青少年的成人起病型糖尿病家系NEUROD1基因突变的筛查与功能解析

doi:10.3969/j.issn.1674-8115.2023.10.005

上海交通大学学报（医学版） ›› 2023, Vol. 43 ›› Issue (10): 1255-1261.doi: 10.3969/j.issn.1674-8115.2023.10.005

• 论著 · 临床研究 • 上一篇

青少年的成人起病型糖尿病家系NEUROD1基因突变的筛查与功能解析

张娟¹(), 葛晓旭²^,³, 张荣², 蒋伏松², 蒋燕燕², 李鸣², 李甜甜², 刘婵薇², 陈亚婷², 刘丽梅²()

^1.黄淮学院医学院形态学教研室，驻马店 463000
^2.上海交通大学医学院附属第六人民医院内分泌代谢科，上海市糖尿病研究所，上海 200233
^3.上海交通大学医学院附属同仁医院内分泌代谢科，上海 200336

收稿日期:2023-08-13 接受日期:2023-09-22 出版日期:2023-10-28 发布日期:2023-10-28
通讯作者: 刘丽梅 E-mail:839329711@qq.com;lmliu@sjtu.edu.cn
作者简介:张娟（1989—），女，讲师，博士；电子信箱：839329711@qq.com。
基金资助:
河南省科技攻关项目(212102310764);国家自然科学基金(81970686)

Screening and functional analysis of mutations in NEUROD1 gene in pedigrees of maturity-onset diabetes of the young

ZHANG Juan¹(), GE Xiaoxu²^,³, ZHANG Rong², JIANG Fusong², JIANG Yanyan², LI Ming², LI Tiantian², LIU Chanwei², CHEN Yating², LIU Limei²()

^1.Department of Morphology, School of Medicine, Huanghuai University, Zhumadian 463000, China
^2.Department of Endocrinology and Metabolism, Shanghai Sixth People's Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai Diabetes Institute, Shanghai 200233, China
^3.Department of Endocrinology and Metabolism, Tongren Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200336, China

Received:2023-08-13 Accepted:2023-09-22 Online:2023-10-28 Published:2023-10-28
Contact: LIU Limei E-mail:839329711@qq.com;lmliu@sjtu.edu.cn
Supported by:
Project of Science and Technology of Henan Province(212102310764);National Natural Science Foundation of China(81970686)

摘要/Abstract

摘要：

目的·筛查青少年的成人起病型糖尿病（maturity-onset diabetes of the young，MODY）家系中NEUROD1基因突变，分析突变与中国人MODY6发病的相关性及其潜在的致病机制。方法·采用PCR-直接测序法对96例GCK/MODY2、HNF1A/MODY3、HNF1B/MODY5突变阴性的中国MODY先证者进行NEUROD1突变筛查，同时比较96例MODY先证者与100例非糖尿病对照者NEUROD1基因变异的基因型频率。采用从头建模法构建NEUROD1蛋白野生型和突变体的3D结构，采用双荧光素酶报告基因系统检测野生型和突变体蛋白对胰岛素基因转录活性的影响。结果·在一个MODY家系中发现NEUROD1基因杂合错义突变Glu59Gln（NM_002500.5，c.175G>C）。3D结构分析发现，该突变将野生型中带负电荷的Glu59转化为突变中不带电荷的Gln59，导致两个盐桥键Glu59-Arg54和Glu59-Lys88缺失，并形成一个新的氢键Gln59- Arg54。与野生型相比，Glu59Gln突变体的胰岛素基因转录活性下降36.3％（P<0.05）。与非糖尿病对照相比，96例MODY先证者中Ala45Thr（G-A）变异的AA+GA基因型频率显著升高（P=0.002）。结论·Glu59Gln突变改变了NEUROD1蛋白N端的分子构象，导致其胰岛素基因转录活性显著下降，是该家系突变携带者胰岛素分泌缺陷的原因。Ala45Thr变异与MODY6先证者糖尿病发病年龄的提前有关。

关键词: 青少年的成人起病型糖尿病, NEUROD1基因, MODY6, Glu59Gln突变, 功能解析

Abstract:

