Prevalence and Impact of HMOX1 Polymorphism (rs2071746: A > T) in Indian Sickle Cell Disease Patients

Introduction

Sickle cell disease (SCD) is a chronic lifelong debilitating disorder that begins in childhood and characterized by point mutation in the β-hemoglobin gene (HBB), resulting in substitution of adenine for thymine (GAG > GTG) at codon 6 of the β-globin gene [NM_000518.5 (HBB):c.20A > T (p.Glu7Val)]. This base pair substitution leads to change in amino acid valine for glutamic acid that results in polymerization of the abnormal sickle-shaped hemoglobin (HbSS)polymers, that is, sickle hemoglobin (HbS) especially when hemoglobin is in deoxygenated state. As the disease progresses for longer duration, there is permanent deformation of the shape of initially reversibly sickled red blood cells into the rigid irreversibly sickle cells that cannot pass through the microcirculation efficiently, resulting in anemia, cell adhesion, vaso-occlusion, and ultimately painful sickle cell crisis.^[1,2] The sickle cell gene is widespread across in India and it is believed that the occurrence of the HbS mutation in India is different and separate from those in Africa. This is known as the Asian haplotype.^[3]

Among the predictors of survival in SCD, fetal hemoglobin (HbF) levels play very important and significant role in these patients as targeted therapy with an aim to raise the HbF levels may be one of the foundation stone in lowering down the morbidity and mortality. HbF inhibits HbS polymerization and its abundance in the red blood cells dilutes down the concentration of HbS, thus leading to amelioration of the symptoms.^[4–6] Different studies have identified that high levels of HbF have a protective role by reducing the severity of SCD. HbF is present at residual levels in healthy adults with over 20-fold possible quantitative variation and studies have shown that 89% of this variation is heritable, although the genetic etiology for this is complex.^[7,8]

A wide range of complications has been reported in SCD inducing microvascular obstruction, abnormal adhesion of leukocytes and platelets, inflammation, and hypercoagulation. The sickle hemoglobin gene haplotype observed among various tribal populations in Central and Southern India is similar to that of the Asian-Indian haplotype and is associated with high HbF levels.^[9]

At present about 05% of the world's population are carriers of any one of the hemoglobin gene mutation, with predominance of heterozygous condition. Every year about 300,000 infants worldwide are born with thalassemia syndrome (30%) and sickle cell anemia (SCA) (70%). Although the percentage of carriers of thalassemia is greater than that of carriers of SCA globally, but because of the higher frequency of the sickle cell gene in certain regions, overall number of affected population is higher than that with thalassemia. Although the general incidence of β-thalassemia trait and SCD varies between 3 to 17% and 1 to 44%, higher consanguinity, caste and area endogamy result in some communities exhibiting greater incidence of SCD, thus making this disease a major public and genetic health problem in India.^[10,11]

As some genes in the β-globin gene cluster can affect disease pathology, other genes outside the gene cluster such as heme oxygenase-1 (HMOX1) and chemokine receptor 5 (CCR5) can modulate disease severity and subsequently reduce the severity of clinical events such as vascular occlusion, pain, chest syndrome, priapism, leg ulcers, and hepatobiliary complications.^[12–14]

HMOX1 gene, present on the long arm of chromosome 22, codes the heme oxygenase 1 (HO-1) enzyme, which acts in the breaking of the porphyrin ring of the heme to produce biliverdin and carbon monoxide.^[15] The HMOX1 gene is over expressed in sickle cell patients.^[8] Few studies have shown that polymorphisms of the HMOX1 gene, including rs2071746: A > T, cause an increase in the HbF level possibly lowering the disease severity. HO-1 also acts as a potential anti-inflammatory and cytoprotective enzyme that is activated by inflammation, hypoxia and ischemia, and plays an important role in establishment of hemostasis.^[16] The elevated oxidative stress in SCD is attributed to loss of HO-1 function because of HMOX1 polymorphisms that lead to excess of free heme in plasma, that auto-oxidizes leading to superoxide formation which results in free radical mediated inflammatory cascade ultimately augmenting the complications.^[17]

After thorough search in literature, we found that there have been very few studies that have addressed the effect of HMOX1 polymorphisms (HMOX1-413A/T (rs2071746) in SCD patients and hence, we planned this study to understand the prevalence of this polymorphism and its effect on HbF levels in Indian SCD patients.

