Association of vitamin D receptor gene polymorphisms with susceptibility to asthma in Tunisian children: A case control study

Human Immunology 74 (2013) 234–240

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Association of vitamin D receptor gene polymorphisms with susceptibility to asthma in Tunisian children: A case control study Haifa Maalmi a,⇑, Fayçal Haj Sassi a, Anissa Berraies a,b, Jamel Ammar a,b, Kamel Hamzaoui a, Agnes Hamzaoui a,b a b

Université de Tunis El Manar, Faculté de médecine de Tunis, 99/08-40 homeostasis and cell dysfunction unit research, 15 Rue Djebel Lakdar 1007, Tunis Tunisia Hopital Abderrahmane MAMI, Service de Pneumologie et Université de Tunis El Manar, Tunisia

a r t i c l e

i n f o

Article history: Received 10 May 2012 Accepted 15 November 2012 Available online 29 November 2012

a b s t r a c t Background: Vitamin D and its nuclear receptor (VDR) are linked to asthma in a genetic and immunologic basis. Polymorphisms in the VDR gene may alter the actions of vitamin D and then influence the development and the severity of asthma. Aims: We aimed at elucidating the genetic association of VDR gene polymorphisms with susceptibility to asthma in Tunisian children and with serum vitamin D levels. Methods: The study included 155 patients recruited from Abderrahmen MAMI hospital in Tunisia and two hundred twenty five healthy individuals matched with patients in age and sex for comparison. VDR genotypes were determined by PCR-RFLP method using endonuclease FokI, BsmI, TaqI and ApaI and vitamin D was assessed with a radioimmunoassay kit. Results: The distribution of genotype frequencies differed significantly between asthmatics and controls (FokI: P = 0.04; BsmI: P = 0.006; TaqI: P = 0.006). Haplotype analyses revealed a significant association between bAt and bat haplotypes and asthma (P = 0.00076, P = 0.016). When patients were stratified according to atopic status and stage of severity, no significant association was detected with VDR variants. No association was found between VDR SNPs and serum 25-hydroxyvitamin D levels. Conclusion: Our study shows a relation between VDR gene polymorphisms and susceptibility to asthma in children. Ó 2012 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.

1. Introduction Asthma is the most common disease of childhood and is in large part attributable to genetic factors [1]. Airway inflammation is the main characteristic of this chronic disease. Dysregulated Th cells and abnormal inflammatory response, including increased levels of IL-4, IL-5, and IL-13 may be responsible of the previous cited inflammation status [2]. The result of this exacerbated response is followed with eosinophils, lymphocytes and macrophages infiltration of mucosal airways tissue [2]. In the last years, vitamin D and its nuclear receptor VDR have emerged as new factors contributing to the pathogenesis of asthma. Vitamin D has potent immunomodulatory properties, on cells Abbreviations: VDR, vitamin D receptor; SNP, single nucleotide polymorphism.

⇑ Corresponding author. Address: Medicine University Tunis, 15 Rue, Djebel Lakdar, 1007 Tunis, Tunisia. Fax: +216 71 569 427. E-mail addresses: [email protected] (H. Maalmi), [email protected] (F. Haj Sassi), [email protected] (A. Berraies), [email protected] (J. Ammar), [email protected] (K. Hamzaoui), agnes.hamzaoui@gmail. com (A. Hamzaoui).

of the innate and adaptive immune system which may modulate the airway inflammation. 1,25(OH)2D stimulates innate immune responses through facilitating production of antimicrobial proteins such as cathelicidin and by inhibiting NF-jB signaling which could result in decreased induction of NF-jB-linked chemokines and cytokines [3]. Therefore modulating chemokines production may have the potential to prevent excessive inflammation. On the other hand, 1,25(OH)2D inhibits the surface expression of MHC-IIcomplexed antigen and of co-stimulatory molecules on APC (like DCs), in addition to production of IL-12 and IL-23 cytokines, thereby indirectly shifting the polarization of T cells from a Th1 and Th17 phenotype towards a Th2 phenotype. In addition, 1,25(OH)2D directly affects T cell responses, by inhibiting the production of Th1 cytokines (IL-2 and IFN-c), Th17 cytokines (IL-17 and IL-21), and by stimulating Th2 cytokine production (IL-4). Moreover, 1,25(OH)2D favors Treg cell development via modulation of DCs and by directly targeting T cells [4]. Further studies demonstrate that asthma may be linked to vitamin D on a molecular genetic basis. Genome scans have identified linkage in several regions, including region q13–23 of chromosome

