High Hb F level is associated with the risk of cerebral vasculopathy in a cohort of sickle cell children in Mayotte.

Background Understanding the genetics underlying the heritable subphenotypes of sickle cell anemia, specific to each population, would be prognostically useful and could inform personalized therapeutics. Methods This study aimed to describe the genetic modulators of sickle cell disease in a cohort of pediatric patients followed in Mayotte between 2007 and 2017. Results We included 190 children with 72% SS, 16% Sβ0-thalassemia and 12% Sβ+ thalassemia. The mean age was 9.5 (1.6-23.4) years; 10% of patients were lost to follow up. The Bantu haplotype was associated with an increase in hospitalizations and transfusions. The alpha-thalassemic mutation was associated with a decrease of hemolysis biological parameters (anemia, reticulocytes), and less cerebral vasculopathy. The SNPs BCL11A rs4671393, BCL11A rs11886868, BCL11A rs1427407 and HMIP rs9399137 were associated with the group of children with HbF> 10%. The survival analysis without occurrence of cerebral vasculopathy showed that the group of patients with HbF> 10% presented a significant risk of early onset of cerebral vasculopathy. Conclusions Although the sub-phenotypes were difficult to clearly distinguish in our study, a genotype-sub-phenotype link is emerging.


Background
Sickle cell disease (SCD) refers to a group of autosomal recessive genetic disorders characterized by the synthesis of an abnormal hemoglobin: sickle hemoglobin S (β s , HbS), results fromthe substitution of a single amino acid (Glu→Val) at the sixth position of βchain of normal hemoglobin (HbA) molecule [1,2]. This single-point mutation leads to the polymerization of the HbS molecule and red cell sickling under deoxygenated conditions.
Homozygous SS (sickle cell anemia or SCA) is generally considered the most severe form of SCD. Compound heterozygotes, in whom HbS is combined with a different mutation in major genetic modulator of the hematologic and clinical features of SCD [8]. Clinical manifestations of sickle cell anemia are generally not apparent until the switch from HbF to HbS occurs after the first months of life. This beneficial effect of HbF has been noted in patients who are compound heterozygotes for HbS and for hereditary persistence of fetal hemoglobin, or for other genetic variants of sickle cell anemia with elevated HbF levels. Fetal hemoglobin genes are genetically regulated, and the level of HbF and its distribution among sickle erythrocytes is highly variable [9,10].
This article aims to describe the genetic modulators of SCD in a cohort of pediatric patients followed in Mayotte between 2007 and 2017.

Study design
This cohort study was conducted between 2016 and 2017 at the Center for SCD of the Mamouzou General Hospital in Mayotte.

Patients
Homozygous SS or Compound heterozygotes Sβ sickle cell patients, younger than 18 years, who were receiving regular treatment at the Center for SCD of the Mamouzou General Hospital in Mayotte, were prospectively included in the database, after giving a statement of patient's non opposition. For 50% of them, SCD has been diagnosed by universal neonatal screening.

SCD clinical history
For each patient, basic information and specific clinical complications were collected from the medical records: age, gender, hemoglobin type, alpha and beta globin genotype, beta globin haplotype, basal HbF level, basal Hb level, glucose-6-phosphate-dehydrogenase (G6PD) status, UGT1A1 gene mutations status, severity and number of prior acute or chronic sickle cell specific complications (acute splenic or hepatic sequestration, acute chest syndrome, sickling related painful vasoocclusive crisis (VOC), neurologic events, severe infections, acute anemia, cholelithiasis), use of opioids for painful events,hydroxyurea treatment, number of transfusions ( or exchange transfusions), and number of hospitalizations.

Definitions
VOC: a painful event that required hospital treatment. Hemolytic crisis: decreases in the concentration of hemoglobin (Hb) and hematocrit. Handfoot syndrome: swelling in the hands and feet with pain and/or local heat, which may also be associated with a decrease in Hb concentration. Infection: fever accompanied by prostration and leukocytosis, with or without other laboratory tests and imaging. Acute splenic sequestration was defined as a sudden increase in the spleen size associated with pain in the left upper quadrant, a decrease in the hemoglobin concentration of at least 2 g/dL and in thrombocytes number.
Acute hepatic sequestration was defined as a sudden increase in liver size associated with pain in the right upper quadrant, a decrease in the hemoglobin concentration of at least 2 g/dL, and more abnormal results of liver-function tests not due to biliary tract disease.

