Abstract
A recent genome-wide association analysis of psoriasis identified IL12B and IL23R as significantly associated with psoriasis. Here we report association test results of a Thai cohort consisting of 206 psoriasis cases and 114 controls. The IL23R SNPs rs7530511 and rs11209026, and IL12B SNPs rs3212227 and rs6887695 were genotyped using Taqman assays. Data were analyzed using a logistic regression model for linear trend of association. One of the IL23R markers, rs7530511, was marginally significant (p=0.017). The other IL23R marker, rs11209026, was not polymorphic. One of the IL12B markers, rs3212227, showed significant association with psoriasis (OR = 1.64, p = 0.0058) while the other, rs6887695, did not (OR = 1.29, p = 0.12). Haplotype analysis of the two IL12B SNPs yielded highly significant association (p=0.00081, OR=1.73). These results show that IL12B is an important genetic factor in psoriasis pathogenesis in the Thai population, with an association strong enough to yield significant confirmatory evidence using a modest sample size. Together with previously reported evidence for IL12B association in Caucasian, Japanese and Chinese psoriatics, our results support the hypothesis that genes encoding components of the IL23-mediated inflammatory pathway are important determinants of psoriasis pathogenesis across multiple racial groups.
Keywords: Psoriasis, genetic association, IL12B, IL23, Thailand, Asia
INTRODUCTION
Psoriasis is a common autoimmune inflammatory skin disease with a multifactorial genetic basis. Genome-wide linkage analyses have identified at least nine psoriasis susceptibility loci in various populations [12]. However, attempts to confirm and fine map these linked loci by linkage and association techniques have yielded reproducibility only for PSORS1 in the major histocompatibility complex (MHC). We showed that HLA-Cw6 is most likely the disease allele at PSORS1 [10]. However, PSORS1 accounts for less than 50% of the familial clustering of psoriasis [16], implying that non-MHC loci remain to be identified. In an effort to identify these loci, Cargill et al. performed a genome-wide association analysis using gene-centric markers, and identified two associated genes: IL12B and IL23R [2]. For IL12B, which encodes the p40 subunit common to the interleukins (IL)-12 and IL-23, the reported association was with the major allele (A-G) of a haplotype defined by SNPs rs3212227 (in the 3′ untranslated region) and rs6887695 (located 60 kb upstream of the gene). For IL23R, the gene encoding a subunit of the IL-23 receptor, the major allele (C-G) of the haplotype defined by exonic SNPs rs7530511 (L310P) and rs11209026 (Q381R) was disease-associated. The association of IL12B with psoriasis was originally reported in a small Japanese study [17]. Associations of IL12B and IL23R with psoriasis have recently been confirmed in additional Caucasian cohorts [1, 8, 11, 13] and in a Chinese cohort [4]. Together with emerging immunologic and pharmacologic data, these findings support an IL23-mediated inflammatory pathway in psoriasis pathogenesis involving IL-17-producing T-cells [7]. In this study we explored genetic association of psoriasis with IL12B and IL23R in a Thai cohort.
MATERIALS AND METHODS
The study samples were collected at the Institute of Dermatology clinic in Bangkok. The case sample consisted of 206 psoriasis cases of Thai ancestry (average and median age at onset: 34 years, age range: birth-81 years, 58% male, 42% female) diagnosed by a single experienced dermatologist (Preya Kullavanijaya). The controls were 114 individuals of Thai ancestry with no family history of psoriasis, collected from the same geographic area as the cases (average age at sampling: 45 years, median age: 44 years, age range: 16–78 years, 42% male, 58% female). All samples were obtained after written informed consent following principles of the Declaration of Helsinki. DNA was prepared from whole blood using previously established methods [9]. The IL23R SNPs rs7530511 and rs11209026, and IL12B SNPs rs3212227 and rs6887695, were genotyped using Taqman assays (Applied Biosystems, Foster City, CA). Data were analyzed for association using logistic regression and p-values were determined by Monte Carlo simulation, as described previously [11]. In all cases, the extra fit afforded by a two-term genotype regression model was not statistically significant, so only results for the simpler one-term linear trend model are discussed here. Posterior probabilities of each 2-SNP IL12B haplotype pair for each individual, which served as weights in regression analysis, were estimated separately for cases and controls by version 2.1 of PHASE [14, 15] with default settings. Confidence intervals for risk allele frequencies were combined over sample strata based on the F-distribution with a rescaling factor [18]. Two weighting schemes were used to combine strata: equal weights and weights proportional to strata sample sizes. Power calculations were carried out with version 3.1 of the G*Power [5] using Fisher’s exact test of allelic association, which is essentially equivalent to the exact ordinal logistic regression test reported here.
