肥厚型心肌病心肌肌球蛋白结合蛋白C基因突变的研究
|
摘要
目的：1．利用分子生物学技术进行山东地区汉族人群中肥厚型心肌病患者中肌球蛋白结合蛋白C基因突变的研究；2．筛选该病的致病突变；3．通过与国外相关研究的比较，探讨中国汉族人群肥厚型心肌病患者在肌球蛋白结合蛋白C基因上发生突变的概率是否具有相似性；4．对基因型与表现型之间的关系进行分析；5．建立一种在基因水平上对肥厚型心肌病患者进行临床前期诊断的方法。
方法：1．临床收集76例肥厚型心肌病患者(均为山东医科大学齐鲁医院门诊及住院患者)，在知情同意的前提下抽取外周静脉血约2mL，提取白细胞，利用酚-氯仿法抽提基因组DNA；2．聚合酶链反应(PCR)扩增MYBPC基因相应的外显子14-15、17、26、27、33，对扩增产物进行琼脂糖凝胶电泳以了解扩增的效率及特异性；3．利用单链构象多态性分析(SSCP)分析PCR扩增产物，处理后的产物行8％及10％非变性聚丙烯酰胺凝胶电泳，银染法显色观察结果；4．根据同一块胶上电泳条带迁移率的差异判断是否为阳性结果，出现异常条带者将目的片段连接T载体并转染细菌，培育后送检测序；5．对基因突变阳性者进行家系调查及临床表型分析。
结果：1．我们成功地对76例肥厚型心肌病患者的肌球蛋白结合蛋白C基因的第14-15、17、26、27、33号外显子进行了PCR扩增；2．全部扩增产物均经过两种条件下的单链构象多态性分析(SSCP)进行基因突变的筛选，通过调整反应条件，使每一个PCR产物均显示清晰的单链条带，与正常人相应的单链带进行了比较分析；3．在一患者的肌球蛋白结合蛋白C基因第26号外显子上发现了一个新的突变位点Arg856fs。
结论：1．PCR-SSCP银染技术对于HCM患者的肌球蛋白结合蛋白C基因突变的检测是适用的，并可以进行推广；2．肌球蛋白结合蛋C基因的第26外显子上发现了一个新的突变位点Arg856fs，经检索世界上尚未见相同的报道；3．山东地区汉族人群肥厚型心肌病患者在肌球蛋白结合蛋白C基因
Objective: 1. To investigate the situation of Cardiac myosin binding protein-C gene mutation in Chinese population of Shandong province with hypertrophic cardiomyopathy (HCM); 2. To research if there are differences between the Chinese people and foreigners in the incidence of Cardiac myosin binding protein-C gene mutation in patients with hypertrophic cardiomyopathy; 3. To analysize the correlation between genotypes and phenotypes; 4. To establish a method to diagnose hypertrophic cardiomyopathy pre-clinically at gene level.
Methods: 1. 76 unrelated clinical patients with HCM were chosen for the study. After informed consent, a venous blood sample for extraction of DNA was obtained. In addition, 100 healthy individuals were examined as normal controls; 2. High molecular weight DNA was extracted from peripheral blood lymphocytes of patients and controls with the SDS proteinase K method and phenol/chloroform extraction; 3. The primers used for amplification of exonl4-15, 17, 26, 27, 33 of Cardiac myosin binding protein-C gene were from adjacent intronic sequence. Protein coding exonsl4-15,17, 26, 27, 33 of Cardiac myosin binding protein-C was amplified using polymerase chain reaction (PCR). The PCR products were examined by means of agar-gel electrophoresis to assay the quantity and specialty; 4. Single strand conformation polymorphism (SSCP) method was used to detect possible gene mutations in the PCR products. Different conditions were used to enhance the sensitivity of the method including different polypropylene concentration, different proportion of PCR products and cushion fluid etc. Sequence the suspicious PCR products for exact mutant point; 5. Perform clinical studies and examine the incidence of sudden cardiac death within
方法：1．临床收集76例肥厚型心肌病患者(均为山东医科大学齐鲁医院门诊及住院患者)，在知情同意的前提下抽取外周静脉血约2mL，提取白细胞，利用酚-氯仿法抽提基因组DNA；2．聚合酶链反应(PCR)扩增MYBPC基因相应的外显子14-15、17、26、27、33，对扩增产物进行琼脂糖凝胶电泳以了解扩增的效率及特异性；3．利用单链构象多态性分析(SSCP)分析PCR扩增产物，处理后的产物行8％及10％非变性聚丙烯酰胺凝胶电泳，银染法显色观察结果；4．根据同一块胶上电泳条带迁移率的差异判断是否为阳性结果，出现异常条带者将目的片段连接T载体并转染细菌，培育后送检测序；5．对基因突变阳性者进行家系调查及临床表型分析。
结果：1．我们成功地对76例肥厚型心肌病患者的肌球蛋白结合蛋白C基因的第14-15、17、26、27、33号外显子进行了PCR扩增；2．全部扩增产物均经过两种条件下的单链构象多态性分析(SSCP)进行基因突变的筛选，通过调整反应条件，使每一个PCR产物均显示清晰的单链条带，与正常人相应的单链带进行了比较分析；3．在一患者的肌球蛋白结合蛋白C基因第26号外显子上发现了一个新的突变位点Arg856fs。
