Abstract
Background: The association between vitamin D receptor (VDR) polymorphisms and the risk of asthma remains unclear. This study aimed to investigate the effect of VDR gene polymorphisms and VDR mRNA expression levels on respiratory function, nitric oxide levels in expiratory air, and serum vitamin D levels in children with asthma.
Materials and Methods: The study included 80 healthy children (control group) and 100 asthmatic children (asthma group) between the age of 5 and 18 years. The VDR genotypes (ApaI, TaqI, and FokI) and VDR mRNA levels were determined in all groups.
Results: There was no statistically significant difference in vitamin D levels between the asthma group and the control group (P > 0.05). A significant association was found between both genotype (CC) of the TaqI polymorphism [odds ratio (OR) = 0.2, 95% confidence interval (CI) (0.07–0.5), P = 0.003] and genotype (CA) of ApaI polymorphisms [OR = 0.2, 95% CI (0.07–0.8), P = 0.02], and asthma risk. In addition, when single-nucleotide polymorphism allelic frequencies between asthma and control groups were compared there is no significant association (P > 0.05). When compared to control group, VDR mRNA expression in asthma group decreased in genotypes CC and CA of ApaI and in genotypes TT and TC of TaqI (P < 0.05).
Conclusion: The results provide supporting evidence for an association between TaqI and ApaI polymorphisms and asthma susceptibility.
Keywords: asthma, vitamin D, exhaled nitric oxide, VDR gene polymorphism, VDR mRNA expression
Introduction
Asthma is a multifactorial disease, with many genetic and environmental factors playing a role in its etiology. In recent years, gene polymorphisms in asthma have been examined, and several sensitivity loci, including 2q, 5q, 6p, 11q, 12q, and 13q regions, have been identified.1 Recent studies reported that vitamin D played a role in the pathogenesis of many inflammatory diseases, including asthma, and that the vitamin D receptor (VDR) appeared to be a candidate gene for asthma. The effects of vitamin D on the immune system are generally in the form of immunological tolerance, immunomodulation, and immunosuppression. Specifically, vitamin D targets genes encoding proinflammatory and anti-inflammatory cytokines, which play a role in the development of airway inflammation.1,2
Risk factors for asthma and vitamin D deficiency, 2 common conditions, are similar and include urban-style life, obesity, and undernutrition.3–5 Due to the link between asthma and vitamin D deficiency, a number of studies have investigated the relationship between genes involved in vitamin D metabolism and asthma phenotypes. Most of these studies provided support for the idea that vitamin D provided protection against asthma and other allergic diseases and a strong association between vitamin D levels and asthma development, asthma severity, and pulmonary function.3,4,6–8 However, some studies found no relationship between asthma and vitamin D levels.9,10
In humans, the VDR is found on the long arm of chromosome 12 and is composed of 8 introns and 9 exons. The most important single-nucleotide polymorphisms (SNPs) in the VDR gene are FokI, ApaI, EcoRV, BsmI, TaqI, Tru9I, 3′ UTR, Cdx2, and polyA. Of these, the most studied SNPs are FokI, BsmI, ApaI, and TaqI.11 Several epidemiological studies reported an association between 1 or more VDR polymorphisms and asthma,12–19 whereas other studies found no such association.20–22
This study aimed to investigate the effect of VDR gene polymorphisms (ApaI, TaqI, and FokI) and VDR mRNA expression levels on respiratory function, nitric oxide levels in expiratory air, and serum vitamin D levels in children with asthma.
Materials and Methods
Patients' study design
This was a case–control study conducted between January 2012 and June 2012 in Turkish Children. The study consisted of 100 children with asthma (asthma group) and 80 healthy children (control group) between 5 and 18 years of age. All the participants in the asthma group had been diagnosed with long-term asthma at least 1 year before and were receiving regular prophylactic treatment. The diagnosis and classification of the clinical severity of asthma were based on clinical symptoms and lung function according to the Global Initiative for Asthma (GINA) guidelines. They were diagnosed according to the clinical manifestations (cough, wheezing, shortness of breath, and exercise intolerance) in agreement with GINA guidelines and confirmed by spirometry.1 Patients receiving vitamin D therapy or antiepileptic drugs and those with chronic diseases (eg, cardiorespiratory, metabolic, or endocrine diseases) other than asthma were excluded from the study. We recruited 80 healthy children as control group from the emergency department of our hospital. The children in the control group did not have any history of chronic disease, including asthma, other respiratory disease, liver disease such as hepatitis, renal/urologic disease, bone disease, and metabolic disease. Neither the asthma nor the control group subjects had received vitamin D supplementation in the past 3 years.
