用位点特异PCR法检测Leber遗传性视神经病家系的mtDNA11778G→A点突变

Detection of mtDNA 11778 (G→A) Point Mutation in a Family with Leber's Hereditary Optic Neuropathy by Site-specific Polymerase Chain Reaction

目的 寻找一种快速、准确、有定量突变比例的简单PCR方法,以识别Leber遗传性视神经病(LHON)患者所携带的mtDNA 11778G→A点突变。方法 根据已知致病突变的碱基变化,设计M(突变)和N(正常)引物,分别与反向引物R配对扩增,严格控制退火温度和其他PCR反应条件达到特异扩增。病例组为LHON家系共10个个体,对照组为40位正常人。结果 在先证者、母系已发病亲属和一个10岁男孩(未发病)体内分别检出突变比例各不相同11778G→A点突变,而在家系的正常配偶、父系子女及40位正常对照组未检出该突变。结论 位点特异PCR是一种不受DNA序列有否限制性内切酶位点的检测突变的方法,适用于LHON等致病突变明确的遗传病的基因诊断。

Objective To find a simple, fast, accurate, and quantitative PCR-based method for mutation detection, so as to identify mitochondrial DNA 11778 G→A point mutation in patients with Leber's hereditary optic neuropathy (LHON). Method On the basis of sequencing of mtDNA from LHON proband, M primer for mutation and N primer for normal were designed to be coupled with reverse primer respectively. Specific PCRs were done on an amplifying condition with high stringency such as a well controlled annealing temperature, low Mg2+ concentration and less thermal cycles. The objective pedigree includes 10 individuals, were against 40 normal control persons. Results Different ratios of indicative mtDNA 11778A→G mutation were checked out from the proband, affected maternal members and a 10 year-old boy (up to now no appearance yet), whereas not appeared on normal spouses, paternal offsprings in the family, neither did on 40 controls. Conclusion This site-specific PCR method is a kind of general mutation analysis way, without the restriction of existence of endonuclease site. It can be applied for the gene diagnosis of known-mutation hereditary diseases such as LHON.