线粒体ND4和ND6基因突变在Leber遗传性视神经病变发病中的作用

发布时间：2018-04-03 04:23

本文选题：Leber遗传性视神经病变　切入点：线粒体DNA　出处：《浙江大学》2015年博士论文

【摘要】：Leber遗传性视神经病变(Leber's Hereditary Optic Neuropathy,LHON)是一种主要累及青年男性,导致双眼快速无痛性视力减退的母系遗传性眼病。线粒体DNA (mitochondria DNA, mtDNA)突变是该病的分子致病基础之一。线粒体ND4基因和ND6基因是与LHON相关的突变热点区域。本研究在全国范围内共收集无血缘关系LHON汉族家系1218个,对收集的1218例先证者及316名正常对照者ND4基因突变筛查显示,ND4基因上共存在149个变异位点,携带已知的m.11253TC、m.11778GA和m.11696GA突变位点家系共466个,占LHON总家系的38.26%,其中携带m.11778GA突变位点的家系440个,占36.12%。此外我们还发现10个携带可能有意义突变位点的家系,占LHON总家系的0.82%。我们对其中一个携带m.11778GA突变位点的5代111人的大家系进行了详细的眼科学、遗传学和生化功能评估。17名母系成员中6名患者表现出全视盘或部分视盘苍白,11名携带者中7名母系成员眼底正常,而4名携带者(V-6,V-7,V-8和V-9)眼底血管迂曲。光学相干断层扫描(OCT)检查显示患者组视网膜神经纤维层(RNFL)厚度在各个象限均变薄,携带者RNFL厚度在颞侧象限较正常组变厚。值得一提的是,携带者V-6的RNFL厚度在颞上侧象限变薄,V-8的RNFL厚度在鼻侧,鼻上侧和颞上侧象限均变薄。对该家系5名患者和5名携带者及具有相同单体型的4名正常对照者建立永生化淋巴细胞系,患者组和携带者的线粒体膜电位分别是对照者组的61.15%和66.46%。在2-DG和丙酮酸存在情况下,患者组氧化磷酸化产生ATP是正常组的49.09%,而携带者组较正常组下降35.08%。此外,患者组和携带组的ROS水平较正常组分别增加85%和56%。患者组和携带者组ND4,ND1,ND5,ND6,NDUFS1和NDUFB8蛋白表达量较正常对照组明显降低。ND6基因突变筛查显示,ND6基因上共存在92个变异位点,包括29个错义突变位点(9个未报道突变和20个已报道突变)和63个沉默突变位点,94个携带ND6基因14484TC,14502TC,14459GA突变位点的家系占总家系的7.7%,其中m.14484TC突变位点家系占4.4%。此外,12个携带可能有意义突变位点的家系占总家系的1.1%,因此,106个携带ND6基因突变位点的家系占总家系的8.7%。线粒体单体型M9,M10,M11和H2在患者中的比例大于正常对照者。我们对单独携带m.14502TC突变位点的患者,单独携带m.11778GA突变位点的患者,同时携带m.14502TC突变位点和m.11778GA突变位点的患者和正常对照者建立永生化淋巴细胞系和转线粒体细胞系(Cybrids)。研究发现,单独携带m..14502TC突变位点的Cybrids其基础OCR值、ATP偶联OCR值及最大OCR值较正常组分别下降18.3%,15.2%和33.7%,而同时携带m.14502TC突变位点和m.11778GA突变位点的Cybrids下降程度更明显。线粒体膜电位和氧化磷酸化产生的ATP在同时携带m.14502TC突变位点和m.11778GA突变位点的Cybrids明显降低。ROS水平在携带双突变组明显高于单独携带m.14502TC突变位点组及单独携带m.11778GA突变位点组。这些结果证实,m.14502TC突变位点可能是与LHON相关的继发突变位点,本身可引起线粒体功能下降,也可协同原发突变,加重线粒体的损伤。
[Abstract]:Leber hereditary optic neuropathy (Leber's Hereditary Optic Neuropathy, LHON) is a major involvement of young men, leading to disease of maternally inherited eyes rapid painless visual deterioration. Mitochondrial DNA (mitochondria DNA mtDNA) is one of the molecular basis of pathogenic mutations in this disease. The mitochondrial ND4 gene and ND6 gene mutation hotspots associated with the LHON. This study collected unrelated LHON families of Han nationality 1218 nationwide, the collection of 1218 probands and 316 normal controls ND4 gene mutation screening showed that the ND4 gene exist in 149 mutation sites with known m.11253TC, m.11778GA and m.11696GA mutation in home a total of 466 lines, accounting for LHON family 38.26%, which carry the m.11778GA mutation in 440 families, accounting for 36.12%., we also found that the 10 may carry meaningful mutations in families, accounting for LHON The total 0.82%. of our family one carrying the m.11778GA mutation of 5 generations of 111 people, we carried out a detailed Ophthalmology, genetics and biochemical evaluation of 6 patients.17 matrilineal relatives showed the whole disc or part of pale optic disc, 11 carriers in 7 maternal members and normal fundus. 