A Natural Variant Sequence in the AHSP Gene May Impact Severity of Beta-Thalassemia.

Alpha-hemoglobin stabilizing protein (AHSP) is an erythroid-specific molecular chaperone that binds the alpha chains of hemoglobin, preventing their precipitation and deleterious effects. Loss of AHSP exacerbates alpha globin precipitation and anemia in a murine model for beta thalassemia. In vitro, recombinant AHSP inhibits the production of reactive oxygen species (ROS) by alpha hemoglobin. To further define the role of AHSP as a modifier of beta-thalassemia, we analyzed AHSP sequences for mutations in a large population of beta-thalassemic and control subjects. A single nucleotide change, from A to T at position 12888 (Genebank accession number AC106.730.2) in the third exon of the AHSP gene was identified, converting asparagine 75 to leucine (N75I). We detected a patient who was heterozygous for both beta-thalassemia (b³⁹/b^A) and the AHSP-N75I mutation. She presented with an unusually severe anemia that required regular blood transfusion. The father is hematologically normal, without any thalassemia mutation and heterozygous for AHSP-N751. The mother is heterozygous for the beta 39 mutation with classical mild hypochromic microcytic anemia and no mutation in the AHSP gene. Of 282 unrelated control subjects tested, one (0.35%) contained the N75I mutation. Analysis of red blood cells from this subject revealed normal hemoglobin indices but a small number of Heinz bodies, suggesting hemoglobin precipitation. This finding is similar to what we observed previously in mice that are heterozygous for an AHSP null allele. To investigate how the N75I mutation affects AHSP function, we studied the biochemical properties of the recombinant mutant protein. Surface plasmon resonance showed that the binding affinity of AHSP N75I for alpha hemoglobin is normal. Importantly, compared to wild type AHSP, the N75I mutant protein exhibited significantly reduced capacity to inhibit ROS production by alpha hemoglobin. Hence, AHSP N75I may be less effective at conferring protection from oxidative-mediated damage by free alpha hemoglobin in erythrocytes. These effects, when coupled with beta thalassemia, could result in more severe anemia, implicating AHSP N75I as a potential genetic modifier.