Genetic Studies On Hypertrophic Cardiomyopathy And Dilated Cardiomyopathy

Background:(1)Hypertrophic cardiomyopathy(HCM)and dilated cardiomyopathy(DCM)are common cardiomyopathic diseases,with a prevalence of 1/500 for HCM and 1/250 for DCM.HCM is the most common cause of sudden death for atheletes and young people(<30 years old).High-risk HCM patients often suffer from dis-ease-related complications,such as sudden cardiac death(SCD),heart failure(HF)and atrial fibrillation(AF).DCM is the leading cause of SCD,HF,and heart trans-plantation in both children and adults.Half the DCM patients will die in 5 years be-cause of SCD and HF after initial diagnosis.(2)HCM is the most-common genically inherited form of heart disease,and over 23 genes have been identified as HCM-causing genes since 1990s.It’s now well recongnized that HCM is a disease of sarcomere because over 70%HCM-causing mutations are found in MYH7 and MYBPC3 genes.However,there is still a substan-tial deficit in our understanding of the molecular mechanisms of HCM.Pharmacolog-ical treatments could not alter the natural progress of HCM because they are not de-signed to target HCM-specific pathways.A more thorough understanding of the mo-lecular mechanisms of HCM is warranted,which will accelerate the development of novel and targeted drugs for HCM.In our premier pedigree study,remarkable differ-ences of clinical manifestations and prognosis were observed in 7 members harboring the same MYBPC3 mutation,indicating the existence of modifying genes to regulate the penetrance of HCM-related phenotypes.(3)In contrast to HCM in which mutations in MYH7 and MYBPC3 could account for 70%of all clinical cases,the fractional contribution of each of the>40 genes identi-fied for DCM significantly varies,with 25%for TTN and<1%to 6%for others.De-spite the large number of genes identified as DCM-causing,only 35%of the genetic causes could be explained.This indicates that additional mutations in other novel genes remain to be discovered.Meanwhile,the overall profile of genetics in sporadic Chinese DCM patients has not ever been addressed,which could be conducted with the help of the newly emerging second-generation sequencing(NGS)technologyMethods:Familial and sporadic cases of HCM and DCM hospitalized in the Affili-ated Drum Tower Hospital,Nanjing University School of Medicine were enrolled.Targeted NGS was performed following the extraction of genomic DNA from blood cells.More than 100 genes,including cardiomyopathy-and channelopathy-related genes,were simultaneously sequenced.Data was filtered through the analysis of mu-tation type,the comparison to reference databases,the co-segregation analysis and functional-predicting analysis,and validation using Sanger sequencing.Whole-exome sequencing(WES)was employed when no consistent result was obtained through targeted NGS.Results:(1)Seven members in a large HCM pedigree were identified harboring the MYBPC3 c.3624del mutation.Remarkable differences were observed regarding clini-cal manifestations in these seven mutation carriers:three had early onset and sever symptoms;one 65-year-old woman had no clinical symptoms but an echocardiog-raphy-confirmed increase in LV wall thickness;the other three had no clinical symp-toms or abnormity in echocardiography.Targeted NGS revealed that all three MYBPC3 mutation carriers with sever symtoms also carried a CACNB2 c.1598C>T mutation,and the other four MYBPC3 mutation carriers with either no phenotype or partial HCM-related phenotype did not have the CACNB2 mutation(2)Targeted NGS on a DCM pedigree identified 4 potential disease-causing mutations,i.e.DSG2 c.1974T>A,DSP c.2360A>G,SCO2 c.274G>A,and AKAP9 c.4870 G>C mutations.None of these 4 mutations co-segregated following overlapping analysis in this pedigree,indicating rare mutations in novel gene existed.Therefore we performed WES in four memebers of this family.Twenty-nine single nucleotide variation and 43 insertion/deletion mutations remained following overlapping analysis.Further analy-sis including referring to worldwide databases,examing the expression files of these proteins,primary function analysis,and validation in another DCM patients in this family whose genomic DNA was not sent for WES because of limited amount of DNA.After these procedures,only 1 mutation,i.e.NBAS c.A5227G,p.M 1743V re-mained.This mutation was not observed in 100 healthy subjects from the same co-hort.(3)Fourty-two "probably disease-causing" mutations were identified following tar-geted NGS in 100 idiopathic DCM patients in China.The majority of disease-causing mutations could be found in TTN(17%)and DMD(10%)genes.Genes encoding sar-comere,cytoskeleton and ion flux had the highest number of mutations,accouting for over 70%of the mutationsConclusions:(1)We first reported that mutaions in sarcomere and Ca2+ channel coexisted in HCM patients.In our HCM pedigree,MYBPC3 c.3624delC was the pri-mary disease-causing mutation and CACNB2 c.1598C>T functioned as a disease modifying mutation to promote the penetrance of HCM-related phenotype(2)In our DCM pedigree,WES revealed that NBAS c.A5227G mutatin co-segerated within this family and proved to be disease-causing following bioinformatic analyses NBAS might be a novel DCM-causing gene,and nonsense-mediated mRNA decay might be the primary mechanism of its damaging function in DCM.(3)We first conducted the genetic study in a large number of Chinese DCM popula-tion using NGS technology.We firstly showed the overall mutation profiles of Chi-nese DCM patients,with TTN and DMD having the largest number of disease-causing mutations.