多种方法结合对一个肾单位肾痨患者家系的基因筛查验证

Validation of genetic screening for a nephronophthisis family with multiple methods

目的:探讨少年型肾单位肾痨1型(NPHP1)的临床特点基因诊断方法,采用多种方法对其家系进行筛查验证。方法:2018年7月我院基因诊断中心收入1例男性肾衰患儿(14岁),收集分析其临床资料,利用二代测序技术对其进行基因检测。确诊后扩增患儿及其父母弟弟的NPHP1基因内多个微卫星标记以及内参标记位点,同时采用琼脂糖凝胶电泳和毛细管电泳技术,对患儿弟弟进行筛查验证。结果:二代测序结果显示该患儿样本NPHP1基因整体纯合缺失,该变异为致病性变异,结合临床表现确诊为NPHP1。琼脂糖凝胶电泳结果显示该患儿NPHP1基因前后内参标记D2S1896,RanBP11/12阳性,微卫星标记del-2,del-5-(5)2,del-9,del-10,del-16均为阴性;该患儿父母和弟弟NPHP1基因内参和微卫星标记均为阳性。内参及微卫星序列的毛细管电泳结果显示该患儿弟弟NPHP1基因同时存在来自于父亲和母亲的序列片段,排除该致病基因的纯合或杂合缺失。结论:利用毛细管电泳技术检测NPHP1基因微卫星标记,判断该位点纯合或杂合性缺失,简单易行,可作为常规筛查手段辅助肾单位肾痨的诊断。

Objective: To investigate the use of various genetic methods in the diagnosis of juvenile nephronophthisis(NPHP1) via screening and verification of a family case.M ethods: A 14-year-old boy with renal failure was admitted in July, 2018. The peripheral blood DNA was extracted from this boy, his parents and younger brother. Firstly, next-generation sequencing(NGS) was used to screen the potential target genes of the boy. Then multiple microsatellite markers located within the known homologous deletion of NPHP1 in this family were confirmed by PCR amplification, and markers outside the deletion were used as controls. At last, heterozygosity of the boy’s brother was confirmed by capillary electrophoresis(CE).Results: Growth retardation was found in the boy at age 11(2015), who was hospitalized for rapidly renal failure in 2018. NGS results showed homozygous deletion of NPHP1 gene in him. Combined with clinical manifestations, the boy was diagnosed as NPHP1. By PCR amplification, the microsatellite markers of del-2, del-5-(5)2, del-9, del-10 and del-16 were all missed in the boy, but positive in his parents and brother, while control markers D2S1896, RanBP11/12 were positive in all of them. Capillary electrophoresis verified that both his father and mother’s sequence fragments of NPHP1 gene existed in the younger brother, thus excluding homozygous and heterozygous deletion of NPHP1 gene.Conclusion: Microsatellite markers detected by normal PCR or capillary electrophoresis are simple and feasible to assist clinical diagnosis.