心肌肌钙蛋白基因突变与肥厚型心肌病发病研究
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摘要
背景：肥厚型心肌病是一种原发性的心肌病，以心室肥厚和肌纤维排列紊乱为特点。家族性肥厚型心肌病以常染色体显性方式遗传。国外报告患病率约为0.2％。江苏省人民医院心血管流行病研究室在1985-1994年间对60岁以下人群进行研究，报告年平均发病率为1.3-1.5／10万。患者临床表现变异较大，可无症状亦可出现严重心律失常、心力衰竭甚至猝死。肥厚型心肌病是健康个体最主要的猝死原因之一。研究肥厚型心肌病病因，发病机制具有重要的临床意义：1．可以更好的定义和诊断该疾病；2．增加对该病病理生理的认识；3．对该病和左心室肥厚(LVH)分子基础的阐明起关键作用；4．可以在出现临床症状前得到诊断及遗传咨询；5．对患者进行危险度分层；6．为探讨新的治疗方法提供理论依据，防止该病在儿童时期的进展或促使该病在成年人中消退。目前已知至少有11种编码肌小节蛋白的基因约近200种突变与肥厚型心肌病有关，即：β-肌球蛋白重链(β-myosin heavy chain，β-MyHC)、心肌肌钙蛋白T(cardiac troponin T，cTnT)、心肌肌钙蛋白I(cardiac troponin I，cTnI)、心肌肌钙蛋白C(cardiac troponin C，cTnC)、肌球蛋白结合蛋白C(myosin binding protein C，MyBP-C)、肌联蛋白(titin)、肌球蛋白必需轻链和调节轻链(myosin essential and regulatory light chains)、α-肌动蛋白(alpha-actin)、α-原肌球蛋白(alpha-tropomyosin，α-TM)、α-肌球蛋白重链(alpha-myosin heavy chain，α-MyHC)。其中约15-20％的肥厚型心肌病患者由心肌肌钙蛋白I、T基因突变引起。目前肥厚型心肌病的病因研究进展很快，但对发病机制的研究相对滞后。
目的：研究肥厚型心肌病与心肌肌钙蛋白I、T基因突变的关系，尝试用多种实验方法检测突变；克隆SD大鼠心肌肌钙蛋白I基因，构建一种肥厚型心肌病相关心肌肌钙蛋白I基因突变类型(Lys184缺失突变，相当于人Lys183)重组腺病毒载体，用于发病机制的研究。
内容与方法：
1．收集临床确诊的肥厚型心肌病病例58例，提取外周血白细胞
DNA。
二．叮R扩增心 肌肌钙蛋白I基因7、8号外显子，产物直接坝序检
测有无突变。
3．KR扩增。G肌肌专蛋白T基因8、9、n号外显子及。G肌肌钙蛋
白1基因5 号外显子，采用单链构型多态性（single七trand
COflfoflfltloflPolymorphism，SSCP）分析法检坝哭变。
一．对新发现的突变设计相应的生物素标记寡核苦酸探针，斑点杂
交进一步验证结果。
5．RT－PCR法克隆洲 大鼠心3几j几钙蛋白 ICDNA，同时在 CDNA两端
弓入 A fi 11 ／N h elF 制性酶切位点。
6．采用定点哭灭枝术卜ite个irectedmutagenesis）构建。U肌肌
钙蛋白ILyS184缺夫突变CDNA，并做测序验证。
7．KR方法在突变及野生型的 心删几钙蛋白IC洲AC端引入6X
HIS标签序歹。
8．牙用A＊11 ＊he双酶切将 突变及野生勺C月肌钙蛋白Ic ＊A
亚克隆至穿梭质粒。
9．P工Scel／I｛ed双酶切重组穿梭质粒，分别将含有突变及野生
的酶切片段亚克隆到腺病毒载体。
10．Pac酶切重组腺病毒DNA，转染HEK2”细胞。扩增收集重组
腺病毒，分另IJ用KR和 wes tern彦迹法鉴定构建的 cDNA和目的
蛋白表达。
11．C S CI梯度超速昏 法纯｛t重组腺病毒。
12．开胸暴露SD大鼠，G脏，直接在心肌内注射腺病毒，2周后注射
％F位。＊肌做免疫组化 检坝及 we。t。r n6迹分析。
结果：
1．肥厚型。。肌病患者58例；其中有家族史2一例，散发性病例N
仔入男36 ｛F入女22例，年龄1卜77岁（平均46．“上17．％岁）。
病程 0刁6年；平均病程 5．57 t 5．40年；发病年龄 5刁6岁；平
士发病年龄叩．48 t侣．22岁。58例病人中2例（3．州）无特
6
殊不适；胸痛、胸闷41例（70．批），。G悸20例（34．5％），晕
厥10例门7．2们，呼吸困难11例门9．0％）；超声』C动图表现：
梗阻型19仑11（u．8％），非梗阻型”例门7．2％），。C尖肥厚型
5 ｛FIJ门．6们；4 0例具有完整的。。电图资料，其中 31例有 S T｛
改变（77．5％），8例出现异常Q波（2＊．0％）。
2．在1仟州巴厚型。删几病病人发现新的心肌肌钙蛋白I基因突变类
型：Arg R14打r卜 100例正常对照人群中未发现该突变类型。
3．未发现目前国外已报道的所有。G肌肌钙蛋白I基因突变类型。
4．生物素标记的寡核酸探针杂交证实新发现的。。肌肌钙蛋白I基
因突变Arg R145Trp。
5，队R扩增。卯几肌钙蛋白I基因S号外显子，产物％n分析未见
异常。
6．nR扩增，G肌肌钙蛋白T基因8号、9号和甘 号外显子，产物
SSCP分析未见异常。
7．jNIJ序证实获得两端带 Af 11州hC限制性酶切位点的汕大鼠
