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SCOT基因“轻微”突变的患者并不总是出现永久性的酮病，这是SCOT缺陷的病原学特征。2)在SCOT缺陷患者中，我们发现外显子6和7的跳跃是由外显子6末端的G&GT；A替换引起的。我们分析了异核RNA和胞质RNA，发现具有单个外显子6跳跃的mRNA被无义介导的RNA降解。由于跳过外显子6和7的mRNA在框架内，虽然它在核RNA中只是少量的转录本，但它成为胞浆部分的主要产物。2)线粒体乙酰乙酰-辅酶A硫解酶(T2)缺陷1)与tiglyl-CoA联用的方法在欧美被广泛用于T2缺陷的酶学诊断。我们发现并证明了在两名T2基因缺陷患者中，使用tiglyl-CoA的偶联检测结果是正常的，这些患者具有“轻微”突变。这意味着具有轻微突变的T2缺陷患者被误诊为正常，使用tiglyl-CoA偶联试验。2)我们发现了一个大的T2基因缺失，包括外显子2-4，这是由不相等的同源重组引起的。3.肝组织特异性抑制人SCOT基因表达。我们分析了HeLa细胞和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CpG岛的甲基化状态。出人意料的是，甚至在肝母细胞瘤细胞系和小鼠肝组织中，Scot基因启动子区域周围的CpG岛也发生了低甲基化，几乎完全抑制了Scot基因。4.人硫解酶家族的三级结构。我们测定了细胞质乙酰乙酰辅酶A硫解酶的晶体三级结构，并利用该结构在酶机制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.