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The increase of Hb A(2) (α2δ2) beyond the upper limit [2.0-2.2/3.3-3.4% of the total hemoglobin (Hb)] is an invaluable tool in the hematological screening of β-thalassemia (β-thal) carriers. Factors decreasing Hb A(2) percentages can hinder correct diagnosis. In order to analyze the genotype-phenotype relationship, we characterized δ-, β- and α-globin genotypes in 190 families where the probands had Hb A(2) values of ≤2.0% or were β-thal heterozygotes with normal Hb A(2) levels. Hb A(2) was measured with cation exchange high performance liquid chromatography (HPLC). Mutations were detected with allele-specific methods or DNA sequencing; two multiplex-ARMS (amplification refractory mutation system) assays were set up. The molecular basis underlying the decrease in Hb A(2) was extremely heterogeneous. Nineteen δ-globin alleles (Hb A(2)-S.N. Garganico was new) were detected; their interaction with α- or β-globin alleles (10 and eight, respectively) led us to observe 52 genotypes in 261 carriers. The type of δ-globin mutations, the relative genotypes, the interaction with α(0)-thal traits, are the most important factors in decreasing the Hb A(2) percentage. These results are extremely useful in addressing the molecular diagnosis of hemoglobinopathies and thalassemias.

We report a new unstable variant identified in three carriers of a family from East Sicily; it was named Hb Bronte after the place from which the family originated. DNA sequencing from nucleotides -181 to +894 (alpha1) and to +884 (alpha2) revealed a GTG-->GGG substitution at codon 93 of the alpha2-globin gene. The MCV and MCH values were at the lower end of the normal range in the carriers. On cation exchange high performance liquid chromatography (HPLC), the Hb A2 level was apparently increased to around 6%, and a small abnormal peak (0.3-0.4%) was detected after Hb A2. Two abnormal bands were detected by cellulose acetate electrophoresis: a major band (about 3-4%) migrated between Hb A and Hb F; a minor band (<1%) migrated between Hb A2 and carbonic anhydrase. Normal values of Hb A2 were detected by DEAE microchromatography. On reversed phase HPLC the variant chain was not detected, and most likely it was eluted with the alpha chain peak. The isopropanol stability test was very slightly positive in the carriers. Hemolytic symptoms were absent with the exception of indirect bilirubin, which was at high borderline in 2/3 carriers. In biosynthesis in vitro, the specific activity of the alpha chains was much higher than that of the beta-globin chains, and the alpha/beta biosynthetic ratio in the mother and proband was of the beta-thalassemia (thal) type (2.24 and 2.54, respectively). Time course experiments showed that the increase of the 3H-specific activity of the peak containing normal and variant alpha chains was not linear and was much higher than that of beta chains; moreover, the alpha/beta biosynthetic ratio varied during the 2 hours incubation.

Hb G-San Josè or beta7(A4)Glu-->Gly has been reported in Southern Italian or Mexican families. We have studied four families from Sicily and Campania, Southern Italy. In six carriers, the hemoglobin variant level ranged from 32 to 38%. In four double heterozygotes for Hb G-San Josè and alpha-thalassemia the variant level showed a strong correlation with the alpha-thalassemia genotype. In fact, the variant level was 15% when interacting with the - (alpha)20.5/alphaalpha, 19.6% with the alphaalpha/alphaPoly Aalpha, and 24.8% with alphaalpha/alpha(-5) ntalpha genotypes. In two double heterozygotes for Hb G-San Josè and beta+ -IVS-I-6 (T-->C) the hemoglobin variant level was 67%. These data show that the reduced synthesis of alpha chains causes drastic reduction of probability to form Hb G-San Josè in favor of the formation of Hb A. Moreover, this reduction, (i) correlates with the type of alpha-thalassemia genotype and with the degree of the alpha chain deficiency, and (ii) is, most probably, more marked than the degree of alpha chain reduction. The minor affinity of the beta chain variant for the alpha chains associated with the reduced synthesis of the alpha chains is probably the principal cause of the variant hemoglobin reduction. Moreover, the rapid removal of the abnormal chains by proteolytic enzymes must have an essential role in order to reduce the chain variant pool. These conclusions are in agreement with the results obtained in reticulocyte and in vitro recombination experiments.