New Inheritance Patterns And Genotype-phenotype Analysis Of Hypertrophic Cardiomyopathy

BackgroundHypertrophic cardiomyopathy（HCM）is the most common inherited cardiac disorder characterized by unexplained left ventricular hypertrophy（LVH）,affecting at least 1 in 500 individuals in the general population.Genetic analysis in the last three decades has established a classic concept that HCM is a dominant Mendelian disease mainly caused by single rare mutations in genes encoding sarcomere proteins in more than HCM patients,including MYH7,MYBPC3,MYL2,MYL3,TNNT2,TNNI3,TPMl and ACTC1.However,the genetic causes in up to 40%of HCM patients remain to be explored.ObjectivesThe present study aimed to explore the potential genetic factors predisposing to HCM in a multicenter case-control study.MethodsThere were a total of 2831 HCM patients and 2113 non-HCM controls from one discovery cohort（986 patients and 761 controls）and three replication cohorts（529 patients and 307 controls,646 patients and 468 control,670 patients and 577 controls）included in the final analysis of this study.We performed whole exome sequencing in patients from discovery cohort.Both single variant association screen for common variants and gene-based association screen for rare variants were performed in the discovery cohort to search for novel genetic factors contributing to HCM.The primary finding was replicated in three independent studies.Variants in 8 sarcomere genes were classified as pathogenic,likely pathogenic,uncertain significant,likely benign or benign using the criteria of American College of Medical Genetics and Genomics and the Association for Molecular Pathology.Then patients with any variant of pathogenic,likely pathogenic,or uncertain significance were grouped as SARC+,and those without such variants were grouped as S ARC-.The families of proband were followed up,and the cosegration of genetic factor and HCM was analyzed.RNA sequencing and transcripts analysis were performed on the myocardial tissue of HCM patients who underwent Morrow septal myectomy to explore the potential mechanism of the found genetic factor for HCM.ResultsIn gene-based association screen,rare variants in the two most common causal genes,MYH7 and MYBPC3,were greatly enriched in cases（P=5.56×10-31 and 3.43×10-9,respectively）.There was no novel gene attaining the genome-wide significance.In the single-variant association screen,there was a relatively common variant Arg79Cys in TNNI3（rs3729712,NC₀00019:g.55667616G>A;NM₀00363:c.235C>T;NP₀00354:p.Arg79Cys）associated with HCM[odds ratio（OR）=3.31,95%confidence interval（CI）:2.12-5.15],exceeding the Bonferroni-corrected threshold for global significance（P=1.3×10-7）.The association was further replicated in three independent studies.The ORs[95%CI]were estimated to be 4.72[1.70,13.06],P=2.8×10-3;5.40[2.70,10.82],P=2.0×10-6;and 8.33[4.31,6.10],P=3.O×10-10.Meta-analysis of all four studies estimated the OR[95%CI]of per risk allele to be 4.62[3.39,6.30]（P=3.7×10-22）.There were 284（10.0%）cases carrying Arg79Cys variant,including 25 homozygotes and 259 heterozygotes.The association signal was mainly driven by the high frequency of heterozygotes in.cases,which accounted for 91.2%of the Arg79Cys carrers in the HCM cases.A total 151 additional familial members,including 72 heterozygotes and 2 homozygotes of Arg79Cys,were collected from 42 families.Except for 3 heterozygotes each carrying a pathogenic variant diagnosed as HCM,none of the other 69 heterozygotes presented LVH.In contrast,the co-segregation of homozygous Arg79Cys with HCM were found in two pedigrees,implying that the variant could lead to HCM in a recessive manner.Moreover,across the four studies there were 25（0.88%）patients with homozygous Arg79Cys of 2831 HCM cases,but none in the 2113 controls（P=1.2×10-6）.The risk allele was more prevalent in SARC-patients than SARC+（OR[95%CI]=2.08[1.56,2.76];P=4.6×10-7）.The Arg79Cys was more frequent in the SARC-group than in the SARC+group（OR[95%CI]=2.08[1.56,2.76];P=4.6×10-7）,and its effect size was more than doubled in the SARC-group（OR[95%CI]=5.27[3.65,7.61];P=7.0×10-19）than controls.Moreover,although carrying causal sarcomere variants,the SARC+patients still had a higher frequency of Arg79Cys than the controls（OR[95%CI]=2.64[1.76,3.96];P=2.8×10-6）.RNA sequencing indicated there was no difference in TNNI3 expression levels between patients with and withour Arg79Cys variant,which indicates there was no disruptive mutation in the region to alter the expression levels of TNNI3.Moreover,there was