Sickle cell haplotypes
Having at least one Bantu allele concerned almost all of our study population. The patients with Bantu / Bantu haplotype had significantly lower hematocrit, higher MCV and MCHC. They were hospitalized and transfused more often. Such patients did not carry the Xmn1 Single Nucleotide Polymorphism (SNP). Regarding the Benin haplotype, it was associated with a high hematocrite and a higher HbF, and a lower MCV. Patients with at least one Benin allele had more neonatal screening and were less hospitalized.
Homozygous Benin was rare (4 cases) and associated with a lower HbF and a high MCHC.
It also was significantly correlated with pathological transcranial Doppler, longer follow-up period, and iron chelation treatment.

Alphathalessemia
Our sickle cell patients with at least one alpha-thalassemic mutation had a significantly higher hemoglobin and hematocrit. They had a lower level of reticulocytes and a lower MCV. The absence of this mutation was significantly associated with cerebral vasculopathy and more frequent RBC transfusions.
Single nucleotide polymorphism (Table 1) The presence of Xmn1 in our cohort was significantly associated with male sex, higher hemoglobin and hematocrit levels, decreased leukocytes, and a higher splenic ratio.
Having two favourable SNP alleles rs4671393 was significantly associated with higher hemoglobin and hematocrit, and a higher HbF for patients under HU treatment, as well as lower HbS. Patients with at least one favorable rs11886868 allele had higher hemoglobin and hematocrit, Patients with at least one favorable rs1427407or rs939913 alleles had higher HbF level.The favourable rs10189857 allele was associated with a low hemoglobin and hematocrit and high leucocytes. Patients with the favourable rs28384513 allele were more frequently diagnosed with the neonatal screening test. The absence of TAC deletion at SNP rs66650371 was significantly associated with higher mortality.

UGT1A1 gene mutations status
The search for a biological, radiological or clinical association with Gilbert's disease did not find any significant result.

Hemoglobin F
According to our working hypothesis, the HbF level could direct us towards a subphenotype of the disease. We therefore looked for a HbF value to determine these two sub-phenotypes. We performed a ROC curve for HbF which allows to distinguish two groups of 101 and 88 patients ( Table 2). The survival analysis without occurrence of cerebral vasculopathy showed that the group of patients with HbF> 10% presented a significant risk of early onset of cerebral vasculopathy, the main complication of the hemolytic sub-phenotype ( Figure 3). The group with low HbF was associated with vasoocclusive complications. Homozygous Bantu patients in the HbF group> 10% were less hospitalized (p= 0.002), less transfused (p =0.025), had less VOC / year (p= 0.039), but they had more cerebralvasculopathy(p= 0.023) than those with <10% HbF. Homozygous Bantu patients in the HbF group <10% had less cholelithiasis (p= 0.021).Patients in both groups, when they carried one or two Benin haplotypes, were less hospitalized (p= 0.002), had less VOC per year (p= 0.039) and their 1st VOC occured less early (p= 0.03) than those that did not have any Benin haplotypes. Patients who do not carry a Benin allele were more transfused (p= 0.018) than those who did. The alpha-thalassemic mutation was associated with an increase in hemoglobin level in patients at risk of vasculopathy (p= 0.023), and an increased leukocyte rate (p= 0.001). Children in the group with an alpha mutation were hospitalized less often (p= 0.004) and were less ikely to havecholelithiasis (p= 0.041) than other children. Children in the <10% HbF group who carried an alpha mutation received fewer transfusions than those >10% (p= 0.048). Multivariate analysis did not find any independent genotypic marker. However, some SNPs were close to significance: BCL11A rs1427407 (p= 0.051) and BCL11A rs11886868 (p= 0.06). BCL11A rs4671393 (p= 0.2) and HMIP rs9399137 (p= 0.24) were not independently associated with the phenotypic groups.A concordance chi-2 test found preferential associations between some SNPs ( Table 3). The linkage imbalance between BCL11A rs66650371 and rs9399137 was highly significant for a large number.

