Vitamin D receptor gene polymorphism and ulcerative colitis susceptibility in Han Chinese

Authors

  • Feng Hua PEI,

    1. Department of Gastroenterology, The First Hospital of Jilin University
    2. Department of Gastroenterology, The Second Affiliated Hospital of Harbin Medical University, Harbin, Heilongjiang Province, China
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  • Yan Jun WANG,

    1. Department of Endocrinology, The Second Hospital of Jilin University, Changchun, Jilin Province
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  • Shan Ling GAO,

    1. Department of Gastroenterology, The Second Affiliated Hospital of Harbin Medical University, Harbin, Heilongjiang Province, China
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  • Bing Rong LIU,

    1. Department of Gastroenterology, The Second Affiliated Hospital of Harbin Medical University, Harbin, Heilongjiang Province, China
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  • Ya Ju DU,

    1. Department of Gastroenterology, The Second Affiliated Hospital of Harbin Medical University, Harbin, Heilongjiang Province, China
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  • Wei LIU,

    1. Department of Gastroenterology, The Second Affiliated Hospital of Harbin Medical University, Harbin, Heilongjiang Province, China
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  • Hong Ying YU,

    1. Department of Gastroenterology, The Second Affiliated Hospital of Harbin Medical University, Harbin, Heilongjiang Province, China
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  • Li Xia ZHAO,

    1. Department of Gastroenterology, The Second Affiliated Hospital of Harbin Medical University, Harbin, Heilongjiang Province, China
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  • Bao Rong CHI

    Corresponding author
    1. Department of Gastroenterology, The First Hospital of Jilin University
      Bao Rong CHI, Department of Gastroenterology, The First Hospital of Jilin University, Changchun, Jilin Province 130021, China. Email: chibr@jlu.edu.cn
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Bao Rong CHI, Department of Gastroenterology, The First Hospital of Jilin University, Changchun, Jilin Province 130021, China. Email: chibr@jlu.edu.cn

Abstract

OBJECTIVE:  Specific polymorphisms in the vitamin D receptor (VDR) gene have been associated with genetic susceptibility to inflammatory bowel disease (IBD) in different ethnic populations.

METHODS:  A total of 218 ulcerative colitis (UC) patients and 251 healthy controls were genotyped for VDR gene polymorphisms using PCR-restriction fragment length polymorphism (PCR-RFLP) assay. VDR gene polymorphisms (Apa I, Taq I, Bsm I and Fok I) were analyzed for both genotypic and phenotypic susceptibilities.

RESULTS:  Among the four examined VDR gene polymorphisms, the Bsm I polymorphism showed a slightly higher distribution in our study population than that in the previous studies. We also found that the increased frequency of the Bb genotype of the Bsm I VDR gene polymorphism was associated with UC in Han Chinese, as compared with healthy controls (28.4% vs. 18.7%, χ2 = 6.044, P = 0.014, OR = 1.739, 95% CI = 1.122–2.697). Moreover, Bsm I polymorphic allele (B) frequency was significantly increased in the UC cases, as compared to the healthy controls (14.7% vs. 7.8% χ2 = 6.222, P = 0.013; OR = 1.670, 95% CI = 1.113–2.506). In contrast, the other three VDR gene polymorphisms (Apa I, Taq I and Fok I) were not associated with UC susceptibility in the Han Chinese cohort. In addition, none of these four VDR polymorphisms had statistical association with clinicopathological parameters of these UC patients.

CONCLUSION:  This study demonstrated a probable association of the Bsm I polymorphism of the VDR gene with ulcerative colitis susceptibility in Han Chinese.

