African Sickle Cell Disease Research Articles

Determinants of the haemoglobin level in patients with sickle cell disease living in sub-Saharan Africa: Major impact of the country of residence and independent effects of leucocyte and platelet counts and haemolysis.

The degree of anaemia in sickle cell disease (SCD) is a well-known contributor to morbidity and mortality. We aimed to explore the factors affecting haemoglobin (Hb) level in African SCD patients, considering haemolysis biomarkers (LDH and bilirubin level, and reticulocyte count), leucocyte and platelet counts and socio-demographic characteristics (gender, age group, country of residence and BMI). The research was part of the CADRE multinational cohort and involved 3699 SCD patients living in Mali, Senegal, Ivory Coast, Democratic Republic of Congo, Gabon and Cameroon: 2936 SS/Sβ0, 587 SC and 176 Sβ + patients with median Hb level of 8, 11.3 and 11.2 g/dL respectively (p < 0.001). In multivariate analysis conducted in 1394 SS/Sβ0 patients, living in Cameroon, female gender, lower BMI, higher haemolysis markers (especially LDH) and higher leucocyte and platelet counts were independently associated with lower Hb level (all p < 0.05). In 497 SC and 156 Sβ + patients, female gender (p < 0.001), lower BMI (p < 0.05) and higher platelet counts (p < 0.001) were independently associated with lower Hb level. Anaemia in African SCD patients is not only associated with haemolysis but also with the country of residence, lower BMI and leucocyte or platelet counts which might reflect inflammation related to infectious burden in the region.

5613165 INVESTIGATING VARIANTS ASSOCIATED WITH QUANTITATIVE RED BLOOD CELL PHENOTYPES FROM TOPMED PREDICTIONS IN AFRICAN SICKLE CELL DISEASE POPULATIONS.

Background: Sickle cell disease (SCD) is caused by a single point mutation (c.20A>T, p.Glu6Val), which results in the formation of mutant haemoglobin S (HbS) instead of haemoglobin A (HbA). It is a life-long inherited recessive disorder that predominantly affects people of African, Indian, and Mediterranean descent. The polymerisation of HbS within red blood cells (RBCs) is the primary pathophysiological alteration in SCD, which results in the sickle-shape of RBCs. Sickle-shaped RBCs are more prone to haemolysis and blocking blood flow, resulting in anaemia and painful episodes. Haematological indices including haematocrit (HCT), haemoglobin concentration (Hb), mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), red blood cell width (RDW) and RBC count indicate the size, development, and haemoglobin content of RBCs. Haematological indices are routinely measured as a means of monitoring SCD. A study recently found associations between 14 novel variants and haematological indices when using whole genome sequencing (WGS) data. The association between these variants and haematological indices have not yet been investigated in African SCD patients. This study aims to determine the associations between these variants and haematological indices in SCD patients from Cameroon and Senegal compared to non-SCD individuals. Aim: Investigating the association of 13 novel variants previously found in the Trans-Omics for Precision Medicine (TOPMed) project to haematological indices in African SCD patients. Methods: The 14 variants from the TOPMed study will be genotyped using a TaqMan Assay platform. Sanger sequencing will then be performed on 10% of the cohort to validate the TaqMan Assay result. The application R will be used for the descriptive statistics using sociodemographic, clinical, and haematological indices to evaluate the cases and controls. The Hardy-Weinberg Equilibrium will be used to ensure quality control. A linear regression analysis will be performed in R to determine the association between the variants and RBC phenotypes. Expected results: We are expecting to find an association between the SNPs and haematologocial indices. Conclusion: The variants of focus in this study were prioritized using WGS data, therefore capturing variants that are usually not included in GWAS arrays or imputation reference panels. The associations of the 13 novel variants with haematological indices have not yet been tested in African SCD populations. The results of this investigation could positively impact future studies into predictive modelling using haematological indices.

Abstract 65: Anemia-related Heart Failure in Sub-saharan African Sickle Cell Disease Patients

