Erythroenzymopathies: Molecular analysis of pyruvate kinase deficiency

1)本实验室98例非球形溶血性贫血中，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(一家系2例)是匈牙利布达佩斯国家血液学和输血研究所苏珊·霍伦博士推荐的匈牙利男孩，以及一名腺苷酸激酶缺乏症男孩。2)1988年，我们成功地克隆并测定了人M_2型蛋白激酶的全碱基序列，从而确定了其氨基酸序列。3)我们分离了人L和M_2型蛋白激酶基因的3‘非编码序列，构建了L和M_2型蛋白激酶的特异性探针。用这些探针，Northern印迹分析表明，肾脏和肝脏都有与L和M探针杂交的mRNAs。结果表明，白细胞中存在少量的L型PKmRNA和大量的M型PK。小肠、骨骼肌、脑、睾丸和肺的PK mRNAs仅与M探针杂交。我们的探针被认为是有用的检测少量表达的PK同工酶的类型，这是很难用传统的PK聚丙烯酰胺凝胶电泳法检测的。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。人基因组PK由10.5kb左右的长度和CAATbox组成，与大鼠基因组PK非常相似。在人L蛋白的5‘端上游有CAAT box和类LF-Bl序列(GTGTTAATTATGGACCC)以及R型PK的第一外显子。4)虽然我们在1例真纯合性PK缺乏症患者中发现了1个碱基替换，但验证性研究尚未完成。

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.