Genetic Polymorphisms of HBS1L-MYB (rs4895441 and rs9376090) in Egyptian Patients with Hemoglobinopathy

Objective: Study the HBS1L-MYB (rs4895441 and rs9376090) genetic polymorphisms in Egyptian patients with β-thalassemia major and sickle cell disease and its relation to Hb F and severity of the disease. Background: Hb F is a predominant modulator for the severity of β-thalassemia major & sickle cell disease. Genetic polymorphism in the intergenic region (HBS1L-MYB) between GTP-binding elongation factor HBS1L and myeloblastosis oncogene MYB on chromosome 6q is associated with high fetal hemoglobin levels. Subjects and Methods: 150 subjects were included in this study. For all studied groups: Complete blood picture and serum ferritin were evaluated. For patients, hemoglobin variants were separated by High-performance liquid chromatography. Genotyping of HBS1L-MYB (rs4895441 & rs9376090) was evaluated by real-time polymerase chain reaction technique using TaqMan probe. Results: AG, CT genotypes, and G, C alleles of HBS1L-MYB (rs4895441 & rs9376090) were significantly high in sickle cell patients [OR (3.400); 95% C.I (1.482 - 7.799)], (p = 0.003) & [OR (4.522); 95% C.I (1.854 -11.029)], (p = 0.001) respectively. Also, a significant association was detected between polymorphisms and disease severity. However, in β-thalassemia major, no significant association was detected. Conclusion: In sickle cell disease patients, Genetic polymorphisms in HBS1L-MYB (rs9376090 & rs4895441) affect the level of Hb F which could improve the prognosis of these patients.


Introduction
Hemoglobinopathies are inherited diseases caused by defects in globin chain synthesis; they include thalassemia syndromes, sickle cell disease & globin chain variants [1]. β-thalassemia results from numerous mutations or rarely deletions of the β globin gene on chromosome 11. These mutations primarily point mutations that affect transcriptional control, translation, and splicing of the Hb B gene and gene product [2]. β-thalassemia is characterized by chronic anemia and iron excess due to blood transfusion and increased gastrointestinal absorption [3]. A comparison of Egyptian β-thalassemia patients with their healthy peers revealed that the former reported lower growth parameters, lower physical, mental, schoolwork, and overall quality of life scores [4].
The intensity of β-thalassemia may be mitigated by the production of Hb F as it may alter the imbalance of the α/β globin chain [5].
Sickle cell disease (SCD) is considered as one of the commonest gene disorders of human beings. In Africa, more than 200,000 infants are born yearly with sickle cell anemia [6]. Sickle cell disease is an inherited genetic disorder, resulting from homozygous and compound heterozygote mutation in the β globin gene as a single base-pair point mutation (GAG to GTG) results in the substitution of glutamic acid (hydrophilic) to Valine (hydrophobic) in the 6 th position of the β-chain of hemoglobin resulting in hemoglobin S formation [7].
The increase of Hb F can decrease the severity of SCD because of its ability to inhibit the polymerization of Hb S [8].
Hb F expression is affected by several quantitative trait loci (QTL) within and outside the β globin gene cluster. The HBG2, BCL11A & HBS1L-MYB are well-known major modifier loci [9]. HBS1L-MYB genes are expressed in the precursor cells of the erythroid. HBS1L encodes a protein with apparent GTP binding activity and is included in different cellular processes while MYB encodes a transcription factor for erythroid differentiation in hematopoiesis [10].
Disturbance in the interval between the HBS1L and MYB suppresses MYB expression and gives rise to the increased level of Hb F [7]. Single nucleotide polymorphisms (SNPs) in the Gγ-globin gene promoter in BCL11A regions and HBS1L-MYB intergenic polymorphism (HMIP) are thought to be associated with Hb F levels. Strongly associated markers in HMIP block 2, are either in intron 1a (rs9376090, rs9399137, rs9402685 and rs11759553), or directly upstream of the 5 UTR of HBS1L exon 1a (rs4895440, rs4895441, rs9376092, rs9389269, rs9402686, rs11154792 and rs9483788) [11].
With the increasing prevalence of β-thalassemia and SCD among Egyptian children, regardless of traditional genetic factors, there are no data related to the association of HBS1L-MYB intergenic polymorphism with these conditions. Samples collection: Before the planned blood transfusion (for β-thalassemia major patients) and under complete aseptic conditions, 5 ml of venous blood were collected, and then divided as follows: 2 ml were collected into an EDTA tube for CBC and HPLC, another EDTA tube with 2 ml blood for genotyping of HBS1L-MYB gene. The remaining 1 ml was collected in a plain tube for measurement of serum ferritin level.
Laboratory investigations: Serum ferritin was done by the auto-analyzer (HITACHI Cobas e 411 High-Technologies Corporation Tokyo, Japan).

