We studied the PK-LR gene in 15 unrelated Italian patients with
congenital hemolytic anemia associated with erythrocyte
pyruvate kinase (
PK) deficiency. Fourteen different mutations were detected among 26 mutated alleles identified: a five-
nucleotide (nt) deletion (227 to 231), two splice-site (1269C and IVS3(-2)c), 10 missense (514C, 787T, 823A, 993A, 994A, 1168A, 1456T, 1529A, 1552A, and 1594T) and one
nonsense mutation(s) (721T). Eight of these (deletion 227-231, 1269C, IVS3(-2)c, 514C, 787T, 823A, 1168A, and 1552A) were novel. Moreover, a new polymorphic site was detected in the
3' untranslated region of the
mRNA (C/T,
nucleotide 1738). The deletion 227-231 causes a stop
codon after
amino acid 77, probably resulting in an unstable gene product. Mutations 1269C and IVS3(-2)c lead to an alteration of the 5' and
3' splice-site consensus sequence, respectively;
cDNA analysis failed to reveal any abnormal transcript, suggesting that these mutations generate an unstable
mRNA that is rapidly degraded. Of the five new missense mutations, 823A (Gly275-Arg) and 1168A (Asp390-Asn) involve highly conserved
amino acids, 514C (Glu172-Gln) and 1552A (Arg518-Ser), although found in less conserved regions, affect the balance of the electric charges of the
protein. Mutation 787T (Gly263-Trp) is likely to determine strong modifications 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 found between mutations, biochemical characteristics of the
enzyme, and
clinical course of the disease.