The HBG2 rs7482144 (C > T) Polymorphism is Linked to HbF Levels but not to the Severity of Sickle Cell Anemia

Abstract

Sickle cell anemia (SCA) is a severe disease characterized by anemia, acute clinical complications, and a relatively short life span. In this disease, abnormal hemoglobin makes the red blood cells deformed, rigid, and sticky. Fetal hemoglobin (HbF) is one of the key modulators of SCA morbidity and mortality. Interindividual HbF variation is a heritable trait that is controlled by polymorphism in genes linked and unlinked to the hemoglobin β gene (HBB). The genetic polymorphisms that determine HbF levels are known to ameliorate acute clinical events. About 190 well-characterized homozygous SCA patients were included in this study. Complete blood count (CBC), high-performance liquid chromatography (HPLC), and clinical investigations were obtained from patient's records. Severity scores were determined by using the combination of anemia, complications, total leucocyte count, and transfusion scores. HBG2 rs7482144 polymorphism was genotyped by using the polymerase chain reaction and restriction fragment length polymorphism. The association between HBG2 rs7482144 polymorphism and HbF levels as well as the disease severity of SCA were assessed. SCA patients carrying TT genotype were found to have higher HbF levels. In addition, SCA patients with increased severity showed significantly lower levels of hemoglobin, HbF, and hematocrit values. However, the genotypes of HBG2 rs7482144 polymorphism were not found to be associated with the risk of disease severity. In summary, this study demonstrated that HBG2 rs7482144 polymorphism is linked with HbF levels, but it does not affect disease severity. The sample sizes used and the pattern of association deduced from our small sample size prevents us from extrapolating our findings further.

Introduction

Sickle cell anemia (SCA) is a widespread and severe single-gene hemoglobin (Hb) disorder. ¹ The substitution of nucleotide T for A in codon 6 of HBB gene results in the substitution Glu to Val amino acid leading to impaired β-globin chain synthesis. ² Sickle cell anemia is characterized by sickle-shaped red blood cells (RBCs) with a relatively short life span and varying degrees of anemia. Complications of SCA includes severe episodes of pain, acute chest syndrome, stroke, hepatomegaly, splenomegaly, renal failure, and susceptibility to bacterial infection. ³ There is a considerable heterogeneity in the severity of SCA. There has been tremendous progress in understanding the molecular basis of disease severity in SCA patients in the recent years. ⁴ Several lines of evidence demonstrate that the genetic factors involved in inflammation, cell–cell interaction, and nitric oxide biology could influence the severity of SCA. ^{5 6 7}

Fetal hemoglobin (HbF) is one of the normal hemoglobin variants and a predominant hemoglobin during the fetal stage. HbF exists at approximately 90 to 95% during birth and gradually drops to <2% by the age of 1 year. Normal concentrations of HbF in adults range from 1 to 2%. ⁸ HbF plays a key role in regulating SCA morbidity and mortality. Several studies have indicated that increased HbF levels have an ameliorating effect on SCA patients and reduced hospitalizations. ^{9 10} Several pharmacological agents such as butyrate, hydroxyurea (HU), 5-azacytidine, and erythropoietin have been shown to increase HbF in SCA patients and reduce severe anemia. ¹¹ Among these, HU is shown to stimulate γ-globin gene expression in vivo. Hydroxyurea-mediated HbF induction occurs through a range of signaling pathways (cAMP/cGMP; p38/MAPK/CREB1; Giα/c-Jun N-terminal kinase/Jun) that modulate γ-globin expression. ^{12 13} Furthermore, HbF is a highly heritable trait that is influenced by variants in HBG2 , BCL11A , HBS1L - MYB , and SAR1 genes. ¹⁴

