Molecular basis of inborn errors of ketone body metabolism : mainly basic studies for responsible genes

1.琥珀酰辅酶a:3-酮酸辅酶a转移酶（SCOT）缺陷1)我们发现并证明了“轻度”突变的SCOT缺陷患者并不总是表现出永久性酮症，而永久性酮症是SCOT缺乏症的病理特征。2)在SCOT缺陷患者中，我们发现外显子6和7跳变，这是由外显子6末端核苷酸的G> a取代引起的。我们分析了异核RNA和胞质RNA，发现单外显子6跳变的mRNA通过无义介导的RNA衰变迅速降解。由于外显子6和7跳跃的mRNA在框架内，虽然它在核RNA中是次要的转录物，但它成为细胞质部分的主要产物。线粒体乙酰乙酰辅酶A硫酶（T2）缺乏症1)在欧美已被广泛应用于酶促诊断T2缺乏症。我们发现并证明了用tigll - coa偶联试验的结果是正常的2例T2缺陷患者的“轻度”突变。这意味着T2缺陷的“轻度”突变患者在使用tigll - coa偶联试验时被误诊为正常。2)我们发现了一个包含2-4外显子的T2基因大缺失，这是由不平等同源重组引起的。这是该机制在T2基因中引起的第一个突变。肝脏特异性抑制人SCOT基因表达。我们分析了HeLa细胞、肝母细胞瘤细胞系以及小鼠肝组织和心肌中SCOT基因启动子区域周围CpG岛的甲基化状态。出乎意料的是，即使在SCOT基因几乎完全被抑制的肝母细胞瘤细胞系和小鼠肝组织中，SCOT启动子区域周围的CpG岛也被低甲基化。人硫醇酶家族的三级结构。我们确定了胞质乙酰乙酰辅酶a硫醇酶的晶体三级结构，并利用该结构在酶的机理上发现了一些新的发现。

1.Succinyl-CoA:3-ketoacid CoA transferase (SCOT) deficiency1)We revealed and proved that SCOT deficient patients with "mild" mutations did not always show permanent ketosis which is pathognomonic feature of SCOT deficiency.2)In a SCOT deficient patient, we found exons 6 and 7 skipping, which was caused by G>A substitution at the end nucleotide of exon 6. We analyzed heteronuclear RNA and cytosolic RNA and revealed that mRNA with single exon 6 skipping was degraded rapidly by nonsense-mediated RNA decay. Since mRNA with exon 6 and 7 skipping was in frame and although it was minor transcript in nuclear RNA, this mRNA became a major product in cytosolic fraction.2.Mitochondrial acetyoacetyl-CoA thiolase (T2) deficiency1)A coupled assay with tiglyl-CoA had been widely used for the enzymatic diagnosis of T2 deficiency in Europe and USA. We revealed and proved that the result of coupled assay using tiglyl-CoA was normal in two T2 deficient patients with "mild" mutations. This means that T2 deficient patients with "mild" mutations had been mis-diagnosed as normal using the coupled assay with tiglyl-CoA.2)We identified a large T2 gene deletion including exons 2-4 caused by unequal homologous recombination. This is the first mutation caused by this mechanism in T2 gene.3.Liver specific suppression of human SCOT gene expression.We analyzed status of methylation in CpG islands around SCOT gene promotor regions in HeLa cells and hepatoblastoma cell lines and murine hepatic tissues and cardiac muscle. Unexpectedly, the CpG islands around SCOT promotor regions were hypomethylated in even hepatoblastoma cell lines and murine hepatic tissues where SCOT gene was almost completely suppressed.4.Tertiary structure of human thiolase family.We determined a crystal tertiary structure of cytosolic acetoacetyl-CoA thiolase and found several new findings in enzyme mechamism using the structure.