Erythroenzymopathies: Molecular analysis of pyruvate kinase deficiency

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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.