结论：1．PCR-SSCP银染技术对于HCM患者的肌球蛋白结合蛋白C基因突变的检测是适用的，并可以进行推广；2．肌球蛋白结合蛋C基因的第26外显子上发现了一个新的突变位点Arg856fs，经检索世界上尚未见相同的报道；3．山东地区汉族人群肥厚型心肌病患者在肌球蛋白结合蛋白C基因
Objective: 1. To investigate the situation of Cardiac myosin binding protein-C gene mutation in Chinese population of Shandong province with hypertrophic cardiomyopathy (HCM); 2. To research if there are differences between the Chinese people and foreigners in the incidence of Cardiac myosin binding protein-C gene mutation in patients with hypertrophic cardiomyopathy; 3. To analysize the correlation between genotypes and phenotypes; 4. To establish a method to diagnose hypertrophic cardiomyopathy pre-clinically at gene level.
Methods: 1. 76 unrelated clinical patients with HCM were chosen for the study. After informed consent, a venous blood sample for extraction of DNA was obtained. In addition, 100 healthy individuals were examined as normal controls; 2. High molecular weight DNA was extracted from peripheral blood lymphocytes of patients and controls with the SDS proteinase K method and phenol/chloroform extraction; 3. The primers used for amplification of exonl4-15, 17, 26, 27, 33 of Cardiac myosin binding protein-C gene were from adjacent intronic sequence. Protein coding exonsl4-15,17, 26, 27, 33 of Cardiac myosin binding protein-C was amplified using polymerase chain reaction (PCR). The PCR products were examined by means of agar-gel electrophoresis to assay the quantity and specialty; 4. Single strand conformation polymorphism (SSCP) method was used to detect possible gene mutations in the PCR products. Different conditions were used to enhance the sensitivity of the method including different polypropylene concentration, different proportion of PCR products and cushion fluid etc. Sequence the suspicious PCR products for exact mutant point; 5. Perform clinical studies and examine the incidence of sudden cardiac death within
引文
1 Rayment I, Holden HM, Sellers JR, et al.Structural interpretation of the mutations in beta cardiac myosin that have been implicated in familial hypertrophic cardiomyopathy.Proe Natl Acad Sci, 1995; 92: 38-64.
2 Sadoul N, de Chillou C, Aliot E, et al.Evaluation of the risk of sudden death in hypertrophic cardiomyopathy.Arch Mal Coeur Vaiss, 1999, 92:65.
3 Maron BJ, Shirani L Poliac LC, et al.Sudden death in young competitive athletes, Clinical、demographic、and pathological profiles[J].JAMA, 1996, 276(3): 199.204.
4 Jarcho JA, MeKenna W, Pare JAP, et al.Mapping a gene for familial hypertrophic cardiomyopathy to chromosome 14q1.N Engl J Med 1989: 321: 1372—8.
5 Weisberg A, Winegrad S.Alteration of myosin cross bridges by phosphorylation of myosin-binding protein C in cardiac muscle.Proc Natl Acad Sci U S A 1996;93:8999-9003.
6 Lucie Carrier; Gisele Bonne; Ellen Bahrend; Bing Yu; et al.rganization and sequence of human cardiac myosin binding protein C gene (MYBPC3) and Identification of Mutations Predicted to Produce Truncated Proteins in Familial Hypertrophic Cardiomyopathy.Circulation Research; Mar 1997; 80,3; ProQuest Medical Library pg.427.