Measures
Lung function was measured by maximum expiratory flow volume loops (ZAN 100; Spiromed, Oberthulba, Germany), recording the test with the highest forced vital capacity (FVC), forced expiratory volume in 1 s (FEV1), peak expiratory flow (PEF), forced expiratory flow between 25% and 75% of vital capacity (FEF 25%–75%), and FEV1/FVC ratio values as the best of 3 tests. The values were expressed as percent of the predicted for the child's height, age, and gender. These measurements were performed according to the standards of the European Respiratory Society (ERS) and the American Thoracic Society (ATS). Fractional exhaled nitric oxid (FeNO) was analyzed with NioxMinor® from Aerocrine (NIOX System; Aerocrine, Sweden) and was performed following the recommendations of the ERS and ATS. In accordance with the ERS/ATS recommendations, low FeNO was defined as a level ≤20 ppb, an intermediate level as 21–34 ppb, and a high level as ≥35 ppb.
Background information regarding the diagnosis, treatment, and monitoring of asthma was obtained from clinical records and parents. Nutritional status was defined according to the patient's body mass index (BMI). The children were categorized as normal weight (BMI in <85th percentile), overweight (BMI in ≥85th and <95th percentiles), or obese (BMI in ≥95th percentile). Serum 25(OH)D was measured using a commercially available kit (Dia Sorin, Saluggia, Italy), and values were reported in nanograms per milliliter. In descriptive analysis, vitamin D levels were categorized as sufficient (>30 ng/mL), insufficient (20–30 ng/mL), or deficient (<20 ng/mL) on the basis of previous recommendations.23
A venous blood sample was collected from all subjects for analysis of VDR gene polymorphisms and VDR gene expression, and serum was separated and stored at −80°C until used in the analysis. DNA was isolated from the blood samples using a Pure Link Genomic DNA isolation kit (Catalog No. K182002; Invitrogen, Carlsbad, CA), and DNA polymorphisms were detected using the ABI Prism StepOnePlus™ Real Time System (Applied Biosystems, Foster City, CA) and TaqMan probes. Three different specific and well-characterized VDR SNPs, namely FokI, ApaI, and TaqI, were assessed. According to allele 1 and allele 2 discrimination, the genotypes were considered homozygous normal (AA), heterozygous mutant (CA), and homozygous mutant (CC) for ApaI; homozygote normal (TT), heterozygote mutant (TC), and homozygote mutant (CC) for TaqI; and homozygote normal (CC), heterozygote mutant (TC), and homozygote mutant (TT) for FokI.
The amount of RNAs obtained for assessing VDR gene expression was measured using a Qubit device (Invitrogen), following separation of DNA samples using an RNA isolation kit (Invitrogen Ambion® RNA Mini Kit-Catalog No. 12183018A; Invitrogen). cDNA synthesis was performed using a high-capacity cDNA synthesis kit (Invitrogen). To analyze VDR gene expression, the real-time polymerase chain reaction was performed using a TaqMan Gene Expression Assay (Invitrogen). Beta-actin was used a housekeeping gene. VDR gene polymorphisms and VDR mRNA expression were studied according to manufacturer's recommendations.23
The clinical research ethics committee of our university approved this study, and written informed consent was obtained from all the subjects and their parents.
Statistical analysis
All statistical analyses were performed using the SPSS statistical package, version 21.0 (IBM, SPSS, Inc., Chicago, IL). The characteristics of the 2 patient groups and control group were compared using a chi-square test and t-test for categorical and continuous variables, respectively. A two-tailed P value of ≤0.05 was considered statistically significant. Genetic risks were assessed by Binary Logistic regression analysis calculating odds ratios (ORs), with their 95% confidence intervals (95% CIs). A chi-square test was performed for Hardy–Weinberg equilibrium (HWE) determination. All genotypes were tested for HWE. The obtained genotype and allele frequencies were compared against the values predicted by the HWE using a chi-square test. Linkage disequilibrium was calculated using Haploview 4.2 software package (Daly Lab, Cambridge, MA). In our study, sample size and formal power calculation were performed as follows: when a power analysis was performed at 80% power and at 0.05 significance level for 25 (OH) vitamin D levels measured in the study, it was calculated that at least 80 optimum 100 cases should be included in the study. An 80% power had been obtained with post hoc power analysis according to the effect size. The effect size for three VDR polymorphisms of 25 (OH) Vitamin D levels was 0.319 according to the F-test.
Results
Among the asthma group, there were 52 (52%) males and 48 (48%) females. The mean age was 9.5 ± 2.8 (5–16) years. In the control group, there were 42 (51%) males and 38 (49%) females, and the mean age was 9.5 ± 2.5 (5–14) years. There was no statistically significant difference in the age, sex, parental relationship, family history, birth pattern, premature birth history, breastfeeding, use of vitamin D, BMI, or serum vitamin D levels between the asthma group and control group (P > 0.05). There was also no statistically significant difference in vitamin D deficiency, insufficiency, or sufficiency between the both groups (P > 0.05) (Table 1).
