Expression studies of delta-globin gene alleles associated with reduced hemoglobin A(2) levels in Greek cypriots

We previously identified five delta-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 --> T change at codon 27 and the A --> G change in IVS-2 both affect RNA splicing, whereas the C --> 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 --> Ile at codon 4 (Hb delta T4I), Ala --> Ser at codon 27 (Hb delta A27S), and Arg --> Cys at codon 116 (Hb delta R116C)) showed decreased heat stability compared with Hb A(2), whereas the Hb delta T4I variant showed highest instability to mechanical agitation, Coexpression in yeast of alpha-globin chain and the delta-chain variant containing a Leu --> 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 delta-chains, which inhibits assembly with alpha-chains, and/or reduced assembly is suggested for the other three alleles.