7 Carrier L, Bonne G, B? hrend E, et al.Organization and sequence of human cardiac myosin binding protein C gene (MYBPC3) and identification of mutations predicated to produce truncated proteins in familial hypertrophic cardiomyopathy.Circ res, 1997, 80: 427-434.
8 Bottinelli R, Coviello DA, Redwood CS,etal.A mutant tropomyosin that causes hypertrophic cardiomyopathy is expressed and associated with an increased calcium sensitivity[J].CircRes, 1998,82: 106.
9 Ohsawa Y, Toko H, Katsura M, et al.Overexpression of P104L mutant caveolin-3 in mice develops hypertrophic cardiomyopathy with enhanced contractility in association with increased endothelial nitric oxide synthase activity[J].Hum Mol Genet, 2004,13: 151.
10 Marian AJ.On genetic and phenotype variability of hypertrophic cardiomyopathy: nature versus nurture.J Am Coll Cardiol, 2001, 38(2): 331-334.
11 YanagaF, MorimotoS, Ohtsuki I. J Biol Chem. 1999; 274(13): 8806-8812
12 Gao WD, Perez NG, Seidman CE, et al.J ain Invest, 1999; 103(5): 661-666.
13 BottinelliR, Coviello DA, Redwood CS.et al.Cire Res, 1998; 82(1):106-115.
14 Gilbert R, Kelly MG, Mikawa T, et al.The carboxyl terminus of myosin binding protein-C (MyBP-C, C-protein) specifies incorporation into the A-band of Striated muscle.J Cell Sci, 1996,109:101-111.
15 Yang Q, Sanbe AJ, Osinska H, et al.A mouse model of myosin binding protein C human familial hypertrophic cardiomyopathy.J Clin Invest, 1998,102: 1292-1300.
16 Cuda G.Fananapazir L, ghu WS, et al.J 121in Invest, 1993; 91(6): 2861-2865.
17 Carrier L, Bonne G, Bahrend E, et al.Organization and sequence of human cardiac myosin binding protein C and identification of mutations predicted to produce truncated proteins in familial hypertrophic cardiomyopathy [J].Cir Red,1997, 80(1):427.
18 Rottbauer Wolfgang, Gautel M, Zehelein J, et al.Novel spice donor sire mutation in the cardiac myosin—binding protein C gene in familial hypertropbic cardiomyopathy[J].J Clin Invest, 1997, 100, 475.
19 Maron BJ, Development of left ventricular hypertrophy in adults with hypertrophic cardomyopathy caused by cardiac myosin-binding protein C gene mutations.J Am Coll Cardiol, 2001, 38(2): 315-321.
20 Niimura H, Patton K, McKenna WJ, et al.Sarcomere protein gene mutations in hypertrophic cardiomyopathy of the elderly.Circulation, 2002,105: 446-451.
21 Erdmann J, Raible J, Maki-Abadi J, et al.Spectrum of clinical phenotypes and gene variants in cardia myosin-binding protein C mutation carriers with hypertrophic cardiomyopathy.J Am Coll Cardiol, 2001, 38(2): 322-330.
22 Harris SP, Rostkova, Gautel M, et al.Binding of myosin binding protein-C to myosin sub-fragment S2 affects contractility independent of a tether mechanism.Circulation Research, 2004,95: 930-936.
23 Van Driest SI , Vasile VC, Ommen SR.et al.Myosin binding protein C mutations and compound heterozygosity in hyper-trophic cardiomyopathy , J Am Coll Cardiol, 2004, 44: 1903-1910.
24 Garcia-Castro M,Reguero JR,Alvarez V,et al. Hypertrophic cardiomyopathy linked to homozygosity for a new mutation in the myosin-binding protein C gene (A627V) suggests a dosage effect.International Journal Cardiology[J]2005, 102: 501-507.
25 Marian AJ. On genetic and phenotype variability of hypertrophic cardiomyopathy: nature and nurture. J Am Coll Cardiol, 2001, 38(2): 331-334.
26 Brito D, Richard P, et al. Familial hypertrophic cardiomyopathy: the same mutation, different prognosis. Comparison of two families with a long follow-up. Rev Port Cardiol. 2003 Dec;22(12): 1445-61.
