|Year : 2021 | Volume
| Issue : 1 | Page : 305-309
Genetic polymorphism of BCL11A (rs11886868) in Egyptian patients with β-thalassemia
Mohamed A Soliman1, Rawhia H El-Edel1, Amal H AbdElhameed2, Seham M Ragab3, Manal Monir Mansour1, Mohamed A Helwa1
1 Department of Clinical Pathology, Faculty of Medicine, Menoufia University, Menoufia, Egypt
2 Department of Clinical Pathology, Faculty of Medicine, Tanta University, Tanta, Egypt
3 Department of Pediatrics, Faculty of Medicine, Menoufia University, Menoufia, Egypt
|Date of Submission||28-Jul-2019|
|Date of Decision||09-Sep-2019|
|Date of Acceptance||14-Sep-2019|
|Date of Web Publication||27-Mar-2021|
Manal Monir Mansour
Department of Clinical Pathology, Faculty of Medicine, Menoufia University, Menoufia
Source of Support: None, Conflict of Interest: None
To study the effect of BCL11A (rs11886868) gene polymorphism on clinical status of Egyptian patients with β-thalassemia.
β-thalassemia is a genetic disorder that results from β-globin gene mutations leading to defective synthesis of adult hemoglobin (Hb A). Clinical consequences vary tremendously from nearly asymptomatic to severely anemic and transfusion-dependent patients. Phenotypic variations have been linked not only to the type of β-globin gene mutations and amount of synthesized Hb A but also to recently discovered genetic modifiers mapping outside the β-globin gene cluster including BCL11A gene polymorphism. These modifiers are associated with increased fetal hemoglobin (Hb F) levels.
Patients and methods
Blood samples were collected from 120 patients with β-thalassemia before transfusion of blood. Complete blood count, quantitative measurement of different Hb types by high-performance liquid chromatography, and analysis of single nucleotide polymorphism rs11886868 in the BCL11A gene by Taqman single nucleotide polymorphism genotyping assay and real-time PCR were done.
The genotype distribution of BCL11A gene polymorphism showed low frequency of CC genotype (15%) among Egyptian patients with β-thalassemia. The CT genotype was 41.7% and TT genotype was 43.3%. There was a significant relation between BCL11A (rs11886868) genotypes and Hb F levels, with the highest Hb F levels were observed in the CC genotype followed by CT genotype, whereas Hb F levels were the lowest in TT genotype. Patients with CC genotype had mild disease phenotype owing to increased Hb F production. Hb F utilizes some of the free α-chain and ameliorates clinical presentation.
Screening of BCL11A gene variants may be used as a marker for severity risk in newly diagnosed patients with β-thalassemia and as a predictor of clinical outcome of this disorder.
Keywords: BCL11A gene, hemoglobin F, polymorphism, β-thalassemia
|How to cite this article:|
Soliman MA, El-Edel RH, AbdElhameed AH, Ragab SM, Mansour MM, Helwa MA. Genetic polymorphism of BCL11A (rs11886868) in Egyptian patients with β-thalassemia. Menoufia Med J 2021;34:305-9
|How to cite this URL:|
Soliman MA, El-Edel RH, AbdElhameed AH, Ragab SM, Mansour MM, Helwa MA. Genetic polymorphism of BCL11A (rs11886868) in Egyptian patients with β-thalassemia. Menoufia Med J [serial online] 2021 [cited 2021 Oct 23];34:305-9. Available from: http://www.mmj.eg.net/text.asp?2021/34/1/305/312022
| Introduction|| |
β-Thalassemia is a congenital hemolytic anemia that is representing a major and serious health problem and having the highest prevalence and incidence rates worldwide . In Egypt, the carrier rate varies between 5.5% and more than or equal to 9%. It is estimated that there are 1000/1.5 million per year live births born with β-thalassemia . It results from mutations of β-globin genes leading to defective synthesis of adult hemoglobin (Hb A). More than 200 mutations have been described so far in the β-globin genes responsible for this disease . The clinical phenotype of β-thalassemia may also be modified by the co-inheritance of other genetic factors mapping outside the β-globin gene cluster. It has been suggested that these factors determine the persistence of fetal Hb production that may ameliorate the clinical and hematological severity in β-thalassemia . Analysis of genome-wide association studies data revealed a new locus on chromosome 2, located in the BCL11A (B-cell lymphoma/leukemia 11A) gene that is linked to Hb F . BCL11A gene is a zinc finger protein that represses the γ-globin gene expression. Hence, it inhibits γ-globin chain synthesis and helps switching from Hb F to Hb A . However, a single nucleotide polymorphism (SNP), rs11886868, in BCL11A has been associated with an up-regulation of fetal Hb synthesis . Other studies could not establish a relation between this SNP and increased Hb F levels in some other ethnic groups, indicating a discrepancy in different populations . The aim of this work was to investigate the effect of SNP rs11886868 in the BCL11A gene on clinical status of Egyptian patients with β-thalassemia.
