Abstract
Fetal hemoglobin (HbF) is a potent genetic modifier of β-thalassemia phenotype. B-cell lymphoma 11A ( BCL11A ) gene results in significant silencing of HbF. The aim of this study was to assess the prevalence of different BCL11A genotypes among a cohort of Egyptian children with β-thalassemia and to correlate them to HbF and clinical severity score. Eighty-two children with β-thalassemia (aged 12.95 ± 3.63 years) were recruited from the Pediatric Hematology Clinic, Ain Shams University. They were divided based on the clinical severity of β-thalassemia into three subgroups: 20 mild (24.4%), 24 moderate (29.3%), and 38 severe (46.3%). Age, gender, age of diagnosis, initial HbF level, transfusion history, and history of splenectomy were assessed. Anthropometric measures, signs of anemia and hemosiderosis, and the severity score were determined. Laboratory investigations such as complete blood picture, ferritin, and single gene polymorphism genotyping of the rs11886868 were also performed. Our findings showed that 16 children had CC genotype (19.5%), 38 had TC genotype (46.3%), and 28 had TT genotype (34.1%) of the rs#. β-thalassemia children with TT genotype had significantly higher severity scoring than the other two groups ( p < 0.001). Moreover, mean initial HbF was found to be lower in children with TT genotype followed by TC and CC genotypes ( p < 0.001). Increased γ-globin expression associated with BCL11A gene polymorphism is associated with better clinical severity of β-thalassemia.
Keywords: BCL11A polymorphism , β-thalassemia, phenotypic heterogeneity
Introduction
More than 800 types of mutations and structural variants in the β-globin gene have been identified. From these mutations, β-thalassemia has more than 200 mutations. 1
The clinical variability of β-thalassemia correlates with the type of mutation present in the β-globin gene; however, variations in the β-globin gene mutations in many instances are not enough to explain the genotype–phenotype correlation in these patients. 2 The phenotypic diversity of this disease is related to the influencing genetic factors, such as the presence of mild/silent β-thalassemia alleles, and coinheritance with α-thalassemia (α-β-thalassemia). Milder clinical symptoms are allegedly caused by a better balance ratio between the α-chain and the non-α-chain. In particular, reactivation of fetal hemoglobin (HbF) or delay of switch to adult Hb has been reported to ameliorate the clinical severity in β-hemoglobin disorders such as β-thalassemia and sickle cell disease. 3
HbF is a potent functional modifier of the hemoglobin molecular stability in β-thalassemia and sickle cell anemia. Differences in the levels of HbF affect the severity of sickle cell disease and the β-thalassemia. 4 The level of HbF that persists in adulthood life is of enormous clinical relevance, given its role in ameliorating the severity of these hemoglobin disorders. 5
B-cell lymphoma 11A (BCL11A) is a potent silencer of HbF. 6 It is a zinc-finger protein encoded by the transcription factor BCL11A gene located on chromosome 2p16.1. It is predominantly expressed in hematopoietic tissue, within multiprotein complexes consisting of erythroid transcription factors, transcriptional corepressors, and chromatin-modifying enzymes. 7 In addition to its prominent role in HbF regulation, BCL11A was found to play a critical for differentiation in diverse contexts, including B- and T-lymphoid cells and brain development. 8
The minor allele “C” of the single gene polymorphism (SNP) rs11886868 in the BCL11A gene has been shown to be a major modifier of HbF level. The different number of variants explained by each marker reflects the heterogeneity of the allele frequencies among different ethnic groups. A significant association of CC genotype and C allele was observed with the milder disease phenotype in β-thalassemia as well as sickle cell disease. 3
However, the prevalence of different BCL11A genotypes and its relation to phenotypic heterogeneity among Egyptian children with β-thalassemia has not been previously explored.
Hence, we aimed to assess the prevalence of different BCL11A genotypes of the SNP (rs11886868) among a cohort of Egyptian children with β-thalassemia and correlate them with the initial HbF levels and clinical disease severity.