。H月JLR］L姜 蛋白IcDNA 克隆。
8．jNIJ序证实经PCR定点突变技术获得。HjL肌钙蛋白 ILysl84缺失
突变CDNA。在突变及野生CDNA C多 弓入6 X
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 developme
nt 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 the
aetiology 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 METHODS
1. 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
目的：研究肥厚型心肌病与心肌肌钙蛋白I、T基因突变的关系，尝试用多种实验方法检测突变；克隆SD大鼠心肌肌钙蛋白I基因，构建一种肥厚型心肌病相关心肌肌钙蛋白I基因突变类型(Lys184缺失突变，相当于人Lys183)重组腺病毒载体，用于发病机制的研究。
内容与方法：
1．收集临床确诊的肥厚型心肌病病例58例，提取外周血白细胞
DNA。
二．叮R扩增心 肌肌钙蛋白I基因7、8号外显子，产物直接坝序检
测有无突变。
3．KR扩增。G肌肌专蛋白T基因8、9、n号外显子及。G肌肌钙蛋
白1基因5 号外显子，采用单链构型多态性（single七trand
COflfoflfltloflPolymorphism，SSCP）分析法检坝哭变。
一．对新发现的突变设计相应的生物素标记寡核苦酸探针，斑点杂
交进一步验证结果。
5．RT－PCR法克隆洲 大鼠心3几j几钙蛋白 ICDNA，同时在 CDNA两端
弓入 A fi 11 ／N h elF 制性酶切位点。
6．采用定点哭灭枝术卜ite个irectedmutagenesis）构建。U肌肌
钙蛋白ILyS184缺夫突变CDNA，并做测序验证。
7．KR方法在突变及野生型的 心删几钙蛋白IC洲AC端引入6X
HIS标签序歹。
8．牙用A＊11 ＊he双酶切将 突变及野生勺C月肌钙蛋白Ic ＊A
亚克隆至穿梭质粒。
9．P工Scel／I｛ed双酶切重组穿梭质粒，分别将含有突变及野生
的酶切片段亚克隆到腺病毒载体。
10．Pac酶切重组腺病毒DNA，转染HEK2”细胞。扩增收集重组
腺病毒，分另IJ用KR和 wes tern彦迹法鉴定构建的 cDNA和目的
蛋白表达。
11．C S CI梯度超速昏 法纯｛t重组腺病毒。
12．开胸暴露SD大鼠，G脏，直接在心肌内注射腺病毒，2周后注射
％F位。＊肌做免疫组化 检坝及 we。t。r n6迹分析。
结果：
1．肥厚型。。肌病患者58例；其中有家族史2一例，散发性病例N
仔入男36 ｛F入女22例，年龄1卜77岁（平均46．“上17．％岁）。
病程 0刁6年；平均病程 5．57 t 5．40年；发病年龄 5刁6岁；平
士发病年龄叩．48 t侣．22岁。58例病人中2例（3．州）无特
6
殊不适；胸痛、胸闷41例（70．批），。G悸20例（34．5％），晕
厥10例门7．2们，呼吸困难11例门9．0％）；超声』C动图表现：
梗阻型19仑11（u．8％），非梗阻型”例门7．2％），。C尖肥厚型
5 ｛FIJ门．6们；4 0例具有完整的。。电图资料，其中 31例有 S T｛
改变（77．5％），8例出现异常Q波（2＊．0％）。
2．在1仟州巴厚型。删几病病人发现新的心肌肌钙蛋白I基因突变类
型：Arg R14打r卜 100例正常对照人群中未发现该突变类型。
3．未发现目前国外已报道的所有。G肌肌钙蛋白I基因突变类型。
4．生物素标记的寡核酸探针杂交证实新发现的。。肌肌钙蛋白I基
因突变Arg R145Trp。
5，队R扩增。卯几肌钙蛋白I基因S号外显子，产物％n分析未见
异常。
6．nR扩增，G肌肌钙蛋白T基因8号、9号和甘 号外显子，产物
SSCP分析未见异常。
7．jNIJ序证实获得两端带 Af 11州hC限制性酶切位点的汕大鼠
。H月JLR］L姜 蛋白IcDNA 克隆。
8．jNIJ序证实经PCR定点突变技术获得。HjL肌钙蛋白 ILysl84缺失
突变CDNA。在突变及野生CDNA C多 弓入6 X
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 developme
nt 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 the
aetiology 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 METHODS
1. 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
引文
1. Report of the 1995 World Health Organization/International Society and Federation of Cardiology Task Force on the Definition and Classification of Cardiomyopathies Circulation 1996 93: 841-842.
