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Published ahead of print on July 28, 2004, doi:10.1164/rccm.200404-447OC
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American Journal of Respiratory and Critical Care Medicine Vol 170. pp. 1057-1065, (2004)
© 2004 American Thoracic Society
doi: 10.1164/rccm.200404-447OC

Original Article

Association of Vitamin D Receptor Gene Polymorphisms with Childhood and Adult Asthma

Benjamin A. Raby, Ross Lazarus, Edwin K. Silverman, Steven Lake, Christoph Lange, Mathias Wjst and Scott T. Weiss

Channing Laboratory and Division of Pulmonary and Critical Care Medicine, Department of Medicine, and Harvard Partners Center for Genomics and Genetics, Brigham and Women's Hospital; Division of Pulmonary and Critical Care Medicine, Beth Israel Deaconess Medical Center; Harvard Medical School; and Harvard School of Public Health, Boston, Massachusetts; and GSF-National Research Center for Environment and Health, Institute of Epidemiology, Neuherberg, Germany

Correspondence and requests for reprints should be addressed to Benjamin Raby, M.D.C.M., M.P.H., Channing Laboratory, Brigham and Women's Hospital, Boston, MA 02115. E-mail: benjamin.raby{at}channing.harvard.edu


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Vitamin D receptor (VDR) polymorphisms have been associated with several immune-related diseases, and VDR and vitamin D itself modulate T cell differentiation. VDR maps to chromosome 12q, near a region commonly linked to asthma. We evaluated VDR as part of a 12q positional candidate survey, and in response to observations of VDR polymorphism associations with asthma and atopy in a founder population of Quebec. Twenty-eight loci in 7 positional candidates (7 in VDR) were genotyped in 582 families. Whereas other candidates demonstrated no association, the VDR ApaI polymorphism demonstrated significant transmission distortion, with undertransmission of the C allele in a ratio of 4:5 (p = 0.01). This association was most prominent in girls, in whom distortion was more marked (p = 0.009). Sex-specific associations between multiple VDR polymorphisms and immunoglobulin E levels were also observed (p = 0.006–0.01). Asthma associations were replicated in a second cohort (517 females with asthma and 519 matched control subjects): 4 of 6 VDR variants demonstrated significant association (p = 0.02–0.04). The direction of association in this second cohort was opposite to the effects seen in the trios, but similar to findings in the Quebec study. These results suggest that VDR influences asthma and allergy susceptibility in a complex manner.

Key Words: association studies • asthma • genetics • replication • VDR

The vitamin D receptor (VDR; OMIM 601769) is the principal receptor that binds 1{alpha},25-dihydroxyvitamin D3 [1,25-(OH)2D3] and is a critical molecule in calcium metabolism and bone turnover. Rare nonsynonymous mutations in VDR cause vitamin D–resistant rickets (Type IIA), and more common variants contribute to variability in bone mineral density and susceptibility to osteoporosis. In addition to the central role of VDR in bone metabolism, it has long been recognized as an important contributor to a diverse array of cellular processes (1). VDR belongs to a family of nuclear proteins that act as transcriptional regulatory factors and is structurally similar to both steroid hormone and thyroid hormone receptors (2). Data suggest that vitamin D and a normally functioning VDR are important for proper development of the immune system (3–5). Vitamin D supplementation in a murine model of pulmonary eosinophilic inflammation alters cytokine expression profiles, immunoglobulin E levels, and the pattern of airway eosinophilia during allergen sensitization, suggesting that the vitamin D pathway may influence the development of allergy and asthma (6). Importantly, VDR polymorphisms have been implicated in several immune and inflammatory disorders, including mycobacterial and human immunodeficiency virus susceptibility (7–9), diabetes (10, 11), psoriasis (12), and Crohn's disease (13), although the precise mechanisms of action of these diverse disease-related effects remain speculative.