INTRODUCTION

Inflammatory bowel disease (IBD) can be divided into two major types, ulcerative colitis (UC) and Crohn's disease (CD), upon the basis of clinical features and histopathology. The exact etiology of IBD remains to be defined; however, IBD development could be affected by multiple factors, including genetic, environmental, and immunological factors.1

Epidemiological studies have suggested that genetic susceptibility is a major contributing factor to IBD development. Both UC and CD are considered to be complex polygenic diseases. Gene-linkage studies have identified several susceptibility genes located in certain regions of the chromosomes, such as IBD 1 to IBD 9.2,3 Among them, NOD2/CARD15 within the IBD 1 locus located at chromosome 16 has been identified as a susceptibility gene for CD,4,5 while IBD2 (VDR, STAT6, interferon γ, and β7 integrin) located on chromosome 12 appears to be a major contributor to UC susceptibility.6 Recently, vitamin D receptor (VDR) gene has gained increased attention from researchers and clinicians7 as it is capable of mediating immune responses and calcium homeostasis in the human body.8,9 The activated form of vitamin D, 1,25-dihydroxyvitamin D3[1,25(OH)2D3], has been shown to be involved in a number of activities, such as regulation of blood calcium and phosphate concentration, whereby it promotes healthy mineralization, growth and remodeling of bone, and prevention of hypocalcemia. Vitamin D also possesses important functions for regulation of cell growth and differentiation, normal immune functions, cardiovascular and mental health.8–10 However, VDR is known to mediate these effects of vitamin D and several genetic variations of VDR genes have been identified.11

The VDR gene is located on chromosome 12 and its encoded protein is a member of the nuclear receptor superfamily. Upon activation by vitamin D, the VDR forms a heterodimer with the retinoid-X receptor and binds to hormone response elements on DNA resulting in expression or trans-repression of target genes. VDR is differentially expressed among different human cells, including those of the immune system: monocytes and activated B and T lymphocytes. 1,25(OH)2D3 is synthesized and released by activated macrophages, where it acts locally as a cytokine, defending the body against microbial invaders; specifically, this form of vitamin D can inhibit nuclear factor (NF)-κB and lead to production of a variety of different cytokines, such as interleukin-2 (IL-2), IL-12 and interferon-γ (IFN-γ).12 VDR-knockout mouse studies showed that loss of VDR expression resulted in perturbed inflammation reactions in the gastrointestinal tract and indicated a critical role for VDR in the control of innate immunity and in the response of colonic tissues to chemical injury.6,9,13–15 Vitamin D deficiency and VDR defects have been shown to exacerbate chronic IBD in IL-10-knockout mice, as evidenced by extreme susceptibility of the gut to chemical injury. Moreover, Kong et al.16 investigated the role of VDR in mucosal barrier homeostasis using the dextran sulfate sodium (DSS)-induced colitis mouse model and found that vitamin D deficiency induced damage in the mucosal barrier, leading to increased susceptibility to IBD risk.

Previous studies have also identified several gene polymorphisms in the VDR gene by using a restrictive enzyme digestion strategy. Those polymorphisms include two single nucleotide polymorphisms (SNPs) located in exon 8 (Taq I and Bsm I), one in exon 9 (Apa I) and one in exon 2 (Fok I).17,18 Further studies demonstrated that the Fok I polymorphism was associated with bone mineral density and osteoporosis,19 while the Taq I was associated with development of prostate cancer.20 In contrast, the homozygote of the Taq I t allele has been demonstrated to contribute to altered susceptibility to a variety of infectious diseases,21 while the Fok I polymorphism was associated with UC and CD susceptibility in an Iranian population.22 The Bsm I polymorphism was associated with increased susceptibility to UC in Jewish Ashkenazi patients,23 and the Taq I homozygote was associated with CD susceptibility.24 However, these kinds of data are lacking among the Han Chinese. Therefore, we conducted a study to determine the significance of the Apa I, Taq I, Bsm I and Fok I polymorphisms of the VDR gene with UC in a northern Han Chinese patient population. These data will likely contribute to our understanding of the heterogeneity of UC in terms of geography, age of onset, clinical course, and predicted response to conventional treatments.

MATERIALS AND METHODS

Study subjects

Cases

Two hundred and eighteen Han Chinese patients with UC were recruited from Harbin Medical University- and Jilin University-affiliated hospitals, during a 3-year period (2008–2010). Among the cases, 64.7% were males and 35.3% were females. The diagnosis of UC was verified on the basis of well-established clinical, endoscopical, radiological and histopathological criteria by the cooperation group IBD of the Chinese Medical Association Digestive Branch in 2007.25 The UC phenotype was determined according to the disease extension, disease type, disease scale division and age of onset.

