Abstract
Background/Aim: Vitamin D plays a critical role in regulating immune function. In literature, several studies have indicated that variations in the vitamin D receptor gene (VDR) may be associated with the risk of asthma risk. However, the findings remain inconsistent. The current study aimed to assess the influence of VDR polymorphisms on both asthma risk and severity.
Patients and Methods: A cohort comprising 453 non-asthmatic healthy controls and 198 asthmatic cases was assembled, and the VDR rs731236, rs1544410, rs2228570, and rs7975232 genotypes were determined utilizing polymerase chain reaction-restriction fragment length polymorphism methodology.
Results: Our findings indicated that people carrying the variant AG and AA genotypes of VDR rs2228570 demonstrated an elevated risk of asthma compared to wild-type GG carriers (odds ratio=1.55 and 2.19, 95% confidence interval=1.02-2.35 and 1.37-3.49; p=0.0507 and 0.0014, respectively). Furthermore, individuals carrying the variant A allele at VDR rs2228570 exhibited a higher risk of asthma than those carrying the wild-type G allele (odds ratio=1.51, 95% confidence interval=1.19-1.92; p=0.0008). Moreover, a significant association was observed between VDR rs2228570 A-carrying genotypes and increased the severity of asthma symptoms among asthmatic patients (p for trend=0.0024).
Conclusion: The variant A allele at VDR rs2228570 was associated with higher asthma susceptibility, and may serve as a predictor for asthma symptom severity. Our findings encouraged further validation in larger and diverse populations to further elucidate the significance of VDR genotype in asthma development.
Introduction
Asthma affects an estimated 300 million individuals globally, with projections indicating that by 2025, an additional 100 million people may develop the condition (1, 2). Vitamin D exhibits immunomodulatory effects, as evidenced by its deficiency being linked to immune-related disorders such as asthma, rhinitis, and atopic dermatitis (3, 4). Studies suggest that vitamin D contributes to immune tolerance of allergens, mitigating immunoglobulin E sensitization, a key factor in the pathogenesis of asthma and other allergic conditions (5, 6). Consequently, inadequate vitamin D levels may be a contributing factor to the rising global prevalence of asthma and allergic diseases. Investigations into the role of vitamin D in asthma pathogenesis often focus on maternal prenatal levels and their potential influence on neonatal respiratory outcomes (7-9). Insufficient vitamin D in early life has been associated with heightened allergic sensitization, increasing the likelihood of asthma, eczema, and allergic reactions in childhood (10, 11). Additionally, lower vitamin D levels in asthmatic individuals correlate with diminished responsiveness to glucocorticoid therapy (7, 12). Chronic vitamin D deficiency is also linked to a higher incidence of inflammatory lung diseases and reduced pulmonary function (13, 14), whereas regular supplementation has been shown to enhance lung function, particularly in individuals with asthma and chronic pulmonary disease (15). Although Vitamin D is well recognized to play a pivotal role in immune homeostasis, its functions in human diseases, such as COVID-19 and atherosclerosis remain of high interest (16, 17).
In circulation, vitamin D binds to vitamin D-binding protein and is transported to the liver, where it undergoes hydroxylation to form 25-hydroxyvitamin D. This metabolite is further converted in the kidneys into 1,25-dihydroxyvitamin D, the biologically active form of vitamin D (18). Upon activation, vitamin D binds to vitamin D receptor (VDR) in the cytoplasm, facilitating its translocation to the nucleus, where it interacts with vitamin D response elements to regulate the expression of target genes (19, 20). In clinical practice, serum 25-hydroxyvitamin D3 levels serve as the primary biomarker for assessing vitamin D status (21). A deficiency in vitamin D, defined as a serum concentration below 30 ng/mL, has been linked to increased asthma severity (22, 23).
The VDR gene, located on chromosome 12, is integral to various physiological functions (24, 25). Several polymorphisms within the VDR gene, including rs731236, rs1544410, rs2228570, and rs7975232, have been widely investigated, with some variants reported to confer a heightened susceptibility to asthma (26, 27). Nevertheless, the precise association between VDR genetic variants and asthma risk or disease severity remains to be fully elucidated.
