Eight novel mutations and consequences on mRNA and protein level in pyruvate kinase-deficient patients with nonspherocytic hemolytic anemia

Pyruvate kinase (PK) deficiency (PKD) is an autosomal recessive disorder wi th the typical manifestation of nonspherocytic hemolytic anemia. We analyze d the mutant enzymes of 10 unrelated patients with PKD, whose symptoms rang ed from a mild, chronic hemolytic anemia to a severe anemia, by sequence an alysis for the presence of alterations in the PKLR gene. In all cases the p atients were shown to be compound heterozygous. Eight novel mutations were identified: 458T-->C (Ile153Thr), 656T-->C (Ile219Thr), 877G-->A (Asp293Asn ), 991G-->A (Asp331Asn), 1055C-->A (Ala352Asp), 1483G-->A (Ala495Thr), 1649 A-->T (Asp550Val), and 183-184ins16bp. This 16 bp duplication produces a fr ameshift and subsequent stop codon resulting in a drastically reduced mRNA level, and probably in an unstable gene product. Surprisingly, the existenc e of M2-type PK could be demonstrated in the patient's red blood cells. The study of different polymorphic sites revealed, with one exception, a stric t linkage of the 1705C, 1738T, IVS5(+51)T, T(10) polymorphisms and the pres ence of 14 ATT repeats in intron ii. Our analyses show the consequences of a distorted structure on enzyme function and we discuss the correlations be tween the mutations identified and the parameters indicative for enzyme fun ction. Hum Mutat 15:261-272, 2000, (C) 2000 Wiley-Liss, Inc.