Tang Sha, Halberg Michelle C, Floyd Kristen C, Wang Jing
Medical Genetics Laboratories, Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, USA.
Methods Mol Biol. 2012;837:259-79. doi: 10.1007/978-1-61779-504-6_18.
Mitochondrial disorders are clinically and genetically heterogeneous. There are a set of recurrent point mutations in the mitochondrial DNA (mtDNA) that are responsible for common mitochondrial diseases, including MELAS (mitochondrial encephalopathy, lactic acidosis, stroke-like episodes), MERRF (myoclonic epilepsy and ragged red fibers), LHON (Leber's hereditary optic neuropathy), NARP (neuropathy, ataxia, retinitis pigmentosa), and Leigh syndrome. Most of the pathogenic mtDNA point mutations are present in the heteroplasmic state, meaning that the wild-type and mutant-containing mtDNA molecules are coexisting. Clinical heterogeneity may be due to the degree of mutant load (heteroplasmy) and distribution of heteroplasmic mutations in affected tissues. Additionally, Kearns-Sayre syndrome and Pearson syndrome are caused by large mtDNA deletions. In this chapter, we describe a multiplex PCR/allele-specific oligonucleotide (ASO) hybridization method for the screening of 13 common point mutations. This method allows the detection of low percentage of mutant heteroplasmy. In addition, a nonradioactive Southern blot hybridization protocol for the analysis of mtDNA large deletions is also described.
线粒体疾病在临床和遗传上具有异质性。线粒体DNA（mtDNA）中存在一组反复出现的点突变，这些突变导致了常见的线粒体疾病，包括MELAS（线粒体脑病、乳酸性酸中毒、卒中样发作）、MERRF（肌阵挛性癫痫伴破碎红纤维）、LHON（Leber遗传性视神经病变）、NARP（神经病变、共济失调、色素性视网膜炎）和Leigh综合征。大多数致病性mtDNA点突变以异质性状态存在，这意味着野生型和含突变的mtDNA分子共存。临床异质性可能归因于突变负荷（异质性）的程度以及受影响组织中异质性突变的分布。此外，Kearns-Sayre综合征和Pearson综合征由mtDNA大片段缺失引起。在本章中，我们描述了一种用于筛查13种常见点突变的多重PCR/等位基因特异性寡核苷酸（ASO）杂交方法。该方法能够检测到低比例的突变异质性。此外，还描述了一种用于分析mtDNA大片段缺失的非放射性Southern印迹杂交方案。