Introduction: Anemia has been implicated in heart failure. Existing literatures, involving predominantly African-Americans, suggests that Sickle Cell Disease (SCD) maybe linked to various cardiovascular complications including pulmonary hypertension and left venticular dysfunction. Peculiarly, our study involves exclusively Sub-Saharan population. Method: We conducted a cross sectional observational study of 208 hydroxyurea-naive consecutive SCD patients aged 10-52 years at steady state and 94 healthy non-matched controls who were studied in an out patient clinic in Sub-Saharan Africa. SCD patients were required to have electrophoretic or liquid chromatography documentation of major sickling phenotypes. Control group was required to have non-sickling phenotypes. Cardiac measurements were performed with TransThoracic Echo according to American Society of Echocardiography guidelines. Hemoglobin level was also obtained. Results: Hemoglobin level in SCD group (8.5+/- 1.5) was significant (P&lt;0.001) compared to control (13.8+/- 1.7). Although SCD group had significantly higher values of left ventricular (LV) size, there was no qualitative evidence of LV dysfunction. SCD group had higher values of Ejection Fraction but not statistically significant. There was no evidence of LV wall stiffening to impair proper filling in SCD group, with the ratio of early to late ventricular filling velocities, E/A ratio elevated (1.7+/-0.4 compared to 1.6+/- 0.4; P=0.010). Right ventricular systolic pressure was determined using the formula of 4x Tricuspid Reugurgitant jet (TRV) square as an indirect measurement of Pulmonary arterial systolic pressure. SCD patients had significantly higher mean±SD values for tricuspid regurgitant jet velocity than did the controls (2.1±0.6 vs. 1.8±0.5; p= 0.001). Within the SCD group, there was no clear pattern of worsening diastolic function with increased TRV. Furthermore, E/A had a significant positive relationship with jet velocity in bivariate analysis (R=0.20; P=0.013). Conclusions: We were unable to demonstrate existence of anemia-associated left ventricular dysfunction in Sub-Saharan African with SCD. Further studies is required to highlight the reason behind this finding.

Association Between Hyperhemolysis and Vascular Complications in Sickle Cell Disease Sub-Saharan African Patients

IntroductionAbout 80 % of sickle cell disease (SCD) patients live in sub-Saharan Africa whereas most studies on SCD are performed in Europe and America. Hyperhemolysis is thought to play a major role in the pathological process of SCD vasculopathy. Nevertheless, the paradigm of hyperhemolysis is controversial and has never been studied in the African context. We aim to analyze the association between hemolysis and clinical vascular complications among SCD African patients.MethodsCADRE is a multinational cohort of SCD African patients aged 3 years and older, included prospectively and explored in a steady state. All patients from the CADRE cohort in Ivory Coast, Cameroun and Mali were included in the present study. Patients were classified in SS-Sβ0 phenotype or SC-Sβ+ phenotype. SCD vascular complications (pulmonary arterial hypertension (PAH), microalbuminuria, leg ulcers, priapism, stroke and osteonecrosis) were assessed using clinical examination, laboratory exams and echocardiography. Hemolysis was measured by a composite score, created with a principal component analysis, that included LDH, hemoglobin and bilirubin rates and clinical icterus. The association between hyperhemolysis (upper quartile of the hemolysis score) and the vascular complications was assessed using multivariate regression analysis in the whole population with further stratification by hemoglobin phenotype.ResultsWe included 2409 patients among which 1751 SS-Sβ0 patients and 658 SC-Sβ+ patients. Compared to SC-Sβ+ patients, SS-Sβ0 patients were younger (15 years old versus 21 years old) and exhibited more leg ulcers (166 (9.5%) versus 24 (3.7%)) and microalbuminuria (573 (42.4%) versus 119 (20.0%)). The hemolysis score was higher in SS-Sβ0 patients as compared to SC-Sβ+ patients (median 1.18 versus 0.43, Figure). After adjustment for age, sex and country, hyperhemolysis was associated with microalbuminuria (OR = 1.59 [1.17-2.16]) and priapism (OR=1.58 [1.01-2.49]) (Table). In SS-Sβ0 patients, hyperhemolysis was associated with microalbuminuria (OR=1.54 [1.10-2.15]) and there was a trend to an association with PAH (OR=1.65 [0.91-2.98]) and priapism (OR=1.50 [0.95-2.39]). In SC-Sβ+ patients, only the association between hyperhemolysis and microalbuminuria remained significant. Sensibility analysis in the adult population showed that hyperhemolysis was significantly associated with all the SCD vascular complications, except for osteonecrosis.ConclusionIn African SCD patients, associations between hyperhemolysis and SCD vascular complications were statistically significant but of modest magnitude, and depended on the hemoglobin phenotype. These results suggest that in the African context, hemolysis is not a major determinant of the development of SCD vasculopathy.Table.Associations between SCD complications and hemolysis score (quartiles 1 to 3 versus quartile 4) in the whole population: multivariate analysis with adjustment for age, sex, country and hemoglobin phenotype% of patients with the complicationMultivariate analysisN event/N totalQuartiles 1 to 3Quartile 4ORIC 95%P valuePAH*115/4316.147.081.580.89-2.830.123Urine albumin/creatinine > 30mg/g724/199124.8845.361.591.17-2.160.004Leg ulcer, lifetime190/24096.8011.011.200.81-1.780.355Priapism, lifetime **160/109513.2717.901.581.01-2.490.046Stroke, lifetime25/24090.791.771.320.49-3.540.578Osteonecrosis, lifetime277/240911.2212.281.000.70-1.431.000PAH: Pulmonary Arterial Hypertension. * Patients from Mali and Cameroun only ** Males only [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Interethnic diversity of the CD209 (rs4804803) gene promoter polymorphism in African but not American sickle cell disease.

