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

doi:10.1074/jbc.271.43.26931

Volume 271, Number 43, Issue of October 25, 1996 pp. 26931-26938
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.

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

(Received for publication, May 30, 1996, and in revised form, July 30, 1996)

Panayiota Trifillis , Kazuhiko Adachi , Takamasa Yamaguchi , Elias Schwartz and Saul Surrey

From the Division of Hematology, Abramson Pediatric Research Center, The Children's Hospital of Philadelphia, Philadelphia, Pennsylvania 19104

We previously identified five $delta$ -globin gene alleles associated with reduced hemoglobin (Hb) A₂ (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 A₂ tetramers were expressed in yeast to assess effects of amino acid changes on oxygen binding and stability to heat and mechanical agitation. The G $right-arrow$ T change at codon 27 and the A $right-arrow$ G change in IVS-2 both affect RNA splicing, whereas the C $right-arrow$ T change at codon 97 and the AT deletion in IVS-2 have no effect. Oxygen equilibrium curves of the Hb A₂ variants expressed in yeast were similar to that of wild type Hb A₂. None of the three variant Hb A₂ tetramers (Thr $right-arrow$ Ile at codon 4 (Hb $delta$ T4I), Ala $right-arrow$ Ser at codon 27 (Hb $delta$ A27S), and Arg $right-arrow$ Cys at codon 116 (Hb $delta$ R116C)) showed decreased heat stability compared with Hb A₂, whereas the Hb $delta$ T4I variant showed highest instability to mechanical agitation. Co-expression in yeast of $alpha$ -globin chain and the $delta$ -chain variant containing a Leu $right-arrow$ 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 A₂ 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.

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