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Association study between vitamin D receptor gene polymorphisms and asthma in the chinese han population: a case-control study
© Saadi et al; licensee BioMed Central Ltd. 2009
Received: 05 March 2009
Accepted: 21 July 2009
Published: 21 July 2009
Modulation of the immune system is one of the principal roles of Vitamin D, for which the effects are exerted via the vitamin D receptor (VDR). Importantly, variants in the VDR gene have been susceptible in the past to raise the risk of asthma in several populations. These effects of VDR allelic markers remain speculative in the Chinese Han population.
A case-control study of 1090 individuals including 567 asthmatic patients was realized on five SNPs within the VDR gene. Only rs7975232 (ApaI) marker showed a significant association with asthma (P = 0.009). Haplotype analysis of the five VDR polymorphisms showed a significant association with asthma (global-p value = 0.012).
Although the susceptibility of VDR gene variants with asthma could not be confirmed for all SNPs tested in this study, the significant association obtained for rs7975232 provides evidence for a previously unknown report about the Chinese Han population and may raise the susceptibility of VDR to be a candidate gene for asthma.
Recent discoveries made in the field of molecular biology have reported that vitamin D derived from diet and supplements is not a vitamin, but a steroid with immunosuppressive properties when elevated . It has also been linked with lessening symptoms of autoimmune diseases like multiple sclerosis , Crohn's disease , and rheumatoid arthritis .
However, the effects of this steroid-like molecule are essentially exerted via its cognate the vitamin D receptor (VDR). In addition, molecular modeling findings have defined an interaction between the hormonal 1, 25-dihydroxyvitamin D3 form and its receptor through a ligand-binding domain (LBD) . The activated VDR plays then a fundamental role in the body by regulating numerous primary target genes. Three years before, a Canadian study has identified on a large scale that VDR may transcribe more than 900 genes . In fact, the interaction of 1, 25(OH)2D3 with VDR and its cofactors modulates many biological activities of the neural, immune, and endocrine systems; including calcium and phosphorous homeostasis, apoptosis and cell differentiation. For instance, a previous study has demonstrated that VDR rs7975232 polymorphism is associated with hip bone mineral density (BMD) in a group of 260 healthy postmenopausal Chinese Han women .
Besides to this classical action, and due to the pleiotropic effect the 1, 25(OH)2D3-VDR complex exerts, its genetic variants have been found to be associated with a variety of diseases/phenotypes, in which the risk can often depend on inter-individual variability or genetic differences within VDR protein; such like the heterogeneous phenotype of asthma. In fact, since VDR is located within the region q13-26 of chromosome 12 previously linked to asthma or related phenotypes in different populations [8–12], it is considered to be one of the candidate genes of asthma. A number of association studies were previously conducted in different populations and ethnic groups. Two of them have suggested associations among VDR polymorphisms and asthma. The first one was a family-based study on a founder French population from northeastern Quebec, accompanied by a second Canadian study, using both the childhood asthma management network as family-based study and the Healthy women from European ancestry as a case-control study [13, 14]. Another research among a German population has also tested the same hypothesis, however, there was no preferential transmission of VDR variants to children with asthma . Assays were also done on animal models, where an experimental allergic asthma was induced in VDR knockout and wild-type (WT) mice. As expected, WT mice developed symptoms of airway inflammation with an influx of eosinophils, elevated Th2 cytokine levels, mucous production, and airway hyperresponsiveness .
Thus, we sought to replicate the association previously described between the VDR gene and asthma among a Chinese Han population. Five Single nucleotide polymorphisms (SNPs) were selected, genotyped and statistically analyzed in a case-control study including 1090 Chinese individuals. Our results demonstrated that VDR rs7975232 (ApaI) variant was associated with asthma susceptibility in the cohort studied.
Baseline characteristics of the asthmatic patients with asthma and controls.
Patients(n = 567)
Controls (n = 523)
Samples origins and
Weifang (n = 424) 195/229
SPH (n = 308) 217/91
Qilu Hospital(n = 143) 58/85
Qilu Hospital (n = 215) 143/72
Age: mean (range)
FEV1, % predicted
56.48 ± 16.8
85.98 ± 12.2
Changes in FEV1 by bronchodilator, %
28.28 ± 14.6
4.38 ± 3.9
Total frequency of females
Total frequency of males
SNPs Selection and Genotyping
Different loci selected within the vitamin D receptor sequence and their corresponding primers used during PCR-RFLP method.
