论文标题:线粒体基因突变与2型糖尿病的研究
Study on the Relationship between the Mutations of Mitochondrial Gene and Type 2 Diabetes
论文作者 于珮
论文导师 于德民,论文学位 硕士,论文专业 内分泌与代谢病学
论文单位 天津医科大学,点击次数 117,论文页数 79页File Size3552k
2002-05-01论文网 http://www.lw23.com/lunwen_34900282/ 线粒体DNA;基因突变;2型糖尿病;聚合酶链式反应;限制性片段长度多态性
mitochondrial DNA (mtDNA);gene mutation;Type 2 diabetes mellitus;polymerase chain reaction (PCR);restriction fragment length polymorphism (RFLP)
目的 线粒体是细胞内能量储存和供给的场所。细胞生命活动中需要的能量约有95％来自线粒体。线粒体DNA是独立于细胞核染色体外的基因组，具有自我复制、转录和翻译功能，可编码13种与细胞氧化磷酸化有关的酶蛋白亚单位。线粒体DNA高度保守地编码蛋白质，由于多种原因使其突变率是核DNA的10-20倍。由于氧化磷酸化在胰岛B细胞分泌胰岛素过程中起重要作用，线粒体基因缺陷与糖尿病发生发展的关系日益受到关注。以随机选取的居住天津、汉族、无亲缘关系的300例2型糖尿病患者为实验组和280例正常者对照组(糖耐量正常)为研究对象，对线粒体基因突变热点区域内4个位点进行筛查，以便了解线粒体基因突变与2型糖尿病间的关系。 方法 1、收集2型糖尿病人及对照者的血液标本。 2、提取血液标本基因组DNA。 3、利用PCR-RFLP方法对300例2型糖尿病人和280例对照者的线粒体DNAnp3243、3316、3394、3426等4个位点进行基因突变筛查。 4、对筛查出的突变基因进行测序分析。 5、分析临床资料。 结果 在300例2型糖尿病患者中，共检出8例np3316G→A突变，突变发生率为2.67％；检出12例np3394T→C突变，突变发生率为4.00％；检出2例np3426A→G突变，突变发生率为0.67％。在280例对照者中，发现2例np3394T→C突变，突变发生率为0.71％；未发现np3316G→A和np3426A 大 学申漾诞士放艾 中文殷要 一G突变。两组中皆未检出 up3243A—G突变。两组间 up331 6G～A突变率 和up3394T，C突变率有显著性差异（均P＜0．05人但up3426AIG突变率无 显著性差异（P＞0刀5人实验组线粒体 DNA NDI基因 up3316和 up3394点突 变阳性者较阴性者有明显的＊M家族史（P＜（．05）和母系遗传倾向（尸＜0．01人 线粒体基因突变糖尿病起病早于无突变者（P＜D．05），微血管病变与突变无关 联（P＞0刀5）。 结论 线粒体 DNA up331 6G—A突变可使非极性丙氨酸被极性的苏氨酸 所取代（GCC—ACC人线粒体 DNA up3394T—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（TAT一C盯h线粒体 DNA nP3426A—G 突变是一个缴氨酸的同义突变（GTA—GTG）。三种突变皆位于 ND基因区， 可能影响NADH脱氢酶活性，导致ATP合成减少，胰岛B细胞能量供应不 足，影响胰岛素分泌，从而参与2型糖尿病的发生。研究较早、报道较多的 线粒体 DNA up3243A—G突变可导致线粒体转运核糖核酸亮氨酸基因异常， 己证实是母系遗传伴耳聋糖尿病的致病原因，但在本研究中未发现。出此可 知，线粒体DNAnp3316G—A突变、up3394T—C突变和up3426A—G突变在 2型糖尿病患者中发生率相对较高。
Objective Mitochondron. is the special organ which stores and supplies energy for cells, mitochondrial DNA (mtDNA) is the only extra-chromosomal genome in human cells, which can replicate, transcript and encode autonomously. It encodes for 13 kinds of essential proteins of the respiratory chain, which are needed for the oxidative phosphorylation (OXPHOS). Since mitochondrial OXPHOS plays an important role in the glucose-induced insulin secretion in pancreatic beta cells, the relationship between mitochondrial gene defect and the development of diabetes mellitus (DM) has been widely attended. In 1997, American diabetes association (ADA) put this type of DM into the subgroup of special diabetes mellitus, which has genetic defects in pancreatic beta cells. In the study, 300 randomly selected, unrelated patients with type 2 DM and 280 normal controls were included. 4 positions in mtDNA1^" (UUR) gene and its adjacent region were screened, which were thought to be genetic hot spots for DM. The aim of this study is to investigate the prevalence of mtDNA gene mutations and find out the relation between mtDNA mutations and microvascular complications. Moreover a simple and sensitive way to be performed in clinical diagnosis of DM caused by mitochondrial gene mutation may be developed.Methods1. Collecting the blood samples according to ADA criteria of type 2 DM.2. Extracting and purifying the genomic DNA from the blood samples.3. Using PCR-RFLP analysis, 4 mtDNA positions (np3243, np3316, np3394, np3426) were screened in 300 type 2 diabetics and 280 normal controls.4. Sequencing the mutation genes screened by RFLP.5. Analysize the clinical data.Results In the study, 8 (2.69%) with the mutation of np3316G- A, 12 (4.00%) with np3394T-C, and 2 (0.67%) with np3426A- G were found in type 2 DM group. In controls, 2 (0.71%) with that of np3394 were secreened. However the well-known mutation of np3243 was not found. The mutations of np3316G- A and np3394T-C had significiant differences between two groups (P<0.05, respectively), and that of np3426A- G was on the contrary. The mutation group showed obvious family history of DM (P<0.05) and tendency of maternal inherition (P<0.01). The age when DM came on was correlative with mtDNA mutations (P<0.05). However, there was no relationship between the microvascular complications and mtDNA mutations (P<0.05).Conclusions The mutations at np3316, np3394 and np3246 are all located in the mtDNA NADH dehydrogenase subunit 1 (ND1) region. It is well known that the np3316 G- A changes a nonpolar alanine to a polar threonine (GCC- ACC), the np3394T-C turns a conserved tyrosine to histidine (TAT -CAT) and np3426A-G is not a missense mutation (GTA- GTG). These point mutations may involve in the development of type 2 DM by decreasing the activity of ND1 . The famous mutation of mtDNA np3243 A-G which can lead to abnormality of tRNALcu(UUR)gene is proven to be the reason of maternally inherited diabetes and deafness (MIDD). However, it was not found in the study. It indicates that the point mutations at np3316, np3394 and np3426 of ND1 may contribute to the occurrence of type 2 DM.
|