Objective ·To screen the mutations of NEUROD1 gene in families of maturity-onset diabetes of the young (MODY), and investigate the correlation between the mutation and MODY6 and its potential pathogenesis in Chinese. Methods ·PCR-direct sequencing was used for screening NEUROD1 mutations from 96 MODY probands who were negative for mutations in the GCK/MODY2, HNF1A/MODY3 and HNF1B/MODY5 genes, and the genotypic frequency of NEUROD1 variations were compared between the 96 MODY probands and 100 non-diabetic control subjects. A de novo modeling method was used to predict the three-dimensional (3D) structures of wild type (WT) and mutated NEUROD1 proteins. Transcriptional activities of both WT and mutant of NEUROD1 on insulin gene were detected by using dual luciferase reporter gene system. Results ·Glu59Gln (NM_002500.5, c.175G>C), a heterozygous missense mutation in the NEUROD1 gene, was identified in a MODY pedigree. 3D structural analysis showed that the mutation transformed the negatively charged Glu59 of WT into uncharged mutation Gln59, leading to the loss of Glu59-Arg54 and Glu59-Lys88, two salt bridge bonds, and the formation of Gln59-Arg54, one new hydrogen bond. Transcriptional activity of Glu59Gln mutant for insulin gene was reduced by 36.3% when compared with that of WT (P<0.05). A common variation Ala45Thr (G-A) was identified, and AA+GA genotypic frequency of the variation was significantly elevated in the 96 MODY probands in comparison to non-diabetic control subjects (P=0.002). Conclusion ·Glu59Gln mutation alters the N-terminal molecular conformation of NEUROD1 protein, resulting in decreased transcriptional activity of insulin gene, which is the cause of the defective insulin secretion in mutation carriers of the MODY6 pedigree. The Ala45Thr variation is associated with earlier age of onset of diabetes in MODY6 probands.

Key words: maturity-onset diabetes of the young (MODY), NEUROD1 gene, MODY6, Glu59Gln mutation, functional analysis

中图分类号:

R587.1

张娟, 葛晓旭, 张荣, 蒋伏松, 蒋燕燕, 李鸣, 李甜甜, 刘婵薇, 陈亚婷, 刘丽梅. 青少年的成人起病型糖尿病家系NEUROD1基因突变的筛查与功能解析[J]. 上海交通大学学报（医学版）, 2023, 43(10): 1255-1261.

ZHANG Juan, GE Xiaoxu, ZHANG Rong, JIANG Fusong, JIANG Yanyan, LI Ming, LI Tiantian, LIU Chanwei, CHEN Yating, LIU Limei. Screening and functional analysis of mutations in NEUROD1 gene in pedigrees of maturity-onset diabetes of the young[J]. Journal of Shanghai Jiao Tong University (Medical Science), 2023, 43(10): 1255-1261.

图/表 4

图1 NEUROD1 基因中Glu59Gln突变的鉴定Note： A. Schematic organization of NEUROD1 protein. The numbers indicate the amino acids bordering the functional domains. Solid and dashed arrows indicate the identified mutation Glu59Gln and variation Ala45Thr in NEUROD1 gene, respectively. B. Alignment of specific regions of NEUROD1 from different mammals using Clustal O. C. DNA sequences of two genotypes of WT (Glu59Glu) and mutant (Glu59Gln) in NEUROD1.

Fig 1 Identification of Glu59Gln mutation in NEUROD1 gene

表1 非糖尿病对照者与MODY先证者 NEUROD1 基因Ala45Thr变异的分布［n（%）］

Tab 1 Distribution of Ala45Thr variation in NEUROD1 gene between non-diabetic controls and MODY probands ［n（%）］

Group	n	Frequency of genotypes			Frequency of alleles
Group	n	AA	GA	GG	A	G
Non-diabetic control	100	0 (0)	9 (9.0)	91 (91.0)	9 (4.5)	191 (95.5)
MODY proband	96	3 (3.1)	22 (22.9)^①	71 (74.0)	28 (14.6)^②	164 (85.4)

图2 NEUROD1野生型和Glu59Gln突变体的3D结构模型Note： A/B. The location of wild type Glu59(A) and mutant Gln59(B) on the global landscape of NEUROD1 protein. C. Interaction of Glu59 with surrounding residues. The side chains of Glu59 formed strong salt bridge bonds with Arg54 and Lys88, and the main chains of Glu59 formed hydrogen bonds with Gly56 and Asp61, respectively. D. Interaction of Gln59 with surrounding amino acids. In the Glu59Gln mutation, the salt-bridge bonds of the side chains of Glu59 were disrupted, while the mutated Gln59 formed a new hydrogen bond with Arg54. Blue dotted lines represent salt bridge bonds formed between amino acids, while green dotted lines represent hydrogen bonds.

