Ueki Isao, Koga Yasutoshi, Povalko Nataliya, Akita Yukihiro, Nishioka Junko, Yatsuga Shuichi, Fukiyama Ryo, Matsuishi Toyojiro
Department of Pediatrics and Child Health, Kurume University School of Medicine, 67 Asahi-Machi, Kurume 830-0011, Japan.
Mitochondrion. 2006 Feb;6(1):29-36. doi: 10.1016/j.mito.2005.10.003. Epub 2005 Dec 5.
Lactic acidosis has been associated with a variety of clinical conditions and can be due to mutation in nuclear or mitochondrial genes. We performed mutations screening of all mitochondrial tRNA genes in 44 patients who referred as hyperlactic acidosis. Patients showed heterogeneous phenotypes including Leigh disease in four, MELAS in six, unclassified mitochondrial myopathy in 10, cardiomyopathy in five, MERRF in one, pure lactic acidosis in six, and others in 12 including facio-scaplo-femoral muscular dystrophy (FSFD), familial cerebellar ataxia, recurrent Reye syndrome, cerebral palsy with mental retardation. We measured enzymatic activities of pyruvate dehydrogenase complex, and respiratory chain enzymes. All mitochondrial tRNA genes and known mutation of ATPase 6 were studied by single strand conformation polymorphism (SSCP), automated DNA sequence and PCR-RFLP methods. We have found one patient with PDHC deficiency and six patients with Complex I+IV deficiency, though the most of the patients showed subnormal to deficient state of respiratory chain enzyme activities. We have identified one of the nucleotide changes in 29 patients. Single nucleotide changes in mitochondrial tRNA genes are found in 27 patients and one in ATPase 6 gene in two patients. One of four pathogenic point mutations (A3243G, C3303T, A8348G, and T8993G) was identified in 12 patients who showed the phenotype of Leigh syndrome, MELAS, cardimyopathy and cerebral palsy with epilepsy. Seventeen patients have one of the normal polymorphisms in the mitochondrial tRNA gene reported before. SSCP and PCR-RFLP could detect the heteroplasmic condition when the percentage of mutant up to 5, however, it cannot be observed by direct sequencing method. It is important to screen the mtDNA mutation not only by direct sequence but also by PCR-RFLP and the other sensitive methods to detect the heroplasmy when lactic acidosis has been documented in the patients who are not fulfilled the criteria of mitochondrial disorders.
乳酸性酸中毒与多种临床病症相关，可能由核基因或线粒体基因突变所致。我们对44例以高乳酸血症转诊的患者进行了所有线粒体tRNA基因的突变筛查。患者表现出异质性表型，包括4例Leigh综合征、6例MELAS、10例未分类的线粒体肌病、5例心肌病、1例MERRF、6例单纯乳酸性酸中毒，以及12例其他病症，包括面肩肱型肌营养不良（FSFD）、家族性小脑共济失调、复发性瑞氏综合征、伴有智力障碍的脑瘫。我们测定了丙酮酸脱氢酶复合体及呼吸链酶的酶活性。采用单链构象多态性（SSCP）、自动DNA测序和PCR-RFLP方法研究了所有线粒体tRNA基因及ATPase 6的已知突变。我们发现1例丙酮酸脱氢酶复合体（PDHC）缺乏患者和6例复合体I+IV缺乏患者，尽管大多数患者的呼吸链酶活性呈亚正常至缺乏状态。我们在29例患者中鉴定出一种核苷酸变化。27例患者的线粒体tRNA基因存在单核苷酸变化，2例患者的ATPase 6基因存在单核苷酸变化。在表现为Leigh综合征、MELAS、心肌病和伴有癫痫的脑瘫表型的12例患者中鉴定出4种致病性点突变（A3243G、C3303T、A8348G和T8993G）之一。17例患者的线粒体tRNA基因具有之前报道的正常多态性之一。当突变比例高达5%时，SSCP和PCR-RFLP能够检测到异质性状态，但直接测序法无法观察到。对于未符合线粒体疾病标准但已记录有乳酸性酸中毒的患者，不仅要通过直接测序，还要通过PCR-RFLP及其他敏感方法筛查mtDNA突变以检测异质性，这一点很重要。
