Genetic polymorphism of novel SNP rs5006884 in OR51B6 and SNP rs4499252 in AHSP among transfusion-dependent and non-transfusion-dependent β-thalassemia/Hb E patients in Thailand: a multivariate analysis of clinical and genetic polymorphism
- Open Access
- 16.05.2025
- Research
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Abstract
Introduction
Beta-thalassemia (β-thalassemia) is a common genetic blood disorder, particularly prevalent in Southeast Asia, including Thailand, characterized by reduced or absent production of β-globin chains. In combination with other hemoglobinopathies, such as hemoglobin E (Hb E), this disorder leads to various clinical manifestations, ranging from mild to severe anemia. One of the most serious forms of compound heterozygous β-thalassemia and Hb E, transfusion-dependent thalassemia (TDT), requires lifelong blood transfusions to manage severe anemia. Non-transfusion-dependent thalassemia (NTDT) is a milder form where the need for transfusions is less frequent but still poses significant health challenges [1‐4]. The clinical severity of compound heterozygous β-thalassemia and Hb E depends on multiple factors, including the specific β-thalassemia mutations inherited, the level of Hb F expression, and the presence of other modifying factors such as co-inherited genetic modifiers [5].
Recent advances in molecular genetics have been discovered with several genetic polymorphisms that influence the clinical outcomes in compound heterozygous β-thalassemia and Hb E patients. Single nucleotide polymorphisms (SNP) rs9399137 on the HBS1L-MYB intergenic region and SNP rs4671393 on BCL11A have been shown to influence hemoglobin F (HbF) levels, a crucial factor in the clinical presentation of thalassemia. The HBS1L-MYB region has been implicated in regulating the production of HbF, with certain variants leading to higher levels of HbF, which can ameliorate the symptoms of thalassemia by compensating for the defective adult hemoglobin. Whereas SNP rs9399137 in particular has been associated with reduced transfusion requirements and less severe forms of the disease [6‐9].
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In addition, OR51B6 and alpha-hemoglobin stabilizing protein (AHSP) genes have also been studied for its potential role in modulating hemoglobin levels and the clinical severity of hemoglobinopathies. However, its role has not been reported in compound heterozygous β-thalassemia and Hb E patients. The SNP rs5006884 is located in the OR51B6 gene on chromosome 11. OR51B6 is a gene encoding an olfactory receptor, part of the G-protein-coupled receptor (GPCR) family primarily involved in odor detection. While its primary function is in the olfactory system, however; olfactory receptors may have roles beyond smell, including potential involvement in hematological and immune-related processes. Cyrus et al. investigated the role of rs5006884 in modulating hemoglobin A₂ (HbA₂) levels among β-thalassemia carriers. The research found that carriers of the rs5006884 variant exhibited elevated HbA₂ levels. Furthermore, a genome-wide association study identified this SNP as significantly associated with elevated fetal hemoglobin (HbF) levels, explaining the variability in HbF. This association was independent of other known genetic factors affecting HbF levels, such as the XmnI polymorphism and β-globin gene cluster haplotypes [10]. The SNP rs4499252 is located in the promoter region of the AHSP gene on chromosome 16. AHSP functions as a molecular chaperone that binds to free α-globin chains, preventing their precipitation and subsequent cellular damage. A case-control study involving Iraqi patients with β-thalassemia major investigated the association between rs4499252 and disease severity. The study found a significant increase in the frequency of the GG genotype among β-thalassemia patients compared to healthy controls, suggesting a potential link between this polymorphism and the clinical manifestation of the disease [10].
Thus, the aim of this study is to explore the role of genetic polymorphisms rs5006884 in OR51B6, rs4499252 in AHSP, rs9399137 in HBS1L-MYB, and rs4671393 in BCL11A in relation to clinical factors such as severity and transfusion dependency in β-thalassemia/Hb E patients in Thailand. Multivariate models incorporating these factors may provide a comprehensive understanding of the determinants of transfusion dependency and clinical outcomes in compound heterozygous β-thalassemia and Hb E patients.
