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目的研究中国人17α-羟化酶/17 ,20-裂解酶缺乏症CYP17A1基因突变特点,以及结合患者的临床表现与基因突变类型初步探讨P450C17酶蛋白的结构与功能的关系。方法收集5例17α-羟化酶/17 ,20-裂解酶缺乏症患者及其部分家属血标本,提取基因组DNA,设计7对引物扩增CYP17A1基因的8个外显子及外显子与内含子的连接区域,琼脂糖凝胶电泳鉴定PCR产物,产物胶回收后直接作为DNA双链模板测序。DNA双链模板不一致的PCR产物经克隆后测序。测序结果在核苷酸序列数据库进行比较分析。结果 5例患者均检测出CYP17A1基因突变,共存在2种新的复合突变,即6436-6438(TAC→AA)导致Y329K,418X和6531-6532(GC→A)导致L361F,418X。这两种突变均形成缺乏酶活性中心的截短蛋白。其中4例患者为Y329K,418X突变纯合子,1例为Y329K,418X/L361F,418X的复合杂合子。5例患者的临床表型为17α-羟化酶/17 ,20-裂解酶的完全性联合缺陷,与其基因突变类型相一致。结论 6436-6438( TAC→AA)这种突变可能在中国人较常见,可能具有种族特异性。将进一步进行突变酶蛋白的功能学研究。
Objective To study the characteristics of CYP17A1 gene mutation in Chinese 17α-hydroxylase / 17, 20-lyase deficiency and to explore the relationship between the structure and function of P450C17 enzyme protein in patients with clinical manifestations and gene mutation types. Methods The blood samples of 5 patients with 17α-hydroxylase / 17, 20-lyase deficiency and their relatives were collected and genomic DNA was extracted. Seven pairs of primers were designed to amplify the eight exons and exons of CYP17A1 gene The ligation region of the intron was identified by agarose gel electrophoresis and the product gel was recovered and directly sequenced as a DNA double-stranded template. DNA duplex template inconsistent PCR products were cloned and sequenced. Sequencing results were compared in the nucleotide sequence database. Results All 5 patients were detected CYP17A1 gene mutation, there are two new co-existence mutations, namely 6436-6438 (TAC → AA) lead to Y329K, 418X and 6531-6532 (GC → A) lead to L361F, 418X. Both of these mutations form truncated proteins that lack enzyme activity. Among them, 4 patients were Y329K and 418X mutant homozygote, and 1 patient was complex heterozygote Y329K, 418X / L361F and 418X. The clinical phenotype of 5 patients was a complete combined defect of 17α-hydroxylase / 17,20-lyase consistent with the type of gene mutation. Conclusions 6436-6438 (TAC → AA) This mutation may be more common in Chinese and may be racially specific. Functional studies of the mutant enzyme proteins will be further carried out.