MODYx家系Pax4基因R192N错义突变的致病作用与机理研究

MODY gene spectrum varies remarkably among different races. Most MODY patients in China are caused by unknown MODY genes i.e. MODYx. PAX4, a paired-homeodomain transcription factor, plays a critical role in pancreatic β-cell development, mature and function through a pair homeobox and homeodomain, and functions as a transcription repressor on insulin and glucagon gene. By screening genetic mutations of Pax4gene in MODYx families (n=96), which were previously identified as negative for the known six MODY genes, we found a missense mutation of R192N (cgt→aat), which co-segregated with diabetes in this MODYx family. Functional study showed that transcriptional repressor activity of R192N mutant protein in glucagon gene is markedly reduced 31.5% than that of wild type, but no significant change in that of insulin gene. R192N mutation resulted in irregular fold from alpha helix of Pax4 secondary structure by prediction of structural bioinformatics, suggesting this mutation has severely influenced function of Pax4. In this study, combining with earlier clinical phenotype studies, results of function analysis and changes in secondary structure of Pax4 in R192N mutation families, by using Pax4-R192N mutation gene knock-in mouse model, we will explore R192N mutation in the following aspects: 1. its effects on development and function of mouse pancreas; 2. how it affects expression of glycometabolism relative genes; 3. how over-expression of Pax4 in gene knock-in mouse will reverse or relieve diabetic phonotypes. In this way, we could provide significant evidence in translational medicine and insights of molecular mechanisms through which R192N mutation involves in regulation of glycometabolism and targets of gene therapy of MODY.

中国人MODY 90%以上系未知MODY（MODYx）基因突变所致。Pax4是胰腺/胰岛β细胞分化、发育及成熟调控的重要转录因子，抑制胰岛素和胰高糖素基因转录。我们对MODYx家系(n=96）筛查Pax4基因突变，发现R192N错义突变家系，该突变与糖尿病呈遗传共分离。研究显示，与野生型相比R192N蛋白导致胰高糖素基因转录抑制活性减弱31.5%，但对胰岛素基因无显著影响；结构预测显示，R192N导致PAX4蛋白二级结构部分α螺旋变成无规则折叠，表明该突变对PAX4蛋白功能影响严重。本项目我们将结合前期研究结果，通过Pax4-R192N突变基因敲入小鼠模型，研究R192N突变：1.对于小鼠胰腺发育和功能的影响；2.对于糖代谢相关基因表达的影响；3.Pax4过表达对于基因敲入小鼠糖尿病表型的逆转、缓解作用。为阐明R192N参与MODY糖代谢调控分子机理及基因制药靶点,提供转化医学的重要依据。

中国人MODY大部分系因未知致病基因（MODYx基因）所致。Pax4抑制胰岛素和胰高糖素基因转录，是胰腺/胰岛β细胞分化、发育及成熟调控的重要转录因子。我们对MODYx家系(n=96）进行Pax4基因突变筛查，发现1个R192N错义突变家系，该突变与糖尿病呈遗传共分离。研究显示，与野生型相比R192N蛋白导致胰高糖素基因转录抑制活性减弱31.5%，但对胰岛素基因无显著影响；结构预测显示，R192N导致PAX4蛋白二级结构部分α螺旋变成无规则折叠，表明该突变对PAX4蛋白功能影响严重。本项目我们将结合前期研究结果，通过Pax4-R192N突变基因敲入小鼠模型，研究R192N突变的致病机理。.本项目完成Pax4-R192N突变转基因小鼠模型制作并建系。Pax4-R192N转基因小鼠研究结果表明：与WT组相比，90天小鼠IPGTT显示，杂合组（Pax4-R192N+/-）和纯合突变（Pax4-R192N+/+）组小鼠血糖（0、15、30、60、120min）及30min胰岛素水平显著提高，提示机体胰岛素抵抗的代偿机制；140天和200天小鼠IPITT显示各时间点血糖显著提高（p﹤0.01），而0、30、120min胰岛素水平显著下降，胰高糖素水平显著提高。 240天小鼠空腹麻醉取组织试验结果显示, 与WT组相比，突变组血浆葡萄糖和胰高糖素水平显著增高、胰岛素水平显著下降；胰岛素和胰高糖素比值显著降低（p﹤0.01）。FFA和TG则呈增高趋势。胰腺组织的mRNA转录组测序结果表明，纯合和杂合转基因小鼠的mRNA转录变化与MODY发生密切相关（p<0.001），亦与MODY转录因子调控网络信号通路显著相关。RT-PCR验证了部分转录组测序的结果：与WT组相比，突变组INS2 mRNA进一步显著下降、GCG mRNA显著提高、Foxa2和IAPP的mRNA显著减少（p﹤0.005），而HNF4A没有显著变化(p﹥0.05)。Western blot证实，与WT组相比，突变组胰腺组织胰岛素表达显著降低，胰高糖素表达显著增加。我们在国际上，首次用转基因动物—Pax4-R192N转基因小鼠研究证实，人类Pax4-R192N突变可导致胰岛素分泌减少的、胰岛β细胞功能缺陷的家族遗传性MODY型糖尿病的发生。用转基因动物证明MODY致病基因的存在及其糖尿病致病机制研究属于国际第二次报道。