Materials and methods
Samples
Ethical approval of the study was obtained from the Institutional Review Board of Srinakharinwirot University (SWU), Thailand (SWUEC663008) based on Declaration of Helsinki, Belmont Report, International Conference on Harmonization in Good Clinical Practice (ICH-GCP), International Guidelines for Human Research, along with laws and regulations of Thailand. This study, we use leftover specimen that informed with broad consent form with the first collecting specimen. A total of 189 leftover DNA samples were recruited, including 58 subjects with TDT -Thalassemia/Hb E, 58 subjects with NTDT -Thalassemia/Hb E, 33 subjects with homozygous Hb E and 40 normal control subjects. Compound heterozygous -thalassemia and Hb E was categorized into TDT and NTDT based on the clinical severity, the need for regular blood transfusions: TDT was recorded with regular transfusions every 2–4 weeks while NTDT was recorded occasional transfusions may be required during stress or for specific indications.
Hematological, hemoglobin and DNA analyses
The hematological parameters were measured using hematology automation (Sysmex XN3000, Sysmex, Kobe, Japan). Hb typing (Hb analysis) was performed on capillary electrophoresis (Capillarys 2; Sebia, Lisses, France). Identification of common β-thalassemia mutations in Thailand was performed with allele specific PCR as previous described [11]. Whereas rare β-thalassemia mutations were further analyzed with samples that were not found to contain common mutations, using direct DNA sequencing on an ABI PRISM™ 3130 XLanalyzer (Applied Biosystems, Foster City, CA, USA), as described [12].
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Genotyping of single nucleotide polymorphism
Genotyping assay for four SNP including rs5006884 in OR51B6, rs4499252 in AHSP, rs9399137 in HBS1L-MYB, and rs4671393 in BCL11A based on the rhAmp SNP Genotyping System, was performed and reported for the first time in this study. The protocol of each SNPs was created using the rhAmp Genotyping Design Tool (https://www.idtdna.com/site/order/designtool/index/GENOTYPING_PREDESIGN). The 5 uL Real-time PCR mixture included 2.65 uL of Combined Master Mix and Reporter Mix, 025 uL of 20X rhAmp SNP Assay (Integrated DNA Technologies, Singapore), 2 uL of 50 ug/uL genomic DNA, with the remainder comprising distilled water. Thermal cycling was performed on real-time PCR QuantStudio5 (Applied Biosystems, California, United States of America), starting with hold stage (95oC for 10 min) for activating the Tag DNA polymerase, followed by 40 cycles of PCR (95oC for 10 s, 60 oC for 30 s, 68 oC for 20 s). The final heat activation was performed at 95oC for 15 min. The visualization of separated genotype each SNP was performed on QaunStudio design and analysis software v1.4.3 (Applied Biosystems, California, United States of America) as shown in Fig. 1.
Fig. 1
Four SNPs were genotyped based on the rhAmp SNP Genotyping assay, including rs4671393 on BCL11A, rs9399137 on HBS1L-MYB, rs5006884 on OR51B6, and rs4499252 on the AHSP gene. The genotypes were clearly categorized by red dots, blue dots, and green dots, corresponding to homozygous wild-type, homozygous mutant, and heterozygous, respectively
Statistical analysis
Statistical analysis and proportion were performed using MINITAB release 14.12.0 statistical software. The multivariate models associated with genetic and clinical factors in compound heterozygous β-thalassemia and Hb E (n = 116) related to transfusion event (NTDT), and (TDT) was applied using multiple regression analysis on MINITAB release 14.12.0 statistical software. In this study, we analyzed two model related to severity of TDT and NTDT based on thalassemia variants model and genetics modifying factors (SNPs). A p-value < 0.05 was considered to be statistically significant.