Building upon the aforementioned findings, this study aimed to investigate the influence of four VDR gene polymorphisms–rs731236, rs1544410, rs2228570, and rs7975232 (illustrated in Figure 1)–on asthma susceptibility and examine their potential role in modulating disease severity in a Taiwanese population. Additionally, a comprehensive review of related literature was conducted to elucidate the association between VDR genetic variants and asthma risk.
The physical map for the vitamin D receptor (VDR) rs731236, rs1544410, rs2228570, and rs7975232 polymorphic sites.
Patients and Methods
Recruitment of asthmatic cases and non-asthmatic controls. This study enrolled 198 individuals diagnosed with asthma, carefully selected from China Medical University Hospital. A control group of 453 non-asthmatic individuals was also included, matched to cases by sex and age (±5 years), following methodologies established in our previous research (28, 29). Ethical approval was obtained from the Research Ethics Committee of China Medical University Hospital (Approval No. CMUH106-REC1-004). To ensure diagnostic accuracy, asthma severity in each case was rigorously evaluated by at least two experienced pulmonary specialists, under the supervision of Dr. Hsia. This evaluation adhered to the guidelines set by the Global Initiative for Asthma (30). All procedures complied with the Declaration of Helsinki.
Based on clinical characteristics, patients were stratified into four asthma severity stages, and were systematically documented and monitored throughout the study period. The clinical parameters assessed are summarized in Table I.
Distributions of baseline characteristics among the 198 asthmatic patients and 453 non-asthmatic controls.
DNA extraction and storage. Genomic DNA was extracted from buffy-coat fractions isolated from peripheral blood samples within 24 hours of collection (31-33). The extracted DNA was preserved at −80°C for long-term storage. To ensure sample integrity and reproducibility, DNA from both asthmatic cases and non-asthmatic controls was diluted, aliquoted, and prepared as a working stock collection, adhering to previously established protocols (34, 35). These 96-well plate DNA stocks were maintained at −20°C until further analysis.
Genotyping of VDR polymorphisms. The genotypic analysis of VDR polymorphisms was conducted following the polymerase chain reaction-restriction fragment length polymorphism methodology detailed in our previous study (36). In brief, amplification by polymerase chain reaction was performed using specific forward and reverse primers (AllBio, Taichung, Taiwan, ROC) (Table II). Subsequently, amplified VDR fragments corresponding to rs731236, rs1544410, rs2228570, and rs7975232 were subjected to restriction enzyme digestion using Taq I, Bsm I, BtsC I, and Hae I, respectively (New England Biolabs, Taipei, Taiwan; Table II). The resulting DNA fragments were then analyzed via 3% agarose gel electrophoresis. To ensure accuracy and reproducibility, all genotyping procedures were conducted in a double-blinded manner by two independent researchers. The results were 100% concordant, with no discrepancies observed between the duplicate analyses.
Summary of the polymorphic sites of vitamin D receptor, paired primer sequences, restriction enzymes and DNA fragments after enzyme digestions.
Statistical analysis. The age distribution in asthmatic and non-asthmatic groups was analyzed using an unpaired Student’s t-test. The genotypic distributions of VDR polymorphisms among subgroups were assessed using Pearson’s chi-square test with Yates’ correction when all expected counts were ≥5, or Fisher’s exact test when an expected count was <5. To evaluate the association between VDR genotypes and asthma susceptibility, odds ratios (ORs) and their corresponding 95% confidence intervals (CIs) were calculated for each genotypic comparison. the relationship between VDR polymorphisms and asthma severity was similarly examined. A value of p<0.05 was considered statistically significant.
