汉族人多囊肾病1型致病基因突变检测体系的建立及初步应用

目的 建立适合筛查汉族人多囊肾病1型致病基因（PKD1）突变的检测体系。方法 利用设计的82对引物8对针对PKD15′端多拷贝区的长链聚合酶链式反应（PCR）引物和57对巢式PCR引物，17对针对3′端单拷贝区PCR引物]分别对PKD1的46个外显子进行扩增，扩增产物通过单链构象多态性（SSCP）分析筛检出异常条带后，再经测序确定基因突变位点。利用建立的PCR－SSCP检测体系对汉族人2个常染色体显性遗传性多囊肾病患者家系进行PKDA1突变检测，健康献血员为对照。结果 用82对PCR引物，可成功扩增PKD1各个外显子区域，并经测序证实为PKD1目的片段。将建立的SSCP－PCR基因突变检测体系，分别从2个汉族人常染色体显性多囊肾病（ADPKD）家系检测出PKD1基因Del 3 bp(G49761-G49763)和C47629T2个突变，其可分别导致编码产物第3827位缺失谷氨酸（Glu3827)和第3555位丝氨酸，而产生由苯丙氨酸(S3555F)替代的改变。结论 本研究建立的PCR－SSCP检测体系，可完成PKD1各外显子区域特异性扩增，并成功检测出汉族人2个ADPKD家系基因突变位点，不仅为PKD1基因突变的致病机理研究提供宝贵资料，而且为下一步汉族人多囊肾病的大规模基因突变筛查和临床诊断试剂盒的研制奠定了基础。

Development and preliminary application of a systems for detecting the Hans PKD1 mutation

Objective To set up a system for detecting the Hans PKD1 mutations and then screen two ADPKD families to testify the system effectiveness. Methods Using the genomic DNA extracted from the Hans as a template, we synthesized 82 pairs of primers, including 8 pairs for the multi copy areas long range PCR and 57 pairs for the nested PCR, 17 pairs for single copy amplifications. The PCR products were analyzed by single strand conformation polymorphism technique (PCR SSCP) and the abnormal bands were selected to be sequenced. Results Using 82 pairs of primers,the Hans PKD1 could be distinctively amplified via Long range and nested PCR,which were suitable for SSCP analysis. At last, we detected two point mutation including Del 3 bp(G 49761 G 49763 )and C47629T] existed in two ADPKD pedigrees of Hans, which led to frameshift after Glu3827 and S3555F in PKD1 translations,respectively. Conclusion The system could be used as a tool for the Hans PKD1 mutations, which will not only supply more data for exploring the mechanism of the ADPKD pathogenesis, but also be helpful to screen the Hans ADPKD in large scale further and to develop the detection kit for the PKD1 mutations.