Controls

Two hundred and fifty-one healthy subjects who matched the UC cases in terms of age, ethnicity, or residency locations were recruited from these same hospitals whereupon they were subjected to routine health check-ups. The control cohort was comprised of 55.6% males. These subjects were verified by endoscopy to be free of any bowel diseases, including IBD, colon carcinoma, colonic polyp, and tuberculosis of intestine. Informed consent, blood samples, and clinicopathological data were collected from all the subjects according to a protocol that was approved by the Institutional Review Board of the Ethics Committee.

DNA extraction

Genomic DNA was extracted from ethylenediaminetetraacetic acid (EDTA) or trisodium citrate anticoagulant peripheral blood samples using a Blood DNA Kit (Tiangen, Beijing, China) and then stored at −20°C until use.

Primer design

The primers to amplify the VDR gene were designed using published VDR sequences (Homo sapiens vitamin D receptor on chromosome 12, NG_008731) and synthesized by Shanghai Chaoshi Co. (Shanghai, China). (Table 1)

Table 1.  The primers of vitamin D receptor (VDR) and band site of genotype
SNPRestriction enzymePrimersFragment size (bp)Band site of genotype (bp)
  1. SNP, single nucleotide polymorphism.

rs7975232Apa I5′-CAGAGCATGGACAGGGAGCAAG-3′
5′-GCAACTCCTCATGGCTGACTGCTCA-3′
745AA: 745
Aa: 745, 528, 217
aa: 528, 217
rs731236Taq ITT: 452, 251
Tt: 452, 293, 251, 201
tt: 293, 251, 201
rs1544410Bsm I5′-TGCCCTTTCTCCACATTCAT-3′
5′-CAAAACGACCCCAAATGACT-3′
512BB: 512
Bb: 512, 315, 197
bb: 315, 197
rs10735810Fok I5′-AGCTGGCCCTGGCACTGACTCTGGCT-3′
5′-ATGGAAACACCTTGCTTCTTCTCCCTC-3′
267FF: 267
Ff: 267, 197, 70
ff: 197, 70

PCR amplification

Genomic DNA from the cases and controls was subjected to PCR analysis of the VDR gene and then restriction enzyme digestion to identify the particular SNPs harbored in each individual's VDR gene. For PCR, 20 µL of reaction mixtures were used, which consisted of 2 µL 10 × PCR buffer (750 mmol/L Tris-Cl, pH 8.8; 200 mmol/L (NH4)2SO4; 0.1% Tween 20), 2 µL MgCl2 (25 mmol/L), 2 µL dNTP (2.5 mmol/L), 1 µL each of forward and reverse primers (10 pmol/µL), 1–2 µg DNA, and 1 µL Taq DNA polymerase (5 U; Fermentas, Burlington, Canada). The PCR amplification was carried out on a BIOER XP cycler with the following cycling parameters. For Apa I and Taq I digestion, the PCR conditions included an initial 94°C for 5 min followed by 35 cycles of 94°C for 45 s, 60°C for 45 s, and 72°C for 60 s and a final extension at 72°C for 10 min. For Bsm I digestion, the PCR conditions included an initial 94°C for 5 min followed by 32 cycles of 94°C for 30 s, 51°C for 30 s, and 72°C for 30 s and a final extension at 72°C for 10 min. For Fok I digestion, the PCR condition was an initial 94°C for 5 min followed by 35 cycles of 94°C for 30 s, 63°C for 30 s, and 72°C for 45 s and a final extension at 72°C for 10 min.

Restriction fragment length polymorphism analysis

After PCR amplification of the VDR gene from blood samples, the PCR products were subjected to restriction fragment length polymorphism (RFLP) analysis. Briefly, the restriction enzyme digestion set for each 20 µL reaction mixture, consisted of 10 µL PCR products, 2 µL 10 × buffer, and enzyme (10 units Apa I, Taq I, Bsm I, or Fok I; Fermentas or New England Biolabs, Ipswich, MA, USA) and incubated at 37°C for 16 h for Apa I, Bsm I, and Fok I digestion and at 65°C for Taq I digestion. After that, the DNA products were electrophoresed at 100 V through 2.0% agarose gels containing 0.5 µg/mL ethidium bromide for approximately 25 min, and then visualized and photographed under UV illumination. The different VDR genotypes were determined by the number of bands generated from the PCR digestion (Table 1).

