Dissecting the role of genetic modi ers of hemoglobinopathies: A futuristic approach towards precision medicine


 Introduction: Hemoglobinopathies though a monogenic disorder, show phenotypic variability. Hence, understanding the genetics underlying the heritable sub-phenotypes of hemoglobinopathies, specific to each population, would be prognostically useful and could inform personalized therapeutics. This study aimed to evaluate the role of genetic modifiers leading to higher HbF production with cumulative impact of the modifiers on disease severity. Materials and methods:200 patients [100 β-thalassemia homozygotes,100 Sickle Cell Anemia], and 50 healthy controls were recruited. Primary screening followed with molecular analysis for confirming the β-hemoglobinopathy was performed. Co-existing α-thalassemia and the polymorphisms located in 3 genetic loci linked to HbF regulation were screened.Results: The most remarkable result was the association of SNPs with clinically relevant phenotypic groups. The γ-globin gene promoter polymorphisms [-158 C→T,+25 G→A],BCL11A rs1427407 G→T,-3 bp HBS1L-MYB rs66650371 and rs9399137 T→C polymorphisms were correlated with higher HbF, in group that has lower disease severity score (P<0.00001), milder clinical presentation, and a significant delay in the age of the first transfusion.Conclusion:Our study emphasizes the complex genetic interactions underlying the disease phenotype that may be a prognostic marker for predicting the clinical severity and assist in disease management.

β-thalassemia and sickle cell anemia (SCA) form a major health burden in India as they cause a high degree of morbidity, moderate to severe hemolytic anemia with the carrier frequency varying from 3 to 17% among different population groups of India [1]. Both the diseases are caused by mutations in the β-globin gene and are inherited as an autosomal recessive single gene disorder. However, despite this apparent genetic simplicity, both the disorders display a remarkable spectrum of phenotypic severity. In βthalassemics, the primary determinant of disease severity is the type of β-globin gene mutation that the patient inherits. Severe β-thalassemia mutation (β 0 /β + ) may completely down-regulate β-globin chain synthesis. However, milder β ++ mutations, present in the conserved 5′ β-globin promoter region and 3′ untranslated region, may alter the mRNA expression, leading to the moderate synthesis of the β-globin chain [2]. It has been observed that patients inheriting the same β-globin gene mutations, display phenotypic heterogeneity. Thus, the clinical variability observed among the patients prompted the search for the additional genetic modulators of these diseases.
Several modi er genes have been identi ed which in uence the severity of hemoglobinopathies. The most common secondary modi er is the co-inheritance of α-thalassemia and the elevated levels of fetal hemoglobin (HbF), both of which directly reduce the globin chain imbalance. An interacting combination of α-thalassemia with hemoglobinopathy has been shown to alleviate the severity by reducing the intracellular precipitation of free alpha-globin chains in β-thalassemia and by reducing HbS concentration in SCA patients [3,4].
The possibility that the severity of hemoglobinopathies could be reduced by induction of HbF was realized as the symptoms of hemoglobinopathies are observed after 6 months of age after the birth when there is a gradual reduction in the HbF levels. Thus, the study of other secondary mechanisms related to the sustained production of HbF in adult life may be important. With the advent of genome-wide association studies, recently several unlinked genetic factors have been identi ed which elevate the HbF levels [5]. Four major quantitative trait loci (QTLs): the HBB locus, the BCL11A gene, the HBSB1L-cMYB inter-region along with KLF1 gene variations bring about 20-50 % variation in HbF levels in hemoglobinopathy patients [5]. Thus, in this study, we have screened for the presence of primary and secondary modi ers in hemoglobinopathy patients and have analysed the cumulative effect of these modi ers on the phenotypic variability in the patient group.

Selection of patients and healthy controls:
The study was approved by the National Institute of Immunohaematology-Institutional Ethics Committee and all methods were performed in accordance with the relevant guidelines and regulations. Unrelated 100 β-thalassemia homozygous and 100 SCA patients were selected for this study. 50 unrelated healthy adult blood donors with normal hematological indices, with no transfusion history and medication, were randomly selected. The blood samples were collected in K2 EDTA vacutainers after informed consent.
2.2 Primary screening and molecular analysis: Primary screening involved complete blood count analysis and hemoglobin electrophoresis on BioRad Variant II high-performance liquid chromatography to evaluate the concentration of different hemoglobin fractions.
Molecular analysis of the β-globin gene was rst carried out to con rm the hemoglobinopathy status in the patient samples by CRDB, AMRS PCR, or by direct DNA sequencing. α-globin gene deletions were detected by multiplex PCR. The γ-globin promoter region was screened by direct DNA sequencing. Five BCL11A polymorphisms which showed the highest correlation with HbF levels [rs11886868 (C→T), rs7557939 (A→G), rs4671393 (A→G), rs1427407 (G→T) and rs7606173 (G→C)] were analyzed by real-time SNP genotyping and by ARMS PCR. 2 polymorphisms in HBS1L-MYB intergenic region [rs66650371 (Intact 'TAC'→ Deletion 'TAC'), rs9399137 (T→C)] were screened by ARMS PCR and by DNA sequencing respectively.
The patients were clinically evaluated and the disease severity score was calculated based on the detailed clinical history of the patient [6,7].