0198-8859/$36.00 - see front matter Ó 2012 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.humimm.2012.11.005

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12, housing the vitamin D receptor (VDR) gene [5–7]. A number of association studies were previously conducted in different populations and ethnic groups. Two different family-based studies in North American subjects reported an association between VDR polymorphisms and asthma [8,9]. However another research involving German subjects did not show this link [10]. The discrepancies are probably due to differences of populations origins and different outcomes measured. This immune and genetic control of vitamin D may influence the pathogenesis of asthma. Epidemiological studies confirmed the existence of a positive link between vitamin D deficiency and asthma clinical characteristics. Lower vitamin D circulating levels are associated with an increase in asthma severity including asthma exacerbation and odds of hospitalizations [11,12], airway responsiveness [11,13,14] and inhaled steroid use [11,15]. These findings indicated that 1.25(OH)2D and its specific nuclear receptor, VDR, may be closely associated with asthma risk. Nevertheless, several genetic variations (single nucleotides polymorphisms; SNP) that have been identified in the VDR gene may influence either its expression or its function which may cause alteration in the vitamin D activity. In addition, many of the VDR target genes are involved in the regulation of vitamin D metabolite concentrations via a classical endocrine feedback loop [16]. Any defect in the VDR expression or function could influence concentrations of vitamin D metabolites. Four single nucleotide polymorphisms (SNPs) in the VDR gene have been well investigated by genetic associations studies, namely FokI F > f (rs2228570), BsmI B > b (rs1544410), ApaI A > a (rs7975232), and TaqI T > t (rs731236). Allele F of the Fok-I SNP creates an alternative ATG initiation codon in exon 2 leading to a three amino acids longer VDR protein. The ApaI, BsmI and TaqI polymorphisms are located near the 30 end of the VDR gene; the BsmI and ApaI SNPs are both located in intron 8, and the TaqI is a silent SNP in exon 9. A number of association studies were previously conducted in different populations and ethnic groups. Some of them have suggested association between one or more of these SNPs and asthma [8,9,17] but others have failed to confirm this finding [10,18]. In this study, we aimed at elucidating the genetic association of VDR polymorphisms with susceptibility to asthma in Tunisian children. We also assessed the eventual association between the VDR gene polymorphisms and serum level of 25(OH)D in asthmatics and healthy subjects.

2. Subjects and methods 2.1. Subjects A total of 155 asthmatic children (59 girls and 96 boys) were recruited from the department of Pediatric and Respiratory Diseases, Abderrahmane MAMI Hospital of Chest Diseases, Ariana, Tunisia. The mean age of the subjects was 9.1 years. The diagnosis and classification of the clinical severity of asthma was based in clinical symptoms and lung function according to the GINA guidelines [19]. Patients were classified as intermittent and mild persistent asthma (N = 82), moderate persistent asthma (N = 63), and severe persistent asthma cases (N = 10) (Table 1). Skin sensitivity to specific allergens was used to define the atopic status of 92 patients. We defined atopy by a positive skin test reaction characterized by a weal of at least 3 mm in diameter, to one or more allergens in the presence of a positive histamine control and a negative uncoated control. A panel of common aeroallergens was used. Seventy patients were considered atopic. In addition, we recruited a group of 225 control children (aged 4–16 years, means 9.5), from the emergency department of Tunis

Table 1 Clinical characteristics of the study sample. Asthmatics

Controls

Number of subjects Mean age (range) Sex ratio (girls/boys) Total frequency of females Total frequency of males

155 9.1 (4–16) 0.6 (59/96) 38% 62%

225 9.5 (2–16) 0.7 (99/126) 44% 56%

Atopic status (n = 92) Atopic (n, %) Non atopic (n, %)

70 (76%) 22 (24%)