Genotype
Our study population was characterized by the predominance of sickle cell anemia, with a severe clinical presentation [12], followed by the compound heterozygous HbS/β thalassemia. The Bantu haplotype, accounting for 80% of the alleles, reflects the East African origin of the Mahoran population [13]. Compared to the previous study conducted in Mayotte [14], haplotypes seemed to diversify: 64.9% of homozygous Bantou in 2017, against 88% in 2011. The Benin, Cameroon and Senegal haplotypes appeared or became more frequent in the past six years. This result should be taken with caution because the inclusion criteria were not the same, the previous study only considering children who had been diagnosed by the neonatal screening. The Bantu haplotype was associated with an increase in hospitalizations and transfusions in our population. It was not directly related to a particular phenotypic group, but increased the risk of cerebral vasculopathy in patients with HbF> 10%. It was probably difficult to highlight a statistical link because of its very high frequency in our population.The Bantu haplotype is classically associated with a more severe prognosis, and appears to be related to greater hemolysis in a study comparing Jamaican and Ugandan populations, and in another involving a Brazilian cohort [15,16]. The Benin haplotype was associated with the vasooclusive phenotype in our study. It corresponded to more severe phenotypes than other haplotypes (Senegal, Arabo-Indian), but is not known to be associated with the risk of cerebral vasculopathy. The G6PD mutation was not associated with biological parameters of hemolysis, but appears to be related to vascular complications. Girls with that mutation also had this risk. This link was not found at the level of phenotypic groups.The studies on this subject obtained different results: G6PD deficiency leads to a hemolytic phenotype according to some Our study investigated three mutations, but did not collect the molecular and clinical expression of G6PD deficiency. It did not take into account the possible presence of other mutations, and possible chromosomal inactivation by lyonization. It would be interesting to specify the residual enzymatic activity and the clinical complications presented by the patients.

HbF associated with a high risk of cerebra vasculopathy
Our survival analysis without occurrence of cerebral vasculopathy showed that the group of patients with HbF> 10% presented a significant risk of early onset of cerebral vasculopathy. Even if predicting sickle cell severity is complex, stroke appears to be the

