摘要
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 precise high-throughput mitochondrial （mt） DNA chip and to investigate the relationship between mtDNA tRNA Leu （UUR） and ND1 gene mutations and diabetes mellitus. Methods A wild-type and mutant probes of 28 loci in tRNA Leu （UUR） and ND1 gene were immobilized on the Hybond N ＋ nylon membrane by UV-crosslinking, then the mtDNA chips were used to detect 28 loci mutation in 200 cases of type 2 diabetes mellitus and 210 matched healthy controls. All the mutations were further confirmed by DNA sequencing. Mfold and Antherprot softwares were used to predict the secondary structures of the mutant gene and protein. Results The mtDNA chip, which could detect 28 loci mutations, was successfully developed. In diabetic group, there were 2 （ 1.0% ） cases of T3 290C mutation, 6 （3.0%） of G3 316A （Ala→Thr） mutation, 5 （2. 5% ） of T 3 394C （Tyr→His） mutation, 1 （0. 5% ） of T3 593 C （Val→Ala） mutation , 1 （0. 5% ） of A3 606G （Leu→Leu） mutation , 8 （4. 0% ） of A4 164G（Met →Met） mutation, 2 （1.0%） of T4 216C （Met→His） mutation. In the controls, 1 （0. 5% ） carrier of G3 316A mutation and 5 （2.4%） carriers of A4 164G mutation were found. There was significant difference between two groups for T3 394C mutation frequencies （P =0. 027）. The secondary structures of the mutant proteins of G3 316A, T3 394C, T3 593C and T4 216C mutations were all different from those of the wild-types＇. Conclusion mtDNA chip is a rapid and reliable high-throughput method for mutations detection, and T3 394C mutation in ND1 gene might contribute to the pathogenesis of mitochondrial diabetes.
出处
《中华医学杂志》
CAS
CSCD
北大核心
2006年第40期2853-2857,共5页
National Medical Journal of China
基金
湖北省自然科学基金资助项目(2004ABA163)
湖北省科技攻关计划资助项目(2006AA301C07)
关键词
糖尿病
DNA
线粒体
突变
Diabetes mellitus
DNA, mitochondrial
Mutation