Statistical analysis

A χ2 test or Fisher's exact test was performed to compare the distributions of genotypic frequency between the cases and controls. The Hardy–Weinberg equilibrium was performed for cases and controls and differential testing was carried out by using χ2 analysis. The odds ratios with 95% confidence interval (CI) were calculated. Statistical significance was defined as P < 0.05. All analyses were performed by using SPSS 13.0 analytical software (SPSS Inc., Chicago, IL, USA).

RESULTS

The case and control groups were well balanced in terms of age and gender. Specifically, the mean age (±standard deviation) of healthy controls and UC patients was 41.56 ± 10.03 years (range 15–72 years) and 39.41 ± 10.37 years (range 17–72 years), respectively (t = 1.627, P = 0.105). Male to female ratio was 1.3:1 and 1.8:1, respectively. The clinical characteristics of UC patients are summarized in Table 2. The dominating disease extension of UC was proctosigmoiditis (27.5%). The type was chronic recidivation (90.8%) and the disease scale division was midrange (51.4%). There was no statistically significant deviation from the expected Hardy–Weinberg equilibrium between the cases and controls for all of the four polymorphisms. (Table 3)

Table 2.  Clinical characteristics of patients with ulcerative colitis
Clinical characteristicsn (%)
Male gender141 (64.7)
Disease extension 
 Proctitis55 (25.2)
 Proctosigmoiditis60 (27.5)
 Left-sided colitis54 (24.8)
 Extensive colitis49 (22.5)
Disease type 
 Initial11 (5.0)
 Chronic continuation9 (4.1)
 Chronic recidivation198 (90.8)
Disease scale division 
 Light14 (6.4)
 Midrange112 (51.4)
 Severe92 (42.2)
Age of onset 
 ≤40 years128 (58.7)
 >40 years90 (41.3)
Table 3.  Hardy–Weinberg equilibrium of vitamin D receptor (VDR) gene polymorphisms
Polymorphism siteGroupnGenotypeHardy-Weinberg equilibrium
χ2P-value
Apa I  AAAaaa  
Control250321061120.7630.383
Case21220106862.4420.118
Taq I  TTTttt  
Control2482202800.8880.346
Case2121822820.6080.435
Bsm I  BBBbbb  
Control2460462002.6170.106
Case2111601503.8120.051
Fok I  FFFfff  
Control25080130401.1330.287
Case21860110480.0330.857

We determined VDR gene SNP distributions by using RFLP assay. Table 4 illustrates the genotypic frequencies of VDR gene in cases and controls. As shown, the heterozygote for the Bsm I (Bb) genotype appeared significantly more frequently in patients with UC than in the controls (28.4% vs. 18.7%, χ2 = 6.044, P = 0.014, OR = 1.739, 95% CI = 1.122–2.697), while the difference in frequency between other homozygote or heterozygote genotypes had not reached statistical significance. RFLP gel illustrations are shown in Figures 1–4.

Table 4.  Restriction enzyme detection of single nucleotide polymorphisms (SNPs) of vitamin D receptor (VDR) polymorphisms and ulcerative colitis (UC) risk
PolymorphismGenotypeCases
n (%)
Controls
n (%)
Odds ratio95% confidence intervalχ2P-value
  • The frequency of the cases compared to the frequency of the controls.

  • The bolded font indicates P-values <0.05.

  • NC, not calculated.

Apa IAA20 (9.4)32 (12.8)1.000 1.3010.254
Aa106 (50.0)106 (42.4)0.4310.185–1.0072.6690.102
aa86 (40.6)112 (44.8)0.6200.265–1.4530.8400.359
No PCR product61    
Taq ITT182 (85.8)220 (88.7)1.000 0.8490.357
Tt28 (13.2)28 (11.3)1.2090.691–2.1150.3930.531
tt2 (0.9)0 (0)NCNC0.6760.411
Tt + tt30281.2950.746–2.2470.8490.357
No PCR product63    
Bsm Ibb150 (71.1)200 (81.3)1.000 6.6040.010
Bb60 (28.4)46 (18.7)1.7391.122–2.6976.0440.014
BB1 (0.5)0 (0)NCNC0.0060.939
BB + Bb61461.7681.412–2.7386.6040.010
No PCR product75    
Fok IFF60 (27.5)80 (32.0)1.000 2.9780.291
Ff110 (50.5)130 (52.0)0.7980.435–1.4620.1110.739
ff48 (22.0)40 (16.0)0.4750.221–1.0192.7630.096
No PCR product01    
Total 218251    
Figure 1.