Table 1.
Demographic and Laboratory Characteristics of the Subjects
| Characteristic | Asthma group (n = 100) | Control group (n = 80) | Pa |
|---|---|---|---|
| Age (year) (mean ± SD) | 9.5 ± 2.8 | 9.5 ± 2.5 | 0.7 |
| Gender, n (%) | |||
| Males | 52 (52) | 42 (52.5) | 0.8 |
| Females | 48 (48) | 38 (47.5) | 0.7 |
| BMI (kg/m2) (mean ± SD) | 17.8 ± 3.4 | 17.4 ± 3.1 | 0.4 |
| Breastfeeding, n (%) | 92 (92) | 70 (87.5) | 0.3 |
| Use of supplemental vitamin Db, n (%) | 59 (59) | 43 (54.4) | 0.6 |
| 25-OH vitamin D (ng/mL) (mean ± SD) | 22.2 ± 12.3 | 21.7 ± 6.9 | 0.8 |
| Vitamin D status, n (%) | |||
| Vitamin D deficiency | 45 (58.4) | 32 (41.6) | 0.2 |
| Vitamin D insufficiency | 35 (47.9) | 38 (52.1) | 0.8 |
| Vitamin D sufficiency | 20 (66.7) | 10 (33.3) | 0.07 |
| Method of birth, n (%) | |||
| Caesarean | 32 (32) | 24 (30) | 0.8 |
| Vaginal delivery | 68 (68) | 56 (70) | 0.8 |
| Consanguinity, n (%) | 42 (42) | 31 (38.8) | 0.2 |
The distribution of the data was normal according to variance analysis test. The independent sample t-test for continuous variables and the Pearson's chi-square test for categorical variables have been used.
Use of supplemental vitamin D in childhood period (400 IU/day).
BMI, body mass index; SD, standard deviation.
Comparison of the genotype frequencies of the 3 polymorphisms (ApaI, TaqI, and FokI) in the asthma and control groups revealed no statistically significant difference in the genotype frequencies of FokI polymorphisms (P > 0.05). However, a significant association was found between both genotype (CC) of the TaqI polymorphism [OR = 0.2, 95% CI (0.07–0.5), P = 0.003] and genotype (CA) of ApaI polymorphisms [OR = 0.2, 95% CI (0.07–0.8), P = 0.02], and asthma risk (Table 2). In addition, there was no statistically significant difference in the allele frequencies of ApaI, TaqI, and FokI polymorphisms in either the asthma group or those in the control group (Table 3) (P = 0.05). There was no statistically significant relationship between vitamin D levels and VDR gene polymorphisms in any of the groups (P > 0.05). ApaI, TaqI, and FokI polymorphisms were not significantly associated with the number of asthma attacks, age at asthma onset, asthma severity, asthma control level, asthma treatment steps, FeNO levels, absolute eosinophil count, serum vitamin D level, positive skin prick test, serum total IgE level, FEV1, FEV1/FVC, and PEF values in the asthma group (P > 0.05).
Table 2.
Frequency and Distribution of Genotypes for the Vitamin D Receptor Polymorphisms in Asthmatic and Control Subjects
|
Asthmatics n (%) |
Controls n (%) |
P | OR (95% CI) | |
|---|---|---|---|---|
| ApaI | ||||
| AA | 18 (18) | 26 (32.5) | 0.2 | 0.6 (0.2–1.4) |
| CA | 60 (60) | 42 (52.5) | 0.02 | 0.2 (0.07–0.8) |
| CC | 22 (22) | 12 (15) | 0.06 | |
| TaqI | ||||
| CC | 8 (8) | 20 (25) | 0.003 | 0.2 (0.07–0.5) |
| TC | 61 (61) | 32 (40) | 0.3 | 0.6 (0.1–1.9) |
| TT | 31 (31) | 28 (35) | 0.002 | |
| FokI | ||||
| TT | 9 (9) | 4 (5) | 0.8 | 0.9 (0.4–1.8) |
| TC | 33 (33) | 28 (35) | 0.2 | 0.4 (0.1–1.7) |
| CC | 58 (58) | 48 (60) | 0.5 | |
The reference genotype for the ApaI polymorphism is CC. The reference genotype for the TaqI polymorphism is TT.
The reference genotype for the FokI polymorphism is CC. The binary logistic regression test for statistical analysis has been used.
A, adenine; C, cytosine; CI, confidence interval; OR, odds ratio; T, thymine.