Results
Table 1 summarizes the hematological parameters, age, and genotype distribution of 116 patients with compound heterozygous β-thalassemia and Hb E, categorized into non-transfusion dependent thalassemia (NTDT, n = 58) and transfusion dependent thalassemia (TDT, n = 58). The combination of Hb E β-thalassemia was found with several genotypes. The HBB:c.59 A > G mutation was the most prevalent genotype in the NTDT group, identified in 41 of 58 individuals (70.7%). Other genotypes, including HBB:c.126_129delCTTT and NC_000011.10:g.5224302_ 5227791del, were less common, accounting for six cases each (10.3%). Rare mutations such as HBB:c.-78 A > G, HBB:c.92 + 1G > T, and HBB:c.316–197 C > T were each observed in one sample. Whereas the TDT group, HBB:c.126_129delCTTT was the most frequently observed mutation, present in 22 of 58 individuals (37.9%). Other genotypes, such as HBB:c.92 + 5G > C (19 cases, 32.8%) and HBB:c.92 + 1G > T (7 cases, 12.1%), were also notable. Rare genotypes like HBB:c.46delT, HBB:c.216dupT, and NC_000011.10:g.5112882_5231358del were identified in one individual each.
Table 1
The data of hematological parameters, and age of compound heterozygous β-thalassemia and Hb E (n = 116). The data are divided as two groups; non-transfusion dependent thalassemia (NTDT) and transfusion dependent thalassemia (TDT). The data are presented as mean ± standard deviation or as Raw data where appropriate
Compound heterozygous β-thalassemia and Hb E (Genotypes) | N (116) | Age (yrs) | Hb (g/dL) | Hct (%) | MCV (fL) | MCH (pg) | MCHC (g/dL) | RDW (%) | |
|---|---|---|---|---|---|---|---|---|---|
Non-transfusion dependent thalassemia (NTDT) (n = 58) | |||||||||
Hb E (HBB: c.79G > A) | HBB: c.-78 A > G | 1 | 2 | 9.1 | 27.4 | 63 | 21 | 33.2 | 34.6 |
HBB: c.59 A > G | 41 | 9.8 ± 6.7 | 9.7 ± 1.3 | 29.4 ± 4.1 | 55.1 ± 4.7 | 18.1 ± 1.5 | 32.1 ± 5.3 | 21.2 ± 2.4 | |
HBB: c.92 + 1G > T | 1 | 28 | 9.3 | 28.2 | 66.7 | 22.2 | 33.0 | 27.0 | |
HBB: c.92 + 5G > C | 2 | 46, 22 | 7.3, 9.8 | 23.7, 31.0 | 63.4, 67.7 | 19.5, 21.4 | 30.8, 31.6 | 27.0, 30.8 | |
HBB: c.126_129delCTTT | 6 | 21.7 ± 11.9 | 8.6 ± 1.0 | 27.6 ± 2.5 | 57.1 ± 6.6 | 17.9 ± 2.4 | 31.3 ± 0.8 | 27.2 ± 2.3 | |
HBB: c.316–197 C > T | 1 | 8 | 8.7 | 26.7 | 47.5 | 15.5 | 32.6 | 25.7 | |
NC_000011.10:g. 5224302_5227791del | 6 | 8.2 | 9.6 | 28.9 | 61.0 | 20.1 | 33.1 | 24.5 | |
Transfusion dependent thalassemia (TDT) (n = 58) | |||||||||
Hb E (HBB: c.79G > A) | HBB: c.27dupG | 1 | 1 | 7.2 | 22.1 | 62.6 | 20.0 | 32.6 | 30.2 |
HBB: c.46delT | 1 | 8 | 6.9 | 21.6 | 69.9 | 22.3 | 31.9 | 23.3 | |
HBB: c.52 A > T | 6 | 3.4 ± 0.5 | 6.6 ± 0.7 | 20.3 ± 2.4 | 63.0 ± 7.2 | 20.8 ± 3.0 | 32.3 ± 1.8 | 28.2 ± 1.7 | |
HBB: c.92 + 1G > T | 7 | 12.6 ± 14.9 | 7.0 ± 1.4 | 22.1 ± 4.0 | 58.3 ± 14.4 | 19.7 ± 1.6 | 32.3 ± 0.7 | 31.3 ± 4.4 | |
HBB: c.92 + 5G > C | 19 | 5.5 ± 4.3 | 6.6 ± 1.1 | 21.1 ± 3.0 | 66.8 ± 8.5 | 21.0 ± 3.8 | 31.3 ± 3.3 | 28.3 ± 4.3 | |
HBB: c.126_129delCTTT | 22 | 11.9 ± 11.2 | 6.9 ± 1.2 | 21.5 ± 3.5 | 63.5 ± 8.1 | 20.4 ± 2.7 | 32.1 ± 1.7 | 29.2 ± 3.6 | |
HBB: c.216dupT | 1 | 6 | 6.9 | 21.0 | 70.0 | 16.8 | 32.9 | 29.5 | |