HBG2 rs7482144 polymorphism results from a C to T nucleotide substitution at position-158 of γ-globin gene. ¹⁵ The HBG2 rs7482144 polymorphism is located close to the locus control region of β-globin (β-LCR) gene. The “T” allele of HBG2 rs7482144 polymorphism leads to a weaker binding of transcription inhibitors to the β-LCR, subsequently resulting in persistent activation of the HBG2 gene in adult life. ¹⁶ Clinical studies have shown that the presence of HBG2 rs7482144 “T” allele in SCA patients is correlated with significantly higher mean HbF as compared with patients with the “C” allele. HBG2 rs7482144 polymorphism is associated with increased HbF in some SCA patients. ¹⁷ The frequency of HBG2 rs7482144 polymorphism in HBG2 gene and its relationship with HbF levels in SCA patients has not yet been investigated in Chhattisgarh, a state situated in central India. In Chhattisgarh, SCA is an endemic and according to the available data, approximately 10% of infants are born with a sickle cell trait. ¹⁸ The main objective of this study is to estimate the frequencies of HBG2 rs7482144 polymorphism and investigate the association between HBG2 rs7482144 polymorphism with the levels of HbF and the severity of disease in SCA patients in Chhattisgarh.

Materials and Methods

Subjects

In the present study, 190 sickle cell homozygous (HbSS) patients were identified and examined. Sickle cell subjects were confirmed by Hb electrophoresis. All SCA patients were recruited from Sickle Cell Institute Chhattisgarh, Raipur. The Institutional Ethics Committee of the Sickle Cell Institute Chhattisgarh, Raipur approved the study. Informed consent was obtained from adult study participants, and the legal guardians provided a written consent on behalf of minors. Subjects were excluded if they had any of the following: low platelet count (<100,000/mm ³ ), neutropenia (polymorphonuclear neutrophil <1,200/mm ³ ), pregnancy, on interferon treatment, on HU treatment, and members of the same family. All patients included in this study were known to be in a steady state. Baseline values of complete blood count (CBC) and hemoglobin HPLC were obtained from patient records. All patients were evaluated for clinical phenotypes such as pain, anemia, jaundice, pneumonia, stroke, and osteonecrosis. Severity scores were determined by using the combination of anemia, complications, total leucocyte count, and transfusion scores. A complete overview of the criteria used for assessing the severity score was given elsewhere. ¹⁹ About 3 mL of blood samples was collected from each participant. DNA was extracted from whole anticoagulated blood following the standard protocol. ²⁰

Genotyping of HBG2 rs7482144 Polymorphism

Genotyping of HBG2 rs7482144 polymorphism was performed by following polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP). A 310-base pairs (bp) fragment in the upstream region of the HBG2 were amplified with primers: 5′-AAC TGT TGC TTT ATA GGA TTT T-3′ and 5′-AGG AGC TTA TTG ATA ACC TCA GAC-3′. The PCR reactions were performed in a total reaction volume of 10 μL containing nearly 40 ng genomic DNA, 1.0 U Taq polymerase in 2× PCR master mix and 5 pmol/µL forward and reverse primers. PCR cycling conditions were as follows: a 5-minute initial denaturation at 95°C, 35 cycles of denaturation at 95°C for 40 seconds, annealing at 58°C for 40 seconds, and extension at 72°C for 40 seconds, which followed by a 7-minute final extension step at 72°C. The PCR amplicons were incubated with the Xmn-1 restriction enzyme as recommended by the manufacturer (Fermentas Thermo Fisher Scientific, United States). The digested products were resolved by electrophoresis on 2% agarose gel. Upon digestion, 657 bp PCR product was cleaved into two fragments of 455 and 202 bp for the T allele. The presence of uncleaved 657 bp product indicates the existence of a “C” allele.

Statistical Analysis

The risk associated with genotypes was calculated by the odds ratio and 95% confidence interval (CI) using Medcalc statistical software ( https://www.medcalc.org/calc/odds_ratio.php ). The Chi-squared test was performed to investigate the association between HBG2 rs7482144 polymorphism and SCA severity. The association between HBG2 rs7482144 genotypes and HbF levels was analyzed by using ANOVA. The p -values <0.05 were deemed to be statistically significant. SPSS software package (Version 20) was used to analyze the data.