Elucidating the genomic diversity of CD209 gene promoter polymorphism could assist in clarifying disease pathophysiology as well as contribution to co-morbidities. CD209 gene promoter polymorphism has been shown to be associated with susceptibility to infection. We hypothesize that CD209 mutant variants occur at a higher frequency among Africans and in sickle cell disease. We analyzed the frequency of the CD209 gene (rs4804803) in healthy control and sickle cell disease (SCD) populations and determined association with disease. Genomic DNA was extracted from blood samples collected from 145 SCD and 231 control Africans (from Mali), 331 SCD and 379 control African Americans and 159 Caucasians. Comparative analysis among and between groups was carried out by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Per ethnic diversification, we found significant disparity in genotypic (23.4% versus 16.9% versus 3.2%) and allelic frequencies (48.7% versus 42.1% versus 19.8%) of the homozygote mutant variant of the CD209 (snp 309A/G) gene promoter between Africans, African Americans and Caucasians respectively. Comparative evaluation between disease and control groups reveal a significant difference in genotypic (10.4% versus 23.4%; p = 0.002) and allelic frequencies (39.7% versus 48.7%; p = 0.02) of the homozygote mutant variant in African SCD and healthy controls respectively, an observation that is completely absent among Americans. Comparing disease groups, we found no difference in the genotypic (p = 0.19) or allelic (p = 0.72) frequencies of CD209 homozygote mutant variant between Africans and Americans with sickle cell disease. The higher frequency of CD209 homozygote mutant variants in the African control group reveals a potential impairment of the capacity to mount an immune response to infectious diseases, and possibly delineate susceptibility to or severity of infectious co-morbidities within and between groups.

Harnessing red cell membrane pathophysiology towards point‐of‐care diagnosis for sickle cell disease

Harnessing red cell membrane pathophysiology towards point‐of‐care diagnosis for sickle cell disease

Rapid Increase of CD8+ T Cell Count in Peripheral Blood of Pediatric Patients After HLA-Identical Stem Cell Transplantation for African Sickle Cell Anemia

Abstract 4769 Background.Sickle Cell anemia (SCA) remain a disease with high risk of morbidity and early death, especially in African patients. Allogeneic haematopoietic stem cell transplantation (HSCT) is the only curative treatment for SCA. To analyze immunohematological reconstitution after transplant, we report our experience concerning 12 geno-identical HSCT for SCA-patients prepared with the same myeloablative conditioning regiment consisting of Busulfan and Cyclophosphamide. Patients and Methods.Twelve patients with a median age of 12 years (range, 2–16), affected by sickle cell anemia (SCA), received hematopoietic stem cell transplantations from HLA-identical, related donors following a myeloablative conditioning regimen. To analyze the mechanisms involved in immunological reconstitution post transplant, we analyzed T cell subsets by flow cytometry at + 60 post transplant. Results.All patients had sustained engraftment and remained free of any SCA-related events after transplantation. Sixty days after the transplant, the patients had significantly lower CD4+ T cells in comparison to the controls (15.6 ± 5.9 % vs. 47.5 ± 6% respectively), whereas CD8+ T cells were the first lymphocytes to repopulate the peripheral blood with up to 45% of these cells being CD8+ T cells (in mean 48.5 ± 14.3 % vs. 20 ± 7%). All patients displayed reduced numbers of B cells versus normal value, and 10/12 patients had only 0% to 1% of control levels of CD19+ cells. CD3-CD56+bright NK cells were 18.6 ± 12.2 %, whereas CD3-CD16+ (with cytotoxic functions) were 16.7 ± 11.8 %. Conclusion.Our primary finding include the following: 1) rapid increase of lymphocytes in peripheral blood after transplant; 2) rapid expansion of CD8+ T cell but not CD4 T cell counts. Probably reactivation of cytomegalovirus (CMV) infection, observed in 11/12 patients on the early stages of T-lymphocyte recovery after transplant, might induce a dramatic increase in CD8 but not in CD4 T-cell counts. Disclosures:No relevant conflicts of interest to declare.

