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

doi:10.3969/j.issn.1674-8115.2023.07.012

上海交通大学学报（医学版） ›› 2023, Vol. 43 ›› Issue (7): 898-905.doi: 10.3969/j.issn.1674-8115.2023.07.012

• 论著 · 临床研究 • 上一篇

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

孟祥雨¹(), 闫丹丹², 陈香慧², 赖思宇², 徐云¹, 耿瑞娜¹, 张红², 张蓉², 胡承², 严婧²()

^1.新乡医学院第一附属医院内分泌科，新乡 453100
^2.上海交通大学医学院附属第六人民医院内分泌代谢科，上海市糖尿病临床医学中心，上海市糖尿病重点实验室，上海市糖尿病研究所，上海 200233

收稿日期:2023-02-17 接受日期:2023-07-21 出版日期:2023-07-28 发布日期:2023-07-28
通讯作者: 严婧 E-mail:mengxiangy@126.com;jingyan_1216@vip.163.com
作者简介:孟祥雨（1988—），男，主治医师，硕士；电子信箱：mengxiangy@126.com。
基金资助:
上海交通大学医学院附属第六人民医院面上培育项目(Ynms202109);上海市内分泌代谢疾病研究中心(2022ZZ01002)

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

MENG Xiangyu¹(), YAN Dandan², CHEN Xianghui², LAI Siyu², XU Yun¹, GENG Ruina¹, ZHANG Hong², ZHANG Rong², HU Cheng², YAN Jing²()

^1.Department of Endocrinology, The First Affiliated Hospital of Xinxiang Medical University, Xinxiang 453100, China
^2.Department of Endocrinology and Metabolism, Shanghai Sixth People′s Hospital, Shanghai Jiao Tong University School of Medicine; Shanghai Clinical Centre for Diabetes; Shanghai Key Laboratory of Diabetes Mellitus; Shanghai Diabetes Institute, Shanghai 200233, China

Received:2023-02-17 Accepted:2023-07-21 Online:2023-07-28 Published:2023-07-28
Contact: YAN Jing E-mail:mengxiangy@126.com;jingyan_1216@vip.163.com
Supported by:
General Cultivation Project of Shanghai Sixth People′s Hospital, Shanghai Jiao Tong University School of Medicine(Ynms202109);Shanghai Research Center for Endocrine and Metabolic Diseases(2022ZZ01002)

摘要/Abstract

摘要：

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。

关键词: 全外显子组测序, Wolfram综合征, 糖尿病家系, 致病突变

Abstract:

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 three-dimensional 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 the α-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.

Key words: whole exome sequencing, Wolfram syndrome, diabetes pedigree, causative mutation

中图分类号:

R446.9

孟祥雨, 闫丹丹, 陈香慧, 赖思宇, 徐云, 耿瑞娜, 张红, 张蓉, 胡承, 严婧. 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[J]. 上海交通大学学报（医学版）, 2023, 43(7): 898-905.

MENG Xiangyu, YAN Dandan, CHEN Xianghui, LAI Siyu, XU Yun, GENG Ruina, ZHANG Hong, ZHANG Rong, HU Cheng, YAN Jing. Identification of pathogenic mutations for a Wolfram syndrome pedigree by whole exome sequencing and analysis of its clinical characteristics[J]. Journal of Shanghai Jiao Tong University (Medical Science), 2023, 43(7): 898-905.

图/表 6

图1 先证者家系图Note： M—mutant allele; N—non-mutant allele. S411Cfs*131 is a frameshift mutation; R558H is a missense mutation. Arrow indicates the proband.

Fig 1 Family tree of the proband

表1 测序引物序列

Tab 1 Sequences of the primers

Target mutation	Forward primer (5′→3′)	Reverse primer (5′→3′)
R558H	TTGGCCAGACCTTCATCACC	CTCCAGAGACGTGAACCACC
S411Cfs*131	GAGAACTTCCGCACCCTCAC	CTGACGTTGAGGACGACCAG

图2 WFS1 基因R558H突变测序图Note： ref—reference allele; alt—alter allele. Ⅲ2, Ⅲ1, Ⅱ3, and Ⅰ1 all carried the G/A heterozygous missense mutation.

Fig 2 Sequencing of R588H mutation of WFS1 gene

图3 WFS1 基因S411Cfs*131突变测序图Note： ref—reference allele; alt—alter allele. Ⅲ2, Ⅲ1, Ⅱ4 and Ⅰ3 all carried the GCT/G heterozygous frameshift mutation.

Fig 3 Sequencing of S411Cfs*131 mutation of WFS1 gene

图4 Wolframin蛋白野生型及突变型三维结构Note： WT—wild-type wolframin protein; R558H—R558H mutant wolframin protein. Dashed boxes indicate the protein structures surrounding the 558 amino acids. Red indicates the α-helix of the secondary structure of wolframin protein; green indicates the β-sheet of the secondary structure of wolframin protein.

Fig 4 Three-dimensional structures of wild-type and mutant wolframin proteins

图5 WFS1 突变区域氨基酸序列保守性分析

Fig 5 Conservative analysis of amino acid sequences of WFS1 mutation region

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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

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