浙江地区汉族人群2型糖尿病患者线粒体ND2基因突变的研究

科研成果详情

题名	浙江地区汉族人群2型糖尿病患者线粒体ND2基因突变的研究
其他题名	Research on the Mutation Sites of the Mitochondrial DNA ND2 Gene in Patients with Type 2 Diabetes in Han People in Zhejiang
作者	吴含
学位类型	硕士
导师	郦卫星
答辩日期	2014-05-23
学位授予单位	温州医科大学
学位专业	临床检验诊断
关键词	2型糖尿病线粒体DNA 错义突变保守系数
摘要	目的1.研究浙江地区2型糖尿病(type 2 diabetes mellitus ，T2DM)患者线粒体ND2基因的多态性，分析与2型糖尿病发病有关的基因突变位点。2.探讨线粒体ND2基因多态性与线粒体糖尿病的关系，寻找与T2DM母系遗传相关的突变位点，为疾病的治疗及遗传危险因素评估提供参考。3.分析线粒体ND2基因突变位点与T2DM患者临床并发症之间的关系。方法1.临床及实验室检查：收集自2010年1月-2011年3月浙江省人民医院住院的T2DM 患者448例，T2DM患者均根据1997年WHO糖尿病诊断标准确诊，并记录患者病程、基本临床资料、常规生化指标、并发症情况、糖尿病家族史、胰岛素使用情况及其他合并症等。以204例健康体检者作为正常对照，两组对象的年龄相匹配。2.提取研究对象的外周血总 DNA，设计三对引物，通过聚合酶链反应（PCR）扩增线粒体 DNA ND2基因，纯化 PCR 产物后直接测序，测序结果用DNAStar、Condone Code Aligner等软件与线粒体基因剑桥参考序列(Cambrige reference sequence， CRS)比对，找出线粒体ND2 基因的突变位点。3.分析mtDNA ND2基因变异位点的氨基酸变化；Clustalx生物软件分析碱基序列保守性；从氨基酸改变和蛋白结构方面分析T2DM组和正常对照组中错义突变的情况。4.分析 T2DM 组 ND2 基因变异位点，筛选出高度保守的错义突变位点，并分析携带这些突变位点患者的临床资料，探讨 ND2 基因变异与T2DM的关系。5.分析448例T2DM患者中疑有家族史患者的mtDNA ND2基因的测序结果，找出可能与T2DM母系遗传相关的突变位点。6.将 T2DM患者分为Mt5178野生组和Mt5178突变组，分析线粒体 ND2 基因C5178A多态性与脑梗塞的关系。结果1．通过对浙江地区448例T2DM患者进行线粒体ND2基因测序分析，共发现102个碱基变异，其中错义突变位点26个，有16个错义突变位点在正常对照组中没出现；204例正常汉族人群线粒体ND2基因中共发现37个碱基变异，错义突变10个。错义突变率在两组中无显著性差异（P>0.05）。2.mtDNA ND2 基因 nt4769 A→G的突变率约为100%。3.A4833G突变位点在两组中的分布有统计学差异（P75%），分别是A4722G、A4740G、A4824G、A4902C和C5124A，其中A4722G、A4740G、A4902C和C5124A只在T2DM组检测到。分析ND2蛋白质二级结构，发现A4722G和 C5124A突变导致蛋白质跨膜区氨基酸的改变。5.分析疑有母系遗传糖尿病家族史的T2DM患者的mtDNA ND2基因突变位点，未发现明确的导致家族性糖尿病的突变位点。6.分析mt5178A基因型患者和mt5178C基因型患者的临床资料，发现在T2DM患者中，mt5178A基因型患者收缩压明显低于mt5178C基因型组，而空腹C肽释放量、高密度脂蛋白（HDL）明显高于mt5178C基因型组，脑梗塞的发病率明显低于mt5178C基因型组（P<0.05）。结论1.mtDNA ND2 基因 nt4769 碱基G可作为研究浙江汉族人线粒体基因的正常参考。2.A4833G的突变频率在T2DM组明显高于正常对照组（P<0.05），可能是T2DM的易感基因位点，在一定的环境影响下会导致T2DM的发生。3.A4722G、A4740G、A4824G、A4902C和C5124A属于高度保守突变位点，经过分析认为其中的A4722G和 C5124A突变可能也与T2DM的发生有关，需要增加样本量并深入探讨其机制来证实。4.mtDNA ND2基因中未发现明确的致家族性糖尿病发病的突变位点。5.在T2DM患者中，mtDNA ND2基因C5178A突变有利于抵抗脑梗塞的发生。
其他摘要	Objective1.The aim is to study the occurrence of mitochondrial DNA(mtDNA) polymorphisms at ND2 region among the subjects with type 2 diabetes mellitus(T2DM) in Chinese people. And to analyze the relevant mutations of type 2 diabetes. 2.The purpose is to discuss the relation between the polymorphism of mitochondrial ND2 genes and mitochondrial diabetes, to find mutations which are relevant to T2DM’s maternal inheritance. It provides the reference for the treatment of diseases and the assessment of genetic risk factors. 3.The goal is to investigate the relationship between the variations of ND2 genes and clinical complications of T2DM.Method1.Those below are clinical and laboratory tests. We have collected 448 individuals who have T2DM in Zhejiang Provincial People’s Hospital from January 2010 to March 2011. The T2DM patients are diagnosed according to a 1997’s WHO diabetes’ diagnosis standard. And we have recorded their courses of disease, basic clinical information, conventional biochemical index, situations of complications, family history of diabetes, insulin usage, and other complications and so on. 204 healthy individuals are treated as normal controls, and two groups of objects have showed age match.2.In order to find out the nucleotide mutations of mitochondrial ND2 gene, we have extracted the peripheral blood total DNA of the study obejects, designed three pairs of primers, amplified mitochondrial DNA ND2 gene through the polymerase chain reaction (PCR), directly sequenced after purifying the PCR products. The sequencing results have been compared with Cambridge Reference Sequence (CRS) of mitochondrial genes by software of DNAStar and Condone Code Aligner etc. 3.The paper analyzes the amino acid change of mtDNA ND2 genes’ mutations. We analyze the conservation of the amino acid sequence by biological software Clustalx and compare the situation of missense mutations between T2DM group and the normal control group in the aspects of amino acid variations and the protein structure. 