27 Andersen PS. Jespersgaard C. Vuust J.et al. High-throughput single strand conformation palymorohism mutation detection by automated capillary array electrophoresis: validation of the method. Hum Mutat 2003: 2(2): 116—22
28 Mogensen J. Bahl A.Kubo T. et al. Comparison of fluorescent SSCP and denaturing HPLC analysis with direct sequencing for mutation screening in hypertrophic cardiomyopathy. J Med Genet 2003: 40: 59.
29 谢文丽，刘文玲，胡大一，等，一个汉族肥厚型心肌病家系中首次发现肌球连接蛋白C基因Arg346fs突变．中华医学杂志，2005，85(14)：963-966。
30 Richard P, Charron P, et al. Hypertrophic cardiomyopathy: distribution of disease genes, spectrum of mutations, and implications for a molecular diagnosis strategy. Circulation. 2003 May 6;107(17):2227-32. Epub 2003 Apr 21.
31 安丰双，张运，et，肥厚型心肌病肌钙蛋白T基因突变的研究．中华医学杂志，2004，84(16)：1340-1343。
32 Song L, Zou Y, Wang Z, et al. Mutations profile in Chinese patients with hypertrophic cardiomyopathy. Clin Chim Acta, 2005, 351(1-2):209-216.
33. Sara L. Van Driest, Vlad C. Vasile, Steve R. Ommen, et al. Myosin Binding Protein C Mutations and Compound Heterozygosity in Hypertrophic Cardiomyopathy, Journal of the American College of Cardiology, 2004,44,(9): 1903-1910.
34. Hiroyuki Morita, Martin G. Larson, Scott C. Barr, et al. Single-Gene Mutations and Increased Left Ventricular Wall Thickness in the Community: The Framingham Heart Study, Circulation, 2006, 113(23): 2697-2705.
2 Sadoul N, de Chillou C, Aliot E, et al.Evaluation of the risk of sudden death in hypertrophic cardiomyopathy.Arch Mal Coeur Vaiss, 1999, 92:65.
3 Maron BJ, Shirani L Poliac LC, et al.Sudden death in young competitive athletes, Clinical、demographic、and pathological profiles[J].JAMA, 1996, 276(3): 199.204.
4 Jarcho JA, MeKenna W, Pare JAP, et al.Mapping a gene for familial hypertrophic cardiomyopathy to chromosome 14q1.N Engl J Med 1989: 321: 1372—8.
5 Weisberg A, Winegrad S.Alteration of myosin cross bridges by phosphorylation of myosin-binding protein C in cardiac muscle.Proc Natl Acad Sci U S A 1996;93:8999-9003.
6 Lucie Carrier; Gisele Bonne; Ellen Bahrend; Bing Yu; et al.rganization and sequence of human cardiac myosin binding protein C gene (MYBPC3) and Identification of Mutations Predicted to Produce Truncated Proteins in Familial Hypertrophic Cardiomyopathy.Circulation Research; Mar 1997; 80,3; ProQuest Medical Library pg.427.
7 Carrier L, Bonne G, B? hrend E, et al.Organization and sequence of human cardiac myosin binding protein C gene (MYBPC3) and identification of mutations predicated to produce truncated proteins in familial hypertrophic cardiomyopathy.Circ res, 1997, 80: 427-434.
8 Bottinelli R, Coviello DA, Redwood CS,etal.A mutant tropomyosin that causes hypertrophic cardiomyopathy is expressed and associated with an increased calcium sensitivity[J].CircRes, 1998,82: 106.
9 Ohsawa Y, Toko H, Katsura M, et al.Overexpression of P104L mutant caveolin-3 in mice develops hypertrophic cardiomyopathy with enhanced contractility in association with increased endothelial nitric oxide synthase activity[J].Hum Mol Genet, 2004,13: 151.
10 Marian AJ.On genetic and phenotype variability of hypertrophic cardiomyopathy: nature versus nurture.J Am Coll Cardiol, 2001, 38(2): 331-334.
11 YanagaF, MorimotoS, Ohtsuki I. J Biol Chem. 1999; 274(13): 8806-8812
12 Gao WD, Perez NG, Seidman CE, et al.J ain Invest, 1999; 103(5): 661-666.
13 BottinelliR, Coviello DA, Redwood CS.et al.Cire Res, 1998; 82(1):106-115.
14 Gilbert R, Kelly MG, Mikawa T, et al.The carboxyl terminus of myosin binding protein-C (MyBP-C, C-protein) specifies incorporation into the A-band of Striated muscle.J Cell Sci, 1996,109:101-111.