Statistical analysis:
Statistical analysis of the data was performed using GraphPad version 6.01 software (Graph Pad Prism Inc, California, U.S.A). The hematological indices among different patient groups and normal controls are represented as Mean ± Standard Deviation (SD). Fischer extract test was used to compare the polymorphism distribution among the patients and the control groups. The comparison of the quantitative variables among the groups and between differing genotypes was carried out by unpaired non-parametric Mann-Whitney U test. The P-value ≤ 0.05 was considered to be statistically signi cant. Generalized Multifactor Dimensionality Reduction (GMDR) software version beta 0.9 was used to analyse the interaction among the SNPs in different patient groups. The Kaplan Meier survival curve analysis was performed to determine the age of presentation by considering the transfusion free survival among the patient groups.

Results:
On the basis of clinical history, the β-thalassemia patients were classi ed into a severe group (50 Thalassemia major: TM) and milder group (50 Thalassemia Intermedia: TI).
3 parameters were considered for clinical analysis in both the hemoglobinopathy patient group. These included the age of presentation, frequency of blood transfusion, and organomegaly. As expected, the patients in the β-thalassemia major group had an early age of presentation (9.2 ± 2.7 months) and recurrent blood transfusion requirement (14.8 ± 4.0 times/ year) as compared to the milder β-thalassemia intermedia group (mean age: 4.5 ± 3.3 years and transfusion frequency: 2.5 ± 3.3/ year). In the SCA, the mean age of presentation was found to be 6.3 ± 5.2 years. Further to know the contribution of HbF levels in the clinical presentation of the SCA patients, they were divided into two groups considering the median HbF level of 17.4 %. It was observed that SCA patients with HbF levels ≤ 17.4 % showed higher mean transfusion frequency (3.3 ± 5.5 times) as compared to patients with HbF > 17.4 % (1.7 ± 5.4 times).
Hepatosplenomegaly was pronounced in β-thalassemia intermedia group as against thalassemia major (P: 0.001). Also, in SCA patients, hepatosplenomegaly was observed. The detailed clinical analysis among the patient groups is shown in Table 1. The hematological parameters of the patient groups are shown in Table 2.    As co-inheritance of α-thalassemia is a well-known disease modi er of β-thalassemia and SCA, the presence of α-globin-gene deletions was screened in the patient groups. A much higher prevalence of single alpha globin gene deletions was observed in SCA patients (51%). Among the β-thalassemia homozygotes, the β-thalassemia intermedia showed a higher prevalence (26%) of α-globin gene deletions as compared to β-thalassemia major (20%). (P:0.47) (Suppl. Table 2) The second powerful modi er of disease severity in hemoglobinopathy patients is elevated HbF levels.
Hence the polymorphisms located in the three loci linked to raised HbF levels: γ-globin promoter region, BCL11A and HBSL1-MYB intergenic region were analysed in this study.
In the G γ globin promoter region, the XmnI polymorphism residing in the − 158 (C→T) position was only detected. In the β-thalassemia homozygous group, the homozygosity for the mutant T allele [T/T, Xmn I +/+ ] was signi cantly higher in TI (44 %) as compared to TM (28%), (P: 0.01).Similarly, in SCA patients, 94% of the patients were homozygous for the T allele. As thalassemia major patients were on recurrent transfusion, the genotypes could not be compared with the HbF levels, however, in thalassemia intermediates, the TT [XmnI: +/+] genotype was found to be signi cantly associated with raised HbF levels (79.1% ± 29.0, p: 0.04) as compared to the CC [XmnI: -/-] genotype (53.08 % ± 35.9). [ Figure 1A] In the A γ-globin promoter region, + 25 (G→A) variation was detected and A allele was found to be the variant allele. The A allele was found to be signi cantly higher in the TI group as compared to TM (P: 0.005). Also, the A allele in β-thalassemia intermediates, was signi cantly associated with increased HbF levels (79.9 % ±28.6, P: 0.03). In SCA patients, 94% of the patients were homozygous for the A allele, an observation similar to the XmnI polymorphism. [ Figure 1B].
Among the 5 intronic polymorphisms in the BCL11A gene screened, the mutant T allele of rs1427407 (G→T) polymorphism, was signi cantly higher in the β-thalassemia intermedia group as compared to the thalassemia major group (P: 0.002, OR: 5.6, 1.84-17.22). In SCA patients the T allele was found to be signi cantly associated with raised HbF levels (HBF > 17.4 %, (P: 0.003, OR: 3.14, 1.46-6.75) as compared to the other group. The T allele was also found to be signi cantly associated with HbF levels in both the patient groups. (P < 0.05). [ Figure 1C] In the sickle cell anemia patients, the C allele of rs11886868 C→ T polymorphism was found to be signi cantly associated with increased HbF levels (P: 0.02, HbF: 20.9 % ± 8.8).
[ Figure 1D] Among the HBS1L and MYB gene polymorphisms, the deletional allele of rs66650371 (Intact TAC → Deletion ---'TAC') polymorphism and the C allele of rs9399137 (T→C) were found to be signi cantly present in the milder βhemoglobinopathy patients. As reported in earlier studies these 2 polymorphisms were found to be in complete linkage disequilibrium. The minor alleles of these polymorphisms were found to be signi cantly associated with the HbF levels. [ Figure 1E and F] Table 3 gives a detailed analysis of the allelic frequency of these polymorphisms determined among the patient and the control group.   Figure 3A] Similarly among the SCA patients, gene-gene interaction between the mutant alleles of rs66650371 and rs1427407 were found to be signi cantly higher in the sickle cell anemia patients with HbF levels > 17.4 % with a testing balance accuracy 66.0 % and cross-validation consistency 10/10. [ Figure 3B] The generation of GMDR models for determining the most in uential SNPs among the 9 SNPs studied in the patient groups is shown in supplement Table 3.
The presence of ameliorating alleles may signi cantly delay the age of presentation and transfusion requirement in β-hemoglobinopathy patients. Hence, for the analysis we included both primary modi ers [the type of β-globin gene mutation in β-thalassemia patients] and secondary modi ers: α-globin genotype and the HbF modulators [γ-globin promoter variations, BCL11A, MYB and KLF1 variations (the KLF1 data from our previous published paper) [8]. Among the SCA patients, a strong negative correlation was observed between the HbF levels and the disease severity score. (Pearson correlation coe cient r: -0.7, P < 0.00001) The patients inheriting the higher numbers of modulating allele showed signi cantly elevated HbF levels (mean HbF: 21.9% ±9.8) as compared to patients with a less number of disease severity modulating alleles (mean HbF:16.5 %±7.1). Also showed a signi cant delay in the age of rst transfusion as compared to the other group. [ Figure 4A and 4B] The β-thalassemia intermedia patients inheriting more number of the disease ameliorating alleles showed elevated HbF levels (mean HbF: 75.1 % ± 29.9), with reduced disease severity score (mean DSS :5.6) as compared to patients with lower numbers of disease severity modulating alleles, who had lower HbF levels (mean HbF: 54.1% ± 36.9). Further, when compared to the age of rst transfusion, however, no signi cant difference was observed among the 2 groups.
[ Figure 4C and 4D] The supplementary table 4 shows the median transfusion free survival and hazard ratio in both the patient groups. The number of modulating alleles, transfusion free survival ratio was inversely associated with the hazard ratio.

