X连锁显性遗传性肾炎一家系相关基因排除定位分析

A Exclusive Mapping Analysis on the Gene Associated with X-linked Dominant Inherited Nephritis in One Pedigree

目的 研究我国X连锁显性遗传性肾炎一家系 (又名Alport综合征 ,Alport’ssyndrome ,AS)编码Ⅳ型胶原α5链的COL4A5基因突变特点。方法 提取 3 5例现存家系成员基因组DNA ,根据COL4A5基因中与外显子相邻的内含子序列设计引物 ,分别作DNA聚合酶链式反应 (PCR) ,将该基因所有 5 1个外显子扩增。以琼脂糖凝胶电泳鉴定产物 ,作单链构象多态性 (SSCP)分析检测PCR产物 ,对迁移率异常者进行DNA直接测序。结果 PCR -SSCP分析发现 17个外显子PCR产物单链DNA迁移率异常 ,通过DNA直接测序发现 9个外显子可疑突变 ,但反向测序均未能证实。结论 该家系COL4A5基因外显子未发生突变 ,其突变可能发生在该基因内含子或其它致病基因。

Objective To study the characteristics of mutations of gene COL4A5 encoding type Ⅳ collagen α 5 chain in one chinese pedigree with X-linked dominant inherited nephritis (Alport's syndrome,AS). Methods Genomic DNA was extracted from 35 members of the pedigree of Alport's syndrome. All of 51 exons of COL4A5 gene were amplified by PCR with the primers synthesized according to the published intron sequences of COL4A5. The PCR products were further analyzed by agarose gel electrophoresis and single strand conformation polymorphism (SSCP). The PCR products showing polymorphism were directly sequenced. Results PCR-SSCP analysis showed that 17 PCR products had abnormal mobility of single strand DNA. DNA sequencing analysis revealed 9 suspicious mutations. But these suspicious mutations were not be confirmed by inverse sequencing analysis. Conclusion The exon mutation of COL4A5 gene of this pedigree did not be found, and the mutations of COL4A5 gene may locate in the its introns.