Table 3.
Frequency and Distribution of Alleles for the Vitamin D Receptor Polymorphisms in Asthmatic and Control Subjects
|
Asthmatics n (%) |
Controls n (%) |
P | OR (95% CI) | |
|---|---|---|---|---|
| ApaI | 0.05 | |||
| A | 96 (49) | 94 (59) | 1.5 (0.9–2.3) | |
| C | 104 (51) | 66 (41) | ||
| TaqI | 0.2 | |||
| T | 123 (61.5) | 88 (55) | 1.3 (0.8–1.9) | |
| C | 77 (38.5) | 72 (45) | ||
| FokI | 0.5 | |||
| C | 149 (74.5) | 124 (77.5) | 1.1 (0.7–1.9) | |
| T | 51 (25.5) | 36 (22.5) |
The binary logistic regression test for statistical analysis has been used.
None of the 3 categories of vitamin D levels showed a statistically significant association with age at onset of asthma, asthma severity, asthma treatment steps, number of asthma attacks, skin prick test positivity, serum IgE level, absolute eosinophil count, and FeNO parameters in asthma groups (P > 0.05). However, the FEV1 and PEF parameter levels were statistically higher in the sufficiency category of Vitamin D level compared to the other 2 groups (P = 0.04 and P = 0.02, respectively) (Table 4).
Table 4.
Relationship Between Demographic, Clinical, and Laboratory Characteristics and 25(OH)D Level Categories
| Parameter | Category of 25(OH)D | P | ||
|---|---|---|---|---|
| Sufficiency | Insufficiency | Deficiency | ||
| Age of asthma onset (year) (mean ± SD) | 6.6 ± 2.7 | 4.9 ± 3.4 | 6.6 ± 3.8 | 0.09 |
| Asthma severity, n (%) | ||||
| Mild | 15 (75) | 17 (48.6) | 28 (62.2) | 0.3 |
| Moderate | 3 (15) | 13 (37.1) | 10 (22.2) | |
| Severe | 2 (10) | 5 (14.3) | 7 (15.6) | |
| Asthma control, n (%) | ||||
| Controlled | 17 (85) | 21 (60) | 27 (60) | 0.3 |
| Partially controlled | 1 (5) | 7 (20) | 10 (22.2) | |
| Uncontrolled | 2 (10) | 7 (20) | 8 (17.8) | |
| Treatment step, n (%) | ||||
| Treatment step 2 | 11 (55) | 15 (42.9) | 20 (44.5) | 0.7 |
| Treatment step 3 | 7 (35) | 11 (31.4) | 15 (33.3) | |
| Treatment step 4 | 2 (10) | 9 (25.7) | 10 (22.2) | |
| Number of asthma attacks (mean ± SD) | 7.8 ± 7.9 | 12.2 ± 8.7 | 11.2 ± 10.7 | 0.8 |
| FEV1 (mean ± SD) (%) | 94.5 ± 19.8 | 81.3 ± 21.7 | 83.7 ± 16.2 | 0.04 |
| PEF (mean ± SD) (%) | 87.0 ± 25.9 | 79.8 ± 24.2 | 71.8 ± 16.1 | 0.02 |
| Positive skin prick test, n (%) | 5 (12.8) | 15 (38.5) | 19 (48.7) | 0.3 |
| Total IgE level (mean ± SD) | 446.2 ± 563.6 | 286.4 ± 370.5 | 341.1 ± 560.9 | 0.5 |
| Peripheral eosinophilia (mean ± SD) | 354.5 ± 157.5 | 364.6 ± 299.2 | 265.8 ± 214.5 | 0.1 |
| FeNO (ppb) (mean ± SD) | 20.0 ± 8.9 | 27.7 ± 17.3 | 27.8 ± 22 | 0.2 |
| FeNO n (%) | ||||
| ≤20 ppb | 12 (60) | 16 (45.7) | 26 (57.8) | 0.6 |
| 21–34 ppb | 5 (25) | 9 (25.7) | 8 (17.8) | |
| ≥35 ppb | 3 (15) | 10 (28.6) | 11 (24.4) | |
The ANOVA and chi-square tests for comparison of more than 2 groups have been used.
ANOVA, analysis of variance; FeNO, fractional nitric oxide in exhaled breath; FEV1, forced expiratory volume in 1 s; PEF, peak expiratory flow.
