Search

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.

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.

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 survey of hemoglobinopathies in northern Sardinia revealed a high frequency (0.3%) of carriers of a hematologic condition characterized by increased expression of fetal hemoglobin during adult life (hereditary persistence of fetal hemoglobin or HPFH). In spite of a normal hematologic phenotype, the heterozygous carriers for this condition display about 12% HbF, almost exclusively of the A gamma type; compound heterozygotes with beta-thalassemia have 20%-26% HbF and run a very mild clinical course. The sequence analysis of the cloned A gamma gene linked to the HPFH determinant revealed the presence of a G----A substitution at position -117 of the A gamma-globin gene promoter; the same mutation occurs also in Greek HPFH, although associated with different restriction polymorphisms. Another hereditary condition characterized by increased HbF (alpha 2 A gamma 2) level and a mild thalassemia phenotype in Sardinia is associated with the -196C----T substitution in the A gamma-globin gene promoter (Sardinian delta beta-thalassemia). Population studies using oligonucleotides complementary both to the -117 G----A and -196C----T mutations and the corresponding normal sequences confirm the presence of these mutations only in HPFH and delta beta-thalassemia chromosomes and exclude these changes being common DNA polymorphisms.

12 thalassaemic patients from Northern Sardinia showing the beta + phenotype were examined by isoelectric focusing and high-performance liquid chromatography techniques for the determination of the variant A gamma T globin chain of the foetal haemoglobin. Two patients (16.7%) were homozygotes for the A gamma T gene variant, 2 (16.7%) were heterozygotes and 8 (66.7%) were homozygotes for the normal A gamma I allele. The A gamma T gene frequency was 0.183, much lower than the observed 0.823 in beta zero homozygosity. These data suggest the presence of at least 2 beta +-thalassaemic chromosomes in Sardinians, one associated with the variant A gamma T allele and one associated with the normal A gamma I. The latter is prevalent among adult patients showing the intermediate form of the thalassaemic disease, which is not transfusion-dependent.

G gamma:A gamma composition of 54 newborns from Sardinia was determined by electrophoresis on polyacrylamide gels containing urea and Triton X-100. G gamma was present at a concentration of 67% with bimodal distribution (with mean values at 65 and 70% respectively). Concentration higher than 76% was not observed. When Hb F in peripheral blood was below 70%, G gamma:A gamma ratio correlates with Hb F%.

A survey of hemoglobinopathies in Northern Sardinia allowed the identification of two subjects heterozygous for a new type of G gamma hereditary persistence of fetal hemoglobin (HPFH). The G gamma-globin gene from the HPFH chromosome shows the presence of a T----C substitution 175 nucleotides upstream of the CAP site, adding a new example of single-point mutations occurring in the promoter region of the gamma-globin genes and linked to HPFH phenotypes. In this case the mutation affects the 3' end nucleotide of a conserved octamer sequence known to be present in other regulatory elements of several genes.

During a study of Sardinian families with hereditary persistence of fetal hemoglobin (HPFH), two unrelated subjects with unusually elevated Hb F levels were identified. By selective amplification of the A gamma gene promoter and hybridization to synthetic oligonucleotides, we demonstrate that these subjects are homozygous for the -117A gamma G----A substitution that is responsible for a form of nondeletional HPFH. The hemoglobin synthetic pattern of these patients is discussed.