中国人17α-羟化酶/17,20-裂解酶缺乏症基因突变研究

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／1361F，418X的复合杂合子。5例患者的临床表型为17Q-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。

Study on the genetic mutations of 17α-hydro-xylase/17,20-lyase deficiency in Chinese patients

OBJECTIVE: To investigate the CYP17A1 gene mutations in Chinese patients with 17 alpha-hydroxylase/17, 20-lyase deficiency. METHODS: Clinical data were retrospectively analyzed. The CYP17A1 gene mutations were detected in 5 cases with 17 alpha-hydroxylase/17, 20-lyase deficiency and their relatives. The genomic DNA of the patients was isolated from whole blood. Seven pairs of primers were used to amplify eight exons and exon-intron boundaries of the CYP17A1 gene. The amplified PCR products were purified by agarose gel and then directly sequenced. In order to confirm the DNA sequences of different alleles, some fragments were inserted into pMD 18-T vector and then sequenced. Sequencing results were compared to the established human CYP17A1 sequence. RESULTS: Briefly, we found 2 kinds of compound mutations, of which were: (1) 6436-6438(TAC-->AA), causing amino acid Y329K, 418X; (2) 6531-6532(GC-->A), causing amino acid L361F, 418X. Among the five cases, four were homozygous for 6436-6438(TAC-->AA), whereas one was compound heterozygous for 6436-6438(TAC-->AA)/6531-6532(GC-->A). The clinical characteristics of 5 cases were all completely combined defects of 17 alpha-hydroxylase/17, 20-lyase, and they all carried two alleles of CYP17A1 gene mutations that all shifted the reading frame and resulted in truncated protein which lack of the activity center site of P450C17, of which corresponding with their clinical feature. CONCLUSION: Nine alleles have the mutation of 6436-6438(TAC-->AA), accounting for 90% of total alleles (9/10). That suggests this kind of mutation may have racial specificity. More study should be done to have better understanding of the function of the truncated P450C17 enzymes.