Determination of HBS1L-MYB (rs 9376090 and rs 4895441) Genotypes by Real Time-PCR
1) Genomic DNA extraction: [12] The genomic double-stranded DNAs were extracted from whole blood using

Statistical Analysis
Results were collected, tabulated, and statistically analyzed by an International Kruskal Wallis, Chi-square, Mann Whitney, Fischer's Exact, Post hoc tests were used. Two-sided P-value of ≤0.05 was considered statistically significant.

Results
In β-thalassemia major patients, age of disease presentation was ranged from (6 -11 months). Thirty-seven β-thalassemia major patients were on regular blood transfusion every 3 weeks, while thirteen of them were on regular blood transfusion every 5 weeks. In SCD, the age of disease presentation was (9 -36 months) and the numbers of crises were (1 -8/year). There was a statistically significant increase in serum ferritin in β-thalassemia than controls, also a significant decrease in Hb level in β-thalassemia compared to other groups (Table 1). HPLC for Hb distribution in β-thalassemia major and sickle cell disease patients showed a significant difference between the two groups regarding Hb A and Hb F ( Table 2).
The independent segregation genotype for the two SNPs in the controls was in agreement with the Hardy-Weinberg equilibrium (p > 0.05) as (AA) and (TT) were the wild types of HBS1L-MYB (rs4895441 and rs9376090) respectively. AG, CT genotypes and, G, C alleles of HBS1L-MYB (rs4895441 and rs9376090) polymorphisms, were significantly higher in sickle cell disease patients. Also sig-   In β-thalassemia major patients, there was no significant association between HBS1L-MYB (rs 9376090 and rs 4895441) genotypes and age of disease presentation, frequency of blood transfusion, splenectomy, Hb level, and Hb F (Table   4). Meanwhile, in the SCD group, CT genotype of HBS1L-MYB (rs 9376090) was significantly associated with a lower number of crises and lower Hb S level, however, no significant association was detected between this polymorphism and age of disease presentation and Hb F, while AG genotype of HBS1L-MYB (rs4895441) was significantly associated with lower number of sickling crises, higher age of disease presentation, higher Hb F and lower Hb S levels ( Table 5).

Discussion
β-thalassemia and SCD are serious autosomal hereditary disorders that occur in several populations around the world. β-thalassemia is associated with genetic mutations that contribute to a reduced or total loss of expression of the β-globin gene. While in the SCD, mutation of the β-globin gene induces alterations in protein structure [14]. In β-thalassemia, the accumulation of unbound alpha-globin chains precipitates in the erythrocyte precursors resulting in bone marrow destruction resulting in ineffective erythropoiesis. As well, excess unbound alpha-globin chains cause membrane damage in mature red thalassemia cells leading to hemolysis [15]. SCD pathogenesis starts when hemoglobin deoxy-sickle molecules are polymerized leading to the deformation of the red blood cell structure. Due to environmental and genetic factors, clinical phenotypes are extremely variable in both diseases, ranging from relatively mild to severe anemia [14].  General genetic HbF modifiers influence disease outcomes such as frequency of painful episodes, the incidence of end-organ complications, the efficacy of hydroxyurea therapy, and life expectancy [16]. Polymorphisms in the intergenic region between HBS1L and MYB are identified as HBS1L-MYB Intergenic Polymorphism (HMIP) is significantly related to variability in expression of Hb F .
The stimulation of Hb F expression can offer alternate treatment for the improvement of these diseases severity [17].

