- Email: [email protected]
Polymorphisms in Genetics of Vitamin D Metabolism Confer Susceptibility to Ocular Behçet Disease in a Chinese Han Population
Accepted Manuscript Polymorphisms in genetics of Vitamin D metabolism confer susceptibility to ocular Behçet disease in a Chinese Han population Jing Fang, MM Shengping Hou, PhD Qin Xiang, MB Jian Qi, MM Hongsong Yu, PhD Yanyun Shi, MM Yan Zhou, MB Aize Kijlstra, PhD Peizeng Yang, PhD PII:
S0002-9394(13)00706-X
DOI:
10.1016/j.ajo.2013.10.010
Reference:
AJOPHT 8711
To appear in:
American Journal of Ophthalmology
Received Date: 12 August 2013 Revised Date:
18 October 2013
Accepted Date: 18 October 2013
Please cite this article as: Fang J, Hou S, Xiang Q, Qi J, Yu H, Shi Y, Zhou Y, Kijlstra A, Yang P, Polymorphisms in genetics of Vitamin D metabolism confer susceptibility to ocular Behçet disease in a Chinese Han population, American Journal of Ophthalmology (2013), doi: 10.1016/j.ajo.2013.10.010. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Polymorphisms in genetics of Vitamin D metabolism
Chinese Han population
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confer susceptibility to ocular Behçet disease in a
Jing Fang, MMa, Shengping Hou, PhDa, Qin Xiang, MBa, Jian Qi, MMa, Hongsong Yu, PhDa, Yanyun Shi, MMa, Yan Zhou, MBa ,Aize Kijlstra, PhDb, Peizeng Yang, PhDa
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a The First Affiliated Hospital of Chongqing Medical University, Chongqing Key Laboratory of Ophthalmology and Chongqing Eye Institute, Chongqing, P. R. China b University Eye Clinic Maastricht, Maastricht, The Netherlands
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Correspondence to: Professor Peizeng Yang, MD, PhD. The First Affiliated Hospital of Chongqing Medical University, Chongqing Key Laboratory of Ophthalmology and Chongqing Eye Institute, Chongqing, P.R. China,400016; Phone: +8623 89012851; FAX: +8623 89012851; Email: [email protected] Key words Vitamin D; DHCR7; CYP2R1; CYP27B1; CYP24A1; Behçet disease; Vogt-Koyanagi-Harada syndrome; ankylosing spondylitis; pediatric uveitis; anterior uveitis
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short title : Vitamin D genes confer susceptibility to Behçet disease
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Supplemental Material available at AJO.com
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Abstract Purpose: To test whether singlenucleotidepolymorphisms(SNPs) of the four Vitamin D family genes(DHCR7, CYP2R1, CYP27B1 and CYP24A1) previously associated with several autoimmune diseases, are associated with ocular Behçet disease, Vogt-Koyanagi-Harada (VKH) syndrome, acute anterior uveitis with ankylosing spondylitis (AAU+ankylosing spondylitis+) or pediatric uveitis in the Chinese Han population. Design: Prospective case control study Methods: Genotyping was performed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP)and the genotypes were verified with direct sequencing. The first stage study comprised 400 ocular Behçet disease patients, 400 VKH syndrome patients, 218 AAU+ankylosing spondylitis+ patients, 400 pediatric uveitis patients and 600 healthy subjects from Chinese Han populations. The second stage included 427 ocular Behçet disease patients and 1000 healthy Chinese Han subjects. Allele and genotype frequencies were compared between patients and controls using the χ2 test. Results: In the first stage study, only the frequencies of the rs12785878/DHCR7 genotype TT and T allele were significantly higher in ocular Behçet disease patients (p Bonferroni correction (pc) = 0.036; pc = 0.008, respectively) compared with controls among six SNPs. No associations could be detected for VKH, AAU+ankylosing spondylitis+ or pediatric uveitis. A second stage and combined study confirmed the association of rs12785878/DHCR7 TT genotype and T allele with ocular Behçet disease(pc = 3.28E-04, OR = 1.506, 95% CI 1.248 to 1.818; pc = 2.82E-05, OR = 1.339, 95% CI 1.188 to 1.508, respectively). Conclusions: This study provides evidence that the DHCR7 gene is involved in the susceptibility to ocular Behçet disease.
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Introduction Uveitis is an intraocular inflammation that can be caused by infectious as well as non-infectious mechanisms and is one of the main global causes of blindness [1, 2]. Uveitis can be manifested as an isolated ocular disease but can also belong to one of the symptoms of a systemic autoimmune or autoinflammatory diseases, such as sarcoidosis, Behçet disease, Vogt-Koyanagi-Harada (VKH) syndrome or acute anterior uveitis with ankylosing spondylitis (AAU+ ankylosing spondylitis +) [3]. Many of these uveitis syndromes are caused by an aberrant innate or adaptive immune response and have a genetic background [4]. Immunogenetic studies in uveitis have focused on genes of the HLA system and cytokine genes, but little attention has been paid to the genetic control of the interaction between the immune and endocrine system. Within the endocrine system, evidence is accumulating for a role of Vitamin D deficiency in several autoimmune disorders including type 1 diabetes mellitus, multiple sclerosis, Crohn Disease, and rheumatoid arthritis [5-7]. 1,25(OH)2D3, as the final active metabolite of vitamin D, can inhibit Th1 and Th17 cells, which are believed to have an important role in the pathogenesis of autoimmune and autoinflammatory disease [8, 9]. Serum levels of 1,25(OH)2D3 were found to be decreased in uveitis patients with Behçet disease, VKH syndrome and AAU+ ankylosing spondylitis+, suggesting a role for 1,25(OH)2D3 in their pathogenesis [10, 11]. In a recent study using lymphocytes obtained from ocular Behçet disease patients, we showed that vitamin D3 inhibits Th1 and Th17 differentiation but upregulates IL-10 [12]. In view of these findings we decided to investigate the role of polymorphisms in a number of genes involved in the synthesis and metabolism of Vitamin D3 on the development of Behçet disease. As a uveitis control we included patients with other uveitis entities including VKH syndrome, acute anterior uveitis associated with ankylosing spondylitis and patients with pediatric uveitis. It is well known that choosing preliminary candidate (single nucleotide polymorphisms) SNPs is critical for a candidate gene association study. So, our chosen strategy was based on previously described associations in various autoimmune diseases. Nine SNPs involving in four Vitamin D metabolism genes were chosen in this study. These SNPs were reported to have association with one or two autoimmune diseases such as type 1 diabetes mellitus, multiple sclerosis and Autoimmune Addison’s disease in the previous studies[13-16]. Materials and methods Patients and healthy controls study population This was a prospective case-control study. The study protocol was approved by the Ethics Committee of the First Affiliated Hospital of Chongqing Medical University, Chongqing, China (Permit Number: 2009-201008). The tenets of the Declaration of Helsinki were followed in all procedures. Written informed consent was given by all participants for this study. This study has registered in Chinese Clinical Trial Registry. The registration number is ChiCTR-CCC-12002184
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A two-stage case-control association study was performed. The first stage studied group comprised 400 ocular Behçet disease patients, 400 VKH syndrome patients, 218 AAU+ ankylosing spondylitis+ patients, 400 pediatric uveitis patients and 600 healthy subjects who were referred to the Zhongshan Ophthalmic Center, Sun Yat-sen University and the First Affiliated Hospital of Chongqing Medical University, Chongqing, China. The second stage included a total of another 427 ocular Behçet disease patients and another 1000 healthy controls. The analysis of stage one and two is independent. All control subjects, which were collected from April 2005 to February 2013, were matched ethnically and geographically with the patients. The patients with ocular Behçet disease fulfilled the criteria of the International Study Group[17]. The clinical features of these patients were recorded during the whole course of follow-up and summarized in Result part. The diagnosis of VKH syndrome was based on the First International Workshop criteria for VKH syndrome [18]. The clinical characteristics of the patients are presented in Supplementary table 1 (Supplemental Material, available at AJO.com). Pediatric uveitis was defined as uveitis (including anterior uveitis, intermediate uveitis, posterior uveitis and panuveitis) first presenting at an age below 16 years. Juvenile idiopathic arthritis was one of the most extraocular manifestations in pediatric uveitis. Among the 400 pediatric uveitis patients, 7.3% had juvenile idiopathic arthritis. The clinical characteristics of patient groups with pediatric uveitis and acute anterior uveitis with ankylosing spondylitis are shown in Supplementary table 2, 3 (Supplemental Material, available at AJO.com). Pediatric patients with Behçet disease, VKH syndrome or with definite infectious uveitis were excluded. To obtain a comparable genetic background, we strictly chose the cases from Chinese Han descendents.
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Single nucleotide polymorphisms selection and genotyping Based on previous reported disease association studies[13-16], we selected one SNP (rs12785878) of 7- dehydrocholesterolreductase (DHCR7), two SNPS (rs10741657, rs12794714) of Cytochrome p450, subfamily IIR, polypeptide 1 (CYP2R1), two SNPs (rs2248359, rs2762932) of Cytochrome p450, family 24, subfamily A, polypeptide 1 (CYP24A1) and four SNPs (rs10877012, rs118204009, rs118204011, rs118204012) of 25-hydroxyvitamin D3-1-alpha-hydroxylase (CYP27B1) as our candidate SNPs to explore the possible association with ocular Behçet disease ,VKH syndrome, AAU+ ankylosing spondylitis+ and pediatric uveitis. Genomic DNA samples of the healthy controls and patients with ocular Behçet disease, VKH syndrome, AAU+ ankylosing spondylitis+ and pediatric uveitis were extracted with the QIAamp DNA Blood Mini Kit (Qiagen, Valencia, CA). The genotyping success rate for the various SNPs ranged between 97.4% and 98.9%. The primers of rs12785878, rs10741657, rs12794714, rs2248359, rs2762932, rs10877012, rs118204009, rs118204011, rs118204012 for amplifying target DNA sequence by PCR are depicted in Table 1. The formed digestion products were separated on a 5% agarose gel (one SNP: rs2762932) or 4% agarose gel (another eight SNPS) and stained with GoldViewTM (SBS Genetech, Beijing, China). The restriction
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enzymes were purchased from Thermo Fisher Scientific. Using randomly selected subjects (5% of all samples) to check the result of the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method used in our study, direct sequencing was performed by the Majorbio Biotechnology Company (Shanghai, China).
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Statistical analysis The χ2 test was used to estimate whether the data were conform the Hardy-Weinberg principle. The patterns of linkage disequilibrium (LD) of the SNPs (rs10741657, rs12794714) of CYP2R1 and the SNPS of CYP24A1 were compared using Haploview 4.0 respectively, except for the SNP (rs12785878) of DHCR7 and the SNP (rs10877012) of CYP27B1. Direct counting estimated genotype frequencies. The χ2 test using SPSS (version 17.0) was used to compare allele and genotype frequencies between patients and controls. The p values were corrected (pc) with the Bonferroni correction by multiplying with the number of analyses performed. The Bonferroni corrections were based on a number of 18 independent comparisons.
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Results We found a significant association with a polymorphism for the gene encoding DHCR7 in ocular Behçet disease patients, but not in the other uveitis entities studied. No association was found with polymorphisms for the genes encoding CYP2R1, CYP24A1 or CYP27B1.