2．中华心血管病杂志编辑委员会心肌炎心肌病对策专题组。关于成人急性病毒性心肌炎诊断标准和采纳世界卫生组织及国际心脏病学会联合会工作组关于心肌病定义和分类的意见。中华心血管病杂志，1999，27(6)：405-407。
3. Teare RD. Asymmetrical hypertrophy of the heart in young adults. Br Heart J. 1958 20: 1-8
4. Hollman A, Goodwin JF, Teare D, Renwick JW. A family with obstructive cardiomyopathy (asymmetrical hypertrophy). Br Heart J. 1960; 22: 449-456.
5. Jarcho JA, McKenna W, Pare JAP, Solomon SD, Holcombe RF, Dickie S, Levi T, Donis-Keller H, Seidman JG, Seidman CE. Mapping a gene for familial hypertrophic cardiomyopathy to chromosome 14ql. N Engl J Med. 1989; 321: 1372-1378.
6. Geisterfer-Lowrance AAT, Kass S, Tanigawa G, Vosberg HP, McKenna W, Seidman CE, Seidman JG. A molecular basis for familial hypertrophic cardiomyopathy: α β cardiac myosin heavy chain gene missense mutation. Cell. 1990; 62: 999-1006.
7. Van Driest SL, Ackerman MJ, Ommen SR, Shakur R, Will ML,
Nishimura RA, Tajik AJ, Gersh BJ. Prevalence and severity of "benign" mutations in the beta-myosin heavy chain, cardiac troponin T, and alpha-tropomyosin genes in hypertrophic cardiomyopathy. Circulation. 2002 Dec 10; 106(24): 3085-90.
8. Kimura A, Harada H, Park JE, Nishi H, Satoh M, Takahashi M, Hiroi S, Sasaoka T, Ohbuchi N, Nakamura T, Koyanagi T, Hwang TH, Choo JA, Chung KS, Hasegawa A, Nagai R, Okazaki O, Nakamura H, Matsuzaki M, Sakamoto T, Toshima H, Koga Y, Imaizumi T, Sasazuki T. Mutations in the cardiac troponin I gene associated with hypertrophic cardiomyopathy. Nat Genet. 1997 Aug;16(4):379-82.
9. Morner S, Richard P, Kazzam E, Hainque B, Schwartz K, Waldenstrom A. Deletion in the cardiac troponin I gene in a family from northern Sweden with hypertrophic cardiomyopathy. J Mol Cell Cardiol. 2000 Mar; 32(3): 521-5.
10. Hoffmann B, Schmidt-Traub H, Perrot A, Osterziel KJ, Gessner R. First mutation in cardiac troponin C, L29Q, in a patient with hypertrophic. Hum Mutat 2001 Jun; 17(6): 524cardiomyopathy.
11. Rottbauer W, Gautel M, Zehelein J, Labeit S, Franz WM, Fischer C, Vollrath B, Mall G, Dietz R, Kubler W, Katus HA. Novel splice donor site mutation in the cardiac myosin-binding protein-C gene in familial hypertrophic cardiomyopathy. Characterization Of cardiac transcript and protein. J Clin Invest. 1997 Jul 15; 100(2):475-82.
