一种新的NADH-细胞色素b5还原酶基因点突变的发现和基因诊断

THE IDENTIFICATION OF POINT MUTATION IN NADH-CYTOCHROME b5 REDUCTASE GENE AND GENE DIAGNOSIS

为了查明中国人遗传性高铁血红蛋白血症(RaVl)患者的NADH-细胞色素b5还原酶(b5R)基因突变类型,探讨RCM发生的分子机制,从已报道的1例RCM I型患者的外周血白细胞中提取RNA.应用反转录PCR法扩增了b5R cDNA,分析了b5R cDNA的全部编码序列的921bp片段.结果显示,b5RcDNA基因的第203位密码子存在(TGC→TAC)Cys→Try的错义突变.经基因组PCR-RFLP分析证实该突变并非PCR反应过程中的错配,而是一种新的基因突变类型.由于此突变减少了一个对酶功能至关重要的带巯基的半胱氨酸,而引入了一个带酚侧链的氨基酸,致使酶的稳定性及活性下降.

To characterize the mutation in b5R gene in a Chinese patient with RCM I and elucidate the molecular basis of the disease, we isolated total RNA from the peripheral leukocytes of the propositus and synthesized cDNA using RT-PCR. The coding region of b5R cDNA (921bp) was analysed by sequencing the RT-PCR products. A novel mutation with replacement of Cys203(TGC) by Tyr(TAC) in exon 7 was found, which awas further confimed by restriction enzyme analysis of the genormic DNA fragment. Replacement of Cys with Tyr at 203 of b5R gene is the molecular basis of the propositus. The Cys203 Tyr mutation abolishes a thiol group critical to enzyme functionality.