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Association of the four vitamin D related gene polymorphisms with susceptibility to ocular Behçet Disease Gene polymorphisms were not observed for three SNPs (rs118204009, rs118204011 and rs118204012) of CYP27B1 in the tested Chinese Han population. A total of 400 patients with ocular Behçet disease and 600 healthy controls were genotyped for the other six SNPS: one SNP (rs12785878) of DHCR7, two SNPS (rs10741657, rs12794714) of CYP2R1, two SNPs (rs2248359, rs2762932) of CYP24A1 and one SNP (rs10877012) of CYP27B1. These six SNPs were successfully genotyped and conformed to the Hardy-Weinberg principle in controls. Frequencies of the rs12785878/DHCR7 genotype TT and T allele were significantly higher in ocular Behçet disease patients (pc = 0.036, OR = 1.550, 95% CI 1.169 to 2.055; pc = 0.008, OR = 1.382, 95% CI 1.155 to 1.655, respectively) compared with controls. The uncorrected P value of the GG genotype of rs12785878/DHCR7 was 0.006(<0.05). However, the significance was lost following Bonferroni correction. Then, we replicated the associated the SNP rs12785878/DHCR7 using other cohort including 427 cases and other 1000 controls. The results showed that frequencies of the rs12785878/DHCR7 genotype TT, GG and T allele were significantly higher in ocular Behçet disease patients (pc = 0.012, OR = 1.450, 95% CI 1.124 to 1,871; pc = 0.009, OR = 0.672, 95% CI 0.518 to 0.872; pc = 0.003, OR = 1.270, 95% CI 1.082 to 1.492, respectively) compared with controls. In the combined stage, after combining the number of ocular Behçet disease cases
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(n=827) and healthy controls (n=1600), we confirmed the association with the rs12785878/DHCR7 genotype TT and T allele (pc = 3.28E-04, OR = 1.506, 95% CI 1.248 to 1.818; pc = 2.82E-05, OR = 1.339, 95% CI 1.188 to 1.508, respectively) (Table 2). No significant differences were found between patients with ocular Behçet disease and controls concerning the frequencies of the other five SNPs (Table 2). Among 827 Bechet disease patients, the age at onset (years ± Standard Deviation(SD)) is 31.4±9.6, 100% had uveitis and recurrent oral ulceration, 51.51% had recurrent genital ulceration, 66.75% had skin lesions, 26.12% had positive pathergy test, 23.34% had hypopyon and 15.11% had arthritis. The proportions of anterior uveitis and panuveitis were 14.4% and 85.6% among the 827 Bechet disease patients, respectively. We also studied the association of polymorphisms of the four vitamin D related genes with the clinical features of ocular Behçet disease entities, using stratified analysis. A significantly higher frequency of TT genotype and T allele of rs12785878/DHCR7 was noticed in ocular Behçet disease patients respectively with recurrent genital ulcers and skin lesions (Table 3 and Supplementary table 4) (Supplemental Material, available at AJO.com). There was no significant statistical association concerning the genotypes of the other five SNPs and clinical manifestations in ocular Behçet disease.
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Association of the four vitamin D related gene polymorphisms with susceptibility to Vogt-Koyanagi-Harada syndrome, acute anterior uveitis + ankylosing spondylitis+ or pediatric uveitis A total of 600 healthy controls, 400 VKH syndrome patients, 218 AAU+ ankylosing spondylitis + patients, and 400 pediatric uveitis patients were genotyped for the six vitamin D related SNPS: one SNP (rs12785878) of DHCR7, two SNPS (rs10741657, rs12794714) of CYP2R1, two SNPs (rs2248359, rs2762932) of CYP24A1 and one SNP (rs10877012) of CYP27B1. The six SNPs were successfully genotyped and conformed to Hardy-Weinberg expectation in controls. The genotype and allele frequencies of the six SNPs examined in healthy controls, VKH syndrome patients, AAU+ ankylosing spondylitis+ patients and pediatric uveitis patients are summarized in supplementary table 5,6,7 (Supplemental Material, available at AJO.com) respectively. No differences were found for the genotype or allele frequencies of the six SNPs between the three diseases and healthy controls except for rs12785878/DHCR7 in pediatric uveitis and rs10877012/CYP27B1 in AAU+ ankylosing spondylitis+. The P value of the GT genotype of rs12785878/DHCR7 in pediatric uveitis was 0.022(<0.05) and that of the GG genotype of rs10877012/CYP27B1 in AAU+ ankylosing spondylitis+ was 0.011(<0.05). After Bonferroni correction, the significance of the investigated genotypes with both uveitis entities was lost. No significant statistical association was found for the six SNPs genotypes and clinical manifestations in VKH syndrome, AAU+ ankylosing spondylitis+ or pediatric uveitis. Discussion The role of vitamin D related gene polymorphisms in the risk of developing uveitis
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was investigated in this study and revealed that rs12785878/DHCR7 was significantly associated with Behçet disease but not with other uveitis entities investigated. The role of genes involved in the regulation and synthesis of vitamin D with uveitis has not yet been reported before. Earlier studies investigated eight SNPs of the vitamin D binding protein and suggested an association of rs4752 with the occurrence of uveitis in Korean ankylosing spondylitis patients [19] but in our view significance would be lost if the data had been corrected for multiple comparisons. The vitamin D binding protein is a highly polymorphic protein involved in the transport of vitamin D but was not yet included in our studies. We focused on a set of genes involved in the synthesis of vitamin D that have been shown to affect the risk of certain immune related disorders [20]. One of the genes we investigated included DHCR7, which controls vitamin D levels. The 7-dehydrocholesterol reductase, which is encoded by DHCR7, can convert 7-dehydrocholesterol (7-DHC) to cholesterol, thus reducing the substratum from the synthetic pathway of vitamin D3, which is a precursor of 25-OH D3 [21]. Mutations in DHCR7 have been shown to influence the serum vitamin D level [22, 23]. Wang et al. [21] recently reported that variants involved in Vitamin D synthesis and metabolism including rs127858 /DHCR7 could identify individuals who were at risk of vitamin D deficiency. These findings are in line with the observations that Vitamin D levels are lower in ocular Behçet disease patients [10] and that Vitamin D3 can inhibit Th1 and Th17 lymphocyte differentiation in Behçet disease [12]. In view of the findings mentioned above, we investigated DHCR7 gene susceptibility to uveitis. The selection of SNPs was principally based on previous studies [14, 21]. A similar association was recently reported for rs12785878/DHCR7 in a British cohort of type 1 diabetes mellitus patients [14]. We failed to find an association of the Vitamin D SNPs with susceptibility to VKH syndrome, AAU+ ankylosing spondylitis+ or pediatric uveitis. The discrepancy between ocular Behçet disease and the other uveitis entities investigated may be due to the fact that the pathogenetic mechanisms for Behçet disease are unique and differ from these other three ocular inflammatory diseases. Evidence is mounting that Behçet disease is an autoinflammatory disease caused by an aberrant response against infectious agents[24]. How vitamin D controls the susceptibility to ocular Behçet disease is not clear but vitamin D synthesis and metabolism can either affect the immune response, but may on the other hand also be involved in bacterial colonization [25]. During recent years three other vitamin D related genes (CYP27B1, CYP2R1, CYP24A1) have emerged as candidate susceptibility factors for autoimmune disease [13-16] . A role for CYP27B1 had been suggested in studies involving British, Canadian and Swedish patients with multiple sclerosis [14, 15, 26]. Similarly, Cooper et al.[14] revealed an association between the two Vitamin D related genes - CYP27B1 (rs10877012) and CYP2R1 (rs10741657 and rs12794714) - and type 1 diabetes patients in British patients. Sawcer et al.[16] showed that the SNPs (rs2248359, rs762932) of the CYP24A1 gene had an influence on the risk of multiple sclerosis in European patients. We examined the association of polymorphisms in CYP27B1,
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CYP2R1, CYP24A1 genes and did not find an association with the four chosen uveitis entities in a Chinese Han population. Similar to our results, no association with these three vitamin D related gene polymorphisms has been reported in multiple sclerosis patients from the US [27]. There are a number of limitations in our study. Our study indicates that the rs12785878/DHCR7 may play a role in the susceptibility for ocular Behçet disease patients in a Chinese Han population. Although we have put forward a hypothesis, the exact mechanism by which rs12785878/DHCR7 exerts its role in the pathogenesis of Behçet disease is not completely understood. The fact that we failed to find an association with Vitamin D genes such as CYP24A1, CYP27B1 and CYP2R1, does not rule out the possibility that other relevant Vitamin D genes polymorphisms can be associated with these four uveitis entities. More studies are needed to clarify this issue. Furthermore we did not test the Vitamin D status in our patients. Earlier studies in Han Chinese have however shown that DHCR7 variants are associated with Vitamin D status [23]. In conclusion, our study for the first time reports an association between the polymorphisms of rs12785878/DHCR7, but not rs10741657, rs12794714/CYP2R1, rs2248359, rs2762932/CYP24A1 and rs10877012/CYP27B1, with susceptibility to ocular Behçet disease in a Chinese Han population.
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Acknowledgments All authors have completed and submitted the ICMJE form for disclosure of potential conflicts of interest and none were reported. Financial disclosures: This study was supported, in part, by Chongqing Key Laboratory of Ophthalmology (CSTC, 2008CA5003), Fund for PAR-EU Scholars Program, Key Project of Natural Science Foundation(81130019), Key Project of Health Bureau of Chongqing(2012-1-003), National Basic Research Program of China (973 Program) (2011CB510200) and Research Fund for the Doctoral Program of Higher Education of China (20115503110-002). Contributions of authors: Design and conduct of the study (PY, JF); Collection, management, analysis, and interpretation of data (JF, SH, QX, JQ, HY, YS, YZ); and Preparation, review, or approval of manuscript (JF, SH, AK, PY) References 1.
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12. Tian Y, Wang C, Ye Z, Xiao X, Kijlstra A, Yang P. Effect of 1, 25-Dihydroxyvitamin D3 on Th17 and Th1 response in patients with Behcet's disease. Invest Ophthalmol Vis Sci 2012;53(10):6434-6441. 13. Lopez ER, Zwermann O, Segni M, et al. A promoter polymorphism of the CYP27B1 gene is associated with Addison's disease, Hashimoto's thyroiditis, Graves' disease and type 1 diabetes mellitus in Germans. Eur J Endocrinol 2004;151(2):193-197.
