Psoriasis genome-wide association study identifies susceptibility variants within LCE gene cluster at 1q21

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1 Psoriasis genome-wide association study identifies susceptibility variants within LCE gene cluster at 1q21 Xue-Jun Zhang 1 3, Wei Huang 4,5, Sen Yang 1 3, Liang-Dan Sun 1 3, Feng-Yu Zhang 1 3, Qi-Xing Zhu 1 3, Fu-Ren Zhang 3,6, Chi Zhang 1 3, Wen-Hui Du 1 3, Xiong-Ming Pu 3,7, Hui Li 1 3, Feng-Li Xiao 1 3, Zai-Xing Wang 1 3, Yong Cui 1 3, Fei Hao 8, Jie Zheng 9, Xue-Qin Yang 3,1, Hui Cheng 1 3, Chun-Di He 11, Xiao-Ming Liu 12, Li-Min Xu 13, Hou-Feng Zheng 1 3, Shu-Mei Zhang 1 3, Jian-Zhong Zhang 14, Hong-Yan Wang 1 3, Yi-Lin Cheng 1 3, Bi-Hua Ji 15, Qiao-Yun Fang 2, Yu-Zhen Li 16, Fu-Sheng Zhou 2, Jian-Wen Han 1 3, Cheng Quan 1 3, Bin Chen 1 3, Jun-Lin Liu 1 3, Da Lin 1 3,LiFan 1 3, An-Ping Zhang 1 3, Sheng-Xiu Liu 1 3, Chun-Jun Yang 1 3, Pei-Guang Wang 1 3, Wen-Ming Zhou 1 3, Guo-Shu Lin 1 3, Wei-Dong Wu 3,7, Xing Fan 1 3, Min Gao 1 3, Bao-Qi Yang 3,6, Wen-Sheng Lu 1 3, Zheng Zhang 1 3, Kun-Ju Zhu 1 3, Song-Ke Shen 1 3, Min Li 1 3, Xiao-Yan Zhang 1 3, Ting-Ting Cao 1 3, Wei Ren 1 3, Xin Zhang 1 3,JunHe 1 3, Xian-Fa Tang 1 3, Shun Lu 1 3, Jian-Qiang Yang 1 3, Lin Zhang 1 3, Dan-Ni Wang 1 3, Feng Yuan 1 3, Xian-Yong Yin 1 3, Hong-Jie Huang 4,5, Hai-Feng Wang 4,5, Xin-Yi Lin 17 & Jian-Jun Liu 1,2,17 We report the first large genome-wide association study (GWAS) in a Chinese population to identify susceptibility variants for psoriasis using a two-stage case-control design. In the first stage, we carried out a genome-wide association analysis in 1,139 cases and 1,132 controls of Chinese Han ancestry using Illumina Human 61-Quad BeadChips. In the second stage, we took top SNPs forward for replication in two independent samples of 5,182 cases and 6,516 controls of Chinese Han ancestry, and 539 cases and 824 controls of Chinese Uygur ancestry. In addition to the strong replication for two known susceptibility loci MHC (rs , P ¼ ,OR¼ 22.62) and IL12B (rs321394, P combined ¼ ,OR¼.78), we identified a new susceptibility locus within the LCE gene cluster on 1q21 (rs485613, P combined ¼ ,OR¼.76). Psoriasis is a common chronic, autoimmune and hyperproliferative skin disease 1, affecting 2 5% of individuals of Western European descent 2 and.1.3% of individuals of East Asian ancestry 3,4. Individuals with psoriasis can be grouped into two types: type 1, characterized by age of onset before 4 y and association with HLA genes, and type 2, characterized by age of onset after 4 y and lack of HLA associations 5. Family study suggested that psoriasis follows a pattern of polygenetic or multifactorial inheritance 6, and linkage and association studies have already shown that the major histocompatibility (MHC) locus is a key susceptibility factor for psoriasis 7,8. A large number of additional candidates genes have been studied 9,1, but only a few genes, such as IL12B and IL23R, have had consistent supporting evidence from multiple studies 2,11. Efforts to identify non-mhc susceptibility loci have proven to be difficult 12 14, and the genetic basis of psoriasis remains poorly understood. In this study, using a two-stage design, we carried out the first GWAS study in a Chinese population to identify additional psoriasis susceptibility loci. In the first stage, we conducted a genome-wide association analysis using the Illumina Human 61-Quad BeadChips. Initially, 1,16 cases and 1,166 controls were genotyped with 62,91 SNPs and CNV probes. After stringent quality control, we excluded SNPs with call rate 1 Institute of Dermatology and Department of Dermatology at No.1 Hospital, Anhui Medical University, Hefei, Anhui 2322, China. 2 The Key Laboratory of Gene Resource Utilization for Severe Diseases, Ministry of Education and Anhui Province, Hefei, Anhui 2332,China. 