Restriction fragment length polymorphism with Apa I. Genomic DNA from the case and control was amplified with vitamin D receptor (VDR) gene primers and digested with Apa I, then separated on 2% agarose gel. PP, PCR product.

Figure 2.

Restriction fragment length polymorphism with Taq I. Genomic DNA from the case and control was amplified with vitamin D receptor (VDR) gene primers and digested with Taq I, then separated on 2% agarose gel. PP, PCR product.

Figure 3.

Restriction fragment length polymorphism (RFLP) with Bsm I. Genomic DNA from the case and control was amplified with vitamin D receptor (VDR) gene primers and digested with Bsm I, then separated on 2% agarose gel. PP, PCR product.

Figure 4.

Restriction fragment length polymorphism (RFLP) with Fok I. Genomic DNA from the case and control was amplified with vitamin D receptor (VDR) gene primers and digested with Fok I, then separated on 2% agarose gels. PCR Product: 1; FF genotype: 2, 5, 7, 10, 17,19, 24; Ff genotype: 3, 4, 6, 8, 9, 11, 12, 13, 14, 15, 16, 18, 20, 21, 23; ff genotype: 22.

The allelic frequencies of the VDR gene polymorphisms are presented in Table 5. The frequency of Bsm I polymorphic allele (B) was found to be significantly higher in the cases than in the controls (14.7% vs. 7.8%, χ2 = 6.222, P = 0.013; OR = 1.670, 95% CI = 1.113–2.506), while those of Apa I, Taq I and Fok I alleles did not exhibit any significant association with UC.

Table 5.  Vitamin D receptor allele frequencies in Han Chinese
AlleleCases n (%)Controls n (%)Odds ratio95% confidence intervalχ2P-value
  1. The bolded font indicates P-values <0.05.

A146 (34.4)170 (34.0)1.000   
a278 (65.6)330 (66.0)1.0190.776–1.3390.0190.890
T392 (92.5)468 (94.4)1.000   
t32 (7.5)28 (5.6)0.7330.434–1.2391.3560.244
B62 (14.7)46 (7.8)1.000   
b360 (85.3)446 (92.2)1.6701.113–2.5066.2220.013
F230 (52.8)290 (58.0)1.000   
f206 (47.2)210 (42.0)0.8090.624–1.0472.5980.107

However, we did not find any statistically significant differences of clinical parameters of the UC patients with VDR gene polymorphisms. Table 6 showed an example of the Bsm I polymorphism (other data not shown).

Table 6.  Clinical characteristics and vitamin D receptor (VDR) Bsm I polymorphism of ulcerative colitis (UC) patients
Clinical characteristicsnBB
n (%)
Bb
n (%)
bb
n (%)
χ2P-value
Gender    3.7420.154
 Male1391 (0.7)45 (32.4)93 (66.9)  
 Female720 (0.0)15 (20.8)57 (79.2)  
Disease extension    4.3050.636
 Proctitis530 (0.0)12 (22.6)41 (77.4)  
 Proctosigmoiditis581 (1.7)18 (31.0)39 (67.2)  
 Left-sided colitis520 (0.0)17 (32.7)35 (67.3)  
 Extensive colitis480 (0.0)13 (27.1)35 (72.9)  
Disease type    0.8060.938
 Initial100 (0.0)4 (40.0)6 (60.0)  
 Chronic continuation80 (0.0)2 (25.0)6 (75.0)  
 Chronic recidivation1931 (0.5)54 (28.0)138 (71.5)  
Disease scale division    1.4960.827
 Light120 (0.0)4 (33.3)8 (66.7)  
 Midrange1070 (0.0)31 (29.0)76 (71.0)  
 Severe921 (1.1)25 (27.2)66 (71.7)  
Age of onset    2.3220.313
 ≤40 years1220 (0.0)38 (31.1)84 (68.9)  
 >40 years891 (1.1)22 (24.7)66 (74.2)  
Total2111 (0.5)60 (28.4)150 (71.1)  