Methodology
Study Design
This cross-sectional study included 82 children with β-thalassemia (aged 12.95 ± 3.63 years), during the interval from January 2019 to December 2019.
They were recruited from the regular attendees of the Pediatric Hematology Clinic, Pediatric Hospital, Ain Shams University. They were classified into three groups according to the clinical severity of β-thalassemia.
The study was conducted in accordance with the ethical standards settled by the ethics committee of the Faculty of Medicine, Ain Shams University and was approved by the same committee. The study adhered to the tenets of the Declaration of Helsinki, and all procedures were explained to all participants, with informed consents obtained from them or their guardians.
Patients' Selection
Diagnosis of β-thalassemia was based on clinical history and examination suggestive of chronic hemolytic anemia including pallor, jaundice, or failure to thrive, hepatosplenomegaly, skeletal deformities, or family history of β thalassemia. 9
This was confirmed by laboratory investigations including complete blood count (CBC) and reticulocytic count confirming microcytic hypochromic anemia as well as a qualitative and quantitative hemoglobin analysis using high performance liquid chromatography for quantification of HbA2 and HbF. 10
Exclusion criteria included patients with other hemoglobinopathies as α-thalassemia or sickle thalassemia patients, as well as patients on HbF-inducing agents such as hydroxyurea and histone deacetylase inhibitors.
Clinical Assessment
All patients were subjected to detailed medical history with special emphasis on age, gender, age of diagnosis of thalassemia, disease duration, and transfusion history including age of first transfusion, regular or occasional transfusion, 11 amount of blood in each transfusion, type of transfused packed red blood cells, frequency of transfusion, transfusion index was calculated as the total volume of red cell transfusion per annum per kilogram body weight, 12 and history of splenectomy (done when the calculated annual transfusion requirement is > 200–220 mL RBCs/kg/y with a hematocrit of 70%). 13 Initial HbF at diagnosis and Hb prior to first transfusion were obtained from medical records of patients.
Assessment of anthropometric measures was done with calculation of standard deviation score for age and gender, measurement of vital data including heart rate and blood pressure, signs of anemia and hemosiderosis (e.g., pallor, jaundice, tachycardia, bronze color, and hepatosplenomegaly), and complete examination including cardiac, chest, abdominal, and neurological examination to assess the presence of any complications (endocrinology, renal, or hepatic).
Patients with β-thalassemia were classified into mild, moderate, and severe according to the severity score based on six clinical parameters weighted with different scores 1 : hemoglobin, 2 age at receiving the first transfusion, 3 requirement for (regular) blood transfusion, 4 size of spleen or history of splenectomy, 5 age at thalassemia presentation, 6 and growth and development (retardation). The total scores range from 0 to 10, such that mild (0–3.5), moderate (4–7), and severe (7.5–10) disease 14 ( Table 1 ).
Table 1. Scoring criteria adopted for children with of β-thalassemia.
| Clinical criteria | Points scored | |||
|---|---|---|---|---|
| 0 | 0.5 | 1 | 2 | |
| Hemoglobin (g/dL) | >7.5 | 6–7.5 | <6 | |
| Age at receiving first blood transfusion (y) | >10 | 5–10 | <5 | |
| Requirement for blood transfusion | None/rare | Occasional | Regular | |
| Size of spleen (cm) | <3 | 3–10 | >10 or splenectomized | |
| Age at thalassemia presentation (y) | >10 | 3–10 | <3 | |
| Growth and development | >25th percentile | 3rd–25th Percentile | <3rd percentile | |
Note: Total severity score: Mild 0 to 3.5, moderate 4 to 7, and severe 7.5 to 10.
Laboratory Investigations
Peripheral blood samples were collected on EDTA tubes and the genomic. Laboratory investigations included CBC using Sysmex XT-1800i (Sysmex, Japan), and measurement of serum ferritin using Immulite 1000 analyzer (Siemens Healthcare Diagnostics, Marburg, Germany). For chemical analysis, clotted samples were obtained and serum was separated by centrifugation for 15 minutes at 1,000× g .
