Study On The Genetic, Pathological And Clinical Characteristics Of Arrhythmogenic Cardiomyopathy

Part 1.Genotype-phenotype Correlation Study of Arrhythmogenic CardiomyopathyAimsTo identify genotype-phenotype correlations in a multicenter Chinese-Swiss cohort of patients with arrhythmogenic cardiomyopathy(ACM)using clinical exome analysis by next-generation sequencing(NGS).MethodsClinical data from 186 unrelated patients with ACM from the Chinese and Swiss ACM cohorts were prospectively studied.NGS covering at least 66 genes being associated with cardiomyopathies/channelopathies was applied to identify pathogenic mutations,including 13 ACM-related genes.Bioinformatic analysis of the 13 ACM related genes was performed and detected variants were confirmed by Sanger sequencing.Primary end-point was defined as heart transplantation or death due to heart failure.Secondary end-point was defined as major arrhythmic cardiac events(MACE).The composite end-point included primary end-point and MACE.A multivariable Cox proportional hazards regression model was employed to assess the significance and independence of factors in prediction of heart transplantation and MACE during lifetime.Characteristics of the patients with specific variants were compared and the cumulative incidence since birth until heart transplantation,MACE,and the composite end-point was determined by the Kaplan-Meier method.ResultsPatients from the Chinese cohort had a higher rate of left ventricular involvement at diagnosis(53%vs.38%,p=0.046).Chinese patients had a lower right ventricular ejection fraction(23.6±12.4%vs.40.2±9.3,p<0.001).Genetic background was different in the two cohorts(p=0.014),with more non-desmosomal variant carriers in patients from the Chinese cohort.More patients in the Chinese cohort received heart transplantation as compared to patients from the Swiss cohort(42%vs.4%,p<0.001),and less had MACE(38%vs.57%,p=0.014).Genetic mutations were identified in 96(52%)patients.Patients who were transplanted had less PKP2 variants(22%vs.2%,p=0.001),but more non-desmosomal variants(5%vs.21%,p=0.001),compared to those who were not.Patients who had MACE had more desmosomal variants(46%vs.31%,p=0.035)than those who did not,especially PKP2 variants(26%vs.8%,p=0.001).We entered gender and genotype in a multivariable proportional hazards Cox regression model.It revealed that non-desmosomal and multigenic variants were both independent risk factors of heart transplantation(HR=2.285,p=0.014 and HR=3.496,p=0.048,respectively),whereas PKP2 variants were a significant risk factor of MACE(HR=3.190,p<0.001)As compared to genotype-negative patients,genotype-positive patients had a higher incidence of arrhythmic syncope(36%vs.21%,p=0.021)and right ventricular outflow tract dimensions in both parasternal long-axis and short-axis(40.0±8.8mm vs.36.3±7.8mm,p=0.006 and 41.3±8.6mm vs.37.7±7.7mm,p=0.005).Genotype-positive patients had a lower MACE-free survival rate(p=0.032)and composite end-point free survival(p=0.011).When compared to patients with desmosomal variants,those with non-desmosomal variants had a higher rate of left ventricular dysfunction at diagnosis(34%vs.61%,p=0.034)and heart transplantation(21%vs.61%,p=0.001(.In addition,they had fewer implantable cardioverter defibrillator(ICD)implantations(52%vs.11%,p=0.002),but a lower heart transplantation free survival rate(p=0.005).Finally,we separated patients with desmosomal variants into three groups according to the location of the proteins in the desmosome.As compared to those with DSG2/DSC2 and those with PKP2 variants,patients with DSP variants had significantly more left ventricular involvement(91%vs.53%and 17%,p<0.001),and more frequently heart transplantation(55%vs.27%and 3%,p=0.001).Patients with DSG2/DSC2 variants had a larger right ventricular end-diastolic volume than those with PKP2 and DSP variants(318.1±126.2ml vs.200.5±73.6ml and 211.1±97.3ml,p=0.011).Patients with PKP2 variants had an increased need for VT ablation than those with DSG21DSC2 and DSP variants(53%vs.23%and 9%,p=0.008),and more MACE(73%vs.40%and 45%,p=0.027),so a lower MACE free survival rate(p=0.022),but a higher heart transplantation free survival rate(p=0.025).ConclusionsACM patients with specific mutations have distinct clinical characteristics and outcomes.PKP2 confers a strong arrhythmogenic phenotype,whereas DSP mutations,multigenic mutations,and non-desmosomal mutations put the patients at risk for left ventricular involvement and heart transplantation.Part 2.Recessive Variants in Plakophilin-2 Contributes to Early-Onset Arrhythmogenic Cardiomyopathy with Severe Heart Failure AimsBy using integrated analysis of whole genome(WGS)and whole transcriptome sequencing(RNAseq)to identify deep intronic variants and coding variants that cause aberrant splicing events in arrhythmogenic cardiomyopathy(ACM)patients,this leads to the hypothesis that recessive variants in PKP2 may lead to heart failure.The objective is to evaluate the clinical and pathological characteristics of recessive PKP2 variants ACM patients.MethodsWe performed WGS and RNAseq in 27 heart transplanted ACM patients.By integrated analysis of WGS/RNAseq we discovered that two patients with PKP2 variants were affected in recessive pattern.Then we screened recessive PKP2 variants in 47 non-heart transplant ACM patients to identify recessive PKP2 variants by captured sequencing.The baseline characteristics