NC_000011.10:g. 5112882_5231358del | 1 | 7 | 8.4 | 25.3 | 68.2 | 22.6 | 33.2 | 29.3 | |
The comparison of hematological data between NTDT and TDT was shown in Table 1. Hemoglobin (Hb) levels and hematocrit (Hct) values were significantly influenced by genotype. For example, NTDT individuals with the HBB:c.59 A > G mutation exhibited the highest mean Hb levels (9.7 ± 1.3 g/dL) and Hct (29.4 ± 4.1%). In contrast, TDT individuals with HBB:c.126_129delCTTT had consistently lower Hb levels (6.9 ± 1.2 g/dL) and Hct (21.5 ± 3.5%), reflecting more severe anemia and transfusion dependence. Furthermore, Red cell distribution width (RDW) was markedly higher in TDT individuals with mutations such as HBB:c.92 + 5G > C (mean RDW 28.3 ± 4.3%) compared to NTDT individuals with the same mutation (27.2 ± 2.3%). This highlights the increased heterogeneity in red cell morphology associated with more severe clinical presentations. The distribution of genotypes showed distinct patterns between NTDT and TDT groups. Mutations such as HBB:c.59 A > G were predominantly associated with NTDT, while HBB:c.126_129delCTTT and HBB:c.92 + 5G > C were more prevalent in TDT. Overall, the results highlight the variability in genotype prevalence and its significant influence on hematological parameters, severity, and transfusion requirements in compound heterozygous β-thalassemia and Hb E.
Figure 1 illustrates the genotyping of four single nucleotide polymorphisms (SNPs) including rs5006884, rs4499252, rs9399137, and rs4671393 performed using the rhAmp SNP Genotyping assay. The genotypes for each SNP were distinctly categorized into three groups: homozygous wild-type, homozygous mutant, and heterozygous. This study is the first to report genotyping for these specific SNPs using the rhAmp assay. The rhAmp technology demonstrated robust and reliable performance in distinguishing genotypes with clear clustering of fluorescence signals. This approach eliminates the need for post-PCR processing steps, and reducing the risk of contamination. The cost-effectiveness of the assay was a notable advantage, with an estimated cost of USD 1.5 per sample, making it suitable for large-scale screening of polymorphisms or genetic variants.
Table 2 represented the distribution of allele counts and proportions for four SNPs was analyzed across wild-type, homozygous Hb E, non-transfusion-dependent thalassemia (NTDT), and transfusion-dependent thalassemia (TDT) groups. For rs4671393, the proportion of the G allele was significantly higher in all groups compared to the wild-type (P < 0.05). Specifically, the G allele frequency was 76.25% in the wild-type group, compared to 89.39%, 87.93%, and 89.66% in the homozygous Hb E, NTDT, and TDT groups, respectively. In the case of rs5006884, the T allele showed significant differences in frequency among the groups (P < 0.05). The T allele was more prevalent in the NTDT group (55.17%) compared to the wild-type (25.00%), homozygous Hb E (36.36%), and TDT (23.28%) groups. No significant differences in allele frequencies were observed for rs9399137 and rs4499252 among the groups.