DNA Isolation and Single-Gene Polymorphism Genotyping
The genomic DNA was extracted from the peripheral leucocytes utilizing human DNA extraction Kit, QIAamp DNA Blood Mini kits (Qiagen, Hilden, Germany), according to the manufacturer protocol. The TaqMan real-time polymerase chain reaction (PCR) technique was used for of the analysis BCL11A (rs11886868) genotypes. The reaction mixture was prepared in 25 μL containing 12.5 mL of 2× TaqMan Genotyping Master Mix with 30 to 80 µg/mL of the genomic DNA (2 μL), 1.25 μL of 20× TaqMan genotyping assay mix (containing BCL11A [rs11886868]) specific primers and FAM and VIC dye-labeled probes; Thermo-Fisher Scientific, Waltham, Massachusetts, United States), and 11.25 μL nuclease-free water. The cycling condition was performed on real-time PCR system (Applied Biosystem, step I version, Foster City, California, United States) as follows: 95°C for 10 minutes, then 40 cycles as 15 seconds at 95°C and 1 minute at 60°C.
The data were analyzed and presented on the multicomponent plot for allelic discrimination of allele 1 versus allele 2 using Applied Biosystem, step I version software analysis modules.
Statistical Analysis
Analysis of data was done using Statistical Package for Social Science (IBM SPSS) version 21 (IBM Corporation, Armonk, New York, United States). Kolmogorov–Smirnov's test was used to examine the normality of data. Quantitative variables were described in the form of range, mean and standard deviation or median, and interquartile range (75th and 25th percentiles). Qualitative variables were described as number and percentage. One-way analysis of variance test was used to compare parametric datasets. Nonparametric variables were compared using Kruskal–Wallis' test. A Tukey's honestly significant difference post hoc test was performed if an overall significance was found. Qualitative variables were compared using chi-square test. Pearson's correlation coefficient was used to assess the association between two normally distributed variables. When a variable was not normally distributed, a Spearman's correlation test was performed. A p -value of less than 0.05 was considered significant and less than 0.01 was considered highly significant.
Results
A total of 82 children with β-thalassemia whose age ranged from 6 to 18 years were studied. Their mean initial HbF at diagnosis was 63.50 ± 26.19%, range 9.6 to 98.8 and their mean Hb level prior to first transfusion was 7.98 ± 1.56 g/dL, range 5.1 to 12.7. They were divided into three subgroups (mild, moderate, and severe) based on severity scoring system. 14 There were 20 mild (24.4%), 24 moderate (29.3%), and 38 severe (46.3%) β-thalassemia children. The clinical and biochemical characteristics of the studied children with β-thalassemia are listed in Table 2 . According to BCL11A gene SNP (rs11886868) genotyping, 16 children had CC genotype (19.5%), 38 had TC genotype (46.3%), and 28 had TT genotype (34.1%) ( Fig. 1 ).
Table 2. Clinicodemographic and biochemical characteristics of the studied children with β-thalassemia.
