早发2型糖尿病家系NeuroD1基因突变筛查与功能研究

Screening and functional analysis of variation in NeuroD1 gene in early-onset type 2 diabetic pedigrees

目的在中国人早发2型糖尿病家系先证者中筛查NeuroD1基因突变／变异，并研究其功能及携带者家系的临床表型和遗传特点。方法用PCR-直接测序法检测85例早发、95例晚发2型糖尿病家系先证者和87名非糖尿病对照者NeuroD1基因突变／变异，比较筛查到的组间突变／变异的基因型及等位基因频率的差异。分别构建含小鼠NeuroD1（mND1）基因cDNA的野生型、突变型表达质粒载体和与人胰岛素基因启动子相连的荧光素酶报告基因质粒载体，共转染至大鼠INS-1细胞。分别检测荧光素酶活性，比较野生型、突变体蛋白对人胰岛素基因转录活性的影响。结果在一早发先证者发现新突变S159P（T→C），它与来自父方的4例携带突变的糖尿病患者遗传上呈共分离；与野生型相比，S159P突变体导致胰岛素基因转录活性下降25％；检测到常见多态A45T（G→A），与非糖尿病组及晚发2型糖尿病组相比，早发组该变异的AA＋GA基因型频率显著增加（P=0．006和P=0．014）。结论NeuroD1基因S159P突变促进该早发2型糖尿病家系的发病。A45T变异增加中国人早发2型糖尿病的易感性，或与之呈连锁不平衡，该变异可能影响中国人2型糖尿病的发病方式，即早发而不是晚发。

Objective To screen the variation in NeuroDl gene and to study its function in vitro and its clinical phenotypes and genetic characteristics in Chinese early-onset type 2 diabetic probands. Methods PCR- direct sequencing of NeuroD1 gene was performed in 85 early-onset type 2 diabetic probands, 95 late-onset type 2 diabetics with strong diabetic history and 87 non-diabetic control subjects. Distributions of the identified variation were calculated and compared among the three groups. Expression vectors with mouse NeuroD1 （ mNDI ） cDNA wild type or mutant type and reporter vectors with human insulin promotor-linked luciferase were constructed. Then the above vectors were co-transfected into rat INS-1 cells. Relative luciferase activities were measured to compare transcriptional activities of insulin gene between WT and MT. Results S159P （T→C）, a new mutation was identified in a proband, which was co-segregated with diabetes in 4 carriers from the paternal side. The functional study showed that the S159P mutant exhibited a 25% reduction in transcriptional activity of insulin gene as compared with the wild type. A45T （G→A）, a common variation was identified. The AA ＋ GA genotypic frequencies were markedly increased in early-onset type 2 diabetic probands as compared with late-onset type 2 diabetic probands and non-diabetic control subjects （P = 0. 006 and P = 0. 014, respectively）. Conclusion The novel S159P mutation in the NeuroD1 gene seems to contribute to the development of diabetes in the Chinese earlyonset type 2 diabetic family. The A45T variation may increase susceptibility to or be in disequilibrium with earlyonset type 2 diabetes mellitus in Chinese population. In addition, the A45T variation may affect the onset pattern of type 2 diabetes mellitus, such as early-onset but not late-onset type 2 diabetes mellitus .