Studies On The Relationship Between Hypertrophic Cardiomyopathy And Cardiac Troponin Gene Mutations

BACKGROUND: Hypertrophic cardiomyopathy(HCM) is an autosomal dominant disease of myocardium characterized by ventricular hypertrophy and myofibrillar disarrays. The prevalence reported abroad is 0.2%, From 1985 to 1994,a study on population under the age of 60 was conducted in institute of epidemic cardiovascular disease of Jiangsu Province Hospital. The results showed that the morbidity of HCM was 1.3-1.5/100,000 per year. Patients with HCM have a variety of clinical features, from no symptoms to serious arrhythmia, heart failure and even sudden death. And it is the most common cause of sudden death in otherwise healthy individuals. Genetic studies in HCM are of significances^ 1) to lead to better definition and diagnosis of the disease.(2) to increase our understanding of its pathophysiology and be critical for the elucidation of the molecular basis of the disease and LVH in general. (3) to permit preclinical diagnosis and genetic counseling. (4) to improve risk stratification and (5) may lead to development of therapies that prevent progression of the disease in children or cause regression of the disease in adults. Up to now ,about 200 mutations in 11 genes encoding sarcomeric proteins have been identified as the causes of the disease .including β-myosin heavy chain(β-MyHC), cardiac troponin T (cTnT), cardiac troponin I (cTnl), cardiac troponin C (cTnC), myosin binding protein C (MyBP-C), titin, myosin essential and regulatory light chains, alpha-actin, alpha-tropomyosin ( a -TM) and alpha-myosin heavy chain (a - MyHC). Approximately 15-20 percent of HCM cases are caused by mutations in cardiac troponin T and I genes. Although theaetiology of hypertrophic cardiomyopathy has been extensively elucidated, its pathogenesis is not completely understood. OBJECTIVE: to study the relationship of hypertrophic cardiomyopathy and mutations in cardiac troponin (I and T) genes; to clone the full-length cDNA of the SD rat and construct a mutant cDNA of the SD rat(deletion Lysl84),then subclone the mutant and wild cDNA into adeno-X?genome to produce recombinant adenovirus. CONTENTS AND METHODS1. 58 patients with hypertrophic cardiomyopathy were enrolled and genomic DNA was prepared from their peripheral blood leukocytes.2. The exons 7 and 8 of cardiac troponin I gene were amplified by PCR, and the products were directly sequenced.3. The exon 5 of cardiac troponin I and exons 8 ,9 and 11 of cardiac troponin T were amplified respectively by PCR and the products analyzed by single-strand conformation polymorphism (SSCP).4. The mutations were verified by specific biotin-labeled olignucleotide probe dot blot hybridization.5. One step method was used to extract total RNA from rat left ventricle tissue, and a pair of primers with restriction endonuclease site, Af1 II /Nhe I ,were used to amplify the cTnl cDNA by reverse transcription- polymerase chain reaction(RT-PCR), then the products were inserted into T-vector .6. The A △lys184 mutation was introduced into the cTnI cDNA by site-directed mutagenesis.7. A 6 × His tag sequence was engineered into the carboxyl-terminus of mutant and wild cTnl cDNA by PCR.8. Digested with Afl II and Nhe I restriction endonuclease, the mutant and wild type cDNA were then subcloned into pShuttle plasmid, respectively.9. Digested with PI-See and I-Ceu restriction endonuclease,the mutant and wild type cDNA were subcloned into Adeno-X?Genome, respectively.10. Digested with Pac I restriction endonuclease and HEK-293 cells were transfected. The recombinant viruses were propagated and collected. Western blotting tested the expressed proteins.11. The recombinant viruses were purified on a CsCl density gradient.12. Two weeks after direct myocardial injection of the recombinant Adenovirus, the recombinant proteins were tested by immunohistochemistry and western blotting.RESULTS:1. Of the 58 patients with HCM(36 male and 22 female,aged 46.05 ± 17.96 yea...