12. Watkins H, Conner D, Thierfelder L, Jarcho JA, MacRae C, McKenna WJ, Maron BJ, Seidman JG, Seidman CE. Mutations in the cardiac myosin binding protein-C gene on chromosome 11 cause familial hypertrophic cardiomyopathy. Nat Genet. 1995 Dec; 11(4): 434-7.
13. Satoh M, Takahashi M, Sakamoto T, Hiroe M, Marumo F, Kimura A. Structural analysis of the titin gene in hypertrophic cardiomyopathy: identification of a novel disease gene. Biochem Biophys Res Commun. 1999 Aug 27; 262(2): 411-7.
14. Andersen PS, Havndrup O, Bundgaard H, Moolman-Smook JC,
Larsen LA, Mogensen J, Brink PA, Borglum AD, Corfield VA, Kjeldsen K, Vuust J, Christiansen M. Myosin light chain mutations in familial hypertrophic cardiomyopathy: phenotypic presentation and frequency in Danish and South African populations. J Med Genet. 2001 Dec; 38(12): E43.
15. Olson TM, Doan TP, Kishimoto NY, Whitby FG, Ackerman MJ, Fananapazir L. Inherited and de novo mutations in the cardiac actin gene cause hypertrophic cardiomyopathy. J Mol Cell Cardiol. 2000 Sep; 32(9): 1687-94.
16. Prabhakar R, Boivin GP, Grupp IL, Hoit B, Arteaga G, Solaro JR, Wieczorek DF. A familial hypertrophic cardiomyopathy alpha-tropomyosin mutation causes severe cardiac hypertrophy and death in mice. J Mol Cell Cardiol. 2001 Oct; 33(10): 1815-28.
17. Niimura H, Patton KK, McKenna WJ, Soults J, Maron BJ, Seidman JG, Seidman CE. Sarcomere protein gene mutations in hypertrophic cardiomyopathy of the elderly. Circulation. 2002 Jan 29;105(4):446-51.
18. Marin-Garcia J, Goldenthal MJ. Mitochondrial DNA defects in cardiomyopathy. Cardiovasc Pathol. 1998; 7(4): 205-213.
19. Blair E, Redwood C, Ashrafian H, Oliveira M, Broxholme J, Kerr B, Salmon A, Ostman-Smith I, Watkins H. Mutations in the gamma(2) subunit of AMP-activated protein kinase cause familial hypertrophic cardiomyopathy: evidence for the central role of energy compromise in disease pathogenesis. Hum Mol Genet. 2001 May 15;10(11):1215-20.
20. Gollob MH, Green MS, Tang AS, Gollob T, Karibe A, Ali Hassan AS, Ahmad F, Lozado R, Shah G, Fananapazir L, Bachinski LL, Roberts R, Hassan AS. Identification of a gene responsible for familial Wolff-Parkinson-White syndrome. N Engl J Med. 2001 Jun 14; 344(24): 1823-31.
21. Geier C, Perrot A, Ozcelik C, Binner P, Counsell D, Hoffmann K, Pilz
B, Martiniak Y, Gehmlich K, van der Ven PF, Furst DO, Vornwald A, von Hodenberg E, Nurnberg P, Scheffold T, Dietz R, Osterziel KJ. Mutations in the human muscle LIM protein gene in families with hypertrophic cardiomyopathy. Circulation. 2003 Mar 18; 107(10): 1390-5.
22. Kuang SQ, Yu JD, Lu L, He LM, Gong LS, Chen SJ, Chen Z. Identification of a novel missense mutation in the cardiac beta-myosin heavy chain gene in a Chinese patient with sporadic hypertrophic cardiomyopathy. J Mol Cell Cardiol. 1996 Sep; 28(9): 1879-83.
23．宋雷，黄晓红，惠汝太，等。心肌球蛋白重链基因Arg719Gln突变基因型与家族性肥厚型心肌病。中华心血管杂志，2001，29：348-352。
24．宋雷，徐荣，吴格茹，等。肌球蛋白重链基因 Arg663Cys和Arg66 3His突变基因型与家族性肥厚型心肌病表型关系研究。中华心血管杂志，2002，30：131-135。
25. Bertinchant JP, Polge A, Juan JM, Oliva-Lauraire MC, Giuliani I, Marty-Double C, Burdy JY, Fabbro-Peray P, Laprade M, Bali JP, Granier C, de la Coussaye JE, Dauzat M. Evaluation of cardiac troponin I and T levels as markers of myocardial damage in doxorubicin-induced cardiomyopathy rats, and their relationship with echocardiographic and histological findings. Clin Chim Acta. 2003 Mar; 329(1-2): 39-51.