VDR maps to the long arm of chromosome 12, a region that has been commonly linked to asthma and allergy-related phenotypes in genome-wide linkage analyses (reviewed in Raby and coworkers [14]). We and others have previously demonstrated linkage with asthma to a genomic region at the centromeric end of chromosome 12q that includes the VDR locus (14–16). In a companion article (17), Poon and colleagues report associations between VDR polymorphisms and haplotypes with asthma and atopy susceptibility in a founder population from northeastern Quebec. As part of our ongoing efforts to identify the genetic determinants of the asthma linkage on chromosome 12q, we screened several biologically relevant positional candidate genes, using a family-based association study design, including VDR (Table 1). Whereas variation in the other candidate genes evaluated did not demonstrate significant associations with asthma, VDR was significantly associated with asthma and related phenotypes. These effects were strongest in girls. Additional testing in a second cohort of women also revealed evidence of association between VDR and asthma. Together with the initial associations described in the Quebec cohort, these findings suggest that VDR polymorphisms contribute to asthma and atopic risk.


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  		TABLE 1. Asthma positional candidate genes in centromeric region of chromosome 12q

 

   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Populations
The Childhood Asthma Management Program (CAMP) is a multicentered North American clinical trial designed to investigate the long-term effects of inhaled antiinflammatory medications in children with mild to moderate asthma (18, 19). Of the 1,041 children enrolled in the original clinical trial, 968 children and 1,518 of their parents contributed DNA samples. Five hundred and eighty-two complete nuclear families (including 55 families with additional affected offspring) are included in the analysis presented here: 471 are of non-Hispanic Caucasian descent, 64 are of African American descent, and 47 are of Hispanic descent. A diagnosis of asthma was based on methacholine hyperreactivity (PC20 [provocative concentration causing a 20% fall in FEV1] no greater than 12.5 mg/ml) and one or more of the following criteria for at least 6 months in the year before recruitment: (1) asthma symptoms at least two times per week, (2) at least two uses per week of an inhaled bronchodilator, and (3) daily asthma medication. Spirometry was performed according to American Thoracic Society recommendations, using a volume displacement spirometer, and methacholine challenge was performed according to the Wright nebulizer tidal breathing technique (18). Total blood eosinophil counts were performed by center-specific methods, and total serum IgE was measured in radioimmunosorbent assays from blood samples collected during the screening sessions of the CAMP study.

The Nurses' Health Study (NHS) is a questionnaire-based longitudinal cohort study of 120,000 women, aged 30–55 years, initiated in 1976 (20–22). Between 1988 and 1996, 1,596 incident physician-diagnosed asthma cases were reported. An evaluation of the validity of a questionnaire-based asthma diagnosis revealed that only 5% of women who originally met diagnostic criteria raised issues suggesting misdiagnosis (23). DNA has been made available for 517 cases and 519 control subjects of European ancestry, matched for age. No lung function or allergy phenotypes are available for this population. To test for population stratification in this cohort, we tested 49 unlinked single-nucleotide polymorphisms (SNPs) for evidence of association, as suggested by Prichard and Rosenberg (24). The global {chi}2 was 60.2, with a corresponding p value of 0.13, given 49 degrees of freedom (the number of loci tested). On the basis of this analysis, there is no evidence of significant population stratification in this data set.

Informed assent and consent were obtained from the study participants and their parents to collect DNA for genetic studies in CAMP. The Institutional Review Board of Brigham and Women's Hospital (Boston, MA), as well as those of the other CAMP study centers, approved the studies. Approval by the Institutional Review Board of Brigham and Women's Hospital was obtained for DNA collection in the NHS as well.

Polymorphism Genotyping
SNPs were selected from the SNP database (dbSNP; see http://www.ncbi.nlm.nih.gov/SNP/) that mapped to unique genomic positions, were double-hit SNPs (i.e., more than one submitter), and were not in repeat regions. Included were three VDR SNPs (TaqI, ApaI, and FokI) that have previously been implicated in other disorders (25, 26). SNP genotyping was performed on the basis of unlabeled minisequencing reactions and mass spectrometry analysis as implemented in the Sequenom MassARRAY platform (Sequenom, San Diego, CA), and using the 5' -> 3' exonuclease assay as implemented in the TaqMan assay (Applied Biosystems, Foster City, CA) (27). Protocol details and primer data are available at http://wchanning.bwh.harvard.edu/epigenetics/Projects. Duplicate genotyping was performed on about 10% of the sample to assess genotype reproducibility. Genotype data quality was assessed on the basis of several criteria including completeness of genotype data, degree of discordant duplicate genotyping, evidence of non-Mendelian marker inheritance, and Hardy–Weinberg disequilibrium among parental data.

