Study on biochemical and molecular evoluation for mitochondrial β-oxidation defects

线粒体脂肪酸β-氧化紊乱(FAOD)最近引起了人们的关注。我们调查了日本FAOD患者的实际情况，并对FAOD患者的生化评价和分子分析进行了研究。1)日本FAOD患者调查：采用问卷、文献和个人交流的方法，在1985年至2000年期间共发现62例日本FAOD患者。CPT2缺乏症患者最多(14例)，其次为戊二酸血症2型(12例)。2)建立简化的GC/MS数据处理系统：FAOD患者在GC/MS筛查有机酸血症时，常表现为非脑血管性二羧酸尿症。我们建立了一个基于个人计算机的自动代谢图谱和诊断系统。3)干血滤纸中游离脂肪酸的测定：为了筛选FAOD，串联质谱仪分析酰卡尼汀已成为一种流行方法。Tandem MS可能无法将此类疾病检测为假阴性。我们建立了一种简单、灵敏的方法来检测血液滤纸中的游离脂肪酸水平。4)利用培养细胞掺入RI标记的脂肪酸来检测FAOD：我们通过掺入RI标记的脂肪酸(包括棕榈酸酯和肉豆蔻酸酯)建立了FOOD的筛选系统，分析了VLCAD缺陷、GA2和CPT2缺陷的细胞。5)VLCAD缺陷和GA2缺陷患者的分子分析：我们在5名VLCAD缺陷患者和3名GA2患者中发现了基因突变。在VLCAD缺陷症中，在检测到的突变中，存在明显的基因/表型相关性和温度敏感性。在GA2中，我们通过脉冲标记和DNA分析在两名患者中确定了首例日本ETF-α亚单位缺乏症患者。

Mitochondrial fatty acid beta-oxidation disorders (FAOD) has atracted attention recently. We investigated the acutual situation of Japanese patients with FAOD, and studied the biochemical evaluation and molecular analysis of FAOD patients.1) Survey of Japanese patients with FAOD : We found 62 Japanese patients with FAOD during the period between 1985 to 2000, using questionnaires, literatures and personal communications. The number of patients was largest in CPT2 deficiency (14), second was glutaric acidemia type 2 (12). The patients in early childhood tend to have acute encephalopathy-like illness, whereas patients in older childhood or adolescence did myopathic illness.2) Establishment of a simplified GC/MS data processing system : FAOD patients often show nonkletotic dicarboxylic aciduria in organic acidemia screening using GC/MS.We established a personal computer-based system of automated metabolic profiling and diagnosis.3) Measurement of free fatty acids in dried blood filter paper : For the screening of FAOD, acylcarnitine analysis by tandem MS has become popular. Tandem MS may potentially fail to detect such disorders as a false-negative. We established a simplified, sensitive method to measure the blood free fatty acid levels in blood filter paper.4) Detection of FAOD by incorporation of RI-labeled fatty acids using cultured cells : We established a screening system for FAOD by incorporation of RI-labeled fatty acids, including palmitate and myristate, analyzing cells from VLCAD deficiency, GA2 and CPT2 deficiency.5) Molecular analysis of Japanese patients with VLCAD deficiency and GA2 : We identified genetic mutations in five VLCAD deficient patients and three GA2 patients. In VLCAD deficiency, the genotype/phenotype correlation was apparent and the temperature sesitivity was seen among mutations detected. In GA2, we identified the first Japanese case of ETF-alpha subunit deficiency in two patients by pulse labeling and DNA analysis.