14. Cooper JD, Smyth DJ, Walker NM, et al. Inherited variation in vitamin D genes is associated with predisposition to autoimmune disease type 1 diabetes. Diabetes 2011;60(5):1624-1631. 15. Ramagopalan SV, Dyment DA, Cader MZ, et al. Rare variants in the CYP27B1 gene are associated
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Table 1. Primers and restriction enzymes used for restricted fragment length polymorphisms analysis of the DHCR7, CYP27B1, CYP2R1, and CYP24A1 gene SNP ID
Primers
DHCR7
rs12785878
5' ACCACCTTCAAATAGGGCTGT 3' 5' CAGCAGACAGGACATGAGGAT 3'
CYP27B1
rs10877012
5’ AACATAGTCGAACTGTCTCTAC 3'
rs118204009
5' T GTGCTTTGCAACCTAGACTGT 3'
rs118204011
5' G GAAGTTTTCTGGGGCTACTTT 3' 5' TTCACATGTTTTTCAGGTGTCC 3'
CYP2R1
rs10741657 rs12794714
CYP24A1
rs2248359
279bp
MluCI
248bp
MnLI
288bp
5' ATGCGCACTCTCTCCTCAAC
3'
5' CTCTGTCCTGGGACTCACCTT
3'
5' G GGAAGAGCAATGACATGGA
3'
5' G CCCTGGAAGACTCATTTTG
3'
5' CCATAAGTCCAACCAGGAAGG
3'
5' AGCTTTGGAGAGCTGAAGA GG
3'
5' TTCTACACCGACTACCTTGTGC
3'
5' CAAAAATCTCCAACTTCCGTTC
3'
5'CCTACATACTAACTGTGATTCTAA GGAGGT 3' 5'GGCCAATGTGGTCATGATGATAAT 3'
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BsmI
3'
*SNP, single-nucleotide polymorphism
(195+103)
281bp
5' TT CTCTGCTATCTCCCTGCTTC
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rs2762932
298bp
BssSI
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rs118204012
HinfI
271bp (151+119)
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5’ TTCAATTCCAGAACTTCAGAGC 3'
Restriction enzyme TaqI
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Gene
(151+105+32) FokI
299bp(161 +137)
SacII
191bp(134+57)
BstEII
204bp(176+28)
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Table 2. Polymorphisms of DHCR7, CYP27B1, CYP2R1, and CYP24A1 genes in ocular Behçet Disease
rs12785878
b
Replication
Combined
CYP27B1 (rs10877012)
First
c
Control n(freq)
P value
Pc value
OR(95%CI)
TT
129(0.323)
141(0.235)
0.002
0.036
1.550(1.169-2.055)
TG
201(0.503)
310(0.517)
0.661
NS
0.945(0.734-1.217)
GG
70(0.175)
149(0.248)
0.006
T
459(0.574)
592(0.493)
4.18E-04
G
341(0.426)
608(0.507)
4.18E-04
TT
127(0.297)
226(0.226)
0.004
TG
195(0.457)
480(0.480)
0.419
CYP24A1 (rs2762932)
First
First
1.450(1.124-1.871)
1.257
0.911(0.725-1.143)
294(0.294)
0.003
0.009
0.672(0.518-0.872)
932(0.466)
0.003
0.003
1.270(1.082-1.492)
G
405(0.474)
1068(0.534)
0.003
0.003
0.787(0.670-0.924)
TT
256(0.310)
367(0.229)
1.82E-05
3.28E-04
1.506(1.248-1.818)
TG
396(0.479)
790(0.494)
0.486
8.750
0.942(0.796-1.114)
GG
175(0.212)
T
908(0.549)
G
746(0.451)
TT
151(0.378)
TG
199(0.498)
GG
50(0.125)
T
501(0.626)
G
299(0.374)
GG
EP
443(0.277)
4.69E-04
0.008
0.701(0.574-0.856)
1524(0.476)
1.56E-06
2.82E-05
1.339(1.188-1.508)
1676(0.524)
1.56E-06
2.82E-05
0.747(0.663-0.842)
247(0.412)
0.28
NS
0.867(0.669-1.123)
284(0.473)
0.454
NS
1.102(0.855-1.419)
69(0.115)
0.632
NS
1.099(0.746-1.621)
778(0.648)
0.314
NS
0.909(0.755-1.095)
422(0.352)
0.314
NS
1.100(0.914-1.325)
52(0.130)
79(0.132)
0.939
NS
0.985(0.677-1.434)
197(0.493)
281(0.468)
0.454
NS
1.102(0.855-1.419)
151(0.378)
240(0.400)
0.475
NS
0.910(0.701-1.180)
301(0.376)
439(0.366)
0.636
NS
1.046(0.869-1.258)
499(0.624)
761(0.634)
0.636
NS
0.956(0.795-1.151)
AA
48(0.120)
91(0.152)
0.156
NS
0.763(0.524-1.110)
AG
193(0.483)
248(0.413)
0.031
NS
1.323(1.026-1.707)
GG
159(0.398)
261(0.435)
0.239
NS
0.857(0.663-1.108)
A
289(0.361)
430(0.358)
0.894
NS
1.013(0.841-1.220)
G
511(0.639)
770(0.642)
0.894
NS
0.987(0.820-1.190)
CC
131(0.328)
223(0.372)
0.152
NS
0.823(0.631-1.075)
CT
195(0.488)
283(0.472)
0.623
NS
1.066(0.827-1.373)
AC C
CYP24A1 (rs2248359)
0.012
105(0.246)
G
First
0.723(0.604-0.866)
449(0.526)
A
CYP2R1 (rs12794714)
0.008
T
AG First
0.642(0.467-0.882)
1.382(1.155-1.655)
GG
AA CYP2R1 (rs10741657)
NS
0.008
RI PT
(DHCR7)
Case n(freq)
SC
First
a
Genotype Allele
M AN U
Stage
TE D
SNPs
TT
74(0.185)
94(0.157)
0.24
NS
1.222(0.874-1.708)
C
457(0.571)
729(0.608)
0.106
NS
0.861(0.718-1.032)
T
343(0.429)
471(0.392)
0.106
NS
1.162(0.969-1.393)
CC
6(0.015)
6(0.010)
0.477
NS
1.508(0.483-4.708)
CT
61(0.153)
103(0.172)
0.423
NS
0.868(0.615-1.227)
TT
333(0.833)
491(0.818)
0.564
NS
1.103(0.790-1.542)
C
73(0.091)
115(0.096)
0.731
NS
0.947(0.696-1.289)
T
727(0.909)
1085(0.904)
0.731
NS
1.056(0.776-1.436)
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
*SNP, single-nucleotide polymorphism; OR, odds ratio; CI, confidence interval; NS, no significant different; freq, frequency; pc, p Bonferroni correction. *a: First stage(stage one) , case: control = 400: 600 b: Replication stage(stage two), case: control = 427: 1000 c: Combined stage(a + b), case: control = 827: 1600
ACCEPTED MANUSCRIPT
Table 3. Polymorphisms of the DHCR7 gene in patients with extraocular features of Behçet disease
Clinical features
Genotype
Behçet disease patients (%)
Controls (%)
Pc
TT
145(0.340)
367(0.229)
6.76E-05
genital ulcer
TG
187(0.439)
790(0.494)
NS
95(0.221)
443(0.277)
T
477(0.56)
1524(0.476)
G
375(0.44)
1676(0.524)
TT
172(0.312)
367(0.229)
NS
1.399(1.202-1.629)
1.01E-04
0.715(0.614-0.832)
0.003
1.521(1.227-1.885)
TG
252(0.457)
790(0.494)
GG
128(0.232)
443(0.277)
NS
T
596(0.54)
1524(0.476)
0.002
1.290(1.125-1.480) 0.775(0.676-0.889)
NS
508(0.46)
1676(0.524)
0.002
TT
65(0.301)
367(0.229)
NS
pathergy test
TG
100(0.463)
367(0.229)
NS
GG
51(0.236)
790(0.494)
NS
Hypopyon
Arthritis
T
230(0.532)
443(0.277)
NS
G
202(0.468)
1524(0.476)
NS
TT
58(0.301)
367(0.229)
NS
TG
93(0.482)
790(0.494)
NS
GG
42(0.218)
443(0.277)
NS
T
209(0.542)
1524(0.476)
NS
G
177(0.459)
1676(0.524)
NS
27(0.216)
367(0.229)
NS
64(0.512)
790(0.494)
NS
TT TG GG
34(0.272)
443(0.277)
NS
118(0.472)
1524(0.476)
NS
132(0.528)
1676(0.524)
NS
TT
30(0.252)
367(0.229)
NS
TG
57(0.479)
790(0.494)
NS
GG
32(0.269)
443(0.277)
NS
T
117(0.492)
1524(0.476)
NS
G
121(0.508)
1676(0.524)
NS
TT
165(0.233)
367(0.229)
NS
TG
341(0.482)
790(0.494)
NS
GG
202(0.285)
443(0.277)
NS
T
671(0.474)
1524(0.476)
NS
G
745(0.526)
1676(0.524)
NS
T
AC C
EP
G anterior uveitis
panuveitis
M AN U
G Positive
rs12785878
1.727(1.370-2.178)
1.01E-04
SC
Skin lesions
GG
OR (95%CI)
RI PT
Recurrent
TE D
SNP
SNP, single-nucleotide polymorphism; OR, odds ratio; CI, confidence interval; NS, no significant different
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT
AC C
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M AN U
SC
RI PT
Jing Fang, MD, is currently studying at the First Affiliated Hospital of Chongqing Medical University, Chongqing Key Laboratory of Ophthalmology and Chongqing Eye Institute, Chongqing, P. R. China. Before MD, he had been a pediatric oculist for eight years in Department of Ophthalmology, Children's Hospital, Chongqing Medical University, Chongqing, P. R. China. His research interests are in ophthalmic genetics, uveitis, and optometry.
ACCEPTED MANUSCRIPT
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Prof. Peizeng Yang is working at the First Affiliated Hospital of Chongqing Medical University. He is an international council representative of International Ocular Inflammation Society, founding executive member of Asia-Pacific Intraocular Inflammation Study Group, member of the International Uveitis Study Group, council member of International Society for Behçet’s Disease and vice president of Chinese Ophthalmological Society. He is mainly engaging in the study on diagnosis and treatment of uveitis and the mechanisms involved in this disease.
ACCEPTED MANUSCRIPT
manuscript number : AJO-13-865R2 Polymorphisms in genetics of Vitamin D metabolism confer susceptibility to ocular Behçet disease in a Chinese Han population. Jing Fang, MM, Shengping Hou, PhD, Qin Xiang, MB, Jian Qi, MM, Hongsong Yu, PhD, Yanyun Shi, MM, Yan Zhou, MB ,Aize Kijlstra, PhD, Peizeng Yang, PhD
AC C
EP
TE D
M AN U
SC
RI PT
Vitamin D family genes have been reported to be associated with several autoimmune diseases. We performed a prospective case control study to test whether these genes are associated with four kinds of uveitis by genotyping. We found the frequencies of rs12785878/DHCR7 genotype TT and T allele were significantly higher in ocular Behçet disease patients compared with controls. This study provides evidence that the DHCR7 gene is involved in the susceptibility to ocular Behçet disease.
ACCEPTED MANUSCRIPT
Supplementary table 1. Clinical features of the investigated Vogt-Koyanagi-Harada syndrome patients Patients with VKH syndrome N (total=400)
%
34.5±8.2
Uveitis
400
100%
Stiffness of the neck and back
52
13.0%
Headache
204
51.0%
Scalp sensitivity
40
10.0%
Tinnitus
156
39.0%
Alopecia
84
21.0%
Poliosis
104
Vitiligo
100
SC
Age at onset (years±SD)
26.0%
25.0%
EP
TE D
M AN U
*SD, Standard Deviation; VKH syndrome, Vogt-Koyanagi-Harada syndrome
AC C
RI PT
Clinical features
ACCEPTED MANUSCRIPT
Supplementary table 2. Clinical features of the investigated pediatric uveitis patients Patients with pediatric uveitis N (total=400)
%
Age at onset (years±SD)
14.08±8.4
Uveitis
400
100%
Juvenile idiopathic arthritis
29
7.3%
Retinal vasculitis
28
7.0%
AC C
EP
TE D
M AN U
SC
*SD, Standard Deviation
RI PT
Clinical features
ACCEPTED MANUSCRIPT
Supplementary table 3. Clinical features of the investigated ankylosing spondylitis patients Patients with ankylosing spondylitis N (total=218)
%
Age at onset (years±SD)
39.2±10.0
uveitis
218
100%
HLA-B27(+)
207
94.8%
HLA-B27(-)
11
5.2%
AC C
EP
TE D
M AN U
SC
*SD, Standard Deviation
RI PT
Clinical features
ACCEPTED MANUSCRIPT
Supplementary table 4. Criteria for diagnosis of Behçet disease* Recurrent Oral ulceration
Minor aphthous, major aphthous, or herpetiform ulceration observed by physician or patient, which recurred at least 3 times in one 12-month period
RI PT
Plus 2 of: Recurrent genital ulceration Eye lesions
M AN U
SC
Skin lesions
Aphthous ulceration or scarring, observed by physician or patient Anterior uveitis, posterior uveitis, or cells in vitreous on slit lamp examination; or Retinal vasculitis observed by ophthalmologist Erythema nodosum observed by physician or patient, pseudofolliculitis, or papulopustular lesions; or Acneiform nodules observed by physician in postadolescent patients not on corticosteroid treatment Read by physician at 24-48 h.
