http://informahealthcare.com/rst ISSN: 1079-9893 (print), 1532-4281 (electronic) J Recept Signal Transduct Res, Early Online: 1–6 ! 2014 Informa Healthcare USA, Inc. DOI: 10.3109/10799893.2014.975248
RESEARCH ARTICLE
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Association of vitamin D receptor gene polymorphisms with the susceptibility to ulcerative colitis in patients from Southeast China Sheng-Long Xia1, Li-Qin Yu1, Hao Chen1, Ding-Yuan Hu1, Xiao-Xiao Shao1, Mao-Dong Guo1, Li-Jia Jiang2, Xin-Xin Lin1, Xiu-Qing Lin3, and Yi Jiang1 1
Department of Gastroenterology, The Second Affiliated Hospital of Wenzhou Medical University, Wenzhou, China, 2Department of Gastroenterology, The Center Hospital of Wenzhou City, Wenzhou, China, and 3Department of Gastroenterology, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou, Zhejiang Province, China Abstract
Keywords
The association studies from different ethnic groups showed that vitamin D receptor (VDR) gene polymorphisms might be connected with the susceptibility to ulcerative colitis (UC); however, the conclusions were less consistent. Our study aimed to analyze the associations of UC with common mutations of VDR in Chinese patients. A total of 382 UC patients and 489 healthy controls were recruited. The genotypes of VDR FokI (rs2228570), BsmI (rs1544410), ApaI (rs7975232) and TaqI (rs731236) were examined by SNaPshot assays. Haplotype analysis was performed in all study subjects. After Bonferroni correction, the mutant alleles and genotypes of VDR FokI, BsmI, ApaI and TaqI did not statistically differ between UC patients and the controls (all p40.0125). However, the mutant allele C and genotype TC + CC of FokI gene were significantly increased in patients with mild and moderate UC compared to those with severe UC (C allele: 54.1% versus 39.3%, OR ¼ 1.83, 95% CI: 1.21–2.75, p ¼ 0.004; TC + CC genotype: 81.6% versus 57.1%, OR ¼ 3.32, 95% CI: 1.83–6.06, p50.001, respectively). Haplotype analysis showed that the VDR BsmI, ApaI and TaqI polymorphic loci were in a strong linkage disequilibrium. Furthermore, the frequency of AAC haplotype was statistically lower in UC patients than that in the controls (3.8 versus 5.9%, OR ¼ 0.63, 95% CI: 0.39–1.01, p ¼ 0.039). In conclusion, the mutation of FokI gene influenced severity of the disease in UC patients. Moreover, the AAC haplotype formed by the VDR BsmI, ApaI and TaqI gene might engender a reduced risk of UC attack.
Gene polymorphism, haplotype, ulcerative colitis, vitamin D receptor
Introduction As one of the main forms of inflammatory bowel diseases (IBDs), ulcerative colitis (UC) is characterized by inflammation of the mucosa and submucosa of the large intestine. Although the specific etiology of UC still remains obscure, growing evidence has corroborated the notion that UC is primarily originated from an inappropriate immune response to intestinal microbial flora in genetically susceptible hosts (1). So far, nearly 47 candidate loci affecting innate or adaptive immune response have been reported to be potentially related to UC (2). The risk of UC attributable to those known genetic factors, nevertheless, is estimated to be less than 16% (2). Therefore, the other underlying susceptibility factors involved in the pathogenesis of UC have received extensive attention. Address for correspondence: Yi Jiang, The Second Affiliated Hospital of Wenzhou Medical University, 109 Xueyuan West Road, Wenzhou 325000, Zhejiang Province, China. Tel: 086-0577-88002715. Fax: 0860577-88816191. E-mail: [email protected]
History Received 9 June 2014 Revised 30 September 2014 Accepted 7 October 2014 Published online 27 October 2014
Vitamin D receptor (VDR) is a member of steroid receptor family, which plays a pivotal role in a variety of immune responses through binding the biologically active form of vitamin D[1,25-(OH)2D]. It has been found that numerous immune cells, including monocytes, macrophages, dendritic cells and activated T lymphocytes, express VDR and vitamin D metabolizing enzymes such as 1-a-hydroxylase (3). Several studies further indicated that VDR signaling stimulated innate immune responses through facilitating production of antimicrobial proteins such as cathelicidin, or decreasing the production of chemokines and cytokines linked to NF-kB signal (4). Recent data even confirmed that VDR signaling was essential for maintaining intestinal immune homeostasis, and enhancing the antibacterial activity, as well as modulating intestinal mucosa abnormal activation of T cell response (5). In addition to many infectious disorders, VDR signaling has also been suggested to be highly correlated with a large range of autoimmune diseases, such as rheumatoid arthritis (RA), type 1 diabetes mellitus (T1DM) and multiple sclerosis (MS) (6,7). With regard to its central roles in immunological
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Table 1. Demographic characteristics of patients with ulcerative colitis (UC) and healthy controls (HC). Characteristics
UC (N ¼ 382)
HC (N ¼ 489)
p
Gender: female/Male Age (M ± SD) Age of onset (M ± SD) Smoking, N (%) Current or ex-smoker Never smoked Disease type, N (%) Initial Chronic continuation Chronic recidivation Extent of disease, N (%)a Distal colitis Extensive colitis Severity of disease, N (%) Mild Moderate Severe Treatment, N (%) SASP/5-ASA Prednisone Antibiotics Immunosuppressive Colectomy
172/210 42.11 ± 14.91 39.46 ± 14.33
225/264 40.96 ± 15.84
0.77 0.28
206 (53.9) 176 (46.1)
267 (54.6) 222 (45.4)
0.84
129 (33.8) 71 (18.6) 182 (47.6) 228 (59.7) 154 (40.3) 139 (36.4) 187 (49.0) 56 (14.6) 307 149 111 10 3
(80.4) (39.0) (29.1) (2.6) (0.8)
a
Distal colitis was defined as disease lesion distal to spleen flexure and extensive colitis was defined as disease lesion location beyond spleen flexure. SASP, sulfasalazine; 5-ASA, 5-aminosalicylic acid.
and inflammatory regulation, furthermore, the association of VDR signaling with UC has been extensively studied in recent years. Numerous studies have demonstrated that the biological functions of VDR were primarily modulated by the genetic polymorphisms of VDR. The human VDR gene is located on chromosome 12q13.11 within one of the IBD candidate regions determined by wide genome scans (8). Moreover, more than 200 single nucleotide polymorphisms (SNPs) have been identified in VDR gene. Of them, the FokI (T/C, rs2228570), BsmI (G/A, rs1544410), ApaI (C/A, rs7975232) and TaqI (T/C, rs731236) are of greatest concern. Association studies from different ethnic populations indicated that the above four SNPs of VDR gene were likely related to the predisposition of UC; however, the conclusions as a whole were lack of consistency (8–10). Therefore, we attempted to investigate the allelic and genotypic distributions of VDR FokI, BsmI, ApaI and TaqI gene in 382 UC patients and 489 healthy controls, who were recruited from Southeast China. As a result, our findings showed that the mutation of FokI gene influenced severity of the disease in UC patients. Moreover, the AAC haplotype formed by the VDR BsmI, ApaI and TaqI gene might engender a reduced risk of UC attack in this cohort of Chinese population.