Decreased Apoptosis in Bone Marrow of Pediatric Patients After HLA-Identical Stem Cell Transplantation for African Sickle Cell Anemia

Abstract 4751 Background.In thalassemia patients ineffective erythropoiesis appears to be caused by accelerated apoptosis, in turn caused primarily by deposition of a-globin chains in erythroid precursors. The bone marrow is the main site of the erythroid cell death. The occurrence of apoptosis at the polychromatophilic stage coincides with the stage where intensive hemoglobinization occurs and could be explained by the increase in a-globin production and precipitation. The mechanism of BM apoptosis is also observed in sickle cell anemia (SCA), but is reduced. We report our experience concerning 12 geno-identical hematopoietic stem cell transplantation (HSCT) for SCA-patients, to analyse the effect of transplant on “normalization” of intramedullary apoptosis. Patients and Methods.Twelve patients with a median age of 12 years (range, 2–16), affected by sickle cell anemia (SCA), received hematopoietic stem cell transplantations from HLA-identical, related donors following a myeloablative conditioning regimen. We studied Fas (CD95+) and caspases-3 expression on erythroblast subpopulation. Using both CD71 and FSC parameters, we obtain 3 principal populations, which we labelled A, B, C erythroblasts (Ery. A, Ery. B, Ery. C). Ery A (CD71high FSChigh) are basophilic, Ery. B (CD71high FSClow) are late basophilic and polychromatic, whereas Ery. C (CD71low FSClow) are orthochromatic erythroblasts and reticulocytes. Results.We observed an expansion of Ery A, Ery B and Ery C population at baseline, probably as an essential process needed to maintain a constant red cell production in SCA patients (42.4% Ery A; 24.7% Ery B; 35.6% Ery C). After BM transplant decreased levels was observed especially on Ery A and Ery B populations (12.9% Ery A; 7% Ery B; 35% Ery C). After BM transplant Fas expression was reduced in all 3 erythroid population, but significantly on more immature Ery A (17% vs 3.5% CD95+ on Ery A; 32.2 vs 24.4 on Ery B; 5% vs 3.3% on Ery C). This observation may suggest that Fas, expressed by early erythroblasts in vivo, might contribute to the cell death of erythroid precursors in bone marrow, but this was corrected after HSCT. In addition, only after transplant we observed an initial increase of caspases 3 on more mature Ery C population (2.3% at baseline vs. 15.3% after transplant), as observed during later steps of “normal” cell maturation. Conclusion.After HSCT in SCA we observed a “normalization” of erythroid populations, in parallel with decreased intramedullary apoptosis rate, suggesting normal erythroid maturation in ex-SCA patients. Disclosures:No relevant conflicts of interest to declare.

Red blood cell alloimmunization and antigen matching in sickle cell disease – the African perspective

In sickle cell disease (SCD), blood transfusion facilitates improved blood and tissue oxygenation, reduces the propensity to sickling by diluting host cells, and suppresses the production of red blood cells (RBCs) containing sickle haemoglobin (HbS). Delivery of RBC transfusions to patients with SCD varies by method (simple vs. exchange) and frequency (episodic vs. chronic). However, due to the genetic differences between blood donors and recipients, repeated transfusions increase the risk of developing alloantibodies to RBC antigens. The antigens most frequently involved belong to the Rh, Kell, Kidd, Duffy, Lewis, and MNS blood group systems. Consequences of RBC alloimmunization include delays and difficulties in obtaining compatible blood for future transfusions, the occurrence of delayed haemolytic transfusion reactions (DHTRs), the hyperhaemolysis syndrome and autoimmunization. In Europe and USA, RBC alloimmunization rates ranging from 18% to 76% have been reported in SCD while other multiply transfused (OMT) patients had alloimmunization rates of 5% to 20% indicating that SCD patients are at a higher risk of developing RBC alloantibodies. To prevent alloimmunization in SCD patients, the standard practice in Europe and USA is to determine their extended RBC phenotype (ABO, Rh, Kell, Kidd, Duffy, Lewis, MNS) before commencing transfusion therapy and perform antigen matching for C, E and K antigens for patients without prior alloantibody formation. However in Africa, lower RBC alloimmunization prevalence rates of 6–10% have been reported in SCD patients and no differences were observed between SCD and OMT patients. This may be explained by the presumed high phenotypic compatibility between donors and SCD patients who were all Black Africans. Also, a low transfusion load (a median 3 U of blood were transfused) in SCD patients might have led to the poor response to alloantigenic challenge. Anti‐K alloimmunization was notably rare among African SCD patients compared to anti‐S. In many African countries, pre‐transfusion immunohaematologic testing includes neither the detection of RBC alloimmunization nor preventive antigen matching. Most transfusion laboratories are understaffed and underequipped; they perform ABO/D typing plus room temperature saline cross‐matches and do not screen for RBC alloantibodies. Hence, immunized SCD patients are not diagnosed and do not have the opportunity of receiving antigen‐negative blood. Furthermore, data on the occurrence of DHTRs are lacking. Introducing pre‐transfusion RBC alloantibody screening in all African countries will significantly improve the transfusion management of SCD patients. A program of limited phenotype matching for C, E and S antigens is recommended to prevent additional alloantibody formation in immunized SCD patients in Africa.

