Using a retrospective cross-sectional descriptive and analytical study, we determined the likely associations between the haptoglobin phenotype and morbidity-adjusted hemoglobin phenotype in a SCD population.

The studied population was a cohort of 170 black African patients, carriers of hemoglobin S. The cohort was built in 2021. Patients were taken care in the hematology department of Yopougon University Hospital (Abidjan, Ivory Coast). The biological analyzes were carried out in the biology laboratory of the Yopougon University Hospital and at the Center for Diagnosis and Research on Aids and other infectious diseases (CeDReS), University Hospital of Treichville.

Selection criteria and morbidity were determined with different types and sub-types of collected variables: social, anthropological, clinical and biological variables (Table 1). An overall morbidity score was defined as following. One point was assigned to each clinical, surgical, or infectious complication. Overall morbidity was determined by cumulated morbidities. The morbidity score range was 0 to 10. The higher the morbidity score was, the higher the morbidity was.

Patients regardless their age and their biological sex consulting for medical follow-up of SCD were included in the study. Included hemoglobin phenotypes were homozygous sickle cell disease (SSFA₂), sickle-β⁺-thalassemia (SAFA₂),

sickle-β˚ thalassemia (SFA₂) and SC hemoglobinosis. Data were collected from patients’ medical files. Were excluded from the study, heterozygous Hb AS patients, patients from which we did not get their consent and those who demanded to quit the study.

The study was designed and conducted following the Declaration of Helsinki. It was reviewed and approved by the scientific committee of the medical biology chair of Pharmaceutical and Biological Sciences faculty (University Felix Houphouet-Boigny) and by the medical committee of the Yopougon University Hospital.

At inclusion in the cohort, blood samples with anticoagulant EDTA were collected from fasting patients for at least 10 hours. Hemoglobin phenotyping was performed on whole blood, that of haptoglobin on plasma.

Phenotypes were determined by electrophoretic methods. Hemoglobin electrophoresis was performed on agarose gels at alkaline and acid pH [13] . Haptoglobin electrophoresis was performed on a non-denaturing 5% polyacrylamide vertical gel. Migrations were revealed by the peroxidase activity of the haptoglobin-hemoglobin complex [14] .

Probabilities of events were determined, and margins of error were calculated using statistical tests of the IBM SPSS^TM v18.0.0 software. Descriptive analyzes described the profile of the studied population. The statistical parameters of the associations between the haptoglobin phenotype and the elements of morbidity or the different phenotypes of SCD were the Pearson’s chi-square test, the odd ratio determined from binary logistic regressions and contingency tables on which were applied the Cochran-Mantel-Haenszel decision test.

A result was considered statistically significant for a p-value < 0.05.

In the cohort, 63.5% were female. Patients’ age ranged from 1 to 67 years. The mean age was 18 and the median was 14. The age distribution was skewed to the right (skewness of 1.30). In the population, 85% was literate.

Clinical disorders of SCD were heterogeneous but the more frequent ones were non-vaso-occlusion hematological disorders (42.4%), non-malarial infectious syndromes (42.3%), vaso-occlusions (28.8%) and severe malaria (18.8%).

The distribution of different hemoglobin phenotypes in the sickle cell population was as follows: homozygous sickle cell disease SSFA₂ (36.5%), hemoglobinosis SC (26.5%), sickle cell-β⁺ thalassemia SAFA₂ (14.1%) and sickle cell-β˚ thalassemia SAFA₂ (22.9%).

The major phenotypes of haptoglobin polymorphism were found in our SCD cohort. Distribution of haptoglobin phenotypes in sickle cell disease population was as follows: Hp 1-1 (24.1%), Hp 2-1 (56.5%), Hp 2-2 (19.4%). The phenotype Hp 0-0 was not found.

Using adjusted logistic regressions, relationships were sought between occurrence of vaso-occlusions, infectious syndromes or hemolytic crises (considered as dependent variables) and explanatory variables of the study (haptoglobin phenotype, hemoglobin phenotype, education level, vaccination status, etc…).

Previous studies have matched the electrophoretic fingerprint of phenotyping with the PCR method of haptoglobin genotyping [15] . Therefore, the electrophoretic phenotyping of haptoglobin makes it possible to highlight the phenotypic polymorphism of the haptoglobin gene.

SCD is a genetic disorder with variable morbidity and mortality depending on the genetic profile and the quality of medical care [16] [17] . This contributes to a right-side skewed age distribution.

According to many studies, the average age of homozygous sickle cell disease (Hb SS) is 25 to 27 years old [18] [19] . However, the average age of our population was 18 years. This difference could be explained on the one hand, by the phenotypic heterogeneity of Hb in our population (SSFA₂, SFA₂, SAFA₂, SC) and on the other hand, by possible differences in the quality of medical follow-up.

Morbidity disorders were dominated by hematological disorders and infectious syndromes. It is known that the literacy rate lowers morbidity [20] [21] . However, morbidity in our study, specifically the frequency of the infectious syndrome, is high despite a literacy rate of 85%. Factors other than the literacy rate seem to be associated with this morbidity.

In the studied population, the clinical and biological symptoms of SCD, even though varied, were dominated by a pathophysiology inducing blood transfusions, vaso-occlusions and infectious syndromes like in previous studies [22] [23] .

Phenotyping by the electrophoretic method detects the Hp 0-0 phenotype, but it does not fit with the differentiation of the Hp 0-0 phenotype by acquired hypohaptoglobinemia from the Hp 0-0 phenotype by congenital anhaptoglobinemia. However, the Hp 0-0 phenotype was not found in our population, although described in black African populations [24] .

Although one of the roles of haptoglobin is to inhibit extracorpuscular hemoglobin, no direct association between the hemoglobin phenotype and the haptoglobin phenotype was revealed in the studied population. However, in multivariate analyses, hemoglobin phenotype-adjusted morbidity appeared to vary with haptoglobin phenotype in SCD (Table 6). When the hemoglobin phenotype was not homozygous, morbidity was lower, with a greater probability for the haptoglobin Hp 2-1 and Hp 2-2 phenotypes. Thereby, the conjunction of a heterozygous SCD (non-SSFA₂ phenotype) and a haptoglobin phenotype different from Hp 1-1 (Hp 2-1 or Hp 2-2) appeared to be a better prognostic factor (based on the morbidity score). On the contrary, in Meher’s study, whose population was only homozygous sickle cell patients (Hb SSFA₂), it was the Hp 2-2 phenotypes that had the worst prognosis [25] . Like our results, Fotsing also showed, in a population of homozygous sickle cell subjects, that subjects with the Hp 1-1 phenotype had a greater tendency to oxidative stress than Hp 2-1 subjects [26] . Since several studies present the Hp² allele associated with phenotypes of lower antioxidant activity, our results suggest further research to understand the reason why the Hp¹ allele is associated with greater morbidity in this study.

The association between haptoglobin phenotype and SCD morbidity may involve other factors not considered in the present study. Simultaneous description of the genetic profile, immunoinflammatory status, and haptoglobin phenotype could enlighten the determinants of morbidity in SCD and other similar genetic conditions.