一个多囊肾家系的PKD2基因变异分析及蛋白定位研究

Analysis of PKD2 gene variant and protein localization in a pedigree affected with polycystic kidney disease

目的检测一个常染色体显性多囊肾家系的基因变异位点,并进行致病性功能验证。方法采集先证者及其家属的外周血标本,提取血液基因组DNA,运用全外显子组测序手段对先证者进行基因变异分析,筛选候选基因变异位点,再用Sanger测序技术对所有家系成员进行验证,并在健康人群中筛查该变异。同时构建PKD2基因的野生型和变异型真核表达载体,转染HEK293T和HeLa细胞,观察蛋白表达及细胞定位情况。结果先证者存在PKD2基因c.2051dupA(p.Tyr684Ter)移码变异,该变异使cDNA序列第2051位碱基A重复,导致终止密码子的形成,产生截短蛋白。免疫荧光实验显示,与野生型相比,变异型蛋白在细胞内的定位发生了改变,这种改变可能由PKD2编码蛋白C端缺失引起。结论PKD2基因c.2051dupA(p.Tyr684Ter)变异可能是该家系患者的致病原因。

Objective To detect the mutation site in a pedigree affected with autosomal dominant polycystic kidney disease(ADPKD)and verify its impact on the protein function.Methods Peripheral blood samples were collected from the proband and his pedigree members for the extraction of genomic DNA.Mutational analysis was performed on the proband through whole-exome sequencing.Suspected variant was verified by Sanger sequencing.A series of molecular methods including PCR amplification,restriction enzyme digestion,ligation and transformation were also used to construct wild-type and mutant eukaryotic expression vectors of the PKD2 gene,which were transfected into HEK293T and HeLa cells for the observation of protein expression and cell localization.Results The proband was found to harbor a c.2051dupA(p.Tyr684Ter)frame shift mutation of the PKD2 gene,which caused repeat of the 2051st nucleotide of its cDNA sequence and a truncated protein.Immunofluorescence experiment showed that the localization of the mutant protein within the cell was altered compared with the wild-type,which may be due to deletion of the C-terminus of the PKD2 gene.Conclusion The c.2051dupA(p.Tyr684Ter)mutation of the PKD2 gene probably underlay the pathogenesis of ADPKD in this pedigree.