26. Niimura H, Patton KK, McKenna WJ, Soults J, Maron BJ, Seidman JG, Seidman CE. Sarcomere protein gene mutations in hypertrophic cardiomyopathy of the elderly. Circulation. 2002; 105(4):446-51.
27. Mogensen J, Klausen IC, Egeblad H, Baandrup U. Sudden cardiac death in familial hypertrophic cardiomyopathy is associated with a novel mutation in the troponin Ⅰ. Circulation. 1999; 100: Ⅰ-618.
28. Shimizu M, Ino H, Okeie K, Yamaguchi M, Nagata M, Hayashi K, Itoh H, Iwaki T, Oe K, Konno T, Mabuchi H. T-peak to T-end interval may be a better predictor of high-risk patients with hypertrophic cardiomyopathy associated with a cardiac troponin Ⅰ mutation than QT
dispersion. Clin Cardiol. 2002 Jul; 25(7): 335-9
29. Shimizu M, Ino H, Okeie K, Yamaguchi M, Hayashi K, Nagata M, Itoh H, Iwaki T, Oe K, Konno T, Mabuchi H. Septal wall thinning and systolic dysfunction in patients with hypertrophic cardiomyopathy caused by a cardiac troponin I gene mutation. Am Heart J 2002 Apr; 143(4): 690-5
30. Shimizu M, Ino H, Yamaguchi M, Terai H, Hayashi K, Kiyama M, Sakata K, Hayashi T, Inoue M, Kaneda T, Mabuchi H. Chronologic electrocardiographic changes in patients with hypertrophic cardiomyopathy associated with cardiac troponin 1 mutation. Am Heart J 2002 Feb; 143(2): 289-93
31. Thierfelder L, Watkins H, MacRae C, Lamas R, McKenna W, Vosberg HP, Seidman JG, Seidman CE. Alpha-tropomyosin and cardiac troponin T mutations cause familial hypertrophic cardiomyopathy: a disease of the sarcomere. Cell. 1994 Jun 3; 77(5): 701-12.
32. Forissier JF, Carrier L, Farza H, Bonne G, Bercovici J, Richard P, Hainque B, Townsend PJ, Yacoub MH, Faure S, Dubourg O, Millaire A, Hagege AA, Desnos M, Komajda M, Schwartz K. Codon 102 of the cardiac troponin T gene is a putative hot spot for mutations in familial hypertrophic cardiomyopathy. Circulation. 1996 Dec 15; 94(12): 3069-73.
31. Watkins H, McKenna WJ, Thierfelder L, Suk HJ, Anan R, O'Donoghue A, Spirito P, Matsumori A, Moravec CS, Seidman JG, et al.Mutations in the genes for cardiac troponin T and alpha-tropomyosin in hypertrophic cardiomyopathy. N Engl J Med. 1995 Apr 20;332(16):1058-64.
32. Thierfelder L, Watkins H, MacRae C, Lamas R, McKenna W, Vosberg HP, Seidman JG, Seidman CE.Alpha-tropomyosin and cardiac troponin T mutations cause familial hypertrophic cardiomyopathy: a disease of the sarcomere. Cell. 1994 Jun 3;77(5):701-12.
33. Moolman JC, Corfield VA, Posen B, Ngumbela K, Seidman C, Brink
PA, Watkins H.Sudden death due to troponin T mutations. J Am Coll Cardiol. 1997 Mar 1;29(3):549-55.
34. Nakajima-Taniguchi C, Matsui H, Fujio Y, Nagata S, Kishimoto T, Yamauchi-Takihara K.Novel missense mutation in cardiac troponin T gene found in Japanese patient with hypertrophic cardiomyopathy. J Mol Cell Cardiol. 1997 Feb;29(2):839-43.
35. Forissier JF, Carrier L, Farza H, Bonne G, Bercovici J, Richard P, Hainque B, Townsend PJ, Yacoub MH, Faure S, Dubourg O, Millaire A, Hagege AA, Desnos M, Komajda M, Schwartz K. Codon 102 of the cardiac troponin T gene is a putative hot spot for mutations in familial hypertrophic cardiomyopathy. Circulation. 1996 Dec 15; 94(12): 3069-73.
36. Fujino N, Shimizu M, Ino H, Yamaguchi M, Yasuda T, Nagata M, Konno T, Mabuchi H. A novel mutation Lys273Glu in the cardiac troponin T gene shows high degree of penetrance and transition from hypertrophic to dilated cardiomyopathy. Am J Cardiol. 2002 Jan 1;89(1):29-33.