15 Yang Q, Sanbe AJ, Osinska H, et al.A mouse model of myosin binding protein C human familial hypertrophic cardiomyopathy.J Clin Invest, 1998,102: 1292-1300.
16 Cuda G.Fananapazir L, ghu WS, et al.J 121in Invest, 1993; 91(6): 2861-2865.
17 Carrier L, Bonne G, Bahrend E, et al.Organization and sequence of human cardiac myosin binding protein C and identification of mutations predicted to produce truncated proteins in familial hypertrophic cardiomyopathy [J].Cir Red,1997, 80(1):427.
18 Rottbauer Wolfgang, Gautel M, Zehelein J, et al.Novel spice donor sire mutation in the cardiac myosin—binding protein C gene in familial hypertropbic cardiomyopathy[J].J Clin Invest, 1997, 100, 475.
19 Maron BJ, Development of left ventricular hypertrophy in adults with hypertrophic cardomyopathy caused by cardiac myosin-binding protein C gene mutations.J Am Coll Cardiol, 2001, 38(2): 315-321.
20 Niimura H, Patton K, McKenna WJ, et al.Sarcomere protein gene mutations in hypertrophic cardiomyopathy of the elderly.Circulation, 2002,105: 446-451.
21 Erdmann J, Raible J, Maki-Abadi J, et al.Spectrum of clinical phenotypes and gene variants in cardia myosin-binding protein C mutation carriers with hypertrophic cardiomyopathy.J Am Coll Cardiol, 2001, 38(2): 322-330.
22 Harris SP, Rostkova, Gautel M, et al.Binding of myosin binding protein-C to myosin sub-fragment S2 affects contractility independent of a tether mechanism.Circulation Research, 2004,95: 930-936.
23 Van Driest SI , Vasile VC, Ommen SR.et al.Myosin binding protein C mutations and compound heterozygosity in hyper-trophic cardiomyopathy , J Am Coll Cardiol, 2004, 44: 1903-1910.
24 Garcia-Castro M,Reguero JR,Alvarez V,et al. Hypertrophic cardiomyopathy linked to homozygosity for a new mutation in the myosin-binding protein C gene (A627V) suggests a dosage effect.International Journal Cardiology[J]2005, 102: 501-507.
25 Marian AJ. On genetic and phenotype variability of hypertrophic cardiomyopathy: nature and nurture. J Am Coll Cardiol, 2001, 38(2): 331-334.
26 Brito D, Richard P, et al. Familial hypertrophic cardiomyopathy: the same mutation, different prognosis. Comparison of two families with a long follow-up. Rev Port Cardiol. 2003 Dec;22(12): 1445-61.
27 Andersen PS. Jespersgaard C. Vuust J.et al. High-throughput single strand conformation palymorohism mutation detection by automated capillary array electrophoresis: validation of the method. Hum Mutat 2003: 2(2): 116—22
28 Mogensen J. Bahl A.Kubo T. et al. Comparison of fluorescent SSCP and denaturing HPLC analysis with direct sequencing for mutation screening in hypertrophic cardiomyopathy. J Med Genet 2003: 40: 59.
29 谢文丽，刘文玲，胡大一，等，一个汉族肥厚型心肌病家系中首次发现肌球连接蛋白C基因Arg346fs突变．中华医学杂志，2005，85(14)：963-966。
30 Richard P, Charron P, et al. Hypertrophic cardiomyopathy: distribution of disease genes, spectrum of mutations, and implications for a molecular diagnosis strategy. Circulation. 2003 May 6;107(17):2227-32. Epub 2003 Apr 21.
31 安丰双，张运，et，肥厚型心肌病肌钙蛋白T基因突变的研究．中华医学杂志，2004，84(16)：1340-1343。
32 Song L, Zou Y, Wang Z, et al. Mutations profile in Chinese patients with hypertrophic cardiomyopathy. Clin Chim Acta, 2005, 351(1-2):209-216.
33. Sara L. Van Driest, Vlad C. Vasile, Steve R. Ommen, et al. Myosin Binding Protein C Mutations and Compound Heterozygosity in Hypertrophic Cardiomyopathy, Journal of the American College of Cardiology, 2004,44,(9): 1903-1910.
34. Hiroyuki Morita, Martin G. Larson, Scott C. Barr, et al. Single-Gene Mutations and Increased Left Ventricular Wall Thickness in the Community: The Framingham Heart Study, Circulation, 2006, 113(23): 2697-2705.