neither different splicing pattern between the two groups nor aberrant splicing event in either group,which indicates there was no intronic mutation causing HCM by alternative splicing.The results of RNA sequencing and analysis of TNNI3 transcript suggested the association signal between Arg79Cys and HCM is not due to linkage disequilibrium between Arg79Cys and another rare pathogenic variant at the TNNI3 locus.Human variation databases showed that Arg79Cys was an East Asian-specific variant In the 1000 Genomes database there are six heterozygotes out of 504 East Asians[minor allele frequency（MAF）=0.0060],but none in other populations.In the Asian Admixed Genomes Consortium database,there are 17 heterozygotes out of 920 East Asians（MAF=0.0092）.In the gnomAD database,there are 120 heterozygotes out of 9,656 East Asians（MAF=0.0062）.ConclusionsThis study demonstrated that a relatively common variant Arg79Cys in TNNI3 can predispose to HCM.Nearly 10.0%Chinese patients with HCM carrying this variant which was East Asian-specific.Our data revealed the existence of a non-Mendelian genetic mechanism of HCM.It provides important insights into the complexity and ethnic differences of the genetic architecture of HCM which provided new theory supporting for understanding the pathogenesis of HCM and the genetic basis of HCM patients in China.BackgroundClinical diagnosis of hypertrophic cardiomyopathy（HCM）is mainly based on cardiac imaging findings of left ventricular hypertrophy（LVH）.A number of phenocopy diseases that only,or mainly,affect the cardiac system can mimic HCM by presenting LVH.These phenocopy diseases include Fabry’s disease（FD）,AMPK associated glycogen-storage cardiomyopathy,Danon disease（DD）and transthyretin amyloid cardiomyopathy.Early diagnosis and differential diagnosis of phenocopy diseases from HCM are critical because of differences in diseases management and treatment strategies.These phenocopy diseases are Mendelian disorders caused by mutations in phenocopy genes,including GLA for FD,PRKA G2 for AMPK associated glycogen-storage cardiomyopathy,LAMP2 for DD,and TTR for transthyretin amyloid cardiomyopathy.Nevertheless,HCM was caused by mutation in sarcomere genes.The genetic testing was reported helpful to discriminate phencopy diseases and HCM.However,the diagnostic accuracy of phenocopies using genetic testing in patients diagnosed as HCM has not been carefully evaluated.ObjectivesThe present study aimed to evaluate the diagnostic accuracy of phenocopies using genetic testing in patients diagnosed as HCM.MethodsWhole exome sequencing was performed in 1000 patients clinically diagnosed as HCM,and the causal genes of HCM phenocopies,including GLA for FD,PRAKG2 for AMPK associated glycogen-storage cardiomyopathy,LAMP2 for DD and TTR for transthyretin amyloid cardiomyopathy,were analyzed.The the identified variants in phenocopy genes were classified as pathogenic（P）,likely pathogenic（LP）,uncertain significance（VUS）,likely benign or benign according the guideline of the American College of Medical Genetics.All cases with pathogenic,likely pathogenic,or uncertain significance variants were carefully evaluated by histopathological analyses,clinical examinations and pedigree analysis,and the specificity of genetic testing was assessed using a head-to-head comparison.Families of patients with phenocopy variants were recruited,and genotype and phenotype of them were assessed.Plasma α-Gal A activity was measured in GLA variant carriers.Heart specimens with GLA or PRKA G2 variants were evaluated after hematoxylin-eosin staining.Immunohistochemical staining in the myocardium of LAMP2 variant carriers using an anti-LAMP2 mouse monoclonal antibody.Congo red staining of myocardium specimens was used to confirm transthyretin amyloid cardiomyopathy.All cases were followed up,and the primary endpoint was cardiovascular death.ResultsA total of ten P/LP and ten VUS phenocopy gene variants were detected in 20 patients,including four in GLA,four in PRKAG2,six in LAMP2,and six in TTR.After clinical evaluation by histopathological analyses,clinical manifestations,or pedigree analyses,thirteen（61.9%）subjects with phenocopy variants were confirmed as having phenocopy diseases,including four with FD,one with AMPK associated glycogen-storage cardiomyopathy,six with DD,and two with transthyretin amyloid cardiomyopathy.Notably,all of the ten P/LP variant carriers were confirmed as phenocopy,whereas only three VUS（one in GLA and two in LAMP2）carriers were consistent with the diagnosis of