Results
Comparison of demographic and clinical characteristics between asthmatic and non-asthmatic groups. Table I presents a detailed summary of age, sex, pulmonary function parameters, and symptom severity for the 198 asthmatic patients and 453 non-asthmatic controls. Given the careful age and sex matching between the two cohorts, no statistically significant differences were observed in these variables (p=0.2972 and 0.9956, respectively). In contrast, pulmonary function parameters exhibited significant differences. The asthmatic group demonstrated a lower mean ratio of forced expiratory volume in 1 s (FEV1) to forced vital capacity (FVC) and a reduced percentage of predicted FEV1 compared to the control group (both p<0.0001). Regarding asthma severity, the distribution among asthmatic patients was as follows: 30.3% in stage 1, 32.8% in stage 2, 17.2% in stage 3, and 19.7% in stage 4, as outlined in Table I.
Association between VDR genotypes and asthma susceptibility in Taiwanese. Table II summarizes the genotypic distributions of VDR rs731236, rs1544410, rs2228570, and rs7975232 among 198 asthmatic cases and 453 non-asthmatic controls. Firstly, the genotypic frequencies of VDR rs731236, rs1544410, rs2228570, and rs7975232 in the non-asthmatic control group were consistent with the Hardy-Weinberg equilibrium (HWE) (p=0.2962, 0.1888, 0.1669, and 0.3910, respectively). Secondly, a significant association was observed for VDR rs2228570, where the genotypic distribution significantly differed between asthmatic and non-asthmatic individuals (p for trend=0.0041; Table III, middle section). Specifically, individuals carrying the heterozygous AG and homozygous AA genotypes exhibited an elevated risk for asthma, with ORs of 1.55 (95% CI=1.02-2.35, p=0.0507) and 2.19 (95% CI=1.37-3.49, p=0.0014), respectively. Thirdly, in a recessive model (comparing the AA genotype to the GG/AG genotypes), individuals with the AA genotype had an OR of 1.66 (95% CI=1.14-2.41, p=0.0111) for asthma (Table III, middle section). Lastly, no significant differences in genotype distributions were identified for VDR rs731236, rs1544410, and rs7975232 under any genetic model (all p>0.05, Table III).
Distribution of genotypes for vitamin D receptor (VDR) single nucleotide polymorphisms (SNP) among the controls and patients with asthma.
Association between VDR allelic frequencies and asthma susceptibility in Taiwan. To further evaluate the impact of VDR polymorphisms on asthma risk, we analyzed allelic frequencies, as summarized in Table IV. The results demonstrated a significant difference in the distribution of the variant A allele of VDR rs2228570 between asthmatic and non-asthmatic individuals (p=0.0008). Notably, individuals carrying the A allele exhibited a 1.51-fold increased risk of developing asthma (OR=1.51, 95% CI=1.19-1.92) compared to those with the wild-type G allele (Table IV). Conversely, no significant differences in allelic frequencies were observed for VDR rs731236, rs1544410, and rs7975232 between asthmatic and non-asthmatic groups across any genetic model (all p>0.05, Table IV).
Allelic frequencies for genotypes for vitamin D receptor (VDR) single nucleotide polymorphisms (SNP) among the controls and patients with asthma.
Stratified analysis of VDR rs2228570 genotypes in relation to severity of asthma symptoms. Given the potential of VDR rs2228570 genotypes as predictors of asthma risk, we sought to investigate the relationship between these genotypes and asthma symptom severity. To this end, asthmatic patients were stratified into two groups based on symptom severity: mild (stages 1 and 2) and severe (stages 3 and 4). The genotypic distribution of VDR rs2228570 in these groups is summarized in Table V. Our analysis revealed that the VDR rs2228570 genotypes were differentially distributed between the mild and severe groups (p for trend=0.0024, Table V). Specifically, individuals with the variant genotypes (AG or AA) at VDR rs2228570 were more likely to experience severe asthma symptoms compared to those with the wild-type GG genotype (24.7% and 2.7% versus 9.6% and 0.0%, p=0.0062 and 0.1059, respectively). Interestingly, the proportion of individuals carrying the AG+AA genotypes was notably higher in the group with severe symptoms (27.4%) than in the mild group (9.6%) compared to those with the GG genotype (p=0.0021) (Table V).
Genotypic frequencies of vitamin D receptor rs2228570 genotypes in patients with mild or severe asthma.
