Mutations Profile In Chinese Patients With Hypertrophic Cardiomyopathy And Correlation Between Genotype And Phenotype

Hypertrophic cardiomyopathy (HCM) is inherited as a Mendelian autosomal dominant trait; about 55% HCM patients have familial history. Clinical manifestations involve ventricular hypertrophy, especially ventricular septum asymmetric hypertrophy, cardiac arrhythmia, exercise intolerance and sudden death. Generally,encoded cardiac sarcomere genes mutations are molecular mechanisms of HCM. At present, 10 encoding cardiac sarcomere genes which mutations caused HCM have already been found. But three of the HCM-caused mutant gene predominated in frequency: β-myosin heavy chain gene(MYH7), cardiac myosin-binding protein C gene (MYBPC3) and troponin T gene (TNNT2).β-MHC gene mutations account for 30%-50%, MYBPC3 gene mutations account for 20%～30%, TNNT2 gene mutations account for 10%～15%. There are more than 1 million patients with hypertrophic cardiomyopathy (HCM) in China. But the genetic basis presently is unknown. This research selected MYH7, MYBPC3 and TNNT2 to be candidate gene. Ten HCM families were chosen to be subjects for the study. Through proband genome DNA to be sequenced Directly,we would knowledge Chinese HCM-caused gene and mutations and the correlation between genotype and phenotype. Part I: Screen cardiacβ-myosin heavy chain gene mutations of Chinese HCM and study the correlation between genotype and phenotype Objective: The purpose of the study is to scan the disease-causing gene mutations of MYH7 gene in Chinese patients with hypertrophic cardiomyopathy (HCM) and to knowledge mutation site distribution of MYH7 gene. The correlation between genotype and phenotype is also analyzed. Methods: 1. Subjects: Ten unrelated Chinese han race HCM families came from Zhejiang, Heilongjiang, Hunan, Anhui and Beijing were enrolled in this study. There were ten probands involved 5 males and 5 females, with age ranged from 9 months to 73 years. Twenty-eight scattered HCM patients(18 males,10 females), aged 23 -87years,(mean 54.8±15.3 years). All patients were examined by echocardiography and electrocardiography (ECG). Clinical data included physical examination, ECG, two-dimensional echocardiography and Doppler echocardiography. The clinical diagnostic criteria of HCM were defined as the follows:ventricular septum or ventricular wall thickness≥13 mm, absence of another cardiac or systemic disease (based on WHO/ISFC 1996 diagnostic criteria). Familial HCM patients were defined, as there were at least two patients in the same family. Control group was consisted of 80 healthy volunteers, with 47 males and 33 females. All of controlled subjects were han race,aged form 20 to 71 years (mean age 56.2±14.1ys). No abnormality was seen with respect to physical examination, ECG, two-dimensional echocardiography and Doppler echocardiography. 2. Test methods: ①Genome DNA was extracted:venous blood was drew from probands, their family members and volunteers, anticoagulated by EDTA. Genome DNA was extracted from leucocytes by salting out. ②HCM family probands genome DNA were amplificated by PCR: primers were designed according to Harvard Medical web, which include flank intron sequences in exons 3～26 of MYH7 gene. Primers were synthesized by Beijing Aoke biotechnology company, PCR amplification response system was 40μl including genome DNA 250ng,primers 9 pmol, dNTP 160 μmol,10×buffer 4μl and TaqDNA polymerase 2U (Dingguo biotechnology company). Amplification condition:1:Pre-denaturating,95℃5min;2 :Denaturating,95℃30s ; 3:Annealling, 50-69℃30s;4:Extension,72℃40s;5:Repeat 2～4 step for 35 circles;6:Extension , 72℃,10min (PCR instrument is PTC-200 made in America MJ Research company). ③PCR products were sequenced and analyzed:All PCR products have been sequenced by dideoxy chain-termination method (American ABI incorporation 3730XL). Every primer was the same in PCR responses. ④Other family membersDNA were amplificated with primer of the exon, which took mutation in proband(whenever were diagnosed HCM or not). PCR products were sequenced by dideoxy chain-termination method. ⑤Twenty-eight scattered HCM patients'genome DNA were amplificated with primer of the exon, which took mutation in the proband. PCR products were sequenced by dideoxy chain-termination method. ⑥Eighty controls genome DNA were amplificated with primer of the exon, which took mutation in the proband. PCR products were sequenced by dideoxy chain-termination method. ⑦Mutation site were determined:Results of DNA sequenced were analyzed with Vector NTI 7.0 Analysis software and were compared with normalit sequence through GenBank, then mutation site had been determined Results: 1. H9,H18 and H21, three families probands have the MYH7 gene missense mutation. Sequencing results diplayed:Three HCM-caused mutations were found in three probands respectively. H18 proband occurred Arg663His in exon 18. H9 proband occurred Glu924Lys in exon 23. H21 proband occurred Ile736Thr in exon 20. 