Fig 2 3D structural models of wild type and Glu59Gln mutant of NEUROD1 protein

图3 野生型和突变型NeuroD1蛋白的转录活性测定Note：A. Relative luciferase activities of the human insulin promoter luciferase reporter gene stimulated by mNeuroD1-WT and mNeuroD1-Glu59Gln in MIN6 cells. B. Expression of mNeuroD1-WT-Flag and mNeuroD1-Glu59Gln-Flag by Western blotting assay in MIN6 cells. ①P=0.000,compared with NC; ②P=0.024, compared with mNeuroD1-WT. NC—pcDNA3.3.

Fig 3 Transcriptional activities of wild type and mutant of NeuroD1 protein

参考文献 18

1	BOHUSLAVOVA R, SMOLIK O, MALFATTI J, et al. NEUROD1 is required for the early α and β endocrine differentiation in the pancreas[J]. Int J Mol Sci, 2021, 22(13): 6713.
2	ARAMATA S, HAN S I, YASUDA K, et al. Synergistic activation of the insulin gene promoter by the beta-cell enriched transcription factors MafA, Beta2, and Pdx1[J]. Biochim Biophys Acta, 2005, 1730(1): 41-46.
3	HORIKAWA Y, ENYA M. Genetic dissection and clinical features of MODY6 (NEUROD1-MODY)[J]. Curr Diab Rep, 2019, 19(3): 12.
4	NAYA F J, HUANG H P, QIU Y, et al. Diabetes, defective pancreatic morphogenesis, and abnormal enteroendocrine differentiation in BETA2/neuroD-deficient mice[J]. Genes Dev, 1997, 11(18): 2323-2334.
5	ROMER A I, SINGER R A, SUI L, et al. Murine perinatal β-cell proliferation and the differentiation of human stem cell-derived insulin-expressing cells require NEUROD1[J]. Diabetes, 2019, 68(12): 2259-2271.
6	BRODOSI L, BARACCO B, MANTOVANI V, et al. NEUROD1 mutation in an Italian patient with maturity onset diabetes of the young 6: a case report[J]. BMC Endocr Disord, 2021, 21(1): 202.
7	RUBIO-CABEZAS O, MINTON J A, KANTOR I, et al. Homozygous mutations in NEUROD1 are responsible for a novel syndrome of permanent neonatal diabetes and neurological abnormalities[J]. Diabetes, 2010, 59(9): 2326-2331.
8	LI Y Y, WANG H, ZHANG Y Y. Neuronal Differentiation 1 gene Ala45Thr polymorphism and type 2 diabetes mellitus: a meta-analysis of 7, 940 subjects[J]. Nutr Metab Cardiovasc Dis, 2021, 31(6): 1809-1821.
9	LIU L M, JIA W P, ZHENG T S, et al. Ala45Thr variation in neuroD1 gene is associated with early-onset type 2 diabetes with or without diabetic pedigree in Chinese[J]. Mol Cell Biochem, 2006, 290(1/2): 199-204.
10	LIU L, FURUTA H, MINAMI A, et al. A novel mutation, Ser159Pro in the NeuroD1/BETA2 gene contributes to the development of diabetes in a Chinese potential MODY family[J]. Mol Cell Biochem, 2007, 303(1/2): 115-120.
11	American Diabetes Association Professional Practice Committee. 2. classification and diagnosis of diabetes: standards of medical care in diabetes-2022[J]. Diabetes Care, 2022, 45(suppl 1): S17-S38.
12	YANG J Y, ZHANG Y. I-TASSER server: new development for protein structure and function predictions[J]. Nucleic Acids Res, 2015, 43(W1): W174-W181.
13	CHAPLA A, MRUTHYUNJAYA M D, ASHA H S, et al. Maturity onset diabetes of the young in India: a distinctive mutation pattern identified through targeted next-generation sequencing[J]. Clin Endocrinol (Oxf), 2015, 82(4): 533-542.
14	GONSORCÍKOVÁ L, PRŮHOVÁ S, CINEK O, et al. Autosomal inheritance of diabetes in two families characterized by obesity and a novel H241Q mutation in NEUROD1[J]. Pediatr Diabetes, 2008, 9(4 pt 2): 367-372.
15	MALECKI M T, JHALA U S, ANTONELLIS A, et al. Mutations in NEUROD1 are associated with the development of type 2 diabetes mellitus[J]. Nat Genet, 1999, 23(3): 323-328.
16	HORIKAWA Y, ENYA M, MABE H, et al. NEUROD1-deficient diabetes (MODY6): identification of the first cases in Japanese and the clinical features[J]. Pediatr Diabetes, 2018, 19(2): 236-242.
17	FAJANS S S, BELL G I. MODY: history, genetics, pathophysiology, and clinical decision making[J]. Diabetes Care, 2011, 34(8): 1878-1884.
18	刘丽梅, 赵蔚菁, 郑泰山, 等. 早发2型糖尿病家系NeuroD1基因突变筛查与功能研究[J]. 中华内分泌代谢杂志, 2009, 25(1): 34-38.
	LIU L M, ZHAO W J, ZHENG T S, et al. Mutation screening and functional study of NeuroD1 gene in pedigrees with early-onset type 2 diabetes mellitus [J]. Chinese Journal of Endocrinology and Metabolism, 2009, 25(1): 34-38.