Statistical Analysis
Parental (in CAMP) and control (in NHS) genotype data were used to assess Hardy–Weinberg equilibrium and pairwise linkage disequilibrium (LD). Hardy–Weinberg equilibrium was tested at each SNP locus, using an exact method (28). LD between each pair of SNP loci was evaluated by a maximum likelihood method (29) to infer phase for dual heterozygotes and was expressed as r2 (30). Haplotypes were inferred by Bayesian methods (31) as implemented in the PHASE package (32). For the familial data, haplotypes were confirmed and determination of haplotype block structure was performed with Haploview (http://www.broad.mit.edu/personal/jcbarret/haplo/). Blocks were defined on the basis of the Gabriel definition (D' > 0.9; minimum allele frequency, 5%) (33).

In CAMP, ethnicity-specific evidence of association with asthma was evaluated by family-based association analysis with FBAT version 1.4 (34, 35), stratified by ethnic group. Quantitative trait analysis, stratified by ethnic group, was performed with PBAT (36, 37) for the following intermediate phenotypes: postbronchodilator FEV1, bronchodilator (BD) response ([post-BD FEV1 – pre-BD FEV1]/pre-BD FEV1), post-BD FEV1/FVC ratio, airway hyperresponsiveness to methacholine (log-transformed PC20), and log-transformed total serum IgE levels and total serum eosinophils. PBAT allows for variable transformation and covariate adjustment, stratified analysis, and tests for gene–covariate interaction. All variables were z-transformed, and the following covariate adjustments were included: (1) FEV1, FEV1/FVC, and BD response were adjusted for age, sex, and height to the second order; and (2) PC20, IgE, and eosinophils were adjusted for age and sex. Gene–sex interactions also were evaluated. Evidence of haplotype-block association with asthma was assessed by the likelihood-ratio score test implemented in TRANSMIT (38). Tests for global significance of all haplotypes were performed. Haplotype analysis was restricted to the Caucasian cohort, given the limited sample size for the other ethnic groups in CAMP.

For the nested case–control association analysis among the NHS population, single-SNP associations were performed with SAS/Genetics (version 8.2; SAS Institute, Cary, NC). SNPs were assessed for both genotypic and allelic associations, as well as the Armitage trend test. Significant associations (p < 0.05) were then explored further by specifying either dominant or recessive models by recoding genotypes 11 versus 12 or 22, and 11 and 12 versus 22, respectively. Haplotype analysis was performed with haplo.stat (39).

SAS/Genetics version 8.2 and Web-based bioinformatic tools (http://innateimmunity.net) were used to manage and analyze the data.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
CAMP Genetics Cohort Characteristics
The baseline characteristics of the CAMP participants genotyped in this study are presented in Table 2. The cohort is primarily Caucasian and 62% male. Ninety percent of the cohort was atopic (defined as either having at least one positive skin-prick test [3 mm or more] or an elevated IgE [100 IU/ml or more]). Age of asthma symptom onset was significantly higher among girls, and girls had higher baseline lung function and tended to be less atopic than boys. There are no significant differences in asthma severity or lung function between those children included in this study compared with the original CAMP cohort (data not shown).


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  		TABLE 2. Baseline characteristics of childhood asthma management program probands included in vitamin d receptor analysis

 
Positional Candidates for Asthma on Chromosome 12q
We genotyped 28 polymorphisms in 7 candidate genes that map to the centromeric region of chromosome 12q and tested for evidence of association with asthma (Table 1). Polymorphisms in IFNG, STAT6, CPM, KITLG, IL22, and IRAK3 did not show significant evidence of transmission distortion from parent to asthmatic offspring, suggesting a lack of association with disease status. Although we have tested only a limited number of SNPs at these loci, other groups have reported negative associations with several of these genes in other asthmatic cohorts, suggesting that variants in these genes are probably not important genetic determinants of asthma susceptibility, despite the documented functional importance of these genes in asthma-related biological pathways (40–42).

