Maximal gamma-globin expression in the compound heterozygous state for -175G gamma HPFH and beta degree 39 nonsense thalassaemia: a case study.

The -175 (T-->C) G gamma hereditary persistence of fetal haemoglobin is a very rare promoter mutation occurring in Caucasians as well as in African-Americans. Heterozygotes for this non-deletional HPFH show 20% HbF, mostly of G gamma type. We describe here a healthy Sardinian man who coinherited -175 (T-->C) G gamma HPFH with the beta-thalassaemia codon 39 nonsense mutation in trans; he showed 64% HbF, 100% of G gamma type. Although the beta-globin haplotype pattern (II/II) was indicative of the presence of the A gamma T allele on both chromosomes, the A gamma T expression was undetectable by HPLC even in red cell populations separated by age. The proband was, moreover, homozygous for the -4 bp deletion at position -225 to -222 of A gamma promoter which has recently been associated with decreased A gamma T globin expression. These findings suggest that this maximal overexpression of G gamma-globin probably reflects intensified stimulation of the mutated G gamma promoter in this hitherto undescribed genetic condition.

Frequency of delta+ 27-thalassaemia in Sardinians.

To determine the incidence of delta+ 27 thalassaemia in Northern Sardinia we examined blood samples from 750 Sardinian schoolboys by PCR-based molecular analysis. The incidence of delta+ 27 mutation was 1.2% in this study, i.e. twice as high as previously described on the basis of phenotypical studies; the frequency of the beta-thalassaemia is 10.5% and their interaction has been calculated at 0.0003. The majority of delta+ 27 carriers are characterized by a HbA2 level lower than 1.9% and the mean HbA2 level is significantly lower than in normal subjects. All compound heterozygotes for delta+ 27 and beta-thalassaemia show a silent beta-thalassaemic phenotype related to normalization of their HbA2 levels. This study suggests that delta+ 27 thalassaemia should be borne in mind in counselling at-risk couples in which one member has the typical high HbA2 beta-thal trait while the other shows normal or borderline HbA2 level. In these subjects, PCR-based ECO O 109 I digestion of the delta globin gene allows rapid detection of the delta+ 27 mutation.

Fetal hemoglobin expression in compound heterozygotes for -117 (G-->A)A gamma HPFH and beta zero 39 nonsense thalassemia.

The -117(G-->A)A gamma hereditary persistence of fetal hemoglobin (Greek HPFH) and beta zero 39-thal mutations are rather frequent in Sardinia so that their interaction is to be expected. Characterization of eight compound heterozygotes for these defects indicated that HPFH was linked to haplotype VII and beta zero 39-thal to haplotype II. Haplotype II beta zero 39-thal chromosome carries the A gamma T gene which is a useful marker of gamma-gene expression. Since the Hb F level in these compound heterozygotes was significantly higher than in 46 -117 HPFH carriers, the A gamma I, A gamma T, and G gamma globin level was determined. A gamma T was underexpressed while G gamma was significantly increased, which suggest that in -117 A gamma HPFH/beta zero 39-thal healthy subjects the increase in Hb F production is determined only by the -117 mutated A gamma gene and the adjacent G gamma gene.

A 12-year preventive program for beta-thalassemia in Northern Sardinia.

From 1980 to 1991, 6.3% of the adult population of the province of Sassari, Northern Sardinia, underwent voluntary beta-thalassemia screening. Of the 28,000 subjects examined, 15.7% proved to be heterozygotes for beta-thalassemia. In addition, the screening of 7500 students in 26 villages in Sassari province fixed the frequency of beta-thalassemia in this part of Sardinia at 10.4%. Of the 539 couples at risk to be expected from this figure, the screening detected 43% (234). The data suggest that inductive screening played a major role in the efficiency of this preventive beta-thalassemia program. Follow up of 221 pregnancies found to be at risk for homozygous beta-thalassemia and referred to the Antenatal Diagnosis Service, Cagliari, Southern Sardinia, showed that antenatal diagnosis was carried out in 80% of them. The overall percentage of couples refusing antenatal diagnosis was 10.8%, but over the years the acceptance rate for the procedure increased from 87% to 96%. Atypical hematological findings in 1.5% of 468 members of the couples at risk required globin chain synthesis and molecular analyses to define the precise beta-thalassemia genotype. Heterogeneous "mild" beta-thalassemia mutations as well as coexisting delta-thalassemia were found in silent type I and type II beta-thalassemia carriers which, without chain synthesis and DNA investigations, would have escaped detection.

Mild beta+(-87)-thalassemia CACCC box mutation is associated with elevated fetal hemoglobin expression in cis.

The beta zero-thalassemia codon 39 nonsense mutation predominant in Sardinia is severe, and homozygotes are transfusion dependent. Two-thirds of beta zero 39 alleles are linked to A gamma T (haplotype II). One-fourth are linked to A gamma I (haplotypes I and IX), as is the mild beta +-thalassemia -87 C-->G mutation (haplotype VIII). beta +/beta zero-Thalassemia VIII/II compound heterozygotes have significantly higher A gamma I:A gamma T (23:7) than beta zero-thalassemia I/II (24:20) or IX/II (16:17) cases. This suggests that the beta + -87 mutation is associated with elevated gamma expression in cis, which may contribute to the lack of transfusion-dependence in beta +/beta zero cases.