Positive pathergy test
AC C
EP
TE D
(Findings applicable only in absence of other clinical explanations.) *The criteria of diagnosis comes from “Criteria for diagnosis of Behçet’s disease. International Study Group for Behçet's Disease.” Lancet 1990;335(8697):1078-1080.
ACCEPTED MANUSCRIPT
Supplementary table 5. Polymorphism of DHCR7, CYP27B1, CYP2R1, and CYP24A1 genes in Vogt-Koyanagi-Harada syndrome
CYP27B1
CYP2R1
rs10877012
rs10741657
rs12794714
Control
Allele
n(freq)
n(freq)
TT
98(0.245)
TG
212(0.530)
AC C
rs2762932
OR(95%CI)
141(0.235)
0.716
NS
1.056(0.786-1.420)
310(0.517)
0.679
NS
1.055(0.819-1.359)
90(0.225)
149(0.248)
0.397
NS
0.879(0.652-1.185)
T
408(0.510)
592(0.493)
0.465
NS
1.069(0.894-1.278)
G
392(0.490)
608(0.507)
0.465
NS
0.936(0.782-1.119)
TT
160(0.400)
247(0.412)
0.713
NS
0.953(0.736-1.233)
TG
180(0.450)
284(0.473)
0.469
NS
0.910(0.706-1.174)
GG
60(0.150)
69(0.115)
0.106
NS
1.358(0.936-1.970)
T
500(0.625)
778(0.648)
0.287
NS
0.904(0.751-1.089)
G
300(0.375)
422(0.352)
0.287
NS
1.106(0.919-1.332)
AA
41(0.103)
79(0.132)
0.164
NS
0.753(0.505-1.124)
AG
179(0.448)
281(0.468)
0.517
NS
0.919(0.713-1.185)
GG
180(0.450)
240(0.400)
0.117
NS
1.227(0.950-1.585)
A
261(0.326)
439(0.366)
0.069
NS
0.839(0.695-1.014)
G
539(0.674)
761(0.634)
0.069
NS
1.191(0.986-1.439)
AA
58(0.145)
91(0.152)
0.772
NS
0.949(0.664-1.355)
AG
189(0.473)
248(0.413)
0.065
NS
1.271(0.985-1.640)
GG
153(0.383)
261(0.435)
0.099
NS
0.805(0.621-1.042)
A
305(0.381)
430(0.358)
0.298
NS
1.103(0.917-1.328)
770(0.642)
0.298
NS
0.906(0.753-1.091)
495(0.619)
CC
151(0.378)
223(0.372)
0.852
NS
1.025(0.789-1.331)
CT
185(0.463)
283(0.472)
0.776
NS
0.964(0.748-1.242)
TT
64(0.160)
94(0.157)
0.887
NS
1.025(0.725-1.450)
C
487(0.609)
729(0.608)
0.955
NS
1.005(0.837-1.207)
T
313(0.391)
471(0.392)
0.955
NS
0.995(0.828-1.195)
CC
4(0.010)
6(0.010)
1.000
NS
1.000(0.280-3.566)
EP
rs2248359
Pc value
GG
G
CYP24A1
P value
RI PT
rs12785878
case
SC
DHCR7
Genotype
M AN U
SNPs
TE D
Gene
CT
71(0.178)
103(0.172)
0.812
NS
1.041(0.746-1.453)
TT
325(0.813)
491(0.818)
0.816
NS
0.962(0.695-1.332)
C
79(0.099)
115(0.096)
0.829
NS
1.034(0.765-1.398)
T
721(0.901)
1085(0.904)
0.829
NS
0.967(0.716-1.308)
*SNP, single-nucleotide polymorphism; OR, odds ratio; CI, confidence interval; NS, no significant different; freq, frequency; pc, p Bonferroni correction
ACCEPTED MANUSCRIPT
Supplementary table 6. Polymorphism of DHCR7, CYP27B1, CYP2R1, and CYP24A1 genes in ocular ankylosing spondylitis
CYP27B1
CYP2R1
rs10877012
rs10741657
rs12794714
Control
Allele
n(freq)
n(freq)
TT
48(0.220)
TG
102(0.468)
AC C
rs2762932
OR(95%CI)
141(0.235)
0.657
NS
0.919(0.634-1.333)
310(0.517)
0.217
NS
0.823(0.603-1.122)
68(0.312)
149(0.248)
0.069
NS
1.372(0.976-1.930)
T
198(0.454)
592(0.493)
0.161
NS
0.854(0.686-1.065)
G
238(0.546)
608(0.507)
0.161
NS
1.170(0.939-1.458)
TT
82(0.376)
247(0.412)
0.36
NS
0.862(0.627-1.185)
TG
96(0.440)
284(0.473)
0.403
NS
0.876(0.641-1.196)
GG
40(0.183)
69(0.115)
0.011
NS
1.729(1.131-2.645)
T
260(0.596)
778(0.648)
0.053
NS
0.801(0.640-1.004)
G
176(0.404)
422(0.352)
0.053
NS
1.248(0.996-1.563)
AA
37(0.170)
79(0.132)
0.168
NS
1.348(0.881-2.063)
AG
86(0.394)
281(0.468)
0.06
NS
0.740(0.540-1.014)
GG
95(0.436)
240(0.400)
0.358
NS
1.159(0.847-1.585)
A
160(0.367)
439(0.366)
0.966
NS
1.005(0.800-1.262)
G
276(0.633)
761(0.634)
0.966
NS
0.995(0.793-1.249)
AA
23(0.106)
91(0.152)
0.092
NS
0.660(0.406-1.073)
AG
97(0.445)
248(0.413)
0.418
NS
1.138(0.832-1.555)
GG
98(0.450)
261(0.435)
0.711
NS
1.061(0.777-1.449)
A
143(0.328)
430(0.358)
0.255
NS
0.874(0.693-1.102)
770(0.642)
0.255
NS
1.144(0.907-1.443)
293(0.672)
CC
78(0.358)
223(0.372)
0.716
NS
0.942(0.682-1.301)
CT
105(0.482)
283(0.472)
0.763
NS
1.041(0.763-1.420)
TT
35(0.161)
94(0.157)
0.893
NS
1.030(0.674-1.572)
C
261(0.599)
729(0.608)
0.745
NS
0.964(0.770-1.205)
T
175(0.401)
471(0.392)
0.745
NS
1.038(0.830-1.298)
CC
2(0.009)
6(0.010)
0.916
NS
0.917(0.184-4.576)
EP
rs2248359
Pc value
GG
G
CYP24A1
P value
RI PT
rs12785878
case
SC
DHCR7
Genotype
M AN U
SNPs
TE D
Gene
CT
41(0.188)
103(0.172)
0.586
NS
1.118(0.749-1.668)
TT
175(0.803)
491(0.818)
0.612
NS
0.903(0.610-1.338)
C
45(0.103)
115(0.096)
0.657
NS
1.086(0.755-1.562)
T
391(0.897)
1085(0.904)
0.657
NS
0.921(0.640-1.325)
*SNP, single-nucleotide polymorphism; OR, odds ratio; CI, confidence interval; NS, no significant different; freq, frequency; pc, p Bonferroni correction.
ACCEPTED MANUSCRIPT
Supplementary table 7. Polymorphism of DHCR7, CYP27B1, CYP2R1, and CYP24A1 genes in pediatric uveitis
CYP27B1
CYP2R1
rs10877012
rs10741657
rs12794714
Control
P
Pc
Allele
n(freq)
n(freq)
value
value
TT
101(0.253)
141(0.235)
0.527
NS
1.100(0.819-1.476)
TG
177(0.443)
310(0.517)
0.022
NS
0.743(0.576-0.957)
GG
122(0.305)
149(0.248)
0.048
NS
T
379(0.474)
592(0.493)
0.391
NS
G
421(0.526)
608(0.507)
0.391
NS
AC C
rs2762932
1.082(0.904-1.294)
TT
167(0.418)
247(0.412)
0.854
NS
183(0.458)
284(0.473)
0.623
NS
GG
50(0.124)
69(0.115)
0.632
NS
1.099(0.746-1.621)
1.024(0.792-1.324)
0.938(0.728-1.209)
T
517(0.646)
778(0.648)
0.924
NS
0.991(0.822-1.195)
G
283(0.354)
422(0.352)
0.924
NS
1.009(0.837-1.217)
AA
54(0.135)
79(0.132)
0.879
NS
1.029(0.710-1.493)
AG
177(0.443)
281(0.468)
0.422
NS
0.901(0.699-1.162)
GG
169(0.423)
240(0.400)
0.478
NS
1.097(0.849-1.419)
A
285(0.356)
439(0.366)
0.662
NS
0.959(0.796-1.156)
G
515(0.644)
761(0.634)
0.662
NS
1.042(0.865-1.256)
AA
63(0.158)
91(0.152)
0.802
NS
1.046(0.737-1.483)
AG
155(0.388)
248(0.413)
0.415
NS
0.898(0.693-1.163)
GG
182(0.455)
261(0.435)
0.533
NS
1.084(0.841-1.399)
A
281(0.351)
430(0.358)
0.746
NS
0.970(0.804-1.169)
770(0.642)
0.746
NS
1.031(0.855-1.244)
519(0.649)
CC
145(0.363)
223(0.372)
0.768
NS
0.961(0.739-1.250)
CT
201(0.503)
283(0.472)
0.339
NS
1.131(0.878-1.457)
TT
54(0.135)
94(0.157)
0.345
NS
0.840(0.585-1.206)
C
491(0.614)
729(0.608)
0.779
NS
1.027(0.855-1.233)
T
309(0.386)
471(0.392)
0.779
NS
0.974(0.811-1.170)
CC
3(0.008)
6(0.010)
0.682
NS
0.748(0.186-3.009)
EP
rs2248359
1.328(1.002-1.761)
0.925(0.773-1.106)
TG
G
CYP24A1
OR(95%CI)
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rs12785878
case
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DHCR7
Genotype
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SNPs
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Gene
CT
86(0.215)
103(0.172)
0.086
NS
1.322(0.960-1.819)
TT
311(0.778)
491(0.818)
0.112
NS
0.776(0.567-1.062)
C
92(0.115)
115(0.096)
0.168
NS
1.226(0.917-1.639)
T
708(0.885)
1085(0.904)
0.168
NS
0.816(0.610-1.090)
*SNP, single-nucleotide polymorphism; OR, odds ratio; CI, confidence interval; freq, frequency; pc, p Bonferroni correction
S0002-9394(13)00706-X
DOI:
10.1016/j.ajo.2013.10.010
Reference:
AJOPHT 8711
To appear in:
American Journal of Ophthalmology
Received Date: 12 August 2013 Revised Date:
18 October 2013
Accepted Date: 18 October 2013
Please cite this article as: Fang J, Hou S, Xiang Q, Qi J, Yu H, Shi Y, Zhou Y, Kijlstra A, Yang P, Polymorphisms in genetics of Vitamin D metabolism confer susceptibility to ocular Behçet disease in a Chinese Han population, American Journal of Ophthalmology (2013), doi: 10.1016/j.ajo.2013.10.010. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Polymorphisms in genetics of Vitamin D metabolism
Chinese Han population
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confer susceptibility to ocular Behçet disease in a
Jing Fang, MMa, Shengping Hou, PhDa, Qin Xiang, MBa, Jian Qi, MMa, Hongsong Yu, PhDa, Yanyun Shi, MMa, Yan Zhou, MBa ,Aize Kijlstra, PhDb, Peizeng Yang, PhDa
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a The First Affiliated Hospital of Chongqing Medical University, Chongqing Key Laboratory of Ophthalmology and Chongqing Eye Institute, Chongqing, P. R. China b University Eye Clinic Maastricht, Maastricht, The Netherlands
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Correspondence to: Professor Peizeng Yang, MD, PhD. The First Affiliated Hospital of Chongqing Medical University, Chongqing Key Laboratory of Ophthalmology and Chongqing Eye Institute, Chongqing, P.R. China,400016; Phone: +8623 89012851; FAX: +8623 89012851; Email: [email protected] Key words Vitamin D; DHCR7; CYP2R1; CYP27B1; CYP24A1; Behçet disease; Vogt-Koyanagi-Harada syndrome; ankylosing spondylitis; pediatric uveitis; anterior uveitis
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short title : Vitamin D genes confer susceptibility to Behçet disease
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Supplemental Material available at AJO.com
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Abstract Purpose: To test whether singlenucleotidepolymorphisms(SNPs) of the four Vitamin D family genes(DHCR7, CYP2R1, CYP27B1 and CYP24A1) previously associated with several autoimmune diseases, are associated with ocular Behçet disease, Vogt-Koyanagi-Harada (VKH) syndrome, acute anterior uveitis with ankylosing spondylitis (AAU+ankylosing spondylitis+) or pediatric uveitis in the Chinese Han population. Design: Prospective case control study Methods: Genotyping was performed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP)and the genotypes were verified with direct sequencing. The first stage study comprised 400 ocular Behçet disease patients, 400 VKH syndrome patients, 218 AAU+ankylosing spondylitis+ patients, 400 pediatric uveitis patients and 600 healthy subjects from Chinese Han populations. The second stage included 427 ocular Behçet disease patients and 1000 healthy Chinese Han subjects. Allele and genotype frequencies were compared between patients and controls using the χ2 test. Results: In the first stage study, only the frequencies of the rs12785878/DHCR7 genotype TT and T allele were significantly higher in ocular Behçet disease patients (p Bonferroni correction (pc) = 0.036; pc = 0.008, respectively) compared with controls among six SNPs. No associations could be detected for VKH, AAU+ankylosing spondylitis+ or pediatric uveitis. A second stage and combined study confirmed the association of rs12785878/DHCR7 TT genotype and T allele with ocular Behçet disease(pc = 3.28E-04, OR = 1.506, 95% CI 1.248 to 1.818; pc = 2.82E-05, OR = 1.339, 95% CI 1.188 to 1.508, respectively). Conclusions: This study provides evidence that the DHCR7 gene is involved in the susceptibility to ocular Behçet disease.