3 Department of Dermatology and Venereology, Anhui Medical University, Hefei, Anhui 2332, China. 4 Chinese National Human Genome Center at Shanghai, Shanghai 2123, China. 5 The National Engineering Center for Biochip Design and Engineer in Shanghai, Shanghai 2123, China. 6 Shandong Provincial Institute of Dermatology and Venereology, Jinan, Shandong 2522, China. 7 Department of Dermatology, People s Hospital of Xinjiang Uygur Autonomous Region, Urumchi, Xinjiang 831, China. 8 Department of Dermatology, Southwest Hospital, Third Military Medical University, Chongqing, 438, China. 9 Department of Dermatology, Ruijin Hospital, Shanghai Jiao Tong University, School of Medicine, Shanghai 225, China. 1 Department of Dermatology, The General Hospital of Air Force, PLA, Beijing 136, China. 11 Department of Dermatology, No. 1 Hospital of China Medical University, Shenyang, Liaoning 111, China. 12 Department of Dermatology, the 1st Affiliated Hospital of Dalian Medical University, Dalian, Liaoning 11611, China. 13 Department of Dermatology, Changzheng Hospital, Tianjin 312, China. 14 Department of Dermatology, Peking University People s Hospital, Beijing 144, China. 15 Department of Dermatology, Yijishan Hospital of Wannan Medical College, Wuhu, Anhui 241, China. 16 Department of Dermatology, Second Affiliated Hospital of Harbin Medical University, Harbin, Heilongjiang 1586, China. 17 Human Genetics, Genome Institute of Singapore, Singapore , Singapore. Correspondence should be addressed to X.-J.Z. (ayzxj@vip.sina.com). Received 28 July 28; accepted 1 November 28; published online 25 January 29; doi:1.138/ng.31 NATURE GENETICS VOLUME 41 [ NUMBER 2 [ FEBRUARY 29 25

2 Table 1 Sample summary of 6,86 cases and 8,472 controls in the genome-wide and replication studies Cases Controls Analysis Population Sample size Mean age (s.d.) Mean age of onset (s.d.) Male/Female Familial/Sporadic Type 1/2 Sample size Mean age (s.d.) Male/Female GWA study Chinese Han 1, (12.31) (9.59) 669/47 431/7 a 1,132/7 1, (13.2) 67/462 Replication 1 Chinese Han 5, (14.49) (13.31) 2,94/2242 1,212/3,97 4,448/734 6, (1.44) 3,863/2,653 Replication 2 Chinese Uygur (14.99) (12.32) 268/ / / (15.62) 397/427 Total 6, ,877/2,983 1,76/5,92 6,68/792 8, ,93/3,542 a Eight samples were missing information on the familial or sporadic status. o9%, minor allele frequency o1%, or significant deviation from Hardy-Weinberg equilibrium (P o 1 7 ) in the controls. We removed 55 samples owing to duplicated samples, first- and second-degree relatives and population outliers. After further removing the SNPs on the X, Y and mitochondrial chromosomes as well as the CNV probes, we used the genotypes of 494,92 SNPs in 1,139 psoriasis cases and 1,132 controls of Chinese Han descent in the GWAS analysis (Table 1). Principal-component analysis (PCA) provided minimal evidence for population stratification (Supplementary Figs. 1 and 2 online). We carried out the Cochran-Armitage trend test to assess the genotype phenotype association. The quantile-quantile plot of the observed P values for association is shown in Figure 1. Under the null hypothesis of no association, the genome-wide P values should follow a uniform distribution. The quantile-quantile plot of the logarithms of our genome-wide P values showed a strong deviation from the null distribution, likely owing to the strong association observed within the MHC locus. After removal of 4,83 SNPs from the MHC region (chr. 6: Mb), the distribution of the logarithms of the observed P values largely fits the null distribution, except at the tail of distribution (P o 1 4 ) where the observed P values are smaller than that expected under the null hypothesis. A moderate genomic control l value of 1.6 (after removal of MHC SNPs) indicates a minimal overall inflation of the genome-wide statistical results due to population stratification. We observed strong association within