山东地区汉族人群肥厚型心肌病患者心肌肌球蛋白结合蛋白C基因突变的研究
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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.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;3.对基因型与表现型之间的关系进行分析;4.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92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、25、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阳性结果,出现异常条带者送公司测序验证;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92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、25、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,通过反应条件的调整,每一个PCR产物均清晰的单链条带,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14-15号外显子上发现了一个新的突变位点T445S。
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14-15外显子上发现了一个新的突变位点
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 analysis the correlation between genotypes and phenotypes; 4.To establish a method to diagnose hypertrophic cardiomyopathy preclinically at gene level.Methods: 1. Ninety-two 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, one hundred 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 amplication of exonl4-15,17, 25, 27, 33of Cardiac myosin binding protein-C gene were from adjacent intronic sequence. Protein coding exons 14-15,17, 25, 27, 33of 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 concention, different propotion of PCR products and cushion fluid etc; 5.Sequence the suspicious PCR products for exact mutant point. Perform clinical studies and examine the incidence of sudden cardiac death within the family.Results: 1. We succeeded in amplifying the five exons of the ninety two patients. 2. All of the PCR products were examined by SSCP method through different conditions and vivid single strand belts have been got. 3. By contrast with normal controls, we have found a missence mutation T445S for the first time in a HCM patient. 4.By contrast to the other HCM patients, we couldn' t find the paticurity of the patient harboring the mutation.Conclusions: 1. We have found a misence mutation for the first time in a HCM patient. 2. The incidence of mutations in gene encoding Cardiac myosin binding protein-C in HCM patients of Chinese population is much lower than what have been previously reported. 3. The genotypes and phenotypes are not simply in according with each other. 4.There is a long way to go to conquer this disease.
方法:1.临床收集92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、25、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阳性结果,出现异常条带者送公司测序验证;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92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、25、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,通过反应条件的调整,每一个PCR产物均清晰的单链条带,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14-15号外显子上发现了一个新的突变位点T445S。
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14-15外显子上发现了一个新的突变位点
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 analysis the correlation between genotypes and phenotypes; 4.To establish a method to diagnose hypertrophic cardiomyopathy preclinically at gene level.Methods: 1. Ninety-two 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, one hundred 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 amplication of exonl4-15,17, 25, 27, 33of Cardiac myosin binding protein-C gene were from adjacent intronic sequence. Protein coding exons 14-15,17, 25, 27, 33of 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 concention, different propotion of PCR products and cushion fluid etc; 5.Sequence the suspicious PCR products for exact mutant point. Perform clinical studies and examine the incidence of sudden cardiac death within the family.Results: 1. We succeeded in amplifying the five exons of the ninety two patients. 2. All of the PCR products were examined by SSCP method through different conditions and vivid single strand belts have been got. 3. By contrast with normal controls, we have found a missence mutation T445S for the first time in a HCM patient. 4.By contrast to the other HCM patients, we couldn' t find the paticurity of the patient harboring the mutation.Conclusions: 1. We have found a misence mutation for the first time in a HCM patient. 2. The incidence of mutations in gene encoding Cardiac myosin binding protein-C in HCM patients of Chinese population is much lower than what have been previously reported. 3. The genotypes and phenotypes are not simply in according with each other. 4.There is a long way to go to conquer this disease.
引文
1 Rayment I, HoldenHM, 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 J, 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 Yamamoto K, Moos C. The C-proteins of rabbit red, white, and cardiac muscles. J Biol Chem 1983; 258: 8395-401.
6 Vaughan KT, Weber FE, Einheber S, Fischman DA. Molecular cloning of chicken myosin-binding protein (MyBP) H (85-kDa protein) reveals extensive homology with MyBP-C (C-protein) with conserved immunoglobulin C2 and fibronectin type Ⅲ motifs. J Biol Chem 1993; 268: 3670-6.
