Though β-thalassemia and sickle cell disease are single-gene disorders with prototypical Mendalian inheritance patterns, both the disorders display a wide spectrum of clinical phenotypes. Thus, the search for the genetic modifiers was triggered, as 5–10 % of β-thalassemia homozygous patients with the same β-globin gene mutation and sickle cell anemia patients showed a variable pattern of clinical expression [9].
In this study, we first classified the β -thalassemia patients according to the clinical severity and then studied the influence of the genetic modifiers. Modell and Berdukas 1984, reported that 60 % of β-thalassemia homozygous patients presented in the first year of life, these patients were segregated as β- thalassemia major and 9 % of the β-thalassemia homozygous patients who presented after 2 years of age, with intermediate clinical severity were classified as β- thalassemia intermedia [10,11]. A similar observation was made in our study, in which the β-thalassemia major patients presented early by 9.2 ± 2.7 months and the patients in the β-thalassemia intermedia group had a delayed age of presentation mean of 4.3 ± 3.3 years. The β-thalassemia intermedia patients also showed a significantly higher mean baseline hemoglobin of 7.8 ± 1.4 g/dL as compared to thalassemia major patients. Similarly, another study showed that in 63 β-thalassemia intermedia patients, the hemoglobin values ranged between 7–9 g/dL with occasional transfusion regimen and splenomegaly [12]. In our study as well, pronounced hepatosplenomegaly was observed in β-thalassemia intermedia patients as compared to β-thalassemia major. Mpalampa L et al., 2012 considering the mean HbF cut-off as 10 %, in 216 sickle cell anemia patients observed a strong negative correlation of HbF levels with the total number of transfusions (r = − 0.181, P: 0.004), hospitalisations rate (r = − 0.173, P : 0.006), and significant positive correlation with the age at diagnosis (r = 0.151, P : 0.013) [13]. In the Indian context, Nayak et al., 2018 studied 60 sickle cell anemia patients and observed fewer episodes of painful crises in children with high baseline HbF level as compared to children with low HbF level [14]. Correspondingly in our study as well, the mean age of diagnosis among 100 SCA patients was found to be 6.3 ± 5.2 years which is very much delayed as compared to the patient cohort studied by Mpalampa L et al., 2012 This observation could be due to inherently elevated HbF levels in Indian patients mainly due to Arab-Indian haplotype which is a major determinant of HbF levels in Indian SCA patients [15]. Further, it was observed that the patients with higher HbF level had a delayed age of presentation (7.1 ± 5.5 years) with less transfusion requirement and sporadic painful crisis compared to patients with HbF level ≤ 17.4 % (age of presentation: 5.2 ± 4.9 years).
As the β-thalassemia alleles inherited by the patient act as a primary modulator of the disease severity in β-thalassemia, Colah et al., 2004 observed that the milder mutations are prevalent in β-thalassemia intermedia group as compared to severe β-thalassemia major patients [16]. Similarly in our study, the presence of milder β-thalassemia alleles were significantly higher in β-thalassemia intermedia as compared to β-thalassemia major patients [P: 0.004, Odds Ratio: 8.6 (95 % CI: 1.9–37.9)]. However, Garewal et al.,2007 described that, in majority of the Indian patients, the beta genotype alone cannot predict the clinical phenotype of the patients [17]. A similar observation was seen in our patients, which suggested the presence of other genetic factors that may play a synergistic role in modifying the disease severity of β-thalassemia. In a previous study by Nadkarni et al., 2001, the associated α-thalassemia was found to be significantly higher in the thalassemia intermedia group (37%) as compared to β-thalassemia major group (5%) (P < 0.025) [18]. A study by Pandey et al., 2011 revealed 32 % sickle cell anemia patients with co-existing α-globin gene deletion, showed a relatively milder clinical course with improved hematological indices and reduced transfusion history [19]. Similarly, Rumaney et al., 2014 observed that in Cameroon sickle cell disease patients, co-inheritance of α-thalassemia showed improved hematological indices with a better survival rate [20]. Similarly in this study, we observed that the coinheritance of α-thalassemia was higher in the milder β-thalassemia patient group as compared to the other group. 51 % of SCA patients also showed presence of α-thalassemia.