the MHC region (PSORS1), with the most significant evidence at rs (P ¼ , OR ¼ 22.62; Fig. 2). This extremely high significance of association is likely due to the enrichment of subjects with early-onset and familial psoriasis in our GWAS sample, as the MHC locus is known to have a strong role in these forms of the disease 5. We also observed significant association at two SNPs within IL12B (rs321394, P ¼ , OR ¼.75; rs779212, P ¼ ,OR¼.75), another known risk gene for psoriasis 2, Besides these two known susceptibility loci, we observed significant association at four SNPs (rs , rs , rs and rs ) on 1q21 (P ¼ to ,OR¼.7) and suggestive evidence at additional ten SNPs (P o 1 5 ; all were outside the MHC region; Fig. 1). We also Figure 1 Genome-wide association results from the initial GWAS analysis. The genome-wide P values of the Cochran-Armitage trend test from 494,92 polymorphic SNPs in 1,139 psoriasis cases and 1,132 controls of Chinese Han ancestry are presented. The chromosomal distribution of all the P values ( log 1 P values) is shown. The red horizontal line presents a liberal threshold of 1 5 for suggestive significance. The inset shows the quantile-quantile (Q-Q) plots of the observed P values for association. The plot in red is for the P values from all the 494,92 SNPs, whereas the plot in blue is for P values for SNPs excluding the 4,83 SNPs within the MHC region (chr. 6: Mb). checked our genome-wide results for other previously reported genes, such as IL23R (P ¼.5), and did not find any evidence in support of an association (Supplementary Table 1 online). To do a fast-track replication analysis, we selected 64 GWAS SNPs and genotyped them in two independent case-control samples. The first sample consisted of 5,182 cases and 6,516 controls of Chinese Han ancestry, and the second one included 539 cases and 824 controls of Chinese Uygur ancestry. Of the 64 SNPs, 4 SNPs on 1q21 and 2 SNPs within IL12B were validated with significant evidence from the two follow-up samples (Table 2). The results of the remaining 58 SNPs are reported in Supplementary Table 2 online. The known association between the PSORS1 locus and psoriasis was supported by our genome-wide study. The strongest association within the PSORS1 locus (P o 1 15 and OR 4 5) was within the region flanked by rs and rs1365, where HLA-C, HCG27, POU5F1, TCF19 and CCHCR1 are located (Fig. 2). Given the known extensive linkage disequilibrium (LD) within the MHC region, we checked the independence of the evidence for multiple associations within the PSORS1 locus by carrying out a conditional logistic regression analysis of all 1,225 SNPs (chr. 6: Mb) with a P value o1 1. After control for the genetic effect of rs , the smallest conditional P value was.33, which is barely significant after correction for the large number of SNPs tested by the conditional analysis. Therefore, the multiple associations within the PSORS1 locus log 1 (P) Chr1 Chr13 LCE3A/D Chromosomal plot (1,139 cases + 1,132 controls) IL12B Chr2 Chr3 Chr4 Chr5 Chr14 Chr15 Chr16 MHC(P = 1.93E-28) log P observed Chr6 Chr7 Chr17 Chr18 Chromosomal location Chr8 Chr19 Chr9 Chr log P expected Chr1 Chr11 Chr12 Chr21 Chr22 26 VOLUME 41 [ NUMBER 2 [ FEBRUARY 29 NATURE GENETICS

3 log 1 (P value) OR > > OR > > OR > rs rs rs rs rs rs rs rs rs72465 Figure 2 Association evidence within the MHC region from the stage 1 GWAS analysis. The P values (trend test) and allelic ORs of 2,87 MHC SNPs (with a P value o1 1 5 and within chr. 6: Mb) from the initial GWAS analysis are presented. The upper panel shows the distribution of log 1 P values across the region, and the log 1 P values are colored according to their corresponding odds ratio. The top nine SNPs showing the strongest evidence (P o 1 15 and allelic OR above 5) are listed. The bottom panel lists the eight known genes and the nine top SNPs around the region where