DISCUSSION

Previous studies have identified an association between IBD and VDR gene polymorphisms in different ethnic populations, such as Japanese and Koreans,26–28 Ashkenazi Jewish,23 Caucasian Europeans29 and African Americans.30 In the current study, we performed PCR-RFLP analysis of VDR gene polymorphisms to associate them with UC and found one polymorphism (Bsm I) of the VDR gene that was associated with UC susceptibility in a Han Chinese population among the four VDR gene polymorphisms (Apa I, Taq I, Bsm I and Fok I) after linkage analysis. In Han Chinese, this polymorphic B allele frequency (Bsm I) of the VDR gene was present in approximately 4.8% and 5.6% of the individuals, according to the two different studies performed.31,32 However, in this current study, we found a slightly higher distribution of this polymorphic B allele in the controls (7.8%). This may be due to our use of a different subpopulation of the Han Chinese from different provinces. We also found that increased frequency of the Bb genotype of the Bsm I VDR gene polymorphism was associated with UC in northern Han Chinese, as compared to that of healthy controls. In addition, the Bsm I polymorphic allele (B) frequency was found to be significantly increased in the UC cases, as compared to that of healthy controls. However, the other three VDR gene polymorphisms (Apa I, Taq I and Fok I) and their corresponding alleles (A, T and F, respectively) were not found to be associated with UC, as compared to the controls.

Our current data are in agreement with a previous study examining the Jewish population.23 In that study, Dresner-Pollak et al. found that the Bsm I VDR gene polymorphism was associated with increased susceptibility to UC in Ashkenazi Jewish patients, but not in CD patients or in UC patients with non-Ashkenazi origin. Furthermore, Martin et al.33 demonstrated increased frequency of the VDR Taq I tt genotype in UC patients from a European population; but, here, we did not observe this association in Han Chinese patients with UC. Naderi et al.34 reported that only the frequency of the Fok I polymorphism was significantly higher in Iranian patients with UC. These discrepant findings emphasize the importance of VDR gene polymorphisms in different populations and ethnicities.

The mechanism by which the VDR gene polymorphism affects the susceptibility to IBD has not been yet defined. Previous studies showed that the Bsm I site is located at the 3′ end of the VDR gene, which is in mutual tight linkage disequilibrium with the other polymorphisms, such as Taq I site.11,31,35,36 Although this polymorphism has yet to yield any evidence to support a functional effect of this polymorphism on alteration of VDR activity,37 the 3′-end polymorphisms, including Bsm I and Taq I, may affect messenger RNA stability and VDR gene transcription regulation.11,38In vitro functional study had demonstrated that the baT (Bsm I/Apa I/Taq I) haplotype mediated a lower reporter gene activity than Bat,39 and that the baT haplotype was associated with low VDR messenger RNA levels.40 These data, together with ours, indicate the importance of VDR in regulation of UC risk. In addition, previous studies also found that Vitamin D and VDR deficiency can result in accelerated IBD,41,42 and the VDR deficiency further prohibits Vitamin D absorption.43–45

In addition, VDR has been shown to affect cell proliferation and differentiation, each of which is likely to play a role in IBD.46 Previous studies demonstrated that VDR was able to arrest cells in the G0/G1 cell cycle phase, and was associated with upregulation of a number of cell cycle inhibitors, including p21 (waf1/cip1) and p27 (kip1).47–49 Expression of VDR was also associated with elevated expression of certain brush-border-associated enzymes, such as alkaline phosphatase, intermediate filaments, vinculin, desmosomes, and E-cadherin, resulting in cell adhesion and inhibition of cell migration.50 Other data linked VDR polymorphisms (Bsm I or Fok I) with risk of breast cancer,51 prostate cancer,20 and malignant melanoma.52

In summary, our current study suggests a relatively weak link between VDR gene polymorphisms and the susceptibility to IBD, and the latter is probably mediated by the combined effects of multiple polymorphic genes, each having only a relatively modest effect for interactions with environmental factors. Further study will investigate how the VDR gene polymorphisms link to IBD or cancer and whether and how the VDR Bsm I polymorphism affects VDR protein expression in UC.

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