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目的建立一种快速、准确的高通量检测线粒体DNA突变的基因芯片技术,并探讨这些突变与糖尿病的关系。方法以尼龙膜为介质,通过紫外交联固定28个待测突变位点的野生型和突变型探针,并采用该基因芯片对200例2型糖尿病患者和210名健康对照的线粒体tRNAleu(UUR)基因及ND1基因进行筛查,DNA测序进一步确证突变位点,Mfold和Antheprot软件预测变异型基因及蛋白质二级结构。结果成功研制可检测28个突变位点的线粒体DNA芯片,在糖尿病组检出tRNAleu(UUR)基因T3290C突变2例(1.0%),ND1基因突变23例G3316A突变6例(3.0%)、T3394C突变5例(2.5%)、A4164G突变8例(4.0%),T4216C突变2例(1.0%),T3593C(0.5%)和A3606G突变各1例(0.5%);对照组检出G3316A突变1例(0.5%)、A4164G突变5例(2.4%)。这些突变位点的测序结果与基因芯片检测结果一致。两组间仅T3394C突变率差异有统计学意义(P=0.027)。G3316A突变导致丙氨酸→苏氨酸,T3394C突变导致酪氨酸→组氨酸,T3593C突变导致缬氨酸→丙氨酸,T4216C突变导致酪氨酸→组氨酸,A3606G和A4164G突变分别为亮氨酸和蛋氨酸的无意义突变,前4种突变型的ND1蛋白二级结构不同于野生型。结论线粒体DNA芯片是一种快速可靠的高通量基因突变检测方法,ND1基因T3394C突变可能参与了线粒体基因缺陷型糖尿病的发病。
Objective To establish a rapid and accurate high-throughput gene chip technique for detecting mitochondrial DNA mutations and to explore the relationship between these mutations and diabetes mellitus. Methods The wild-type and mutant-type probes of 28 sites to be tested were immobilized by nylon membrane as a medium. The mitochondrial tRNAleu (UUR) of 200 type 2 diabetic patients and 210 healthy controls ) Gene and ND1 gene were screened. DNA sequencing further confirmed the mutation sites. Mfold and Antheprot software predicted the variant gene and protein secondary structure. Results Twenty-eight mitochondrial DNA (DNA) fragments were detected successfully in two groups. T3290C mutation of tRNAleu (UUR) gene was detected in 2 cases (1.0%), 23 cases of ND1 gene mutation in 6 cases (3.0%), T3394C mutation 5 cases (2.5%), A4164G mutation in 8 cases (4.0%), T4216C mutation in 2 cases (1.0%), T3593C (0.5%) and A3606G mutation in 1 case 0.5%), A4164G mutation in 5 cases (2.4%). The sequencing results of these mutation sites are consistent with the results of the gene chip test. Only T3394C mutation rate between the two groups was statistically significant (P = 0.027). G3316A mutation leads to alanine → threonine, T3394C mutation leads to tyrosine → histidine, T3593C mutation leads to valine → alanine, T4216C mutation leads to tyrosine → histidine, A3606G and A4164G mutations are Leucine and methionine, and the first four kinds of mutant ND1 proteins have different secondary structure from the wild type. Conclusion Mitochondrial DNA chip is a rapid and reliable method for detecting high-throughput gene mutations. The T3394C mutation of ND1 gene may be involved in the pathogenesis of mitochondrial gene-deficient diabetes.