MODY-x家系MAPK8IP1基因M7V错义突变的致病机理研究

Maturity Onset Diabetes of the Young (MODY) is a monogenic form of diabetes. Accurate diagnosis of MODY is considered challenging but is key for precise treatment of MODY. Our previous results suggested that MAPK8IP1 (M7V) is the pathogenic variant in MAPK8IP1 of a suspected MODY in a Chinese Han family, and the anti-apoptotic effect of MAPK8IP1 (M7V) in pancreatic β-cells was significantly decreased. However, the role and molecular mechanism by which MAPK8IP1 (M7V) causes MODY remain unclear. Therefore, we hypothesized that MAPK8IP1 (M7V) mutation reduces its ability to bind to c-Jun N-terminal kinase (JNK), and increases the amount of JNK that bound to c-Jun, thereby increases the phosphorylation of c-Jun and accelerates the rate of pancreatic β-cell apoptosis, resulting in diabetes. Thus, in this study, we aimed to establish three models of overexpression of MAPK8IP1 (M7V) in INS-1 cells, as well as MAPK8IP1 (M7V) mutations in mouse and pancreatic β-cells derived from the above-mentioned mutant mice. We evaluated the ability of MAPK8IP1 (M7V) mutation to bind to JNK, phosphorylated c-Jun, and the rate of pancreatic β-cell apoptosis and diabetic phenotype. In addition, we studied the molecular mechanism involved to rectify that the point mutation improved the binding ability with JNK and improved β-cell apoptosis by using Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/ CRISPR-associated 9 (Cas9) gene editing technology. Unraveling this mechanism of action will provide a novel strategy for precise treatment of this type of MODY.

MODY是单基因突变引起青少年发病的成人型糖尿病，对MODY的精确诊断是MODY精准治疗的难题及关键。申请人前期发现MAPK8IP1(M7V)是一个疑似MODY家系致病性突变，且MAPK8IP1(M7V)突变抗胰岛细胞凋亡作用显著下降，然而其在该病中的作用及机制尚不清楚。由此我们假设MAPK8IP1(M7V)突变与JNK结合能力下降，JNK与c-Jun结合增多，c-Jun磷酸化增强，胰岛β细胞凋亡增加而诱发糖尿病。本项目拟在过表达MAPK8IP1(M7V)细胞模型、MAPK8IP1(M7V)点突变小鼠及该点突变小鼠胰岛β细胞模型，观察MAPK8IP1(M7V)突变对与JNK结合能力、c-Jun磷酸化、细胞凋亡或糖尿病表型的影响，通过CRISPR/Cas9基因编辑技术研究对该点突变进行修复后改善其与JNK结合能力进而改善细胞凋亡的分子机制。阐明该机制，将为该类型MODY的精准治疗提供新策略。

单基因糖尿病是由单个基因发生稀少突变，改变基因的表达或蛋白的结构与功能而致病。MODY作为单基因糖尿病的典型代表，已知的14种MODY亚型的临床表现具有高度异质性，其治疗方案差异较大，而我国MODY基因绝大部分为MODY-x型，因此，对MODY的精确诊断及致病基因的筛查可以制定精准的治疗方案及明确疾病的预后，对提高患者生活质量及改善血糖水平，同时对家族成员的遗传指导具有重要意义。本项目围绕单基因糖尿病的发病机制、精准治疗进行多视角研究，目前共计发表论文5篇，其中SCI4篇，北大核心期刊1篇，获湖北省科技进步奖2等奖1项。本项目主要实验结果有⑴MAPK8IP1为MODY致病基因，MAPK8IP1(M7V)突变为MODY致病性突变，系统阐明了MAPK8IP1(M7V)突变使其与JNK结合能力下降，c-Jun竞争性结合JNK，激活JNK信号通路，c-Jun磷酸化增强而诱导胰岛β细胞凋亡增加而诱发糖尿病的分子机制；⑵MODY2（GCK，c.1160C>T，p.Ala387Val）型糖尿病可以通过饮食、锻炼加以控制血糖，而无需使用降糖药物（妊娠期除外），可减少不必要的药物治疗和药物不良反应，并且可以提高临床医师的诊疗水平；⑶发现IRS1与已知MODY致病基因存在互作，并且参与2型糖尿病与胰岛素信号通路中，IRS1（c.2137C>T，p.His387Tyr）错义突变与早发2型糖尿病密切相关；⑷ATP7B基因突变引起铜离子沉积于胰腺而引起胰岛β细胞凋亡而诱发糖尿病，在使用铜离子螯合剂药物（青霉胺）后，患者的神经症状与糖尿病表型都得到明显改善；⑸应用CRISPR-Cas9技术可在体纠正ob/ob小鼠Leptin单基因点突变，可使小鼠脂肪细胞产生的正常LEPTIN蛋白，从而减轻ob/ob小鼠的糖脂代谢紊乱；⑹系统综述了当前已知的14种MODY亚型的流行现状、发病机制及精准治疗策略，为MODY的精准治疗提供参考。综上，单基因遗传病致病基因的鉴定及其致病机理的探索，对于明确病因、精准诊断以及精准治疗至关重要，运用CRISPR-Cas9基因编辑技术对于单基因遗传病（包括MODY）的精准治疗提供了新策略。