Targeted Gene Panel Sequencing In Chinese Children With Sporadic Non-syndromic Congenital Heart Disease And Primary Cardiomyopathy

Background:Congenital heart disease(CHD)is a genetically heterogeneous cardiac malformation disease that resluts in significant morbidity and mortality in infants.Approximatly 80%of CHD patients are non-syndromic,occuring sporadically with various gene mutations.However,the genetic causes of sporadic non-syndromic CHD(NS-CHD)are largely unkonwn.In this study,we systematically identified the mutation in 510 children with NS-CHD,and investigated the frequency of mutated known CHD-associated genes.Moreover,attempt to identify novel potential pathogenic CHD-associated genes.Methods:We designed a gene panel comprising of 232 known and candidate CHD-associated genes and performed a deep targeted sequencing in 510 Chinese Han children with sporadic NS-CHD.Firstly,genomic DNA were isolated followed by library preparation,probe hybridzation,PCR enrichment,and high-quality sequencing data were acquired unilizing an Illumina Hiseq 4000 platform.Secondly,we mapped the reads to the reference human GRch37 with BWA-MEM(version 0.7.9a),called variants with GATK HaplotypeCaller(version 3.2),filtered out variants failing the variant quality score recalibration(VQSR)criteria and annotated called variants with ANNOVAR(version 20141112).Thirdly,we filtered out the potential deleterious variants and analysised the mutation frequency in known CHD-associated genes.Finally,we utilized four in silico programs(Polyphen2,SIFT,Mutation assessor and LRT)and applied strict standards to predict functional consequence in candidate CHD-associated genes.The cutoff scores of four computational programs were set as follows:Polyphen2 score>0.9,SIFT socre<0.06,Mutation Assesser>3.5,LRT<0.000117.In order to identify genes enriched with variants,we used 1000g East Asians exome dataset as a control population.In addition,we combined tools from CLAMMS.GATK4,DNAcopy and CNTools to detect the copy number variations across 510 NS-CHD samples.Results:A total of 23 potential heterozygous causative variants were identified in?4.5%patients,including 15 unique novel loss-of function mutations(CITED2 c.-273delG,CRELD1 c.620delG,MYH6 c.735+1G>A,MYH6 Y583X,SMAD6 Q218X,TDGF1 S48fs,MYH7 c.4519+2T>A,MYH7 N911fs,MYBPC3 E611X,MYIBPC3 comp.het,MYOCD c.972-1G>A,MYOCD c.121+1>T,NOTCH1 S2353fs,NOTCH1 F993fs,PBX4 R135X).We identified two pathogenic/likely pathogenic variants ZFPM2 M703L and NKX2-5 E203G in-2.5%patients.Among them,the known pathogenic variant M703L of ZFPM2 gene was significantly enriched in our cohort(p=2.7017×10-8).We observed a large amount of novel or rare missense in CHD-associated genes that are functionally deleterious,envolutionarily conservative.Among them,the NOTCH1,MYBPC3,CHD7 and KMT2D are the frequently mutated genes.The mutation frequencies are 7%,5.1%,4.9%and 4.5%respectively.The rare mutations of NOTCH1 are significantly enriched in our NS-CHD cohort comparing with control(p=0.010514).Moreover,our analysis indicated a high prevalence of mutations in signaling transduction genes.In addition,we identified two novel potential causative CHD genes,AARS2 and COX15.There is no big CNV changes cross samples.There are a few 1 copy deletions and the highest copy number among genes across all patients is 3.05.Conclusion:We identified 15 novel potential causative LoF variants and 2 novel CHD associated genes(AARS2 and COX15).We further demonstrated that NOTCH1 is the most frequently mutant known CHD-associated gene and the high mutation frequencies in genes encoding signal transduction proteins.Targeted next-generation sequencing of currently well-selected genes is a dependable approach to identify potential deleterious variants and informative genetic factors to sporadic NS-CHD.Objectives:Primary cardiomyopathy have a substantial inherited etiology and predispose to sudden cardiac death,which supplied a great inducement to identify pathogenic variants in patients.The purpose of this study is to evaluate the diagnostic value of targeted gene panel sequencing in Chinese patients with primary cardiomyopathy.Methods:A total of three Chinese Han inherited cardiomyopathy families and 45 unrelated cardiomyopathy patients were collected,and drew 2-4ml peripheral bloods in patients before informed consent.We designed a panel including 88 known CM-associated genes and 144 candidate CM-associated genes.Genomic DNA were isolated followed by quality control,library preparation,probe hybridazation,cBot cluster preparation and sequencing on Illumina Hiseq 4000 platform.We achieved a high-quality sequencing raw data,which were filtered to clean data by FASTQC software.Next,we mapped the reads to the reference human hg19 with BWA-MEM(version 0.7.9a),called variants with GATK HaplotypeCaller(version 3.2),filtered out variants failing the variant quality score recalibration(VQSR)criteria and annotated called variants with ANNOVAR(version 20141112).The pathogenic or potential causative variants were filtered by minor allele frequency(MAF)in large databases,regional and molecular consequence.All causative variants were compared to databases(dbSNP142,ESP6500,ExAC,ExAC_eas,1000g,1000g_eas,Clinvar and OMIM,etc.)for evaluating the minor allele frequency,pathogenicity and whether novel or known pathogenic variants.We adopted Blueprint Genetics' variant classification scheme to evaluate all candidate rare variants.Results:We identified a heterozygous dominant nonsense variants MYBPC3 c.G1831T p.E611X in a three generation's familial hypertrophic cardiomyopathy family.MYBPC3 E611X co-segregated with cardiac phenotype.The IGV were performing for validated MYBPC3 E611X.MYBPC3 E611X was absent from dbSNP142,ExAC,ESP6500 and 1000 genomes,therefore it is a novel pathogenic site in MYBPC3 gene.Secondly,a heterozygous dominant nonsynonymous variants MYL2 c.G172A p.R58Q were observed in a trio family of hypertrophic cardiomyopathy.MYL2 R58Q co-segregated with cardiac phenotype and were reported in Clinvar databases as a pathogenic variants.Previously reported showed that R58Q mutant directly intervened myofibrillogenesis,affected MYL2 gene on phosphorylation and Ca(2+)binding.However,we firstly reported MYL2 R58Q and supported pathogenic evidence of R58Q in Chinese Han hypertrophic family.At last,we identified a heterozygous deleterious potentially causative variant SDHA c.T341G p.M114R in a identical male twins with dilated cardiomyopathy.We utilized four in silico programs(Polyphen2,SIFT,Mutation assessor and LRT)and applied strict standards to predict functional consequence.The cutoff scores of four computational programs were set as follows:Polyphen2 score>0.9,SIFT socre<0.06,Mutation Assesser>3.5,LRT<0.000117.In total,we identified 13 pathogenic/likely pathogenic variants(3 nonsense,2 frameshift,1 splicing,7 rare missense)in 45 unrelated cardiomyopathy patients.Of the causative genes in our study,63%(12/19)were sarcomere genes(MYBPC3 11.3%,MYH7 5.7%,MYL2 3.8%,TTN 3.8%,TNNT2 1.9%),21%were Z-disc and desmosomal genes(NEXN 3.8%,PKP2 3.8%)?In addition,one pathogenic variant was observed in mitochondrial gene.Conclusion:Our diagnostic yield in Chinese patients with primary cardiomyopathy was 35.8%.Diagnostic yield was significantly higher in familial vs.sporadic cases(75 vs.28.9%,p=0.019).In summary,panel-based,high-quality targeted next generation sequencing enables high diagnostic yield especially in familial form of cardiomyopathy.