Discussion
Severe asthma, affecting approximately 4-10% of asthma patients, is characterized by a poor prognosis and necessitates meticulous management to optimize patient outcomes (37, 38). Emerging evidence suggests a crucial role of vitamin D in both the development and management of asthma. Specifically, vitamin D modulates immune responses by activating key immune cells involved in regulating the severity and frequency of asthma exacerbations (39-41). Multiple studies have demonstrated a strong association between vitamin D deficiency and an increased risk of asthma (42, 43), with a particularly pronounced effect in pediatric populations (44). Notably, 68.1% of asthmatic children were reported to have serum vitamin D levels of ≤15 ng/ml, underscoring a potential link between vitamin D insufficiency and asthma susceptibility (44, 45). Furthermore, higher serum vitamin D levels have been correlated with reduced inflammatory markers and improved asthma symptom control (46). However, the relationship between vitamin D levels and asthma severity remains controversial. While some studies have found no significant correlation (47, 48), others have reported a clear association between vitamin D deficiency and worsening asthma symptoms (22, 23, 49). To establish a definitive role of vitamin D in asthma severity and control, large-scale multinational studies with standardized definitions of asthma severity are warranted.
In 2004, Raby and colleagues conducted the first investigation into the association between the VDR rs2228570 polymorphism and asthma risk in a German population but found no significant correlation (50). Similarly, in 2009, Fang and colleagues found no association between this polymorphism and asthma risk in a smaller Chinese cohort, consisting of 206 controls and 101 asthmatic cases (51). Around the same time, Saadi et al. (52) and Li et al. (53) also reported no significant association in Chinese populations (52, 53). Additionally, a study by Pillai and colleagues in 2011, involving 122 asthmatic and 74 non-asthmatic African American children, found no significant correlation between the VDR rs2228570 polymorphism and asthma risk (54). However, in 2013, Maalmi and colleagues observed a unique association between the VDR rs2228570 polymorphism and childhood asthma, revealing that the AA genotype was linked to a reduced risk of asthma in children (55). In 2014, a study by Iordanidou and colleagues in a Greek cohort, comprising 91 controls and 127 asthmatic cases, reported no significant association (56), aligning with earlier studies. In 2017, Zhang and colleagues also failed to find any association between the VDR rs2228570 genotype and asthma in a relatively small Chinese pediatric cohort (57). In contrast, Despotovic and colleagues examined the role of VDR rs2228570 polymorphisms in asthma in a Serbian cohort. Their findings suggested that the AG and AA genotypes were associated with an increased risk of asthma (58). Most recently in early 2025, Uehara and colleagues evaluated the impact of VDR rs2228570 genotypes on asthma severity in a Japanese population and reported that the homozygous variant AA genotype was linked to more severe asthma symptoms (59). The literature mentioned revealing the role of VDR rs2228570 genotypes to asthma risk are concisely summarized in Table VI.
Literature-reported studies of genotypes of vitamin D receptor single nucleotide polymorphism rs2228570 among asthmatic patients.
The genotypic distribution for VDR rs2228570 in our control group adheres to HWE (p=0.1669, Table III). We carefully reviewed all the relevant literature, summarized in Table VI, excluding studies with small sample sizes or those that did not align with HWE. Data from a large cohort of 4,716 East Asian samples indicates that the G allele and A allele occur at frequencies of 56.85% and 43.15%, respectively (60). In our study, the frequencies of the major G allele and minor A allele in the non-asthmatic control group were 55.70% and 44.30%, respectively. This concordance supports the notion that our cohort’s genotyping not only represents the Taiwanese population but also aligns well with East Asian populations. The current study showed a significant association between the VDR rs2228570 polymorphism and asthma risk in the Taiwanese cohort (Table III and Table IV), which is consistent with the findings of Despotovic et al. in Serbia (58). However, our results diverge from those of several other studies, particularly those by Zhang et al. (51), Li et al. (52), Saadi et al. (53), and Fang et al. (57). One possible explanation for these inconsistencies is the differences in sampling methodology and criteria. Additionally, environmental, lifestyle, and cultural factors may influence the outcomes. Another significant factor might be whether the studies focused on adult or pediatric populations, as this may contribute to variability in the association between VDR polymorphisms and asthma risk.
