Molecular characterization of the PK‐LR gene in sixteen pyruvate kinase‐deficient patients

Abstract

We studied the PK‐LR gene in 16 unrelated patients with congenital haemolytic anaemia associated with erythrocyte pyruvate kinase deficiency. Fifteen different mutations were detected among the 28 mutated alleles identified: two deletions (del 1010G, del 1042–1044); one four nucleotide duplication (nt 1515–1518, GGTC); one splice site [IVS6(−2)t]; nine missense (991A, 1003A, 1151T, 1160G, 1181T, 1181A, 1456T, 1483A, 1529A); and two nonsense (721T, 1675T) mutations. Eight of them [del 1010G, del 1042–1044, dupl 1515–1518, IVS6(−2)t, 1003A, 1160G, 1181T, 1181A] were novel. The deletion 1042–1044 causes the loss of Lys 348. Deletion 1010G and duplication 1515–1518 determine a frameshift and the creation of a stop codon at nucleotides 1019 and 1554 respectively. Mutation IVS6(−2)t leads to an alteration of the 5′ and 3′ splice site consensus sequence; the cDNA analysis shows a 67‐bp deletion in the first part of exon 11 (del 1437–1503). All the four new missense mutations involve highly conserved amino acids. The most frequent mutation in Italy would appear to be 1456T. Correlation was made between mutations, biochemical characteristics of the enzyme and clinical course of the disease.

Fifteen novel mutations inPKLRassociated with pyruvate kinase (PK) deficiency: Structural implications of amino acid substitutions in PK

“…Amino acid substitutions in the B domain are rare and only seven mutations have been described thus far Kugler et al, 2000;Neubauer et al, 1991;Pissard et al, 2006;Zanella et al, 1997]. The side chain of Arg163 points into the cleft between the A and B domains where the active site is located.…”

Human erythrocyte pyruvate kinase: characterization of the recombinant enzyme and a mutant form (R510Q) causing nonspherocytic hemolytic anemia

“…23 Two patients carrying the R510Q mutation have been followed and characterized by us (see Zanella et al 19,33 ). A summary of the relevant biochemical data concerning RPK of these patients is reported in Table 3.…”

“…In addition, contamination with the isozyme from leukocytes and presence of hybrid enzyme from heterozygous or compound heterozygous patients also hamper the enzymatic characterization. Although detection of mutations at the gene level has been advancing rapidly, 17,19,21,22 the lack of protein characterization makes it difficult to predict the phenotypic consequence of specific mutations. To analyze the mutations at the protein level and eventually to elucidate the protein crystal structure, we cloned the full-length human R-type PK complementary DNA (cDNA) and expressed, purified, and characterized the recombinant enzyme.…”

“…Nonsense mutations, deletions, and alterations of the splicing sites have also been reported. [17][18][19] Patients carrying these mutations have been primarily found in Japan, Europe, and the United States, but increasing evidence indicates that the disease is spread worldwide. Clinical diagnosis of PK deficiency relies on the demonstration of quantitative and/or qualitative enzyme abnormalities in the cells.…”

AG-348 enhances pyruvate kinase activity in red blood cells from patients with pyruvate kinase deficiency

“…9 Hundreds of mutations, most of which are single nucleotide missense mutations, have been described, which can have deleterious effects on PK-R catalytic activity, protein stability, or protein expression. 6 For example, the R486W mutation, present in ;25% of patients with PK deficiency in southern Europe, 10 is thought to affect the catalytic efficiency of the enzyme, and the R510Q mutation, found in ;40% of northern European patients with PK deficiency, significantly destabilizes the active tetrameric species. 11 PK deficiency is an autosomal recessive disease, and patients are either homozygous, or more commonly, compound heterozygous for 2 mutant PK-R (mtPK-R) alleles.…”