The subjects with mRNA expression of ≥2 μg/mL in the VDR gene were evaluated for gene expression. Among the asthmatics, mRNA expression was ≥2 μg/mL in 71 patients (30/39 allergic asthma and 41/61 nonallergic asthma), and it was ≥2 μg/mL in 37 cases in the control group. mRNA expression was 4.82 ± 0.97 (2.45–6.87) μg/mL in the asthmatics and 5.86 ± 1.63 (2.33–7.87) μg/mL in the controls. Compared with the control group, we observed that mRNA expression of VDR decreased in the asthma group (P = 0.0001). In addition, we analyzed the relationship between SNPs and VDR mRNA expression in the asthmatics and controls. The mRNA expression levels in the CC and CA genotypes of ApaI decreased significantly statistically low in the asthmatic group compared to the control group (P = 0.01 and P = 0.02, respectively). Similarly, mRNA expression levels in the TT and TC genotypes of TaqI were significantly lower in the asthma group compared with that in the controls (P = 0.0001 and P = 0.0001, respectively). However, there was no statistically significant relationship for VDR mRNA expression levels in the FokI genotype between asthma and control groups (P > 0.05) (Table 5).
Table 5.
The Evaluation of Vitamin D Receptor mRNA Expression Levels (μg/mL) According to Single-Nucleotide Polymorphism Genotypes in Asthma and Control Groups
| SNP |
Asthma (n = 71) (mean ± SD) |
Control (n = 37) (mean ± SD) |
P |
|---|---|---|---|
| ApaI (n) | |||
| AA | (15) 4.63 ± 0.75 | (10) 4.97 ± 0.90 | 0.7 |
| CA | (42) 4.74 ± 1.02 | (21) 6.43 ± 1.65 | 0.02 |
| CC | (14) 5.26 ± 0.94 | (6) 5.44 ± 1.91 | 0.01 |
| TaqI (n) | |||
| TT | (20) 5.22 ± 0.90 | (12) 6.76 ± 1.14 | 0.0001 |
| TC | (46) 4.72 ± 0.98 | (17) 5.96 ± 1.47 | 0.0001 |
| CC | (5) 4.14 ± 0.61 | (8) 4.37 ± 1.66 | 0.7 |
| FokI (n) | |||
| CC | (7) 4.87 ± 0.78 | — | — |
| TC | (21) 4.66 ± 0.97 | (8) 4.84 ± 2.10 | 0.5 |
| TT | (43) 4.69 ± 1.00 | (29) 4.77 ± 1.39 | 0.6 |
The independent sample t-test for data analysis has been used.
SNP, single-nucleotide polymorphism.
Discussion
In this study, we found no significant difference between the asthma and control groups in terms of FokI polymorphism genotypes. However, a significant association was found between both genotype (CC) of the TaqI polymorphism and genotype (CA) of ApaI polymorphisms, and asthma risk. A number of previous studies also reported a statistically significant association between asthma and TaqI and ApaI polymorphisms.12–19,24,25 In agreement with the results of this study, Arababadi et al.26 showed that TaqI polymorphism was more frequent in asthmatic patients compared with that in healthy controls. In a case–control study in Cypriot adolescents, the TaqI homozygous genotype was associated with wheezing and asthma.18 Maalmi et al.14 evaluated VDR SNPs in Tunisian children with asthma and reported that FokI, BsmI, and TaqI polymorphisms were associated with asthma. Hutchinson et al.25 found a relationship between asthma and TaqI and ApaI polymorphisms in Irish children.
Wjst22 investigated 13 SNPs, including FokI, TaqI, and ApaI polymorphisms, and failed to find a significant association between asthma and any polymorphism. Pillai et al.20 also found no association between VDR genetic polymorphisms and asthma development in a study of African Americans. The Childhood Asthma Management Program study examined 7 VDR SNPs in 3 different ethnic groups (Caucasians, African Americans, and Hispanics) and found a significant association between ApaI and asthma in a genetic analysis stratified according to the ethnic group.13 The family-based Nurses' Health Study identified a significant relationship between the TaqI polymorphism and asthma.15 The same study reported that the TaqI polymorphism was significantly associated with serum IgE levels. Han et al.17 suggested that FokI, ApaI, TaqI, and rs3782905 polymorphisms in the VDR may contribute to asthma development. Saadi et al.16 studied FokI, DdeI, BsmI, ApaI, and TaqI polymorphisms in a Han population in China and reported that the ApaI polymorphism was associated with asthma. A case–control study in Canada of 12 VDR SNPs in individuals 3–80 years of age found a statistically significant relationship among 6 of the SNPs (rs3782905, rs1540339, rs2239185, rs2239182, BsmI, and TaqI) with asthma. In this study, researchers also reported that the T-allele of the TaqI polymorphism showed a statistically significant relationship with asthma and atopy.12 In the majority of the aforementioned studies, VDR polymorphisms were considered independent risk factors for asthma susceptibility.