the strongest evidence for association was identified. The position of the coding SNP rs1365 is indicated by a red arrow, and the positions of the other eight SNPs are indicated by black arrows POU5F1 TCF19 CCHCR1 CDSN PSORS1C HCG27 HLA-C Position (Mb) are not independent and, as has been the case with many other studies, our analysis could not resolve the primary origin of the association, owing to the extensive LD within the region. It is noteworthy to point out that CDSN, one of the suggested susceptibility genes within the PSORS1 locus, is outside this critical region of strongest association (it is about 3 kb away from rs1365) (Fig. 2). Outside the MHC region, the strongest association evidence was identified at four SNPs (rs , rs , rs485613, rs ) within the LCE gene cluster on 1q21. In the genome-wide analysis, all four SNPs showed significant association (P ¼ to ,OR¼.7). In the replication study, both of the follow-up samples provided significant evidence at these four SNPs, yielding P values from to in Chinese Han subjects (OR ¼.78 to.8) and from to in Chinese Uygur subject (OR ¼.68). The joint analysis of the initial and followup samples provided confirmation for this association (rs485613, P combined ¼ ,OR¼.76). All four SNPs are located within the same linkage disequilibrium block and in almost complete LD (pairwise r ; Fig. 3). In the combined samples, after control for the genetic effect at rs485613, the association evidence for the remaining three SNPs was no longer significant (smallest P conditional ¼.47). The four SNPs therefore do not seem to be independently associated, and the association signal may be due to a single unidentified risk allele in the region. Our finding within the LCE gene cluster on 1q21 is a new association. Suggestive association evidence was recently reported at rs (within the LCE1C gene) 11, which is about 2 kb away from the four SNPs identified in this study. According to the LD data from the HapMap project, rs and the four SNPs are in different LD blocks in Chinese population and not correlated (r 2 o.5). Although the SNPs are within one long LD block in population of European ancestry (Fig. 3), rs and the four SNPs are still not correlated (r 2 o.5). In our genome-wide analysis, very weak evidence, if any, was observed at rs (P ¼.2). Furthermore, the minor allele of rs was shown to be associated with a risk effect in the population of European ancestry 11,whereastheminor alleles of four SNPs are shown to be associated with a protective effect in our study. Taken together, these results indicate that our finding within the LCE gene cluster is a new association and distinct from the previous evidence reported at rs Chr. 1 Genotyped SNPs158k CRCT1 LCE3D LCE3E LCE3C LCE3B LCE3A 159k LCE2D LCE2C LCE2B LCE2A LCE4A 151k KPRP LCE2F LCE1E LCE1D LCE1C LCE1B Figure 3 The LD patterns of the LCE gene cluster region on 1q21 in populations of Chinese and European ancestry. The LD patterns (D ) were created in the Haploview by using the genotyping data (only SNPs with MAF 4.1) from the HapMap project. The top panel shows all the genes and transcripts within the region. The middle panel shows the D pattern in Chinese population; and the bottom panel shows the D pattern in the European-descent population. The positions of the four SNPs (rs485613, rs , rs and rs ) identified in this study are indicated by red arrows, whereas rs reported in a previous study 11 is indicated by a yellow arrow. Position (kb) D.5 1 NATURE GENETICS VOLUME 41 [ NUMBER 2 [ FEBRUARY 29 27