4.The highly conservative missense mutations have been filtered by analysis of mtDNA ND2 gene variations of T2DM groupr. Moreover, we have analyzed the family history and clinical data of patients who carry these mutations in order to discuss the relationship between ND2 gene variation and T2DM. 5.For finding out mutations that may be relevant to maternal inheritance, we have analyzed the mtDNA ND2’s sequencing results of those who may be patients having a family history among 448 cases of T2DM patients.6.T2DM patients are divided into Mt5178 wild group and Mt5178 mutation group and we have studied the relationship between the polymorphism of C5178A and cerebral infarction in T2DM patients.Result1.102 bases variations are foud, among which there are 26 missense mutation sites, and 16 missense mutation sites can’t be fond in normal control group through mitochondrial ND2 gene’s sequencing analysis of T2DM patients in Zhejiang area. There are 37 bases variations in the mitochondrial ND2 genes of 204 normal people, among which there are 10 missense mutations. The missense mutation rate has no significant difference between two groups (P>0.05). 2.The rate of nt4769 A-G mutation of mtDNA ND2 gene is about 100%.3.The distribution of A4833G mutations in two groups has statistically difference (P 75%) which are A4722G, A4740G, A4824G, A4902C and C5124A are selected according to the amino acids’ conservatism among species in the mitochondrial ND2 genes’ mutations of 448 T2DM patients. A4722G, A4740G, A4902C and C5124A can only be found in the T2DM group. We find that A4722G and C5124A mutations lead to changes of the amino acids in the protein transmembrane region by ananlysis of the ND2 protein’s secondary structure. 5.The mutation that can obviously lead to familial diabetes isn’t found after analyzing mtDNA ND2 gene mutations of those who are suspected of family history of maternal inheritance diabetes. 6.Among T2DM patients, analyzing the clinical materials of mt5178A genotype patients and mt5178C genotypes patients, on the index of systolic blood pressure, the former is obviously lower than the latter, about hollow c-peptide release quantity and high-density lipoprotein (HDL), the former is significantly higher than the latter, and about cerebral infarction, the latter is obviously higher than tha former(P <0.05).Conclusion1.The MtDNA ND2 gene nt4769 base G can be a normal reference for mitochondrial gene of Zhejiang people who are Han Chinese.2.The frequency of A4833G variations is significantly higher in T2DM group than normal control group (P <0.05). Probably T2DM susceptibility genes sites lead to the the occurrence of T2DM in the right environment. 3.A4722G, A4740G, A4824G, A4902C and C5124A are highly conservative mutations. After analysis we think that A4722G and C5124A mutations may be relevant to the occurrence of T2DM, which should be confirmed through increasing the sample size and also and thoroughly discussing the mechanism. 4.The relative mutation that can clearly result in familial diabetes can’t be found in mtDNA ND2 gene. 5.The C5178A mutation of mtDNA ND2 gene is helpful to resist the occurrence of cerebral infarction in the T2DM patients
语种	中文
学号	2009010337
发布年限	2010-05-27
毕业论文分类号	0R04
原始专题	检验医学院暨生命科学院
学位论文研究方向	生物化学与分子诊断
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全文文件名	2009010337吴含2012临床检验诊断.pdf\|2009010337吴含2012临床检验诊断.pdf
文献类型	学位论文
条目标识符	https://kms.wmu.edu.cn/handle/3ETUA0LF/118145
专题	温州医科大学
作者单位	溫州医科大学检验医学院暨生命科学院
推荐引用方式 GB/T 7714	吴含. 浙江地区汉族人群2型糖尿病患者线粒体ND2基因突变的研究[D]. 温州医科大学,2014.