| Children with β thalassemia N = 82 |
||
|---|---|---|
| Gender | Female | 38 (46.3%) |
| Male | 44 (53.7%) | |
| Age (y) | Mean ± SD | 12.95 ± 3.63 |
| Range | 6–18 | |
| Wt (kg) | Mean ± SD | 35.11 ± 11.19 |
| Range | 15–60 | |
| Wt (z-score) | Median (IQR) | −1.53 (−2.5 to −1.12) |
| Range | −4.93 to 1.3 | |
| Ht (cm) | Mean ± SD | 136.79 ± 26.65 |
| Range | -1.7–165 | |
| Ht (z score) | Median (IQR) | −1.97 (−2.56 to −0.95) |
| Range | −3.75 to 1.84 | |
| Disease duration (y) | Mean ± SD | 12.06 ± 3.72 |
| Range | 3–17.5 | |
| Age at first transfusion (mo) | Median (IQR) | 7 (6–12) |
| Range | 2–48 | |
| Transfusion interval (wk) | Median (IQR) | 2 (2–4) |
| Range | 2–4.3 | |
| Transfusion index (mL/kg/y) | Median (IQR) | 270 (231–360) |
| Range | 140–500 | |
| Chelation therapy | No | 0 (0.0%) |
| Single | 36 (43.9%) | |
| Combined | 46 (56.1%) | |
| Serum ferritin (ng/mL) | Median (IQR) | 1,686 (1,000–4,000) |
| Range | 120–9,500 | |
| Splenectomy | Negative | 42 (51.2%) |
| Positive | 40 (48.8%) | |
| Hb prior to first transfusion (g/dL) | Mean ± SD | 7.98 ± 1.56 |
| Range | 5.1–12.7 | |
| Initial HbF at diagnosis (%) | Mean ± SD | 63.50 ± 26.19 |
| Range | 9.6–98.8 | |
| Severity scoring | Median (IQR) | 7 (4–8.5) |
| Range | 2–9.5 | |
| BCL11A genotype | CC | 16 (19.5%) |
| TC | 38 (46.3%) | |
| TT | 28 (34.1%) | |
Abbreviations: BCL11A, B-cell lymphoma 11A; Hb, hemoglobin; HbF, fetal hemoglobin; Ht, height; IQR, interquartile range; SD, standard deviation; Wt, weight.
Fig. 1.
Detection of Taqman real-time polymerase chain reaction product showing: ( a ) Rising for the curve of VIC stain (green) at cycle number 28 indicating wild (CC) B-cell lymphoma 11A (BCL11A) genotype. ( b ) Two curves rising for both FAM stain (blue) and VIC stain (green) at cycle number 28 indicating heterozygous (TC) BCL11A genotype. ( c ) Rising for the curve of FAM stain (blue) at cycle number 22 that indicating mutant (TT) BCL11A genotype.
Upon comparing the three different subgroups (mild, moderate, and severe), a significant difference in the genotypic distribution and allele frequency was found between the mild, moderate, and severe groups of β-thalassemia ( p < 0.001).
Fourteen children from the mild group had CC genotype (87.5%), whereas 20 children from the moderate group had TC genotype (52.6%) and 26 children from the severe group had TT genotype (92.9%) ( Table 3 ).
Table 3. Comparison of children with mild, moderate, and severe β-thalassemia regarding various clinic-laboratory data.
| Mild N = 20 |
Moderate N = 24 |
Severe N = 38 |
Test value | p -Value | ||
|---|---|---|---|---|---|---|
| Gender | Female | 10 (50.0%) | 8 (33.3%) | 20 (52.6%) | 2.345 a | 0.310 |
| Male | 10 (50.0%) | 16 (66.7%) | 18 (47.4%) | |||
| Age (y) | Mean ± SD | 12.15 ± 4.58 | 12.92 ± 2.97 | 13.39 ± 3.57 | 0.779 b | 0.462 |
| Range | 6–18 | 8–18 | 6–18 | |||
| Wt Z score | Median (IQR) | −1.46 (−2.74 to −1.13) | −1.27 (−2.4 to −0.69) | −1.54 (−2.26 to −1.25) | 1.263 c | 0.532 |
| Range | −3.26 to 0.48 | −3.18 to −0.36 | −4.93 to 1.3 | |||
| Ht z score | Median (IQR) | −1.48 (−2.49 to −0.9) | −1.76 (−2.36 to −1.04) | −2.24 (−3.24 to −0.47) | 0.971 c | 0.615 |
| Range | −3.75 to −0.45 | −3.32 to 0.2 | −3.73 to 1.84 | |||
| Ferritin (ng/mL) | Median (IQR) | 1,125 (625–2,077) | 1,459 (1,077–3,043.5) | 3,680 (1,400–5,000) | 9.449 c | 0.009 |
| Range | 120–3,979 | 202–7,646 | 274.8–9,500 | |||
| BCL11A genotype | CC | 14 (87.5%) | 2 (12.5%) | 0 (0.0%) | 69.691 a | < 0.001 |
| TC | 6 (15.8%) | 20 (52.6%) | 12 (31.6%) | |||
| TT | 0 (0.0%) | 2 (7.1%) | 26 (92.9%) | |||
Abbreviations: Ht, height; IQR, interquartile range; SD, standard deviation; Wt, weight.