Global arginine bioavailability in Tanzanian sickle cell anaemia patients at steady‐state: a nested case control study of deaths versus survivors

Global arginine bioavailability in Tanzanian sickle cell anaemia patients at steady‐state: a nested case control study of deaths <i>versus</i> survivors

Pulmonary hypertension of sickle cell disease: More than just another lung disease

Pulmonary hypertension (PH) of sickle cell disease (SCD), as defined by a tricuspid regurgitant jet velocity by echocardiogram of >or=2.5 m/sec, occurs in approximately 1/3 of HbSS adults and is an independent risk factor for mortality. Although studies of the past few years have greatly expanded our knowledge of the clinical manifestations and pathogenesis of PH in SCD, many questions remain. Two of the key issues addressed by manuscripts in the issue of the American Journal of Hematology are the prevalence of PH in the large African SCD population and the association of PH with renal disease and systemic hypertension. Because of its high impact on mortality, the assessment of PH in the African population is crucial from a public health standpoint. The association of PH with systemic disease in SCD suggests that a more widespread vasculopathy occurs in these patients that may be one of the mechanisms responsible for the observed increase in mortality. It is through studies such as these that a greater understanding of the pathogenesis and treatment of pulmonary hypertension is sickle cell disease can ve achieved.

Spleen in sickle cell anemia: comparative studies of Nigerian and U.S. patients.

Anecdotal reports have attributed persistent splenomegaly in African sickle cell anemia (SS) patients to the effects of malaria. However, no comparative studies of patients in malarial and nonmalarial regions have been conducted, and few studies of malaria antibody titers have been reported. In the present study, age- and sex-matched Nigerian patients (n = 310), while it was found only in 8% of U.S. patients (n = 100) from Georgia. There was significant linear correlation between spleen size and Hb levels and with serum immunoglobulins in the Nigerian group. However, serum complement levels (C3 and C4) were not affected by spleen size. In both groups, patients with splenomegaly had fewer circulating pitted red cells than their counterparts without splenomegaly. The mean +/- SE of IgG-specific malaria antibody titer among the Nigerian patients without palpable spleens was 9,386 +/- 2,036; 9,334 +/- 2,980 in those with spleens between 1 and 5 cm, 16,201 +/- 4,502 in those with spleens between 6 and 10 cm, and 22,445 +/- 8,456 in those with spleens above 10 cm. Coexistent alpha-thalassemia did not influence the prevalence of splenomegaly among the Nigerian SS patients. This study provides additional evidence that malaria plays a significant role in the persistence of splenomegaly in African patients.

The Senegal DNA haplotype is associated with the amelioration of anemia in African-American sickle cell anemia patients

We have previously determined that in African sickle cell anemia (SS) patients three different beta-like globin gene cluster haplotypes are associated with different percent G gamma (one of the two types of non- alpha chains comprising hemoglobin F [HbF]), mean percent HbF, and percent dense cells. We report now that in adult New York SS patients, the presence of at least one chromosome with the Senegal haplotype is associated with higher Hb levels (1.2 g/dL higher) than is found for any other non-Senegal haplotype (P less than .004). The percent reticulocytes and the serum bilirubin levels were lower in these patients. When the effect of alpha-gene number was analyzed by examining a sample of SS patients with concomitant alpha-thalassemia, the same results were obtained. Because the HbF level is significantly higher among the Senegal haplotype carriers in this sample, the inhibitory effect on sickling of this Hb variant may be one of the reasons for the haplotype effect. We conclude that the Senegal beta- like globin gene cluster haplotype is associated with an amelioration of the hemolytic anemia that characterizes sickle cell disease.