MOLECULAR CHARACTERIZATION OF PK-LR GENE IN PYRUVATE KINASE-DEFICIENTITALIAN PATIENTS

We studied the PK-LR gene in 15 unrelated Italian patients with congen ital hemolytic anemia associated with erythrocyte pyruvate kinase (PK) deficiency. Fourteen different mutations were detected among 26 mutat ed alleles identified: a five-nucleotide (nt) deletion (227 to 231), t wo splice-site (1269C and IVS3(-2)c), 10 missense (514C, 787T, 823A, 9 93A, 994A, 1168A, 1456T, 1529A, 1552A, and 1594T) and one nonsense mut ation(s) (721T). Eight of these (deletion 227-231, 1269C, IVS3(-2)c, 5 14C, 787T, 823A, 1168A, and 1552A) were novel. Moreover, a new polymor phic site was detected in the 3' untranslated region of the mRNA (C/T, nucleotide 1738). The deletion 227-231 causes a stop codon after amin o acid 77, probably resulting in an unstable gene product. Mutations 1 269C and IVS3(-2)c lead to an alteration of the 5' and 3' splice-site consensus sequence, respectively; cDNA analysis failed to reveal any a bnormal transcript, suggesting that these mutations generate an unstab le mRNA that is rapidly degraded. Of the five new missense mutations, 823A (Gly275-Arg) and 1168A (Asp39O-Asn) involve highly conserved amin o acids, 514C (Glu172-Gln) and 1552A (Arg518-Ser), although found in l ess conserved regions, affect the balance of the electric charges of t he protein. Mutation 787T (Gly263-Trp) is likely to determine strong m odifications in the local structure of the molecule. The most frequent mutation in Italy appears to be 1456T (seven of 30 alleles), followed by 1529A (three of 30) and 994A (three of 30). A correlation was foun d between mutations, biochemical characteristics of the enzyme, and cl inical course of the disease. (C) 1997 by The American Society of Hema tology.