Chi-square test.
One way analysis of variance test.
Kruskall–Wallis' test.
Regarding BCL11A (rs11886868) genotypes, TT genotype had a significantly higher severity scoring than the other two groups ( p < 0.001) with a lower initial HbF % at diagnosis ( p < 0.001) ( Table 4 ).
Table 4. Comparison of children with β-thalassemia having different BCL11A genotypes regarding various clinic-laboratory data.
| CC N = 16 |
TC N = 38 |
TT N = 28 |
Test value | p -Value | ||
|---|---|---|---|---|---|---|
| Gender | Female | 4 (50.0%) | 7 (36.8%) | 8 (57.1%) | 1.389 a | 0.499 |
| Male | 4 (50.0%) | 12 (63.2%) | 6 (42.9%) | |||
| Age (y) | Mean ± SD | 11.94 ± 4.43 | 12.16 ± 3.77 | 14.61 ± 2.39 | 2.381 b | 0.106 |
| Range | 6–17 | 6–18 | 11.5–18 | |||
| Wt (Z score) | Median (IQR) | −1.37 (−2.27 to −0.8) | −1.53 (−2.5 to −0.9) | −1.54 (−2.98 to −1.26) | −0.785 c | 0.432 |
| Range | −3.26 to 0.48 | −3.18 to 1.3 | −4.93 to 0.09 | |||
| Ht (z score) | Median (IQR) | −1.01 (−.97 to −0.75) | −1.97 (−2.49 to −1) | −2.34 (−3.24 to −0.99) | −1.536 c | 0.125 |
| Range | −2.72 to −0.45 | −-3.75 to 1.84 | −3.73 to −0.3 | |||
| Ferritin (ng/mL) | Median (IQR) | 947.5 (685–2,926.5) | 1,566 (1,100–3,680) | 4,193.5 (990–5,893) | −1.775 c | 0.076 |
| Range | 120–3,979 | 202–7,646 | 274.8–9,500 | |||
| Initial HbF at diagnosis (%) | Mean ± SD | 90.93 ± 7.08 | 59.69 ± 13.01 | 24.55 ± 10.71 | 96.899 b | <0.001 |
| Range | 73–98.8 | 32–79.5 | 9.6–38.3 | |||
| Severity scoring | Median (IQR) | 1 (1–1) | 2 (2–3) | 3 (3–3) | −3.873 c | <0.001 |
| Range | 2–4 | 3–8.5 | 6–9.5 | |||
Abbreviations: HbF, fetal hemoglobin; Ht, height; IQR, interquartile range; SD, standard deviation; Wt, weight.
Chi-square test.
One way analysis of variance test.
Kruskall–Wallis' test.
Discussion
HbF is a potent functional modifier of the hemoglobin molecular stability in hemoglobinopathies especially β-thalassemia and sickle cell disease. Differences in the levels of HbF affect the severity of sickle cell disease and β-thalassemia. Induction of HbF is a promising therapeutic approach to ameliorate the clinical severity of β-hemoglobin disorders. 6
BCL11A is a major suppressor of HbF. Downregulation of BCL11A expression in primary adult erythroid cells from people with β-thalassemia and controls led to robust HbF expression, without major perturbation of erythropoiesis. 15 Moreover, mice lacking BCL11A appeared to have normal erythropoiesis, but failed to silence the embryonic globin genes in definitive erythroid cells, allowing persistent expression of HbF. 16 However, the exact mechanism through which BCL11A acts to silence HbF remains unraveled.