RESULTS
None of the SNPs had genotype frequencies that deviated significantly from Hardy-Weinberg equilibrium in controls (exact test p > 0.6). One of the IL23R markers, rs7530511, had only four occurrences of the minor allele but association with the major allele (C) was still marginally significant (p=0.017, Table 1). The other IL23R marker, rs11209026, was not polymorphic in our sample. The IL12B marker rs3212227 showed significant association with psoriasis (OR = 1.64, p =0.0058) while the other, rs6887695, did not (OR = 1.29, p = 0.12). Haplotype analysis of the two IL12B SNPs yielded strongly significant association (global p=0.00086), with the associated A-G haplotype (p = 0.00081, OR = 1.73) being the same as that found in Caucasians (Table 2). Interestingly, frequencies of the IL12B risk alleles and haplotype in Thais and other Eastern Asians are much lower than in Caucasians, whereas the frequencies of IL23R risk alleles and haplotype are substantially higher (Table 3). Similar results were seen when strata were weighted equally rather than in proportion to their sample size (data not shown). These frequency differences hold true for both cases and controls, which results in an effect size for IL12B SNP rs3212227 in Thais (OR = 1.64, 95% CI = 1.14–2.38) that is similar to that in Caucasians (meta-OR = 1.48 and meta-95% CI = 1.37–1.60; for data from Cargill et al. [2], Nair et al. [11], Capon et al. [1], Smith et al. [13]). On the other hand, the effect size for IL23R SNP rs7530511 in Thais (OR = 9.77, 95% CI = 1.21–∞) appears larger than for Caucasians (meta-OR = 1.43, 95% CI = 1.33–1.53), but a much larger sample is needed to robustly estimate this parameter in the Thai population. Although our study provides confirmatory evidence for the association of these two SNPs in Thais, much larger samples would be required to independently establish significance at a genome-wide level. For IL12B SNP rs3212217, a sample size of approximately 1,900 each of cases and controls provides 80% power to detect association for a type I error rate of 1 × 10−7, assuming a population frequency of 0.51 for the risk allele and an OR of 1.50. For IL23R SNP rs7530511, assuming a risk allele frequency of 0.98, enormous samples in excess of 35,000 each of cases and controls would be needed to attain genome-wide significance if the effect size is similar to that of Caucasians (OR = 1.43), and even at a very large OR of 10 (the imprecise estimate of our sample), 3,100 each of cases and controls would be required.
Table 1.
Association of IL12B and IL23R SNPs in Thai psoriatics.
| Frequency in |
||||||
|---|---|---|---|---|---|---|
| Gene | SNP | Genotypea | Cases | Controls | OR (95% CI)b | Pc |
| IL12B | rs3212227 | AA | 0.363 | 0.255 | 1.64 (1.14–2.38) | 0.0058 |
| AC | 0.517 | 0.509 | ||||
| CC | 0.119 | 0.236 | ||||
| rs6887695 | GG | 0.393 | 0.289 | 1.29 (0.94–1.79) | 0.12 | |
| GC | 0.408 | 0.474 | ||||
| CC | 0.199 | 0.237 | ||||
| IL23R | rs7530511 | CC | 1.000 | 0.965 | 9.77 (1.21–∞) | 0.017 |
| CT | 0.000 | 0.035 | ||||
| TT | 0.000 | 0.000 | ||||
| rs11209026 | GG | 1.000 | 1.000 | — | — | |
| GA | 0.000 | 0.000 | ||||
| AA | 0.000 | 0.000 | ||||
For all SNPs, the homozygous risk genotype is given first.
Odds ratios and their 95% confidence intervals for an ordinal logistic model, which are calculated by permutation without recourse to asymptotic assumptions. A median unbiased estimate of the OR for SNP rs7530511 is provided because its conditional maximum likelihood estimate is infinite.
Monte Carlo p-value (based on 10,000 permutations) for likelihood ratio test of linear trend of association using an ordinal logistic regression model.
Table 2.