The effect of the genetic modifiers of fetal hemoglobin was also analysed in this study. A study in the Egyptian β-thalassemia patients showed that 83.3 % of β-thalassemia intermedia cases were heterozygous for XmnI polymorphism as compared to β-thalassemia major (57.6 %) and that β-thalassemia intermedia with single T allele of XmnI showed delayed age of diagnosis, raised HbF levels and milder disease phenotype as compared patients negative for the XmnI polymorphism [21]. In another study, it was also determined that the patients with homozygosity for the mutant T allele of XmnI polymorphism significantly showed higher mean HbF levels (85.5 ± 6.8 %) as compared to the thalassemia intermedia patients homozygous for XmnI CC genotype (19.5 % ± 29.3) [22]. A similar result was observed in our patient group where in the β-thalassemia intermedia patients homozygous for variant allele T showed significantly higher HbF level.
+ 25 G→A polymorphism in Aγ-globin promoter region was found to be significantly associated with elevated HbF levels in the β-thalassemia intermedia group. This polymorphism was first reported by Bianchi et al., 2016 and a strong linkage of this polymorphism with the − 158 C→T (XmnI polymorphism) was observed in their study as well [23]. It has been reported that + 25 G→A polymorphism reduces the binding efficacy of LYAR transcription factor (repressor of γ-globin gene expression) and abolishes the binding of 2 negative epigenetic regulators [DNA methyltransferase 3 alpha (DNMT3A) and protein arginine methyltransferase 5 (PRMT5)] to this promoter region [23, 24]. Thus, it could be speculated that there could be a cumulative effect of mutant alleles of both XmnI polymorphism (T allele) and + 25 G→A polymorphism (A allele) in synergistically elevating the HbF levels.
The association of BCL11A polymorphisms with elevated HbF levels and their effect on amelioration of the disease phenotype was studied by Uda et al., 2008 in Sardinian β-thalassemia homozygous patients [25]. They showed that the mutant C allele of rs11886868 (C→T) formed the major allele in Sardinian population and was significantly associated with elevated HbF levels in β-thalassemia intermedia patient group. Similarly, in Indian patients Dadheech et al., 2016, determined that the C allele was significantly associated with the raised HbF levels and delayed the age of presentation in both thalassemia homozygous and SCA groups [26]. In our study, the mutant CC genotype was found to be significantly associated with HbF levels only in the sickle cell anemia patients.
Similarly, in Indonesian HbE-β-thalassemia patients inheriting variant alleles of rs11886868, rs766432 in the BCL11A gene, showed higher HbF levels and reduced disease severity as compared to patients with wild type alleles [27]. The second SNP that was found to be significantly associated with the HbF levels is rs1427407 (G→T) polymorphism in the BCL11A gene. Our results were found to be consistent with the earlier report by Bhanushali et al., 2014, who showed a similar distribution of allelic frequency of rs1427407 in Indian SCA patients [28]. Studies have demonstrated that the patients with the mutant T allele of rs1427407 (G→T) showed significantly higher HbF level, the results of which are concordant with our study, thus suggesting a crucial role of this SNP in modulating the HbF levels [28, 29].
Similarly, Chaouch et al., 2016 observed that co inheritance of the mutant C allele of the rs11886868 and the mutant A allele of the rs46713939 ameliorated the clinical phenotype of SCA patients [30]. In our study, though the A allele of rs46713939 was found to be higher in the TI group, no significant difference in the allelic frequencies among the milder and severe groups could be observed. Studies have identified a restricted 14 kb region in BCL11A intron 2 to be associated with H3-acetylation, RNA pol II activity as well as a strong GATA-I, TAL-1 binding site, all of which indicated the presence of a regulatory sequence in this region [31, 32]. Thus, suggesting that the presence of a polymorphism could potentially alter the recruitment of transcription factors to this region.