Li et al (2018) suggested that downregulation of BCL11A induces production of c-globin in K562 cells and primary adult erythroid cells from people with β-thalassemia and controls without major perturbation in erythropoiesis. 6 In addition, Menzel et al speculated that dysregulated BCL11A expression may influence HbF by affecting the kinetics of erythropoiesis. They stated that BCL11A loci have a major influence on the quantitative variation of the trait in healthy adults and possibly on the “erythropoietic stress” responses underlying variability in β-thalassemia and sickle cell disease severity and on the capacity of affected individuals to respond to pharmacologic inducers of HbF. 17
In the current study, the most common BCL11A (rs11886868) genotype in the studied cohort was TC being present in 38 children with β-thalassemia (46.3%), followed by homozygous CC mutation being present in 28 children (34.1%) and TT (wild) genotype in 16 children (19.5%). Similarly, Dadheech et al (2016) reported the most common genotype for the same SNP in a cohort of Indian people with β-thalassemia to be the TC genotype being present in 51.2%, followed by CC genotype present in 40% of the studied cohort. 3 The frequency of CC variant genotype was found to be 28.8% in an Iranian population and 61.2% in a Sardinian population. 18 19 This goes in concordance with the data from the HapMap project, which states that CC alleles are widespread in the Asian population, including China and Japan (80–95%), whereas the TT alleles are most widely documented in the populations of Europe and Latin American plains. The frequency of BCL11A (rs11886868) genotypes in the European populations were 4.5% for CC genotype, 31.2% for TC genotype, and 64.3% for TT genotype, while the frequency of these genotypes in the Asian populations were 2.4 to 12.8% for CC, 38 to 42.9% for CT, and 48 54% for TT genotypes. 20
Children with β-thalassemia having TT genotype had a significantly higher severity scoring than the other two groups. Similarly, Dadheech et al found a significant association between CC genotype and observed milder disease phenotype in people with β-thalassemia. 3 In addition, Danjou et al found that rs11886868 CC genotype was associated with milder disease severity and delayed first transfusion ( p < 0.001). 21
This might be attributed to the suppression of HbF by the TT genotype. Cantor and Orkin (2002) suggested that BCL11A interacts with the hematopoietic transcription factors GATA1, SOX6, and ZFPM1/FOG1, as well as the nucleosome remodeling and deacetylase chromatin remodeling and repressor complex. 22 Moreover, BCL11A has been shown to bind to several transcriptional corepressors, including the lysine-specific demethylase/corepressor to repressor element 1—silencing transcription factor), SWI/SNF (mating-type switching/sucrose nonfermenting), and NCoR/SMRT (nuclear receptor corepressor/silencing mediator for retinoid and thyroid receptors) complexes 23 leading to silencing of HbF.
In the present study, a significant positive relation was found between wild rs11886868 CC genotype and initial HbF level at diagnosis. In concordance with these results, studies on populations in Europe and Central Asia showed that BCL11A (rs11886868) polymorphism had a strong relationship with the increase in HbF, in both normal individuals and thalassemia patients. 24 25 On the other side, Rujito et al (2016) showed no significant relationship between BCL11A quantitative trait locus in people with β-thalassemia and HbF level or clinical severity. This might be attributed to the fact that the studied cohort of people with β-thalassemia were all severe. 26
The role of BCL11A as a potent silencer of γ-globin gene has also been demonstrated experimentally by increased production of HbF in developing adult erythroblasts after small hairpin RNA-mediated knocked down. 27 This supports the hypothesis that BCL11A plays an integral role in silencing γ-globin genes during the developmental switching, in addition to reactivation of HbF in adult erythroblasts.
One limitation of our study is its cross-sectional nature which could not imply causality and the relatively small sample size. The lack of β-globin gene mutation analysis in the studied children with β-thalassemia is also an important limitation. Therefore, further larger longitudinal studies are needed to identify the role of different BCL11A (rs11886868) genotypes in predicting disease severity in β-thalassemia. Moreover, studies should address the role of BCL11A gene polymorphism induction as potential therapy for ameliorating the severity of β-thalassemia.
Conclusion
Increased γ-globin expression associated with BCL11A gene (rs11886868) polymorphism is associated with better clinical severity of β-thalassemia in the studied cohort. Assessment of rs11886868 could help in stratification of children with β-thalassemia into different severity groups.
Footnotes
Conflict of Interest None declared.
References
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