A. Pujades

Heterogeneity of “Mediterranean type” glucose-6-phosphate dehydrogenase (G6PD) deficiency in Spain and description of two new variants associated with favism

SummaryGlucose-6-phosphate dehydrogenase (G6PD; EC 1.1.1.49) from thirty-six unrelated spanish males was partially purified from blood, and the variants were characterized biochemically and electrophoretically according to the methods recommended by the World Health Organization. Subjects were from multiple geographic regions within Spain, and all suffered from hemolytic anemia, either acute (34 cases) or chronic nonspherocytic (2 cases). Almost all the variants studied presented residual erythrocyte G6PD activity ranging from 0 to 10% of normal, and five different mutants were responsible for the deficient phenotype. Three variants were similar to others previously described: G6PD Mediterranean (11 cases), G6PD Athens-like (3 cases), and G6PD Union (2 cases). The remaining variants were different from the numerous variants already reported and have been considered as new mutants. Provisionally they are called G6PD Betica (19 cases) and G6PD Menorca (1 case).The present study constitutes the first attempt to characterize the deficient G6PD variants found in Spain and supplies new data on the relationship between molecular characteristics of deficient variants and their clinical manifestations. The most important findings can be summarized as follows: (1) The Spanish population is characterized by an important heterogeneity in G6PD deficiency. (2) Although G6PD Mediterranean is very frequent, it presents a relatively high degree of polymorphism. (3) Favism has been observed associated with all kinds of variants described here. (4) G6PD Betica, which is the most frequent variant found in subjects of Southern Spanish origin, has been observed associated with favism in all cases except one.

Hereditary non-spherocytic haemolytic anaemia due to red blood cell glutathione synthetase deficiency in four unrelated patients from Spain: clinical and molecular studies

In four unrelated patients with chronic haemolysis and markedly reduced red blood cell (RBC) glutathione (49·5%, 12·6%, 11·5% and 15% of the normal concentration respectively), a severe glutathione synthetase (GSH‐S, EC 6.3.2.3) deficiency was found. One case exhibited a neonatal haemolytic anaemia associated with oxoprolinuria, but without neurological manifestations. The family study revealed GSH‐S activity in both parents to be around half the normal level, a finding consistent with the presumed autosomal recessive mode of inheritance of this enzymopathy. Two cases exhibited a well‐compensated haemolytic syndrome without anaemia or splenomegaly at steady state. One of these cases was diagnosed after an episode of acute haemolytic anaemia after fava bean ingestion. The remaining patient suffered from moderate to severe chronic non‐spherocytic haemolytic anaemia and splenomegaly, and required occasional blood transfusion for a haemolytic crisis associated with drug ingestion. In this patient, the anaemia was corrected by splenectomy. In addition to GSH‐S, a panel of 16 other RBC enzyme activities was also studied in all the patients. Hexokinase, aldolase, glucose‐6‐phosphate dehydrogenase and pyruvate kinase activities all increased; these increases were to be expected, given the rise in the number of circulating reticulocytes. In two patients, the incubation of RBCs with hydrogen peroxide revealed an enhanced production of malonyldialdehyde. DNA analysis showed a homozygous state for 656 A→G mutation in patients 2 and 3. The GSH‐S gene of patient 1, studied elsewhere, revealed an 808 T→C. The GSH‐S gene of patient 4 was not available for study. The present study demonstrates that GSH‐S deficiency is also present in Spain and further supports the molecular and clinical heterogeneity of this enzymopathy

Haemoglobin Lleida: a new α2-globin variant (12 bp deletion) with mild thalassaemic phenotype

Molecular studies of α‐thalassaemias have revealed defects at different steps in the process of α‐gene expression. It is not surprising, therefore, that in some cases a single mutation or small deletion can result in a structurally abnormal haemoglobin that produces the α‐thalassaemia phenotype. In this report we describe a new unstable α‐globin variant, Hb Lleida, in a Spanish patient with α‐thalassaemia trait. The mutation was detected by single‐strand conformation polymorphism in the third exon of the α2‐globin gene. Direct sequence analysis of the α‐globin gene showed a 12 bp deletion as the only defect of the α2‐ and α1‐globin genes. The propositus was revealed to be a heterozygous carrier, and two alleles were separated by electrophoresis. This deletion causes the loss of four aminoacid residues (from codon 113 to 116) and would be expected to produce an unstable haemoglobin, as a shorter α‐globin chain variant is created with 137 amino acids instead of 141 amino acids present in a normal α‐globin chain. However, no abnormal haemoglobin was found by either isoelectric focusing or haemoglobin electrophoresis. Since the deletion affects an aminoacid residue (114 Pro) involved in α1‐β1‐globin chain contacts, the interaction required for efficient Hb assembly is also compromised. The resulting unstable α‐globin chain is rapidly catabolized and unsuitable for haemoglobin tetramer formation, causing an α‐thalassaemia trait phenotype in the heterozygous patient.