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Introduction Uveitis is an intraocular inflammation that can be caused by infectious as well as non-infectious mechanisms and is one of the main global causes of blindness [1, 2]. Uveitis can be manifested as an isolated ocular disease but can also belong to one of the symptoms of a systemic autoimmune or autoinflammatory diseases, such as sarcoidosis, Behçet disease, Vogt-Koyanagi-Harada (VKH) syndrome or acute anterior uveitis with ankylosing spondylitis (AAU+ ankylosing spondylitis +) [3]. Many of these uveitis syndromes are caused by an aberrant innate or adaptive immune response and have a genetic background [4]. Immunogenetic studies in uveitis have focused on genes of the HLA system and cytokine genes, but little attention has been paid to the genetic control of the interaction between the immune and endocrine system. Within the endocrine system, evidence is accumulating for a role of Vitamin D deficiency in several autoimmune disorders including type 1 diabetes mellitus, multiple sclerosis, Crohn Disease, and rheumatoid arthritis [5-7]. 1,25(OH)2D3, as the final active metabolite of vitamin D, can inhibit Th1 and Th17 cells, which are believed to have an important role in the pathogenesis of autoimmune and autoinflammatory disease [8, 9]. Serum levels of 1,25(OH)2D3 were found to be decreased in uveitis patients with Behçet disease, VKH syndrome and AAU+ ankylosing spondylitis+, suggesting a role for 1,25(OH)2D3 in their pathogenesis [10, 11]. In a recent study using lymphocytes obtained from ocular Behçet disease patients, we showed that vitamin D3 inhibits Th1 and Th17 differentiation but upregulates IL-10 [12]. In view of these findings we decided to investigate the role of polymorphisms in a number of genes involved in the synthesis and metabolism of Vitamin D3 on the development of Behçet disease. As a uveitis control we included patients with other uveitis entities including VKH syndrome, acute anterior uveitis associated with ankylosing spondylitis and patients with pediatric uveitis. It is well known that choosing preliminary candidate (single nucleotide polymorphisms) SNPs is critical for a candidate gene association study. So, our chosen strategy was based on previously described associations in various autoimmune diseases. Nine SNPs involving in four Vitamin D metabolism genes were chosen in this study. These SNPs were reported to have association with one or two autoimmune diseases such as type 1 diabetes mellitus, multiple sclerosis and Autoimmune Addison’s disease in the previous studies[13-16]. Materials and methods Patients and healthy controls study population This was a prospective case-control study. The study protocol was approved by the Ethics Committee of the First Affiliated Hospital of Chongqing Medical University, Chongqing, China (Permit Number: 2009-201008). The tenets of the Declaration of Helsinki were followed in all procedures. Written informed consent was given by all participants for this study. This study has registered in Chinese Clinical Trial Registry. The registration number is ChiCTR-CCC-12002184
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A two-stage case-control association study was performed. The first stage studied group comprised 400 ocular Behçet disease patients, 400 VKH syndrome patients, 218 AAU+ ankylosing spondylitis+ patients, 400 pediatric uveitis patients and 600 healthy subjects who were referred to the Zhongshan Ophthalmic Center, Sun Yat-sen University and the First Affiliated Hospital of Chongqing Medical University, Chongqing, China. The second stage included a total of another 427 ocular Behçet disease patients and another 1000 healthy controls. The analysis of stage one and two is independent. All control subjects, which were collected from April 2005 to February 2013, were matched ethnically and geographically with the patients. The patients with ocular Behçet disease fulfilled the criteria of the International Study Group[17]. The clinical features of these patients were recorded during the whole course of follow-up and summarized in Result part. The diagnosis of VKH syndrome was based on the First International Workshop criteria for VKH syndrome [18]. The clinical characteristics of the patients are presented in Supplementary table 1 (Supplemental Material, available at AJO.com). Pediatric uveitis was defined as uveitis (including anterior uveitis, intermediate uveitis, posterior uveitis and panuveitis) first presenting at an age below 16 years. Juvenile idiopathic arthritis was one of the most extraocular manifestations in pediatric uveitis. Among the 400 pediatric uveitis patients, 7.3% had juvenile idiopathic arthritis. The clinical characteristics of patient groups with pediatric uveitis and acute anterior uveitis with ankylosing spondylitis are shown in Supplementary table 2, 3 (Supplemental Material, available at AJO.com). Pediatric patients with Behçet disease, VKH syndrome or with definite infectious uveitis were excluded. To obtain a comparable genetic background, we strictly chose the cases from Chinese Han descendents.
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Single nucleotide polymorphisms selection and genotyping Based on previous reported disease association studies[13-16], we selected one SNP (rs12785878) of 7- dehydrocholesterolreductase (DHCR7), two SNPS (rs10741657, rs12794714) of Cytochrome p450, subfamily IIR, polypeptide 1 (CYP2R1), two SNPs (rs2248359, rs2762932) of Cytochrome p450, family 24, subfamily A, polypeptide 1 (CYP24A1) and four SNPs (rs10877012, rs118204009, rs118204011, rs118204012) of 25-hydroxyvitamin D3-1-alpha-hydroxylase (CYP27B1) as our candidate SNPs to explore the possible association with ocular Behçet disease ,VKH syndrome, AAU+ ankylosing spondylitis+ and pediatric uveitis. Genomic DNA samples of the healthy controls and patients with ocular Behçet disease, VKH syndrome, AAU+ ankylosing spondylitis+ and pediatric uveitis were extracted with the QIAamp DNA Blood Mini Kit (Qiagen, Valencia, CA). The genotyping success rate for the various SNPs ranged between 97.4% and 98.9%. The primers of rs12785878, rs10741657, rs12794714, rs2248359, rs2762932, rs10877012, rs118204009, rs118204011, rs118204012 for amplifying target DNA sequence by PCR are depicted in Table 1. The formed digestion products were separated on a 5% agarose gel (one SNP: rs2762932) or 4% agarose gel (another eight SNPS) and stained with GoldViewTM (SBS Genetech, Beijing, China). The restriction
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enzymes were purchased from Thermo Fisher Scientific. Using randomly selected subjects (5% of all samples) to check the result of the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method used in our study, direct sequencing was performed by the Majorbio Biotechnology Company (Shanghai, China).
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Statistical analysis The χ2 test was used to estimate whether the data were conform the Hardy-Weinberg principle. The patterns of linkage disequilibrium (LD) of the SNPs (rs10741657, rs12794714) of CYP2R1 and the SNPS of CYP24A1 were compared using Haploview 4.0 respectively, except for the SNP (rs12785878) of DHCR7 and the SNP (rs10877012) of CYP27B1. Direct counting estimated genotype frequencies. The χ2 test using SPSS (version 17.0) was used to compare allele and genotype frequencies between patients and controls. The p values were corrected (pc) with the Bonferroni correction by multiplying with the number of analyses performed. The Bonferroni corrections were based on a number of 18 independent comparisons.
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Results We found a significant association with a polymorphism for the gene encoding DHCR7 in ocular Behçet disease patients, but not in the other uveitis entities studied. No association was found with polymorphisms for the genes encoding CYP2R1, CYP24A1 or CYP27B1.
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Association of the four vitamin D related gene polymorphisms with susceptibility to ocular Behçet Disease Gene polymorphisms were not observed for three SNPs (rs118204009, rs118204011 and rs118204012) of CYP27B1 in the tested Chinese Han population. A total of 400 patients with ocular Behçet disease and 600 healthy controls were genotyped for the other six SNPS: one SNP (rs12785878) of DHCR7, two SNPS (rs10741657, rs12794714) of CYP2R1, two SNPs (rs2248359, rs2762932) of CYP24A1 and one SNP (rs10877012) of CYP27B1. These six SNPs were successfully genotyped and conformed to the Hardy-Weinberg principle in controls. Frequencies of the rs12785878/DHCR7 genotype TT and T allele were significantly higher in ocular Behçet disease patients (pc = 0.036, OR = 1.550, 95% CI 1.169 to 2.055; pc = 0.008, OR = 1.382, 95% CI 1.155 to 1.655, respectively) compared with controls. The uncorrected P value of the GG genotype of rs12785878/DHCR7 was 0.006(<0.05). However, the significance was lost following Bonferroni correction. Then, we replicated the associated the SNP rs12785878/DHCR7 using other cohort including 427 cases and other 1000 controls. The results showed that frequencies of the rs12785878/DHCR7 genotype TT, GG and T allele were significantly higher in ocular Behçet disease patients (pc = 0.012, OR = 1.450, 95% CI 1.124 to 1,871; pc = 0.009, OR = 0.672, 95% CI 0.518 to 0.872; pc = 0.003, OR = 1.270, 95% CI 1.082 to 1.492, respectively) compared with controls. In the combined stage, after combining the number of ocular Behçet disease cases
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(n=827) and healthy controls (n=1600), we confirmed the association with the rs12785878/DHCR7 genotype TT and T allele (pc = 3.28E-04, OR = 1.506, 95% CI 1.248 to 1.818; pc = 2.82E-05, OR = 1.339, 95% CI 1.188 to 1.508, respectively) (Table 2). No significant differences were found between patients with ocular Behçet disease and controls concerning the frequencies of the other five SNPs (Table 2). Among 827 Bechet disease patients, the age at onset (years ± Standard Deviation(SD)) is 31.4±9.6, 100% had uveitis and recurrent oral ulceration, 51.51% had recurrent genital ulceration, 66.75% had skin lesions, 26.12% had positive pathergy test, 23.34% had hypopyon and 15.11% had arthritis. The proportions of anterior uveitis and panuveitis were 14.4% and 85.6% among the 827 Bechet disease patients, respectively. We also studied the association of polymorphisms of the four vitamin D related genes with the clinical features of ocular Behçet disease entities, using stratified analysis. A significantly higher frequency of TT genotype and T allele of rs12785878/DHCR7 was noticed in ocular Behçet disease patients respectively with recurrent genital ulcers and skin lesions (Table 3 and Supplementary table 4) (Supplemental Material, available at AJO.com). There was no significant statistical association concerning the genotypes of the other five SNPs and clinical manifestations in ocular Behçet disease.
