PK‐LR gene mutations in pyruvate kinase deficient Portuguese patients

Abstract

Summary.In nine unrelated Portuguese patients with pyruvate kinase (PK) deficient anaemia, whose symptoms ranged from a mild chronic haemolytic anaemia to a severe anaemia presenting at birth and requiring multiple transfusions, the PK-LR gene mutations were identified and correlated with their phenotypes. Five different mutations were identified, three of them for the first time: a missense mutation 1670G → C on exon 12 and two 5 0 splice donor site (GT) mutations on intron 8 [IVS8(þ2)T → G] and intron 10 [IVS10(þ1)G → C]. Two previously described missense mutations, 1456C → T and 993C → A, were also found. The genotype/phenotype correlation showed that patients with two missense mutations or with a missense mutation and a splicing mutation had a mild haemolytic anaemia. The three patients with severe anaemia, who were transfusion dependent until splenectomy, were homozygous for the splicing site mutations IVS10(þ1)G → C or IVS8(þ2)T → G.Keywords: pyruvate kinase deficiency, human PK-LR gene mutations, haemolytic anaemia, splice site, Portugal.Pyruvate kinase (PK EC 2.7.1.40) deficiency is the most common enzyme abnormality in the erythrocyte glycolytic pathway causing hereditary nonspherocytic haemolytic anaemia (HNSHA). The PK-deficient anaemia is transmitted as an autosomal recessive disorder with a heterogenous clinical phenotype, ranging from a mild chronic haemolytic anaemia to a severe anaemia presenting at birth and requiring exchange transfusion. Splenectomy is effective in decreasing haemolysis in most patients. Heterozygotes are usually asymptomatic, with a 30-50% decrease in the PK activity levels (Miwa et al, 1979). PK deficiency was first described by Valentine et al (1961) and since then more than 400 cases have been reported, with the biochemical characteristics of the residual enzyme differing markedly between patients. The International Committee for Standardization in Haematology (ICSH) established the standard methods for enzymatic studies; however, the low activity, different stability and the hybrid tetramers formed in the compound heterozygous causes difficulty in characterization of the PK variants (Miwa et al, 1979). A molecular approach, identifying the gene mutations, enables a better characterization of the PK-deficient patients with a more precise genotype/phenotype correlation.It is also useful for diagnosis among transfusion-dependent patients with chronic haemolytic anaemia and prenatal diagnosis.Four PK isoenzymes have been described in mammals, M1, M2, L and R, which are tissue specific and differ in their enzymatic properties (Imamura & Tanaka, 1972). In mammals there are only two different PK genes: the PK-M gene that codes for the isoenzymes M1 (muscle and brain) and M2 (fetal and most adult tissues) using alternative RNA splicing (Noguchi et al, 1986) and the PK-LR gene, that codes for the L-type (liver) and R-type (erythrocyte) isoenzymes using two different tissue-specific promoters (Noguchi et al, 1987). More than 70 mutations have been identified in the coding and flank...

Analysis of malaria associated genetic traits in Cabo Verde, a melting pot of European and sub Saharan settlers

“…These PK-deficient individuals were previously diagnosed by PK enzyme assay and molecular genetic analysis [28,29].…”

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

“…In PKD, 9 nonsense mutations, 10 frameshift mutations, and 12 splice site mutations have been identified [Baronciani et al, 1998;Zarza et al, 1998;Manco et al, 1999], of which only three have been analyzed at the cDNA level [Kanno et al, 1997]. In this study, we describe three such mutations of the human PKLR gene associated with PKD.…”

“…This substantiates the hypothesis of a common ancestor gene for this mutation. Recent data suggest regional differences, with a Northern/Western European and US Caucasian prevalence of the 1529G→A and a Southern European prevalence of the 1456C→T mutation [Lenzner et al, 1997;Zanella et al, 1997;Zarza et al, 1998;Manco et al, 1999]. Interestingly, in patient 6 the 1456T mutation was found both with the 15 or the 16 ATT repeat rather than the 14 ATT repeat usually observed.…”

“…Apparently, in most cases PKD is caused by the production of mutant enzymes with abnormal biochemical characteristics. To date, at the nucleotide level a total of 125 mutations have been reported in the PKLR gene [Baronciani et al, 1998;Zarza et al, 1998;Manco et al, 1999].…”

Pyruvate kinase deficiency

“…mutations resulting in the absence of a functional protein product) displayed intrauterine growth retardation, severe anemia at birth with need of exchange blood transfusion, transfusion dependence and, in rare cases, intrauterine death or death in the first days of life. 11,12,[18][19][20][21] The production and characterization of the recombinant mutant proteins of human R-PK have recently enabled the effects of amino acid replacements on the enzyme molecular properties to be determined and helped to correlate genotype to clinical phenotype.…”