Correlation Of Primary Cardiomyopathy And Mitochondrial Mutations In Southwest China

Objective:dilated cardiomyopathy (Dilated Cardiomyopathy, DCM) and hypertrophic cardiomyopathy (Hypertrophic Cardiomyopathy, HCM) are the most common types of primary cardiomyopathies,which are all belong to the category of inherited cardiomyopathy and have obvious genetic background. The present study has found that more than 30 kinds of genes associated with DCM, more than 20 kinds of genes, more than 1400 kinds of mutations associated with HCM. As the particularly dependence of heart to oxidative metabolism,mitochondrial’s structure and function change in the role of cardiac damage paid more and more attention as the key part of oxidative phosphorylation system (oxidative Phosphorylation, OXPHOS).So far, more than 300 pathogenic mitochondrial genes (mt) DNA point mutations were found, including protein-coding genes, tRNA genes, rRNA genes, and most commonly in mitochondria tRNA (mt tRNA) and mitochondrial protein-coding genes cytb (mt cytb). As China reported poor mitochondrial mutations,whether the mitochondrial gene mutations or polymorphisms existed in southern China,or even in China;whether the mutations or polymorphisms are related to primary cadiomyopathy or not remain no sufficient evidence. Therefore, this experiment screening the mitochondrial genome of the most prone to cause change on mt cytb, mt tRNA of Gln, Ile, Leu, Lys, Pro, Val, Ser gene to find the presence or absence of mitochondrial mutations or polymorphisms in DCM and HCM,and compare whether it is hot or new mutation to investigate the incidence of mitochondrial genetic changes in patient condition in southwest China, and analyzes its relevance to the two kinds of myocardial disease, thus provide more evidence and directions of genetic correlation of primary cardiomyopathy with mitochondrial genes in southwest China;which is likely to provide a theoretical basis for future gene therapy as well. Methods:The study selected Han population in southwest China, of which 120 patients with DCM, one case of a clear pedigree;HCM patients 65 cases, two case of clear pedigree; 200 cases of healthy controls. Subjects underwent cardiac color Doppler, ECG examination and genetic mapping for family members. Reaserch using case-control study,the mt cytb, mt tRNA Gln, mt tRNA lle, mt tRNA Leu,mt tRNA Lys, mt tRNA Pro, mt tRNA Val, mt tRNA Ser entire gene sequence as the target sequence amplification using DNA reagents Box to extract DNA from peripheral blood of patients and healthy controls as template,using primer primer 5 software todesign specific primers, amplifying target sequences by polymerase chain reaction (PCR) and put the amplification products to agarose gel electrophoresis, Goldview staining technology, ultraviolet spectrometer measurement, gel imaging, Sanger dideoxy chain termination method to determine the nucleotide sequence of all of the amplification product, analyzing the obtained sequence diagram, and the nucleotide sequence determined in the above-described database with the complete genome sequence alignment, obtained with DCM or HCM-related polymorphisms (Single Nucleotide Polymorphisms, SNP) or mutations, calculate SNP frequency between patients and healthy control subjects, using independent samples t test to compare patients with statistically significant difference between polymorphic loci healthy controls. Clinical hearing impairment associated with early-onset DCM direct line mitochondrial genome sequencing and analysis; mutation point and 200 healthy control subjects for comparison and analysis of sequences. Results:1. Clinical characteristics of the study: myocardial thickening in patients with HCM, and majority of HCM patients with mild heart failure, ECG changes are priority to T wave changes. DCM patients with heart significantly increased, decreased left ventricular function, cardiac function in the majority of patients-severe heart failure based on NYHA stages, ECG changes are priority to QRS widening.2. Mitochondrial DNA results analysis in HCM patients: ①one mutation (m.4395A> G) on the mt tRNA Gln gene is found in HCM family 2, HCM family 1 and the remaining patients with HCM, DCM patients and 200 healthy controls were no such site change. This site is mutated to A at nucleotide G, the position of the mutation is located in exon mt tRNA Gln, Gln functions belonging to the conserved region, the type of mutation is a point mutation. Mutation of this site is consistent with a sporadic HCM patients found in Tunisia and it causes mt tRNA Gln structure change, affects protein translation process, for pathogenic mutations may be great.