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Association of the four vitamin D related gene polymorphisms with susceptibility to Vogt-Koyanagi-Harada syndrome, acute anterior uveitis + ankylosing spondylitis+ or pediatric uveitis A total of 600 healthy controls, 400 VKH syndrome patients, 218 AAU+ ankylosing spondylitis + patients, and 400 pediatric uveitis patients were genotyped for the six vitamin D related SNPS: one SNP (rs12785878) of DHCR7, two SNPS (rs10741657, rs12794714) of CYP2R1, two SNPs (rs2248359, rs2762932) of CYP24A1 and one SNP (rs10877012) of CYP27B1. The six SNPs were successfully genotyped and conformed to Hardy-Weinberg expectation in controls. The genotype and allele frequencies of the six SNPs examined in healthy controls, VKH syndrome patients, AAU+ ankylosing spondylitis+ patients and pediatric uveitis patients are summarized in supplementary table 5,6,7 (Supplemental Material, available at AJO.com) respectively. No differences were found for the genotype or allele frequencies of the six SNPs between the three diseases and healthy controls except for rs12785878/DHCR7 in pediatric uveitis and rs10877012/CYP27B1 in AAU+ ankylosing spondylitis+. The P value of the GT genotype of rs12785878/DHCR7 in pediatric uveitis was 0.022(<0.05) and that of the GG genotype of rs10877012/CYP27B1 in AAU+ ankylosing spondylitis+ was 0.011(<0.05). After Bonferroni correction, the significance of the investigated genotypes with both uveitis entities was lost. No significant statistical association was found for the six SNPs genotypes and clinical manifestations in VKH syndrome, AAU+ ankylosing spondylitis+ or pediatric uveitis. Discussion The role of vitamin D related gene polymorphisms in the risk of developing uveitis
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was investigated in this study and revealed that rs12785878/DHCR7 was significantly associated with Behçet disease but not with other uveitis entities investigated. The role of genes involved in the regulation and synthesis of vitamin D with uveitis has not yet been reported before. Earlier studies investigated eight SNPs of the vitamin D binding protein and suggested an association of rs4752 with the occurrence of uveitis in Korean ankylosing spondylitis patients [19] but in our view significance would be lost if the data had been corrected for multiple comparisons. The vitamin D binding protein is a highly polymorphic protein involved in the transport of vitamin D but was not yet included in our studies. We focused on a set of genes involved in the synthesis of vitamin D that have been shown to affect the risk of certain immune related disorders [20]. One of the genes we investigated included DHCR7, which controls vitamin D levels. The 7-dehydrocholesterol reductase, which is encoded by DHCR7, can convert 7-dehydrocholesterol (7-DHC) to cholesterol, thus reducing the substratum from the synthetic pathway of vitamin D3, which is a precursor of 25-OH D3 [21]. Mutations in DHCR7 have been shown to influence the serum vitamin D level [22, 23]. Wang et al. [21] recently reported that variants involved in Vitamin D synthesis and metabolism including rs127858 /DHCR7 could identify individuals who were at risk of vitamin D deficiency. These findings are in line with the observations that Vitamin D levels are lower in ocular Behçet disease patients [10] and that Vitamin D3 can inhibit Th1 and Th17 lymphocyte differentiation in Behçet disease [12]. In view of the findings mentioned above, we investigated DHCR7 gene susceptibility to uveitis. The selection of SNPs was principally based on previous studies [14, 21]. A similar association was recently reported for rs12785878/DHCR7 in a British cohort of type 1 diabetes mellitus patients [14]. We failed to find an association of the Vitamin D SNPs with susceptibility to VKH syndrome, AAU+ ankylosing spondylitis+ or pediatric uveitis. The discrepancy between ocular Behçet disease and the other uveitis entities investigated may be due to the fact that the pathogenetic mechanisms for Behçet disease are unique and differ from these other three ocular inflammatory diseases. Evidence is mounting that Behçet disease is an autoinflammatory disease caused by an aberrant response against infectious agents[24]. How vitamin D controls the susceptibility to ocular Behçet disease is not clear but vitamin D synthesis and metabolism can either affect the immune response, but may on the other hand also be involved in bacterial colonization [25]. During recent years three other vitamin D related genes (CYP27B1, CYP2R1, CYP24A1) have emerged as candidate susceptibility factors for autoimmune disease [13-16] . A role for CYP27B1 had been suggested in studies involving British, Canadian and Swedish patients with multiple sclerosis [14, 15, 26]. Similarly, Cooper et al.[14] revealed an association between the two Vitamin D related genes - CYP27B1 (rs10877012) and CYP2R1 (rs10741657 and rs12794714) - and type 1 diabetes patients in British patients. Sawcer et al.[16] showed that the SNPs (rs2248359, rs762932) of the CYP24A1 gene had an influence on the risk of multiple sclerosis in European patients. We examined the association of polymorphisms in CYP27B1,
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CYP2R1, CYP24A1 genes and did not find an association with the four chosen uveitis entities in a Chinese Han population. Similar to our results, no association with these three vitamin D related gene polymorphisms has been reported in multiple sclerosis patients from the US [27]. There are a number of limitations in our study. Our study indicates that the rs12785878/DHCR7 may play a role in the susceptibility for ocular Behçet disease patients in a Chinese Han population. Although we have put forward a hypothesis, the exact mechanism by which rs12785878/DHCR7 exerts its role in the pathogenesis of Behçet disease is not completely understood. The fact that we failed to find an association with Vitamin D genes such as CYP24A1, CYP27B1 and CYP2R1, does not rule out the possibility that other relevant Vitamin D genes polymorphisms can be associated with these four uveitis entities. More studies are needed to clarify this issue. Furthermore we did not test the Vitamin D status in our patients. Earlier studies in Han Chinese have however shown that DHCR7 variants are associated with Vitamin D status [23]. In conclusion, our study for the first time reports an association between the polymorphisms of rs12785878/DHCR7, but not rs10741657, rs12794714/CYP2R1, rs2248359, rs2762932/CYP24A1 and rs10877012/CYP27B1, with susceptibility to ocular Behçet disease in a Chinese Han population.
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Acknowledgments All authors have completed and submitted the ICMJE form for disclosure of potential conflicts of interest and none were reported. Financial disclosures: This study was supported, in part, by Chongqing Key Laboratory of Ophthalmology (CSTC, 2008CA5003), Fund for PAR-EU Scholars Program, Key Project of Natural Science Foundation(81130019), Key Project of Health Bureau of Chongqing(2012-1-003), National Basic Research Program of China (973 Program) (2011CB510200) and Research Fund for the Doctoral Program of Higher Education of China (20115503110-002). Contributions of authors: Design and conduct of the study (PY, JF); Collection, management, analysis, and interpretation of data (JF, SH, QX, JQ, HY, YS, YZ); and Preparation, review, or approval of manuscript (JF, SH, AK, PY) References 1.
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Do J, Kwon S, Park S, Lee ES. Effects of vitamin D on expression of Toll-like receptors of monocytes
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12. Tian Y, Wang C, Ye Z, Xiao X, Kijlstra A, Yang P. Effect of 1, 25-Dihydroxyvitamin D3 on Th17 and Th1 response in patients with Behcet's disease. Invest Ophthalmol Vis Sci 2012;53(10):6434-6441. 13. Lopez ER, Zwermann O, Segni M, et al. A promoter polymorphism of the CYP27B1 gene is associated with Addison's disease, Hashimoto's thyroiditis, Graves' disease and type 1 diabetes mellitus in Germans. Eur J Endocrinol 2004;151(2):193-197.
14. Cooper JD, Smyth DJ, Walker NM, et al. Inherited variation in vitamin D genes is associated with predisposition to autoimmune disease type 1 diabetes. Diabetes 2011;60(5):1624-1631. 15. Ramagopalan SV, Dyment DA, Cader MZ, et al. Rare variants in the CYP27B1 gene are associated
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with multiple sclerosis. Ann Neurol 2011;70(6):881-886. 16. Sawcer S, Hellenthal G, Pirinen M, et al. Genetic risk and a primary role for cell-mediated immune mechanisms in multiple sclerosis. Nature 2011;476214-219. 17. Criteria for diagnosis of Behcet’s disease. International Study Group for Behçet's Disease. Lancet 1990;335(8697):1078-1080.