[1]	薛彦斌, 齐季瑛, 张子政, 经仁洁, 孙文, 姚华彦, 何萍, 崔斌, 宁光. 上海糖尿病临床专病大数据库建设与真实世界研究[J]. 上海交通大学学报（医学版）, 2023, 43(9): 1145-1152.
[2]	吴佳晋, 钟晨, 李大伟, 陈若洋, 瞿俊文, 张明. 甲基转移酶3调控pri-miR-21甲基化修饰在糖尿病肾病肾脏纤维化中的作用[J]. 上海交通大学学报（医学版）, 2023, 43(1): 1-7.
[3]	贾伟平. 血糖监测技术的进步与展望[J]. 上海交通大学学报（医学版）, 2022, 42(9): 1171-1175.
[4]	张蓉, 陆丽, 王亚昕, 董文倩, 张宇, 周健. 糖尿病患者血糖波动异常与认知功能障碍关系的研究进展[J]. 上海交通大学学报(医学版), 2022, 42(2): 235-240.
[5]	张静静, 祝超瑜, 肖元元, 蒋伏松, 高清歌, 方云云, 魏丽. 胰高血糖素样肽1受体基因rs3765467变异与2型糖尿病的关联研究[J]. 上海交通大学学报(医学版), 2021, 41(9): 1215-1221.
[6]	丁贤彬, 毛德强, 焦艳, 陈莉玲, 许杰. 重庆市糖尿病患病率、死亡率及伤残调整寿命年率分析[J]. 上海交通大学学报(医学版), 2021, 41(1): 78-81.
[7]	姜梦迪, 张文. 糖尿病肾病中的组蛋白修饰与靶向干预的研究进展[J]. 上海交通大学学报(医学版), 2021, 41(1): 103-107.
[8]	姜梦迪, 张文. 糖尿病肾病中的组蛋白修饰与靶向干预的研究进展[J]. 上海交通大学学报(医学版), 0, (): 1-5.
[9]	余梦娜1，杨标2，仰礼真1. 糖尿病相关hsa-miR-223-3p靶基因预测及生物信息学分析[J]. 上海交通大学学报(医学版), 2020, 40(11): 1477-1484.
[10]	蒋毅弘1，刘伟1，周峘1，徐华1，麻静1, 2. 糖尿病患者胃排空速率的临床观察[J]. 上海交通大学学报(医学版), 2020, 40(9): 1249-1255.
[11]	陈宁欣，韩亭亭，郑爽，刘伟，胡耀敏. 基因芯片筛选脂蛋白脂酶基因杂合敲除小鼠的差异表达基因和通路[J]. 上海交通大学学报(医学版), 2020, 40(6): 736-743.
[12]	李宇佳，陆红丽，黄旭，许文燮. 蛋白酶激活受体2对糖尿病小鼠结肠运动的影响[J]. 上海交通大学学报（医学版）, 2019, 39(11): 1255-.
[13]	苏杭，马晓静，应令雯，贺星星，朱玮，唐峻岭，王煜非，包玉倩，周健. 1,5-脱水葡萄糖醇或糖化白蛋白联合空腹血糖筛查糖尿病的效率比较[J]. 上海交通大学学报（医学版）, 2019, 39(9): 1077-.
[14]	王婷婷 1, 2，李明杰 1，林宁 1，钮忆欣 1，简蔚霞 1，苏青 1. 血清高尿酸水平与住院糖尿病患者白蛋白尿短期进展的关系研究[J]. 上海交通大学学报（医学版）, 2019, 39(7): 754-.
[15]	赵芳雅 1，陆俊茜 2，张磊 2，陈海冰 1. 上海地区糖尿病人群高尿酸血症的易患因素及肾功能预后分析[J]. 上海交通大学学报（医学版）, 2019, 39(7): 783-.

青少年的成人起病型糖尿病家系NEUROD1基因突变的筛查与功能解析

Screening and functional analysis of mutations in NEUROD1 gene in pedigrees of maturity-onset diabetes of the young

RichHTML

PDF (PC)

可视化

摘要/Abstract

引用本文

使用本文

图/表 4

参考文献 18

相关文章 15

编辑推荐

Metrics