Results

The Baseline characteristics of subjects are outlined in Table 1 . Out of all patients, 6 families were known to have 3 affected siblings, 53 families with 2 affected siblings, and 131 families with no affected siblings. Amongst 190 SCA patients, 106 (55.8%) were males, while 84 (44.2%) were females with a mean age of 16.5 ± 9.3 (age = 2–65 years). The mean HbF level for SCA patients was 19.57 ± 6.81, while the mean age for males (19.31) was slightly lower than the mean age for females (19.90), although not statistically significant ( p  = 0.557). Mean HbF in different age groups and genotypes of HBG2 rs7482144 (C > T) in SCA patients is outlined in Table 2 . SCA patients with TT genotype were found to have a higher HbF levels than the CT and CC genotypes, which is a statistically significant ( p  < 0.001) distribution. About 164 (86.2%) SCA patients revealed to have their HbF levels within the range of 10 to 30%. HbF <10% and >30% was found in 14 (7.3%) and 12 (6.3%) of SCA patients, respectively ( Table 3 ).

Table 1. Baseline characteristics of the study subjects.

Variable	Measures ( n  = 190)
Age (y)	16.52 ± 9.3
Sex (male/female)	106 (55.8%)/84 (44.1%)
BMI (kg/m 2 )	16.07 ± 3.27
HbF (%)	19.6 ± 6.8
Hb (g/dL)	8.5 ± 1.8
Hematocrit (%)	25.13 ± 5.29
MCV (fL)	86.23 ± 10.25
MCH (pg)	29.03 ± 3.98
No. of hospitalizations	8.24 ± 17.80
No. of blood transfusions	6.98 ± 13.9
Family history
Three affected siblings	6 (3.2%)
Two affected siblings	53 (27.9%
No affected siblings	131 (68.9%)

Abbreviations: BMI, body mass index; Hb, hemoglobin; HbF, fetal hemoglobin; MCH, mean corpuscular hemoglobin; MCV, mean corpuscular volume.

Table 2. Mean HbF in different age groups and genotypes of HBG2 rs7482144 (C > T) in sickle cell anemia patients .

Variable	Frequency n (%)	Mean HbF (%)	p -Value
Age groups
2–10 y	58 (30.5)	19.73 ± 6.30	0.501
11–20 y	79 (41.6)	20.24 ± 7.43
21–30 y	34 (17.9)	18.51 ± 6.25
>30 y	19 (10.0)	18.21 ± 6.75
HBG2 rs7482144 (C > T) genotypes
CC	3 (1.6)	7.50 ± 2.95	0.003
CT	38 (20.0)	18.41 ± 6.66
TT	149 (78.4)	20.09 ± 6.72

Abbreviation: HbF, fetal hemoglobin.

Table 3. Stratification of levels of HbF according to degree of elevation and age group in sickle cell anemia patients.

Age	HbF <10% ( n  = 14)	HbF 10.1–20% ( n  = 82)	HbF 20.1–30% ( n  = 82)	HbF >30% ( n  = 12)	p -Value
2–10 y, ( n  = 58)	2 (3.4)	28 (48.3)	25 (43.1)	3 (5.2)	0.635
10.1–20 y, ( n  = 79)	8 (10.1)	27 (34.2)	37 (46.8)	7 (8.9)
20.1–30 y, ( n  = 34)	3 (8.8)	17 (50.0)	13 (38.2)	1 (2.9)
>30 y, ( n  = 19)	1 (5.3)	10 (52.6)	7 (36.8)	1 (5.3)

Abbreviation: HbF, fetal hemoglobin.