Materials and methods Study population From May 2007 to December 2012, a total of 382 unrelated patients with UC were recruited from The Second Affiliated Hospital and The First Affiliated Hospital of Wenzhou Medical University as well as The Center Hospital of Wenzhou City in Zhejiang Province of Southeast China.
The diagnosis of UC was based on clinical, endoscopic, radiological and histological findings in accordance with Lennard-Jones criteria (11). The severity of UC was evaluated by the Truelove and Witts criteria (12), and the location was defined according to the Montreal classification (13). Four-hundred eighty-nine age- and sex-matched healthy controls were recruited at the Health Examination Center of The Second Affiliated Hospital of Wenzhou Medical University. Individuals with any other immunerelated diseases, including T1DM, MS and RA were excluded from the study. Demographic data of UC patients and the controls are listed in Table 1. All participants selfreported Chinese Han ancestry. The written informed consents were obtained from all study subjects. This study protocol was in line with the Declaration of Helsinki and was approved by Ethics Committees of the three hospitals mentioned above. Genomic DNA extraction and genotype analysis Genomic DNA was extracted from 3 ml EDTA anticoagulated blood samples by DNeasy Blood & Tissue kit (Qiagen GmbH, Hilden, Germany) according to the manufacturer’s instructions and then stored at 20 C pending analysis. Genotyping of VDR FokI, BsmI, ApaI and TaqI gene was performed using SNaPshot assays by ABI (Applied Biosystems, Carlsbad, CA). The details of the primers used for polymerase chain reaction (PCR) and single-base extension are listed in Table 2. The multiplex PCR was carried out in a 10 ml reaction volumes containing 1 ml DNA, 1 ml 10 PCR buffer with MgCl2 (Roche, Basel, Switzerland), 1 ml dNTPs
Associations of UC with common mutations of VDR in Chinese patients
DOI: 10.3109/10799893.2014.975248
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Table 2. Amplification and extension primers of VDR gene.
VDR gene FokI (rs2228570) BsmI (rs1544410) ApaI (rs7975232)
Amplification primer
Size Conc. (bp) (mmol/L)
182 F: 50 -GCAGCCTTCACAGGTCATAGCATT-30 R: 50 -CTGGCACTGACTCTGGCTCTGA-30 F: 50 -AGAACCATCTCTCAGGCTCCAAAGT-30 186 R: 50 -GAGTGTGCAGGCGATTCGTAGG-30 F: 50 -TACTGCTTGGAGTGCTCCTCATTGA-30 281 R: 50 -GTGCTGCCGTTGAGTGTCTGTG-30
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TaqI (rs731236)
(Promega, Madison, WI), 0.5 U FastStart Taq DNA polymerase (Roche) and a defined concentration of each amplification primer. Thermocycling was performed at 95 C for 5 min, followed by 35 cycles of 95 C for 30 s, 65 C for 30 s and 72 C for 1 min, with a final incubation at 72 C for 10 min. After the multiple amplification was completed, the PCR products were purified by shrimp alkaline phosphatase (SAP, New England Biolabs, Ipswich, MA) and exonuclease I (TaKaRa, Dalian, China). The mixture, including 10 ml PCR products, 2U Exo I and 1.5U SAP, was incubated at 37 C for 80 min and subsequently 80 C for 15 min. The SNaPshot extension reaction mixture contained 2 ml PCR purification, 1 ml SNaPshot Multiplex Mix, 1 ml 5 seq buffer and 0.2 mM of each specific primer. The cycling protocol were as follows: 96 C for 1 min, followed by 28 cycles of 96 C for 10 s, 52 C for 5 s and 60 C for 30 s. Purification was carried out by adding 1 U of SAP, followed by an incubation at 37 C for 60 min and 75 C for 15 min. Then samples were mixed with 8 ml of HiDiÔ formamide and 0.3 ml of GeneScan-120 Liz Size Standard (Applied Biosystems) and sequenced by capillary electrophoresis in a 3730xl Genetic Analyzer (Applied Biosystems). Finally, the data were analyzed by GeneMapper 4.0 (Carlsbad, CA). For quality control, 20 cases of the samples were tested repetitively by direct sequencing, and all the results were completely concordant. Statistical analysis The SPSS 17.0 software for Windows (SPSS for Windows version 17.0, Chicago, IL) was used. For each SNP, Chisquare test was employed to evaluate the Hardy–Weinberg equilibrium rule. Comparison of categorical variables (such as gender) between groups was performed by Chi-square test, and age was compared by Student’s t-test. After adjusted for age, gender and smoking, an unconditional logistic regression analysis was employed to assess the associations between the VDR FokI, BsmI, ApaI and TaqI gene polymorphisms and the overall risk of UC attack as well as the clinical features of UC patients. Odds radios (OR) and 95% confidence intervals (CIs) were applied for logistic regression analysis. The Bonferroni correction for multiple comparisons was applied, and the threshold was calculated as 0.05/4 ¼ 0.0125. Linkage disequilibrium between SNPs were determined by Haploview 4.2 software (Cambridge, MA), and haplotype analysis was further carried out by HAPLO.SCORE in R software (Rochester, MA). A two-tailed p value less than 0.05 was considered significant.
Extension primer
size Conc. (bp) (mmol/L)
0.05
50 -45T-TGCTGGCCGCCATTGCCTCC-30
65
0.05
0.1
50 -4T-GAGCCTGAGTATTGGGAATG-30
24
0.1
0.1
50 -32T-GGTGGGATTGAGCAGTGAGG-30
52
0.1
0.1
50 -17T-GCGGTCCTGGATGGCCTC-30
38
0.1
Results Relationship between UC and VDR FokI, BsmI, ApaI and TaqI gene polymorphisms In this study, we found that the genotypic distributions of VDR FokI, BsmI, ApaI and TaqI met the Hardy–Weinberg equilibrium rule in the controls (p ¼ 0.078, 0.359, 0.511 and 0.657, respectively). After Bonferroni correction, the allelic and genotypic frequencies of VDR FokI, BsmI, ApaI and TaqI were not statistically different between UC patients and the controls (all p40.0125) (Table 3). According to the severity of UC, we further stratified the patients into two subgroups. By logistic regression analysis, we found that the mutant allele C and genotype TC + CC of FokI gene were significantly increased in the patients with mild and moderate UC in contrast to those with severe UC (C allele: 54.1% versus 39.3%, OR ¼ 1.83, 95% CI: 1.21–2.75, p ¼ 0.004; TC + CC genotype: 81.6% versus 57.1%, OR ¼ 3.32, 95% CI: 1.83– 6.06, p50.001, respectively). However, the alleles and genotypes of BsmI, ApaI and TaqI were not statistically different between the two subgroups stratified by the severity of UC (all p40.0125). Besides, we also revealed that each of the four SNPs in VDR gene was not significantly associated with the locations of UC in this cohort of patients (all p40.0125; Table 4).
