Molecular analysis and gene therapy of the hereditary blood disorders

为了解遗传性血液病的分子机制，发展遗传学诊断。我调查了β-地中海贫血（一种由奢侈基因（珠蛋白基因）功能障碍引起的疾病）和遗传性高铁血红蛋白血症（一种由管家基因（NADH细胞色素b5还原酶（b5 R）基因）受损引起的疾病），分析了泰国71个β-地中海贫血等位基因、马来西亚45个等位基因、台湾22个等位基因和日本17个等位基因。在泰国、马来西亚、台湾和日本分别鉴定出9、11、3和8种不同的突变。建立了非放射性PCR DNA诊断体系。分析了遗传性高铁血红蛋白血症的一般形式，并确定了b5 R基因第127位氨基酸的T-C取代。这种突变导致核苷酸结合结构域发生显著的构象变化，影响电子传递，导致疾病。为了发展血红蛋白病的基因治疗，我鉴定了从慢性粒细胞白血病患者中分离的培养细胞。我建立了胎儿和成人珠蛋白基因在KMOE细胞中差异表达的条件。我发现在KU 812细胞中连续的红系分化没有添加诱导剂。我用人胎儿珠蛋白基因和成人珠蛋白基因并置的DNA片段产生转基因小鼠，发现这些基因以组织特异性和发育阶段特异性的方式表达。这些观察结果可能有助于建立基于激活胎儿型基因或用外源基因替代的基因治疗。

In order to understand molecular mechanisms of hereditary blood disorders and develop the genetic diagnosis. I investigated beta-thalassemia as a disorder caused by dysfunction of the luxury gene, the globin gene, and hereditary methemoglobinemia as a disorder caused by impairment of the housekeeping gene, the NADH cytochrome b5 reducatase (b5R) gene.Seventy-one alleles of beta-thalassemia in Thailand, 45 alleles in Malaysia, 22 alleles in Taiwan and 17 alleles in Japan were analyzed. Nine, eleven, three and eight different mutations were identified in Thailand, Malaysia, Taiwan and Japan, respectively. I established the nonradioactive DNA diagnosis system using the PCR method. Generalized from of hereditary methemoglobinemia was analyzed and a T-C substitution at amino acid 127 was identified in the b5R gene. This mutation causes a significant conformation change in the nucleotide binding domain that affects electron transport, resulting in the disorder. For development of gene therapy of hemoglobinopathy, I characterized cultured cells isolated from patients of chronic myelogenous leukemia. I established the condition in which the fetal and adult globin genes were differentially expressed in KMOE cells. I found continuous erythroid differentiation in KU812 Cells without the addition of an inducer. I generated transgenic mice with the DNA fragment in which the human fetal and adult globin genes are juxtaposed and oriented in the same direction and found these genes were expressed in tissue-specific and developmental stage specific manner. These observations could be useful to set up gene therapy based on either activation of the fetal type gene or replacing with the exogenous gene.