of the patients with recessive PKP2 variants were collected and we followed these patients.We performed family screening for the patients with recessive PKP2 variants and proband-specific DNA sequencing in their family members by Sanger sequencing.We compared the clinical characteristics of recessive PKP2 variant carrier and heterozygous PKP2 carrier.Immunofluorescence and western blot was performed in the heart section of heart transplanted ACM patients.ResultsAmong the two patients with PKP2 recessive variants who had heart transplantation,one patient had aberrant splicing arising from two intronic variants that led to exon skipping and exon retention events respectively.Among the non-heart transplant ACM patients,3 recessive PKP2 variant carriers were identified.All recessive PKP2 variant carriers had at least one truncating PKP2 variant.When compared with patients with PKP2 heterozygous variant,patients with recessive PKP2 variants showed significantly more prevalent early-onset of ACM-related symptoms(11.8 ± 5.9 years vs.27.4 ± 8.4 years,p<0.001),with a higher rate of abdominal distension(40%vs.0,p=0.040)and bilateral edema(60%vs.0,p=0.006),but a lower rate of palpitations(0 vs.100%,p<0.001).Moreover,patients with recessive PKP2 variants had more severe impaired LV structure and function,with a higher rate of left ventricular dilation(40%vs.0.p=0.060)and dyskinesia(100%vs 0,p<0.001),and a lower left ventricular ejection fraction(27.4 ± 9.8%vs.61.9 ± 6.0%,p<0.001).Patients with PKP2 recessive variants had both worse heart transplantation/death-free and major arrhythmia cardiac events-free survival rates as compared to patients with a single PKP2 heterozygous variant(p<0.001 and p=0.003,respectively).We examined truncating PKP2 variants in explanted heart and confirmed truncated PKP2 was not translated.The morphology of intercalated disc in myocardium derived from recessive PKP2 variants carriers was similar to normal heart suggesting little intercalated disc remodeling.ConclusionsRecessive variants in PKP2 could lead to advanced ACM with severe heart failure.Therefore,genetic testing would be of great utility in assisting risk stratification.Part 3.Clinical and Pathological Study of TTN and Sarcomere Variants in Arrhythmogenic CardiomyopathyAimsArrhythmogenic cardiomyopathy(ACM)is an inheritable heart disease characterized by fibro-fatty replacement of the myocardium.TTN variants and sarcomere variants were previously reported as a primary pathogenic factor for dilated cardiomyopathy(DCM)and hypertrophic cardiomyopathy(HCM),respectively.We detected TTN variants and sarcomere variants in ACM patients.The objective is to evaluate the clinical and pathological characteristics of TTN variants and sarcomere variants in ACM patients.MethodsWe performed captured sequencing in 84 ACM patients,including WGS in 35 patients who had heart transplantation.We analyzed TTN variants in 35 patients who received heart transplantation,and sarcomere variants in all ACM patients who had captured sequencing.Family screening was performed in all patients who had above variants,and specific variants testing in all available family members.Baseline characteristics of all the patients were collected and follow-up was performed for the probands.For the patients who had heart transplantation,we quantified myocardium,fibrous and adipose tissue in blocks of explanted heart.Clinical and pathological characteristics were compared between patients with TTN variants and others.Besides,we performed family screening,variants-specific DNA sequencing in screened family members and co-segregation analysis in all available families.ResultsTTN variants were detected in 11 patients(all missense,9 heterozygous and 2 oligogenic form).TTN truncating variant was absent in the cohort.Patients with TTN variants had a late onset age of the disease(31±13 years vs.17±3 years,P=0.049)and age of heart transplantation(41 ± 14 years vs.24±9 years,P=0.027),larger left ventricle end-diastolic diameter(62±10 mm vs.45± 10 mm,P=0.019),smaller right ventricular outflow tract(34± 14 mm vs 50±15 mm,P=0.046),more myocardium(40.8±29.4%vs.13.8±11.0%,P=0.017)and less adipose tissue(43.0±30.9%vs.66.9±18.5%,P=0.036)in right ventricle than those with desmosomal variants.There were few differences in between patients with TTN variants and those without variants.Pedigrees showed none of the family members with TTN missense variants had a disease phenotype,indicating a very low penetranceWe identified 6 sarcomere variants in 6(7%)patients,which were all definite ACM.Sarcomere variants were detected in NEBL,MYH7,MYH6 and TNNI3,with low prevalence in controls and predicted pathogenic in silico.Among these patients,three had previous detected PKP2 variants.Patients with sarcomere variants all experienced major arrhythmic cardiac event(MACE)with the average age of the first documented MACE being 41.2±11.0 years.Pedigrees analysis showed none of the sarcomere variants carriers among the family members were affected,indicating very low penetrance.ConclusionsTTN missense variants was commonly identified in ACM patients in this cohort,but hardly played a primary role in ACM as causative variants.We detected some sarcomere variants in our ACM cohort.Although those patients with sarcomere variants had severe arrhythmic burden,family co-segregation analysis didn't strongly support a primary role in the pathogenesis of ACM.