According to the disease severity, SCA patients demographic and hematological variables are shown in Table 4 . The distribution of baseline hemoglobin, hematocrit, and HbF levels were found to be statistically different between mild, moderate, and severe clinical severity groups. The distribution of SCA patients with different clinical severities amongst the HBG2 rs7482144 genotypes and alleles are documented in Table 5 . The genotypes and alleles of HBG2 rs7482144 are not statistically significant among different severity groups ( Table 5 ). SCA severity risk associated with HBG2 rs7482144 genotypes in different genetic models is presented in Table 6 . No significant difference in the risk of SCA severity among HBG2 rs7482144 genotypes and alleles was observed. The distribution of HBG2 rs7482144 genotypes in low HbF (<10%) and high HbF (>10%) levels is shown in Table 7 . Individuals with TT genotype were found to have a higher HbF concentration as compared with the CT and CC genotypes among SCA patients; however, this distribution is not statistically significant.

Table 4. Demographic and hematological variables according to severity of patients with sickle cell anemia.

	Mild (27)	Moderate (67)	Severe (96)	p -Value
Male/female	13/14	39/28	54/42	0.668
Age (y)	18.9 ± 8.2	19.5 ± 11.8	13.8 ± 6.3	<0.001
Weight	41.1 ± 10.4	36.3 ± 15.6	30.3 ± 12.4	<0.001
Baseline Hb	9.95 ± 1.48	8.77 ± 1.57	7.83 ± 1.67	<0.001
Hematocrit (%)	28.9 ± 3.8	26.0 ± 5.22	23.5 ± 5.05	<0.001
HbF (%)	22.74 ± 7.19	20.64 ± 6.10	17.93 ± 6.77	0.001

Abbreviation: HbF, fetal hemoglobin.

Table 5. Alleles and genotypes frequencies of HBG2 rs7482144 (C > T) polymorphism according to severity of patients with sickle cell anemia .

HBG2 rs7482144 (C > T)	Mild n (%)	Moderate n (%)	Severe n (%)	p -Value
Genotypes
CC	1 (33.3)	0 (0.0)	2 (66.7)	0.630
CT	4 (10.5)	15 (39.5)	19 (50.0)
TT	22 (14.8)	52 (34.9)	75 (50.3)
Alleles
C allele	6 (13.6)	15 (34.1)	23 (52.3)	0.969
T allele	48 (14.3)	119 (35.4)	169 (50.3)

Table 6. HBG2 rs7482144 (C > T) polymorphism and risk of patient-reported mild, moderate and severe outcomes among sickle cell anemia patients .

Genetic model	Chi-square	OR (95% CI)	p -Value
Mild vs. moderate
TT vs. CC	2.261	7.0 (0.27–178.5)	0.133
TT vs. CT + CC	0.170	0.79 (0.26–2.44)	0.680
T vs. C	0.003	0.99 (0.36–2.71)	0.987
Mild vs. severe
TT vs. CC	0.184	1.71 (0.15–19.70)	0.667
TT vs. CT + CC	0.141	0.81 (0.27–2.40)	0.707
T vs. C	0.030	0.92 (0.35–2.38)	0.862
Moderate vs. severe
TT vs. CC	0.070	0.72 (0.06–8.16)	0.792
TT vs. CT + CC	0.006	1.03 (0.49–2.18)	0.938
T vs. C	0.047	0.93 (0.46–1.85)	0.828

Abbreviations: CI, confidence interval; OR, odds ratio.

Table 7. The effect of HBG2 rs7482144 (C > T) polymorphism on high and low HbF levels of sickle cell anemia patients .

HbF genotypes	CC	CT	TT	p -Value
High HbF, n (%)		36 (20.5)	140 (79.5)
High HbF (mean ± SD)		18.93 ± 6.43	20.92 ± 6.01	0.084
Low HbF, n (%)	3 (21.4)	2 (14.3)	9 (64.3)
Low HbF (mean ± SD)	7.50 ± 2.95	9.0 ± 1.27	7.5 ± 2.11	0.691

Abbreviation: HbF, fetal hemoglobin.