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18. Read RW, Holland GN, Rao NA, et al. Revised diagnostic criteria for Vogt-Koyanagi-Harada disease: report of an international committee on nomenclature. Am J Ophthalmol 2001;131(5):647-652. 19. Jung KH, Kim TH, Sheen DH, et al. Associations of vitamin d binding protein gene polymorphisms with the development of peripheral arthritis and uveitis in ankylosing spondylitis. J Rheumatol
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21. Wang TJ, Zhang F, Richards JB, et al. Common genetic determinants of vitamin D insufficiency: a genome-wide association study. Lancet 2010;376(9736):180-188. 22. Porter FD. Malformation syndromes due to inborn errors of cholesterol synthesis. J Clin Invest 2002;110(6):715-724. 23. Zhang Y, Wang X, Liu Y, et al. The GC, CYP2R1 and DHCR7 genes are associated with vitamin D levels in northeastern Han Chinese children. Swiss Med Wkly 2012;142w13636. 24. Zierhut M, Mizuki N, Ohno S, et al. Immunology and functional genomics of Behcet's disease. Cell Mol Life Sci 2003;60(9):1903-1922. 25. Chalmers JD, McHugh BJ, Docherty C, Govan JRW, Hill AT. Vitamin-D deficiency is associated with
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chronic bacterial colonisation and disease severity in bronchiectasis. Thorax 2013;68(1):39-47. 26. Sundqvist E, Bäärnhielm M, Alfredsson L, Hillert J, Olsson T, Kockum I. Confirmation of association between multiple sclerosis and CYP27B1. Eur J Hum Genet 2010;18(12):1349-1352. 27. Simon KC, Munger KL, Yang X, Ascherio A. Polymorphisms in vitamin D metabolism related genes
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Table 1. Primers and restriction enzymes used for restricted fragment length polymorphisms analysis of the DHCR7, CYP27B1, CYP2R1, and CYP24A1 gene SNP ID
Primers
DHCR7
rs12785878
5' ACCACCTTCAAATAGGGCTGT 3' 5' CAGCAGACAGGACATGAGGAT 3'
CYP27B1
rs10877012
5’ AACATAGTCGAACTGTCTCTAC 3'
rs118204009
5' T GTGCTTTGCAACCTAGACTGT 3'
rs118204011
5' G GAAGTTTTCTGGGGCTACTTT 3' 5' TTCACATGTTTTTCAGGTGTCC 3'
CYP2R1
rs10741657 rs12794714
CYP24A1
rs2248359
279bp
MluCI
248bp
MnLI
288bp
5' ATGCGCACTCTCTCCTCAAC
3'
5' CTCTGTCCTGGGACTCACCTT
3'
5' G GGAAGAGCAATGACATGGA
3'
5' G CCCTGGAAGACTCATTTTG
3'
5' CCATAAGTCCAACCAGGAAGG
3'
5' AGCTTTGGAGAGCTGAAGA GG
3'
5' TTCTACACCGACTACCTTGTGC
3'
5' CAAAAATCTCCAACTTCCGTTC
3'
5'CCTACATACTAACTGTGATTCTAA GGAGGT 3' 5'GGCCAATGTGGTCATGATGATAAT 3'
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BsmI
3'
*SNP, single-nucleotide polymorphism
(195+103)
281bp
5' TT CTCTGCTATCTCCCTGCTTC
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rs2762932
298bp
BssSI
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rs118204012
HinfI
271bp (151+119)
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5’ TTCAATTCCAGAACTTCAGAGC 3'
Restriction enzyme TaqI
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Gene
(151+105+32) FokI
299bp(161 +137)
SacII
191bp(134+57)
BstEII
204bp(176+28)
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Table 2. Polymorphisms of DHCR7, CYP27B1, CYP2R1, and CYP24A1 genes in ocular Behçet Disease
rs12785878
b
Replication
Combined
CYP27B1 (rs10877012)
First
c
Control n(freq)
P value
Pc value
OR(95%CI)
TT
129(0.323)
141(0.235)
0.002
0.036
1.550(1.169-2.055)
TG
201(0.503)
310(0.517)
0.661
NS
0.945(0.734-1.217)
GG
70(0.175)
149(0.248)
0.006
T
459(0.574)
592(0.493)
4.18E-04
G
341(0.426)
608(0.507)
4.18E-04
TT
127(0.297)
226(0.226)
0.004
TG
195(0.457)
480(0.480)
0.419
CYP24A1 (rs2762932)
First
First
1.450(1.124-1.871)
1.257
0.911(0.725-1.143)
294(0.294)
0.003
0.009
0.672(0.518-0.872)
932(0.466)
0.003
0.003
1.270(1.082-1.492)
G
405(0.474)
1068(0.534)
0.003
0.003
0.787(0.670-0.924)
TT
256(0.310)
367(0.229)
1.82E-05
3.28E-04
1.506(1.248-1.818)
TG
396(0.479)
790(0.494)
0.486
8.750
0.942(0.796-1.114)
GG
175(0.212)
T
908(0.549)
G
746(0.451)
TT
151(0.378)
TG
199(0.498)
GG
50(0.125)
T
501(0.626)
G
299(0.374)
GG
EP
443(0.277)
4.69E-04
0.008
0.701(0.574-0.856)
1524(0.476)
1.56E-06
2.82E-05
1.339(1.188-1.508)
1676(0.524)
1.56E-06
2.82E-05
0.747(0.663-0.842)
247(0.412)
0.28
NS
0.867(0.669-1.123)
284(0.473)
0.454
NS
1.102(0.855-1.419)
69(0.115)
0.632
NS
1.099(0.746-1.621)
778(0.648)
0.314
NS
0.909(0.755-1.095)
422(0.352)
0.314
NS
1.100(0.914-1.325)
52(0.130)
79(0.132)
0.939
NS
0.985(0.677-1.434)
197(0.493)
281(0.468)
0.454
NS
1.102(0.855-1.419)
151(0.378)
240(0.400)
0.475
NS
0.910(0.701-1.180)
301(0.376)
439(0.366)
0.636
NS
1.046(0.869-1.258)
499(0.624)
761(0.634)
0.636
NS
0.956(0.795-1.151)
AA
48(0.120)
91(0.152)
0.156
NS
0.763(0.524-1.110)
AG
193(0.483)
248(0.413)
0.031
NS
1.323(1.026-1.707)
GG
159(0.398)
261(0.435)
0.239
NS
0.857(0.663-1.108)
A
289(0.361)
430(0.358)
0.894
NS
1.013(0.841-1.220)
G
511(0.639)
770(0.642)
0.894
NS
0.987(0.820-1.190)
CC
131(0.328)
223(0.372)
0.152
NS
0.823(0.631-1.075)
CT
195(0.488)
283(0.472)
0.623
NS
1.066(0.827-1.373)
AC C
CYP24A1 (rs2248359)
0.012
105(0.246)
G
First
0.723(0.604-0.866)
449(0.526)
A
CYP2R1 (rs12794714)
0.008
T
AG First
0.642(0.467-0.882)
1.382(1.155-1.655)
GG
AA CYP2R1 (rs10741657)
NS
0.008
RI PT
(DHCR7)
Case n(freq)
SC
First
a
Genotype Allele
M AN U
Stage
TE D
SNPs
TT
74(0.185)
94(0.157)
0.24
NS
1.222(0.874-1.708)
C
457(0.571)
729(0.608)
0.106
NS
0.861(0.718-1.032)
T
343(0.429)
471(0.392)
0.106
NS
1.162(0.969-1.393)
CC
6(0.015)
6(0.010)
0.477
NS
1.508(0.483-4.708)
CT
61(0.153)
103(0.172)
0.423
NS
0.868(0.615-1.227)
TT
333(0.833)
491(0.818)
0.564
NS
1.103(0.790-1.542)
C
73(0.091)
115(0.096)
0.731
NS
0.947(0.696-1.289)
T
727(0.909)
1085(0.904)
0.731
NS
1.056(0.776-1.436)
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
*SNP, single-nucleotide polymorphism; OR, odds ratio; CI, confidence interval; NS, no significant different; freq, frequency; pc, p Bonferroni correction. *a: First stage(stage one) , case: control = 400: 600 b: Replication stage(stage two), case: control = 427: 1000 c: Combined stage(a + b), case: control = 827: 1600
ACCEPTED MANUSCRIPT
Table 3. Polymorphisms of the DHCR7 gene in patients with extraocular features of Behçet disease
Clinical features
Genotype
Behçet disease patients (%)
Controls (%)
Pc
TT
145(0.340)
367(0.229)
6.76E-05
genital ulcer
TG
187(0.439)
790(0.494)
NS
95(0.221)
443(0.277)
T
477(0.56)
1524(0.476)
G
375(0.44)
1676(0.524)
TT
172(0.312)
367(0.229)
NS
1.399(1.202-1.629)
1.01E-04
0.715(0.614-0.832)
0.003
1.521(1.227-1.885)
TG
252(0.457)
790(0.494)
GG
128(0.232)
443(0.277)
NS
T
596(0.54)
1524(0.476)
0.002
1.290(1.125-1.480) 0.775(0.676-0.889)
NS
508(0.46)
1676(0.524)
0.002
TT
65(0.301)
367(0.229)
NS
pathergy test
TG
100(0.463)
367(0.229)
NS
GG
51(0.236)
790(0.494)
NS
Hypopyon
Arthritis
T
230(0.532)
443(0.277)
NS
G
202(0.468)
1524(0.476)
NS
TT
58(0.301)
367(0.229)
NS
TG
93(0.482)
790(0.494)
NS
GG
42(0.218)
443(0.277)
NS
T
209(0.542)
1524(0.476)
NS
G
177(0.459)
1676(0.524)
NS
27(0.216)
367(0.229)
NS
64(0.512)
790(0.494)
NS
TT TG GG
34(0.272)
443(0.277)
NS
118(0.472)
1524(0.476)
NS
132(0.528)
1676(0.524)
NS
TT
30(0.252)
367(0.229)
NS
TG
57(0.479)
790(0.494)
NS
GG
32(0.269)
443(0.277)
NS
T
117(0.492)
1524(0.476)
NS
G
121(0.508)
1676(0.524)
NS
TT
165(0.233)
367(0.229)
NS
TG
341(0.482)
790(0.494)
NS
GG
202(0.285)
443(0.277)
NS
T
671(0.474)
1524(0.476)
NS
G
745(0.526)
1676(0.524)
NS
T
AC C
EP
G anterior uveitis
panuveitis
M AN U
G Positive
rs12785878
1.727(1.370-2.178)
1.01E-04
SC
Skin lesions
GG
OR (95%CI)
RI PT
Recurrent
TE D
SNP
SNP, single-nucleotide polymorphism; OR, odds ratio; CI, confidence interval; NS, no significant different
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
Jing Fang, MD, is currently studying at the First Affiliated Hospital of Chongqing Medical University, Chongqing Key Laboratory of Ophthalmology and Chongqing Eye Institute, Chongqing, P. R. China. Before MD, he had been a pediatric oculist for eight years in Department of Ophthalmology, Children's Hospital, Chongqing Medical University, Chongqing, P. R. China. His research interests are in ophthalmic genetics, uveitis, and optometry.
ACCEPTED MANUSCRIPT
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Prof. Peizeng Yang is working at the First Affiliated Hospital of Chongqing Medical University. He is an international council representative of International Ocular Inflammation Society, founding executive member of Asia-Pacific Intraocular Inflammation Study Group, member of the International Uveitis Study Group, council member of International Society for Behçet’s Disease and vice president of Chinese Ophthalmological Society. He is mainly engaging in the study on diagnosis and treatment of uveitis and the mechanisms involved in this disease.
ACCEPTED MANUSCRIPT
manuscript number : AJO-13-865R2 Polymorphisms in genetics of Vitamin D metabolism confer susceptibility to ocular Behçet disease in a Chinese Han population. Jing Fang, MM, Shengping Hou, PhD, Qin Xiang, MB, Jian Qi, MM, Hongsong Yu, PhD, Yanyun Shi, MM, Yan Zhou, MB ,Aize Kijlstra, PhD, Peizeng Yang, PhD
AC C
EP
TE D
M AN U
SC
RI PT
Vitamin D family genes have been reported to be associated with several autoimmune diseases. We performed a prospective case control study to test whether these genes are associated with four kinds of uveitis by genotyping. We found the frequencies of rs12785878/DHCR7 genotype TT and T allele were significantly higher in ocular Behçet disease patients compared with controls. This study provides evidence that the DHCR7 gene is involved in the susceptibility to ocular Behçet disease.
ACCEPTED MANUSCRIPT
Supplementary table 1. Clinical features of the investigated Vogt-Koyanagi-Harada syndrome patients Patients with VKH syndrome N (total=400)
%
34.5±8.2
Uveitis
400
100%
Stiffness of the neck and back
52
13.0%
Headache
204
51.0%
Scalp sensitivity
40
10.0%
Tinnitus
156
39.0%
Alopecia
84
21.0%
Poliosis
104
Vitiligo
100
SC
Age at onset (years±SD)
26.0%
25.0%
EP
TE D
M AN U
*SD, Standard Deviation; VKH syndrome, Vogt-Koyanagi-Harada syndrome
AC C
RI PT
Clinical features
ACCEPTED MANUSCRIPT
Supplementary table 2. Clinical features of the investigated pediatric uveitis patients Patients with pediatric uveitis N (total=400)
%
Age at onset (years±SD)
14.08±8.4
Uveitis
400
100%
Juvenile idiopathic arthritis
29
7.3%
Retinal vasculitis
28
7.0%
AC C
EP
TE D
M AN U
SC
*SD, Standard Deviation
RI PT
Clinical features
ACCEPTED MANUSCRIPT
Supplementary table 3. Clinical features of the investigated ankylosing spondylitis patients Patients with ankylosing spondylitis N (total=218)
%
Age at onset (years±SD)
39.2±10.0
uveitis
218
100%
HLA-B27(+)
207
94.8%
HLA-B27(-)
11
5.2%
AC C
EP
TE D
M AN U
SC
*SD, Standard Deviation
RI PT
Clinical features
ACCEPTED MANUSCRIPT
Supplementary table 4. Criteria for diagnosis of Behçet disease* Recurrent Oral ulceration
Minor aphthous, major aphthous, or herpetiform ulceration observed by physician or patient, which recurred at least 3 times in one 12-month period
RI PT
Plus 2 of: Recurrent genital ulceration Eye lesions
M AN U
SC
Skin lesions
Aphthous ulceration or scarring, observed by physician or patient Anterior uveitis, posterior uveitis, or cells in vitreous on slit lamp examination; or Retinal vasculitis observed by ophthalmologist Erythema nodosum observed by physician or patient, pseudofolliculitis, or papulopustular lesions; or Acneiform nodules observed by physician in postadolescent patients not on corticosteroid treatment Read by physician at 24-48 h.