4 carriers (V-6, V-7, V-8 and V-9) of fundus vascular tortuosity. Optical coherence tomography (OCT) examination showed that patients with retinal nerve fiber layer (RNFL) thickness in each quadrant were thinner, the thickness of RNFL carriers in the temporal quadrant than the normal group becomes thick. It is worth mentioning that the thickness of RNFL carriers V-6 in the upper temporal quadrant thinning, V-8 RNFL thickness in nasal, nasal and temporal side upper quadrant were thinner. The families of 5 patients and 5 carriers with the same haplotype and 4 normal controls to establish immortalized cell lines, patients and carry The mitochondrial membrane potential were the control group and 61.15% 66.46%. in the presence of 2-DG and pyruvate cases, patients with oxidative phosphorylation of ATP is produced in normal group 49.09%, and the carrier group decreased compared to the normal group 35.08%. in patients group and ROS group compared with normal group level carry respectively increased by 85% and the group of patients with 56%. and the carriers of group ND4, ND1, ND5, ND6, NDUFS1 and NDUFB8 protein expression was lower than the control group.ND6 gene mutation screening showed that there were 92 mutation sites in ND6 gene, including 29 missense mutations (9 unreported mutations and 20 reported mutations and 63 silent mutations) 94, carrying ND6 gene 14484TC, 14502TC, 14459GA mutation families accounted for 7.7% families, in which m.14484TC mutation family accounted for 4.4%. in addition, 12 may carry meaningful mutations in families in total families 1.1%, therefore, 106 Carrying mutations in ND6 gene family total family mitochondrial 8.7%. haplotype M9, M10, M11 and H2 in the proportion of patients than in normal controls. We separate carrying m.14502TC mutation in patients carrying the m.11778GA mutation alone in patients carrying m.14502TC mutation and m.11778GA mutation in patients with normal controls establish immortalized lymphocyte lines and transmitochondrial cell line (Cybrids). The study found that individual carrying m..14502TC mutations of the Cybrids based on the OCR value, compared with the normal group respectively decreased by 18.3% and the maximum value of ATP coupling OCR value OCR, 15.2% and 33.7%, while carrying the m.14502TC mutation and m.11778GA mutation in Cybrids the degree of decline is more obvious. The mitochondrial membrane potential and oxidative phosphorylation of ATP at the same time carrying m.14502TC mutation and m.11778GA mutation in Cybrids significantly decreased.ROS In carrying double mutant group was significantly higher than that of m.14502TC alone and carrying mutations alone carrying m.11778GA mutation site group group. These results demonstrated that m.14502TC mutations may be secondary associated with LHON mutations, can cause a decline in mitochondrial function, but also coordinated the primary mutation, increased mitochondrial damage.

【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R774.6

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