37. Ho CY, Lever HM, DeSanctis R, Farver CF, Seidman JG, Seidman CE.Homozygous mutation in cardiac troponin T: implications for hypertrophic cardiomyopathy. Circulation. 2000 Oct 17; 102(16): 1950-5.
38. Varnava A, Baboonian C, Davison F, de Cruz L, Elliott PM, Davies MJ, McKenna WJ.A new mutation of the cardiac troponin T gene causing familial hypertrophic cardiomyopathy without left ventricular hypertrophy. Heart. 1999 Nov;82(5):621-4.
2．中华心血管病杂志编辑委员会心肌炎心肌病对策专题组。关于成人急性病毒性心肌炎诊断标准和采纳世界卫生组织及国际心脏病学会联合会工作组关于心肌病定义和分类的意见。中华心血管病杂志，1999，27(6)：405-407。
3. Teare RD. Asymmetrical hypertrophy of the heart in young adults. Br Heart J. 1958 20: 1-8
4. Hollman A, Goodwin JF, Teare D, Renwick JW. A family with obstructive cardiomyopathy (asymmetrical hypertrophy). Br Heart J. 1960; 22: 449-456.
5. Jarcho JA, McKenna W, Pare JAP, Solomon SD, Holcombe RF, Dickie S, Levi T, Donis-Keller H, Seidman JG, Seidman CE. Mapping a gene for familial hypertrophic cardiomyopathy to chromosome 14ql. N Engl J Med. 1989; 321: 1372-1378.
6. Geisterfer-Lowrance AAT, Kass S, Tanigawa G, Vosberg HP, McKenna W, Seidman CE, Seidman JG. A molecular basis for familial hypertrophic cardiomyopathy: α β cardiac myosin heavy chain gene missense mutation. Cell. 1990; 62: 999-1006.
7. Van Driest SL, Ackerman MJ, Ommen SR, Shakur R, Will ML,
Nishimura RA, Tajik AJ, Gersh BJ. Prevalence and severity of "benign" mutations in the beta-myosin heavy chain, cardiac troponin T, and alpha-tropomyosin genes in hypertrophic cardiomyopathy. Circulation. 2002 Dec 10; 106(24): 3085-90.
8. Kimura A, Harada H, Park JE, Nishi H, Satoh M, Takahashi M, Hiroi S, Sasaoka T, Ohbuchi N, Nakamura T, Koyanagi T, Hwang TH, Choo JA, Chung KS, Hasegawa A, Nagai R, Okazaki O, Nakamura H, Matsuzaki M, Sakamoto T, Toshima H, Koga Y, Imaizumi T, Sasazuki T. Mutations in the cardiac troponin I gene associated with hypertrophic cardiomyopathy. Nat Genet. 1997 Aug;16(4):379-82.
9. Morner S, Richard P, Kazzam E, Hainque B, Schwartz K, Waldenstrom A. Deletion in the cardiac troponin I gene in a family from northern Sweden with hypertrophic cardiomyopathy. J Mol Cell Cardiol. 2000 Mar; 32(3): 521-5.
10. Hoffmann B, Schmidt-Traub H, Perrot A, Osterziel KJ, Gessner R. First mutation in cardiac troponin C, L29Q, in a patient with hypertrophic. Hum Mutat 2001 Jun; 17(6): 524cardiomyopathy.
11. Rottbauer W, Gautel M, Zehelein J, Labeit S, Franz WM, Fischer C, Vollrath B, Mall G, Dietz R, Kubler W, Katus HA. Novel splice donor site mutation in the cardiac myosin-binding protein-C gene in familial hypertrophic cardiomyopathy. Characterization Of cardiac transcript and protein. J Clin Invest. 1997 Jul 15; 100(2):475-82.
12. Watkins H, Conner D, Thierfelder L, Jarcho JA, MacRae C, McKenna WJ, Maron BJ, Seidman JG, Seidman CE. Mutations in the cardiac myosin binding protein-C gene on chromosome 11 cause familial hypertrophic cardiomyopathy. Nat Genet. 1995 Dec; 11(4): 434-7.
13. Satoh M, Takahashi M, Sakamoto T, Hiroe M, Marumo F, Kimura A. Structural analysis of the titin gene in hypertrophic cardiomyopathy: identification of a novel disease gene. Biochem Biophys Res Commun. 1999 Aug 27; 262(2): 411-7.