Association of two-marker haplotypes for the IL12B region in cases and controls.
| Diplotypeb | GlobalPa = 0.00086 |
|||
|---|---|---|---|---|
| Frequency in |
OR (95% CI)c | Pd | ||
| Cases | Controls | |||
| AG/AG | 0.279 | 0.132 | 1.73 (1.25–2.39) | 0.00081 |
| AG/other | 0.469 | 0.487 | ||
| other/other | 0.252 | 0.381 | ||
| CC/CC | 0.085 | 0.132 | 0.81 (0.58–1.15) | 0.24 |
| CC/other | 0.414 | 0.411 | ||
| other/other | 0.501 | 0.457 | ||
| CG/CG | 0.005 | 0.000 | 0.49 (0.29–0.82) | 0.0061 |
| CG/other | 0.163 | 0.306 | ||
| other/other | 0.832 | 0.694 | ||
| AC/AC | 0.005 | 0.019 | 0.78 (0.48–1.26) | 0.31 |
| AC/other | 0.208 | 0.231 | ||
| other/other | 0.788 | 0.751 | ||
Global P-value for Wald test of association of psoriasis and all biallelic diplotypes calculated using a weighted logistic model for linear trend of association. P-value was determined by first generating a set of 10,000 permutational test statistics by randomly shuffling the case and control labels, and then fitting a gamma distribution to the set of permutation test statistics and evaluating its cumulative distribution function.
In each case, the homozygous diplotype for the haplotype being tested is given first; haplotype consists of SNPs rs3212227 and rs6887695.
Odds ratios and their 95% confidence intervals are for the weighted logistic model for linear trend of association, with a robust variance estimator.
P-value for Wald test of association of psoriasis and diplotype using a weighted logistic model for linear trend of association. P-values were determined by simulation as described in footnote a.
Table 3.
Frequencies of risk alleles and haplotypes for IL12B and IL23R among Caucasians, Thais, and Eastern Asians.
| Gene | SNP | Risk allele | Psoriasis Affection | Caucasian |
Thai |
Eastern Asian |
|||
|---|---|---|---|---|---|---|---|---|---|
| No. of indiv./strataa | Frequency (95% CI) b | No. of indiv. a | Frequency (95% CI) b | No. of indiv./strataa | Frequency (95% CI) b | ||||
| IL12B | rs3212227 | A | case | 4163/7 | 0.851 (0.843–0.858) | 201 | 0.622 (0.574–0.668) | 313/2 | 0.617 (0.577–0.655) |
| control | 8931/8 | 0.799 (0.793–0.805) | 106 | 0.509 (0.443–0.576) | 399/4 | 0.540 (0.504–0.575) | |||
| rs6887695 | G | case | 3365/5 | 0.754 (0.744–0.764) | 206 | 0.597 (0.549–0.643) | — | — | |
| control | 6421/6 | 0.683 (0.675–0.691) | 114 | 0.526 (0.462–0.590) | 90/2 | 0.611 (0.534–0.683) | |||
| haplotype | AG | case | 2752/4 | 0.719 (0.707–0.731) | 201 | 0.510 (0.461–0.559) | — | — | |
| control | 3473/5 | 0.637 (0.626–0.649) | 106 | 0.373 (0.310–0.440) | 89/2 | 0.466 (0.391–0.543) | |||
| IL23R | rs7530511 | C | case | 3329/5 | 0.895 (0.887–0.902) | 206 | 1.000 (0.989–1.000) | — | — |
| control | 5130/14 | 0.866 (0.858–0.872) | 114 | 0.982 (0.954–0.995) | 335/21 | 0.978 (0.950–0.982) | |||
| rs11209026 | G | case | 4183/7 | 0.954 (0.949–0.958) | 206 | 1.000 (0.989–1.000) | — | — | |
| control | 7362/16 | 0.933 (0.928–0.937) | 109 | 1.000 (0.979–1.000) | 765/22 | 0.999 (0.985–0.999) | |||
| haplotype | CG | case | 2777/4 | 0.842 (0.832–0.851) | 206 | 1.000 (0.989–1.000) | — | — | |
| control | 3649/13 | 0.791 (0.781–0.800) | 109 | 0.982 (0.952–0.995) | 334/21 | 0.975 (0.946–0.980) | |||
Number of individuals and strata (samples) for the combination of marker, affection and race. Genotypes for Caucasians of European descent were drawn from this study and other case-control studies [1] [2] [11] [13], as well as from the CEU sample of the HapMap project [6] and eight European populations sampled by the Human Genome Diversity Project (HGDP) [3]. The discovery sample of Cargill et al. [2] was excluded to avoid biasing estimates due to the winner’s curse. Genotypes for Thai are from this study, and those for Eastern Asians are from other case-control studies [4] [17] [19], as well as the HCB and JPT samples from HapMap and 19 qualifying population samples from the HGDP.
