PDF(Pubmed)
Abstract：
Objective: Variants of the polycystic kidney and hepatic disease 1 (PKHD1) gene are associated with autosomal recessive polycystic kidney disease (ARPKD). This study aimed to identify the genetic causes in a Chinese pedigree with ARPKD and design a minigene construct of the PKHD1 gene to investigate the impact of its variants on splicing. Methods: Umbilical cord samples from the proband and peripheral blood samples from his parents were collected, and genomic DNA was extracted for whole-exome sequencing (WES). Bioinformatic analysis was used to identify potential genetic causes, and Sanger sequencing confirmed the existence of variants within the pedigree. A minigene assay was performed to validate the effects of an intronic variant on mRNA splicing. Results: Two variants, c.9455del (p.N3152Tfs*10) and c.2408-13C>G, were identified in the PKHD1 gene (NM_138694.4) by WES; the latter has not been previously reported. In silico analysis predicted that this intronic variant is potentially pathogenic. Bioinformatic splice prediction tools revealed that the variant is likely to strongly impact splice site function. An in vitro minigene assay revealed that c.2408-13C>G can cause aberrant splicing, resulting in the retention of 12 bp of intron 23. Conclusion: A novel pathogenic variant of PKHD1, c.2408-13C>G, was found in a fetus with ARPKD, which enriches the variant spectrum of the PKHD1 gene and provides a basis for genetic counseling and the diagnosis of ARPKD. Minigenes are optimal to determine whether intron variants can cause aberrant splicing.
摘要：
目的：多囊肾和肝病1（PKHD1）基因的变异与常染色体隐性遗传多囊肾病（ARPKD）有关。本研究旨在确定中国ARPKD家系的遗传原因，并设计PKHD1基因的小基因构建体，以研究其变体对剪接的影响。方法：收集先证者的脐带样本和其父母的外周血样本，提取基因组DNA进行全外显子组测序(WES)。生物信息学分析用于确定潜在的遗传原因，和Sanger测序证实了谱系中存在变体。进行小基因测定以验证内含子变体对mRNA剪接的影响。结果：两种变体，c.9455del（p。N3152Tfs*10）和c.2408-13C>G，通过WES在PKHD1基因（NM_138694.4）中鉴定；后者以前没有报道过。计算机模拟分析预测该内含子变体具有潜在的致病性。生物信息学剪接预测工具显示，该变体可能会强烈影响剪接位点的功能。体外小基因分析显示c.2408-13C>G可引起异常剪接，导致保留12bp的内含子23。结论：PKHD1的一种新的致病变异，c.2408-13C>G，在一个患有ARPKD的胎儿身上发现,丰富了PKHD1基因的变异谱,为遗传咨询和ARPKD的诊断提供了依据。小基因是确定内含子变体是否可以引起异常剪接的最佳选择。