7 Gilbert R, Kelly MG, Mikawa T, Fischman DA. 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: 101-11.
8 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.
9 Hartzell HC. Effects of phosphorylated and unphosphorylated C-protein on cardiac actomyosin ATPase. J Mol Biol 1985; 186: 185-95
10 Watkins H, Conner D, Thierfelder L, et al. Mutations in the cardiac myosin binding protein-C gene on chromosome 11 cause familial hypertrophic cardiomyopathy. Nat Genet 1995;11:434-7
11 Bonne G, Carrier L, Bercovici J, et al. Cardiac myosin binding proteinC gene splice acceptor site mutation is associated with familial hypertrophic cardiomyopathy. Nat Genet 1995;11:438-40.
12 Carrier L, Hengstenberg C, Beckmann JS, et al. Mapping of a novel gene for familial hyperaophic cardiomyopathy to chromosome 11. Nat Genet 1993;4:311-3.
13 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
14 Yanaga F, Morimoto S, Ohtsuki I. J Biol Chem. 1999; 274 (13): 8806—8812
15 Gao TO, Perez NG,Seidman CE, et al. J ain Invest, 1999; 103(5): 661—666
16 BottinelliR, Coviello DA, Redwood CS. et al. Cire Res, 1998; 82(1) :106- 115
17 Cuda G. Fananapazir L, ghu WS, et al. J 121in Invest, 1993; 91(6): 2861-2865.
18 Offer G, et al .A new protein of the thick filaments of vertebrate skeletal myofibrils. Extractions, purification and characterization. J Mol Biol. 1973 Mar 15;74(4):653-76. No abstract available.
19 Starr R, Almond G, Offer G. Location of C protein and X protein in rabbit skeletal muscle fiber types. J Muscle Cell Motil. 1985;6:227-256.
20 Carrier L, Bonne G, Bahrend E, et al. Organization and sequenc of human
cardiac myosin bindlng protein C and identification of mutations predicted to produce truncated proteins in familial hypertrophic cardiomyopathy[J]. Cir Red, 1997, 80(1): 427.
21 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.
22 Marian A, Roberts R. Molecular genetic basis of hypertrophic cardiomyopathy: genetics for sudden death. J Cardiovasc Electrophysiol, 1998, 9: 88-99.
23 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. English, Portuguese. Erratum in: Rev Port Cardiol. 2004 Jan, 23(1): 142
24 Orba M, lwahana H, Kanazawa H, et, al. Detection of polymorphism of human DNA hy gel eletrophoresis as single-strand conformation polymor-phism. Proc, Natl Acad Sci USA 1989: 86f8): 2766—70
25 Carl W.Dieffenbach, Gabriela S. Dveksler. PCR Primer: A Laboratory Manual. Cold Spring Harbor Laboratory Press, 1995: 173-176.
26 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 Murat 2003: 2(2): 116—22
27 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
28 J Mogensen, A Baht, T Kabo, N Elanko, et al. Comparison of fluorescent SSCP and denaturing HPLC analysis with direct sequencing for mutation screening in hypertrophic cardiomyopathy. J Med Genet, 2003, 40. e59 (http://www.imedgenet.com/cgi/content/full/40/5/e59).
29 Mutations in the gene for cardiac myosin-binding protein C and late-onset familial hypertrophic cardiomyopathy.
30 ichard 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 李莉.肥厚型心肌病的分子遗传学基础及致病机制[J].医学综述,2002,8(10):567-569。
32 Bashyam MD, Savithri GR, Kumar MS, et al. Molecular genetics of familial hypertrophic cardiomyopathy(FHC). J Hum Genet, 2003, 48(2): 55-64.
33 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。
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 J, 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 Yamamoto K, Moos C. The C-proteins of rabbit red, white, and cardiac muscles. J Biol Chem 1983; 258: 8395-401.