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Table 2
The allele counts and proportions of four SNPs were compared across the following groups: wild-type, homozygous Hb E, non-transfusion dependent thalassemia (NTDT), and transfusion dependent thalassemia (TDT). The analysis highlights the differences in allele distribution among these groups
Genes | BCL11A | HBS1L-MYB | OR51B6 | AHSP | ||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|
SNPs ID | rs4671393 | rs9399137 | rs5006884 | rs4499252 | ||||||||
Alleles | A | G | G allele proportion (%) | C | T | T allele proportion (%) | C | T | T allele proportion (%) | A | G | G allele proportion (%) |
Wild type (n = 80) | 19 | 61 | 76.25* | 8 | 72 | 90.00 | 60 | 20 | 25.00 | 54 | 26 | 32.50 |
Homozygous Hb E (n = 66) | 7 | 59 | 89.39 | 7 | 59 | 89.39 | 42 | 24 | 36.36 | 47 | 19 | 28.79 |
NTDT (n = 116) | 14 | 102 | 87.93 | 18 | 98 | 84.48 | 52 | 64 | 55.17** | 74 | 42 | 36.21 |
TDT (n = 116) | 12 | 104 | 89.66 | 16 | 100 | 86.21 | 89 | 27 | 23.28 | 78 | 38 | 32.76 |
The genotype frequencies of four SNPs were compared among wild-type, homozygous Hb E, NTDT, and TDT groups (Table 3). For rs5006884 in the OR51B6 gene, the TT genotype frequency was significantly higher in the NTDT group (32.76%) compared to the wild-type (5.00%), homozygous Hb E (12.12%), and TDT (5.17%) groups (P < 0.05). Whereas the AHSP gene, the GG genotype of rs4499252 was significantly less frequent in the homozygous Hb E group (3.03%) compared to the wild-type (12.50%), NTDT (13.79%), and TDT (12.07%) groups (P < 0.05). No significant differences in genotype frequencies were observed for rs4671393 in the BCL11A gene or rs9399137 in the HBS1L-MYB gene among the groups. However, the GG genotype of rs4671393 was predominant across all groups, with frequencies ranging from 75.86 to 81.82%. Similarly, the TT genotype of rs9399137 was observed at high frequencies, ranging from 72.41 to 80.00% across all groups. Furthermore, we performed a sub-analysis of the SNP genotypes in individuals carrying the HBB:c.126_129delCTTT mutation but with different clinical presentations. There were 22 cases with TDT and 6 cases with NTDT. The results showed no significant differences in the SNP genotypes, allele proportion between TDT and NTDT patients.
A multivariate analysis was performed to assess the association of genetic variants and clinical factors with transfusion status in compound heterozygous β-thalassemia and Hb E patients (Table 4). Two models were evaluated to highlight key determinants and represented P-value < 0.001 with both models. In Model A, which included thalassemia-specific variants, hemoglobin (Hb) levels were identified as a significant predictor, with lower Hb levels correlating strongly with transfusion dependence (P < 0.001). Among the genetic variants, several showed significant associations. The HBB:c.59 A > G, HBB:c.-78 A > G and HBB:c.316–197 C > T mutations were significantly associated with an increased likelihood of non-transfusion event, with P-values of 0.002, 0.018 and 0.014, respectively. Additionally, the NC_000011.10:g. 5224302_5227791del variant also demonstrated a significant against transfusion dependence (P = 0.003). Whereas model B incorporated modifying genetic factors and confirmed the strong influence of Hb levels (P < 0.001). Among the SNPs analyzed, the TT genotype of rs5006884 in the OR51B6 gene emerged as a significant factor, showing an association with non-transfusion dependence (P = 0.002). No significant associations were found for other SNPs in BCL11A (rs4671393), HBS1L-MYB (rs9399137), or AHSP (rs4499252) genes. Overall, the results underscore the critical role of Hb levels and specific genetic variants, particularly in the HBB gene and rs5006884 in the OR51B6 gene, in predicting transfusion among patients.
