家系全外显子组测序鉴定Wolfram综合征致病突变及其临床性状分析

Identification of pathogenic mutations for a Wolfram syndrome pedigree by whole exome sequencing and analysis of its clinical characteristics

目的·拟鉴定一个可疑Wolfram综合征的中国糖尿病家系的致病基因和突变位点,并进行相关临床性状分析。方法·纳入糖尿病家系共12名成员。先证者于2013年5月到新乡医学院第一附属医院内分泌科初诊,并于2022年7月和2023年4月先后2次来院复诊;该家系其他成员分别为先证者的姐姐、父亲、母亲、祖父、祖母、伯伯、姑姑及外祖父、外祖母、大舅和小舅。收集研究对象的临床资料。利用全外显子组测序对该家系6名成员的致病基因及其突变位点进行筛查,并用Sanger测序进行验证;采用CADD、DANN、MetaSVM、Polyphen-2、SIFT和M-CAP生物信息学软件预测Wolfram综合征致病基因WFS1突变对其编码蛋白wolframin结构和功能的影响;使用Swiss-Model软件构建野生型和突变型wolframin蛋白的三维结构,并用PyMOL软件进行可视化;用Clustal Omega软件进行WFS1基因突变位点物种保守性估测;采用JNetPRED软件进行wolframin蛋白二级结构在线预测。结果·该家系中,先证者及先证者的姐姐均携带Wolfram综合征致病基因WFS1的复合杂合突变R558H和S411Cfs*131;先证者的父亲和祖父均携带R558H突变;先证者的母亲和外祖父均携带S411Cfs*131突变。R558H为罕见错义突变,而S411Cfs*131是一个尚未报道过的移码突变。生物信息学分析提示R558H位于wolframin蛋白α螺旋结构域上,为损害突变,且该突变区域的氨基酸序列在包括人类在内的12个进化程度不同的物种间具有较高的保守性。结论·利用家系全外显子组测序鉴定出Wolfram综合征的2个致病突变,可对Wolfram综合征现有基因型和表型谱进行补充,实现对糖尿病家系的早期诊断,并有助于对患者及时开展随访,以实现对疾病的早期干预和治疗。

Objective·To identify the causative gene and mutations and describe the clinical traits in a Chinese diabetes pedigree suspected of Wolfram syndrome.Methods·A total of 12 subjects from one family were included.The proband was admitted to the Department of Endocrinology,The First Affiliated Hospital of Xinxiang Medical University,for the first time in May 2013.Then he visited the hospital for follow-up in July 2022 and in April 2023,respectively.The other members of this family included the proband′s sister,father,mother,paternal grandfather,paternal grandmother,uncle,aunt,as well as maternal grandfather,maternal grandmother,and two brothers of the proband′s mother.Clinical data of all subjects were collected.The whole exome sequencing was used to screen the pathogenic genes and mutation sites of six members of the family,and Sanger sequencing was used to verify the above results.Effects of the mutation of the pathogenic gene WFS1 in Wolfram syndrome on the function of the wolframin protein were evaluated by bioinformatics softwares,including CADD,DANN,MetaSVM,Polyphen-2,SIFT and M-CAP.The threedimensional structures of wild-type and mutant wolframin proteins were constructed with Swiss-Model software,and visualized with PyMOL software.Cluster Omega software was used for evaluating species conservation of WFS1 gene mutation sites.JNetPRED software was used for online prediction of wolframin protein secondary structure.Results·The proband and his sister both carried R558H and S411Cfs*131 mutations,two compound heterozygous mutations of the Wolfram syndrome pathogenic gene WFS1.The proband′s father and parental grandfather both carried the R558H mutation,while the proband′s mother and maternal grandfather both carried the S411Cfs*131 mutation.The R558H mutation was a rare missense mutation,and the S411Cfs*131 mutation was a novel frameshift mutation.Bioinformatics analysis softwares predicted that the R558H mutation located in theα-helical structure of the wolframin protein.This mutation was a damage mutation and the amino acid sequence of the mutation region was highly conservative among 12 species with varying degrees of evolution,including humans.Conclusion·Two causative mutations of WFS1 gene are identified in a Chinese diabetes pedigree by whole exome sequencing.The study supplements the existing genotype and phenotype profiles of Wolfram syndrome,which can realize early diagnosis of diabetes pedigrees and help in performing timely follow-up of patients,so as to achieve early intervention and treatment of this disease.