Analyses for linkage disequilibrium and haplotype of VDR FokI, BsmI, ApaI and TaqI gene polymorphisms We also made an analysis of linkage disequilibrium among the above four SNPs of VDR gene in this study. The BsmI, ApaI and TaqI three polymorphic loci were shown to be in a strong linkage disequilibrium (BsmI/ApaI, D0 ¼ 0.98, r2 ¼ 0.16; ApaI/TaqI, D0 ¼ 0.93, r2 ¼ 0.14; BsmI/TaqI, D0 ¼ 0.83, r2 ¼ 0.68, respectively) (Figure 1). Nevertheless, the FokI locus was not in strong linkage disequilibrium with each of the other three loci. The haplotype frequencies are listed in Table 5. Of the eight haplotypes, the frequencies simultaneously less than 3% in both UC patients and the controls were not taken into account. As a result, the GCT, GAT and AAC haplotypes were retained and analyzed. Although the frequencies of GCT (71.3%), together with GAT (21.0%), accounted for 92% of all haplotypes in the controls, neither of the two haplotypes differed significantly between UC patients and the controls (all p40.05). The AAC haplotype was shown to be statistically decreased in UC
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Table 3. Genotypic and allelic frequencies of VDR gene polymorphisms in patients with ulcerative colitis (UC) and healthy controls (HC). UC (N ¼ 382) N (%)
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VDR gene FokI (rs2228570) TT TC CC TC + CC T C BsmI (rs1544410) GG GA AA GA + AA G A ApaI (rs7975232) CC CA AA CA + AA C A TaqI (rs731236) TT TC CC TC + CC T C
HC (N ¼ 489) N (%)
OR [95% CI)]a
84 199 99 298 367 397
(22.0) (52.1) (25.9) (78.0) (48.0) (52.0)
98 263 128 391 459 519
(20.0) (53.8) (26.2) (80.0) (46.9) (53.1)
Ref 0.88 [0.63–1.25] 0.90 [0.61–1.34] 0.89 [0.64–1.23] ref 0.96 [0.79–1.16]
344 38 0 38 726 38
(90.0) (10.0) (0) (10.0) (95.0) (5.0)
423 65 1 66 911 67
(86.5) (13.3) (0.2) (13.5) (93.1) (6.9)
Ref 0.72 [0.47–1.10] NA 0.71 [0.46–1.08] Ref 0.71 [0.47–1.07]
186 174 22 196 546 218
(48.7) (45.5) (5.8) (51.3) (71.5) (28.5)
249 204 36 240 702 276
(50.9) (41.7) (7.4) (49.1) (72.8) (28.2)
Ref 1.14 [0.87–1.51] 0.82 [0.47–1.44] 1.09 [0.84–1.43] Ref 1.02 [0.82–1.25]
344 38 0 38 726 38
(90.0) (10.0) (0) (10.0) (95.0) (5.0)
424 62 3 65 910 68
(86.7) (13.7) (0.6) (13.3) (93.0) (7.0)
ref 0.76 [0.49–1.16] NA 0.72 [0.47–1.10] ref 0.70 [0.47–1.05]
pa
0.48 0.61 0.48 0.65 0.13 0.11 0.10 0.35 0.49 0.51 0.89 0.20 0.13 0.09
NA: not calculated due to the low frequencies in both groups. a Bonferroni multiple testing correction was used and the threshold was calculated as 0.05/4 ¼ 0.0125.
Table 4. The associations of VDR gene polymorphisms with clinicopathologic features of ulcerative colitis (UC). Severity of disease VDR gene
Mild/moderate (N ¼ 326) N (%)
FokI (rs2228570) TT 60 TC + CC 266 T 299 C 353 BsmI (rs1544410) GG 292 GA + AA 34 G 618 A 34 ApaI (rs7975232) CC 165 CA + AA 161 C 474 A 178 TaqI (rs731236) TT 295 TC + CC 31 T 621 C 31 a
Severea (N ¼ 56) N (%)
(18.4) (81.6) (45.9) (54.1)
24 32 68 44
(42.9) (57.1) (60.7) (39.3)
(89.6) (10.4) (94.8) (5.2)
52 4 108 4
(92.9) (7.1) (96.4) (4.6)
(50.6) (49.4) (72.7) (27.3)
21 35 72 40
(37.5) (62.5) (64.3) (35.7)
(90.5) (9.5) (95.3) (4.7)
49 7 105 7
(87.5) (12.5) (93.7) (6.3)
Extent of disease
pb
OR [95% CI]b
0.000
3.32 [1.83–6.06]
0.004
1.83 [1.21–2.75]
0.45
1.51 [0.52–4.44]
0.46
1.49 [0.52–4.27]
0.07
0.59 [0.33–1.05]
0.07
0.68 [0.44–1.03]
0.49
0.74 [0.31–1.76]
0.50
0.75 [0.32–1.75]
Distal colitis (N ¼ 228) N (%) 48 180 220 236
(21.1) (78.9) (48.3) (51.7)
36 118 147 161
(23.4) (76.6) (47.7) (52.3)
206 22 434 22
(90.4) (9.6) (95.2) (4.8)
138 16 292 16
(89.6) (10.4) (94.8) (5.2)
117 111 332 124
(51.3) (48.7) (72.8) (27.2)
69 85 214 94
(44.8) (55.2) (69.5) (30.5)
208 20 436 20
(91.2) (8.8) (95.6) (4.4)
136 18 290 18
(88.3) (11.7) (94.2) (5.8)
Serve as reference. Bonferroni multiple testing correction was used and the threshold was calculated as 0.05/4 ¼ 0.0125.
b
Extensive colitisa (N ¼ 154) N (%)
pb
OR [95% CI]b
0.59
1.14 [0.70–1.87]
0.89
0.98 [0.73–1.31]
0.81
0.92 [0.47–1.82]
0.82
0.93 [0.48–1.79]
0.21
0.77 [0.51–1.16]
0.32
0.85 [0.62–1.17]
0.35
0.73 [0.37–1.42]
0.37
0.74 [0.38–1.42]
Associations of UC with common mutations of VDR in Chinese patients
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DOI: 10.3109/10799893.2014.975248
Figure 1. Linkage disequilibrium pattern between the four SNPs of VDR gene were determined by Haploview 4.2 software. Numbers inside the boxes represent D0 value for Linkage disequilibrium. Selected region in the Linkage disequilibrium plot indicate the strong Linkage disequilibrium between the VDR BsmI (rs1544410), ApaI (rs7975232) and TaqI (rs731236) polymorphic loci.
Table 5. The frequencies of haplotypes in VDR BsmI, ApaI and TaqI genes between patients with ulcerative colitis (UC) and healthy controls (HC). haplotypes
GCT AACa GAT GAC ACC GCC AAT ACT
UC (N ¼ 764) HC (N ¼ 978)
71.5 71.3
3.8 5.9
22.4 21.0
1.2 0.6
0.0 0.1
0.0 0.4
1.2 0.8
0.0 0.0
a
p ¼ 0.039, OR ¼ 0.63, 95% CI: 0.39–1.01.
patients compared to the controls (3.8% versus 5.9%, OR ¼ 0.63, 95% CI: 0.39–1.01, p ¼ 0.039).