In a meta-analysis study evaluating the relationship between asthma and VDR SNP, including ApaI, BsmI, FokI, and TaqI, they found a statistically significant relationship between the homozygous genotype of ApaI polymorphism and childhood asthma. Stratification by ethnicity revealed a statistical association in Asians. However, the authors reported that there may be no relationship between TaqI polymorphism and the risk of childhood asthma. In addition, they have reported that FokI polymorphism may be connected with pediatric asthma in Caucasian population and BsmI polymorphism marginally contributes to childhood asthma susceptibility.27 In another meta-analysis evaluating 8 case–control studies, FokI, TaqI, and BsmI VDR polymorphisms were significantly associated with an increased risk of asthma in some populations. In this meta-analysis, the authors suggested that serum 25(OH)D levels and various environmental factors could modify the effect of these polymorphisms on asthma development.15
In our study, VDR SNPs were not significantly associated with FeNO, serum 25(OH)D, IgE level, eosinophil counts, skin prick test positivity, respiratory function test parameters, or acute asthma attacks. However, some studies established a relationship between asthma and the TaqI polymorphism, reporting a statistically significant association between this polymorphism and FeNO, IgE levels, eosinophil counts, skin test positivity, respiratory function test parameters, and acute asthma attacks.12–14 Poon et al.12 reported a relationship between 4 SNP alleles (rs2239185C, BsmIG, ApaIC, and TaqIT) and atopy. They also found a significant association between high IgE levels and rs2239185C, ApaIC, and TaqIT alleles. Maalmi et al.14 reported that VDR polymorphisms showed a statistically significant association with serum vitamin D levels and asthma severity.
A number of factors may explain the variable results of VDR SNP studies in different populations. Although the effects of VDR polymorphisms on bone metabolism are similar, gene–gene and gene–environment interactions vary in different populations,28,29 and these variations largely determine the relationship between VDR polymorphisms and asthma.30 Due to stratification in the population, it is often difficult to detect the actual effect of genes in case–control samples, with stratification reversing, altering, or masking the true genetic effect of gene polymorphisms in research. In addition, other variables, such as ethnicity, climate, geographical features of the place, and lifestyle, may explain the inconsistent findings of different VDR SNP studies.15,20
According to the vitamin D workshop consensus on vitamin D nutritional guidelines published by Henry et al.,31 ∼50% of North America and Western Europe and two-thirds of the world's remaining population have vitamin D deficiency, and the prevalence of vitamin D deficiency is increasing worldwide. According to the CAMP study by Brehm et al.,32 of 1,024 North American children with moderate to severe asthma, vitamin D levels were insufficient in 35% of these children. In a study conducted in Qatar, vitamin D levels were lower than 20 ng/mL in 68.1% of asthmatic children and in 36.1% of children in a control group, with a statistically significant difference between the 2 groups.4 In this study, 76% of asthmatic patients and 87% of healthy controls had vitamin D insufficiency (<30 ng/mL), but the between-group difference was not statistically significant. Some studies reported similar results to our data.5,10,33 Vitamin D deficiency in asthmatic patients may be due to many factors, such as obesity, drug use, chronic illnesses, poor nutrition, climate features, and low sun exposure. The latter may be the result of geographical location, season, lifestyle (eg, an excessive time spent remaining indoors), and type of clothing worn due to religious beliefs.34
Previous research reported that the FokI polymorphism in the VDR gene resulted in reduced protein expression of 3 amino acids, but 1.7 times higher activity of transcription. Although the TaqI and ApaI SNPs are not functional, they are thought to be associated with other polymorphisms that are functional and to participate in a complex gene network, which enhances or inhibits the expression of VDR target genes.35 Previous research revealed a relationship between VDR SNPs and VDR mRNA expression levels, but found no relationship between VDR mRNA expression levels and vitamin D levels.36 Based on our results, we speculate that a significant reduction of mRNA gene expression in TaqI and ApaI polymorphisms may contribute to asthma physiopathology by inhibiting vitamin D immunomodulation and immunosuppression functions. Within the cell, active 1-25(OH)2D3 exerts its effects by 2 pathways: genomic and nongenomic. Most of the biological effects of vitamin D involve the genomic pathway. The extent of these biological effects depends on the cellular VDR content, VDR genetic polymorphisms, and nuclear transcriptional mRNA. As the function of a gene can be assessed by measuring the level of mRNA expression, mRNA (RNA transcripts) can serve as an important biomarker for the diagnosis and course of diseases.35,37
This study has some limitations. First, we did not evaluate the effect of various factors, such as nutritional habits, number of hours of sunshine exposure per day, and type of clothing worn, that can influence vitamin D levels. Second, among the asthmatic patients, there were only a few cases of uncontrolled asthma and severe persistent asthma. Third, despite the presence of many functional SNPs in the VDR gene, we studied only 3 polymorphisms. We did not examine the cumulative effect of all SNPs thought to play a role in the development of asthma, other genes involved in vitamin D metabolism, or environmental risk factors that influence asthma development. These topics can be considered subjects for future study.