PCR Product (bp)
R: 5'-GGCTCCCTTCATGGA AACACCT-3'
Single nucleotide polymorphisms were assessed for both genotypic and allelic association analysis among asthma patients and healthy controls. The genotype data of all tested SNPs were then used to estimate Hardy-Weinberg equilibrium by comparison of genotype frequencies within the two groups by a χ2-test. P-values were calculated with SPSS statistical software (SPSS Statistics 17.0; 2008 SPSS Inc, Chicago, IL), where Fisher's exact test was applied to analyze the comparison of the frequencies of discrete variables between cases and controls. The odds ratios (ORs) with 95% confidence intervals (95%CI) were also calculated during the single site analysis to estimate risk of asthma associated with the VDR polymorphic genotypes. An adjustment for multiple tests by Bonferroni correction in which the P-values are multiplied by the number of comparisons was later used to control the false discovery rate. Haplotypes and their frequencies were also constructed using the online software SHEsis http://analysis.bio-x.cn/myAnalysis.php, in which differences of haplotype distribution between patients and controls is assessed by the Monte Carlo method. The differences in pairwise distribution between respective pairs of SNPs tested were assessed by the statistical measure of LD based on calculating D' using Haploview 4.1. Further, a graphical representation of different genotype data obtained with SNP rs7975232 was produced, using a powerful program for analyzing graphs (Graphical Analysis 3.4), in which the genotype frequencies of cases and controls obtained with this site were diagrammatically compared.
Genotypic and allelic association analysis of vitamin D receptor single-nucleotide polymorphisms in the Chinese asthma study.
Controls (n = 523)
Asthmatics (n = 567)
OR (95% CI)
Controls (n = 523)
Asthmatics (n = 567)
Detailed statistical analysis of VDR ApaI site in the Chinese cohort.
Five-locus haplotype analysis for transmission disequilibrium test.
This research directs an issue to test the hypothesis described in the previous association studies between VDR variants and asthma. Though the susceptibility for asthma disease could not totally be confirmed for all SNPs tested in this study, the significant result obtained with the non-coding polymorphism rs7975232 has reported previously unknown information about the relation existing between VDR gene and asthma in the Chinese Han population. This may also increase the probability of VDR to be a candidate gene for asthma.
Our results represent also a replication of the previous findings in the two U.S. studies, where SNP rs7975232 polymorphism has also shown a significant association with asthma. In the CAMP, seven VDR polymorphisms were genotyped in complete nuclear families from three different ethnic groups included in the analysis. After a stratified genetic analysis by ethnic groups, only intronic variant rs7975232 has shown a significant association with asthma (P = 0.01) . However, the NHS on 517 cases and 519 controls has shown evidence for association with asthma of four out of six VDR SNPs (rs7975232, rs731236, rs2239185, and rs3782905) . These results have demonstrated a replication of rs7975232 VDR-locus association with asthma among the Canadian women samples. However, the two polymorphisms rs731236 and rs3782905, which have reported a significant association in the NHS, have not shown any association with asthma in both Chinese population and Canadian families from the CAMP study. Elsewhere, Poon and colleagues addressed a study which involved a total of 223 independent French Northeastern Quebec families . In contrast to our result, their results have not demonstrated any association between VDR rs7975232 association and asthma; however, six other VDR variants widely studied were significantly overtransmitted to both asthmatic and atopic offspring (p < 0.05) in the French families. Another study addresses the previously described association of VDR variants with asthma and related phenotypic traits, in which 951 German individuals from 224 pedigrees were included in the analysis . Thirteen SNPs were tested and all reported no associations with asthma, including three non-significant SNPs also typed in our study (rs3782905, rs1544410 and the coding polymorphism rs731236).
Recently, researchers are following a key challenge to understand the functionality of different VDR polymorphisms, by the fact that most of these markers are synonymous. Most investigators have focused on the 3' regulatory region, because it is close to the synonymous markers tested mostly in association studies, such as BsmI, ApaI, and TaqI. The importance of N-terminal regions of the nuclear hormone receptors is increasingly recognized, especially through regulation of mRNA stability . Only several papers have tried to identify the relation existing between significant associations and functional sequence variations in the VDR gene. Although no consistent association studies support those functionality findings in asthma disease.
In summary, we identified a significant association between a genetic variant at the VDR locus and asthma in Chinese Han population. However, further studies are needed to find out the genuine functional variants in this region.
We address a special thank to both asthmatic patients and healthy subjects for their participation in the study. This work was financially supported by grants of the national Natural Science Foundation of China (No. 30300310, 30671956) for Dr. Qiji Liu, National Hightech Research and Development Program of China (No. 2006AA02A406) and Cultivation Fund of the Key Scientific and Technical Innovation Project, Ministry of Education of China (No. 704030).
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