Discussion The genetic polymorphism of FokI is positioned at exon 2 of VDR gene, which has been identified as one of the most important functional loci in VDR gene. Previous studies demonstrated that the mutation of FokI altered the translation initiation sites of VDR gene and led to the production of a VDR protein, which was three amino acids shorter (14). Furthermore, it has been suggested that the shorter version of VDR protein was more transcriptionally active and had a greater capacity to transactivate VDR target genes, mainly resulted from its increased binding affinity with the basal transcription factor IIb (15). Recent data also indicate that FokI polymorphism could influence the behaviors of some immune cells (16), thus be probably implicated in many immune-mediated diseases, such as Grave’s disease (17) and SLE (18). In our study, there were no statistical differences in the allelic and genotypic distributions of FokI gene between UC patients and the controls, whereas the mutant allele and genotype frequencies of FokI gene were significantly higher in the patients with mild and moderate UC than those with
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severe UC. It indicated that the mutation of FokI gene decreased the severity of UC and played a protective role in this cohort of UC patients recruited from Southeast China. This finding seemed to be distinct from a previous study by Pei et al., in which the polymorphism of FokI was found not to be associated with the risk of UC as well as the clinical features of UC patients from Northern China (10). In Iranian population, moreover, Naderi et al. revealed that individuals with the mutant allele C were less susceptible to UC compared with those carrying the wild allele T of FokI gene (8). In the further genotype–phenotype analysis, nevertheless, they failed to detect any association of FokI polymorphism with the severity or location of UC in those patients (8). In addition, both Hughes et al. (9) and Simmons et al. (19) reported that the mutation of FokI gene was not markedly linked to the predisposition of UC in European Caucasian population. Furthermore, this study showed that the genetic polymorphisms of VDR BsmI, ApaI and TaqI were not connected with the susceptibility to UC as well as the clinical features of UC patients. These conclusions were somewhat different from a previous study on UC patients from Northern China, which showed that the mutation of BsmI gene might increase the overall risk of UC attack, whereas there were no significant associations of UC with the polymorphisms of ApaI and TaqI (10). However, Li et al. revealed that the mutation of BsmI gene appeared not to significantly affect the risk of UC in a total of 300 UC patients collected from both Northern and Southern China (20). We further compared the above findings with similar literature conducted by foreign scholars. As a result, we noticed that they were lack of consistency. For example, a case–control study in Iranian population showed that the mutations of VDR BsmI, ApaI and TaqI gene were not linked to the risk of UC (8). Similar conclusions were drawn from European Caucasians by Hughes et al. (9) and Pluskiewicz et al. (21). In addition, Simmons et al. (19) and Noble et al. (22) found no connection of UC with the VDR ApaI and TaqI polymorphisms in European Caucasians as well. A previous report from German population, however, showed that the genotypic distribution of TaqI gene in UC patients was statistically different from the controls (23). Another study performed by Dresner-Pollak et al. indicated that the mutation of BsmI gene engendered the increased risk of UC in Ashkenazi Jewish patients (24). Summarizing these divergent observations, we conjectured that the impacts of VDR Fok I, BsmI, ApaI and TaqI gene polymorphisms on UC were to some extent dependent on the differences of study subject’s genetic backgrounds, in addition to the study sample sizes as well as the ratio of clinical features in UC patients. In the further haplotype analysis, we noticed that the BsmI, ApaI and TaqI three polymorphic loci were in a strong linkage disequilibrium in all study subjects. Moreover, AAC haplotype formed by the three SNPs might engender a reduced risk of UC. In theory, the BsmI and ApaI are located in intron 8, and the TaqI is in exon 9 of VDR gene (25). Although a single mutation of the three SNPs can not give rise to an amino acid coding change, evidence from different ethnic groups has indicated that there is a strong linkage disequilibrium among the three polymorphic loci (26). Morrison et al., furthermore, found that AAC haplotype had 140% more excessive receptor
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activity than GCT haplotype (27). Carling T et al. reported that GCT haplotype was linked to the lower levels of VDR mRNA (28). Recent research data from Caucasians further indicated that GCT and AAC haplotypes were in linkage disequilibrium with the long and short alleles, respectively, of the poly(A) sequence in the 30 untranslated region of VDR, thereby probably associated with VDR gene transcription, mRNA stability and mRNA and protein levels (29). In summary, our study suggested that the mutation of FokI gene could affect the severity of UC. Moreover, the AAC haplotype formed by VDR BsmI, ApaI and TaqI might engender a lower risk of UC. Combined with the previous studies on the associations of VDR with IBD, the VDR signal pathway has been shown to be a potentially therapeutic target. Evidence from animal models consistently showed that treatment of 1,25-(OH)2D provided benefits in both IL-10 knockout and chemically induced colitis (30). A random, placebo-controlled trial in patients with Crohn’s disease (CD) suggested that supplementation with 1200 IU 1,25-(OH)2D insignificantly reduced the risk of relapse from 29% to 13% (p ¼ 0.06) (31). Another prospective study subsequently implicated that administration of 1,25-(OH)2D (provided by 0.5 mg alfacalidol) might decrease the disease activity of CD and improve quality of life score in a short-term at six weeks (32). Furthermore, as far as we know, few studies have been carried out on treatment of 1,25-(OH)2D in UC patients. Therefore, more high-quality randomized controlled trials are essential for evaluating the efficiency of 1,25-(OH)2D in the treatment of IBD.
Declaration of interest The authors declare no competing interests. This project was supported by grants from Zhejiang Provincial Natural Science Foundation (Grant number: LY14H030012) and Zhejiang Provincial Health Bureau (Grant number: 2014KYB157).
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10. Pei FH, Wang YJ, Gao SL, et al. Vitamin D receptor gene polymorphism and ulcerative colitis susceptibility in Han Chinese. J Dig Dis 2011;12:90–8. 11. Lennard-Jones JE. Classification of inflammatory bowel disease. Scand J Gastroenterol Suppl 1989;170:2–6; discussion 16–19. 12. Truelove SC, Witts LJ. Cortisone in ulcerative colitis; final report on a therapeutic trial. Br Med J 1955;2:1041–8. 13. Satsangi J, Silverberg MS, Vermeire S, et al. The Montreal classification of inflammatory bowel disease: controversies, consensus, and implications. Gut 2006;55:749–53. 14. Xue LN, Xu KQ, Zhang W, et al. Associations between vitamin D receptor polymorphisms and susceptibility to ulcerative colitis and Crohn’s disease: a meta-analysis. Inflamm Bowel Dis 2013;19: 54–60. 15. Ogunkolade BW, Boucher BJ, Prahl JM, et al. Vitamin D receptor (VDR) mRNA and VDR protein levels in relation to vitamin D status, insulin secretory capacity, and VDR genotype in Bangladeshi Asians. Diabetes 2002;51:2294–300. 16. van Etten E, Verlinden L, Giulietti A, et al. The vitamin D receptor gene FokI polymorphism: functional impact on the immune system. Eur J Immunol 2007;37:395–405. 17. Zhou H, Xu C, Gu M. Vitamin D receptor (VDR) gene polymorphisms and Graves’ disease: a meta-analysis. Clin Endocrinol (Oxf) 2009;70:938–45. 18. Zhou TB, Jiang ZP, Lin ZJ, et al. Association of vitamin D receptor gene polymorphism with the risk of systemic lupus erythematosus. J Recept Signal Transduct Res 2014;22:1–7. 