Abstract

PK deficiency is the most common glycolytic enzyme defect associated with chronic non-spherocytic hemolytic anemia. To date about 150 different mutations have been identified in the PK-LR gene. Among them only one large deletion has been described in Gipsy resulting in the loss of exon 11. We report 10 new variants of LR-PK gene in 8 families with pyruvate kinase deficiency. The entire coding region and intronic flanking regions were analyzed by direct sequencing. The results of the molecular analysis are reported in the table:

Pt Origin Hb (g/dL) Tx (n.) PK Activity (IU/gHb) Mutation Aminoacidic substitution 
SD Italy 15.1 10.6 107G / ? Ala36Gly/? 
TT Australia 8.9 nd 409A / del5006bp( IVS3-nt 1431) Ala137Thr / del ex 4-11 
NR Italy >50 5.5 661A /1209A Asp221Asn /Met403Ile 
NA Italy >50 5.3 661A /1209A Asp221Asn /Met403Ile 
SA Italy 12 5.5 1456T/ 1209A Arg486Trp/ Met403Ile 
SC Italy nd nd 5.6 1529A/ 859C Arg510Gln/ Phe287Leu 
CM Italy 9.5 13.6 1456T/ 958A Arg486Trp/ Val320Met 
PS Italy 13.2 1094T /? Lys365Met /? 
VR Italy 10.5 10.7 1706A / ? Arg569Gln /? 
GR Guinea 14.4 7.6 1269A/ IVS9+43c Splice site/? 
Ref. Values  12.2–16  11.1–15.5   
Pt Origin Hb (g/dL) Tx (n.) PK Activity (IU/gHb) Mutation Aminoacidic substitution 
SD Italy 15.1 10.6 107G / ? Ala36Gly/? 
TT Australia 8.9 nd 409A / del5006bp( IVS3-nt 1431) Ala137Thr / del ex 4-11 
NR Italy >50 5.5 661A /1209A Asp221Asn /Met403Ile 
NA Italy >50 5.3 661A /1209A Asp221Asn /Met403Ile 
SA Italy 12 5.5 1456T/ 1209A Arg486Trp/ Met403Ile 
SC Italy nd nd 5.6 1529A/ 859C Arg510Gln/ Phe287Leu 
CM Italy 9.5 13.6 1456T/ 958A Arg486Trp/ Val320Met 
PS Italy 13.2 1094T /? Lys365Met /? 
VR Italy 10.5 10.7 1706A / ? Arg569Gln /? 
GR Guinea 14.4 7.6 1269A/ IVS9+43c Splice site/? 
Ref. Values  12.2–16  11.1–15.5   

Mutations reported in bold are new. By comparing the amino acids sequences among several species (cat M1, chicken M, rat L, yeast and human), we found that mutations 661A, 859C, 958A, 1094T and 1209A involve highly conserved residues. Mutation 1209A when present in association with 661A (cases NA and NR) results in a severe clinical pattern with need of transfusion support, whereas in compound heterozygosity with 1456T (case SA, mother of NA and NR) is associated with a less severe clinical pattern. The variant 1706A was found in a patient carrying the polymorphism 1705C at the homozygous level; the mutation in association with the polymorphism determines the aminoacidic substitution Arg596Gln.

A deletion of 5006 nucleotides extending from intron 3 to the last 3 nucleotides of exon 10 has been found in an Australian baby dead at birth (case TT); the mutation results in a large cDNA deletion encompassing exon 4 and exon11 included. This is the largest abnormality so far detected in LR-PK gene.

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