Association of VDR ApaI SNP with Asthma in CAMP
Seven VDR polymorphisms were genotyped in 582 nuclear families. Genotype quality was high, with an average completion rate of 97.3%, no discordances on repeat genotyping of a random 10% of the cohort, and a low rate of Mendelian inconsistencies (less than 1/1,000 transmissions).

Table 3 shows the results of the family-based associations for the seven VDR SNPs in the Caucasian subgroup. In contrast to the lack of association with the other candidate genes tested, the intronic ApaI polymorphism demonstrated evidence of association with asthma: among informative matings, the C allele was undertransmitted to affected Caucasian offspring in a ratio approaching 4:5 (transmitted:untransmitted [T:U] ratio for the C allele, 199:251; pFBAT = 0.01). Similar results were observed when the analysis was performed excluding the 38 Caucasian families with multiple affected offspring (pFBAT = 0.01), confirming that the results were due to association, rather than to linkage at this region. Similar results were also observed when the analysis was performed with all ethnic groups together (pFBAT = 0.01), but the subsets of other ethnicities were insufficiently powered to detect these effects when analyzed individually. Stratified analysis by sex revealed that the association was essentially restricted to girls: despite a marked reduction in sample size (with only 159 informative trios compared with 450 in the unstratified data), the ApaI C allele demonstrated more striking transmission distortion, with a T:U ratio approaching 2:3 (pFBAT = 0.009). In contrast, the degree of transmission distortion to the male offspring was minimal (T:U ratio, 136:155; pFBAT = 0.27). No other VDR SNP demonstrated evidence of significant association with asthma (unstratified or sex stratified), although the rs2239185 C allele demonstrated trends of transmission distortion similar to those of the ApaI C allele (T:U ratio of 4:4.8 in unstratified analysis, pFBAT = 0.09; and T:U ratio of 2:2.6, pFBAT = 0.12 among girls).


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  		TABLE 3. Association analysis of vitamin d receptor within childhood asthma management program caucasian families with asthma

 
Family-based quantitative analyses for intermediate asthma-related phenotypes were performed with PBAT, which can accommodate adjustment for covariates and gene-by-environment interactions. Among the Caucasians, there were statistically significant (but weak) associations between VDR polymorphisms and asthma severity phenotypes. The FokI C allele was associated with greater baseline airways obstruction (i.e., lower prebronchodilator FEV1/FVC ratio, p = 0.009), whereas G alleles in SNPs rs2239179 and rs1540339 were associated with lower bronchodilator responsiveness (p = 0.009 and p = 0.02, respectively). However, these effects were small, with locus-specific heritability estimates less than 1% each (data not shown). There were no significant associations detected in the other ethnic groups, but power was limited because of the small sample size. There were no significant associations between VDR polymorphisms and other tested phenotypes in unstratified analysis, including baseline FEV1, methacholine responsiveness, total serum IgE levels, or total blood eosinophil counts.

Like the single-SNP associations with the binary asthma phenotype, stratification of the cohort by sex in the quantitative analysis revealed striking differences between boys and girls in the relationships between VDR SNPs and asthma-related intermediate phenotypes (Table 4). The most notable differences were seen in the impact of VDR polymorphisms on serum IgE levels. Whereas the unstratified analysis showed no significant relationship, four of seven VDR polymorphisms contribute to the variance in serum IgE levels in girls (p = 0.006–0.03), but not in boys (p = 0.21–0.81). To assess the stability of these results, we repeated the quantitative analysis, using IgE measurements from a follow-up visit during Year 4 of the clinical trial. Several SNPs (rs1540339 and TaqI) again demonstrated significant relationships to IgE levels in girls, but not in boys, suggesting that the relationship is consistent over time. Sex differences in the relationship between VDR and eosinophil level were also observed, but with fewer SNPs and without evidence of reproducibility at the Year 4 follow-up visit.