Similarly, in our population, a 100 % linkage was observed between rs66650371 and rs9399137 polymorphism. A similar observation was seen in the Tanzanian SCD patients where, both these polymorphisms in HMIP 2A block were strongly associated with HbF levels and showed a strong linkage [33]. Similarly, Lai et al., 2017 in β-thalassemia intermedia patients showed that the mutant alleles of rs9376090, rs7776054, rs9399137, rs9389268, rs9402685 in the HbS1L-MYB intergenic region and rs189984760 in the BCL11A locus, showed significant association with high HbF level [34]. Bioinformatic characterization of the 3 bp deletion polymorphism showed that this region acts as a binding site for 4 transcription factors TAL1/SCL, E47, GATA-2 and RUNX1/ AML1 all of which are important erythroid differentiation, erythropoiesis and the presence of the mutant allele may also disrupt the MYB gene expression, which is a negative regulator of the γ-globin gene [35, 36].
In our study, it was observed that in SCA patients, the prevalence of the T allele of rs1427407 (G→T) and the 3 bp deletional allele of rs66650371 both were significantly higher in the SCA patient group possessing higher HbF levels (HbF > 17.4 %) as compared to the SCA patients, with lower HbF level (HbF ≤ 17.4 %). A similar result was shown by Adeyemo et al., 2018, where in patients with the mutant alleles of these 2 polymorphisms had a milder form of the disease, with improved hemoglobin levels [37].
Similarly, in β-thalassemia homozygous patients, the cumulative effect of 3 HbF associated mutant alleles of the SNPs − 158(C→T), rs11886868 (C→T) and rs1427407 (G→T), were observed significantly in the β-thalassemia intermedia group as compared to β-thalassemia major group. A comparable result was reported by Allawi et al., 2015, where they determined the main factors leading to milder phenotypes were the attenuated β-thalassemia alleles, the T allele of XmnI polymorphism and the minor allele of BCL11A rs10189857 [38]. Cardoso et al., 2014, studied the influence of three known major loci on the HbF trait (HBG2, rs748214; BCL11A, rs4671393; and HBS1L-MYB, rs28384513, rs489544 and rs9399137) in north Brazilian SCA patients and they showed that the raised HbF trait was primarily influenced by mutant alleles of BCL11A [39].
Further to predict the disease severity in presence of these genetic modifiers, Badens et al., 2011 studied 5 genetic modifiers of β-thalassemia. By regression analysis, all 5 types of favorable allele were found to be significantly associated with thalassemia intermedia phenotype. The β-globin gene mutations and XmnI polymorphism were the most influential modifiers of the disease severity [5]. A similar observation was reported by Danjou et al., 2012, wherein they further evaluated the age of the first transfusion with respect to the inheritance of HbF boosting alleles and observed that the age of transfusion was found to be delayed in presence of more number of HbF inducing alleles [40]. Similarly, in our study, it was observed that, 8% of thalassemia intermedia patients (HbF: 82.7 % ± 22.6) had inherited more than 10 disease ameliorating alleles as compared to none in thalassemia major patients. Thalassemia major patients (64 %) showed less number of disease modifying alleles as compared to the thalassemia intermedia patients (36%). Similarly, in SCA patients, the ameliorating alleles was found to be higher in the raised HbF group (HbF > 17.4) (31.3 %) (mean HbF: 26.2%±8.3) as compared to patients in low HbF group (HbF ≤ 17.4%) (4 %) (mean HbF: 12.1 %± 3.5).
These observations suggest that the presence of increased number of an ameliorating allele, may help in reducing the disease severity in hemoglobinopathy patients mainly by restoring the globin chain imbalance. The precise identification of the polymorphisms associated with elevated HbF levels may help in developing a molecular chip that may assist in predicting the disease severity.