– –

Severity Intermittent and mild Moderate Severe

82 63 10

– – –

Children Hospital with no respiratory nor allergy manifestations. All data and sample collections for this study were approved by Local Ethics Committee. Informed parental consent was obtained for all children. 2.2. VDR polymorphisms genotyping Genomic DNA was extracted from peripheral blood leukocytes using the salting out procedure as described [20]. BsmI, TaqI and ApaI VDR polymorphisms were assessed in 155 patients and 225 controls and FokI VDR polymorphism was assessed for 155 patients and only 152 controls. All of the genotyping was done by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Primer sequences used for amplification of FokI, BsmI, TaqI and ApaI polymorphisms were as previously described and are listed in Table 2 [21]. The PCR was performed in a 50 ll reaction volume containing 2.4 lM of each primer, 10 mM of each dNTP, 1x PCR buffer, 1.5 mM MgCl2, 2 U of Taq DNA polymerase (Fermentas), and 100 ng of genomic DNA, diluted to the final volume with H2O. The running conditions were: pre-denaturation at 95 °C for 5 min, followed by 30 cycles of denaturation at 94 °C, annealing temperature at 60 °C, and extension at 72 °C for each 1 min, respectively. Finally, extension was conducted at 72 °C for 5 min. Amplified fragments were digested with the appropriate restriction enzyme (Fermentas) according to the manufacturer’s instructions and visualized on a 3% agarose gel. The usual nomenclature for restriction fragment length polymorphism alleles was used in this study [21,22]. The lowercase allele represents the presence of the restriction site (f, b, a, or t) and the uppercase allele represents the absence of the restriction site (F, B, A, or T). 2.3. Serum 25(OH)D levels The collected serum was immediately shaded from direct light and stored at 20 °C. All samples were analyzed simultaneously at the same laboratory, using a same technique conducted by one technician. Serum concentrations of 25(OH)D were measured with a radioimmunoassay kit (Dia-Sorin, Stillwater, MN, USA) [23] and values were reported in nanograms per milliliter. In descriptive analyses, vitamin D levels were categorized as sufficient (P30 ng/ml), insufficient (P20 and <30 ng/ml) and deficient (<20 ng/ml) on the basis of previous recommendations [24]. 2.4. Statistical analysis All genotypes were tested for Hardy–Weinberg equilibrium. Association analysis was performed using standard Chi-squared test (Epistat statistical package, Epi Info Version 6) to detect

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Table 2 Different loci selected within the VDR sequence and their corresponding primers. SNP

PCR primer 0

0

PCR product (bp)

Restriction enzyme

RFLP products (bp)

265-bp

Fok-I (37 °C)

FF: 265bp Ff: 265, 196 and 69bp ff: 196 and 69bp BB: 825bp Bb: 825, 650 and 175bp bb: 650 and 175bp TT: 245 and 495 bp Tt: 495, 290, 245, and 205 bp tt: 290, 245, and 205 bp AA: 740bp Aa: 740, 530 and 210 bp aa: 530 and 210 bp

Fok-I

F: 5 -AGC TGG CCC TGG CAC TGA CTC TGC TCT-3 R: 50 -ATG GAAACA CCT TGC TTC TTC TCC CTC-30

Bsm-I

F: 50 -CAACCAAGACTACAAGTACCGCGTCAGTGA-30 R: 50 -AACCAG CGGGAA GAG GTCAAGGG-30

825-bp

Bsm-I (37 °C)

Taq-I/Apa-I

F: 50 -CAG AGC ATG GAC AGG GAG CAA-30 R: 50 -GCAACTCCTCATGGCTGAGGTCTC-30

740-bp

Taq-I (50 °C) Apa-I (50 °C)

differences in genotypes, alleles and haplotypes distribution among our groups. P levels smaller than 0.05 were considered significant. The strength of a gene association is indicated by the odds ratio (OR). The odds ratio and the 95% confidence intervals (CI) were calculated whenever applicable. Haplotypes analyses were determined on a Bayesian algorithm basis using the ‘phase’ software. An adjustment for multiple tests by Bonferroni correction in which the P-values are multiplied by the number of comparisons was later applied to control the false finding rate. The linkage disequilibrium (LD) values for the three pairs of SNPs have been calculated using ‘arlequin’ software based on EM algorithm. 25-hydroxyvitamin D levels are presented as means values ± standard deviation (SD). The data were statistically analyzed using Student’s t-test and one way ANOVA. 3. Results The distribution of genotypes and alleles frequencies of VDR gene polymorphisms in cases and control subjects is shown in Table 3. The genotype frequencies of the four SNPs were in agreement with Hardy–Weinberg equilibrium. Significant differences were noted in FokI, TaqI and BsmI genotype distribution between cases and controls. For FokI polymorphism, the frequency of ‘FF ’ genotype in the asthmatic group was 57%, which was significantly higher than the corresponding value for the control group (46%) (v2 = 6.34, P = 0.04). In addition, the alleles frequencies were significantly different, indicated by a 75% prevalence of ‘F ’ allele in cases and 65% in controls (P = 0.01). The Odd Ratio was calculated, and children carrying ‘F ’ allele were once and a half as likely to develop asthma [OR = 1.57, 95%CI (1.09–2.26)] than healthy individuals.