19. Simmons JD, Mullighan C, Welsh KI, et al. Vitamin D receptor gene polymorphism: association with Crohn’s disease susceptibility. Gut 2000;47:211–14. 20. Li P, Lv H, Yang H, et al. IRF5, but not TLR4, DEFEB1, or VDR, is associated with the risk of ulcerative colitis in a Han Chinese population. Scand J Gastroenterol 2013;48:1145–51. 21. Pluskiewicz W, Zdrzalek J, Karasek D. Spine bone mineral density and VDR polymorphism in subjects with ulcerative colitis. J Bone Miner Metab 2009;27:567–73. 22. Noble CL, McCullough J, Ho W, et al. Low body mass not vitamin D receptor polymorphisms predict osteoporosis in patients with inflammatory bowel disease. Aliment Pharmacol Ther 2008;27: 588–96. 23. Martin K, Radlmayr M, Borchers R, et al. Candidate genes colocalized to linkage regions in inflammatory bowel disease. Digestion 2002;66:121–6. 24. Dresner-Pollak R, Ackerman Z, Eliakim R, et al. The BsmI vitamin D receptor gene polymorphism is associated with ulcerative colitis in Jewish Ashkenazi patients. Genet Test 2004;8:417–20. 25. Panierakis C, Goulielmos G, Mamoulakis D, et al. Vitamin D receptor gene polymorphisms and susceptibility to type 1 diabetes in Crete, Greece. Clin Immunol 2009;133:276–81. 26. Fang Y, van Meurs JBJ, d’Alesio A, et al. Promoter and 30 untranslated-region haplotypes in the vitamin D receptor gene predispose to osteoporotic fracture: the Rotterdam study. Am J Hum Genet 2005;77:807–23. 27. Morrison NA, Qi JC, Tokita A, et al. Prediction of bone density from vitamin D receptor alleles. Nature 1994;367:284–7. 28. Carling T, Rastad J, Akerstrom G, et al. Vitamin D receptor (VDR) and parathyroid hormone messenger ribonucleic acid levels correspond to polymorphic VDR alleles in human parathyroid tumors. J Clin Endocrinol Metab 1998;83:2255–9. 29. Ferrarezi DA, Bellili-Mun˜oz N, Dubois-Laforgue D, et al. Allelic variations of the vitamin D receptor (VDR) gene are associated with increased risk of coronary artery disease in type 2 diabetics: the DIABHYCAR prospective study. Diabetes Metab 2013;39: 263–70. 30. Nicholson I, Dalzell A M, El-Matary W. Vitamin D as a therapy for colitis: a systematic review. J Crohns Colitis 2012;6:405–11. 31. Jørgensen S P, Agnholt J, Glerup H, et al. Clinical trial: vitamin D3 treatment in Crohn’s disease-a randomized double-blind placebocontrolled study. Aliment Pharmacol Ther 2010;32:377–83. 32. Miheller P, Muzes G, Hritz I, et al. Comparison of the effects of 1, 25 dihydroxyvitamin D and 25 hydroxyvitamin D on bone pathology and disease activity in Crohn’s disease patients. Inflamm Bowel Dis 2009;15:1656–62.
RESEARCH ARTICLE
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Association of vitamin D receptor gene polymorphisms with the susceptibility to ulcerative colitis in patients from Southeast China Sheng-Long Xia1, Li-Qin Yu1, Hao Chen1, Ding-Yuan Hu1, Xiao-Xiao Shao1, Mao-Dong Guo1, Li-Jia Jiang2, Xin-Xin Lin1, Xiu-Qing Lin3, and Yi Jiang1 1
Department of Gastroenterology, The Second Affiliated Hospital of Wenzhou Medical University, Wenzhou, China, 2Department of Gastroenterology, The Center Hospital of Wenzhou City, Wenzhou, China, and 3Department of Gastroenterology, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou, Zhejiang Province, China Abstract
Keywords
The association studies from different ethnic groups showed that vitamin D receptor (VDR) gene polymorphisms might be connected with the susceptibility to ulcerative colitis (UC); however, the conclusions were less consistent. Our study aimed to analyze the associations of UC with common mutations of VDR in Chinese patients. A total of 382 UC patients and 489 healthy controls were recruited. The genotypes of VDR FokI (rs2228570), BsmI (rs1544410), ApaI (rs7975232) and TaqI (rs731236) were examined by SNaPshot assays. Haplotype analysis was performed in all study subjects. After Bonferroni correction, the mutant alleles and genotypes of VDR FokI, BsmI, ApaI and TaqI did not statistically differ between UC patients and the controls (all p40.0125). However, the mutant allele C and genotype TC + CC of FokI gene were significantly increased in patients with mild and moderate UC compared to those with severe UC (C allele: 54.1% versus 39.3%, OR ¼ 1.83, 95% CI: 1.21–2.75, p ¼ 0.004; TC + CC genotype: 81.6% versus 57.1%, OR ¼ 3.32, 95% CI: 1.83–6.06, p50.001, respectively). Haplotype analysis showed that the VDR BsmI, ApaI and TaqI polymorphic loci were in a strong linkage disequilibrium. Furthermore, the frequency of AAC haplotype was statistically lower in UC patients than that in the controls (3.8 versus 5.9%, OR ¼ 0.63, 95% CI: 0.39–1.01, p ¼ 0.039). In conclusion, the mutation of FokI gene influenced severity of the disease in UC patients. Moreover, the AAC haplotype formed by the VDR BsmI, ApaI and TaqI gene might engender a reduced risk of UC attack.
Gene polymorphism, haplotype, ulcerative colitis, vitamin D receptor
Introduction As one of the main forms of inflammatory bowel diseases (IBDs), ulcerative colitis (UC) is characterized by inflammation of the mucosa and submucosa of the large intestine. Although the specific etiology of UC still remains obscure, growing evidence has corroborated the notion that UC is primarily originated from an inappropriate immune response to intestinal microbial flora in genetically susceptible hosts (1). So far, nearly 47 candidate loci affecting innate or adaptive immune response have been reported to be potentially related to UC (2). The risk of UC attributable to those known genetic factors, nevertheless, is estimated to be less than 16% (2). Therefore, the other underlying susceptibility factors involved in the pathogenesis of UC have received extensive attention. Address for correspondence: Yi Jiang, The Second Affiliated Hospital of Wenzhou Medical University, 109 Xueyuan West Road, Wenzhou 325000, Zhejiang Province, China. Tel: 086-0577-88002715. Fax: 0860577-88816191. E-mail: [email protected]
History Received 9 June 2014 Revised 30 September 2014 Accepted 7 October 2014 Published online 27 October 2014
Vitamin D receptor (VDR) is a member of steroid receptor family, which plays a pivotal role in a variety of immune responses through binding the biologically active form of vitamin D[1,25-(OH)2D]. It has been found that numerous immune cells, including monocytes, macrophages, dendritic cells and activated T lymphocytes, express VDR and vitamin D metabolizing enzymes such as 1-a-hydroxylase (3). Several studies further indicated that VDR signaling stimulated innate immune responses through facilitating production of antimicrobial proteins such as cathelicidin, or decreasing the production of chemokines and cytokines linked to NF-kB signal (4). Recent data even confirmed that VDR signaling was essential for maintaining intestinal immune homeostasis, and enhancing the antibacterial activity, as well as modulating intestinal mucosa abnormal activation of T cell response (5). In addition to many infectious disorders, VDR signaling has also been suggested to be highly correlated with a large range of autoimmune diseases, such as rheumatoid arthritis (RA), type 1 diabetes mellitus (T1DM) and multiple sclerosis (MS) (6,7). With regard to its central roles in immunological
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Table 1. Demographic characteristics of patients with ulcerative colitis (UC) and healthy controls (HC). Characteristics
UC (N ¼ 382)
HC (N ¼ 489)
p
Gender: female/Male Age (M ± SD) Age of onset (M ± SD) Smoking, N (%) Current or ex-smoker Never smoked Disease type, N (%) Initial Chronic continuation Chronic recidivation Extent of disease, N (%)a Distal colitis Extensive colitis Severity of disease, N (%) Mild Moderate Severe Treatment, N (%) SASP/5-ASA Prednisone Antibiotics Immunosuppressive Colectomy
172/210 42.11 ± 14.91 39.46 ± 14.33
225/264 40.96 ± 15.84
0.77 0.28
206 (53.9) 176 (46.1)
267 (54.6) 222 (45.4)
0.84
129 (33.8) 71 (18.6) 182 (47.6) 228 (59.7) 154 (40.3) 139 (36.4) 187 (49.0) 56 (14.6) 307 149 111 10 3
(80.4) (39.0) (29.1) (2.6) (0.8)
a
Distal colitis was defined as disease lesion distal to spleen flexure and extensive colitis was defined as disease lesion location beyond spleen flexure. SASP, sulfasalazine; 5-ASA, 5-aminosalicylic acid.