Nondeletional α-thalassemia: First description of αHphα and αNcoα mutations in a Spanish population

Several different deletions underlie the molecular basis of α‐thalassemia. The most common α‐thalassemia determinant in Spain is the rightward deletion (−α3.7). To our knowledge, however, no cases of α‐thalassemia due to nondeletional mutations have so far been described in this particular Mediterranean area. Here, we report the existence of nondeletional forms of α‐thalassemia in ten Spanish families. The α2‐globin gene was characterized in ten unrelated patients and their relatives only when the presence of deletional α‐thalassemia was ruled out. The α2‐globin gene analysis was performed using the polymerase chain reaction (PCR) followed by restriction enzyme analysis or by allele‐specific priming. This allowed the identification of a 5‐base pair (bp) deletion at the donor site of IVS I (αHphα) in 9 cases and the α2 initiation codon mutation (αNcoα) in one case. Although these α2‐globin gene mutations are found in other Mediterranean areas, our results demonstrate their presence in the Spanish population and suggest that the αHphα/αα genotype is probably the most common nondeletional form of α‐thalassemia in Spain.

Two new mutations of the glucose-6-phospate dehydrogenase (G6PD) gene associated with haemolytic anaemia: clinical, biochemical and molecular relationships

In two unrelated Spanish males with glucose‐6‐phosphate dehydrogenase (G6PD) deficiency and haemolytic anaemia, and two different novel point mutations in the G6PD gene, have been identified. A C to T transition at nucleotide 406 resulting in a (136) Arg to Cys substitution and a C to G transition at nucleotide 1155 resulting in a (385) Cys to Trp substitution. These two molecular defects have not been described before and are designated G6PD Valladolid406 C→T and G6PD Madrid1155 C→G .

Erythrocyte Fructose 2,6-Bisphosphate Content in Congenital Hemolytic Anemias

We have investigated the levels of fructose 2,6-bisphosphate and its synthesizing enzyme 6-phosphofructo-2-kinase in red blood cells from different congenital anemias. Fructose 2,6-bisphosphate concentration and 6-phosphofructo-2-kinase activity are markedly influenced by the number of reticulocytes in all the cases studied with the exception of homozygous pyruvate kinase deficiency, where no correlation was observed with the percentage of reticulocytes.

Heterozygous pyruvate kinase deficiency and severe hemolytic anemia in a pregnant woman with concomitant, glucose-6-phosphate dehydrogenase deficiency

SummaryThe aim of this paper is to describe the clinical and hematological characteristics of a 32-year-old woman with concomitant heterozygous pyruvate kinase (PK) and glucose-6-phosphate dehydrogenase (G6PD) deficiencies and severe hemolytic anemia during pregnancy. In 1964, Oski et al. [16] described a family in which a clinically healthy woman was heterozygous for both PK and G6PD deficiencies. To our knowledge, the present case is the first described in which the same condition is associated with hemolysis. A heterozygous condition for both enzymopathies was clearly demonstrated by family study criteria, and all other causes of hemolytic anemia were eliminated. No evidence of genetic relationship between the two disorders was demonstrated. Since late onset of hemolysis in heterozygous PK-deficient women has been observed in association with pregnancy and the molecular characteristics of the concomitant deficient G6PD enzyme were kinetically favorable, partial PK deficiency is suggested as the major cause of hemolysis in this patient.

Relationship between Lymphocyte Size and Enzyme Activities in Two Morphological Variants of B-Chronic Lymphocytic Leukaemia

The activities of the key glycolytic enzymes phosphofructokinase (PFK), pyruvate kinase (PK) and hexokinase in addition to adenosine deaminase, purine nucleoside phosphorylase (PNP) and lactate dehydrogenase (LDH) have been measured in lymphocytes from 39 cases with B-chronic lymphocytic leukaemia (B-CLL). According to the percentage of circulating large non-granular atypical lymphocytes (AL) the B-CLL cases were classified as: typical (less than 10% of AL; 28 cases) and atypical (10-25% AL; 11 cases). In both groups the median lymphocyte volume (MLV) was assessed and correlated with the correspondent enzyme activities. The MLV of B-CLL lymphocytes was significantly (p less than 0.001) decreased (149.9 +/- 19.4 fl) as compared to normal B lymphocytes (175.1 +/- 14.5 fl) and it was significantly (p less than 0.001) lower in typical B-CLL (141.8 +/- 12.2 fl) than in atypical B-CLL (172.0 +/- 17.2 fl). Furthermore, in patients with typical B-CLL, all enzyme activities when expressed as U/10(9) cells were, with the exception of PFK, significantly decreased compared to normal B lymphocytes. However, when the results were expressed as U/ml cells, only PK, PNP and LDH remained significantly low. These findings demonstrate that the determination of MLV in addition to morphology may be a useful tool to distinguish the two previously described morphological B-CLL variants (typical and atypical) and that these two different B-CLL groups are also distinguishable on the basis of three enzyme activities, PK, PNP and LDH which have been shown to be less dependent on cell size than the other enzymes, also studied here.