For BsmI polymorphism, the ‘B’ allele was significantly related to the increasing risk of developing childhood asthma (OR = 1.34, 95% CI = 0.99–1.82, P = 0.04). In addition, children with homozygous ‘B’ allele were 2.15 times more likely to have asthma than others (95% CI = 1.19–3.90, P = 0.006). For TaqI polymorphism, individuals with ‘TT ’ and ‘Tt’ genotype showed a statistically significant association with asthma compared with those sharing the ‘tt’ genotype (P = 0.006). The OR was calculated, and carriers with one or two copies of ‘T’ allele were 2.33 times as likely to develop asthma (OR = 2.33, 95%CI = 1.20–4.58). The distribution of alleles was not significantly different comparing total patients with controls. For ApaI, no difference was observed in genotype distribution and alleles frequencies between cases and controls. Linkage disequilibrium (LD) analysis of the 3 VDR SNPs located in the 30 UTR region was conducted and illustrated in Table 4. The three 30 SNPs (BsmI-ApaI-TaqI) showed significant LD with each other, as it has been shown in previous studies [15,29], especially BsmI-TaqI. We have also estimated haplotypes for the VDR 30 UTR polymorphisms. VDR haplotype frequencies are depicted in Table 5. The most frequent haplotype in our population were bAT (36.6%) and BAT (16%). The frequency of the haplotype bAt was found significantly (P = 0.00076) higher in healthy individuals (13.3%) than in cases (4.5%). Similarly, bat haplotype was found significantly (P = 0.016) more frequent in controls (9.5%) than in cases (3.54%). This result confirms the genotypic association where b and t alleles are protective. We analyzed the correlation of these polymorphisms with atopic status (Table 6). The genotypes and the alleles frequencies of the four VDR polymorphisms did not differ significantly between the atopic and non-atopic asthmatic groups (P = 0.9 for FokI,

Table 3 Frequency of alleles and distribution of genotypes of VDR polymorphisms in asthmatic and control subjects. SNP

Genotypes Asthmatics n(%)

Fok-I FF Ff ff Bsm-I BB Bb bb Taq-I TT Tt tt Apa-I AA Aa aa

88 (57) 56 (36) 11 (7)

Alleles Controls n(%) 70 (46) 59 (39) 23 (15)

v2

P value

6.34

0.04

OR (95%CI)

Asthmatics n(%)

Controls n(%)

v2

P value

OR (95%CI)

F 232 (75) f 78 (25)

199 (65) 105 (35)

6.45

0.01

1.57 (1.09–2.26)

34 (22) 72 (46) 49 (32)

26 (11.5) 119 (53) 80 (35.5)

7.44

0.006

2.15 (1.19–3.90)

B 140 (45) b 170 (55)

171 (38) 279 (62)

3.89

0.04

1.34 (0.99–1.82)

59 (38) 81 (52) 15 (10)

79 (35) 101 (45) 45 (20)

7.35

0.006

2.33 (1.20–4.58)

T 199 (64) t 111 (36)

259 (58) 191 (42)

3.38

0.06

1.32 (0.97–1.80)

92 (59) 57 (37) 6 (4)

142 (63) 70 (31) 13 (6)

1.76

0.41

A 241 (78) a 69 (22)

354 (79) 96 (21)

0.09

0.76

0.95 (0.66–1.36)

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H. Maalmi et al. / Human Immunology 74 (2013) 234–240 Table 4 Linkage disequilibrium test between 30 UTR VDR gene polymorphisms. SNP1

