Point mutations in the L-type pyruvate kinase gene of two children with hemolytic anemia caused by pyruvate kinase deficiency [see comments]

Abstract

The molecular alterations responsible for the characteristic enzyme abnormalities in pyruvate kinase (PK) deficiency were investigated in two unrelated children homozygous for PK deficiency. Both variant enzymes were characterized according to the recommendations of the International Committee for Standardization in Haematology. Genomic DNA was specifically amplified by the polymerase chain reaction. Normal and mutant alleles of the L-type PK gene were analyzed by nucleotide sequencing. Heterozygosity of the parents was confirmed by allele- specific oligonucleotide hybridization. In PK Linz a C to T base exchange at position 394 of the L-type PK gene was found. As a result, the 132nd amino acid of the mutant enzyme, arginine (CGC), is replaced by cysteine (TGC). The affected amino acid residue is located within the deduced active site of the protein and the enzyme variant shows strongly altered allosteric properties. PK Beirut shows a C for T substitution at position 1058, changing the 353 amino acid from threonine (ACG) to methionine (ATG). In contrast to PK Linz, this amino acid lies outside the deduced substrate binding site and kinetic parameters of PK Beirut are close to normal. Both enzyme variants show a markedly reduced specific activity and thermolability.

Five unknown mutations in the LR pyruvate kinase gene associated with severe hereditary nonspherocytic haemolytic anaemia in France

“…Results previously published (Baronciani & Beutler, 1993a, b;Kanno et al, 1991Kanno et al, , 1992Kanno et al, , 1993aLenzner et al, 1994;Neubauer et al, 1991), as well as our own, show that most PK deficiencies arise from heterogenous mutations and not from a limited number of ancestral mutations, with the exception of the 509 substitution.…”

“…In 1988 a full-length PK-L cDNA was isolated and its sequence determined (Tani et al, 1988). Several mutations in the PK-LR gene have been described as involved in PK deficiency (Baronciani & Beutler, 1993a, b;Kanno et al, 1991Kanno et al, , 1992Kanno et al, , 1993aLenzner et al, 1994;Neubauer et al, 1991). The spatial structure of cat muscle (Muirhead et al, 1986) and that of rabbit muscle PK (Larsen et al, 1994) were resolved respectively at 2 .…”

Erythrocyte pyruvate kinase deficiency: Relations of residual enzyme activity, altered regulation of defective enzymes and concentrations of high‐energy phosphates with the severity of clinical manifestation3

“…34, a point mutation inside the active centre leads to an enzyme exhibiting purely negative cooperative behaviour, whereas in the case of patient No. 36 a point mutation outside the active site of the enzyme shows that normally occurring positive cooperative PEP binding (38). Interestingly enough, Kanno et al (39) described the same point mutation as found in our patient No.…”

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

“…The cloning of the R‐type cDNA has enabled the study of this enzymopathy at the DNA level ( Tani et al , 1988 ; Kanno et al , 1991 ), and, up to now, about 80 mutations involved in the deficient PK‐LR gene have been described ( Baronciani et al , 1996 ; Baronciani & Beutler, 1993a, b; Kanno et al , 1991 , 1992, 1993a, b, c; Lakomek et al , 1994 ; Lenzner et al , 1994 , 1997; Neubauer et al , 1991 ; Bianchi et al , 1994 , 1995; Rouger et al , 1996 ). This is the first report of a complete mutation analysis of the PK‐LR gene performed in 12 unrelated PKD patients from Spain.…”