Expression Studies of δ-Globin Gene Alleles Associated with Reduced Hemoglobin A2 Levels in Greek Cypriots

Abstract

We previously identified five ␦-globin gene alleles associated with reduced hemoglobin (Hb) A 2 (Trifillis, P., Ioannou, P., Schwartz, E., and Surrey, S. (1991) Blood 78, 3298 -3305). We have now evaluated functional consequences of the changes after expression in COS-1 cells to monitor effects on RNA splicing. In addition, variant Hb A 2 tetramers were expressed in yeast to assess effects of amino acid changes on oxygen binding and stability to heat and mechanical agitation. The G 3 T change at codon 27 and the A 3 G change in IVS-2 both affect RNA splicing, whereas the C 3 T change at codon 97 and the AT deletion in IVS-2 have no effect. Oxygen equilibrium curves of the Hb A 2 variants expressed in yeast were similar to that of wild type Hb A 2 . None of the three variant Hb A 2 tetramers (Thr 3 Ile at codon 4 (Hb ␦T4I), Ala 3 Ser at codon 27 (Hb ␦A27S), and Arg 3 Cys at codon 116 (Hb ␦R116C)) showed decreased heat stability compared with Hb A 2 , whereas the Hb ␦T4I variant showed highest instability to mechanical agitation. Coexpression in yeast of ␣-globin chain and the ␦-chain variant containing a Leu 3 Pro change at codon 141 yielded no identifiable tetramers, suggesting lack of assembly or severe tetramer instability. These studies show the probable cause for decreased Hb A 2 for two alleles is due to defective splicing, whereas decreased protein stability, increased tetramer association with red cell membranes, increased interdisulfide bond formation of ␦-chains, which inhibits assembly with ␣-chains, and/or reduced assembly is suggested for the other three alleles.