SNP2

Distance between two polymorphisms

D0

P value

BsmI ApaI BsmI

ApaI TaqI TaqI

998 80 1078

0.52 0.47 0.58

<0.0001 <0.0001 <0.0001

P = 0.39 for BsmI, P = 0.42 for both TaqI and ApaI). Eventually, we looked for a possible difference in the allele and genotype frequencies among severity groups of children with asthma according to their severity status (Table 7). We did not find a relationship between the genotypes, alleles and groups of asthma severity. It seems that VDR polymorphisms were not correlated with the severity of asthma. In relation to serum 25-hydroxyvitamin D levels, a significant difference (P < 0.0001) was observed between asthmatic patients (18.87 ± 6.68) and healthy children (30.29 ± 5.96). Categorization of vitamin D levels revealed that 61.3% of the asthmatic children had deficient vitamin D levels, 29.7% had insufficient levels and only 9% had sufficient levels. However, in non asthmatic children, vitamin D deficiency and insufficiency was present in only 3.6% and 44%, respectively. The major percentage of those children (52%) had sufficient serum vitamin D levels. The difference between the two groups was statistically significant (P < 0.0001). This result indicates that hypovitaminosis D is a reel problem in asthmatic children. Furthermore, we analyzed the distribution of 25(OH)D levels in each cases and controls genotype (Table 8). FokI, BsmI, ApaI and TaqI genotype polymorphisms were not associated with serum 25(OH)D levels. VDR variants are not involved in the regulation of serum 25(OH)D levels.

4. Discussion The VDR locus has been studied in different populations and immune-mediated diseases. The findings in asthma are contrasting. To clarify the contribution of VDR polymorphisms to genetic susceptibility to childhood asthma among Tunisian patients, we conducted a case-control study by analyzing four well-characterized VDR polymorphisms. To our knowledge, this is the first study that aimed at elucidating the relationship between VDR variants and asthma in Africa. In our study, we reported a significant association between VDR FokI, BsmI and TaqI polymorphisms and asthma (P = 0.04, P = 0.006, P = 0.006) in Tunisian children. No significant difference was found in the distribution of genotypes and alleles frequencies between patients and controls for the ApaI polymorphism (P = 0.41). A summary of the literature verifying the association between single nucleotide polymorphisms (SNPs) in the vitamin D receptor (VDR) and asthma is presented in Table 9. FokI is the only polymorphism located in a coding region and our results demonstrated that

its distribution between cases and controls was different and statistically significant. Association between FokI polymorphism and asthma was detected in our study only. For the polymorphisms located in the 30 untranslated region of the VDR gene, our results represent a replication of the previous findings of Poon et al. in the northeastern Quebec population where BsmI and TaqI polymorphisms are associated with asthma in children while ApaI variant is not [9]. The Chinese population failed to demonstrate any association between BsmI polymorphism and asthma in two independent studies [17,25]. For the TaqI polymorphism, our findings are also in concordance with the results of Raby et al. which reported association between this polymorphism and asthma in the Nurses’ Health Study (NHS) population [8]. However, the same study denied any association between this polymorphism and asthma in the Childhood Asthma Management Program (CAMP) population. Moreover, the study of Saadi et al. in the Chinese population was in discordance with our results [17]. Another study addresses the previously described association of VDR variants with asthma, in which 951 German individuals from 224 families were analyzed. Thirteen SNPs were tested and all reported no association with asthma, including three non significant SNPs also typed in our study (FokI, TaqI and BsmI). For the three SNP, results from the German population are in discordance with ours [10]. In our study, we were unable to detect any association between VDR gene polymorphisms and atopy or asthma severity. Our results are inconsistent with the results of Poon et al. [9] where significant association was found between variants in the 30 region and atopy. Similarly, Pillai et al. demonstrated recently that FokI variant was associated with one or more positive aeroallergen skin test (P = 0.003), and increased immunoglobulin E levels (P < 0.001). In addition, it was associated with higher nighttime asthma morbidity scores (P = 0.04), and lower baseline spirometric measures (P < 0.05), that are severity markers [26]. Several factors could influence the discrepancy of associations found between studies of different populations: First of all, in addition to ethnic characteristics, geographical differences and living habits might be the important reasons for these inconsistent results. Second, a genetic influence of the VDR gene, if present, is probably weak and thus the statistical power of association studies might be too low to draw reliable conclusions, in particular with an insufficient number of patients enrolled. It is well appreciated that population stratification, often difficult to recognize within case-control samples can mask, change, or even reverse the true genetic effects for genes underlying complex traits [27]. Third, the interactions between different genes and/or environmental factors play a role in the action of VDR [28]. Gene–gene or gene–environment interactions most likely differ between populations. VDR is a ‘‘master’’ transcription factor that influences several endocrine pathways [29]. Similar to the role of VDR variants in bone biology [30], the association between VDR polymorphisms and asthma is most likely confounded

Table 5 Haplotypes frequencies for VDR 30 UTR polymorphisms between asthmatic cases and controls.

