythmic events and SCD. These results support the use of comprehensive genetic screening of desmosomal genes for arrhythmic risk stratification in ARVC.

Mutations in the area composita protein αT-catenin are associated with arrhythmogenic right ventricular cardiomyopathy

AIMS Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a major cause of juvenile sudden death and is characterized by fibro-fatty replacement of the right ventricle. Mutations in several genes encoding desmosomal proteins have been identified in ARVC. We speculated that αT-catenin, encoded by CTNNA3, might also carry mutations in ARVC patients. Alpha-T-catenin binds plakophilins and this binding contributes to the formation of the area composita, which strengthens cell-cell adhesion in contractile cardiomyocytes. METHODS AND RESULTS We used denaturing high-performance liquid chromatography and direct sequencing to screen CTNNA3 in 76 ARVC patients who did not carry any mutations in the desmosomal genes commonly mutated in ARVC. Mutations c.281T > A (p.V94D) and c.2293_2295delTTG (p.del765L) were identified in two probands. They are located in important domains of αT-catenin. Yeast two-hybrid and cell transfection studies showed that the interaction between the p.V94D mutant protein and β-catenin was affected, whereas the p.del765L mutant protein showed a much stronger dimerization potential and formed aggresomes in HEK293T cells. CONCLUSION These findings might point to a causal relationship between CTNNA3 mutations and ARVC. This first report on the involvement of an area composita gene in ARVC shows that the pathogenesis of this disease extends beyond desmosomes. Since the frequency of CTNNA3 mutations in ARVC patients is not rare, systematic screening for this gene should be considered to improve the clinical management of ARVC families.

Missense mutations in desmocollin-2 N-terminus, associated with arrhythmogenic right ventricular cardiomyopathy, affect intracellular localization of desmocollin-2 in vitro.

BackgroundMutations in genes encoding desmosomal proteins have been reported to cause arrhythmogenic right ventricular cardiomyopathy (ARVC), an autosomal dominant disease characterised by progressive myocardial atrophy with fibro-fatty replacement.We screened 54 ARVC probands for mutations in desmocollin-2 (DSC2), the only desmocollin isoform expressed in cardiac tissue.MethodsMutation screening was performed by denaturing high-performance liquid chromatography and direct sequencing.To evaluate the pathogenic potentials of the DSC2 mutations detected in patients affected with ARVC, full-length wild-type and mutated cDNAs were cloned in eukaryotic expression vectors to obtain a fusion protein with green fluorescence protein (GFP); constructs were transfected in neonatal rat cardiomyocytes and in HL-1 cells.ResultsWe identified two heterozygous mutations (c.304G>A (p.E102K) and c.1034T>C (p.I345T)) in two probands and in four family members. The two mutations p.E102K and p.I345T map to the N-terminal region, relevant to adhesive interactions.In vitro functional studies demonstrated that, unlike wild-type DSC2, the two N-terminal mutants are predominantly localised in the cytoplasm.ConclusionThe two missense mutations in the N-terminal domain affect the normal localisation of DSC2, thus suggesting the potential pathogenic effect of the reported mutations. Identification of additional DSC2 mutations associated with ARVC may result in increased diagnostic accuracy with implications for genetic counseling.

Clinical phenotype and diagnosis of arrhythmogenic right ventricular cardiomyopathy in pediatric patients carrying desmosomal gene mutations

Background Arrhythmogenic right ventricular cardiomyopathy (ARVC) is an inherited heart muscle disease carrying a risk of sudden death. Information about the clinical features during childhood and the age at disease onset is scanty. Objective The aim of the study was to describe the ARVC phenotype as its initial clinical manifestation in a pediatric population (<18 years) with desmosomal gene mutations. Methods Fifty-three ARVC desmosomal gene mutation carriers (mean age 12.3 ± 3.9 years) were investigated by electrocardiogram (ECG), signal-averaged ECG, 24-hour Holter, echocardiogram, and contrast-enhanced cardiac magnetic resonance (CMR). Results None of the children ≤10 years old fulfilled the 1994 criteria, as opposed to six (33%) aged 11–14 years and eight aged >14 years (42%). At the end of follow-up (9 ± 7 years), 21 (40%) fulfilled the 1994 diagnostic criteria (mean age 16 ± 4 years). By using the 2010 criteria in subjects aged ≤18 years, 53% were unaffected, versus 62% by using the traditional criteria. More than two-thirds of affected subjects had moderate-severe forms of the disease. Contrast-enhanced CMR was performed in 21 (40%); of 13 unaffected gene mutation carriers, six showed ARVC morphological and/or tissue abnormalities. Conclusion In pediatric ARVC mutation carriers, a diagnosis was achieved in 40% of cases, confirming that the disease usually develops during adolescence and young adulthood. The 2010 modified criteria seem to be more sensitive than the 1994 ones in identifying familial pediatric cases. Contrast-enhanced CMR can provide diagnostic information on gene mutation carriers not fulfilling either traditional or modified criteria. Management of asymptomatic gene mutation carriers remains the main clinical challenge.

Desmin Mutations and Arrhythmogenic Right Ventricular Cardiomyopathy

Arrhythmogenic right ventricular cardiomyopathy (ARVC) is an inherited heart muscle disease characterized by fibrofatty replacement of the myocardium and ventricular arrhythmias, associated with mutations in the desmosomal genes. Only a missense mutation in the DES gene coding for desmin, the intermediate filament protein expressed by cardiac and skeletal muscle cells, has been recently associated with ARVC. We screened 91 ARVC index cases (53 negative for mutations in desmosomal genes and an additional 38 carrying desmosomal gene mutations) for DES mutations. Two rare missense variants were identified. The heterozygous p.K241E substitution was detected in 1 patient affected with a severe form of ARVC who also carried the p.T816RfsX10 mutation in plakophilin-2 gene. This DES substitution, showing an allele frequency of <0.01 in the control population, is predicted to cause an intolerant amino acid change in a highly conserved protein domain. Thus, it can be considered a rare variant with a possible modifier effect on the phenotypic expression of the concomitant mutation. The previously known p.A213V substitution was identified in 1 patient with ARVC who was negative for mutations in the desmosomal genes. Because a greater prevalence of p.A213V has been reported in patients with heart dilation than in control subjects, the hypothesis that this rare variant could have an unfavorable effect on cardiac remodeling cannot be ruled out. In conclusion, our data help to establish that, in the absence of skeletal muscle involvement suggestive of a desminopathy, the probability of DES mutations in ARVC is very low. These findings have important implications in the mutation screening strategy for patients with ARVC.

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.

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.