Micro-RNA Binding Site Polymorphisms in the WFS1 Gene Are Risk Factors of Diabetes Mellitus

The absolute or relative lack of insulin is the key factor in the pathogenesis of diabetes mellitus. Although the connection between loss of function mutations of the WFS1 gene and DIDMOAD-syndrome including diabetes mellitus underpins the significance of wolframin in the pathogenesis, exact role of WFS1 polymorphic variants in the development of type 1 and type 2 diabetes has not been discovered yet. In this analysis, 787 patients with diabetes and 900 healthy people participated. Genotyping of the 7 WFS1 SNPs was carried out by TaqMan assays. Association study was performed by χ2-test in combination with correction for multiple testing. For functional analysis, the entire 3’ UTR of the WFS1 gene was subcloned in a pMIR-Report plasmid and relative luciferase activities were determined. Linkage disequilibrium analysis showed a generally high LD within the investigated region, however the rs1046322 locus was not in LD with the other SNPs. The two miR-SNPs, rs1046322 and rs9457 showed significant association with T1DM and T2DM, respectively. Haplotype analysis also confirmed the association between the 3’ UTR loci and both disease types. In vitro experiments showed that miR-185 reduces the amount of the resulting protein, and rs9457 miRSNP significantly influences the rate of reduction in a luciferase reporter assay. Genetic variants of the WFS1 gene might contribute to the genetic risk of T1DM and T2DM. Furthermore demonstrating the effect of rs9457 in binding of miR-185, we suggest that the optimal level of wolframin protein, potentially influenced by miR-regulation, is crucial in normal beta cell function.

胰岛素绝对或相对缺乏是糖尿病（Diabetes Mellitus）发病的核心致病因素。尽管WFS1基因功能丧失突变与包含糖尿病在内的DIDMOAD综合征（DIDMOAD Syndrome）之间的关联，证实了沃弗林（wolframin）在糖尿病发病机制中的重要性，但WFS1基因多态变异在1型糖尿病与2型糖尿病发生发展中的具体作用仍未明确。本研究共纳入787名糖尿病患者与900名健康对照个体参与分析。采用TaqMan检测法对7个WFS1基因单核苷酸多态性（Single Nucleotide Polymorphism, SNP）位点进行基因分型，采用χ²检验结合多重检验校正开展关联分析。功能分析环节中，研究人员将WFS1基因全长3'非翻译区（3' Untranslated Region, 3' UTR）亚克隆至pMIR-Report质粒中，并检测相对荧光素酶活性。连锁不平衡（Linkage Disequilibrium, LD）分析结果显示，研究区域内整体呈现较高水平的连锁不平衡，但rs1046322位点与其余SNP位点不存在连锁不平衡关系。两个miRNA相关单核苷酸多态性（miRNA-associated Single Nucleotide Polymorphisms, miR-SNPs）位点rs1046322与rs9457，分别与1型糖尿病（Type 1 Diabetes Mellitus, T1DM）和2型糖尿病（Type 2 Diabetes Mellitus, T2DM）存在显著关联。单倍型分析同样证实了3' UTR区域位点与两种糖尿病类型的关联。体外实验结果显示，miR-185可降低沃弗林蛋白的表达量；而在荧光素酶报告基因实验中，rs9457 miR-SNP可显著影响该蛋白表达的抑制效率。WFS1基因的遗传变异可能增加个体罹患1型与2型糖尿病的遗传风险。本研究进一步证实rs9457位点可影响miR-185的结合能力，提示受miRNA调控的沃弗林蛋白适宜表达水平，对于维持正常β细胞功能至关重要。