phenocopy.Phenocopies were not determined in four VUS carriers and were excluded in three VUS（one in TTR and two in PRKAG2）carriers.Compared with the HCM group,the phenocopy patients were younger（38.5±20.0 years vs.48.0±14.5 years,P=0.020）,more were female（76.9%vs.35%,P=0.002）,more had a family history（53.8%vs.19.3%,P=0.002）,and they had a larger left atrial diameter（41.8±7.1 mm vs.36.4±11.2 mm,P=0.007）.Furthermore,we found patients with phenocopy diseases had a higher risk of cardiovascular death than did the remaining patients（hazard ratio=6.10,95%confidence interval 1.88-19.79,P=0.003）.ConclusionsOur study demonstrates that genetic testing can discriminate phenocopy diseases from HCM with a high specificity by a detection of P/LP variants in phenocopy genes.Clinical evaluation should be carefully performed for accurate diagnosis of phenocopy diseases in those with VUS variants.Owing to the malignant outcomes of phenocopy patients,the genetic testing of phenocopy diseases is especially important for disease identification for early intervention or special therapy.BackgroundAs the most common inherited cardiovascular disease,hypertrophic cardiomyopathy（HCM）is mainly caused by mutations in genes encoding structural components of the sarcomere and it exhibits an autosomal dominant inheritance pattern.However,nearly a half of patients have identifiable disease-causing variants in these genes,suggesting that new disease-associated genes remained to be discovered.Mutations in genes encoding desmosomal proteins play a crucial role in the pathologic mechanism of fibrofatty replacement,including DSP encoding desmoplakin,PKP2 encoding plakophilin-2,DSG2 encoding desmoglein-2,DSC2 encoding desmocollin-2,and JUP encoding plakoglobin.Deleterious rare variants in genes encoding desmosome proteins have been identified as the essential basis of arrhythmogenic cardiomyopathy（ACM）,but the relationship between desmosomal variants and HCM remains unknown.ObjectivesThe present study aimed to evaluate the association of desmosomal variants with HCM,and to systematically analyze the phenotypic characteristics of HCM patients with desmosomal variants.MethodsWhole exome sequencing was performed in 1000 HCM patients and 761 non-HCM controls to search for deleterious rare variants in genes encoding desmosomal proteins including PKP2,JUP,DSC2,DSG2,and DSP.The truncating variants,including nonsense,frameshift,and splice-site mutations affecting positions ±1 and ±2,with minor allele frequency<0.01%in the Genome Aggregation Database and Exome Aggregation Consortium database were labled deleterious rare truncating variants.Deleterious rare missense variants were defined according to the following criteria:1)all multiple bioinformatic predictors agreed the variants would be deleterious,including Sorting Intolerant from Tolerant score,Polymorphism Phenotyping v2 score,and Combined Annotation Dependent Depletion（produces a phred-like score）;2)variants were absence in the Genome Aggregation Database or Exome Aggregation Consortium database;3)variants were conservative with Genomic Evolutionary Rate Profiling>2.Clinical phenotypes were assessed in all HCM patients,and patients with deleterious rare desmosomal variants underwent evaluation of ACM according to the 2010 revised Task Force Criteria.ResultsThere were 13 truncating variants of the desmosome genes identified in the study cohort,including eight variants in PKP2,one in DSC2,three in DSG2,and one in JUP.In detail,there were four nonsense variants,seven frameshift variants,and two variants located in the splicing regions.Besides the truncating variants,there were 14 deleterious rare missense variants identified,consisting of two variants in DSC2,two in DSG2,eight in DSP,and two in JUP.A total of 27 deleterious rare desmosomal variants were present in 24（2.4%）HCM patients and 5（0.66%）controls.The variants were more prevalent in the HCM patients than in the controls（P=0.004）.The majority of patients possessing deleterious rare desmosomal variants could not be diagnosed as ACM.Moreover,the patients with deleterious rare desmosomal variants possessed several distinctive clinical features comparing to patients without such variants,including a higher incidence of non-sustained ventricular tachycardia（29.2%vs.4.5%,P<0.001）,left bundle branch block（33.3%vs.1.6%,P<0.001）,and right ventricular involvement（29.2%vs.0.30%,P<0.001）.ConclusionsOur study indicated deleterious rare desmosomal variants contributed to HCM,and were associated with distinctive clinical features of HCM.