Several different deletions underlie the molecular basis of a-thalassemia. The most common a-thalassemia determinant in Spain is the rightward deletion (-a".'). To our knowledge, however, no cases of a-thalassemia due to nondeletional mutations have so far been described in this particular Mediterranean area. Here, we report the existence of nondeletional forms of a-thalassemia in ten Spanish families. The a,-globin gene was characterized in ten unrelated patients and their relatives only when the presence of deletional a-thalassemia was ruled out. The a,-globin gene analysis was performed using the polymerase chain reaction (PCR) followed by restriction enzyme analysis or by allelespecific priming. This allowed the identification of a 5-base pair (bp) deletion at the donor site of IVS I (aHPha) in 9 cases and the a, initiation codon mutation (aNcoa) in one case. Although these a2-globin gene mutations are found in other Mediterranean areas, our results demonstrate their presence in the Spanish population and suggest that the a H P h d genotype is probably the most common nondeletional form of a-thalassemia in Spain. o 1996 Wiley-Liss, Inc.

Summary. 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 A3G mutation in patients 2 and 3. The GSH-S gene of patient 1, studied elsewhere, revealed an 808 T3C. 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

Glucose-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.

Clinical and metabolic studies were performed in four members of a Spanish family with partial (50%) 6 phosphogluconate dehydrogenase (6PGD) deficiency. In all cases the activities of 6 phosphogluconolactone (6PGL) and glutathione reductase (GR) were normal, and the molecular characterization performed in the partially purified 6PGD from the propositus showed normal kinetic and electrophoretic patterns. Two females (the propositus and her sister) suffered from a well-compensated chronic nonspherocytic hemolytic anemia (CNSHA) and exhibited decreased RBC glutathione (GSH) stability with increased oxidative susceptibility, defined by enhanced malonyldialdehyde (MDA) generation "in vitro." The other two members of the family (the propositus's mother and brother) were clinically asymptomatic. In the propositus and her sister, RBC metabolism exhibited a markedly abnormal concentration of glycolytic intermediates, mainly characterized by striking increases in fructose 1,6 bisphosphate (50-fold), dihydroxiacetone-phosphate (20-fold) and glyceraldehyde 3-phosphate (tenfold). Although the precise mechanism of the hemolysis in the two patients is unknown, the enhanced oxidative threat observed in their RBCs may interfere in some way with the glycolytic pathway function, leading to a marked increase in certain metabolic intermediates located before the glyceraldehyde 3 phosphate dehydrogenase (GA3PD) step. Since it seems that GA3PD half-life is modulated by fluctuations of the cytosolic redox status, an "in situ" approach was simulated by using permeabilized RBCs. In these conditions, GA3PD activity was significantly lower in the propositus and her sister than in the asymptomatic members of the family and the simultaneous normal control.