应用改良的方法检测常染色体显性遗传多囊肾基因突变

Establishment and application of gene mutation method based on autosomal dominant polycystic kidney disease

目的:常染色体显性遗传性多囊肾(ADPKD)是常见的遗传性肾脏疾病,主要是由PKD1和PKD2基因突变引起的。其中,PKD1包含46个外显子,编码区域大概为13 kb,而PKD2有约3 kb的编码区域,含有15个外显子。然而传统的ADPKD基因检测方法繁琐、费时,本研究即旨在建立方便快捷的突变筛查方法。方法:根据临床和实验室检查入选3个互不相关的中国汉族ADPKD家系。利用新一代测序技术(NGS),针对PKD1和PKD2外显子,剪接位点和10 bp内含子侧翼序列进行基因芯片捕获,检出的变异经过筛选,并与单核苷酸多肽数据库进行比对。结果:通过与人类基因突变数据库核对,本研究新鉴定的PKD1中c.7318del G,c.5884C>T和继往报道的PKD2中c.916C>T杂合变异可能是这3个家系的致病性突变。PCR联合Sanger序列分析证实家系中上述杂合突变的存在,在家系中呈现基因型与表现型共分离;并分别导致PKD1编码的氨基酸发生移码(p.Asp2440Thrfs*180)及无义突变引发PKD1(p.Gln1962*)和PKD2(p.Arg306*)蛋白的提前中止。结论:我们利用NGS技术建立了快速分析ADPKD遗传变异的方法,并成功应用于3个家系,鉴定两个新发突变,可作为疑似ADPKD及家系成员的常规筛查手段。

Objective： Autosomal dominant polycystic kidney disease（ ADPKD） is a common hereditary kidney disease,ADPKD is thought to be caused by mutations in the PKD1 and PKD2 genes,PKD1 is comprises 46 exons and coding region is about 13 kb,PKD2 has 15 exons and a coding region of about 3 kb. Traditional analysis methods are tedious and waste time. The purpose of the study was to establish a convenient and rapid detection method to detect the ADPKD mutation position and the type of mutation. Methodology： According to the clinical and laboratory examination were selected for the three unrelated Chinese Han ADPKD families. The probands were detected that using the next generation sequencing technology（ NGS） for gene mutation analysis. The exons,splicing sites and 10 bp flanking intron sequences of PKD1 and PKD2 were captured by a gene chip. Variants（ single nucleotide variants and small insertion） were identified using the GATK Genotyper. Results： The PKD sequence variants were checked by published in the Human Gene Mutation Database. NGS sequencing showed two novel frameshift mutations（ c. 7318 del G,p. Asp2440Thrfs＊ 180）and nonsense mutations（ c. 5884 C〉T,p. Gln1962＊） in PKD1 mutations,one nonsense mutations（ c. 916 C〉T,p. Arg306＊） has been reported in PKD2 gene mutation. PCR combined with Sanger sequence analysis confirmed the existence of theheterozygous mutations in the ADPKD families. The genotype and phenotype were co-segregation in the pedigree. Lead to PKD1 encoded amino acid shift code（ p. Asp2440Thrfs＊ 180） and nonsense mutations triggered PKD1（ p. Gln1962 ＊）and PKD2（ p. Arg306 ＊） protein in the early termination,respectively. Conclusion： Using NGS technology,we established a rapid analysis of ADPKD genetic variation method,successfully applied to three families,and identified two novel mutations. NGS technology can be applied to the routine screening of suspected ADPKD and family members.