Materials and Methods

Results

A total 120 SCD cases and 50 healthy controls were recruited. Out of the 120 cases of SCD, 65 (41 male and 24 female) cases with mean age of 11.91 ± 9.0 (range: 3–23 years) were HbSS, and 55 (34 male and 21 females) cases with mean age of 8.80 ± 7.87 (range: 3–19 years) were compound heterozygous for sickle-beta thalassemia (HbSβ). The mean age of controls was 11.5 ± 7.78 (range: 10–35 years). The hematological profile of cases and controls was given in ►Table 1. Most of the cases of HbSS had normocytic, normochromic anemia and those of HbSβ were microcytic, hypochromic anemia. Of the 65 patients of HbSS, 34 (53%) had mild clinical presentation, whereas 31(48%) had a severe clinical presentation with a history of vaso-occlusive crises and chest syndrome.

HMOX1 polymorphism was studied in all subjects. Out of 65 HbSS patients, 29 (44.61%) patients were heterozygous (AT), 20 (30.76%) were homozygous (TT), and 16 (24.61%) were found wild-type (AA) genotype. Out of 55 HbSβ heterozygous patients, 22 (40%) were heterozygous, 18 (32%) were homozygous, and 15 (28%) were wild type. The incidence of AA, AT, and TT genotype was 20(36%), 27(50%), and 8(14%) in controls.

A comparison of hematological parameters in different genotypes of HMOX1 polymorphism for HbSS and HbSβ is given in ►Tables 2 and 3, respectively. The mean HbF level was significantly higher in AT and TT genotypes when compared to AA (wild-type) genotype both in HbSS and HbSβ cases. The frequency of HMOX-1 polymorphism was higher in among sickle homozygous than sickle-beta thalassemia patients. The impact of genotypes on the clinical features is shown in ►Fig. 1. Clinical severity was improved with variant genotype in sickle cell anemia as well as sickle-beta patients. Painful crisis, jaundice, gall stone, and blood transfusion were lesser in mutant variant than nonvariant. HbF level was found higher in variant (TT) genotype than wild-type and was statistically significant (p-value <0.001). ►Tables 4 and 5 depict the allelic frequency of HbSS and HbSβ cases.

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Fig. 1

Comparative clinical parameter with carrier and noncarrier heme oxygenase-1 (HMOX-1) in hemoglobin sickle shaped (n = 65).

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Discussion

The phenotypic–genotypic diversity and genetic heterogeneity of SCD along with multifactorial influence complicates the assessment of prognostication in these patients. Various factors implicated in modification of this disorder include HbF concentration, β-globin gene cluster haplotype that will ultimately influence its clinical severity.^[18] Intracellular distribution of HbF is a strong modifier of SCD and studies have shown that elevated HbF levels tend to reduce the severity of SCD by reducing the sickling and its manifestations.^[19] Higher levels of HbF (mean 22.3 ± 6.9%; range: 3.9–40.9%) are found in Indian SCD patients.^[20] The βS gene is linked to an Asian haplotype and the XmnI restriction site, but these factors alone cannot explain the high HbF concentration.^[21] Limited data is available regarding the co-expression of HbF modifier like HMOX1 with SCD and its influence on the disease severity and phenotype. To best of our knowledge, this is the first study in HbSS and HbSβ patients to investigate the prevalence and impact of HMOX1-polymorphisms on HbF levels and disease severity in Indian population.