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  		TABLE 4. Quantitative trait analysis of vitamin d receptor polymorphisms in childhood asthma management program caucasian families

 
Haplotype Structure of VDR in CAMP
To better understand the relationship between the asthma-associated and IgE-associated SNPs, linkage disequilibrium analysis was assessed in the CAMP Caucasian parents. Strong evidence for LD was observed at the 3' end of the gene, with r2 = 0.97 for SNPs rs2239185 and ApaI (both with r2 of 0.6 with TaqI). These three SNPs showed associations with asthma and IgE, and form a haplotype block associated with asthma (see below). SNPs rs3782905, rs2239179, and rs1540339 demonstrated some evidence of LD with each other and with three more 3' SNPs, but these relationships were weak (r2 = 0.14–0.57). Two of these three SNPs (rs1540339 and rs2239179) showed evidence of association with IgE. The FokI SNP was not linked to the other six in this cohort and showed no evidence of association with either asthma or IgE. This suggests an important haplotype block structure at the 3' end of VDR.

Haplotype block analysis of the seven VDR SNPs was performed with Haploview and the block definition proposed by Gabriel and coworkers (33). Two major blocks were observed, each with three major haplotypes, accounting for more than 90% of all haplotypes detected (Figure 1). This structure is similar to that observed by Poon and coworkers in a French Canadian cohort of patients with asthma (17), suggesting a conserved haplotype structure of the VDR locus in Caucasians. Identical block structure was observed in the Hispanic group, although the frequency distributions of haplotypes within each block were different. Linkage disequilibrium in the African Americans differed slightly, with much weaker LD between rs2239185 and ApaI. As a result, the block structure among the African Americans was different, with less block structure at the 3' end of the gene (Figure 1).



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  		Figure 1. Haplotype block structure of vitamin D receptor in Childhood Asthma Management Program (CAMP). Inter-SNP (single-nucleotide polymorphism) distance is expressed as kilobases (kb). Haplotypes observed at greater than 1% are presented. Fractions in parentheses denote haplotype frequency observed in CAMP parents. Line thickness reflects frequency of adjacent block haplotype distribution (thick lines, > 10%; thin lines, > 1%).

 
On the basis of this haplotype structure, we tested the VDR blocks for evidence of association with asthma, using TRANSMIT. Because of the limited number of non-Caucasian trios, haplotype analysis was limited to the Caucasian trios. The terminal three-SNP haplotype block (rs2239185–ApaI–TaqI) was associated with asthma in the Caucasian trios (global test of significance, p = 0.02), with overtransmission of haplotypes TAT and TAC, and with undertransmission of haplotype CCT. The consistency of these results with the single SNP analysis is not surprising, given that the ApaI C allele (shown to be significantly undertransmitted in the single SNP analysis) resides only on the CCT haplotype.

Linkage Disequilibrium, Haplotype Block Structure, and Association Testing in VDR Genomic Region
Within 200 kb of VDR reside other attractive biological candidates for asthma susceptibility genes (see Figure 2). Most notable is Type IIA collagen (COL2A1), located within 100 kb of VDR. Collagens (particularly Types I and III) are critical molecules in extracellular matrix repair and play important roles in airway remodeling—a process that contributes to asthma severity and progression (43). Although Type II collagen has not been implicated in asthma previously, the associations observed with the VDR SNPs here could be explained by linkage disequilibrium between these associated VDR SNPs and polymorphisms in adjacent asthma-related genes. To better understand the relationship between VDR and adjacent genes, and because the proximal portion of chromosome 12q has been linked to asthma in several genome-wide and regional linkage studies of asthma, we genotyped a set of polymorphisms across a 330-kb region centered around VDR to evaluate the extent of regional LD and the haplotype block structure and tested these polymorphisms for association with asthma in the CAMP cohort. Twenty-nine polymorphisms were selected from the dbSNP and genotyped in the nuclear families (a complete list of genotyped SNPs can be found with supplemental information at http://wchanning.bwh.harvard.edu/epigenetics/Projects). The median intermarker distance was 4.1 kb, with several large gaps flanking VDR (about 55 kb upstream and 89 kb downstream) because of reduced dbSNP availability of SNP data between genes in this region.