6 Vaughan KT, Weber FE, Einheber S, Fischman DA. Molecular cloning of chicken myosin-binding protein (MyBP) H (85-kDa protein) reveals extensive homology with MyBP-C (C-protein) with conserved immunoglobulin C2 and fibronectin type Ⅲ motifs. J Biol Chem 1993; 268: 3670-6.
7 Gilbert R, Kelly MG, Mikawa T, Fischman DA. 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: 101-11.
8 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.
9 Hartzell HC. Effects of phosphorylated and unphosphorylated C-protein on cardiac actomyosin ATPase. J Mol Biol 1985; 186: 185-95
10 Watkins H, Conner D, Thierfelder L, et al. Mutations in the cardiac myosin binding protein-C gene on chromosome 11 cause familial hypertrophic cardiomyopathy. Nat Genet 1995;11:434-7
11 Bonne G, Carrier L, Bercovici J, et al. Cardiac myosin binding proteinC gene splice acceptor site mutation is associated with familial hypertrophic cardiomyopathy. Nat Genet 1995;11:438-40.
12 Carrier L, Hengstenberg C, Beckmann JS, et al. Mapping of a novel gene for familial hyperaophic cardiomyopathy to chromosome 11. Nat Genet 1993;4:311-3.
13 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
14 Yanaga F, Morimoto S, Ohtsuki I. J Biol Chem. 1999; 274 (13): 8806—8812
15 Gao TO, Perez NG,Seidman CE, et al. J ain Invest, 1999; 103(5): 661—666
16 BottinelliR, Coviello DA, Redwood CS. et al. Cire Res, 1998; 82(1) :106- 115
17 Cuda G. Fananapazir L, ghu WS, et al. J 121in Invest, 1993; 91(6): 2861-2865.
18 Offer G, et al .A new protein of the thick filaments of vertebrate skeletal myofibrils. Extractions, purification and characterization. J Mol Biol. 1973 Mar 15;74(4):653-76. No abstract available.
19 Starr R, Almond G, Offer G. Location of C protein and X protein in rabbit skeletal muscle fiber types. J Muscle Cell Motil. 1985;6:227-256.
20 Carrier L, Bonne G, Bahrend E, et al. Organization and sequenc of human
cardiac myosin bindlng protein C and identification of mutations predicted to produce truncated proteins in familial hypertrophic cardiomyopathy[J]. Cir Red, 1997, 80(1): 427.
21 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.
22 Marian A, Roberts R. Molecular genetic basis of hypertrophic cardiomyopathy: genetics for sudden death. J Cardiovasc Electrophysiol, 1998, 9: 88-99.
23 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. English, Portuguese. Erratum in: Rev Port Cardiol. 2004 Jan, 23(1): 142
24 Orba M, lwahana H, Kanazawa H, et, al. Detection of polymorphism of human DNA hy gel eletrophoresis as single-strand conformation polymor-phism. Proc, Natl Acad Sci USA 1989: 86f8): 2766—70
25 Carl W.Dieffenbach, Gabriela S. Dveksler. PCR Primer: A Laboratory Manual. Cold Spring Harbor Laboratory Press, 1995: 173-176.
26 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 Murat 2003: 2(2): 116—22
27 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
28 J Mogensen, A Baht, T Kabo, N Elanko, et al. Comparison of fluorescent SSCP and denaturing HPLC analysis with direct sequencing for mutation screening in hypertrophic cardiomyopathy. J Med Genet, 2003, 40. e59 (http://www.imedgenet.com/cgi/content/full/40/5/e59).
29 Mutations in the gene for cardiac myosin-binding protein C and late-onset familial hypertrophic cardiomyopathy.
30 ichard 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 李莉.肥厚型心肌病的分子遗传学基础及致病机制[J].医学综述,2002,8(10):567-569。
32 Bashyam MD, Savithri GR, Kumar MS, et al. Molecular genetics of familial hypertrophic cardiomyopathy(FHC). J Hum Genet, 2003, 48(2): 55-64.
33 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。