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紊乱。1)血红素生物合成的遗传紊乱。我们报道了两种类型的卟啉症的分子分析：遗传性伴发卟啉症(HCP)和肝红细胞生成性卟啉症(HEP)。在1例肝细胞癌患者中发现3个突变，其中1个等位基因发生G172H突变，另一个等位基因发生G89S和V194I突变。进一步分析表明，含有G172H的CPO活性很低。我们还在1例HEP患者中发现了两个突变：A T^&lt；417&gt；G^&lt；418&gt；T^&lt；419&gt；一个等位基因存在CCA突变，而另一个等位基因存在A^&lt；677&gt；利用中国仓鼠卵巢细胞，我们阐明了前一种突变和后一种突变通过…降低了UROD活性现在，我们继续努力治疗遗传性铁粒母细胞贫血、杂色性卟啉症和三角洲氨基酮戊酸脱水酶缺乏症。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紊乱的因素(S)，我们启动了红细胞型血红素合成的项目。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，核因子-E2的大亚基和小亚基都是红系特异基因激活所必需的，并且核因子-E2的大亚基受血红素调节。我们还证明了血红素是铁络合酶mRNA所必需的。因此，遗传障碍导致的血红素供应不足不仅会导致铁络合酶mRNA水平的异常，而且还会导致红系血红素生物合成的异常调节。我们还利用UT-7细胞株，研究了巨核细胞成熟和红系分化的机制。这些发现对于更好地了解该途径遗传性障碍患者的血红素代谢具有重要意义。较少

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