*

Haplotype

Cases (freq)

Controls (freq)

OR (95%CI)

P value

P valuec

BsmI/ApaI/TaqI BAT BAt BaT Bat bAT bAt baT bat

N = 310 38 (12.25) 51 (16.45) 16 (5.16) 35 (11.29) 138 (44.5) 14 (4.5) 7 (2.25) 11 (3.54)

N = 450 72 (16) 54 (12) 9 (1) 35 (7) 165 (36.6) 60 (13.3) 12 (2.6) 43 (9.5)

0.73 (0.48–1.12) 1.44 (0.95–2.1) 2.6 (1.15–5.86) 1.5 (0.92–2.46) 1.38 (1.03–1.85) 0.3 (0.17–0.57) 0.8 (0.34–2.16) 0.35 (0.18–0.7)

0.18 0.1 0.02 0.12 0.03 0.000095 0.9 0.002

– – 0.16 – 0.24 0.00076⁄ – 0.016⁄

Signification, the number of tests for Bonferroni correction is 8.

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H. Maalmi et al. / Human Immunology 74 (2013) 234–240

Table 6 Distribution of different genotypes and alleles for the VDR polymorphisms in the atopic and non atopic groups of asthmatic children. SNP

Genotypes

Alleles

Atopic asthmatics N = 70

Non atopic asthmatics N = 22

v2

P value

Atopic asthmatics

Non atopic asthmatics

n(%)

n(%)

v2

P value

OR (95%CI)

n(%)

n(%)

Fok-I FF Ff ff

38 (54) 24 (34) 8 (12)

10 (45) 11 (50) 1 (5)

2.15

0.34

F 100 (71) f 40(29)

31 (70) 13 (30)

0.02

0.9

1.05 (0.47–2.34)

Bsm-I BB Bb bb

14 (20) 35 (50) 21 (30)

8 (36) 7 (32) 7 (32)

3.10

0.21

B 63 (45) b 77 (55)

23 (52) 21 (48)

0.71

0.39

0.75 (0.36–1.55)

Taq-I TT Tt tt

33 (47) 32 (46) 5 (7)

7 (32) 14 (64) 1 (4)

2.15

0.34

T 98 (70) t 42 (30)

28 (64) 16 (36)

0.63

0.42

1.33 (0.61–2.88)

Apa-I AA Aa aa

41 (58) 25 (36) 4 (6)

10 (45) 11 (50) 1 (5)

1.43

0.48

A 107 (76) a 33 (24)

31 (70) 13 (30)

0.64

0.42

1.36 (0.6–3.08)

Table 7 Distribution of VDR polymorphisms according to asthma severity. SNP

Severity Intermittent and mild N = 82

Moderate and severe N = 73

v2

p value

n(%)

n(%)

Fok-I FF Ff ff

48 (59) 29 (35) 5 (6)

39 (53) 26 (36) 8 (11)

1.27

0.53

Bsm-I BB Bb bb

16 (20) 37 (45) 29 (35)

18 (25) 35 (48) 20 (27)

1.31

0.51

Taq-I TT Tt tt

35 (43) 40 (49) 7 (8)

24 (33) 41 (56) 8 (11)

1.61

0.44

Apa-I AA Aa aa

51 (62) 27 (33) 4 (5)

39 (53) 32 (44) 2 (3)

2.18

0.33

Table 8 Relationship between VDR polymorphisms and vitamin D levels. PATIENTS Parameter