| Patients and methods|| |
This prospective study was carried out at Clinical Pathology Department, Faculty of Medicine, Menoufia University, during the period from September 2017 to March 2019. This study was approved by the ethical committee of the Faculty of Medicine, Menoufia University. All participants' parents or guardians provided a written informed consent to collect a blood sample and to review the medical record for research purposes. The study included 120 patients with β-thalassemia. The patients were β-thalassemia intermedia and major, either newly diagnosed or on regular blood transfusion. Blood samples were collected before blood transfusion.
β-thalassemia carriers and heterozygous patients for β-thalassemia/sickle cell disease were excluded. Full history taking, clinical assessment, and clinical scoring of thalassemia severity by phenotypic scoring system proposed by Nadkarni et al.  were done. The parameters for calculating severity index of β-thalassemia included age of presentation, hepatosplenomegaly, and baseline Hb level before transfusion or at the time of diagnosis. Severity index more than 8 was considered as thalassemia major and less than or equal to 8 as thalassemia intermedia. Blood samples were collected to perform complete blood count (ABX Pentra XL 80, California, USA) and measure Hb F levels and other Hb fractions by high-performance liquid chromatography (HPLC) (Arkray Adams A1c HA-8180T HPLC Analyzer, Kyoto, Japan). Hb fraction value (g/dl) was calculated as follow: Hb fraction percentage (obtained by HPLC)×total Hb (g/dl)/100. DNA was extracted by using the Gene JET Whole Blood Genomic DNA Purification Mini Kit manufactured by Thermo Fisher Scientific Baltics (Vilnius, Lithuania). DNA concentration and purity were determined by nanodrop spectrophotometer. DNA absorbance was measured at 260 nm to calculate DNA concentration and at 260 and 280 nm to calculate the ratio and determine DNA purity. The detection of BCL11A gene SNP rs11886868 was made by Taqman SNP genotyping assay (Applied Biosystems by Thermo Fisher Scientific, California, USA). The final reaction mixture was 20 μl: 10 μl Taqman master mix, 0.5 μl Taqman SNP assay (40×), 5 μl DNA, and 4.5 μl DNase-free water. The thermal cycling program was 95°C for 10 min, 40 cycles of 92°C for 15 s, and 60°C for 1 min. Allelic discrimination, multicomponent, and amplification plots were generated by real-time PCR (Applied Biosystems 7500 System) [Figure 1] and [Figure 2].
|Figure 1: Allelic discrimination plot of Taqman genotyping assay for BCL11A SNP rs11886868 at Applied Biosystems 7500 showing VIC dye fluorescence (C allele) at x-axis and FAM dye fluorescence (T allele) at y-axis. The dot color of homozygous CC is red, homozygous TT is blue, and heterozygous CT is green. SNP, single nucleotide polymorphism.|
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|Figure 2: Multicomponent plots showing (a) homozygous alleles for VIC dye (CC), (b) homozygous alleles for FAM dye (TT), and (c) heterozygous alleles for VIC and FAM dyes (CT).|
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Data were analyzed using IBM SPSS software package version 20.0 (IBM Corp., Armonk, New York, USA). The association between genotypes and Hb F level was evaluated by analysis of variance (F test). χ2 test was used to detect the relation between genotypes and clinical score. Genotypes and allelic frequencies were calculated by Hardy–Weinberg equation.