14. Andersen PS, Havndrup O, Bundgaard H, Moolman-Smook JC,
Larsen LA, Mogensen J, Brink PA, Borglum AD, Corfield VA, Kjeldsen K, Vuust J, Christiansen M. Myosin light chain mutations in familial hypertrophic cardiomyopathy: phenotypic presentation and frequency in Danish and South African populations. J Med Genet. 2001 Dec; 38(12): E43.
15. Olson TM, Doan TP, Kishimoto NY, Whitby FG, Ackerman MJ, Fananapazir L. Inherited and de novo mutations in the cardiac actin gene cause hypertrophic cardiomyopathy. J Mol Cell Cardiol. 2000 Sep; 32(9): 1687-94.
16. Prabhakar R, Boivin GP, Grupp IL, Hoit B, Arteaga G, Solaro JR, Wieczorek DF. A familial hypertrophic cardiomyopathy alpha-tropomyosin mutation causes severe cardiac hypertrophy and death in mice. J Mol Cell Cardiol. 2001 Oct; 33(10): 1815-28.
17. Niimura H, Patton KK, McKenna WJ, Soults J, Maron BJ, Seidman JG, Seidman CE. Sarcomere protein gene mutations in hypertrophic cardiomyopathy of the elderly. Circulation. 2002 Jan 29;105(4):446-51.
18. Marin-Garcia J, Goldenthal MJ. Mitochondrial DNA defects in cardiomyopathy. Cardiovasc Pathol. 1998; 7(4): 205-213.
19. Blair E, Redwood C, Ashrafian H, Oliveira M, Broxholme J, Kerr B, Salmon A, Ostman-Smith I, Watkins H. Mutations in the gamma(2) subunit of AMP-activated protein kinase cause familial hypertrophic cardiomyopathy: evidence for the central role of energy compromise in disease pathogenesis. Hum Mol Genet. 2001 May 15;10(11):1215-20.
20. Gollob MH, Green MS, Tang AS, Gollob T, Karibe A, Ali Hassan AS, Ahmad F, Lozado R, Shah G, Fananapazir L, Bachinski LL, Roberts R, Hassan AS. Identification of a gene responsible for familial Wolff-Parkinson-White syndrome. N Engl J Med. 2001 Jun 14; 344(24): 1823-31.
21. Geier C, Perrot A, Ozcelik C, Binner P, Counsell D, Hoffmann K, Pilz
B, Martiniak Y, Gehmlich K, van der Ven PF, Furst DO, Vornwald A, von Hodenberg E, Nurnberg P, Scheffold T, Dietz R, Osterziel KJ. Mutations in the human muscle LIM protein gene in families with hypertrophic cardiomyopathy. Circulation. 2003 Mar 18; 107(10): 1390-5.
22. Kuang SQ, Yu JD, Lu L, He LM, Gong LS, Chen SJ, Chen Z. Identification of a novel missense mutation in the cardiac beta-myosin heavy chain gene in a Chinese patient with sporadic hypertrophic cardiomyopathy. J Mol Cell Cardiol. 1996 Sep; 28(9): 1879-83.
23．宋雷，黄晓红，惠汝太，等。心肌球蛋白重链基因Arg719Gln突变基因型与家族性肥厚型心肌病。中华心血管杂志，2001，29：348-352。
24．宋雷，徐荣，吴格茹，等。肌球蛋白重链基因 Arg663Cys和Arg66 3His突变基因型与家族性肥厚型心肌病表型关系研究。中华心血管杂志，2002，30：131-135。
25. Bertinchant JP, Polge A, Juan JM, Oliva-Lauraire MC, Giuliani I, Marty-Double C, Burdy JY, Fabbro-Peray P, Laprade M, Bali JP, Granier C, de la Coussaye JE, Dauzat M. Evaluation of cardiac troponin I and T levels as markers of myocardial damage in doxorubicin-induced cardiomyopathy rats, and their relationship with echocardiographic and histological findings. Clin Chim Acta. 2003 Mar; 329(1-2): 39-51.
26. Niimura H, Patton KK, McKenna WJ, Soults J, Maron BJ, Seidman JG, Seidman CE. Sarcomere protein gene mutations in hypertrophic cardiomyopathy of the elderly. Circulation. 2002; 105(4):446-51.
27. Mogensen J, Klausen IC, Egeblad H, Baandrup U. Sudden cardiac death in familial hypertrophic cardiomyopathy is associated with a novel mutation in the troponin Ⅰ. Circulation. 1999; 100: Ⅰ-618.
