M. Bianchi and M. Magnani Blood Cells, Molecules & Diseases (1995) 21(1) Jan. 15: 2-8 Institute of Biological Chemistry, G. Fornaini, University of Urbino, Italy 1 Reprint requests to: Professor M. Magnani, Instituto Chimica Biologica, "G. Fornaini," Università degli Studi Urbino, Via Saffi 2, 61029-Urbino, ITALY; 2 phone +39-722-305211, fax +39-722-320188 2 Hexokinase Mutations that Produce Nonspherocytic Hemolytic Anemia M. BIANCHI and M. MAGNANI 1 1,2 Institute of Biological Chemistry "G. Fornaini," University of Urbino, Italy Submitted 12/23/94 (communicated by E. Beutler, M.D., 01/04/95) ABSTRACT. Among glycolytic enzyme defects, hexokinase (ATP: D-hexose 6-phosphotransferase, EC 2.7.1.1; HK) deficiency is a very rare disease where the predominant clinical effect is nonspherocytic hemolytic anemia. Here we report the characterization at molecular level of the HK type I cDNA from a patient with hemolytic anemia due to hexokinase deficiency. PCR amplification and sequence of the cDNA revealed the presence of a deletion and of a single nucleotide substitution, both in heterozygous form. In particular, the deletion, 96 bp long, concerns nucleotides 577 to 672 in the HK cDNA sequence and was never found in the cDNAs of 14 unrelated normal subjects. The sequence of the HK allele without deletion showed a single nucleotide substitution from T to C at position 1667 which causes the amino acid change from Leu 529 to Ser. This heterozygous mutation at nt 1667 was confirmed by direct sequencing of the patient genomic DNA, but when DNAs from 10 normal controls were examined by this technique the substitution at nt 1667 was never found. From these results we concluded that the patient is carrying a point mutation at nt 1667 of one HK allele and a 96 nt deletion in the other allele. In normal subjects two differences from the published cDNA sequence were documented. INTRODUCTION HK-Napoli", an increased K for glucose 1,6- Hexokinase(ATP:D-hexose6- different cells of the patient carrying the "HK- phosphotransferase, EC 2.7.1.1; HK) catalyzes Melzo" variant showed that the HK deficiency the phosphorylation of glucose to glucose 6- was expressed not only in erythrocytes but also phosphate by MgATP as phosphate donor and is in platelets, lymphocytes (6) and fibroblasts (8). . considered one of the rate-limiting enzymes of the All these types of cells contain HK type I as the glycolytic pathway (1-3). In mammals, there are predominant glucose phosphorylating enzyme four isozymes of hexokinase named type I, II, III and in particular platelets and erythrocytes share and IV or glucokinase, which vary in their tissue a strict dependence upon glucose utilization for distribution and kinetic properties (4). their physiological functions. In this paper we Hexokinase deficiency is a rare disease where the report the characterization of the hexokinase predominant clinical effect is nonspherocytic variant named "HK-Melzo" by studying the hemolytic anemia. To our knowledge, only 14 enzymatic deficiency in a lymphoblastic cell line cases have been described so far (5), two of established from the lymphocytes of the patient. which have been studied in our laboratory (6, 7): The sequence of the HK coding cDNA was in one case, designated "HK-Melzo", the residual carried out in order to discover enzyme activity was heat-unstable while in the other, named i bisphosphate was found. Further studies on