Unstable Alpha Hemoglobin Stabilizing Protein as a Cause of Thalassemia: Proof of Concept.

Abstract 462

Alpha hemoglobin stabilizing protein (AHSP), a small 102-residue protein previously known as erythroid differentiation-related factor (EDRF), is present only in erythroid cells, where it acts as a chaperone of α-Hb by forming a stable complex which prevents the aggregation of excess α-Hb that by precipitation would damage membrane structure and trigger cell death. AHSP knock-out mice exhibit ineffective erythropoiesis with reticulocytosis, abnormal erythrocyte morphology, intracellular inclusion bodies, and increased production of reactive oxygen species (ROS) with subsequent cellular oxidative damage as observed in thalassemias. A similar disorder involving AHSP has not yet been found in humans as a cause of thalasssemia. We report here a first case presenting such a syndrome, for a family originating from SE Asia. Their second child was referred to the hospital one month after birth, because of palor and anemia. Biological tests demonstrated a hemolytic microcytic anemia (Hb 6.9 g/dl, MCV : 73.2 fL and MCH 25 pg) and 3% Hb Bart's detected by CE-HPLC. No abnormality was revealed for the molecular screening for α-thal, including detection of common and rare α locus deletions using Gap-PCR and MLPA analysis, the sequencing of α1 and α2 genes, and the sequencing of the HS-40 core sequence. We considered thus the possibility for an abnormality of a modulator gene; the AHSP gene and its promoter region were sequenced. The proband was found to be homozygous for a mutation in exon 3 (c.167 T>G), while the parents and the first son were heterozygous for it. This mutation changes Val 56 to a Gly: the modified residue is not in direct contact with α-Hb but located in the corner between the three helices of the AHSP molecule and could play some role in the stability of the protein. To demonstrate that this abnormal AHSP could be the cause of the thalassemic syndrome, this molecule was produced as a Glutathione-S-transferase (GST) fusion protein by protein engineering in E.coli using both the pGEX6P-AHSP and the pGEX6P-α-AHSP co-expression vectors after site-directed mutagenesis. The yield of the mutated AHSP was lower than that of the wild type when solubilized suggesting that it was less stable than the normal. The kinetics of protein interaction indicate a factor of 2-3 decrease in the affinity. In addition a 20%-30% decrease in capture within GST micro-column of normal α-globin by the mutated recombinant GST-AHSP was observed. Studying the intragenic polymorphisms of the AHSP gene in this family, we observe that the mutated allele belongs to more weakly expressed “clade B” group and the two normal alleles to the “clade A” group as defined by Lai et al (BJH, 2006, 133, 675-682). Up to now, only 3 mutants have been described in the AHSP gene but none was found to impair the association with normal α-Hb chain. This case is thus the first evidence of an isolated α-thal not linked with the α locus but rather with a mutated AHSP. Heterozygous carriers for this AHSP mutation are clinically normal, but the homozygous patient having only the low expressed unstable AHSP presents a more severe α-thal syndrome than that caused by simple α-gene deletion(s).