Lesional Mechanism of Alpha Thalassemias : Towards a Common Mechanism Alpha Thal / Beta Thal

Context and Objectives: Erythropoiesis is a complex process based on the balance between proliferation / maturation and apoptosis, which produces more than 10⁹ red blood cells per day. The lesional mechanism of thalassemia (thal) appears during the synthesis of hemoglobin (Hb) and the classical hypothesis proposes that the clinical severity is proportional to the imbalance of production of alpha / non-alpha-globin chains. However, the severe alpha thal (HbH) phenotypic expression does not adhere to this model since the HbH linked to the association of deletions (alpha0 / alpha +) are less severe than HbH associating deletion and point mutants (alpha0 / alphaCS alpha) while the deficiency is less important, and that there are severe alpha thal linked to homozygosity for an alpha variant without deletion. In order to understand the lesional mechanisms involved, and in light of recent work on beta thal pathophysiology, we have carried out cultures of erythroid progenitors from patients with these two types of « deletional » HbH (alpha⁰/ -3,7) and « non deletional » HbH (alpha⁰/ CS) as well as from a homozygous patient of an Agrinio variant (alpha2 : p29 Leu®Pro). We have characterized the amount of alpha globin aggregates in the progenitors of the different patients. In addition, the Agrinio mutation was produced in E. coli to study the consequences of the mutation on interactions between α-HbAgrinio and the alpha-Hemoglobin Stabilizing Protein (AHSP) chaperone.

Materials and Methods: Erythroid progenitors were analyzed by multiparametric cytometry (Amnis®) and confocal microscopy to quantify and characterize cytoplasmic aggregates. "Non-thalassemic red cells" (csp) and progenitors from a beta thal major patient were used as negative and positive controls, respectively. Normal α-Hb proteins (α-HbWT) and α-HbAgrinio were produced as glutathione S transferase (GST) fusion proteins associated with their chaperones (GST-AHSP / GST-α -Hb). After cleavage of the GST moiety, the characterization of the AHSP / α-HbAgrinio complex was carried out by fluorescence spectroscopy.

Results: The proportion of cells with cellular aggregates analyzed by multiparametric cytometry is proportional to the clinical severity of the patient with an order csp < alpha°/ alpha⁺ < alpha°/alpha^CS < alpha^Agrinio alpha / alpha^Agrinio alpha < beta thal. The expression in E.coli of α-HbAgrinio is similar to that observed for α-Hb^WT, but the amount of α-HbAgrinio obtained after purification is greatly decreased indicating a significant instability of α-HbAgrinio. The absorbance spectroscopy shows that the amplitude of the Soret band, characteristic of the heme molecule, is highly decreased for the AHSP / α-HbAgrinio complex compared to that observed for the AHSP / α-HbWT complex. The fluorescence emission spectrum of the AHSP / α-HbAgrinio complex is increased compared to that of the normal complex indicating that the tryptophan AHSP fluorescence signal is less attenuated since fewer heme molecules are present. These results show that α-HbAgrinio is unstable probably due to an alteration of interaction with its chaperone and to a lack of insertion of heme in the globin for this mutant.

Conclusion: Recent work has shown that one of the mechanisms responsible for dyserythropoiesis in beta thal major progenitors is the sequestration of the HSP70 chaperone in the cytosol by alpha globin precipitates. HSP70 can therefore no longer protect the nuclear GATA1 factor from cleavage by the activated caspase3 at the beginning of terminal differentiation. This leads to inachieved differentiation and apoptosis. Our results indicate that in HbH and non-deletional alpha thal, a comparable mechanism would be an important element in the pathophysiology unifying the lesional mechanism of two apparently opposite pathologies. Studies in the bacterial model confirm the strong instability of α-HbAgrinio and in vitro studies show that this mutant is partially heminized.