Elevated soluble α‐hemoglobin pool in sickle cell anemia
Authors: Corinne Vasseur, Elisa Domingues‐Hamdi, Sadaf Pakdaman, Caroline Barau, Serge Pissard, Philippe Le Corvoisier, France Pirenne, Frédéric Galactéros, Véronique Baudin‐Creuza
Journal: American Journal of Hematology (2017)

Sickle cell anemia (SCA) exhibits broad inter-individual variability as well as intra-individual variability during the patient's lifetime, ranging from mild phenotype to severe disease. The gravity of SCA is modulated by the remaining fetal hemoglobin (HbF α2γ2) synthesis or/and the coinheritance of α-thalassemia (α-thal) which lead to decreased abnormal sickle hemoglobin (HbS α2βS2) amount and concentration in red blood cells (RBCs) and thus reducing its polymerization tendency.1-3 A distinguishing characteristic of the abnormal βS is its lower affinity, relative to normal βA, for α-chain during Hb assembly thus modulating the HbS concentration.4 Hence, there is a problem of a relative inefficiency of α2β2 tetramer formation with βS chains, that could lead to a relative and/or a dynamic excess of free α-Hb. In sickle cell disease, some cytosolic RBC proteins are upregulated such Αlpha-Hemoglobin Stabilizing Protein (AHSP)5 which not only specifically interacts with free α-Hb but also in the assembly of normal Hb tetramers.6 Thus, the α-Hb pool could be a resultant of various genetic factors (HbF, α-thal, AHSP, HS-40…) modulating the SCA phenotype. We postulated that the free soluble α-Hb (α-Hb pool) is increased in SCA patients (SS patients) and that the coinheritance of α-thal could significantly modulate this α-Hb pool. The study was conducted in 21 SS patients and 27 AA controls followed at the French Sickle Cell Referral Center in Mondor Hospital, Creteil, France. All subjects were at least 18 years old. Whole peripheral blood collections on EDTA-anticoagulated were achieved at a 3 months of any clinical or/and therapeutic event such as transfusion. The exclusion criteria for the volunteers was to be hemoglobinopathy carriers. An informed consent was obtained from all subjects in accordance with the Declaration of Helsinki. All subjects underwent blood sampling for genotype determination and biological assessment-blood cell count, Hb electrophoresis. For all SS patients, the βS-globin gene mutation was screened by Real-Time PCR assay. The RBC density, assessed by measuring the percentage of dense RBCs (DRBC: density >1.120) and the median density of RBCs (D50) were measured. In vitro α-Hb pool dosing assay was assessed on the lysed RBCs of all subjects by affinity chromatography support on which recombinant AHSP was beforehand bound as described previously in detail.7 The value of α-Hb pool is expressed in ppm equivalent to ng α-Hb per mg of total Hb subunits contained per mL of hemolysate in order to take account of the total concentration of Hb varying from one patient to another. Mann-Whitney tests were used for group comparisons. Potential relations between α-Hb pool and biologic parameters were analyzed by Spearman's rho and Pearson correlation coefficients. Hematologic and molecular data of 21 SS patients and 27 AA controls (Supporting Information Table S1) indicated that a representative sample of the SS patient population was studied.8 Among 21 SS patients, 12 have 4 α genes (αα/αα) and 9 have a heterozygous α+-thalassemia (αα/α−3.7). Regardless of α-globin genotype, the average Hb and HbF levels in SS patients were similar to those previously observed in adult SS patients.8 The HbA2 values tended to be higher in SS patients with 3 α-globin genes compared to those observed in SS patients with 4 α-globin genes as previously reported8 and higher than HbA2 level observed in control group. No significant difference was seen in the α-Hb pool according to the gender and age in patients and controls. A highly significant elevation of α-Hb pool values was observed in the RBCs of SS patients in comparison to that observed in the RBCs of AA controls (243 ± 66 ppm vs. 92 ± 21 ppm, P < .0001) (Figure 1, Supporting Information Table S1). The analysis of the α-Hb pool according to α-globin status in the SS patients showed that the values of the α-Hb pool were higher in patients with 4 presumably active α-globin genes than that observed in the patients with 3 α-globin genes (288 ± 42 ppm vs. 183 ± 39 ppm) (Figure 1), this difference being highly significant (P < .0001). These results are consistent firstly with a lower affinity of βS compared to βA for α-Hb and secondly with a previous study which showed that the α-Hb pool is decreased in α-thalassemic patients compared to that observed in AA controls.7 Free α-Hb pool in 21 SS patients compared to that observed in 27 AA controls. The SS patients with 4 α genes (n = 12) and with 3 α genes (n = 9) are represented by filled square and open square, respectively. The medians for each group are indicated by the horizontal line It is interesting to note that α-Hb pool value observed in SS patients are within the low range previously observed in β-thalassemia patients,9 as if SCA could be considered as a light β-thalassemia condition, which could explain another aspect of α-thal beneficial effects. Despite the small number of patients, the α-Hb pool was significantly correlated with 11 parameters (Supporting Information Table S2 and Figure S1). An inverse correlation found between α-Hb pool and RBCs (r = −0.6259, P = .0024) and % of HbA2 (r = −0.5149, P = .0169) and also a positive correlation with MCV (r = 0.6157, P = .0030), MCH (r = 0.6351, P = .0020), reticulocyte percent (rho = 0.5179, P = .0162), mean HbS content per RBC (r = 0.6509, P = .0014), %DRBCs (r = 0.6093, P = .0043), D50 (r = 0.5878, P = .0051), lactate dehydrogenase (rho = 0.5145, P = .0170), and bilirubin (rho =0.7388, P = .0001). Among these different parameters, the mean HbS content per RBC, %DRBCs and D50 are important features of SCA, likewise bilirubin and LDH levels, markers of hemolysis in SCA. By contrast, no significant correlation was observed between α-Hb pool and %HbS or %HbF. Also no associations were observed between the α-Hb pool and the Red cell Distribution Width, platelets or ferritin. In summary, this study demonstrates that the α-Hb pool value is increased in RBC lysates of SCA patients and the dispersion of this pool could reflect of the variability of the SCA clinical expression which depends on multiple polygenic factors. Thus, the α-Hb pool could be a new RBC biomarker resulting from α and βS synthesis and HbS tetramer assembly disturbances. The authors thank the patients, nurses, and physicians at the French Sickle Cell Referral Center who contributed to the management of patient care, A. Hamadouche of the "Plateforme de Recherche Clinique" for preparing patient samples, and Dr S. Moutereau for performing RBC density distributions. This work was funded by the Inserm and the UPEC, France. The authors declare no competing financial interests. Corinne Vasseur and Elisa Domingues-Hamdi performed the measurement of the α-Hb pool and analyzed the data. Sadaf Pakdaman collected the data of SS patients and AA controls. Caroline Barau carried out the lysates of RBCs and the DNA samples of SS patients. Serge Pissard performed the α-globin locus studies. Frédéric Galactéros and Philippe Le Corvoisier were in charge of the selection of patients and analyzed the data. Véronique Baudin-Creuza designed the study, collected and analyzed the data. Corinne Vasseur, Véronique Baudin-Creuza and Frédéric Galactéros wrote the paper. All authors critically revised the manuscript and approved the final version of the manuscript. Additional Supporting Information may be found in the online version of this article. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.