【作者】 王康；

【导师】 王国相； 娄晋宁；

【作者基本信息】 中国协和医科大学 ， 病理学与病理生理学， 2008， 博士

【摘要】 Leber遗传性视神经病伴肌张力障碍是一种独特的氧化磷酸化疾病。表现为青中年发病的双侧视力下降,伴儿童发病的全身性进行性肌张力障碍。研究发现Leber遗传性视神经病伴肌张力障碍由线粒体ND6、ND4或ND1基因突变导致。一个安徽的包括18例病人的汉族家系的线粒体基因组被分析。该家系中的患者在14-30岁出现无痛性双侧视力下降、视神经萎缩,部分伴有蓝绿色盲,部分患者在3-8岁出现进行性的全身性肌张力障碍、锥体系及其他神经系统损害,神经影像学提示双侧基底节区对称性新月形坏死。对两例典型LDYT患者进行了肌肉活检。在排除了已知的常见线粒体突变后,该家系患者的全线粒体基因组用线粒体DNA芯片(Affymetrix^TM)结合直接测序法进行了序列分析,对发现的突变进行直接测序再证实。并对101例具有相同遗传背景的健康对照和70例线粒体病患者进行突变片段序列分析。应用基于遗传分析仪的SSCP和RFLP方法对突变的异质状态进行检测。该家系患者的活检肌肉光镜下无显著改变,组织化学染色未发现氧化磷酸化酶活性异常,超薄切片电子显微镜下线粒体结构、形态无显著改变。对该家系4例有代表性成员的线粒体基因组分析发现了51个散布的变异（参照rCRS）,8个为新的变异。其中只有G10197A是非同义突变,改变了线粒体ND3第47位氨基酸残基,一个高度保守的丙氨酸被具有羟基链的苏氨酸取代,在该家系的所有母系亲属中都发现了这一突变,其中2人表型正常（不外显的突变基因携带者）。在101例健康对照者和70例其他线粒体病患者（包括一个具有2例同胞患者的LDYT家系）中都未发现携带该突变者。异质状态研究提示所有母系成员的突变是同质性的。该突变发生在线粒体haplogroup D4b亚型的背景下。ND3基因已经被报道与Leigh综合征或Leigh样脑病相关,并有关于ND3突变导致LHON的报道。本文是首次关于ND3^*10197A导致LDYT的报道。相同线粒体突变背景下表型的变异提示其发病受到了haplogroup亚型及核基因的影响。更多还原

【Abstract】 Leber hereditary optic neuropathy and dystonia （LDYT） characterized by maternal hereditary optic neuropathy variably associated with dystonia and other systemic disorders has been recognized as a unique oxidative phosphorylation disorder caused by mutations in mitochondrial ND6, ND4 or ND1 gene. A Chinese Han family lived in Anhui province with 18 patients and 46 family members was investigated. The patients developed visual loss and optic atrophy from teenage to the third decades, variably associated with generalized dystonia with onset in childhood and prominent symmetric lucencies in the basal ganglia revealed by CT and MRI. Blue-green achromat, pyramidal signs and other central nerve system disorders were found in certain family members. Histochemistry stain of biopsied muscles from two patients with generalized dystonia did not show evidence of abnormal oxidative phosphorylation. Electrical microscopy did not reveal prominent changes on amount or construction of mitochondrion. After analyzed the high frequency mutations of LHON named as ’primary mutation’ and ntl4459A which is a well-known LDYT causative mutation, the nucleotide sequences of the entire mitochondrial genomes in four selected family members （one LHON, one LDYT, one asymptomatic maternal relative and one asymptomatic paternal relative） were determined by employing a DNA microarray designed for mitochondrial DNA (Affymetrix^TM). The base changes identified by the microarray were further confirmed by conventional direct nucleotide sequence analysis. The mutant loading was analyzed by SSCP and RFLP based on genetic analysis system. There are 51 mitochondrial DNA variants were identified in the entire mitochondrial DNA among the four family members. But 50 variants are synonymous polymorphisms referring to rCRS （Human mtDNA Revised Cambridge Reference Sequence）. Only one nonsynonymous mutation （G10197A） substituting a hydroxymethyl-sided threonine for a highly conserved hydrophobic alanine at the codon 47 of mtND3 on the background of mitochondrial haplogroup D4b was identified in the mitochondrial DNA of all the affected members and two asymptomatic maternal relatives, while it was not present in paternal relatives or 101 normal individuals with same genetic background or 70 patients with mitochondriopathies including two siblings with LDYT phenotype. SSCP and RFLP demonstrated the mutant loading in all maternal relatives was 100%, which means it is a homoplasmic mutation at the site of nt10197. Although absent direct evidence of respiratory chain defect in the family until now, there are several lines of evidence to supporting the pathological role of nt10197A mutation. Although several mutations around MT10197 involving ND3 gene have been reported as the causes of Leigh syndrome or Leigh-like encephalopathy, this is the first description of the ND3 mutation causing LDYT. The reported The variable phenotype on the background of same mitochondrial mutation suggests that mitochondrial haplogroup and nuclear factors affect the onset of LDYT.更多还原