and inflammatory regulation, furthermore, the association of VDR signaling with UC has been extensively studied in recent years. Numerous studies have demonstrated that the biological functions of VDR were primarily modulated by the genetic polymorphisms of VDR. The human VDR gene is located on chromosome 12q13.11 within one of the IBD candidate regions determined by wide genome scans (8). Moreover, more than 200 single nucleotide polymorphisms (SNPs) have been identified in VDR gene. Of them, the FokI (T/C, rs2228570), BsmI (G/A, rs1544410), ApaI (C/A, rs7975232) and TaqI (T/C, rs731236) are of greatest concern. Association studies from different ethnic populations indicated that the above four SNPs of VDR gene were likely related to the predisposition of UC; however, the conclusions as a whole were lack of consistency (8–10). Therefore, we attempted to investigate the allelic and genotypic distributions of VDR FokI, BsmI, ApaI and TaqI gene in 382 UC patients and 489 healthy controls, who were recruited from Southeast China. As a result, our findings showed that the mutation of FokI gene influenced severity of the disease in UC patients. Moreover, the AAC haplotype formed by the VDR BsmI, ApaI and TaqI gene might engender a reduced risk of UC attack in this cohort of Chinese population.
Materials and methods Study population From May 2007 to December 2012, a total of 382 unrelated patients with UC were recruited from The Second Affiliated Hospital and The First Affiliated Hospital of Wenzhou Medical University as well as The Center Hospital of Wenzhou City in Zhejiang Province of Southeast China.
The diagnosis of UC was based on clinical, endoscopic, radiological and histological findings in accordance with Lennard-Jones criteria (11). The severity of UC was evaluated by the Truelove and Witts criteria (12), and the location was defined according to the Montreal classification (13). Four-hundred eighty-nine age- and sex-matched healthy controls were recruited at the Health Examination Center of The Second Affiliated Hospital of Wenzhou Medical University. Individuals with any other immunerelated diseases, including T1DM, MS and RA were excluded from the study. Demographic data of UC patients and the controls are listed in Table 1. All participants selfreported Chinese Han ancestry. The written informed consents were obtained from all study subjects. This study protocol was in line with the Declaration of Helsinki and was approved by Ethics Committees of the three hospitals mentioned above. Genomic DNA extraction and genotype analysis Genomic DNA was extracted from 3 ml EDTA anticoagulated blood samples by DNeasy Blood & Tissue kit (Qiagen GmbH, Hilden, Germany) according to the manufacturer’s instructions and then stored at 20 C pending analysis. Genotyping of VDR FokI, BsmI, ApaI and TaqI gene was performed using SNaPshot assays by ABI (Applied Biosystems, Carlsbad, CA). The details of the primers used for polymerase chain reaction (PCR) and single-base extension are listed in Table 2. The multiplex PCR was carried out in a 10 ml reaction volumes containing 1 ml DNA, 1 ml 10 PCR buffer with MgCl2 (Roche, Basel, Switzerland), 1 ml dNTPs
Associations of UC with common mutations of VDR in Chinese patients
DOI: 10.3109/10799893.2014.975248
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Table 2. Amplification and extension primers of VDR gene.
VDR gene FokI (rs2228570) BsmI (rs1544410) ApaI (rs7975232)
Amplification primer
Size Conc. (bp) (mmol/L)
182 F: 50 -GCAGCCTTCACAGGTCATAGCATT-30 R: 50 -CTGGCACTGACTCTGGCTCTGA-30 F: 50 -AGAACCATCTCTCAGGCTCCAAAGT-30 186 R: 50 -GAGTGTGCAGGCGATTCGTAGG-30 F: 50 -TACTGCTTGGAGTGCTCCTCATTGA-30 281 R: 50 -GTGCTGCCGTTGAGTGTCTGTG-30
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TaqI (rs731236)
(Promega, Madison, WI), 0.5 U FastStart Taq DNA polymerase (Roche) and a defined concentration of each amplification primer. Thermocycling was performed at 95 C for 5 min, followed by 35 cycles of 95 C for 30 s, 65 C for 30 s and 72 C for 1 min, with a final incubation at 72 C for 10 min. After the multiple amplification was completed, the PCR products were purified by shrimp alkaline phosphatase (SAP, New England Biolabs, Ipswich, MA) and exonuclease I (TaKaRa, Dalian, China). The mixture, including 10 ml PCR products, 2U Exo I and 1.5U SAP, was incubated at 37 C for 80 min and subsequently 80 C for 15 min. The SNaPshot extension reaction mixture contained 2 ml PCR purification, 1 ml SNaPshot Multiplex Mix, 1 ml 5 seq buffer and 0.2 mM of each specific primer. The cycling protocol were as follows: 96 C for 1 min, followed by 28 cycles of 96 C for 10 s, 52 C for 5 s and 60 C for 30 s. Purification was carried out by adding 1 U of SAP, followed by an incubation at 37 C for 60 min and 75 C for 15 min. Then samples were mixed with 8 ml of HiDiÔ formamide and 0.3 ml of GeneScan-120 Liz Size Standard (Applied Biosystems) and sequenced by capillary electrophoresis in a 3730xl Genetic Analyzer (Applied Biosystems). Finally, the data were analyzed by GeneMapper 4.0 (Carlsbad, CA). For quality control, 20 cases of the samples were tested repetitively by direct sequencing, and all the results were completely concordant. Statistical analysis The SPSS 17.0 software for Windows (SPSS for Windows version 17.0, Chicago, IL) was used. For each SNP, Chisquare test was employed to evaluate the Hardy–Weinberg equilibrium rule. Comparison of categorical variables (such as gender) between groups was performed by Chi-square test, and age was compared by Student’s t-test. After adjusted for age, gender and smoking, an unconditional logistic regression analysis was employed to assess the associations between the VDR FokI, BsmI, ApaI and TaqI gene polymorphisms and the overall risk of UC attack as well as the clinical features of UC patients. Odds radios (OR) and 95% confidence intervals (CIs) were applied for logistic regression analysis. The Bonferroni correction for multiple comparisons was applied, and the threshold was calculated as 0.05/4 ¼ 0.0125. Linkage disequilibrium between SNPs were determined by Haploview 4.2 software (Cambridge, MA), and haplotype analysis was further carried out by HAPLO.SCORE in R software (Rochester, MA). A two-tailed p value less than 0.05 was considered significant.