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  		Figure 2. Linkage disequilibrium map across the genomic region surrounding VDR. Known genes mapped to chromosome 12q13.11 (bp 46,389,340–46,722,774) in July 2003 freeze of the human genome. Gene symbols: P11 = 26 serine protease; EPAC = Rap1 guanine-nucleotide-exchange factor directly activated by cyclic AMP; FLJ20489 = hypothetical protein FLJ20489; HDAC7A = histone deacetylase 7A; VDR = vitamin D (1,25-dihydroxyvitamin D3) receptor; MGC5576 = hypothetical protein MGC5576; COL2A1 = collagen (Type II, {alpha}1). Relative orientation of genes is denoted by intronic arrows. Strength of pairwise linkage disequilibrium (expressed as r2) for parental data is plotted for the 36 SNPs typed in the CAMP cohort.

 
As can be seen in Figure 2, linkage disequilibrium across this 330-kb region was discrete, with clusters of LD observed within three gene clusters (P11–HDAC7A, VDR, and COL2A1), but not between them. Because of the lack of genotype information for the region immediately surrounding VDR, we were unable to characterize the precise extent of VDR blocks. However, there was no evidence of meaningful LD between VDR SNPs and those mapping near or within other candidate genes along the segment, indicating historical recombination around VDR, and suggesting that the VDR locus can be considered a distinct genetic unit in disease association studies. In keeping with this notion, testing of the surrounding non-VDR SNPs for association with asthma did not reveal any significant evidence of transmission distortion (data not shown), suggesting that the VDR single SNP and haplotype associations observed are unlikely to be due to effects of variation in neighboring genes.

Replication of VDR–Locus Association with Asthma among Women
Given that we tested multiple candidate genes, multiple polymorphisms, and multiple phenotypes, our initial evidence of associations may be due to chance. To address this possibility and to evaluate whether VDR associations with asthma are more broadly generalizable, we genotyped six of the seven VDR SNPs in a second cohort of adult women with asthma and age-matched control subjects ascertained through the NHS. We were unable to reliably genotype the seventh, rs2239179. In contrast to the single SNP association noted in the CAMP cohort, four of the six SNPs tested demonstrated evidence of association with asthma in the NHS asthmatic cohort, particularly under an additive model of genetic risk, as assessed by the Armitage trend test (Table 5). The risk conferred by VDR polymorphisms was modest, and the greatest risk was noted among subjects carrying two copies of risk alleles. For example, homozygotes for a C allele at either ApaI or the tightly linked rs2239185 were at 1.4 times the risk of asthma compared with those with one or fewer copies (95% confidence interval, 1.03–1.91; p = 0.02). Interestingly, the minor allele of TaqI demonstrated protective effects in a recessive model (odds ratio, 0.68; 95% confidence interval, 0.47–0.98; p = 0.04), suggesting important haplotype effects. In fact, the rs2239185–ApaI–TaqI haplotype block associations seen in the CAMP cohort were also evident in this NHS cohort (Table 6), shedding some light on the single SNP associations: the CCT haplotype (which is the only one bearing the C alleles at rs2239185 or ApaI) was significantly overrepresented in the cases, whereas the TAC haplotype (the only one bearing the TaqI C allele) was significantly underrepresented.


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  		TABLE 5. Association analysis of vitamin d receptor single-nucleotide polymorphisms in nurses' health study asthma cohort

 

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  		TABLE 6. Haplotype association analysis in nurses' health study asthma cohort

 
Although significant associations are evident in both the CAMP and NHS cohorts, noteworthy differences in the nature of the associations were observed. Most importantly, compared with the associations in the CAMP cohort, the direction of the SNP and haplotype associations in the NHS appear to be reversed. For SNP ApaI, the C allele is overrepresented among NHS cases (49.9 versus 44.6%, p = 0.03), appearing to confer asthma risk, whereas this same allele is undertransmitted to affected asthmatic offspring in CAMP (thereby appearing protective). This difference is also observed at the haplotype level, with the CCT haplotype conferring risk in the NHS cohort, while undertransmitted in CAMP cohort. Of note, the direction of these SNP and haplotype associations in the NHS cohort is consistent with those observed by Poon and colleagues, who observe strong SNP and haplotype associations with asthma and atopy in asthma families from northeastern Quebec (17). Together these results suggest that although the VDR locus harbors variants that contribute to the development of asthma and allergy, the relationships are complex.