4 Table 2 Association evidences of the 4 SNPs within the LCE gene cluster and 2 SNPs in IL12B from the genome-wide and validation studies GWAS (Han) Replication 1 (Han) Replication 2 (Uygur) Combined MAF OR (95% CI) P MAF OR P MAF OR Ptrend OR P Case Ctrl Case Ctrl Case SNP Gene Chr Allele Ctrl.79 (.75.83) 1.8E (.58.8) 4.12E 6.77 (.74.81) 7.13E (.76.84) 5.91E (.57.8) 2.5E 6.76 (.72.8) 6.69E 3.78 (.74.83) 5.44E (.58.8) 3.4E 6.76 (.72.8) 4.35E (.74.83) 1.4E (.57.79) 2.11E 6.76 (.73.8) 2.18E (.78.86) 1.22E (.64.89) 6.97E 4.8 (.77.84) 1.12E (.75.83) 1.56E (.6.85) 1.17E 4.78 (.74.81) 2.58E (.61.79) 6.27E (.62.79) 9.9E (.62.79) 9.83E (.62.79) 1.7E (.66.84) 2.3E (.67.84) 3.39E rs LCE3D 1 T/C.35 rs LCE3D 1 A/C.35 rs LCE3A 1 C/T.35 rs LCE3A 1 T/G.35 rs IL12B 5 C/T rs IL12B 5 A/G OR, per-allele odds ratio in trend test; 95% CI, 95% confidence interval; P, P values from the Cochran-Armitage trend test. Our study also identified highly significant association at the two SNPs within the IL12B gene: rs (P combined ¼ , OR ¼.78) and rs (P combined ¼ ,OR¼.8) (Table 2). The signals at the two SNPs are not independent, because the two SNPs are in LD (r 2 ¼.5) (Supplementary Fig. 3 online) and the association evidence at rs was no longer significant after adjustment for the genetic effect at rs (P conditional ¼.3, uncorrected). Association evidence for IL12B was previously reported at rs (refs. 2,11), but not in a Chinese population. rs was not genotyped in our genome-wide analysis, but it is in the same LD block and in complete LD with rs (r 2 ¼ 1) (according to the LD data from the HapMap project) identified in this study. Our study has therefore confirmed IL12B to be a susceptibility gene in Chinese population. We further evaluated the role of the three confirmed susceptibility loci (rs in LCE, rs in IL12B and rs in MHC) in different clinical subgroups of psoriasis. The subgroup analysis of rs and rs was done in the combined Chinese samples by classifying the cases into familial versus sporadic and type 1 versus type 2, and the subgroup analysis of rs was done in the familial versus sporadic cases of the GWAS sample. As shown in Table 3, we found significant evidence that rs has a stronger genetic effect in the familial cases than the sporadic ones (heterogeneity test P ¼ ), but no evidence that the other two SNPs have a differential role in different clinical subgroups. The newly identified susceptibility locus within the LCE gene cluster has biological implications. The LCE genes encode the stratumcorneum proteins of cornified envelope that have an important role in epidermal terminal differentiation Psoriasis is a disease of interfollicular epidermis in which keratinocytes proliferate rapidly. In psoriatic skin, the epidermal differentiation cycle takes only 3 to 5 d instead of 28 to 3 d as in normal skin 9. This rapid keratinocyte proliferation may cause the production of parakeratotic keratinocytes in psoriatic skin and thus the formation of poorly adherent stratum corneum, which in turn results in the characteristic scales or flakes of psoriasis lesions 22,23. We postulate that by causing the production of abnormal stratum-corneum proteins and thus abnormal cornified envelop, genetic susceptibility variant(s) within the LCE genes may influence the development of psoriasis by interrupting the terminal differentiation of keratinocytes. In summary, we have conducted the first GWAS in the Chinese population, with replication in different Chinese population groups, and report the identification a new susceptibility locus for psoriasis within the LCE gene cluster. METHODS Study populations. The 1,16 cases and 1,166 controls used in the initial GWAS were recruited from the Chinese Han population through multiple hospitals in China. The clinical diagnosis of all subjects was confirmed by at least two dermatologists. Additional clinical information was collected from the subjects through a full clinical checkup, and additional demographic information was also collected from both the cases and the controls through questionnaire. The 1,16 selected cases all had at least two skin lesions. To enrich the genetic risk alleles, we selected cases that mostly had type 1 psoriasis (early