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Molecular analysis and gene therapy of the hereditary blood disorders

遗传性血液疾病的分子分析和基因治疗

基本信息

批准号：
63480137
负责人：
FUKUMAKI Yasuyuki
金额：
$ 3.46万
依托单位：
Kyushu University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (B)
财政年份：
1988
资助国家：
日本
起止时间：
1988 至 1989
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-63480137/
关键词：
Thalassemia Mutations Globin gene KMOE cell KU812 cell Transgenic mice Hereditary Methemoglobinemia NADH cytochrome b5 reducatase 多型遺伝子発現遺伝性メトヘモグロビン血症 NADH-cytochrome b5還元酵素

项目摘要

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.

目的了解遗传性血液病的分子机制，开展遗传学诊断。我调查了由奢侈基因珠蛋白基因功能障碍引起的β-地中海贫血和由看家基因NADH细胞色素b5还原酶(B5R)基因缺陷引起的遗传性高铁血红蛋白血症。分析了泰国的71个等位基因、马来西亚的45个等位基因、台湾的22个等位基因和日本的17个等位基因。在泰国、马来西亚、台湾和日本分别发现了9、11、3和8种不同的突变。建立了用聚合酶链式反应方法进行非放射性DNA诊断的体系。对遗传性高铁血红蛋白血症的遗传易感性进行了分析，发现b5R基因第127位氨基酸存在T-C突变。这种突变会导致核苷酸结合域的显著构象变化，从而影响电子传递，导致疾病。为了发展血红蛋白病的基因治疗，我对从慢性粒细胞白血病患者中分离的培养细胞进行了鉴定。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KU812细胞在没有添加诱导剂的情况下持续向红系分化。我用人类胎儿和成人珠蛋白基因并列并向同一方向的DNA片段生成转基因小鼠，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胎儿型基因激活或替换为外源基因的基因治疗。