Extension primer
size Conc. (bp) (mmol/L)
0.05
50 -45T-TGCTGGCCGCCATTGCCTCC-30
65
0.05
0.1
50 -4T-GAGCCTGAGTATTGGGAATG-30
24
0.1
0.1
50 -32T-GGTGGGATTGAGCAGTGAGG-30
52
0.1
0.1
50 -17T-GCGGTCCTGGATGGCCTC-30
38
0.1
Results Relationship between UC and VDR FokI, BsmI, ApaI and TaqI gene polymorphisms In this study, we found that the genotypic distributions of VDR FokI, BsmI, ApaI and TaqI met the Hardy–Weinberg equilibrium rule in the controls (p ¼ 0.078, 0.359, 0.511 and 0.657, respectively). After Bonferroni correction, the allelic and genotypic frequencies of VDR FokI, BsmI, ApaI and TaqI were not statistically different between UC patients and the controls (all p40.0125) (Table 3). According to the severity of UC, we further stratified the patients into two subgroups. By logistic regression analysis, we found that the mutant allele C and genotype TC + CC of FokI gene were significantly increased in the patients with mild and moderate UC in contrast to those with severe UC (C allele: 54.1% versus 39.3%, OR ¼ 1.83, 95% CI: 1.21–2.75, p ¼ 0.004; TC + CC genotype: 81.6% versus 57.1%, OR ¼ 3.32, 95% CI: 1.83– 6.06, p50.001, respectively). However, the alleles and genotypes of BsmI, ApaI and TaqI were not statistically different between the two subgroups stratified by the severity of UC (all p40.0125). Besides, we also revealed that each of the four SNPs in VDR gene was not significantly associated with the locations of UC in this cohort of patients (all p40.0125; Table 4).
Analyses for linkage disequilibrium and haplotype of VDR FokI, BsmI, ApaI and TaqI gene polymorphisms We also made an analysis of linkage disequilibrium among the above four SNPs of VDR gene in this study. The BsmI, ApaI and TaqI three polymorphic loci were shown to be in a strong linkage disequilibrium (BsmI/ApaI, D0 ¼ 0.98, r2 ¼ 0.16; ApaI/TaqI, D0 ¼ 0.93, r2 ¼ 0.14; BsmI/TaqI, D0 ¼ 0.83, r2 ¼ 0.68, respectively) (Figure 1). Nevertheless, the FokI locus was not in strong linkage disequilibrium with each of the other three loci. The haplotype frequencies are listed in Table 5. Of the eight haplotypes, the frequencies simultaneously less than 3% in both UC patients and the controls were not taken into account. As a result, the GCT, GAT and AAC haplotypes were retained and analyzed. Although the frequencies of GCT (71.3%), together with GAT (21.0%), accounted for 92% of all haplotypes in the controls, neither of the two haplotypes differed significantly between UC patients and the controls (all p40.05). The AAC haplotype was shown to be statistically decreased in UC
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Table 3. Genotypic and allelic frequencies of VDR gene polymorphisms in patients with ulcerative colitis (UC) and healthy controls (HC). UC (N ¼ 382) N (%)
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VDR gene FokI (rs2228570) TT TC CC TC + CC T C BsmI (rs1544410) GG GA AA GA + AA G A ApaI (rs7975232) CC CA AA CA + AA C A TaqI (rs731236) TT TC CC TC + CC T C
HC (N ¼ 489) N (%)
OR [95% CI)]a
84 199 99 298 367 397
(22.0) (52.1) (25.9) (78.0) (48.0) (52.0)
98 263 128 391 459 519
(20.0) (53.8) (26.2) (80.0) (46.9) (53.1)
Ref 0.88 [0.63–1.25] 0.90 [0.61–1.34] 0.89 [0.64–1.23] ref 0.96 [0.79–1.16]
344 38 0 38 726 38
(90.0) (10.0) (0) (10.0) (95.0) (5.0)
423 65 1 66 911 67
(86.5) (13.3) (0.2) (13.5) (93.1) (6.9)
Ref 0.72 [0.47–1.10] NA 0.71 [0.46–1.08] Ref 0.71 [0.47–1.07]
186 174 22 196 546 218
(48.7) (45.5) (5.8) (51.3) (71.5) (28.5)
249 204 36 240 702 276
(50.9) (41.7) (7.4) (49.1) (72.8) (28.2)
Ref 1.14 [0.87–1.51] 0.82 [0.47–1.44] 1.09 [0.84–1.43] Ref 1.02 [0.82–1.25]
344 38 0 38 726 38
(90.0) (10.0) (0) (10.0) (95.0) (5.0)
424 62 3 65 910 68
(86.7) (13.7) (0.6) (13.3) (93.0) (7.0)
ref 0.76 [0.49–1.16] NA 0.72 [0.47–1.10] ref 0.70 [0.47–1.05]
pa
0.48 0.61 0.48 0.65 0.13 0.11 0.10 0.35 0.49 0.51 0.89 0.20 0.13 0.09
NA: not calculated due to the low frequencies in both groups. a Bonferroni multiple testing correction was used and the threshold was calculated as 0.05/4 ¼ 0.0125.
Table 4. The associations of VDR gene polymorphisms with clinicopathologic features of ulcerative colitis (UC). Severity of disease VDR gene
Mild/moderate (N ¼ 326) N (%)
FokI (rs2228570) TT 60 TC + CC 266 T 299 C 353 BsmI (rs1544410) GG 292 GA + AA 34 G 618 A 34 ApaI (rs7975232) CC 165 CA + AA 161 C 474 A 178 TaqI (rs731236) TT 295 TC + CC 31 T 621 C 31 a
Severea (N ¼ 56) N (%)
(18.4) (81.6) (45.9) (54.1)
24 32 68 44
(42.9) (57.1) (60.7) (39.3)
(89.6) (10.4) (94.8) (5.2)
52 4 108 4
(92.9) (7.1) (96.4) (4.6)
(50.6) (49.4) (72.7) (27.3)
21 35 72 40
(37.5) (62.5) (64.3) (35.7)
(90.5) (9.5) (95.3) (4.7)
49 7 105 7
(87.5) (12.5) (93.7) (6.3)
Extent of disease
pb
OR [95% CI]b
0.000
3.32 [1.83–6.06]
0.004
1.83 [1.21–2.75]
0.45
1.51 [0.52–4.44]
0.46
1.49 [0.52–4.27]
0.07
0.59 [0.33–1.05]
0.07
0.68 [0.44–1.03]
0.49
0.74 [0.31–1.76]
0.50
0.75 [0.32–1.75]
Distal colitis (N ¼ 228) N (%) 48 180 220 236
(21.1) (78.9) (48.3) (51.7)
36 118 147 161
(23.4) (76.6) (47.7) (52.3)
206 22 434 22
(90.4) (9.6) (95.2) (4.8)
138 16 292 16
(89.6) (10.4) (94.8) (5.2)
117 111 332 124
(51.3) (48.7) (72.8) (27.2)
69 85 214 94
(44.8) (55.2) (69.5) (30.5)
208 20 436 20
(91.2) (8.8) (95.6) (4.4)
136 18 290 18
(88.3) (11.7) (94.2) (5.8)
Serve as reference. Bonferroni multiple testing correction was used and the threshold was calculated as 0.05/4 ¼ 0.0125.
b
Extensive colitisa (N ¼ 154) N (%)
pb
OR [95% CI]b
0.59
1.14 [0.70–1.87]
0.89
0.98 [0.73–1.31]
0.81
0.92 [0.47–1.82]
0.82
0.93 [0.48–1.79]
0.21
0.77 [0.51–1.16]
0.32
0.85 [0.62–1.17]
0.35
0.73 [0.37–1.42]
0.37
0.74 [0.38–1.42]
Associations of UC with common mutations of VDR in Chinese patients
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DOI: 10.3109/10799893.2014.975248
Figure 1. Linkage disequilibrium pattern between the four SNPs of VDR gene were determined by Haploview 4.2 software. Numbers inside the boxes represent D0 value for Linkage disequilibrium. Selected region in the Linkage disequilibrium plot indicate the strong Linkage disequilibrium between the VDR BsmI (rs1544410), ApaI (rs7975232) and TaqI (rs731236) polymorphic loci.