age of onset), and many of them had family history of disease. All the controls used in the GWAS were individuals without psoriasis, any autoimmune disorders systemic disorders and any family history of psoriasis (including first-, second- and third-degree relatives). Cases and controls were matched on age and sex. Two independent samples were used in the validation study. The first one included 5,182 cases and 6,516 controls of the Chinese Han population recruited through collaboration among multiple hospitals in China. The second 28 VOLUME 41 [ NUMBER 2 [ FEBRUARY 29 NATURE GENETICS

5 Table 3 Subgroup analysis of the three risk loci (rs485613, rs and rs ) in Chinese Han samples rs (chr. 1: ) rs (chr. 5: ) rs (chr. 6: ) Sample group Size MAF OR (95% CI) P P het MAF OR (95% CI) P P het Size MAF OR (95% CI) P P het Controls 7, ,132.1 Familial cases 1, (.71.83) 2.89E (.74.87) 3.81E ( ) 9.1E 14 Sporadic cases 4, E E E 149 (.75.83) (.73.82) ( ) Type 1 cases 5, E E 2.19 (.74.81) (.75.83) Type 2 cases E 8 (.76.95) (.65.82) 2.8E-5 OR, OR per allele (from trend test); 95% CI, 95% confidence interval; P, P value from trend test, P het, P value from heterogeneity test. included 539 cases and 824 matched controls of Chinese Uygurs, collected in Xinjiang Uygur Autonomous Region in China. All the cases and controls were recruited using uniform criteria, and the clinical and demographic information were collected using the same questionnaire as that used for the samples used in the GWAS. Informed consent was obtained from all the participants. The study was approved by each institutional ethical committee and conducted according to Declaration of Helsinki principles. Genotyping analysis. Genomic DNAs were extracted using established method. The genotyping analysis of the GWAS was done using the Illumina Human 61- Quad BeadChips. As a part of quality control, SNPs were excluded if they had a call rate lower than 9%, a minor allele frequency o1% and/or significant deviation from Hardy-Weinberg equilibrium in the controls (P o 1 7 ). Similarly, we removed all the samples with genotyping rate less than 98% from further analysis. Furthermore, all SNPs on the X, Y and mitochondrial chromosomes as well as the CNV-related SNPs and probes were excluded from statistical analysis. The genotyping analysis of the validation study was done by using the MassArray system from the Sequenom. We subjected the genotype data to the same quality control analysis. To further evaluate the quality of the genotype data for the validation analysis, we selected six SNPs (showing significant association evidence in both the GWAS and validation studies) to be re-genotyped in 1 randomly selected samples of the GWAS by using the Sequenom system. The concordance rate between the genotypes from the Illumina and the Sequenom analyses was 99.9%. For all the 64 SNPs analyzed in the validation study, the cluster patterns of the genotyping data from the Illumina and Sequenom analyses were checked to confirm their good quality. The genotype data of the 64 SNPs are available in Supplementary Table 2. Statistical analysis. As a part of quality control, we examined potential genetic relatedness based on pairwise identity by state for all the successfully genotyped samples using the Plink 1.2 software 24. Upon identification of a first- or second-degree relative pair, we removed the one of two related individuals (the sample with lower call rate was removed). The remaining samples were subsequently assessed for population outlier and stratification using a PCAbased approach 25. For all PCA analyses, all MHC SNPs on chromosome 6 from Mb were removed. First, all samples were analyzed together with the 26 reference samples from the International HapMap Project: 57 Yoruba in Ibadan, Nigeria (YRI), 44 Japanese in Tokyo, Japan (JPT), 45 Han Chinese in Beijing, China (CHB) and 6 CEPH (Utah residents with ancestry from northern and western Europe) (CEU). Five population outliers were identified and removed. Afterwards, we carried out PCA for all the remaining case and control samples. Detailed description of the identity-by-state calculation and PCA results is provided in the Supplementary Methods online. After all the SNP and sample quality control analyses, we used genotype data of 494,92 SNPs in 1,139 cases and 1,132 controls in the genome-wide association analysis. We used the Cochran-Armitage trend test for genotype association with disease phenotype and calculated OR per allele (from trend test). We used the quantile-quantile plot to evaluate the overall significance of the genome-wide association results and the potential impact of population stratification. The impact of population stratification was also evaluated by calculating the genomic control inflation factor (excluding 4,83 MHC SNPs on chr. 6:25 34 Mb). Given that the impact of population stratification was found to be minimal, all the statistical results were reported without genomic control correction. Replication analysis was done first by analyzing two follow-up samples separately and then analyzing the combined sample of all the cases and controls. Association analysis of the combined samples was done using Cochran-Mantel-Hanezel stratification analysis 26.AllP values from the validation analysis were reported without correction for multiple testing. We tested the independence of association evidence using a conditional logistic regression analysis where the genotypes of the SNP to be adjusted for are used as strata and the common genetic effect of the primary SNP is estimated and tested within strata. To test for heterogeneity between the odds ratios for different clinical subtypes, we used the logistic regression analysis of the cases (case-only analysis) where subclinical phenotypes were used as the outcome variable 27. Additional information on the genotyping and statistical analyses are provided in the Supplementary Methods. Note: Supplementary information is available on the Nature Genetics website. ACKNOWLEDGMENTS We thank G.P. Zhao, G.Y. Xu, L. Jin, X. Zhang, S.J. Xu and Z.G. Lin for assistance. And we would like to thank all the volunteers who took part in this work. We also want to thank L. Yi and E. Tantoso for their assistance in data analysis. This work was funded by the Anhui Provincial Special Scientific Program (27-7), the High-Tech Research and Development Program of China (863 Plan) (27AA2Z161), the Key Project of Natural Science Foundation of China (35367), National Basic Research Program of China (973 Plan) (27B51681), General Program of National Natural Science Foundation of China ( , , ) and Agency for Science & Technology and Research of Singapore (A*STAR) (for J.J.L. and X.Y.L.). AUTHOR CONTRIBUTIONS X.-J.Z. conceived of this study and obtained financial support. X.-J.Z., S.Y. and J.-J.L. designed it. S.Y., Q.-X.Z., F.-L.X., Y.C., W.-H.D., H.L., S.-M.Z., H.C., Z.-X.W., D.L., L.F., A.-P.Z., H.-Y.W., S.-X.L., C.-J.Y., P.-G.W., W.-M.Z., G.-S.L. and X.F. conducted sample selection and data management. W.-S.L., Z.Z., K.-J.Z., J.-L.L., S.-K.S., M.L., X.-Y.Z., T.-T.C., W.R., X.Z., J.H., X.-F.T., S.L., J.-Q.Y., L.Z., D.-N.W., F.Y., M.G. and X.-Y.Y. coordinated and/or undertook recruitment, collected phenotype data, undertook related data handling and calculation, managed recruitment and obtained biological samples. The following authors from the various collaborating groups undertook assembly of case/control series in their respective regions and collected data and samples: F.-R.Z. and B.-Q.Y. in Shandong province; X.-M.P. and W.-D.W. in Xinjiang Uygur Autonomous Region; F.H. in Chongqing city; X.-Q.Y. and J.-Z.Z. in Beijing city; C.-D.H. and X.-M.L. in Liaoning province; Y.-Z.L. in Heilongjiang province; L.-M.X. in Tianjin city; B.-H.J. in Wuhu city; J.Z. in Shanghai city; L.-D.S., C.Z., Q.-Y.F., F.-S.Z. Y.-L.C., J.-W.H., C.Q., B.C., H.-J.H., H.-F.W., W.H. performed the two-stage genotyping and sequencing. F.-Y.Z., H.-F.Z. and X.-Y.L. undertook data manipulation, statistical analysis and bio-informatic interrogations. All authors contributed to the final paper, with X.-J.Z., S.Y., J.-J.L., F.-Y.Z., L.-D.S. and C.Z. playing key roles. NATURE GENETICS VOLUME 41 [ NUMBER 2 [ FEBRUARY 29 29