   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The results presented here provide independent replication of association of genetic variation at the VDR locus with asthma and related phenotypes in two cohorts. The results are similar to those observed in a family-based study in a French–Canadian founder population (17), and together suggest that VDR has a role in the development of asthma and allergy. This is in keeping with the growing body of evidence that VDR and its ligand, 1{alpha},25-dihydroxyvitamin D3 [1,25-(OH)2D3], are important mediators of immunity and play a central role in the differentiation of naive T lymphocytes toward a helper T cell Type 2 (Th2; allergy-related) lineage. Experimental data suggest that 1,25-(OH)2D3 inhibits macrophage production of interleukin-12 (3), inhibits peripheral blood lymphocyte production of interferon-{gamma} (44), and suppresses helper T cell Type 1 (Th1) cytokine production while preserving Th2 cytokine expression, including interleukin-4 (4), thus favoring an allergic cytokine profile. VDR is expressed in activated T lymphocytes (45, 46), and splenocytes from mice deficient in VDR demonstrate a Th2 cytokine profile (reduced interferon-{gamma}, increased interleukin-4), likely in part due to a shift in T cell lineage expansion from Th1 to Th2, supporting a critical role for VDR in immune regulation (5). Finally, circumstantial epidemiologic evidence suggests that the increase in allergic disease in developed nations may be due in part to vitamin D fortification of maternal and newborn diets (47), although more direct evaluation of this hypothesis is necessary.

The VDR associations observed in the CAMP cohort appear to be most pronounced in girls. Sex affects many important features of asthma and allergy, including lung growth and development (48, 49), age of symptom onset, and disease severity (50–52). There is evidence from twin studies that sex influences the heritability of asthma (53). Modification of genotype–phenotype correlation by sex is well documented, including the sex-specific effects of VDR polymorphisms on bone mineral density and skeletal growth (54, 55). However, sex also appears to influence the effects of VDR polymorphisms on other immune-mediated disease phenotypes, including Type 1 diabetes mellitus (10). These effects may be hormonally mediated, and the effects of VDR on the development of allergy and asthma also may be mediated in a similar fashion. In this study, the case–control population studied was composed of women only. Although positive associations were observed in this cohort as well, replicating the observations of VDR SNP association with asthma in women, we were not able to evaluate the role of VDR in asthma susceptibility in a second cohort of men, thereby precluding any further investigation for sex-specific effects in this study.

An important discrepancy in our findings is the direction of asthma risk conferred by given VDR alleles in the CAMP cohort compared with those observed in the NHS cohort (and the Quebec cohort). Whereas the ApaI C allele seems to confer increased risk in both the Quebec and NHS cohorts (the C allele is over-transmitted to affected offspring in the Quebec cohort and is overrepresented in NHS asthma cases compared with control subjects), it is undertransmitted to affected offspring and thus appears protective in CAMP. Conflicting results such as these are unfortunately common in genetic association studies (56, 57). In one meta-analysis, reports of significant allelic effects in a direction opposite to the original report of association were observed in 11 of 25 gene associations examined (57). Moreover, similar contradictory results have been observed in studies of the vitamin D receptor and its role as a determinant of bone mineral density (25, 54, 58, 59) and susceptibility to tuberculosis (reviewed by Poon and coworkers [17]). Although these outlier studies may be due to chance and publication bias (of positive results, regardless of direction of association), it is possible that both sets of associations are real and are dependent on other important (and unrecognized) covariates or variants.