Mean frequency and 95% confidence interval of the risk allele, combined across strata using the F-distribution with a rescaling factor [18]; weights were proportional to stratum sample size.
DISCUSSION
Our results show that IL12B is an important genetic determinant of psoriasis pathogenesis in the Thai population, with an effect robust enough to yield significant confirmatory evidence of association in a relatively small sample. IL23R also showed modest evidence of association. Much larger samples would be necessary to establish genome-wide levels of significance for the association of these two genes with psoriasis in the Thai population. Together with previously reported evidence for IL12B association in Caucasian, Japanese and Chinese psoriatics, our results support the hypothesis that genes encoding components of the IL-23-mediated inflammatory pathway are important determinants of psoriasis pathogenesis across multiple racial groups, despite significant differences in the prevalence of the risk alleles for IL12B and IL23R in these different populations. Resequencing these loci and/or genotyping additional closely spaced markers followed by haplotype analysis will be required to precisely define the disease-predisposing allele(s).
Acknowledgments
We thank the psoriasis patients and controls who volunteered to participate in this study. This work was supported by awards (R01 AR042742 and R01 AR050511) from the National Institute of Arthritis, Musculoskeletal, and Skin Diseases, National Institutes of Health, and by the Babcock Memorial Trust.
References
- 1.Capon F, Di Meglio P, Szaub J, Prescott NJ, Dunster C, Baumber L, Timms K, Gutin A, Abkevic V, Burden AD, Lanchbury J, Barker JN, Trembath RC, Nestle FO. Sequence variants in the genes for the interleukin-23 receptor (IL23R) and its ligand (IL12B) confer protection against psoriasis. Hum Genet. 2007;122:201–206. doi: 10.1007/s00439-007-0397-0. [DOI] [PubMed] [Google Scholar]
- 2.Cargill M, Schrodi SJ, Chang M, Garcia VE, Brandon R, Callis KP, Matsunami N, Ardlie KG, Civello D, Catanese JJ, Leong DU, Panko JM, McAllister LB, Hansen CB, Papenfuss J, Prescott SM, White TJ, Leppert MF, Krueger GG, Begovich AB. A large-scale genetic association study confirms IL12B and leads to the identification of IL23R as psoriasis-risk genes. Am J Hum Genet. 2007;80:273–290. doi: 10.1086/511051. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 3.Cavalli-Sforza LL. The Human Genome Diversity Project: past, present and future. Nat Rev Genet. 2005;6:333–340. doi: 10.1038/nrg1596. [DOI] [PubMed] [Google Scholar]
- 4.Chang YT, Chou CT, Yu CW, Lin MW, Shiao YM, Chen CC, Huang CH, Lee DD, Liu HN, Wang WJ, Tsai SF. Cytokine gene polymorphisms in Chinese patients with psoriasis. Br J Dermatol. 2007;156:899–905. doi: 10.1111/j.1365-2133.2007.07820.x. [DOI] [PubMed] [Google Scholar]
- 5.Faul F, Erdfelder E, Lang AG, Buchner A. G*Power 3: a flexible statistical power analysis program for the social, behavioral, and biomedical sciences. Behav Res Methods. 2007;39:175–191. doi: 10.3758/bf03193146. [DOI] [PubMed] [Google Scholar]
- 6.International Hapmap Consortium. The International HapMap Project. Nature. 2003;426:789–796. doi: 10.1038/nature02168. [DOI] [PubMed] [Google Scholar]