In conclusion, we found no statistically significant difference in vitamin D levels between the asthmatic patients and the control group. However, a significant association was found between both genotype (CC) of the TaqI polymorphism and genotype (CA) of ApaI polymorphisms, and asthma risk. In addition, mRNA gene expression was significantly reduced in ApaI and TaqI polymorphisms in asthmatic group. The results of this study provide supporting evidence for the association of TaqI and ApaI polymorphisms with asthma susceptibility. Improved knowledge of the association of ApaI and TaqI polymorphisms with asthma may contribute to a better understanding of asthma genetics.
Author Disclosure Statement
No competing financial interests exist.
References
- 1. Global Initiative for Asthma (GINA). Global strategy for asthma management and prevention, 2018. www.ginasthma.org/ Accessed March3, 2019
- 2. Wöbke TK, Sorg BL, Steinhilber D. Vitamin D in inflammatory diseases. Front Physiol 2014; 5:244. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 3. van Oeffelen AA, Bekkers MB, Smit HA, et al. . Serum micronutrient concentrations and childhood asthma: the PIAMA birth cohort study. Pediatr Allergy Immunol 2011; 22:784–793 [DOI] [PubMed] [Google Scholar]
- 4. Bener A, Ehlayel MS, Tulic MK, et al. . Vitamin D deficiency as a strong predictor of asthma in children. Int Arch Allergy Immunol 2012; 157:168–175 [DOI] [PubMed] [Google Scholar]
- 5. Brehm JM, Acosta-Perez E, Klei L, et al. . Vitamin D insufficiency and severe asthma exacerbations in Puerto Rican children. Am J Respir Crit Care Med 2012; 186:140–146 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 6. Hollams EM, Hart PH, Holt BJ, et al. . Vitamin D and atopy and asthma phenotypes in children: a longitudinal cohort study. Eur Respir J 2011; 38:1320–1327 [DOI] [PubMed] [Google Scholar]
- 7. Checkley W, Robinson CL, Baumann LM, et al. . 25-Hydroxy vitamin D levels are associated with childhood asthma in a population-based study in Peru. Clin Exp Allergy 2015; 45:273–282 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 8. Wu AC, Tantisira K, Li L, et al. . Effect of vitamin D and inhaled corticosteroid treatment on lung function in children. Am J Respir Crit Care Med 2012; 186:508–513 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 9. Tolppanen AM, Sayers A, Granell R, et al. . Prospective association of 25-hydroxyvitamin D3 and D2 with childhood lung function, asthma, wheezing, and flexural dermatitis. Epidemiology 2013; 24:310–319 [DOI] [PubMed] [Google Scholar]
- 10. Yao TC, Tu YL, Chang SW, et al. . Suboptimal vitamin D status in a population-based study of Asian children: prevalence and relation to allergic diseases and atopy. PLoS One 2014; 9:e99105. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 11. Wang TT, Tavera-Mendoza LE, Laperriere D, et al. . Large-scale in silico and microarray-based identification of direct 1,25-dihydroxyvitamin D3 target genes. Mol Endocrinol 2005; 19:2685–2695 [DOI] [PubMed] [Google Scholar]
- 12. Poon AH, Laprise C, Lemire M, et al. . Association of vitamin D receptor genetic variants with susceptibility to asthma and atopy. Am J Respir Crit Care Med 2004; 170:967–973 [DOI] [PubMed] [Google Scholar]
- 13. Raby BA, Lazarus R, Silverman EK, et al. . Association of vitamin D receptor gene polymorphisms with childhood and adult asthma. Am J Respir Crit Care Med 2004; 170:1057–1065 [DOI] [PubMed] [Google Scholar]
- 14. Maalmi H, Sassi FH, Berraies A, et al. . Association of vitamin D receptor gene polymorphisms with susceptibility to asthma in Tunisian children: a case control study. Hum Immunol 2013; 74:234–240 [DOI] [PubMed] [Google Scholar]
- 15. Tizaoui K, Berraies A, Hamdi B, et al. . Association of Vitamin D receptor gene polymorphisms with asthma risk: systematic review and updated meta-analysis of casecontrol studies. Lung 2014; 192:955–965 [DOI] [PubMed] [Google Scholar]
