[The molecular basis of dominantly inherited beta-thalassemia]. / Base molecular de uma beta-talassémia de transmissão dominante.

In this study, we sought to clarity the molecular basis of a dominant inherited beta-thalassemia, found in heterozygosity in a northern Portuguese family with thalassemia intermedia. We characterized: i) the alpha-globin gene cluster structure; ii) the beta-globin gene cluster haplotype; and iii) the beta-thalassemia mutation. The alpha-globin gene cluster was structurally normal. The G-->T transversion at codon 121 of the beta-globin gene was found in the affected individuals in association with Orkin's haplotype V. This is an uncommon, though ubiquitous, mutation. Which has also been found, in association with different haplotypes, in several distant populations. It has only been observed in this three-generation family, in the Portuguese population. We suggest a mechanism to explain the genotype/phenotype correlation.

Combined effect of two different polymorphic sequences within the beta globin gene cluster on the level of HbF.

Beta thalassemia and Hb Lepore heterozygotes included in this study exhibit fetal hemoglobin levels varying from trace quantities to 14% (1.74 g/dl) of total hemoglobin in the adult. In this work, we have examined the correlation of DNA sequence polymorphisms with the observed HbF level. The analysis of polymorphic markers within the beta globin cluster in 39 individuals heterozygous for beta thalassemia or Hb Lepore confirms the previous findings for homozygous beta thalassemia: the presence of both an (AT)9 T5 sequence configuration at position -540 of the beta globin gene and a (C --> T) variation at -158 of the Ggamma globin gene is associated with elevated expression of HbF. However, at least one defective beta globin gene is required to reveal this association. The best evidence is from the study of individuals heterozygous for Hb Lepore with various levels of HbF. In these individuals it was possible to explore the effect of a single (AT)x Ty motif (the other being absent from the rearranged Lepore chromosome) on HbF expression. The presence of the (AT)9 T5 configuration increases HbF level from a median of 0.515 g/dl observed in (AT)7 T7 subjects, to 1.39 g/dl. We confirm the existence of linkage disequilibrium between the (C --> T) variation at -158 of Ggamma gene and the (TG)13 configuration at the second intervening sequence (IVS-2) of Agamma gene and identify two new polymorphisms in this region: (TG)7 (CG)5 (TG)8 linked to haplotype V and (TG)8 (CG)5 (TG)10 linked to haplotype II. This study suggests that two distinct regions of the beta cluster, whether in cis or in trans to each other, can interact to enhance HbF expression when a beta thalassemic determinant is present in heterozigosity.

[Neonatal screening of hemoglobinopathies in a population residing in Portugal]. / Rastreio neonatal de hemoglobinopatias numa população residente em Portugal.

The primary objective of newborn screening of hemoglobinopathies is the early identification of infants with sickle cell disease, as they are at increased clinical risk. Other goals include the identification of other types of clinically significant hemoglobinopathies and the detection of heterozygous carriers followed by the screening and counselling of family members. We performed a pilot study for the neonatal screening of hemoglobinopathies in 400 samples of cord blood taken from a maternity in Lisbon. We did not find any newborn with sickle cell disease. Six samples were from sickle cell heterozygotes, the respective families were studied and informed. We looked for the presence of alpha-thalassemia at birth in 100 consecutive samples of cord blood, by the presence of Hb Bart's, abnormal red blood cell indices and alpha-globin genotype. The results show an incidence of 10% of alpha-thalassemia (-alpha) carriers and 4% of triple alpha-globin gene carriers. The authors discuss the feasibility of neonatal screening of hemoglobinopathies in a Portuguese-speaking population consisting of a low prevalence of Hb S trait autoclonous group and a high prevalence immigrant minority.

Beta-thalassaemia unlinked to the beta-globin gene interacts with sickle-cell trait in a Portuguese family.

