变性高效液相色谱检测PKD2基因突变

Mutation detection of PKD2 gene in Chinese by denaturing high-performance liquid chromatograph

目的 利用变性高效液相色谱分析技术 ( denaturing high- performance liquidchromatography,DHPL C) ,检测 2型常染色体显性遗传性多囊肾病致病基因 ( polycystic kidney diseasegene 2 ,PKD2 )突变。方法 收集临床确诊的汉族常染色体显性遗传性多囊肾病 ( autosomal dominantpolycystic kidney disease,ADPKD) 94个家系 ,提取外周血白细胞 DNA,用聚合酶链反应 ( polymerasechain reaction,PCR)扩增目的基因的全编码区 ,DHPL C对 PCR产物进行突变筛选 ,出现异常峰型的DNA片段进行核苷酸序列测定 ,明确突变位点和类型。结果 以 5 0名健康志愿者为正常对照 ,从 94例患者家系中成功检测出 8种突变 ,包括 2种无义突变、3种移码突变、3种错义突变。无义突变分别位于第 5和13外显子 ( 12 4 9C→ T,2 4 0 7C→ T) ,编码氨基酸分别在 4 17和 80 3位形成终止密码子。移码突变分别位于第2、12和 13外显子 ( 6 36 - 6 37ins T,2 348- 2 35 1del AGAA,2 4 0 1del A)。错义突变分别位于第 1、4和 5外显子 ( 5 6 8G→ A,96 4 C→T,116 8G→A) ,其编码氨基酸发生改变 ( 190 Ala→ Thr,32 2 Arg→Trp,390 Gly→ Ser)。结论 所检测出的 8种突变 ,为 ADPKD患者的基因诊断。

Objective To detect the mutations of autosomal dominant polycystic kidney disease gene 2(PKD2)in Chinese. Methods The white blood cell genomic DNA from patients of 94 Chinese autosomal dominant polycystic kidney disease(ADPKD) pedigrees was isolated and amplified by polymerase chain reaction(PCR).The PCR products were analyzed by denaturing high-performance liquid chromatography(DHPLC). The samples with abnormal profiles were sequenced. Results Eight mutations were identified, including 2 nonsense mutations, 2 deletion mutations,1 insertion mutation and 3 missense mutations. Two nonsense mutations occurred in exon 5(1249C→T) and exon 13(2407C→T),both resulted in a stop codon. The insertion was in exon 2(636-637 ins T),and the deletion mutations were in exons 12(2348-2351 del AGAA) and 13(2401 delete A),resulting in the reading frame shift. Three missense mutations were in exons 1(G568→A),4(C964→T),and 5(G1168→A),which caused amino acid changes(190Ala→Thr,322Arg→Trp,390Gly→Ser). Conclusion The method of DHPLC was used in detecting PKD2 mutations successfully and 8 mutations in PKD2 were identified. It will be useful in the molecular diagnosis of ADPKD in advance of the cysts formation and birth.