There are differences between these cohorts that could explain these discrepant results. Although differences in the genetic composition of these cohorts are possible, this is an unlikely explanation for the observed results, for several reasons. Although the Quebec cohort represents a more homogeneous population (and may be genetically distinct), the associations observed in that cohort also were seen in the more heterogeneous NHS cohort. In addition, the allele frequencies, linkage disequilibrium patterns, and haplotype distributions across the three populations are similar, and the significant associations with common variants were seen in all three cohorts. Finally, although Poon and colleagues resequenced the coding regions of VDR, they were not able to identify additional variants that could explain their associations. However, we are not at this time able to conclude which (if any) of the genotyped variants are disease-causing, or whether these are in linkage disequilibrium with the true disease susceptibility variants.

Phenotypic heterogeneity and differing ascertainment schema are another explanation for the discrepancy. The most obvious difference between the CAMP and NHS (and Quebec) cohorts is the age of the populations: CAMP consisted only of children, NHS consisted of adults (with reported symptom onset in adulthood), and the Quebec cohort was mixed. It is difficult to speculate how age of cohort ascertainment might affect the results, although age-dependent effects may arise if there are important gene–hormone interactions, as proposed above to explain the observed sex-specific effects. It is also conceivable that VDR interacts with environmental exposures in a time-dependent manner to affect disease expression. Such factors may include determinants of circulating vitamin D levels (including sunlight exposure and milk intake) or exposure to atypical mycobacteria.

Although these results support a role for VDR in the development of asthma and allergy, it is evident that the precise relationship between VDR polymorphisms and asthma development remains unclear. Moreover, it is likely that variation at this locus is not the only asthma susceptibility gene on chromosome 12q. As described above, chromosome 12q is among the most frequently replicated regions of asthma linkage, with more than 10 groups reporting evidence of linkage with either asthma- or allergy-related phenotypes (reviewed in Reference 14). Two broad peaks of linkage have been identified, one centromeric (centered around interferon-{gamma}), the other telomeric (surrounding nitric oxide synthase-1). VDR localizes to one end of the centromeric linkage, which spans more than 30 Mb. A gene with relatively weak effects is unlikely to have been observed in linkage studies so frequently, and would not generate such broad linkage signals. Moreover, the asthma linkage to centromeric 12q observed by most groups does not include VDR (42, 60–62). We have evaluated multiple polymorphisms in six other candidate genes across the broad linkage peak, but have not identified other associated variants. Although the positional candidate approach has helped in identifying VDR, we suspect that a more comprehensive screen of the region, using high-density linkage disequilibrium mapping, is required to localize the other asthma genes on chromosome 12q.


   Acknowledgments
 
The authors thank all families for their enthusiastic participation in the CAMP Genetics Ancillary Study. The authors also acknowledge the CAMP investigators and research team for collection of CAMP Genetic Ancillary Study data. The authors thank Jody Senter Sylvia, Michael Hagar, and Maura Regan for assistance with sample management and genotyping. The NHS data were provided by Drs. C. Camargo, G. Colditz, and F. Speizer. Dr. Speizer reviewed the manuscript for the NHS group.


   FOOTNOTES
 
Supported by the National Institutes of Health and the National Heart, Lung, and Blood Institute (K08 HL074193, NO1-HR-16049, P50 HL67664, and T32 HL07427). B.A.R. is a recipient of a Clinician Scientist Award from the Canadian Institutes of Health Research (MC1-40745).

This article is a companion article to Poon AH, Laprise C, Lemire M, Montpetit A, Sinnett D, Schurr E, Hudson TJ. Association of vitamin D receptor genetic variants with susceptibility to asthma and atopy (Am J Respir Crit Care Med 2004;170:967–973). This article appeared in the November 1 issue of the Journal. It may be accessed at http://dx.doi.org/10.1164/rccm.200403-412OC.

Conflict of Interest Statement: B.A.R. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; R.L. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; E.K.S. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; S.L. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; C.L. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; M.W. served as a speaker at scientific meetings of GlaxoSmithKline, Wyeth, Boehringer, and Roche and has been doing joint research projects with Sequenom and Illumina; S.T.W. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript.

Received in original form April 2, 2004; accepted in final form July 27, 2004


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