The Research On Molecular Genetic Characteristics Of Cardiomyopathy And Their Correlation With Environmental Factors In Yunnan

Based on ventricular morphology and function,inherited cardiomyopathies are classified as a group of cardiovascular disorders,including dilated cardiomyopathy(DCM),hypertrophic cardiomyopathy(HCM),arrhythmogenic right ventricular cardiomyopathy(ARVC),and restrictive cardiomyopathy(RCM).Of them,HCM and DCM are the major types.As the leading cause of sudden death in young athletes and adolescents,HCM was highly related with gene mutation with autosomal dominant inheritance,at least 30 genes are associated with HCM.DCM is characterized by remarkable genetic heterogeneity;it is the leading cause of heart failure and heart transplantation.It has also been documented that more than 50 genes are associated with dilated cardiomyopathy,in Mendelian autosomal dominant patterns;however,mitochondrial DNA,recessive as well as X-linked inheritance may also be involved.Gene sequencing is the major method for detecting genetic diseases.Inherited cardiomyopathy has been associated wuth multiple gene mutions that could be sequenced by using conventional capillary electrophoresis.However,this kind of method is time-consuming,laborious and expensive.Next-generation semiconductor sequencing technology(Ion torrent PGM)can simultaneously sequencing on multiple target genes,with the advantages of high throughput,fast and cheap,etc.,has became the best choice for small genome sequencing,genetic variation of multiple genes.As a result of genetic and environmental factors,and it has a significant ethnic difference.Inherited cardiomyopathy was widely prevalent in a variety of ethnic in Yunnan Province,the special geographical environment and climate.To our knowledge,this is the first systematic screening for patients with HCM and DCM in Yunnan;it is also necessary for the controlling and prevation of HCM and DCM.In current study,based on Ion torrent PGM sequencing platform,we established a method for sequencing on 12 human genes including MYH7,MYBPC3,TNNT2,TNNI3,MYH6,TPM1,ACTC1,PRKAG2,MYL2 and MYL3,which highly related with HCM and DCM.Using this method,these genes from 127 and 44 recruited and unrelated HCM and DCM patients were sequenced,respectively.As the results,(1)8 novel mutations of TNNT2-p.Gly180Ala,MYBPC3-p.Cys1124Phe,MYH6p.Arg1047Cys,MYH6-p.Thr1253Met,MYL3-p.Asp126Gly,MYH7-p.Asn885Thr,MYH7-p.Arg54Gln and TNNT2-p.Arg296His were identified with molecular genetic analyzing.Except for MYL3-p.Asp126Gly,the other gene mutations are statistically related with the occurrence of dilated and/or hypertrophic cardiomyopathies.(2)According to spectrum of gene mutations,17.3%patients with DCM and 36.4%patients with HCM was identified to harbor one or more mutations.Positive mutations are associated with 36.4%of HCM patients,which consistent with previously reported.In paralle,DCM has a low detection rates in comparion with previous documents.This indicates that environmental factors may be lead to the occurrence of HCM and DCM,but the exact cause remains unclear.(3)To elucidate the association between genotype and phenotype in patients with DCM and HCM,clinical characteristics are compared between groups of patients with and without gene mutations.It was demonstrated that patients with DCM and HCM harboured DCM or HCM-associated mutations had been diagnosed with their condition at a significantly younger age than patients with DCM and HCM without the mutations,and positive of gene mutations patients with DCM and HCM have a severe clincal phenotype.An analysis of the altitude and climate were performed in DCM and HCM of patients with gene mutation,the result shown that high altitude and low temperature environment can exacerbate the DCM cardiac dysfunction.(4)One familial DCM and seven HCM patients were geneticed testing by ion torrent PGM sequencer,and the result shown that 5 pairs of heterozygous mutations were detected in five diferent(Miao,Hani,Yi,Naxi and Pumi)ethnic groups,respectively.It exhibited a severe clinical phenotype in patients with familal cardiomyopathies patients that carried a double heterozygous mutation;and then it is required a multiple genes that suffered genetic testing in HCM patients.Those results illustrated that it has a special genetic mutations in patients with familial HCM in Yunnan.Furthermore,taken high occurenced mutations of MYH7-c.1987C>T,TNNI3-c.370G>C,MYH7-c2155C>T,TNNI3-c.433C>G and PRKAG2-c.298G>A,TaqMan-MGB probe was designed and real time PCR method was established.The method is economy,fast and accurate,lower requirements for equipment,which provided a technical methods and molecular diagnostic platform for genetic testing in patients with HCM.In summary,we have established a high-throughput genetic testing method on DMC and HCM by using next generation semiconductor sequencing(NGS)technology platform.It is the first time to clarify the genetic characteristics of HCM and DCM in Yunnan,and to elucidate the relationship of genotype and clinical phenotype.Finally,we established a method that was used for testing the "hot spot"mutaions in patients with HCM,which based on real time PCR with TaqMan-MGB.This research is the first systematically study of inherited cardiomyopathy in Yunnan;it provides an important theoretical data and technology platform for genetic testing and risk evaluation of individuals with HCM and DCM.