Molecular studies on beta-ketothiolase deficiency

3-酮硫醇酶缺乏症是一种遗传性有机酸代谢紊乱，表现为常染色体隐性遗传。许多患者近期出现严重的酮症酸中毒。它是由迄今在哺乳动物中发现的四种硫解酶中线粒体乙酰乙酰辅酶A硫解酶(T2)的生物合成缺陷引起的。为了从蛋白质和基因水平研究该病，我们利用免疫化学方法、Northern blotting或PCR方法分析了12例该病患者的成纤维细胞，并克隆了人T2基因和T2基因。结果如下：(1)在脉冲追逐实验中，12例患者观察到的T2生物合成缺陷在分子大小、数量或与T2的交叉反应蛋白的稳定性方面至少可分为六组。(2)证实了T2的酶分析和免疫印迹可以利用非侵袭性获得的外周淋巴细胞和直肠粘膜进行。(3)克隆了人T2基因和T2基因。人T2基因有一个1281碱基的开放阅读框，编码427个氨基酸。人类T2基因全长约27kb，包括12个外显子。(4)以T2基因为探针的Northern印迹分析也显示了基因表达的异质性。(5)应用聚合酶链式反应方法对1例患者进行分子分析，发现该患者为复合杂合子，其中一个等位基因由单核苷酸替换引起氨基酸替换，另一个等位基因跳过外显子8。外显子8的跳跃是由内含子8的5‘剪接点的单核苷酸替换引起的，这些研究将有助于阐明该病的分子基础。

3-Ketothiolase deficiency is an inherited metabolic disorder of organic acids and shows an autosomal recessive inheritance. Many patients present severe ketoacidosis reccurrently. It is caused by a defect in biosynthesis of mitochondrial acetoacetyl-CoA thiolase (T2) among four thiolases identified in mammals known up to now. For the study of the disease at the protein and gene levels, we analyzed fibroblasts from 12 patients with the disease, using immunochemical procedures, Northern blotting, or PCR method, and cloned the human T2 cDNA and T2 gene. The results were as follows :(1) In the pulse-chase experiments, the pattern of defects of T2 biosynthesis observed in 12 patients appeared to be separated into at least six groups in respect to the molecular size, the amount, or the stability of the cross reactive protein to T2. (2) It was confirmed that enzyme assay and immunoblotting of T2 can be performed using peripheral lymphcytes and rectal mucosa that are obtainable less-invasively. (3) We cloned human T2cDNA and T2 gene. Human T2 cDNA was found to have a 1281-base open reading frame encoding 427 amnio acids. Human T2 gene was found to span approximately 27 kilobases, including twelve exons. (4) Heterogeneity in mRNA expression was also revealed by Northern blot analysis using T2 cDNA as a probe. (5) Molecular analysis of a patient using a PCR method revealed that he is a compound heterozygote, consisting of a single nucleotide replacement causing an amino acid substitution in one allele, and a skip of exon 8 in another allele. The exon 8 skipping was found to be caused by a single nucleotide replacement at the 5' splicing site of intron 8. These studies should contribute elucidation in the molecular basis of this disease.