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目的在16p13-12．1位点20 cm的区域内明确致病基因,了解基因型和临床表型的关系。方法采用双向DNA直接测序法筛选候选基因;运用免疫组化和蛋白免疫印迹法等了解突变基因的表达情况。运用质粒重组及体外表达方法获得野生型和突变型蛋白,使用免疫共沉淀法了解这两者的结合程度。结果在两个家系中发现编码平滑肌肌球蛋白的MYH11基因突变,所有TAAD和PDA患者均为突变基因携带者。在法国家系中发现两个突变。前者为剪切点供体杂合子突变,导致编码71个氨基酸的第32外显子完全缺失:第2个突变为第37外显子上的1个点突变,导致精氨酸变为谷氨酸。在美国家系中发现1个杂合子突变,为第28外显子上编码24个氨基酸的72个碱基缺失。在法国患者的病变动脉标本上运用蛋白免疫印迹法,发现野生型和突变型肌球蛋白蛋白共存,但有异常免疫组化表现。体外实验显示重组野生型和突变型蛋白不能正常结合。结论MYH11基因突变导致家族性TAAD合并PDA,野生型和突变型平滑肌肌球蛋白不能正常结合,推论杂合子突变导致“Dominant-negative”作用。
Objective To identify the causative genes in the region of 20 cm from 16p13-12.1 and to understand the relationship between genotypes and clinical phenotypes. Methods Candidate genes were screened by direct DNA sequencing. The expression of mutant genes was analyzed by immunohistochemistry and Western blotting. Wild-type and mutant proteins were obtained by plasmid recombination and in vitro expression methods, and the co-immunoprecipitation method was used to understand the degree of binding between the two. Results Mutations in the MYH11 gene encoding smooth muscle myosin were found in both pedigrees, and all TAAD and PDA patients were carriers of the mutated gene. Two mutations were found in the French pedigree. The former is a donor heterozygous mutation at the point of cleavage that results in the complete deletion of the 32nd exon encoding the 71 amino acids: the second mutation is a point mutation on exon 37 leading to the conversion of arginine to glutamine acid. A heterozygous mutation was found in the U.S. pedigrees, with a 72-base deletion of 24 amino acids on exon 28. Western blotting was used to detect the presence of wild-type and mutant myosin proteins in diseased specimens of French patients, but abnormal immunohistochemical findings were observed. In vitro experiments show that the recombinant wild-type and mutant proteins can not be normally combined. Conclusion Mutations of MYH11 gene lead to the failure of familial TAAD with PDA, wild-type and mutant smooth muscle myosin to bind normally, suggesting that heterozygous mutation leads to “Dominant-negative” effect.