| Results|| |
This study included 120 patients with β-thalassemia, comprising 71 males and 49 females. The mean patients' age was 8.59 years, and mean age at diagnosis of β-thalassemia was 1.77 years. The mean of β-thalassemia score by phenotypic scoring system was 7.66 and the mean of blood transfusion requirements was 11.89 times per year.
The CC genotype and C allele were less frequent among Egyptian patients with β-thalassemia. The genotype frequencies were consistent with Hardy–Weinberg equation [Table 1]. The relation between BCL11A (rs11886868) genotypes and total baseline Hb levels was nonsignificant. However, the BCL11A (rs11886868) had statistically significant associations with Hb F levels. To accurately describe Hb F levels, the mere Hb F percentage values were not included (notice that in a patient with markedly low/absent Hb A production, Hb F percentage can be very high. This may be misleading because the actual Hb F production may be low). Instead, Hb F levels, expressed in g/dl, were stratified according to the levels of total Hb and levels of Hb A. The Hb F levels when normalized to total Hb levels and to Hb A levels were significantly higher in patients with the CC genotype followed by patients with CT genotype and was the lowest in patients with TT genotype. Based on total baseline Hb levels, patients were categorized into patients with Hb less than 5, 5–less than 8, and more than or equal to 8 g/dl. In each of these groups, Hb F levels showed highly significant and significant differences among different BCL11A genotypes. Similarly, patients were grouped based on Hb A levels into patients with Hb A 0–less than 4, 4–less than 6, and more than or equal to 6 g/dl. Hb F levels showed highly statistical significance among different BCL11A genotypes at different Hb A levels [Table 2] and [Table 3].
|Table 1: A simple calculator to determine whether the observed genotype frequencies are consistent with Hardy–Weinberg equation|
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|Table 2: Relation between BCL11A (rs11886868) genotypes and hemoglobin F levels at different total baseline hemoglobin levels (N = 120)|
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|Table 3: Relation between BCL11A (rs11886868) genotypes and hemoglobin F levels at certain hemoglobin A levels (N = 120)|
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All cases of CC genotype had phenotypic score of less than or equal to 8 that correspond to thalassemia intermedia, whereas most cases of TT genotype had score of more than 8 that correspond to thalassemia major with highly significant P value less than 0.001. Hence, CC genotype is associated with higher Hb F level and milder disease phenotype. Regarding the age of disease presentation, CC genotyped patients had later onset of disease presentation compared with early onset of TT genotype. The patients were also assessed for organomegaly; cases with CC genotype showed normal or mildly enlarged liver and spleen compared with TT genotype cases, which showed moderate and huge organomegaly or splenectomy. Moreover, CC genotyped patients showed low blood transfusion requirements/year compared with TT genotype, who showed higher transfusion requirements. However, genotypes had no relation with serum ferritin levels.
