湖北汉族人群2型糖尿病患者线粒体基因突变检测及临床意义

Clinical significance and detection of mitochondnal DNA mutation in type 2 diabetes

目的研究线粒体DNA(mtDNA)16S rRNA基因(T3200C、C3206T)和ND1基因(A4136G、A4164G和T4216C)突变在湖北汉族2型糖尿病人群中的发生率及临床意义。方法采用等位基因特异性PCR(AS-PCR)、PCR-限制性酶切片段长度多态性(PCR-RFLP)分析及PCR产物直接测序,对175例2型糖尿病患者和200例正常对照的线粒体基因突变进行检测,并用Primer、Antherprot和Mfold软件对检出的突变位点进行分析,对RNA突变后最小自由能下的二级结构变化和ND1基因突变后蛋白质的二级结构改变进行预测。结果2型糖尿病组检出3200(T→C)突变2例(1．14％),3206(C→T)突变11例(6．28％),4216(T→C)突变3例(1．71％),4136位点未发现突变。发现2个未曾报道的新突变位点4164(A→G)7例(4．00％),4200(A→T)1例(0．57％)。对照组中检出3206(C→T)突变8例(3．92％),4164(A→G)突变5例(2．45％),两位点突变率在两组中比较差异无显著性(P>0．05)。RNA二级结构预测显示3200(T→C)突变引起16S rRNA基因最小自由能和二级结构的改变,可能引起疾病。4164(A→G)和4200(A→T)突变分别为Met和Leu的无义突变,4216(T→C)突变可使线粒体呼吸链中一个中性酪氨酸被亲水性组氨酸取代,蛋白质二级结构预测显示该突变可引起NDl蛋白质二级结构改变。研究还显示线粒体基因突变率在≥40岁个体呈上升趋势。结论mtDNA 3200(T→C)和4216(T→C)突变可能增加糖尿病的易感性;3206(C→T)和4164(A→G)为基因多态性改变;4136位点未发现突变。以上结果提示糖尿病的发生在线粒体基因突变上存在一定的异质性。

Objective To study the clinical significance and mutation rate at point 3200,3206 of mtDNA 16srRNA and point 4136,4164,4216 of ND1 gene in the patients with type 2 diabetes （T2DM） in Han population of Hubei Province. Methods Mitochondria was detected using PCR restriction fragment length polymorphism （PCR-RFLP） analysis with allele specific polymerase chain reaction （ASPCR） and DNA sequencing. All mutations were analyzed by DNAstar, Primer, mfold and Antherprot softwares. Five nucleotide substitutions of mtDNA （nt3200,nt3306,nt4136,nt4164,nt4216） were screened in 175 diabetics and 200 non-diabetic subjects. Results In diabetic group,there were 2 carriers （1.14%） of 3200 T→C,11 （6.28%） of 3206 C→T and 3 （1.71%） of4216T→C （Tyr→His）.Meanwhile,two novel mutations,7 cases （4%） of 4164 A→G and 1 case （0.57%） of 4200 A→T,were found. In control group,therewere 8 cases （4%） of 3206 C→T,5 cases （2.5%） of 4164 A→G. The point mutation 4136 A→G was not found in the two groups.The 3200 T→C mutation caused a great alteration in the minimal free energy and secondary structure mode, while the 3206 mutation didnot altered normal structure. The secondary structure prediction of protein revealed that there were differences between 4216 mutant andwild-type ND1 protein. Conclusions MtDNA 16SrRNA and ND1 gene mutations at nt3200 （T→C） and nt4216 （T→C） might contribute to the pathogenesis of Type 2 DM with other genetic factors and environment factors, while 3206 （C→T） and 4164 （A→G）weregene polymorphism. No mutation at point 4136 suggested that there was certain heterogeneity in mitochondrial heritage in the development of T2DM.