Gene Mutations Screening Highly Related With Cardiomyopathies In Yunnan And The Establishment Of TaqMan-MGB Detection Method

Cardiomyopathy is an inherited heart disease with abnormal cardiac structure and function.It has divided cardiomyopathy into two categories:Hypertrophic cardiomyopathy(HCM)and dilated cardiomyopathy(DCM).Dilated cardiomyopathy is a disease of myocardial systolic blood dysfunction,and its clinical manifestations are arrhythmia,heart failure,arterial embolism and so on.It is clinically characterized by left ventricular dilation with systolic dysfunction and affects at least 1/2500 of the general population worldwide.It has no other treatment except heart transplant operation.The mortality rate of this disease is very high and it can reach 15%-50%in 5 years.It has been reported more than 50 mutation of pathogenic genemutation which has a connection with the incidence of DCM.Pathogenic gene mutations play an important role in DCM pathogenesis and mostly follow Mendelian autosomal dominant patterns;however,recessive as well as X-linked inheritance and mitochondrial DNA may also be involved.Hypertrophic cardiomyopathy as a common inherited cardiomyopathy,its basic feature is the left ventricular and asymmetric septum hypertrophy;it is one of the most common causes of sudden cardiac death(SCD),especially in adolescents and young athletes.Gene mutation is the main molecular genetic basis of HCM,it mainly follows the autosomal dominant inheritance pattern,and the major of pathogenic genes can encode sarcomeric proteins.It encoded cardiac β myosin heavy chain gene(MYH7)which is the main pathogenic gene.MYH7 locates on 14q12,MYH7 gene has an important role in energy supply and maintaining the concentration of Ca2+ in the cardiac muscle cells,and it is one of the most important subunit of myosin.MYH7 gene was mainly expressed in heart muscle and skeletal muscle,it is first detected which has associated with pathogenesis gene of HCM,approximately 30%-50%of patients with HCM is associated with MYH7 gene.Hypertrophic cardiomyopathy was concerned by researcher in China,however,it is not enough.So it is significant that detected of MYH7 gene mutations.Considerable evidence indicates that cardiomyopathy is difference in different ethnic groups.Yunnan Province,it located in the southwest of China,which has a special race,it was reported fewer about genetic testing with cardiomyopathy.therefore,this study established a database of patients with hypertrophic cardiomyopathy and dilated cardiomyopathy in Yunnan Province,Gene mutation screening on 24 patients with dilated cardiomyopathy and one familial hypertrophic cardiomyopathy by using Sanger sequencing,and then the bioinformatics and molecular genetic analysis of the gene mutaion,and the results,four mutations was detected,which are associated with cardiomyopathy;and also we described for the first time the double heterozygous mutations of MYH7,c.77C>T plus c.3134G>A in familial hypertrophic cardiomyopathy patients;Finally,We have established a method of double(MYH7,c.77C>T plus c.3134G>A)mutaion with hypertrophic cardiomyopathy by using TaqMan-MGB.The results of this study are as follows:(1)it was not detected pathogenic mutantion by high throughput sequencing of 11 pathogenic genes(MYH7,MYBPC3,TNNT2,TNNI3,MYH6,TPM1,ACTC,SCN5A,LMNA,MYL2 and MYL3)with 24 sporadic DCM patients,then these four genes(VCL,DES,TNNC1 and MYPN)was detected by using Sanger sequencing.The results shown that four novel mutations(TNNC1,c.8A>T,VCL,c.625A>T,DES,c.1157G>A and MYPN,c.1888G>A)was found,which were highly correlated with the dilated cardiomyopathy by bioinformatics,molecular genetics,and case control studies;(2)It had detected one double heterozygous hot spot mutations(MYH7,c.77C>T and c.3134G>A)in a familial hypertrophic cardiomyopathy;(3)it was established method of hot spot mutation(MYH7,c.77C>T and c.3134G>A)by TaqMan-MGB,this method is convenient,economical,fast and accurate,so it is suitable for wide popularization.It can provide a gene detection technology and method for molecular diagnosis on patients with hypertrophic cardiomyopathy.In conclusion,Sanger sequencing technology was used to screen the gene mutation with dilated and hypertrophic cardiomyopathy in this study.The molecular genetic basis of hypertrophic and dilated cardiomyopathy in Yunnan Province was expounded to a certain degree,and between the mutation genotype and clinical phenotype was confirmed.Also the TaqMan-MGB fluorescence real-time PCR detection method was established for the detection of target genes.These results first time provided the information for the prevention and treatment of cardiomyopathy in Yunnan Province,but also provide a technical platform,which can used to detect of the pathogenic gene mutation(MYH7,c.77C>T plus c.3134G>A)in hypertrophic cardiomyopathy.