HO enzyme encoded by HMOX1 gene located in long arm of chromosome 22 plays pivotal role common as an antioxidant enzyme in neutralizing the toxic free heme into three biologically active by products, that is, biliverdin, free iron, and carbon monoxide and plays a vital role in the protection of cells and DNA from reactive oxygen species damage. HMOX1 polymorphism rs2071746: A > T has been studied in various diseases to evaluate its role in ischemia and hypertension.^[22] However, there is sparse data on the association of this polymorphism with SCD as a result of its impact on HbF. βs globin gene haplotypes are evaluated to assess the severity of disease. Some studies have shown that SNPs in some genes in HBBP1 and out of HMOX1 β-globin gene cluster can affect disease severity.^[23,24]

A previous study from Egyptian population in year 2019 reported the prevalence of HMOX1 polymorphic AT and TT genotypes in patient and control group as 92 and 85% respectively.^[25] Furthermore, a study in the Brazilian population in year 2020 showed that the HMOX1 rs2071746 genotype frequencies were 24.3% (AA), 48.6% (AT), and 27.0% (TT) in the SCA patient group and 28.2% (AA), 52.7% (AT), 19.1% (TT) in the control group.^[26]

In this study, 29/65 (44.61%) heterozygous, 20/65 (30%) homozygous, and 16/65 (24.61%) were found wild genotype. However, AT was the most common genotype in HbSS patients and healthy individuals with 44.61 and 50% prevalence, respectively. This was concordance with Jaseb et al.^[27] Out of 55 HbSβ, 22 cases (40%) were heterozygous, 18 (32%) were homozygous, and 15 (28%) cases were wild-type and this is in contrast to Bakr et al 2019, who reported a very low frequency of wild genotype (6.1%) and a much higher frequency of mutant genotype (93.9%) in Egyptian patients.^[26] Furthermore, our study results suggest that SCD patients harboring mutant HMOX1 had less frequent vaso-occlusive crises, less incidence of blood transfusion, stroke, and hospitalization with improved hematological as well as clinical parameters. This is also in accordance with the data reported by Bakr et al.^[26]

HMOX1 can ameliorate vaso-occlusion and prevent vascular inflammation via converting damaging heme into biliverdin and carbon monoxide molecules that have antioxidant, anti-inflammatory properties. Studies have shown that the T allele of rs2071746: A > T polymorphism leads to decreased expression of HMOX1 gene. There are two hypotheses regarding influence of this polymorphism on HbF levels; first this polymorphism in promoter region causes decreased production of HO protein, which increases the concentration of free heme in circulation. This leads to slow erythropoiesis and reduced production of erythroblasts which result in an augmented production of HbF, and second the homozygous mutant TT genotype may indirectly affect the genes associated with it, causing increased HbF production.^[28–30]

Our findings showed the higher levels of HbF in SCD patients with HMOX1 gene polymorphism that was in line with the results of Bhagat et al and Keikhaei et al.^[31,32] In our study population, TT genotype in HMOX1 gene (rs2071746: A > T polymorphism) had a significant correlation in relation to increasing HbF level in SCD (p < 0.001). This was in line with Gil et al 2013 and Jaseb et al, who found association of this genotype with increased levels of HbF (p = 0.0131, p < 0.019). However, the mean steady state HbF concentrations in our study was higher in the all three genotypes when compared to Bakr et al^[26] and Gil et al^[13] possibly due to the Arab-Indian haplotype of βS gene that is associated with higher HbF levels.

In conclusion, we found significant association between the TT genotype of the rs2071746:A > T polymorphism of HMOX1 gene, and increased levels of HbF. We suggest this polymorphism can be considered as novel marker related to HbF levels in Indian SCD patients so that it can serve as a new prognostic marker for guiding the clinicians to formulate management protocol among Indian SCD patients. However, further studies in larger subset of patients are suggested to strengthen our findings.