An autosomally transmitted hypochromic microcytic mild anaemia with elevated haemoglobin (Hb) A2 and globin chain imbalance has been observed in a three-generation family of Portuguese origin. Extensive DNA analysis of the beta-globin gene cluster, including the complete sequencing of the beta-globin gene and flanking regions, failed to reveal any genetic alteration. The co-segregation of sickle-cell trait in this family enabled us to postulate a defective erythroid trans-acting factor was playing a role in the down-regulation of both beta A- and beta S-globin genes. Among the transcription factors that could possibly have caused the reported phenotype, NF-E2 is unlikely to be implicated, whereas Nrf1 and Nrf2 cannot be ruled out. Thus, this family carries a novel beta-thalassaemia autosomal determinant unlinked to the beta-globin gene. This observation reinforces the notion of the haemoglobinopathies as single gene disorders under polygenic regulation.

Molecular basis of alpha-thalassemia in Portugal.

We have estimated the incidence and molecular basis of alpha-thalassemia in a Portuguese population, mostly from the Greater Lisbon area. In a group of 100 consecutive cord blood samples, the gene frequency of the rightward deletion (-alpha 3.7) was 0.035, and the leftward deletion (-alpha 4.2) was 0.015. In this group, we have also found four heterozygotes for the triple alpha-globin gene rearrangement (alpha alpha alpha anti 3.7. gene frequency 0.020). We have characterized the subtypes of -alpha 3.7 and alpha alpha alpha anti 3.7 rearrangements. On the whole, these results give an incidence of 10% for deletional alpha-thalassemia carriers in the studied Portuguese population. In a group of 342 subjects presenting beta-thalassemia, or Hb S trait, beta-thalassemia major sickle cell disease or low red blood cell indices, the -alpha 3.7, -alpha 4.2, -SEA, -MED, (alpha alpha)MM, and alpha alpha alpha anti 3.7 haplotypes were found in different combinations. Only one nondeletional alpha-thalassemia determinant (a 5 nucleotide deletion in the alpha 2-globin gene in the second intervening sequence donor site) was detected, which might suggest a low incidence of these defects in the Portuguese population.

Importation route of the sickle cell trait into Portugal: contribution of molecular epidemiology.

To elucidate the origin and spread of the sickle cell trait into the Portuguese population, we examined nine polymorphic DNA markers within the beta globin gene cluster defining the haplotype. The population sample included 64 sickle-cell-gene-bearing individuals from defined Portuguese-speaking white, black, and Asian Indian populations. The nature and geographic distribution of the different beta S haplotypes in Portugal suggest that the sickle cell trait has been imported twice: between the eighth and the thirteenth centuries from the Mediterranean basin (in association with the Benin haplotype) and after the fifteenth century from black Africa over an Atlantic route (Senegal and Bantu haplotypes).

Hemoglobin Redondo [beta 92(F8) His----Asn]: an unstable hemoglobin variant associated with heme loss which occurs in two forms.

Hb Redondo [beta 92(F8) His----Asn] illustrates how post-translational structural modifications may modify the phenotypic expression of an unstable hemoglobin. This variant was found in a Portuguese patient suffering from a chronic hemolytic anemia. The electrophoretic pattern demonstrated that it occurred in two forms, both being semi-hemoglobins: the fastest one migrating like HbS and the other like HbA2; after a few days of storage at 4 degrees C the intensity of the slowest Hb fraction decreased while that of the other increased proportionally. In both cases, the RP-HPLC analysis of the tryptic digest of the aminoethylated beta chains demonstrated the presence of an abnormal beta T10 peptide carrying a His----Asx substitution. Microsequence study of these two peptides demonstrated that the slowest abnormal Hb fraction had a beta 92 His----Asn substitution and the fastest a His----Asp at the same site. All these results suggest that the beta 92 His----Asn variant loses readily its heme group and that a deamidation occurs rapidly in vitro, yielding a beta 92 Asp semi-hemoglobin. The oxygen affinity of the patient's red blood cells was increased, leading to a stimulation of erythropoiesis and to a macrocytic hemolytic disease.