2. 28 scattered HCM patients did not have the Arg663His, Glu924Lys and Ile736Thr mutation. 3. 80 controls did not have the Arg663His, Glu924Lys and Ile736Thr mutation. Conclusion: This study had confirmed 3 mutations of MYH7 gene among 10 HCM families. MYH7 accounts for 30%. It was identical to foreign reports. The other families had not been found any disease-cause mutation of MYH7. It reflected that HCM have genetic heterogeneity. In this study, Glu924Lys or Ile736Thr is mutation that identified firstly in Chinese. Part II: Screen cardiac Troponin T gene mutations of Chinese HCM and study the correlation between genotype and phenotype Objective: This study was to scanning the disease-causing gene mutation of TNNT2 gene in Chinese patients with familial hypertrophic cardiomyopathy (HCM), which had been scanned in MYH7 gene and had no mutaions found. Purpose is to knowledge mutation site distribution of disease-causing gene. The correlation between genotype and phenotype were also studied.Methods: Subjects: Seven un-relationship Chinese han race HCM families came from Zhejiang, Heilongjiang, Hunan, Anhui and Beijing respectively were enrolled in this study. There were seven probands involved 3 males and 4 females, with age ranged from 9 months to 73 years. All of them are han race. Conditions of 28 scattered HCM patients and 80 healthy volunteers were same as PartⅠ. Test method: 1. Genome DNA were extracted:Details were same as Part I. 2. HCM family probands genome DNA were amplificated by PCR: primers were designed according to Harvard medical web, which included flank intron sequences in exons 2～16 of TNNT2 gene. Primers were synthesised in Beijing Aoke Biotechnology Company. PCR condition were same as Part I. 3. PCR products were sequenced and analysed: Details were same as Part I. Results: The study could not find any mutation in TNNT2 gene. But it found an insertion/deletion polymorphism in intron 3 which seem have a higher tendence for DD genotype in familial HCM. Conclusion: This study did not find any HCM-caused mutation in TNNT2 gene, which was much lower than 10%-15% that foreign reported. This research displayed that different race had different distribution of HCM-caused genes. Part Ⅲ: Screening of cardiac myosin-binding protein C gene mutations of Chinese HCM and study of the correlation between genotype and phenotype Objective: This study was to research the disease-causing gene mutation of MYBPC3 gene in Chinese patients with familial hypertrophic cardiomyopathy (HCM), which had been scanning in MYH7 and TNNT2 gene and no mutaions were found. In order to knowledge the mutation site distribution of MYBPC3. The correlation between genotype and phenotype was also analyzed. Methods: Subjects: Seven un-relationship Chinese han race HCM families came from Zhejiang, Heilongjiang, Hunan, Anhui and Beijing respectively were enrolled in this study. There were 7 probands involved 3males and 4 females, with age ranged from 9 months to 73 years. All of them are han race. Conditions of 28 scattered HCM patients and 80 healthy volunteers were same as Part I. Test method: 1. Genome DNA was extracted:Details were same as Part I. 2. HCM family probands genome DNA were amplificated by PCR:Primers were designed according to Harvard medical web, which included flank intron sequences in exons 2～16 of TNNT2 gene. Primers were synthesised in Beijing Aoke Biotechnology Company. PCR conditions were same as Part I. 3. PCR products were sequenced and analysed: Details were same as Part I. Results: 1. H13,H23, two families probands had the MYBPC3 gene missense mutation,splicing mutation and frameshift mutation. H13 proband took MYBPC3 gene missense mutation Arg502Trp in exon 18, splicing mutation IVS27+12C>T in intron 27 and same-sense mutation V849V (C16203T, GTT →GTC) in exon 26 simultaneously. His mother had Arg502Trp in exon 18, His father had IVS27+12C>T in intron 27 and same-sense mutation V849V in exon 26. H23 proband took 5 bases (CGGCA) insertion in exon 13 that frameshift mutation Arg346fs. 2. 28 scattered HCM patients did not have the Arg502Trp, IVS27+12C>T and Arg346fs. 3. 80 controls did not have the Arg502Trp, IVS27+12C>T and Arg346fs. Conclusions: 1. Two families probands had the MYBPC3 gene missense mutation,splicing mutation and frameshift mutation. This research had found 2 families taking MYBPC3 gene mutations among ten families. MYBPC3 accounts for 20%. It was identical to foreign reports. 2.This study had confirmed 3 mutations of MYBPC3 gene among 7 HCM families, which have been scanning in MYH7 and TNNT2 gene and no mutaions had been found. The other families had not found any disease-cause mutation of MYBPC3. It reflected that HCM have genetic heterogeneity. Arg346fs frameshift mutation is reported in Chinese people firstly and we have not seen any report in foreign reports. Part ⅣStudy of 5-base insertion /deletion polymorphism of cardiac troponin T gene intron 3 in Chinese HCM patients...