Influence of Haptoglobin and Hemoglobin Phenotypic Polymorphisms on Sickle Cell Disease Morbidity

Objectives: Sickle cell disease (SCD) has a varied clinical and biological expression depending on the hemoglobin phenotype: SSFA 2 , SFA 2 , SAFA 2 and SC. Considering the antioxidant properties of the different haptoglobin phenotypes (Hp 1-1, Hp 2-1, Hp 2-2), it seemed relevant to know their influence on the morbidity of the different hemoglobin phenotype of SCD. Thus, the objective of this study was to identify associations between haptoglobin phenotype and morbidity of different SCD phenotypes. Methods: In a retrospective cross-sectional descriptive and analytical study, with a cohort of 170 black African carriers of hemoglobin S, in Ivory Coast, West Africa, hemoglobin and haptoglobin phenotypes were determined by electrophoretic me-thods. Results: The three major phenotypes of haptoglobin polymorphism were found in the SCD cohort: Hp 1-1 (24.1%), Hp 2-1 (56.5%), Hp 2-2 (19.4%). Vaso-occlusions were associated with haptoglobin phenotype Hp 1-1, (OR = 2.03; CI 95% = [1.06 - 3.9]; p < 0.05). Probability of worse morbidity score was 4.55 times greater for hemoglobin phenotype different from SSFA 2 (CI 95% = [1.43 - 14.44]) and the probability of having the Hp 1-1 phenotype was lower (CI 95% = [0.170 - 0.705]). Conclusions: Haptoglobin phenotype was associated to morbidity-adjusted hemoglobin phenotype. The study revealed a greater probability of a worse morbidity when the hemoglobin phenotype is homozygous. Unexpectedly, the worse morbidity is associated to


Introduction
Sickle cell disease (SCD) is the most widespread genetic disease in the world: the prevalence of the S gene varies within 2% and 30% depending on the population [1].It is associated with high morbidity and mortality.In Côte d'Ivoire, 12% of the population carries hemoglobin (Hb) S, making this disease a public health problem [2].
The morbid events of the pathology are due to polymerization of globular Hb which induces vascular occlusions and chronic hemolysis.This hemolysis exposes all tissues to the deleterious oxidative effects of hemoglobin [3].The most common hemoglobin phenotypes that experience Hb S polymerization are SSFA 2 , SFA 2 , SAFA 2 and SC.SCD has a varied clinical and biological expression depending on the patient and the hemoglobin phenotype [4].
Haptoglobin (Hp) is a protein with a potent antioxidant activity.The intensity of the antioxidant activity varies according to the haptoglobin phenotypes (Hp 1-1, Hp 2-1, Hp 2-2) [5].Since haptoglobin binds to extra-globular hemoglobin to attenuate hemoglobin deleterious oxidative stress on tissues, the morbidity of the major forms of sickle cell disease could depend on the phenotype of haptoglobin [6] [7].Consequently, persons with some haptoglobin phenotypes seem to be more sensitive to some diseases and/or they could have specific prognosis [8] [9] [10].To date, the determinants of the associations between the haptoglobin phenotype and the clinical and biological manifestations of sickle cell disease are unclear [11] [12].Therefore, it would be relevant to know the influence of haptoglobin phenotypes on the morbidity of the different profiles of SCD.
The objective of this study was to identify associations between the haptoglobin phenotype and the morbidity of sickle cell diseases.In a specific way, we described the distributions of the clinical and biological profiles of sickle cell disease patients according to their respective hemoglobin and haptoglobin phenotypes.

Study Design
Using a retrospective cross-sectional descriptive and analytical study, we determined the likely associations between the haptoglobin phenotype and morbidi-Advances in Biological Chemistry ty-adjusted hemoglobin phenotype in a SCD population.

Population, Variables and Definitions
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 (SSFA 2 ), sickle-β + -thalassemia (SAFA 2 ), sickle-β˚ thalassemia (SFA 2 ) 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.

Ethical Approval
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.

Phenotyping
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].

Statistics
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.

Social and Anthropological Description of the Population
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.

Interdependence of Studied Variables
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…).
In univariate analyses, no relationships were found between the occurrence of vaso-occlusions and respectively homozygous phenotype of sickle cell disease, severe malaria and vaccination status.However, relationships appeared between the occurrence of vaso-occlusions and respectively sickle-β + thalassemia phenotype (SAFA 2 ) and the infectious syndromes (Table 3).

Factors Influencing Occurrences of Infectious Syndrome
In univariate analyses, no relationship was found between the occurrence of infectious episodes and the haptoglobin phenotype, neither homozygous sickle cell disease.However, there were inverse relationships between the occurrence of an infectious syndrome and respectively the fact of being literate or the vaccination status (Table 4).

Factors Affecting the Occurrence of Acute Hemolytic Crises
In univariate analyses, there were significant relationships between the occurrence of acute hemolytic crises and respectively the hemoglobin phenotype, the occurrence of vaso-occlusive episodes or the occurrence of infectious syndrome (Table 5).

Relationship between Haptoglobin and Hemoglobin Phenotypes
In univariate analyses, no direct relationships appeared between haptoglobin phenotype and hemoglobin phenotype.
Multivariate analyses revealed that haptoglobin phenotype was associated to morbidity-adjusted hemoglobin phenotype.When the hemoglobin phenotype was not SSFA 2 (homozygous sickle cell disease), the probability of having a lower morbidity score (≤5) was 4.55 times greater.When the hemoglobin phenotype was not SSFA 2 (homozygous sickle cell disease), the probability of having the Hp 1-1 phenotype was lower (Table 6).

About Analytical Methods
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.

About the Studied Population
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 (SSFA 2 , SFA 2 , SAFA 2 , 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].

About Haptoglobin Phenotypes
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-SSFA 2 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 SSFA 2 ), 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 Hp 2 allele associated with phenotypes of lower antioxidant activity, our results suggest further research to understand the reason why the Hp 1 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.

Conclusion
The major haptoglobin phenotypes were found in the SCD population.An asso-ciation between morbidity and the haptoglobin phenotype appeared.In a SCD, there was a greater probability of presenting a worse morbidity when the hemoglobin phenotype is homozygous and when the haptoglobin phenotype is Hp 1-1.However, the associations found were not systematic and need further studies to provide more insight in the determinism of SCD morbidity.

Table 1 .
Variables for selection criteria and morbidity disorders.

Table 2 .
Distribution of vaso-occlusions according to haptoglobin phenotype.

Table 3 .
Relationships between vaso-occlusions and different explanatory variables.

Table 4 .
Relationships between the occurrence of infectious syndromes and different explanatory variables.

Table 5 .
Relationships between the occurrence of hemolytic crises and different explanatory variables.

Table 6 .
Distribution of haptoglobin phenotype according to morbidity-adjusted hemoglobin phenotype.