T. A. Omar et al. Open Journal of Blood Diseases
This research investigated HBS1L-MYB (rs 4895441 & rs 9376090) gene polymorphisms in Egyptian patients with hemoglobinopathy (Beta thalassemia major & sickle cell disease) and its relation to Hb F and severity of the disease.
In β-thalassemia major, age of disease presentation was (6 -11 months), 74% of patients were on regular blood transfusion every 3 weeks and 26% every 5 weeks. These results were in agreement with Tubman et al. [18] who reported that β-thalassemia major patients remain asymptomatic until 3 -6 months of age or more when Hb F production falls and adequate Hb A can't be produced. According to blood transfusion, Langhi et al. [19] indicated that β-thalassemia major patients received regular blood transfusion throughout life, usually administered every 2 -5 weeks depending on the transfusion needs of each individual. While in SCD patients' age of disease presentation was (9 -36 months) and the number of crises was (1 -8/year). Strouse, [20] reported that SCD patients during the first 6 months of life are protected by elevated levels of Hb F and when reduced symptoms of SCD appear and clarified that the frequency of crises is extremely variable as crises have many triggers as hypoxemia, dehydration & change in body temperature. In β-thalassemia major, the serum ferritin level was significantly high. Taher and Saliba [21] explained this increase in transfusion-dependent thalassemia patients by the process of iron loading secondary to transfusion therapy and in non-transfusion-dependent thalassemia patients by ineffective erythropoiesis, enhanced intestinal absorption, and hepcidin suppression.
In SCD, AG genotype & G allele of HBS1L-MYB (rs4895441) were significantly higher than controls. Also, AG genotype was significantly associated with higher age of disease presentation, lower number of crises, higher Hb F, and lower Hb S level. These results were in agreement with Cardoso et al. [11] who demonstrated a significant association between AG genotype in African SCD patients and higher Hb F level, better clinical picture, and lower incidence of pain.
Also, Menzel et al. [22] reported that polymorphisms in HBS1L-MYB (rs4895441) explained 9.2% of the variation in Hb F present in sickle cell disease patients.
No significant difference was observed between β-thalassemia major patients and controls regarding HBS1L-MYB (rs4895441) gene polymorphism and allele frequency and no association was detected between gene and disease severity (age of disease presentation, frequency of blood transfusion, splenectomy and Hb F). This agreed with Nguyen et al. [23] who explained that the high level of ineffective erythropoiesis observed in beta-thalassemia major could mask or inactivate the biological expression of HBS1L-MYB polymorphisms, another explanation that the levels of multiple cytokines and/or chemokines are much higher in beta-thalassemia patients than normal subjects, these small molecular weight substances in peripheral blood are likely to exert diverse effects on erythropoiesis including globin gene expression. On the contrary, Cyrus et al. [17] reported a significant association between beta-thalassemia major and HBS1L-MYB (rs 4895441 and rs 9376090) genetic polymorphism in Saudi Arabian patients. This difference may be due to marked heterogeneity in the preva-lence of the HBS1L-MYB allele among ethnic groups. In SCD, CT genotype & C allele of HBS1L-MYB (rs 9376090) were significantly higher when compared to other groups. Also, CT genotype was significantly associated with a lower number of crises & lower Hb S levels. Sales et al. [24] reported a significant impact of HBS1L-MYB (rs 9376090) polymorphism on sickle cell disease phenotype and the minor allele of HBS1L-MYB (rs 9376090) was associated with decreasing the odds of having a stroke by 3.7 folds. On the contrary, Wonkam et al. [25] reported a non-significant association between sickle cell disease severity and HBS1L-MYB (rs9376090) polymorphism. This difference may be due to marked heterogeneity in the prevalence of the HBS1L-MYB allele among ethnic groups.
No significant difference was detected between β-thalassemia major patients and controls regarding HBS1L-MYB (rs 9376090) gene polymorphism and allele frequency and no significant association was detected between gene polymorphism and age of disease presentation, frequency of blood transfusion, splenectomy, Hb level, the severity of disease nor Hb F. This result was in agreement with Yunyan et al. [26], while Cyrus et al. [17] reported a significant difference in HBS1L-MYB (rs9376090) genotyping between β-thalassemia major patients and controls.

Conclusion
Lastly, we concluded that the severity of certain genetic disorders could be controlled by the expression of many genes that could decrease disease severity. In SCD, genotypes and alleles distribution of HBS1L-MYB (rs4895441&rs9376090) were associated with lower sickling crises numbers, higher Hb F, and lower Hb S. So this finding supports the development of innovative therapeutic targets.

Recommendations
Other studies including a large sample size and more HBS1L-MYB gene SNPs to obtain more conclusive results about the distribution of the HBS1L-MYB gene in Egyptian patients with hemoglobinopathy are recommended.