| Discussion|| |
β-thalassemia is a genetic disorder characterized by total absence or reduction in the synthesis of β-globin chains . The clinical presentation of β-thalassemia is extremely variable in severity. The presence of genetic variants that sustain Hb F production has a strong effect on ameliorating the clinical phenotype . In this study, the effect of SNP rs11886868 in the BCL11A gene on clinical status of β-thalassemia in Egyptian patients was investigated. Low frequency of CC genotype (15%) compared with CT genotype (41.7%) and TT genotype (43.3%) was observed. The C allele frequency was 86 (35.8%), and T allele frequency was 154 (64.2%). Thus, T allele is more common among Egyptian patients with β-thalassemia. Hb F levels, expressed in g/dl, were stratified according to the levels of total Hb and levels of Hb A in g/dl. There was a significant relationship between BCL11A (rs11886868) genotypes and Hb F levels when evaluated at different total baseline Hb levels and at certain Hb A levels as well. Hb F levels were significantly higher in patients with the CC genotype followed by patients with CT genotype and was the lowest in patients with TT genotype. All cases of CC genotype had milder clinical presentation with phenotypic score less than or equal to 8 (that correspond to thalassemia intermedia), whereas most cases of TT genotype had severe clinical presentation with phenotypic score more than 8 (that correspond to thalassemia major), with highly significant P value less than 0.001. This is explained by the effect of CC genotype and C allele in BCL11A (rs11886868), which reactivate production of γ-chains, which bind to unpaired α-chains forming Hb F and ameliorating the clinical severity of the disease.
The age of presentation of β-thalassemia was also assessed in this study. The CC genotype had later onset of disease presentation compared with TT genotype. The CC genotype had normal liver and spleen or mild enlargement compared with moderate and huge enlargement or splenectomy in most cases that had TT genotype. Regarding blood transfusion requirements, CC genotyped patients showed low requirements for blood transfusion/year compared with TT genotype. However, the genotypes had no relation with serum ferritin levels.
Similarly, Dadheech et al.  observed a significant difference in the mean Hb F levels between the three genotypes in β-thalassemia. Hb F levels were found to be higher in patients with CC genotype followed by TC then TT genotype. The CC genotype and C allele were associated with milder disease phenotype, and ~70% of CC-bearing patients were diagnosed after 1 year of age, whereas the remaining 30% were diagnosed at less than 1 year of age. Galanello et al.  found that the frequency of the minor allele C of SNP rs11886868 in the BCL11A gene was associated with an increase in the production of Hb F and was significantly higher in the patients with thalassemia intermedia (P < 0.001). Moreover, the study by Uda et al.  of BCLL11A rs11886868 polymorphism revealed that the C allele was associated with high Hb F percentage and was significantly more frequent in patients with thalassemia intermedia. On the contrary, the C allele was more frequent among Indonesian, Iranian, and Indian patients with thalassemia ,,. The difference in the frequency of variant genotype might be owing to ethnicity. Rujito et al.  demonstrated that there was no significant relationship between the quantitative trait locus on BCL11A rs1188686, Hb F level, expressed in percentage, and clinical outcome in patients with β-thalassemia. Their explanation was that most of the patients with β-thalassemia were of severe type (β°/β+). However, in Hb E/β-thalassemia, there was a significant relation with Hb F levels. Neishabury et al.  reported that the frequency of the ameliorating allele (C) for BCL11A SNP, rs11886868, was not significantly higher in patients with milder phenotype. The different BCL11A alleles in the homozygous state had no effect neither on the mean Hb F values, expressed in percentage, nor the clinical phenotypes in patients with thalassemia. They explained these finding by co-inheritance of other modifying factors that had influence on BCL11A polymorphism outcome.
In addition, studies on populations in Europe and Central Asia showed that BCL11A rs11886868 had a strong relationship with the increase in Hb F level in normal individuals . BCL11A variants and Hb F levels have been linked to the moderation of pain crisis rate in patients with sickle cell disease as well . Chaouch et al.  considered the C allele as the normal allele and the T allele as the mutant one and observed that most patients with sickle cell anemia presented with Hb F more than 15% had CC genotype and ameliorated phenotype. Whereas, the CT genotype and mutant allele T were associated with reduced Hb F level and higher risk of occurrence of clinical events.
| Conclusion|| |
The present study has shown that Hb F-associated SNP (CC genotype) and the C allele were associated with increase production of Hb F in patients with β-thalassemia contributing to milder disease phenotype. Thus, screening of BCL11A gene variants may be used as a marker for severity risk in newly diagnosed patients with β-thalassemia and as a predictor of clinical outcome of this disorder.
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Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2]
[Table 1], [Table 2], [Table 3]