Table 5. The frequencies of haplotypes in VDR BsmI, ApaI and TaqI genes between patients with ulcerative colitis (UC) and healthy controls (HC). haplotypes
GCT AACa GAT GAC ACC GCC AAT ACT
UC (N ¼ 764) HC (N ¼ 978)
71.5 71.3
3.8 5.9
22.4 21.0
1.2 0.6
0.0 0.1
0.0 0.4
1.2 0.8
0.0 0.0
a
p ¼ 0.039, OR ¼ 0.63, 95% CI: 0.39–1.01.
patients compared to the controls (3.8% versus 5.9%, OR ¼ 0.63, 95% CI: 0.39–1.01, p ¼ 0.039).
Discussion The genetic polymorphism of FokI is positioned at exon 2 of VDR gene, which has been identified as one of the most important functional loci in VDR gene. Previous studies demonstrated that the mutation of FokI altered the translation initiation sites of VDR gene and led to the production of a VDR protein, which was three amino acids shorter (14). Furthermore, it has been suggested that the shorter version of VDR protein was more transcriptionally active and had a greater capacity to transactivate VDR target genes, mainly resulted from its increased binding affinity with the basal transcription factor IIb (15). Recent data also indicate that FokI polymorphism could influence the behaviors of some immune cells (16), thus be probably implicated in many immune-mediated diseases, such as Grave’s disease (17) and SLE (18). In our study, there were no statistical differences in the allelic and genotypic distributions of FokI gene between UC patients and the controls, whereas the mutant allele and genotype frequencies of FokI gene were significantly higher in the patients with mild and moderate UC than those with
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severe UC. It indicated that the mutation of FokI gene decreased the severity of UC and played a protective role in this cohort of UC patients recruited from Southeast China. This finding seemed to be distinct from a previous study by Pei et al., in which the polymorphism of FokI was found not to be associated with the risk of UC as well as the clinical features of UC patients from Northern China (10). In Iranian population, moreover, Naderi et al. revealed that individuals with the mutant allele C were less susceptible to UC compared with those carrying the wild allele T of FokI gene (8). In the further genotype–phenotype analysis, nevertheless, they failed to detect any association of FokI polymorphism with the severity or location of UC in those patients (8). In addition, both Hughes et al. (9) and Simmons et al. (19) reported that the mutation of FokI gene was not markedly linked to the predisposition of UC in European Caucasian population. Furthermore, this study showed that the genetic polymorphisms of VDR BsmI, ApaI and TaqI were not connected with the susceptibility to UC as well as the clinical features of UC patients. These conclusions were somewhat different from a previous study on UC patients from Northern China, which showed that the mutation of BsmI gene might increase the overall risk of UC attack, whereas there were no significant associations of UC with the polymorphisms of ApaI and TaqI (10). However, Li et al. revealed that the mutation of BsmI gene appeared not to significantly affect the risk of UC in a total of 300 UC patients collected from both Northern and Southern China (20). We further compared the above findings with similar literature conducted by foreign scholars. As a result, we noticed that they were lack of consistency. For example, a case–control study in Iranian population showed that the mutations of VDR BsmI, ApaI and TaqI gene were not linked to the risk of UC (8). Similar conclusions were drawn from European Caucasians by Hughes et al. (9) and Pluskiewicz et al. (21). In addition, Simmons et al. (19) and Noble et al. (22) found no connection of UC with the VDR ApaI and TaqI polymorphisms in European Caucasians as well. A previous report from German population, however, showed that the genotypic distribution of TaqI gene in UC patients was statistically different from the controls (23). Another study performed by Dresner-Pollak et al. indicated that the mutation of BsmI gene engendered the increased risk of UC in Ashkenazi Jewish patients (24). Summarizing these divergent observations, we conjectured that the impacts of VDR Fok I, BsmI, ApaI and TaqI gene polymorphisms on UC were to some extent dependent on the differences of study subject’s genetic backgrounds, in addition to the study sample sizes as well as the ratio of clinical features in UC patients. In the further haplotype analysis, we noticed that the BsmI, ApaI and TaqI three polymorphic loci were in a strong linkage disequilibrium in all study subjects. Moreover, AAC haplotype formed by the three SNPs might engender a reduced risk of UC. In theory, the BsmI and ApaI are located in intron 8, and the TaqI is in exon 9 of VDR gene (25). Although a single mutation of the three SNPs can not give rise to an amino acid coding change, evidence from different ethnic groups has indicated that there is a strong linkage disequilibrium among the three polymorphic loci (26). Morrison et al., furthermore, found that AAC haplotype had 140% more excessive receptor
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activity than GCT haplotype (27). Carling T et al. reported that GCT haplotype was linked to the lower levels of VDR mRNA (28). Recent research data from Caucasians further indicated that GCT and AAC haplotypes were in linkage disequilibrium with the long and short alleles, respectively, of the poly(A) sequence in the 30 untranslated region of VDR, thereby probably associated with VDR gene transcription, mRNA stability and mRNA and protein levels (29). In summary, our study suggested that the mutation of FokI gene could affect the severity of UC. Moreover, the AAC haplotype formed by VDR BsmI, ApaI and TaqI might engender a lower risk of UC. Combined with the previous studies on the associations of VDR with IBD, the VDR signal pathway has been shown to be a potentially therapeutic target. Evidence from animal models consistently showed that treatment of 1,25-(OH)2D provided benefits in both IL-10 knockout and chemically induced colitis (30). A random, placebo-controlled trial in patients with Crohn’s disease (CD) suggested that supplementation with 1200 IU 1,25-(OH)2D insignificantly reduced the risk of relapse from 29% to 13% (p ¼ 0.06) (31). Another prospective study subsequently implicated that administration of 1,25-(OH)2D (provided by 0.5 mg alfacalidol) might decrease the disease activity of CD and improve quality of life score in a short-term at six weeks (32). Furthermore, as far as we know, few studies have been carried out on treatment of 1,25-(OH)2D in UC patients. Therefore, more high-quality randomized controlled trials are essential for evaluating the efficiency of 1,25-(OH)2D in the treatment of IBD.
Declaration of interest The authors declare no competing interests. This project was supported by grants from Zhejiang Provincial Natural Science Foundation (Grant number: LY14H030012) and Zhejiang Provincial Health Bureau (Grant number: 2014KYB157).
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