SNPs is associated with high Hb F level
Investigation of genetic variants has identified several genes as principal influencers of HbF regulation. In our study, the alleles BCL11A rs1427407, HMIP rs4895441 and HMIP rs9399137 were significantly associated with an increase in HbF. In the literature, these The mechanism of action is not explained. The association of SNPs with HbF varies between populations of different origins, so some SNPs have no effect in some populations. This was the case for Xmn1 in our cohort, which may have resulted from its rarity. A study comparing two cohorts of European and African origin observed differences in allele frequency and correlation with HbF [38]. Another study, conducted in Cameroon, showed identicallelic frequencies between a Cameroonian population and the African-American cohort, but a lower impact on HbF among Africans [39]. These results show the interest of looking for SNPs in a given population by performing GWAS, and not simply extrapolating the polymorphisms found in another population. The African continent in particular could benefit from more GWAS polymorphisms Xmn1. BCL11A rs4671393 and BCL11A rs11886868 are associated with elevated hemoglobin. This result is found in other African studies [36,40]. HMIP rs66650371 is correlated with a decrease in mortality on a small population in our cohort, which is not reported (to our knowledge) in the literature.
The most remarkable result of our study was the association of SNPs with the phenotypic groups that we aimed to determine. BCL11A rs4671393, BCL11A rs11886868, BCL11A rs1427407 and HMIP rs9399137 were correlated with the HbF group> 10%, which presents a higher risk of cerebral vasculopathy and would be oriented towards the hemolytic subphenotype. BCL11A rs1427407 was the most strongly associated in our population, which corresponds to its strong correlation with HbF found in the Tanzanian GWAS [36]. HMIP rs9399137 is the HMIP polymorphism most strongly associated with HbF levels in African populations [41]. Multivariate analysis found no independent association of these SNPs with clinical profiles, BCL11A rs1427407 being close to significance. There are therefore unknown factors (interactions, intermediate factors, or other SNPs in linkage disequilibrium) that intervene in this genotype-phenotype correlation HMIP rs66650371 was not associated with either HbF or a phenotypic group in our cohort. This deletion of 3 bases, in linkage disequilibrium with rs9399137 in the literature as in our study, is located at the binding sites of four essential transcription factors in erythroid differentiation. It inhibits the expression of MYB, and thus leads to both an acceleration of differentiation (responsible for an increase in HbF) and a decrease in erythrocyte proliferation (which could cause a decrease in hemoglobin) [25,41,42]. These two effects could explain the lack of correlation with the clinical phenotype. The favorable SNP rs66650371 is less common in African populations and particularly in our cohort, which may also explain the lack of observed link.We also did not find any clinical phenotypic association for the SNP Xmn1, which is also infrequent in our population. This geno-phenotypic clinical association in sickle cell disease is interesting because it is poorly described in the literature. In 2008, Lettre found a significant link between the association of 5 SNPs (BCL11A rs4671393, HMIP rs28384513, rs9399137 and rs4895441, and XmnI rs7482144) and the reduction of VOCs in the SCD cohort [43]. These SNPs are also associated with a less severe clinical phenotype in an other pathology of hemoglobin, beta-thalassemia [44]. The results of Lettre and other studies show a stronger geno-phenotypic correlation when several SNPs are associated [43,45]. It would therefore be interesting to study the link between these sets of specific polymorphisms and the sub-phenotypes of sickle cell disease. In conclusion, our study found an association between some SNPs and the risk of cerebral vasculopathy; this link depends on the frequency of the polymorphism, the correlation rate according to the population, and could be amplified by the association of these SNPs.

The alpha-thalassemic mutation is a vaso-occlusive profile
The alpha-thalassemic mutation was associated with a decrease of hemolysis biological parameters (anemia, reticulocytes), and less cerebral vasculopathy. In the literature, it is also associated with fewer vascular complications [28,46]. This mutation decreases the parameters and complications of hemolysis in the at-risk group of vasculopathy. It protects against vascular complications, even in patients who are at high risk.This is due to the decrease in HbS concentration in erythrocytes, which leads to a decrease in

Limitations and interests of our study
Our determination of the sickle cell sub-phenotypes from the HbF level did not yield the expected result, although some trends have emerged. Difficulties in monitoring the Mahoran pediatric patients lead to poor control of environmental prognostic factors. This may have impacted some results of our study. However, we relied on the fact that environmental factors do not appear to affect the type of expression of the disease [14].The number of missing data, which is too high for some parameters, requires further study. The studied SNPs were not, for some, the most frequent or the most strongly associated with the HbF level in an African population. GWAS and genotype-phenotype correlation research must be adapted to different types of populations for a better global understanding of SCD. Our results, to be further developed, could make it possible to predict early (in utero or during the neonatal period) the type of complication that the sickle cell child will present, and thus to predict the type of surveillance and treatment required for each patient. They could help in the decision of intensive interventions such as bone marrow transplantations.

Conclusion
Our study allowed a description of the Mahoran pediatric population, reflecting the need to continue to improve monitoring. In our cohort, the SNPs BCL11A rs4671393, BCL11A rs11886868, BCL11A rs1427407 and HMIP rs9399137 were associated with the group of children with HbF> 10% between 2 and 10 years, and which seemed to present a high risk of occurrence of cerebral vasculopathy. This link was not found independently for each SNP. Beta-globin haplotypes and alpha-thalassemic mutations also influence the clinical expression of the disease. Although the sub-phenotypes were difficult to clearly distinguish in our study, a genotype-sub-phenotype link is emerging. Flow chart describing how the cohort was identified.