- 7.Kryczek I, Bruce AT, Gudjonsson JE, Johnston A, Vatan L, Szeliga W, Wang YH, Liu Y, Aphale A, Welling TH, Elder JT, Zou W. Induction of memory IL-17+ T cell trafficking and expansion by IFN-gamma: Mechanism and pathological relevance. Journal of Immunology. 2008;181:4733–4741. doi: 10.4049/jimmunol.181.7.4733. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 8.Liu Y, Helms C, Liao W, Zaba LC, Duan S, Gardner J, Wise C, Miner A, Malloy MJ, Pullinger CR, Kane JP, Saccone S, Worthington J, Bruce I, Kwok PY, Menter A, Krueger J, Barton A, Saccone NL, Bowcock AM. A genome-wide association study of psoriasis and psoriatic arthritis identifies new disease loci. PLoS Genet. 2008;4:e1000041. doi: 10.1371/journal.pgen.1000041. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 9.Nair R, Guo S, Jenisch S, Henseler T, Lange E, Terhune M, Westphal E, Christophers E, Voorhees J, Elder J. Scanning chromosome 17 for psoriasis susceptibility: lack of evidence for a distal 17q locus. Hum Hered. 1995;45:219–230. doi: 10.1159/000154293. [DOI] [PubMed] [Google Scholar]
- 10.Nair RP, Stuart PE, Nistor I, Hiremagalore R, Chia NV, Jenisch S, Weichenthal M, Abecasis GR, Lim HW, Christophers E, Voorhees JJ, Elder JT. Sequence and haplotype analysis supports HLA-C as the psoriasis susceptibility 1 gene. Am J Hum Genet. 2006;78:827–851. doi: 10.1086/503821. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 11.Nair RP, Ruether A, Stuart PE, Jenisch S, Tejasvi T, Hiremagalore R, Schreiber S, Kabelitz D, Lim HW, Voorhees JJ, Christophers E, Elder JT, Weichenthal M. Polymorphisms of the IL12B and IL23R genes are associated with psoriasis. J Invest Dermatol. 2008;128:1653–1661. doi: 10.1038/sj.jid.5701255. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 12.Rahman P, Elder JT. Genetic epidemiology of psoriasis and psoriatic arthritis. Ann Rheum Dis. 2005;64(Suppl 2):ii37–39. doi: 10.1136/ard.2004.030775. discussion ii40–31. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 13.Smith RL, Warren RB, Eyre S, Ho P, Ke X, Young HS, Griffiths CE, Worthington J. Polymorphisms in the IL-12beta and IL-23R genes are associated with psoriasis of early onset in a UK cohort. J Invest Dermatol. 2008;128:1325–1327. doi: 10.1038/sj.jid.5701140. [DOI] [PubMed] [Google Scholar]
- 14.Stephens M, Donnelly P. A comparison of bayesian methods for haplotype reconstruction from population genotype data. Am J Hum Genet. 2003;73:1162–1169. doi: 10.1086/379378. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 15.Stephens M, Smith NJ, Donnelly P. A new statistical method for haplotype reconstruction from population data. Am J Hum Genet. 2001;68:978–989. doi: 10.1086/319501. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 16.Trembath RC, Clough RL, Rosbotham JL, Jones AB, Camp RDR, Frodsham A, Browne J, Barber R, Terwilliger J, Lathrop GM, Barker JNWN. Identification of a major susceptibility locus on chromosome 6p and evidence for further disease loci revealed by a two stage genome-wide search in psoriasis. Hum Mol Genet. 1997;6:813–820. doi: 10.1093/hmg/6.5.813. [DOI] [PubMed] [Google Scholar]
- 17.Tsunemi Y, Saeki H, Nakamura K, Sekiya T, Hirai K, Fujita H, Asano N, Kishimoto M, Tanida Y, Kakinuma T, Mitsui H, Tada Y, Wakugawa M, Torii H, Komine M, Asahina A, Tamaki K. Interleukin-12 p40 gene (IL12B) 3′-untranslated region polymorphism is associated with susceptibility to atopic dermatitis and psoriasis vulgaris. J Dermatol Sci. 2002;30:161–166. doi: 10.1016/s0923-1811(02)00072-5. [DOI] [PubMed] [Google Scholar]
- 18.Waller JL, Addy CL, Jackson KL, Garrison CZ. Confidence intervals for weighted proportions. Stat Med. 1994;13:1071–1082. doi: 10.1002/sim.4780131009. [DOI] [PubMed] [Google Scholar]
- 19.Yamazaki K, Onouchi Y, Takazoe M, Kubo M, Nakamura Y, Hata A. Association analysis of genetic variants in IL23R, ATG16L1 and 5p13.1 loci with Crohn’s disease in Japanese patients. J Hum Genet. 2007;52:575–583. doi: 10.1007/s10038-007-0156-z. [DOI] [PubMed] [Google Scholar]