Positive pathergy test
AC C
EP
TE D
(Findings applicable only in absence of other clinical explanations.) *The criteria of diagnosis comes from “Criteria for diagnosis of Behçet’s disease. International Study Group for Behçet's Disease.” Lancet 1990;335(8697):1078-1080.
ACCEPTED MANUSCRIPT
Supplementary table 5. Polymorphism of DHCR7, CYP27B1, CYP2R1, and CYP24A1 genes in Vogt-Koyanagi-Harada syndrome
CYP27B1
CYP2R1
rs10877012
rs10741657
rs12794714
Control
Allele
n(freq)
n(freq)
TT
98(0.245)
TG
212(0.530)
AC C
rs2762932
OR(95%CI)
141(0.235)
0.716
NS
1.056(0.786-1.420)
310(0.517)
0.679
NS
1.055(0.819-1.359)
90(0.225)
149(0.248)
0.397
NS
0.879(0.652-1.185)
T
408(0.510)
592(0.493)
0.465
NS
1.069(0.894-1.278)
G
392(0.490)
608(0.507)
0.465
NS
0.936(0.782-1.119)
TT
160(0.400)
247(0.412)
0.713
NS
0.953(0.736-1.233)
TG
180(0.450)
284(0.473)
0.469
NS
0.910(0.706-1.174)
GG
60(0.150)
69(0.115)
0.106
NS
1.358(0.936-1.970)
T
500(0.625)
778(0.648)
0.287
NS
0.904(0.751-1.089)
G
300(0.375)
422(0.352)
0.287
NS
1.106(0.919-1.332)
AA
41(0.103)
79(0.132)
0.164
NS
0.753(0.505-1.124)
AG
179(0.448)
281(0.468)
0.517
NS
0.919(0.713-1.185)
GG
180(0.450)
240(0.400)
0.117
NS
1.227(0.950-1.585)
A
261(0.326)
439(0.366)
0.069
NS
0.839(0.695-1.014)
G
539(0.674)
761(0.634)
0.069
NS
1.191(0.986-1.439)
AA
58(0.145)
91(0.152)
0.772
NS
0.949(0.664-1.355)
AG
189(0.473)
248(0.413)
0.065
NS
1.271(0.985-1.640)
GG
153(0.383)
261(0.435)
0.099
NS
0.805(0.621-1.042)
A
305(0.381)
430(0.358)
0.298
NS
1.103(0.917-1.328)
770(0.642)
0.298
NS
0.906(0.753-1.091)
495(0.619)
CC
151(0.378)
223(0.372)
0.852
NS
1.025(0.789-1.331)
CT
185(0.463)
283(0.472)
0.776
NS
0.964(0.748-1.242)
TT
64(0.160)
94(0.157)
0.887
NS
1.025(0.725-1.450)
C
487(0.609)
729(0.608)
0.955
NS
1.005(0.837-1.207)
T
313(0.391)
471(0.392)
0.955
NS
0.995(0.828-1.195)
CC
4(0.010)
6(0.010)
1.000
NS
1.000(0.280-3.566)
EP
rs2248359
Pc value
GG
G
CYP24A1
P value
RI PT
rs12785878
case
SC
DHCR7
Genotype
M AN U
SNPs
TE D
Gene
CT
71(0.178)
103(0.172)
0.812
NS
1.041(0.746-1.453)
TT
325(0.813)
491(0.818)
0.816
NS
0.962(0.695-1.332)
C
79(0.099)
115(0.096)
0.829
NS
1.034(0.765-1.398)
T
721(0.901)
1085(0.904)
0.829
NS
0.967(0.716-1.308)
*SNP, single-nucleotide polymorphism; OR, odds ratio; CI, confidence interval; NS, no significant different; freq, frequency; pc, p Bonferroni correction
ACCEPTED MANUSCRIPT
Supplementary table 6. Polymorphism of DHCR7, CYP27B1, CYP2R1, and CYP24A1 genes in ocular ankylosing spondylitis
CYP27B1
CYP2R1
rs10877012
rs10741657
rs12794714
Control
Allele
n(freq)
n(freq)
TT
48(0.220)
TG
102(0.468)
AC C
rs2762932
OR(95%CI)
141(0.235)
0.657
NS
0.919(0.634-1.333)
310(0.517)
0.217
NS
0.823(0.603-1.122)
68(0.312)
149(0.248)
0.069
NS
1.372(0.976-1.930)
T
198(0.454)
592(0.493)
0.161
NS
0.854(0.686-1.065)
G
238(0.546)
608(0.507)
0.161
NS
1.170(0.939-1.458)
TT
82(0.376)
247(0.412)
0.36
NS
0.862(0.627-1.185)
TG
96(0.440)
284(0.473)
0.403
NS
0.876(0.641-1.196)
GG
40(0.183)
69(0.115)
0.011
NS
1.729(1.131-2.645)
T
260(0.596)
778(0.648)
0.053
NS
0.801(0.640-1.004)
G
176(0.404)
422(0.352)
0.053
NS
1.248(0.996-1.563)
AA
37(0.170)
79(0.132)
0.168
NS
1.348(0.881-2.063)
AG
86(0.394)
281(0.468)
0.06
NS
0.740(0.540-1.014)
GG
95(0.436)
240(0.400)
0.358
NS
1.159(0.847-1.585)
A
160(0.367)
439(0.366)
0.966
NS
1.005(0.800-1.262)
G
276(0.633)
761(0.634)
0.966
NS
0.995(0.793-1.249)
AA
23(0.106)
91(0.152)
0.092
NS
0.660(0.406-1.073)
AG
97(0.445)
248(0.413)
0.418
NS
1.138(0.832-1.555)
GG
98(0.450)
261(0.435)
0.711
NS
1.061(0.777-1.449)
A
143(0.328)
430(0.358)
0.255
NS
0.874(0.693-1.102)
770(0.642)
0.255
NS
1.144(0.907-1.443)
293(0.672)
CC
78(0.358)
223(0.372)
0.716
NS
0.942(0.682-1.301)
CT
105(0.482)
283(0.472)
0.763
NS
1.041(0.763-1.420)
TT
35(0.161)
94(0.157)
0.893
NS
1.030(0.674-1.572)
C
261(0.599)
729(0.608)
0.745
NS
0.964(0.770-1.205)
T
175(0.401)
471(0.392)
0.745
NS
1.038(0.830-1.298)
CC
2(0.009)
6(0.010)
0.916
NS
0.917(0.184-4.576)
EP
rs2248359
Pc value
GG
G
CYP24A1
P value
RI PT
rs12785878
case
SC
DHCR7
Genotype
M AN U
SNPs
TE D
Gene
CT
41(0.188)
103(0.172)
0.586
NS
1.118(0.749-1.668)
TT
175(0.803)
491(0.818)
0.612
NS
0.903(0.610-1.338)
C
45(0.103)
115(0.096)
0.657
NS
1.086(0.755-1.562)
T
391(0.897)
1085(0.904)
0.657
NS
0.921(0.640-1.325)
*SNP, single-nucleotide polymorphism; OR, odds ratio; CI, confidence interval; NS, no significant different; freq, frequency; pc, p Bonferroni correction.
ACCEPTED MANUSCRIPT
Supplementary table 7. Polymorphism of DHCR7, CYP27B1, CYP2R1, and CYP24A1 genes in pediatric uveitis
CYP27B1
CYP2R1
rs10877012
rs10741657
rs12794714
Control
P
Pc
Allele
n(freq)
n(freq)
value
value
TT
101(0.253)
141(0.235)
0.527
NS
1.100(0.819-1.476)
TG
177(0.443)
310(0.517)
0.022
NS
0.743(0.576-0.957)
GG
122(0.305)
149(0.248)
0.048
NS
T
379(0.474)
592(0.493)
0.391
NS
G
421(0.526)
608(0.507)
0.391
NS
AC C
rs2762932
1.082(0.904-1.294)
TT
167(0.418)
247(0.412)
0.854
NS
183(0.458)
284(0.473)
0.623
NS
GG
50(0.124)
69(0.115)
0.632
NS
1.099(0.746-1.621)
1.024(0.792-1.324)
0.938(0.728-1.209)
T
517(0.646)
778(0.648)
0.924
NS
0.991(0.822-1.195)
G
283(0.354)
422(0.352)
0.924
NS
1.009(0.837-1.217)
AA
54(0.135)
79(0.132)
0.879
NS
1.029(0.710-1.493)
AG
177(0.443)
281(0.468)
0.422
NS
0.901(0.699-1.162)
GG
169(0.423)
240(0.400)
0.478
NS
1.097(0.849-1.419)
A
285(0.356)
439(0.366)
0.662
NS
0.959(0.796-1.156)
G
515(0.644)
761(0.634)
0.662
NS
1.042(0.865-1.256)
AA
63(0.158)
91(0.152)
0.802
NS
1.046(0.737-1.483)
AG
155(0.388)
248(0.413)
0.415
NS
0.898(0.693-1.163)
GG
182(0.455)
261(0.435)
0.533
NS
1.084(0.841-1.399)
A
281(0.351)
430(0.358)
0.746
NS
0.970(0.804-1.169)
770(0.642)
0.746
NS
1.031(0.855-1.244)
519(0.649)
CC
145(0.363)
223(0.372)
0.768
NS
0.961(0.739-1.250)
CT
201(0.503)
283(0.472)
0.339
NS
1.131(0.878-1.457)
TT
54(0.135)
94(0.157)
0.345
NS
0.840(0.585-1.206)
C
491(0.614)
729(0.608)
0.779
NS
1.027(0.855-1.233)
T
309(0.386)
471(0.392)
0.779
NS
0.974(0.811-1.170)
CC
3(0.008)
6(0.010)
0.682
NS
0.748(0.186-3.009)
EP
rs2248359
1.328(1.002-1.761)
0.925(0.773-1.106)
TG
G
CYP24A1
OR(95%CI)
RI PT
rs12785878
case
SC
DHCR7
Genotype
M AN U
SNPs
TE D
Gene
CT
86(0.215)
103(0.172)
0.086
NS
1.322(0.960-1.819)
TT
311(0.778)
491(0.818)
0.112
NS
0.776(0.567-1.062)
C
92(0.115)
115(0.096)
0.168
NS
1.226(0.917-1.639)
T
708(0.885)
1085(0.904)
0.168
NS
0.816(0.610-1.090)
*SNP, single-nucleotide polymorphism; OR, odds ratio; CI, confidence interval; freq, frequency; pc, p Bonferroni correction