A novel Mediterranean "delta beta-thalassemia" determinant containing the delta (+) 27 and beta (0) 39 point mutations in cis.

The term delta beta-thalassemia with normal HbF has been recently proposed to define heterogenous delta and beta globin gene molecular defects involving the same chromosome in cis. Here, we describe a Sardinian family in which three members showing microcytosis, border-line HbA2 levels and normal HbF proved to be heterozygotes for delta(+) 27 and beta(0) 39 point mutations in cis by allele specific oligonucleotide hybridization as well as by ECO 0 109 I endonuclease digestion and electrophoresis. As some of these beta-thalassemia carriers shows normal HbA2 levels, knowledge of the molecular basis of this novel delta beta-thalassemia silent phenotype would be useful in thalassemia screening and genetic counselling.

[Current views of thalassemia intermedia]. / Vedute attuali sulla talassemia intermedia.

Recently the molecular bases of thalassemia intermedia have been elucidated in several populations. In general this attenuated, non-transfusion dependent form of homozygous beta-thalassemia is mainly determined by a) the co-inheritance of deletion alpha-thalassemia; b) the presence of the so-called mild beta-thalassemia mutations; and more rarely, c) the inheritance of genetic conditions able to enhance the gamma-globin chain expression in adult life. Although there are several complex genetic and acquired interactions involved in the wide clinical heterogeneity of thalassemia intermedia, data in Italians indicate a definite genotype-phenotype relationship in conditions such as the co-inheritance of at least two alpha-thalassemia genes in severe and mild homozygous beta-thalassemia; the molecular homozygosity or double heterozygosity for the -87, -101 and IVS1(nt6) beta(+)-thalassemia mutations; and the coexistence of structural gamma-globin gene defects, i.e. Sicilian and Sardinian delta beta-thalassemias, deletional and non-deletional hereditary persistence of fetal hemoglobin and the polymorphism for the -158 XmnI G gamma restriction site. Thalassemia intermedia resulting from the inheritance in heterozygous beta-thalassemia of triple alpha-globin gene complex or the presence of dominant beta-thalassemia is also described and the role of these new informations in genetic counselling is discussed.

Haematological phenotypes in a family with triplicated alpha-globin gene, beta zero 39 and delta+27 thalassaemia mutations.

In this paper we report an unusual Sardinian family, in which the heterozygosity for beta zero 39-thalassaemia and for triple alpha-globin gene complex have been found in two members: the former showing a high HbA2 mild thalassaemia intermedia syndrome, the latter, her daughter, showing a normal HbA2 thalassaemia trait. Molecular analysis revealed the daughter to also be a carrier of a delta+27-thalassaemia point mutation, which in trans to the beta zero 39 defect invariably normalizes the HbA2 levels.

[Differential diagnosis of thalassemia syndromes]. / Diagnosi differenziale delle sindromi talassemiche.

In Sardinia, as in other areas with a high incidence of thalassemia syndromes, a prevention program based on the detection of healthy carriers through mass screening and on prenatal diagnosis in the at-risk couples has been in course for several years. The commonly adopted beta-thalassemia flow-chart consists of a first operative step involving simple and widely standardized tests: the estimation of red cell indices, the measurement of Hb A2 and Hb electrophoresis. These investigations permit the identification of the majority of the at-risk couples for beta-thalassemia. However, the not infrequent evidence of Hb A2 borderline levels, with or without microcytosis, isolated microcytosis or Hb F increased values, causes some problems in differential diagnosis, because these findings can indicate the presence of silent beta-thalassemic traits or other beta-thalassemic like states. A diagnostic definition of these unusual hematological phenotypes is particularly important for the identification of eventual at-risk couples. In this paper we report our data concerning the voluntary screening for beta-thalassemia carried out in North Sardinia. The operative flow-chart is shown. In a population with a high incidence of phenotypically heterogeneous thalassemic syndromes, such as that of Sardinia, differential diagnosis of thalassemic traits can require molecular studies. This molecular characterization, which could be carried out in specialized reference centers, is today absolutely necessary both for exact identification of at-risk couples and eventual prenatal diagnosis.

Homozygous beta zero-39 mutation with thalassemia intermedia in northern Sardinia: clinical, hematological and molecular analysis.

In this study, we investigated the clinical and hematological features and carried out alpha- and beta-globin gene analyses in 11 Sardinian adult beta zero-thalassemia homozygotes from Northern Sardinia who were not transfusion-dependent. Oligonucleotide analysis revealed in nine out of 11 patients the nonsense mutation at codon 39, which was associated either with haplotype II or IX (14/16 and 2/16 chromosomes, respectively). Haplotype II was linked to the A gamma T mutation. The G gamma globin level ranged from 50 to 70%. Four out of nine patients (44%) were heterozygous and 3/9 (33%) homozygous for the rightward deletional type of alpha-thalassemia; two (22%) had the normal alpha-gene complement. Patients who were alpha-thalassemia homozygotes (-alpha/-alpha) showed a more balanced globin chain synthesis ratio. This study confirms that alpha-thalassemia may ameliorate the clinical picture of homozygous beta zero-thalassemia.