Molecular analyzes of heme regulation and its disorders.

在本项目中，我们研究了血红素代谢的分子调控及其遗传障碍。1)血红素生物合成的遗传性疾病。我们报道了两种卟啉症的分子分析：遗传性同比例卟啉症（HCP）和肝红细胞生成性卟啉症（HEP）。从HCP患者中鉴定出三种coproportion phyrinogen oxidase （CPO）突变。在一个等位基因上发现了G172H的替换，而在另一个等位基因上发现了G89S和V194I的替换。进一步分析表明，含G172H的CPO活性较低。其余的替换为多态性。我们还从HEP患者身上发现了两种尿卟啉原脱羧酶（UROD）突变。其中一个等位基因存在A^< 417>G^<418>T^<419> - CCA突变，另一个等位基因存在A^<677> - C突变。利用中国仓鼠卵巢细胞，我们发现前者和后者突变分别使UROD活性降低了20%和80%以上。现在，我们继续研究遗传性铁母细胞性贫血、多种卟啉症和氨基乙酰酸脱水酶缺乏症。2)血红素代谢的分子调控。为了了解可能影响遗传性血红素疾病的因素，我们启动了红系血红素合成项目。到目前为止，我们已经阐明了红系转录因子GATA-1不仅在红系细胞中表达，而且在睾丸中也表达，NF-E2的大亚基和小亚基是红系特异性基因激活所必需的，NF-E2的大亚基受血红素的调节。我们还证明血红素对于铁螯合酶mRNA是必需的。因此，遗传性疾病导致血红素供应不足，不仅会导致铁螯合酶mRNA水平异常，还会导致红系血红素生物合成的异常调节。利用UT-7细胞系，我们还研究了巨核细胞成熟和红细胞分化的机制。这些发现对于更好地理解血红素代谢的遗传疾病患者的途径是重要的。少

In the present project, we have investigated the molecular regulation of heme metabolism and its genetic disorder.1) Genetic disorder of heme biosynthesis. We have reported molecular analyzes of two types of porphyria : hereditary coproporphyria (HCP) and hepatoerythropoietic porphyria (HEP). Three mutations were identified in coproporphyrinogen oxydase (CPO) from a patient with HCP.A G172H substitution was observed in one allele, while G89S as well as V194I substitutions were demonstrated in the other allele. Further analysis indicated that CPO with G172H has little activity. The other substitutions were revealed to be polymorphism.We have also identified two mutations in uroporphyrinogen decarboxylase (UROD) from a patient with HEP.A T^<417>G^<418>T^<419> to CCA mutation was existed in one allele, whereas A^<677> to C mutation was investigated in the other allele. Using Chinese hamster ovary cells, we have elucidated that the former and the latter mutation decreased UROD activity by … More 20% and by 80%, respectively.Now, we continued our effort on hereditary sideroblastic anemia, variegate porphyria, and delta-aminolevulinate dehydratase deficient porphyria.2) Molecular regulation of heme metabolism. To understand the factor (s) that might influence the hereditary heme disorder, we have started the project on erythroid type of heme synthesis. Until today, we have elucidated that a erythroid transcriptional factor, GATA-1, is expressed not only in erythroid cells but also in the testis, that the large subunit as well as the small subunit of NF-E2 is necessary for erythroid specific gene activation, and that the large subunit of NF-E2 is regulated by heme. We also demonstrated that heme is necessary for ferrochelatase mRNA.Thus, the insufficient supply of heme by genetic disorder will result in not only the abnormal level of ferrochelatase mRNA but also the abnormal regulation of erythroid heme biosynthesis.Using a cell line, UT-7, we also investigated the mechanisms to undergo megakaryocytic maturation as well as to undergo erythroid differentiation. These findings are important for the better understanding of heme metabolism in patients with genetic disorder of the pathway. Less