Discussion:
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 modi ers 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 rst classi ed the β -thalassemia patients according to the clinical severity and then studied the in uence of the genetic modi ers. Modell and Berdukas 1984, reported that 60 % of βthalassemia homozygous patients presented in the rst year of life, these patients were segregated as βthalassemia major and 9 % of the β-thalassemia homozygous patients who presented after 2 years of age, with intermediate clinical severity were classi ed 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 signi cantly 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 signi cant 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 signi cantly 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 signi cantly 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 αthalassemia 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 modi ers 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 βthalassemia 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 signi cantly 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 signi cantly higher HbF level. + 25 G→A polymorphism in A γ-globin promoter region was found to be signi cantly associated with elevated HbF levels in the β-thalassemia intermedia group. This polymorphism was rst 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 e cacy 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 signi cantly 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 signi cantly 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 signi cantly 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 signi cantly 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  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 identi cation of the polymorphisms associated with elevated HbF levels may help in developing a molecular chip that may assist in predicting the disease severity.

Conclusions:
The present study expands the knowledge of the frequency of the genetic modi ers (primary and secondary modi ers) and the independent effect of individual predictor genes on HbF levels in hemoglobinopathy patients. The analysis of the cumulative effect of the HbF modulators may help in identifying the strongest response gene to the HbF level in both β-thalassemia and sickle cell anemia patients in the population. The predictions based on genetic modi ers thus can foresee the severity of βthalassemia and SCA. This study may assist the clinicians, to predict the clinical phenotype of hemoglobinopathy patients at an early stage and thus may help in the e cient management of the disease. This may contribute towards molecular mechanisms of HbF regulation and the development of therapeutic approaches for β-hemoglobinopathies.

Declarations
Acknowledgment: We thank the individuals for participating in the study. We also thank University of Mumbai for their support. Declarations: Funding: This work was funded by Indian Council of Medical Research (ICMR).
Con icts of interest: The authors have no con icts of interest to declare that are relevant to the content of this article.
Availability of data and material: Not applicable Code availability: Not applicable Authors' contributions: PH: Laboratory work and wrote the manuscript.
MG, PS, PM: Laboratory work and data collection.
AN: Designed the research study and edited the manuscript.
Ethics approval: The study was approved by the National Institute of Immunohaematology-Institutional Ethics Committee.
Animal Research: Not applicable Consent to participate: Informed consent was obtained from all individual participants included in the study.