Leber视神经萎缩（LHON）家系线粒体DNA突变的分析

发布时间：2018-08-07 18:23

【摘要】： 研究目的:对我国的一个LHON家系的线粒体基因组全序列进行测序分析,探讨其致病的分子基础。研究对象:一个包括11例患者的6代LHON家系,其中5个患者和4个表型正常的家系成员纳入研究,另外,50例无任何亲缘关系的正常健康人作对照。研究方法:(1)抽取患者和正常健康对照的外周血(抗凝);(2)利用Wizard Genomic DNA Purification试剂盒从外周血中提取和纯化基因组DNA;(3)应用等位基因特异性多重PCR(MAS-PCR)方法体系排查3个原发性LHON突变位点;(4)利用两套PCR体系扩增线粒体DNA(mtDNA)全序列,进行DNA测序分析并与最新剑桥标准mtDNA序列比对。研究结果:该LHON家系未发现3个LHON原发性突变位点(G3460A, G11778A和T14484C),mtDNA全序列分析结果显示该家系一个罕见的原发突变C4171突变及29个其他位点变异.其中多态性位点150T, 5231A, 12358G, 12372A,16257A, 16261T, 16129A, 4386C, 12007A,和16111T确定为东亚单倍型N9a1。原发突变C4171A使得ND1基因的289个密码子由高度保守的亮氨酸变成了蛋氨酸。29个其他位点变异中包括27个为多态性(SNP),一个新的突变位点A14841G和一个已发现的A15095G。A14841G突变导致细胞色素b(Cytb)蛋白32位置上高度保守的天门冬氨酸变为丝氨酸,推测该突变位点可能与C4171A突变在LHON表现上起着协同作用。结论:该家系所有患者携带纯合的C4171A突变位点,结果进一步验证了C4171A突变是LHON罕见原发突变。发现了LHON一个新的突变A14841G,并推测可能是LHON的一个继发突变位点,使得具有相同C4171A突变的LHON患者具有较高的外显率和较低的视力恢复率。
[Abstract]:Objective: to study the molecular basis of mitochondrial genome sequence in a LHON family in China. Participants: a six-generation LHON pedigree including 11 patients, 5 patients and 4 normal phenotypic family members were included in the study, and 50 healthy individuals without any kinship were selected as controls. Methods: (1) extracting and purifying genomic DNA from peripheral blood (anticoagulant); (_ 2) of patients and healthy controls using Wizard Genomic DNA Purification kit; (3) screening three primary DNA by allele-specific multiplex PCR (MAS-PCR) method (4) two sets of PCR systems were used to amplify the whole mitochondrial DNA (mtDNA) sequence. DNA sequencing was performed and aligned with the latest Cambridge standard mtDNA sequence. Results: in this LHON pedigree, three LHON primary mutation loci (G3460A, G11778A and T14484C) were not found. The results showed that this family had a rare primary mutation C4171 and 29 other mutations. The polymorphism loci were 150T, 5231A, 12358G, 12372A 16257A, 16261T, 16129A, 4386C, 12007A, and 16111T were identified as East Asian haplotype N9a1. The primary mutation C4171A transformed 289 codon of ND1 gene from highly conserved leucine to methionine. Twenty-seven of the 29 other mutations, including 27 for polymorphic (SNP), a new mutation site A14841G, and a discovered A15095G.A14841G mutation, caused the mutation. The highly conserved aspartic acid of cytochrome b (Cytb) protein 32 was transformed into serine. It is speculated that this mutation site may play a synergistic role with C 4171A mutation in LHON expression. Conclusion: all patients in this family carry homozygous C4171A mutation, which further confirms that C4171A mutation is a rare primary mutation in LHON. A new mutation A14841G of LHON was found, and it was speculated that it might be a secondary mutation of LHON, which makes LHON patients with the same C4171A mutation have higher explicit rate and lower visual recovery rate.
【学位授予单位】：福建医科大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R774.6

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