N (%) 25(OH)D

TEMOINS

FokI

P

FF

Ff

ff

FF

Ff

ff

88 (57) 18.57±6.72

56 (36) 19.69±6.91

11 (7) 17.1±4.98

0.4

70 (46) 31.65±6.27

59 (39) 30.15±6.4

23 (15) 31.52±7.03

P

BsmI

BsmI

N (%) 25(OH)D

BB

Bb

bb

34 (22) 17.92±6.27

72 (46) 19.41±7.12

49 (32) 18.73±6.34

TaqI

N (%) 25(OH)D

BB

Bb

bb

0.5

26 (11.5) 30.22±5.16

119 (53) 30.33±6.42

80 (35.5) 30.26±5.54

P

TaqI

0.3

0.9 P

TT

Tt

tt

TT

Tt

tt

81 (52) 18.98±6.98

15 (10) 19.92±5.65

0.7

79 (35) 30.26±6.31

101 (45) 30.27±5.82

45 (20) 30.39±5.77

P

ApaI

0.9 P

AA

Aa

aa

AA

Aa

aa

92 (59) 19.11±6.65

57 (37) 18.85±7.01

6 (4) 15.38±2.11

142 (63) 29.79±5.69

70 (31) 31.39±6.54

13 (6) 29.84±5.22

0.4

P

P

59 (38) 18.45±6.57 ApaI

N (%) 25(OH)D

FokI

0.1

239

H. Maalmi et al. / Human Immunology 74 (2013) 234–240 Table 9 A summary of the literature concerning the association between single-nucleotide polymorphisms (SNPs) in the vitamin D receptor (VDR) and asthma. Variant

dbSNPrs–

Bsm-I Taq-I Apa-I Fok-I

rs1544410 rs731236 rs7975232 rs2228570

Population Tunisian

Quebec

CAMP

NHS

German

Chinese (1)

Chinese (2)

Chinese (3)

African American

S S NS S

S S NS NS

– NS S NS

– S S NS

NS NS – NS

NS NS – NS

NS – – NS

– – – NS

– – – NS

Tunisian: our study (155 patients and 225 controls). Quebec: Poon et al. 2004 (223 independent families). CAMP, NHS: Raby et al. 2004 (582 families for the CAMP population and 517 cases and 519 controls for the NHS population). German: Wjst 2005 (224 pedigrees). Chinese 1: Saadi et al. 2009 (567 asthmatics and 523 controls). Chinese 2: Fang et al. 2009 (101 asthmatics and 206 controls). Chinese 3: Li et al. 2011 (467 cases and 288 unrelated healthy controls). African American: Pillai et al. 2011 (139 subjects with asthma and 74 controls).

by numerous potential gene–gene and/or gene–environment interactions. Finally, many of the SNPs used in association studies, with the potential exception of the FokI variant, are nonfunctional and are more likely markers of truly causative polymorphisms. The FokI polymorphism creates an alternative start codon ATG leading to a three-amino acid longer VDR protein. Most of the experiments conducted so far point to the fact that the shorter form of the protein (424aa) is more active than the long form (427aa) in terms of its transactivation activity as a transcription factor [31,32]. BsmI and ApaI polymorphisms are located in intron 8 of the VDR gene and TaqI is located in exon 9 and leads to a silent codon change, with ATT and ATC, both coding for Isoleucine and associated with increased VDR mRNA stability [30]. Therefore, VDR RFLPs appear to be markers in the linkage disequilibrium with other relevant polymorphisms elsewhere in VDR gene or in its proximity [33], rather than primary susceptibility loci in asthma disease. Since LD may differ between populations, the associations of nonfunctional variants which depend on LD are heterogenic. In this study, BsmI and TaqI polymorphisms are in LD (D0 = 0.58) which confirms previous findings [10,17]. Given these underlying problems, genetic association studies need to be performed by replication in multiple samples, avoidance of population stratification and recommendation of metaanalysis of all similar studies [34]. Genetic studies indicated that VDR polymorphisms might be implicated in the pathogenesis of asthma and related phenotypes. Genes encoding for pro-inflammatory and anti-inflammatory cytokines involved in the development of airway inflammation are target of vitamin D and reduced activity of VDR-dependent signaling pathways caused by VDR polymorphisms would ultimately influence the rate of transcription of these genes and then affect asthma. Other polymorphisms of genes encoding components of the vitamin D pathway have been reported to be associated with the risk of asthma [26,28,35]. Another important aspect of the study was the lack of correlation between FokI, BsmI, TaqI, ApaI and 25(OH)D levels (Table 8) upon Anova test analysis (P > 0.05). Similar results were obtained by Ahn et al. [36] and Orton et al. [37] in multiple sclerosis patients. More recently, Hibler et al. [38] confirmed that genetic variation in VDR was not associated with 25(OH)D or 1,25(OH)2D serum levels. However, one study found an association between FokI and serum 25(OH)D levels [39], where F allele was associated with lower winter and summer serum 25(OH)D levels in multiple sclerosis patients and healthy individuals. Vitamin D deficiency is considered as a risk factor for asthma. Epidemiological studies support the fact that this last could be the onset element of asthma. The demonstrated deficiency could even be implicated in severity and poor lung function. However, the deficiency of vitamin D could be due to multiple factors such

