Alpha Hemoglobin Stabilizing Protein (AHSP) Is a Susceptibility Gene to Drug /Infection-Induced Hemolytic Anemia.

Approximately 500 patients are diagnosed as having congenital hemolytic anemia every year in Japan, and the etiology in 75% of these patients involves red cell membrane disorders. The remaining cases are categorized as congenital non-spherocytic hemolytic anemia (CNSHA) and only about 25 % of CNSHA patients are diagnosed as either unstable hemoglobinopathies or erythroenzymopathies. As a result, up to 100 subjects remained diagnosed as having idiopathic hemolytic anemia. Recently, α-hemoglobin stabilizing protein (AHSP) has been identified, and found to play an important role as molecular chaperones in the stabilization of unstable free α-globin. Intact α-globin monomer generates reactive oxygen species, resulting in damage to red cell protein and lipids. AHSP knockout mice showed compensated hemolysis with Heinz bodies. Genetic defects in protection against oxidative stress such as glucose-6-phosphate dehydrogenase deficiency may account for acute hemolysis only when subjects are exposed to either oxidative drugs or infection, but chronic hemolysis is not evident in those cases. These observations led us to hypothesize that AHSP gene defects might account for either Heinz body-positive or drug/infection-induced hemolytic anemia.

We examined 25 CNSHA patients with unknown etiology: 6 cases showing Heinz body-positive red cells and 19 cases showing episodes of drug/infection-induced acute hemolysis. Erythroenzymopathies and unstable hemoglobinopathies were ruled out using red cell enzyme analysis, isopropanol stability tests and DNA sequencing of globin genes. Seven SNPs (single nucleotide polymorphisms) surrounding the AHSP gene (rs 4889672, rs 8046452, rs 8050390, rs 4296276, rs 17677, rs 10843, and a novel SNP at 92-bp upstream of rs 4889672) were examined, and compared with 100 control subjects. We found that a certain haplotype, 5′-GTCGA**-3′, was significantly prevalent in CNSHA cases (p=0.0086). Each SNP within the haplotype is located in the 5′-flanking region of AHSP gene, suggesting that the SNP(s) might account for decreased expression of AHSP gene. The reporter gene assay using K562 cells revealed that a construct containing 5′-GTCGA**-3′ had about 30% of transcriptional activity compared to a major haplotype 5′-ACCAG**-3′. Taken together, we conclude that the AHSP gene is correlated to disease-susceptibility in a group of hemolytic anemia with either Heinz body-positive or drug/infection-induced episodes.