- 16. Saadi A, Gao G, Li H, et al. . Association study between vitamin D receptor gene polymorphisms and asthma in the Chinese Han population: a case-control study. BMC Med Genet 2009; 10:71. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 17. Han JC, Du J, Zhang YJ, et al. . Vitamin D receptor polymorphisms may contribute to asthma risk. J Asthma 2016; 53:790–800 [DOI] [PubMed] [Google Scholar]
- 18. Papadopoulou A, Kouis P, Middleton N, et al. . Association of vitamin D receptor gene polymorphisms and vitamin D levels with asthma and atopy in Cypriot adolescents: a case-control study. Multidiscip Respir Med 2015; 10:26. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 19. Einisman H, Reyes ML, Angulo J, et al. . Vitamin D levels and vitamin D receptor gene polymorphisms in asthmatic children: a case-control study. Pediatr Allergy Immunol 2015; 26:545–550 [DOI] [PubMed] [Google Scholar]
- 20. Pillai DK, Iqbal SF, Benton AS, et al. . Associations between genetic variants in vitamin D metabolism and asthma characteristics in young African Americans: a pilot study. J Investig Med 2011; 59:938–946 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 21. Fang WL, Gao LB, Liang WB, et al. . Association analysis of vitamin D receptor gene polymorphisms in chinese population with asthma. Iran J Allergy Asthma Immunol 2009; 8:141–147 [PubMed] [Google Scholar]
- 22. Wjst M. Variants in the vitamin D receptor gene and asthma. BMC Genet 2005; 6:2. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 23. ThermoFisher Scientific. https://www.thermofisher.com/order/catalog/product/12183018A and K18200 Accessed March3, 2019
- 24. Vieth R, Bischoff-Ferrari H, Boucher BJ, et al. . The urgent need to recommend an intake of vitamin D that is effective. Am J Clin Nutr 2007; 85:649–650 [DOI] [PubMed] [Google Scholar]
- 25. Hutchinson K, Kerley CP, Faul J, et al. . Vitamin D receptor variants and uncontrolled asthma. Eur Ann Allergy Clin Immunol 2018;50:108–116 [DOI] [PubMed] [Google Scholar]
- 26. Arababadi MK, Abousaidi H, Hassanshahi G, et al. . Polymorphisms within Exon 9, but not intron 8, of the vitamin D receptor gene are associated with asthma. Iran J Basic Med Sci 2011; 14:225–230 [Google Scholar]
- 27. Zhao DD, Yu DD, Ren QQ, et al. . Association of vitamin D receptor gene polymorphisms with susceptibility to childhood asthma: a meta-analysis. Pediatr Pulmonol 2017; 52:423–429 [DOI] [PubMed] [Google Scholar]
- 28. Bossé Y, Lemire M, Poon AH, et al. . Asthma and genes encoding components of the vitamin D pathway. Respir Res 2009; 10:98. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 29. Deng HW. Population admixture may appear to mask, change or reverse genetic effects of genes underlying complex traits. Genetics 2001; 159:1319–1323 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 30. Wjst M, Fischer G, Immervoll T, et al. . A genome-wide search for linkage to asthma. Genomics 1999; 58:1–8 [DOI] [PubMed] [Google Scholar]
- 31. Henry HL, Bouillon R, Norman AW, et al. . 14th Vitamin D Workshop consensus on vitamin D nutritional guidelines. J Steroid Biochem Mol Biol 2010; 121:4–6 [DOI] [PubMed] [Google Scholar]
- 32. Brehm JM, Schuemann B, Fuhlbrigge AL, et al. . Serum vitamin D levels and severe asthma exacerbations in the Childhood Asthma Management Program study. J Allergy Clin Immunol 2010; 126:52–58 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 33. Dogru M, Kirmizibekmez H, Yesiltepe Mutlu RG, et al. . Clinical effects of vitamin D in children with asthma. Int Arch Allergy Immunol 2014; 164:319–325 [DOI] [PubMed] [Google Scholar]
- 34. Holic MF. Vitamin D deficiency. N Engl J Med 2007; 357:266–281 [DOI] [PubMed] [Google Scholar]
- 35. Uitterlinden AG, Fang Y, Van Meurs JB, et al. . Genetics and biology of vitamin D receptor polymorphisms. Gene 2004; 338:143–156 [DOI] [PubMed] [Google Scholar]
- 36. Ogunkolade BW, Boucher BJ. Vitamin D receptor (VDR) mRNA and VDR protein levels in relation to vitamin D status, insülin secretory capacity, and VDR genotype in Bangladeshi Asians. Diabetes 2002; 51:2294–300 [DOI] [PubMed] [Google Scholar]
- 37. Whitfield GK, Remus LS, Jurutka PW, et al. . Functionally relevant polymorphisms in the human nuclear vitamin D receptor gene. Mol Cell Endocrinol 2001; 177:145–159 [DOI] [PubMed] [Google Scholar]