Another significant finding in the current study is that the VDR rs2228570 variant AG and AA genotypes appear to serve as predictors for more severe asthma symptoms (Table V). This observation is consistent with Uehara et al.’s findings, which specifically identified the AA genotype as contributing to severe asthma symptoms (59). However, the exact influence of VDR rs2228570 genotypes on asthma severity needs further validation in larger and more diverse populations. Moreover, it is important for future studies to refine the definitions of asthma severity, as the impact of VDR polymorphisms may vary depending on how severity is classified and assessed. This will allow for a more comprehensive understanding of the role of VDR genotypes in determining asthma outcomes across different populations.
Several limitations in the current study must be acknowledged. Firstly, it had a relatively limited sample size. The relatively small number of asthmatic cases and non-asthmatic controls may have introduced potential bias and limit the generalizability of the findings. A larger cohort would be essential to confirm the results and reduce sampling bias. Secondly, there were unadjusted parameters–the analyses were conducted without adjusting for potential confounding variables such as age, sex, and lifestyle factors. Future studies should incorporate these factors for a more comprehensive analysis and to better control for potential biases in the associations observed. Thirdly, the SNP evaluation was limited. This study focused on only four SNPs within the VDR gene. However, there are several other functional SNPs in the VDR gene that might potentially contribute to asthma susceptibility and severity. A broader assessment of other genetic variants would provide a more complete understanding of the role of VDR in asthma. Last, there was a lack of functional analysis. The study did not assess RNA or protein expression levels of VDR in the serum of asthmatic patients and controls. Understanding the functional consequences of the identified genetic variants would enhance the interpretation of how these polymorphisms contribute to asthma pathology. Moreover, it was not possible to analyze genotype–phenotype correlations, limiting the ability to directly connect the genetic findings with specific clinical outcomes.
In conclusion, our study provides compelling evidence suggesting that the variant A allele of the VDR rs2228570 polymorphism may play a significant role in predicting asthma susceptibility in adults in Taiwan. Additionally, our findings indicate that the VDR rs2228570 AG and AA genotypes are associated with exacerbated asthma symptom severity, potentially serving as predictive markers for asthma severity. While these preliminary findings are promising, further validation in larger cohorts and across diverse ethnic populations is essential. Additionally, more in-depth studies are needed to better understand the genotype–phenotype relationship in VDR and its contribution to asthma etiology.
Acknowledgements
The Authors are grateful to Yu-Cheng Luo and Yu-Hsin Yen for their excellent technical assistance. All the participants in this study are highly appreciated. This study was supported by China Medical University and Hospital (DMR-112-026), Taichung Armed Forces General Hospital (TCAFGH-D-114005), Asia and China Medical University (CMU113-ASIA-06). The funders had no role in the study design, data collection and analysis, decision to publish or preparation of the manuscript.
Footnotes
Authors’ Contributions
Research design: Wang SC, He JL, Bau DT, and Hsia TC; patient and questionnaire summaries: Wang SC, Hsia TC; experimental work: He JL, Chang WS, Shih HY, Wang YC, and Tsai CW; data clearance and identification: Chen JC, He JL, Shih HY, and Hsia TC; statistical analysis: Chen JC, Wang SC, Shih HY, Chang WS, and Tsai CW; manuscript writing: Hsia TC, and Bau DT; review and revision: Tsai CW, Chang WS, Hsia TC, and Bau DT.
Conflicts of Interest
The Authors declare no conflicts of interest regarding this study.
- Received March 25, 2025.
- Revision received April 11, 2025.
- Accepted April 15, 2025.
- Copyright © 2025 The Author(s). Published by the International Institute of Anticancer Research.
This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY-NC-ND) 4.0 international license (https://creativecommons.org/licenses/by-nc-nd/4.0).
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