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Erythroenzymopathies: Molecular analysis of pyruvate kinase deficiency

红酶病：丙酮酸激酶缺乏症的分子分析

基本信息

批准号：
63480281
负责人：
MIWA Shiro
金额：
$ 0.96万
依托单位：
Okinaka Memorial Institute for Medical Research
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

项目摘要

1) Out of 98 cases with nonspherocytic hemolytic anemia referred to our laboratory, 4 cases with pyruvate (PK) deficiency, 4 with glucose 6-phosphate dehydrogenase (G6PD) deficiency, 2 with triosephosphate isomerase (TPI) deficiency, and 1 with adenylate kinase deficiency were found. TPI deficiency cases (2 cases in one family) are Hungarian boys referred from Dr. Susan Hollan, National Institute of Haematology and Blood Transfusion, Budapest, Hungary, and so was one boy with adenylate kinase deficiency..2) In 1988, we have succeeded to clone and determine complete base sequence, and hence, amino acid sequence of human M_2- type PK.3) We isolated 3'-noncoding sequences of both human L- and M_2- type PK cDNA to construct L-type and M-type PK specific probes. With these probes, Northern blot analysis showed that both kidney and liver had mRNAs that hybridized with both the L and M probts. It was clarified that leukocytes had small amount of L-type PK mRNA as well as large amount of M-type PK. Small intestine, skeletal muscle, brain, testis and.lung PK mRNAs hybridized only with the M probe. Our probes are considered useful for the detection of the types of PK isozymes expressed in small amounts, which are very difficult to detect using the .conventional PK polyacrylamide gel electrophoretic method.4).In 1989, we cloned genomic PK DNA from leukocyte DNA and analyzed it. Human genomic PK was composed of about 10.5 kilo-base pairs in length and CAATbox and was found to be quite similar to that of rat genomic PK. There are CAAT box and LF-Bl-like sequences (GTGTTAATTATGGACCC) as well as presumable first exon of R(red cell)-type PK on upstream of 5' end of human L-type PK.4) Although we found one base substitution in one case with true homozygous PK deficiency, confirmatory study is not completed as yet.

1）本实验室转诊的98例非球细胞性溶血性贫血患者中，丙酮酸（PK）缺乏症4例，葡萄糖6-磷酸脱氢酶（G6PD）缺乏症4例，磷酸丙糖异构酶（TPI）缺乏症2例，腺苷酸激酶缺乏症1例。 TPI缺乏症病例（一个家庭2例）是由匈牙利布达佩斯国家血液学和输血研究所Susan Hollan博士转介的匈牙利男孩，其中一名男孩患有腺苷酸激酶缺乏症。2）1988年，我们成功克隆并确定了完整的碱基序列，从而确定了人M_2型PK的氨基酸序列。3）我们分离了3'-非编码序列人 L 型和 M_2 型 PK cDNA 构建 L 型和 M 型 PK 特异性探针。使用这些探针，Northern 印迹分析显示肾脏和肝脏都有与 L 和 M 探针杂交的 mRNA。明确白细胞中含有少量的L型PK mRNA以及大量的M型PK mRNA。小肠、骨骼肌、脑、睾丸和肺 PK mRNA 仅与 M 探针杂交。我们的探针被认为可用于检测少量表达的PK同工酶类型，而使用传统的PK聚丙烯酰胺凝胶电泳方法很难检测到这些类型。4)。1989年，我们从白细胞DNA中克隆了基因组PK DNA并对其进行了分析。人类基因组 PK 由长度约为 10.5 kb 的碱基对和 CAATbox 组成，与大鼠基因组 PK 非常相似。在人L型PK的5'端上游存在CAAT盒和LF-Bl样序列（GTGTTAATTATGGACCC）以及推测的R（红细胞）型PK的第一个外显子。4）虽然我们在一个具有真正纯合性PK缺陷的病例中发现了一个碱基替换，但验证研究尚未完成。