Table 3
Comparison of genotype frequencies for four SNPs in non-transfusion dependent thalassemia (NTDT) and transfusion dependent thalassemia (TDT. The analysis includes comparisons between wild-type and homozygous Hb E groups
Genes | BCL11A | HBS1L-MYB | OR51B6 | AHSP | ||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|
SNPs ID | rs4671393 | rs9399137 | rs5006884 | rs4499252 | ||||||||
Genotypes | AA | GA | GG | CC | CT | TT | CC | CT | TT | AA | AG | GG |
Wild type (n = 40) | 0.0750 | 0.3250 | 0.6000 | 0.0000 | 0.2000 | 0.8000 | 0.5500 | 0.4000 | 0.0500 | 0.4750 | 0.4000 | 0.1250 |
Homozygous Hb E (n = 33) | 0.0303 | 0.1515 | 0.8182 | 0.0000 | 0.2121 | 0.7878 | 0.3940 | 0.4849 | 0.1212 | 0.4545 | 0.5156 | 0.0303* |
NTDT (n = 58) | 0.0000 | 0.2413 | 0.7586 | 0.0345 | 0.2414 | 0.7241 | 0.2241 | 0.4483 | 0.3276* | 0.4138 | 0.4482 | 0.1379 |
TDT (n = 58) | 0.0172 | 0.1724 | 0.8103 | 0.0172 | 0.2414 | 0.7414 | 0.5861 | 0.3621 | 0.0517 | 0.4155 | 0.4138 | 0.1207 |
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Table 4
Multivariate models associated with genetic and clinical factors in compound heterozygous β-thalassemia and Hb E (n = 116) related to transfusion event (NTDT), and (TDT)
Gene/ variants/ variable | Coefficients | SE Coefficients | P-value |
|---|---|---|---|
Model A: Thalassemia variants (P < 0.001) | |||
Hb | -0.07678 | 0.01852 | < 0.001 |
HBB | |||
HBB: c.-78 A > G | -0.8541 | 0.3539 | 0.018 |
HBB: c.46delT | -0.0230 | 0.3522 | 0.948 |
HBB: c.52 A > T | -0.0435 | 0.2691 | 0.872 |
HBB: c.59 A > G | -0.8059 | 0.2564 | 0.002 |
HBB: c.92 + 1G > T | -0.1374 | 0.2662 | 0.607 |
HBB: c.92 + 5G > C | -0.1188 | 0.2547 | 0.642 |
HBB: c.126_129delCTTT | -0.2121 | 0.2534 | 0.405 |
HBB: c.216dupT | -0.0230 | 0.3522 | 0.948 |
HBB: c.316–197 C > T | -0.8848 | 0.3532 | 0.014 |
NC_000011.10:g.5224302_5227791del | 0.8170 | 0.2725 | 0.003 |
NC_000011.10:g.5112882_5231358del | 0.0921 | 0.3528 | 0.795 |
Model B: Modifying factors (P < 0.001) | |||
Hb | -0.19182 | 0.01836 | < 0.001 |
BCL11A | |||
rs4671393 (AG) | -0.2603 | 0.3511 | 0.460 |
rs4671393 (GG) | -0.3676 | 0.3453 | 0.289 |
HBS1L-MYB | |||
rs9399137 (CT) | 0.0351 | 0.2028 | 0.863 |
rs9399137 (TT) | -0.0845 | 0.1968 | 0.669 |
OR51B6 | |||
rs5006884 (CT) | -0.10017 | 0.07148 | 0.164 |
rs5006884 (TT) | -0.29372 | 0.09447 | 0.002 |
AHSP | |||
rs4499252 (AG) | -0.01706 | 0.06631 | 0.797 |
rs4499252 (GG) | -0.0220 | 0.1034 | 0.832 |
Discussion
This study provided the detailed insights into the hematological parameters, age distribution, and genotype profiles of patients with compound heterozygous β-thalassemia and Hb E, emphasizing the differences between NTDT and TDT groups. The findings found the heterogeneity of genotypes and their potential impact on clinical phenotypes and transfusion requirements. The predominance of the HBB:c.59 A > G mutation in the NTDT group (70.7%) aligns with its association with a milder clinical phenotype. This mutation, commonly found in Southeast Asian populations, may preserve residual β-globin production, contributing to less severe anemia and reducing the need for regular transfusions [13‐15]. In contrast, genotypes such as HBB:c.126_129delCTTT, which were more frequently observed in the TDT group (37.9%), are known to result in significant reductions in β-globin synthesis, leading to more severe anemia and necessitating regular transfusions. As expected, individuals in the NTDT group generally were maintained higher Hb levels and hematocrit values, which reflect better erythropoietic compensation compared to the TDT group. The elevated red cell distribution width (RDW) was observed in both groups, particularly in TDT, the significant degree of ineffective erythropoiesis and anisopoikilocytosis, which are hallmarks of β-thalassemia. Comprehensive genotyping could guide clinicians in predicting disease severity, transfusion requirements, and potential complications [16‐18].
