Molecular characterization of β-thalassemia intermedia in patients of Italian descent and identification of three novel β-thalassemia mutations

In this study, we have defined by dot-blot analysis with allelic specific oligonucleotide probes or direct sequencing on amplified DNA the β-thalassemia mutations in a large group of patients (23) of Italian descent with thalassemia intermedia. These patients had one parent with either the silent β-thalassemia carrier phenotype or borderline-normal hemoglobin A₂ (HbA₂) levels (2.5% to 3.5%). Nearly all were genetic compounds for a severe β-thalassemia mutation and a β-thalassemia mutation associated with high residual output of β-globin chains (β+ intervening sequence [IVS]-I-nt6, β^-87, β^-101), indicating that inheritance of a mild β-thalassemia allele, even in a single dose, is the most common molecular mechanism producing thalassemia intermedia in the Italian population. In three cases, in whom we failed to define by dot-blot analysis the mutations, we sequenced the β+globin gene and found three novel β-thalassemia mutations, which are certainly very rare because they have been hitherto detected solely in a single patient. These mutations consist of: (1) a T-A substitution at position 2 of IVS-I, in a patient compound heterozygote for this mutation and the -87 promoter mutation; (2) a G-C substitution at position 844 of IVS-II, in a patient heterozygous for this mutation who showed normal sequences at the in trans β-globin gene (The reason for the presence of clinical manifestations in a β-thalassemia heterozygote has not been defined.); and (3) a deletion of one nucleotide (-T) at codon 126, resulting in a frameshift and readthrough of the 5 untranslated region and most likely producing an elongated Hb molecule of 156 amino acid residues, in a patient heterozygous for this mutation with normal β-globin gene sequences at the other locus.