Hereditary xerocytosis (HX) is a rare haemolytic disease due to dehydrated red blood cells (RBCs). A unique feature of this syndrome is that affected members often show normal or near normal haemoglobin levels despite clinical and laboratory evidence of mild to moderate haemolysis. The diagnostic clue is the association of markedly increased RBC Na+ + K+ fluxes with low total cation (Na+ + K+) content. 11 patients of six unrelated families of Spanish origin with HX have been studied from clinical, genetical and biological points of view. In addition, we have investigated the sensitivity of RBC membrane to heat at three different incubation times (15, 30 and 60 min) and two different temperature values (46 degrees C and 49 degrees C). Under these conditions control RBCs (50 normal subjects) exhibited at 49 degrees C and 30 min a maximum of 30% fragmented RBCs. This value increased to 80% after 60 min of incubation. In contrast, patients with HX showed significantly lower percentages of fragmented RBCs at both 30 and 60 min of incubation (maximum 10% and 30%, respectively). In an attempt to determine if increased heat stability was unique to HX RBCs, several other congenital membranopathies with haemolytic anaemia were also studied. The degree of fragmentation, except in one case of HPP (which was strongly increased), did not differ from the control group. Electrophoretic studies of membrane proteins performed in RBCs of all the patients with HX did not explain any qualitative nor quantitative abnormality. In addition to its physiopathological interest, study of RBC heat stability, together with other haematological parameters (increased MCHC and decreased RBC osmotic fragility), may be useful for HX diagnosis, especially in laboratories which are not equipped to evaluate RBC membrane permeability.

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.

Molecular studies of alpha-thalassaemias have revealed defects at different steps in the process of alpha-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 alpha-thalassaemia phenotype. In this report we describe a new unstable alpha-globin variant, Hb Lleida, in a Spanish patient with alpha-thalassaemia trait. The mutation was detected by single-strand conformation polymorphism in the third exon of the alpha 2-globin gene. Direct sequence analysis of the alpha-globin gene showed a 12 bp deletion as the only defect of the alpha 2- and alpha 1-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 alpha-globin chain variant is created with 137 amino acids instead of 141 amino acids present in a normal alpha-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 alpha 1-beta 1-globin chain contacts, the interaction required for efficient Hb assembly is also compromised. The resulting unstable alpha-globin chain is rapidly catabolized and unsuitable for haemoglobin tetramer formation, causing an alpha-thalassaemia trait phenotype in the heterozygous patient.

Summary.A frameshift mutation in the a 1 -globin gene, responsible for a clinically mild a-thalassaemia phenotype, has been characterized in a Spanish woman. After excluding the most common forms of a-thalassaemia found in the Mediterranean area, both a-globin genes (a 1 and a 2 ) were amplified and analysed selectively by non-radioactive singlestrand conformation polymorphism (SSCP). An abnormal SSCP mobility was present in the second exon of the a 1 -globin gene and direct sequence analysis revealed a 13 bp deletion (between codons 51 and 55) affecting a single allele.The consequence of this mutation is a reading frameshift leading to a novel amino acid coding sequence from codons 51-61 and a premature stop signal at new position 62, which results in a net reduction of the affected a-globin chain output. The presence of this new mutation was confirmed by restriction enzyme analysis of the specific PCR product.

A new unstable alpha-globin chain associated with alpha-thalassemia phenotype has been found in a Spanish patient. Molecular analysis of the alpha-globin gene complex using PCR and non-radioactive single-strand conformation analysis, allowed to identify a new mutation in the second exon of the alpha-globin gene. Direct sequencing of the abnormal fragment revealed a 3 bp deletion, which led to the loss of a single codon corresponding to a Lys (K) residue at position 60 or 61 DK60 or DK61. Theoretical structural analysis, performed by computational methods, indicated that the loss of an amino acid residue at this position disturbed the contact region between the B and E-helices, affecting the overall stability of the molecule. Therefore, the DK60 and DK61 results in a structurally abnormal alpha-globin chain, not previously described, named Hb Clinic, which leads to the alpha-thalassemia phenotype in the heterozygote patient. No abnormal hemoglobin was detected by standard electrophoretic procedures, suggesting that this alpha-globin chain variant is so unstable that it may be catabolized immediately after its synthesis. This mutation was confirmed by PCR using an allele specific primer.