②HCM family 1 gene mutation was not found above, also found no mt cytb, mt tRNA Gln, mt tRNA lle, mt tRNA Leu,mt tRNA Lys, mt tRNA Pro, mt tRNA Val, mt tRNA Ser mutation or gene loci. ③ The rest were not found in patients with HCM on mt cytb, mt tRNA Gln, mt tRNA lle,mt tRNA Leu, mt tRNA Lys, mt tRNA Pro, mt tRNA Val, mt tRNA Ser mutation or gene loci.3.DCM patients with genetic analysis results: ① Compared by dbSNPC database of mitochondiral(mitochondrial genome reference version "NC-012920.1"), in southwest China crowd found 5 mt cytb mitochondrial gene polymorphism loci, namely:m.14766C> G (rs527236041), m.14783C> T (rs527236042), m.15043G> A(rs527236043), m.15301A> G (rs527236045), m.15326 A> G (rs2853508). mt cytb gene is located on the mitochondrial genome 14747-15887 bit,both of the entire sequence are in the exons, three conserved sites in its functional area, domestic no outside m.15043G> A, m.15301A> G two sites with HCM, DCM-related reports, m.14766C> G, m.15326 A> G two sites with the Danish population HCM polymorphic loci consistent, m.15043G> A consistent with the American people in DCM polymorphic loci,②calculating over 5 mt cytb gene loci frequency in DCM patients and healthy controls, using SPSS 19 for both two independent samples t test frequency show a significant difference (P<0.05); and the multiple Status bit frequency in DCM patients was significantly higher than healthy controls. Because mitochondrial genetic follow strict maternal inheritance and genetic bottleneck effect, it does not apply to its Hardy-Weinberg equilibrium and test results of this experiment with a representative patient population. ③ Found five new mutations in mt cytb gene of DCM patients, respectively m.15263C> T, m.15290C> A, m.15330T> G, m.15338C> G, m.15386T> G,which are located in mt cytb exons and its function conserved region.200 healthy controls are no changes in the above sites. These mutations are single base mutation, and causes a change in the corresponding amino acid sequence mutations occur with non-hybrid map. ④The study of the DCM pedigree patients who are associated with hearing impairment line mitochondrial genome sequencing test results found on mt tRNA Glu gene mutation sites:m.14696A> G, all HCM patients in this study and the rest of the DCM’s and 200 healthy controls are no change in this locus. This site base mutation from A to G, sequencing analysis of the mutation rate was 95.98%, the mutation is a point mutation type. Finnish people have reported mutations agree with mitochondrial myopathy, but this mutation was not found associated with primary cardiomyopathy. ⑤mt cytb, mt tRNA Ile, mt tRNA Leu,mt tRNA Lys, mt tRNA Pro, mt tRNA Val, mt tRNA Ser mutation or gene loci are not found patients of DCM in southwest China.Conclusion: The clinical features of an object of this study indicate that in southwest China HCM, DCM patients with heart failure and arrhythmia as the main clinical manifestations, most patients are sporadic, some are hereditary.2. Southwest China HCM population and changes in mitochondrial gene correlation analysis: ① the same patient with Tunisia presence of mitochondrial gene mutations in patients with HCM pedigrees in southwest China (mt tRNA Gln 4395A> G), has confirmed that the mutation site HCM pathogenesis and significant correlation in the Chinese people whose genetic background may be one causative gene of familial HCM (Familiar Hypertrophic Cardiomyopathy, FHCM).② HCM population in southwest China may not exist mt cytb, mt tRNA Ile, mt tRNA Leu, mt tRNA Lys, mt tRNA Pro, mt tRNA Val, mt tRNA Ser gene mutations or polymorphisms.3. DCM and mitochondrial genes in southwest China changed Correlation Analysis:①We have firstly discovered 5 mt cytb mitochondrial gene polymorphism loci DCM crowd in southwest China, where m.15043G> A, m.15301A> G, m.15326 A> G polymorphism loci for new hair can be used as a genetic suggestion of DCM and mt cytb polymorphic loci analysis of; m:14766C> G, m.15326 A> G two polymorphic loci genera common polymorphic loci HCM, DCM, indicating that mitochondrial gene polymorphism also has the characteristics of genetic heterogeneity. ②Five polymorphic loci mt cytb gene frequency in DCM patients are significantly higher than healthy controls, indicating that the the incidence of existence of five polymorphic loci are correlated with DCM in southwest China.③ mt cytb genes found in a five novo mutations, sequencing pattern prompt mutations are homozygous mutation, the mutation site is located five conserved regions most likely through oxidative phosphorylation, the formation of the respiratory chain, reducing ATP generation, causing mitochondrial dysfunction associated with DCM.④The novo mutations on mt tRNA Glu14696A> G is associated with the onset of DCM in southwest China’s population, combined with the results of the Finnish study, we hypothesize that mutation m.14696A> G may be shared with mitochondrial myopathy and DCM; at the same time Clinical characteristics of patients with this mutation analysis sources speculate that the mutation may be one associated with early-onset hearing loss in patients with DCM disease genes. In addition, the genetic characteristics of apparent familial DCM mitochondrial gene mutation has a significant threshold effect,⑤mt tRNA Ile,mt tRNA Leu,mt tRNA Lys, mt tRNA Pro, mt tRNA Val, mt tRNA Ser gene mutations or polymorphisms may not existed on DCM patients of southwest China’s population.