The SNP rs4671393, located in the BCL11A gene, showed a higher frequency of the G allele in the homozygous Hb E, NTDT, and TDT groups compared to the wild-type group, with proportions of approximately 89% in all thalassemia-related groups. This result suggests a potential association between the G allele and thalassemia, especially considering that BCL11A is known to regulate fetal hemoglobin production and may be involved in the pathology of thalassemia. However, while the allele frequency differences were statistically significant (P < 0.05), no significant differences were found in genotype frequencies, with the GG genotype remaining predominant across all groups. Phanrahan P et al. also reported a GG genotype frequency of 68.8% in NTDT patients, which is similar to the findings of the study [17]. This suggests that the G allele may not substantially affect the genotype distribution but may still contribute to phenotypic variations in thalassemia.
In contrast, for SNP rs5006884 was located on the OR51B6 gene, the T allele was significantly more prevalent in the NTDT group compared to the other groups. The T allele frequency was notably higher in NTDT (55.17%) than in the wild-type (25.00%), homozygous Hb E (36.36%), and TDT (23.28%) groups. This observation was supported by a significantly higher frequency of the TT genotype in the NTDT group (32.76%), suggesting that the presence of the T allele in this SNP may be associated with the NTDT form of compound heterozygous β-thalassemia and Hb E. The role of OR51B6 in thalassemia remains unclear, but our findings warrant further investigation into its involvement in the regulation of hemoglobin production or other pathways that may contribute to thalassemia severity [10]. The potential relationship between the HBB:c.59 A > G mutation and the T SNP in OR51B6 in the context of non-transfusion dependence was further investigated. Given that both OR51B6 and the beta-globin gene are located on chromosome 11p15.4, we considered the possibility of linkage disequilibrium (LD) between these loci. To assess this, we calculated the pairwise LD using HAPLOVIEW 4.2 software. This analysis revealed a strong LD (D′ value = 0.899, 95% CI: 0.71–0.97) between the HBB:c.59 A > G mutation and the T SNP in OR51B6. This strong LD suggests that the inheritance of the TT genotype of this SNP may be influenced by the HBB:c.59 A > G mutation and may contribute to the association with non-transfusion dependence.
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For SNP rs4499252 in the AHSP gene, the GG genotype was significantly less frequent in the homozygous Hb E group compared to the other groups. The AHSP gene plays a critical role in the stability and regulation of alpha-globin chains, and this result may suggest that genetic variations in AHSP are influencing the manifestation of thalassemia, particularly in patients with homozygous Hb E [19]. Finally, no significant differences in allele and genotype frequencies were found for SNPs rs9399137 in the HBS1L-MYB gene. Despite high frequencies of the TT genotype across all groups, these findings indicated that this SNP might not play a substantial role in the genetic variability of thalassemia in the populations studied. However, the C allele of rs9399137 of the HBS1L-MYB intergenic region were observed among NTDT patients in previous study [17]. This data is consistent with a previous finding that indicated a minor effect this intergenic region on phenotypic expression of the patients [17, 20].
