δ-Thalassemia due to a Mutation in an Erythroid-Specific Binding Protein Sequence 3’ to the δ-Globin Gene

We have previously described a family of Northern Sardinian descent in which the propositus was affected by thalassemia major resulting from compound heterozygosity for codon 39 nonsense mutation and the β+IVS II nt 745 mutation and in which all heterozygotes for the β+IVS II nt 745 mutation had normal hemoglobin (Hb) A2levels. To define the reasons for normal HbA2levels in otherwise typical β-thalassemia het-erozygotes, we cloned and sequenced the δ-thalassemia gene in cis to the β+IVS II nt 745 mutation. The sequence analysis showed a single nucleotide substitution (G → A) at position 69 nts (δ+69) downstream to the polyA addition site. Dot blot analysis with an oligonucleotide probe complementary to the δ+69 mutation detected this mutation in several heterozygotes for the β+IVS II nt 745 mutation from the proband's family, but failed to show it either in a group of normal individuals of the same origin or in nonrelated heterozygotes for the β+IVS II nt 745 mutation of the same or different descent from the proband. The δ+69 (G → A) mutation may be responsible for the low δ-globin output from the β+IVS II nt 745 chromosome or could be a silent polymorphism not affecting the function of the δ-globin gene. The normal G at position 69 is part of a sequence very similar to the core DNA (A/T)GATA(A/G) motif (GATA box) that is a binding site for the GATA-1 protein. Gel-retardation assay has shown that a DNA fragment containing the GATA motif with the G → A at position +69 has increased binding affinity for erythroid-specific DNA binding protein(s) as compared with the wild-type sequence. These findings may suggest that the δ+69 mutation is responsible for the deficient function of the in cis δ-globin gene.