Discussion

HbF analysis in SCA patients showed that the mean HbF level is 16.5 ± 9.3% and no significant difference in HbF level persists between males and females. Further, SCA patients with HBG2 rs7482144 TT genotype were found to exhibit higher HbF levels, while patients with severe disease showed significantly lower levels of Hb, HbF, and hematocrit levels. However, genotypes HBG2 rs7482144 polymorphism were not found to be associated with the risk of disease severity in different genetic models.

Although SCA is a monogenic disorder, its clinical features are extremely variable in severity. ²¹ This clinical heterogeneity is a result of the dynamic interplay between multiple genetic and environmental factors. However, the genetic etiology of variable disease expression is still unclear. ¹⁴ Several lines of evidence indicate that age, coinheritance of a-thalassemia, polymorphisms in genes linked, and unlinked to the β-globin gene are involved in modulating the disease phenotype. ^{22 23} Mild to moderate advancement of HbF in SCA patients is noticed due to hereditary persistence of fetal hemoglobin. The HbF levels have been shown to influence SCA severity. In the erythrocytes of SCA patients, the higher HbF does not participate in the formation of HbS polymer and prevent polymerization. ²⁴ The distribution of fetal hemoglobin (HbF) among sickle erythrocytes is not uniform, and few erythrocytes produce higher HbF levels referred to as F cells. Patients with large deletional thalassemia exhibit homocellular HbF distribution, whereas SCA and delta β thalassemia exhibit hetero-cellular HbF distribution. ²⁵

SCA patients in Africa who carried the HBG2 rs7482144 polymorphism revealed higher levels of HbF compared with patients without the polymorphism. Further, SCA patients from Atlantic West Africa (Senegalese) were found to have a lower proportion of dense red cells, higher levels of HbF due to the presence of HBG2 rs7482144 polymorphism. ²⁶ SCA patients with Arab-Indian haplotype reveal HbF levels and confer a mild disease course. ²⁷ A significant effect of HBG2 rs7482144 polymorphisms on baseline HbF was documented in the BABY HUG trial. ²⁸ Several independent studies have reported the association between HBG2 rs7482144 polymorphism and HbF levels in SCA patients. ^{15 29 30 31} In another study, the combined linkage and association analysis of the β-globin complex revealed a strong association between HBG2 rs7482144 polymorphism and the levels of fetal cell. ³² However, HBG2 rs7482144 T allele does not consistently represent high HbF levels in SCA patients. The African American Cooperative Study of Sickle Cell Disease (CSSCD) cohort study demonstrated that the TT genotypes of HBG2 rs7482144 polymorphism account for only 2.2% of the variations in HbF levels. ³³ In the present study, SCA patients carrying HBG2 rs7482144 T allele were not associated with milder disease. However, the SCA patients carrying HBG2 rs7482144 TT genotype showed higher HbF levels than the other genotypes. Despite of having higher HbF levels, Indian SCA patients are known to exhibit severe disease, indicative of additional factors leading to clinical severity. Adhesion, inflammation, coagulation, oxidative stress, nutrition, growth, and several other processes play a vital role in determining the clinical severity of SCA patients.

Multiple genetic loci largely control the HbF variation between individuals. The primary limitation of this investigation is that the known trans-acting QTLs, such as FCP locus (Xp22), HBS1L - MYB intergenic polymorphism (HMIP) locus (6q22.3–24), TOX (8q), and BCL11A gene, as well as the polymorphisms in the globin gene (HBB) complex were not included in this study (2p16.1). ¹⁶ However, the strength of the present study is the involvement of well-characterized SCA patients in a large number more than the previous studies. Further, SCA patients with complete clinical and hematological variables assisted us in evaluating the association between HBG2 rs7482144 polymorphism and the levels of HbF in different severity groups. In summary, HBG2 rs7482144 polymorphism is associated with the HbF levels, but it does not affect the disease severity. The sample sizes used and the patterns of association deduced from our small sample sizes prevent us from extrapolating our findings further.

Funding Statement

Funding This work was supported by Chhattisgarh Council of Science & Technology (CCOST) project (No.2740/CCOST/MRP/2015).