28. Shimizu M, Ino H, Okeie K, Yamaguchi M, Nagata M, Hayashi K, Itoh H, Iwaki T, Oe K, Konno T, Mabuchi H. T-peak to T-end interval may be a better predictor of high-risk patients with hypertrophic cardiomyopathy associated with a cardiac troponin Ⅰ mutation than QT
dispersion. Clin Cardiol. 2002 Jul; 25(7): 335-9
29. Shimizu M, Ino H, Okeie K, Yamaguchi M, Hayashi K, Nagata M, Itoh H, Iwaki T, Oe K, Konno T, Mabuchi H. Septal wall thinning and systolic dysfunction in patients with hypertrophic cardiomyopathy caused by a cardiac troponin I gene mutation. Am Heart J 2002 Apr; 143(4): 690-5
30. Shimizu M, Ino H, Yamaguchi M, Terai H, Hayashi K, Kiyama M, Sakata K, Hayashi T, Inoue M, Kaneda T, Mabuchi H. Chronologic electrocardiographic changes in patients with hypertrophic cardiomyopathy associated with cardiac troponin 1 mutation. Am Heart J 2002 Feb; 143(2): 289-93
31. Thierfelder L, Watkins H, MacRae C, Lamas R, McKenna W, Vosberg HP, Seidman JG, Seidman CE. Alpha-tropomyosin and cardiac troponin T mutations cause familial hypertrophic cardiomyopathy: a disease of the sarcomere. Cell. 1994 Jun 3; 77(5): 701-12.
32. Forissier JF, Carrier L, Farza H, Bonne G, Bercovici J, Richard P, Hainque B, Townsend PJ, Yacoub MH, Faure S, Dubourg O, Millaire A, Hagege AA, Desnos M, Komajda M, Schwartz K. Codon 102 of the cardiac troponin T gene is a putative hot spot for mutations in familial hypertrophic cardiomyopathy. Circulation. 1996 Dec 15; 94(12): 3069-73.
31. Watkins H, McKenna WJ, Thierfelder L, Suk HJ, Anan R, O'Donoghue A, Spirito P, Matsumori A, Moravec CS, Seidman JG, et al.Mutations in the genes for cardiac troponin T and alpha-tropomyosin in hypertrophic cardiomyopathy. N Engl J Med. 1995 Apr 20;332(16):1058-64.
32. Thierfelder L, Watkins H, MacRae C, Lamas R, McKenna W, Vosberg HP, Seidman JG, Seidman CE.Alpha-tropomyosin and cardiac troponin T mutations cause familial hypertrophic cardiomyopathy: a disease of the sarcomere. Cell. 1994 Jun 3;77(5):701-12.
33. Moolman JC, Corfield VA, Posen B, Ngumbela K, Seidman C, Brink
PA, Watkins H.Sudden death due to troponin T mutations. J Am Coll Cardiol. 1997 Mar 1;29(3):549-55.
34. Nakajima-Taniguchi C, Matsui H, Fujio Y, Nagata S, Kishimoto T, Yamauchi-Takihara K.Novel missense mutation in cardiac troponin T gene found in Japanese patient with hypertrophic cardiomyopathy. J Mol Cell Cardiol. 1997 Feb;29(2):839-43.
35. Forissier JF, Carrier L, Farza H, Bonne G, Bercovici J, Richard P, Hainque B, Townsend PJ, Yacoub MH, Faure S, Dubourg O, Millaire A, Hagege AA, Desnos M, Komajda M, Schwartz K. Codon 102 of the cardiac troponin T gene is a putative hot spot for mutations in familial hypertrophic cardiomyopathy. Circulation. 1996 Dec 15; 94(12): 3069-73.
36. Fujino N, Shimizu M, Ino H, Yamaguchi M, Yasuda T, Nagata M, Konno T, Mabuchi H. A novel mutation Lys273Glu in the cardiac troponin T gene shows high degree of penetrance and transition from hypertrophic to dilated cardiomyopathy. Am J Cardiol. 2002 Jan 1;89(1):29-33.
37. Ho CY, Lever HM, DeSanctis R, Farver CF, Seidman JG, Seidman CE.Homozygous mutation in cardiac troponin T: implications for hypertrophic cardiomyopathy. Circulation. 2000 Oct 17; 102(16): 1950-5.
38. Varnava A, Baboonian C, Davison F, de Cruz L, Elliott PM, Davies MJ, McKenna WJ.A new mutation of the cardiac troponin T gene causing familial hypertrophic cardiomyopathy without left ventricular hypertrophy. Heart. 1999 Nov;82(5):621-4.