Multivariate analysis represented the significant role of hemoglobin (Hb) levels and specific genetic variants in predicting transfusion requirements, offering potential targets for personalized treatment strategies. In both models evaluated, Hb levels were consistently identified as a strong predictor of transfusion dependence. Specifically, lower Hb levels were strongly correlated with an increased likelihood of requiring regular transfusions (P < 0.001). This finding is consistent with the well-established clinical understanding that β-thalassemia and Hb E patients with more severe anemia typically require more frequent transfusions. Thus, monitoring Hb levels remains a crucial aspect of clinical management in thalassemia [17, 18]. Model A, which focused on thalassemia-specific genetic variants, revealed significant associations of the HBB:c.59 A > G HBB:c.-78 A > G, HBB:c.316–197 C > T mutations, and the NC_000011.10:g.5224302_5227791del variant, was found to be significantly associated with non-transfusion dependence (P < 0.05). Further investigation would be needed to fully understand the functional implications of these variants and how it may contribute to the clinical variability seen in thalassemia patients. However, this finding has been expected and extensively reported in the several literature. In the study aimed to explore additional genetic factors that may contribute to variability in clinical severity, particularly the novel association of SNP rs5006884 in OR51B6.
Model B, which incorporated additional modifying genetic factors, confirmed the strong influence of Hb levels. It also was highlighted the significant role of the rs5006884 variant in the OR51B6 gene. The TT genotype of this SNP was associated with an increased likelihood of non-transfusion dependence (P = 0.002). Although the precise mechanism remains unclear, the association of rs5006884 with non-transfusion dependence could open up new avenues for investigating the role of non-coding genes and genetic modifiers in thalassemia. Although, previous studies on NTDT have reported an association of BCL11A (rs4671393) and HBS1L-MYB (rs9399137) with mild phenotypic expression. However, these findings have not been compared with TDT patients. Therefore, this study aims to demonstrate the lack of significant differences between the two groups [16, 20, 21].
Finally, no significant associations were found for SNPs in the BCL11A, HBS1L-MYB, or AHSP genes in Model B, suggesting that these variants do not have a strong independent effect on transfusion in the population studied. While these genes are known to be involved in the regulation of hemoglobin production, their role in transfusion event may be less direct or may be influenced by other factors not found in this study.
The limitation of the study was the sample size. We acknowledge the relatively small sample size, which is a limitation due to the rarity of β-thalassemia/Hb E in the studied population. However, we believe that our findings still provide meaningful insights, as they align with previous literature and highlight novel genetic associations that warrant further investigation.
In conclusion, the two novel SNPs in the OR51B6 and AHSP genes provided valuable insight into potential genetic factors that may influence the clinical outcomes of thalassemia, such as the need for transfusions or the severity of symptoms. Furthermore, multivariate models identified Hb levels and specific genetic variants, particularly in the HBB and the OR51B6 genes, as predictors of transfusion events in compound heterozygous β-thalassemia and Hb E patients. These results contribute to our understanding of the genetic factors that influence clinical outcomes in thalassemia and could guide the development of more personalized treatment strategies aimed at reducing transfusion dependency. Further research is needed to explore the functional mechanisms underlying these genetic associations and to assess their potential applications in clinical practice.
Acknowledgements
This study was supported by a research grant from HRH Princess Maha Chakri Sirindhorn Medical Center, Faculty of Medicine, Srinakharinwirot University (Contract No. 093/2567). WJ, RK, PS, NM, and TT were supported by a research cluster in hematology and genetic diseases, as well as a research grant from HRH Princess Maha Chakri Sirindhorn Medical Center, Faculty of Medicine, Srinakharinwirot University (Contract No. 443/2567).
Declarations
Institutional review board statement
Ethical approval of the study was obtained from the Institutional Review Board of Srinakharinwirot University, Thailand (SWUEC663008) based on Declaration of Helsinki, Belmont Report, International Conference on Harmonization in Good Clinical Practice (ICH-GCP), International Guidelines for Human Research, along with laws and regulations of Thailand.
Competing interests
The authors declare no competing interests.
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