as poor diet, lack in sun exhibition, latitude, season, or the staying indoors at home behavior of the asthmatics. Genetic factors may also contribute to vitamin D deficiency status by feedback loop regulation. In fact, VDR and its ligand 1.25-dihydroxyvitamin D and the retinoid X receptor (RXR) generate an heterodimer which bind to the VDRE located in the promoter regions of genes play a regulating loop on vitamin D metabolites levels [16]. This endocrine feedback loop exerted by vitamin D to self-regulate its concentrations may be deregulated if the VDR, RXR, or any enzyme of vitamin D metabolism is affected. In conclusion, we observed an association of VDR variants FokI, BsmI and TaqI with asthma in Tunisian children. Further studies with a larger sample of patients will improve our understanding of the contribution of this and other genes to asthma status and atopy and allow preventive and/or therapeutic interventions in asthmatic patients. Acknowledgments This study was supported by a grant from the Ministry of Higher Education and Scientific Research. References [1] Bijanzadeh M, Mahesh PA, Ramachandra NB. An understanding of the genetic basis of asthma. Indian J Med Res 2011;134:149–61. [2] Bousquet J, Jeffery PK, Busse WW, Johnson M, Vignola AM. Asthma: from bronchoconstriction to airways inflammation and remodeling. Am J Respir Crit Care Med 2000;161(5):1720–45. [3] Hansdottir S, Monick MM, Lovan N, Powers L, Gerke A, Hunninghake GW. Vitamin D decreases respiratory syncytial virus induction of NF-kappaB-linked chemokines and cytokines in airway epithelium while maintaining the antiviral state. J Immunol 2010;184:965–74. [4] Baeke F, Takiishi T, Korf H, Gysemans C, Mathieu C. Vitamin D modulator of the immune system. Curr Opin Pharmacol 2010;10(4):482–96. [5] Raby BA, Silverman EK, Lazarus R, Lange C, Kwiatkowski DJ, Weiss ST. Chromosome 12q harbors multiple genetic loci related to asthma and asthmarelated phenotypes. Hum Mol Genet 2003;12:1973–9. [6] Wjst M, Fischer G, Immervoll T, Jung M, Saar K, Rueschendorf F, et al. A genome-wide search for linkage to asthma. Genomics 1999;58:1–8. [7] Malerba G, Lauciello MC, Scherpbier T, Trabetti E, Galavotti R, Cusin V, et al. Linkage analysis of chromosome 12 markers in Italian families with atopic asthmatic children. Am J Respir Crit Care Med 2000;162:1587–90. [8] Raby BA, Lazarus R, Silverman EK, Lake S, Lange C, Wjst M, et al. Association of vitamin D receptor gene polymorphisms with childhood and adult asthma. Am J Respir Crit Care Med 2004;170(10):1057–65. [9] Poon AH, Laprise C, Lemire M, Montpetit A, Sinnett D, Schurr E, et al. Association of vitamin D receptor genetic variants with susceptibility to asthma and atopy. Am J Respir Crit Care Med 2004;170(9):967–73. [10] Wjst M. Variants in the vitamin D receptor gene and asthma. BMC Genet 2005;15(6):2. [11] Brehm JM, Celedón JC, Soto-Quiros ME, Avila L, Hunninghake GM, Forno E, et al. Serum vitamin D levels and markers of severity of childhood asthma in Costa Rica. Am J Respir Crit Care Med 2009;179:765–71. [12] Brehm JM, Schuemann B, Fuhlbrigge AL, Hollis BW, Strunk RC, Zeiger RS, et al. Serum vitamin D levels and severe asthma exacerbations in the childhood

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