6 Published online at Reprints and permissions information is available online at reprintsandpermissions/ 1. Bhalerao, J. & Bowcock, A.M. The genetics of psoriasis: a complex disorder of the skin and immune system. Hum. Mol. Genet. 7, (1998). 2. Cargill, M. et al. A large-scale genetic association study confirms IL12B and leads to the identification of IL23R as psoriasis-risk genes. Am. J. Hum. Genet. 8, (27). 3. Simons, R.D. Additional studies on psoriasis in the tropics and in starvation camps. J. Invest. Dermatol. 12, (1949). 4. Yip, S.Y. The prevalence of psoriasis in the Mongoloid race. J. Am. Acad. Dermatol. 1, (1984). 5. Henseler, T. & Christophers, E. Psoriasis of early and late onset: characterization of two types of psoriasis vulgaris. J. Am. Acad. Dermatol. 13, (1985). 6. Zhang, X., Wang, H., Te-Shao, H., Yang, S. & Chen, S. The genetic epidemiology of psoriasis vulgaris in Chinese Han. Int. J. Dermatol. 41, (22). 7. Nair, R.P. et al. Sequence and haplotype analysis supports HLA-C as the psoriasis susceptibility 1 gene. Am. J. Hum. Genet. 78, (26). 8. Fan, X. et al. Fine mapping of the psoriasis susceptibility locus PSORS1 supports HLA-C as the susceptibility gene in the Han Chinese population. PLoS Genet. 4, e138 (28). 9. Schön, M.P. & Boehncke, W.H. Psoriasis. N. Engl. J. Med. 352, (25). 1. Zhang, X.J. et al. Polymorphisms in interleukin-15 gene on chromosome 4q31.2 are associated with psoriasis vulgaris in Chinese population. J. Invest. Dermatol. 127, (27). 11. Liu, Y. et al. A genome-wide association study of psoriasis and psoriatic arthritis identifies new disease loci. PLoS Genet. 4, e141 (28). 12. Bowcock, A.M. Psoriasis genetics: the way forward. J. Invest. Dermatol. 122, xv xvii (24). 13. Lesueur, F. et al. ADAM33, a new candidate for psoriasis susceptibility. PLoS ONE 2, e96 (27). 14. Capon, F. et al. Identification of ZNF313/RNF114 as a novel psoriasis susceptibility gene. Hum. Mol. Genet. 17, (28). 15. Capon, F. et al. Sequence variants in the genes for the interleukin-23 receptor (IL23R) and its ligand (IL12B) confer protection against psoriasis. Hum. Genet. 122, (27). 16. Chang, Y.T. et al. Cytokine gene polymorphisms in Chinese patients with psoriasis. Br. J. Dermatol. 156, (27). 17. Tsunemi, Y. et al. Interleukin-12 p4 gene (IL12B) 3 -untranslated region polymorphism is associated with susceptibility to atopic dermatitis and psoriasis vulgaris. J. Dermatol. Sci. 3, (22). 18. Backendorf, C. & Hohl, D. A common origin for cornified envelope proteins? Nat. Genet. 2, 91(1992). 19. Engelkamp, D., Schafer, B.W., Mattei, M.G., Erne, P. & Heizmann, C.W. Six S1 genes are clustered on human chromosome 1q21: identification of two genes coding for the two previously unreported calcium-binding proteins S1D and S1E. Proc. Natl. Acad. Sci. USA 9, (1993). 2. Mischke, D., Korge, B.P., Marenholz, I., Volz, A. & Ziegler, A. Genes encoding structural proteins of epidermal cornification and S1 calcium-binding proteins form a gene complex ( epidermal differentiation complex ) on human chromosome 1q21. J. Invest. Dermatol. 16, (1996). 21. Wang, A., Johnson, D.G. & MacLeod, M.C. Molecular cloning and characterization of a novel mouse epidermal differentiation gene and its promoter. Genomics 73, (21). 22. Bowcock, A.M. & Krueger, J.G. Getting under the skin: the immunogenetics of psoriasis. Nat. Rev. Immunol. 5, (25). 23. Krueger, J.G. & Bowcock, A. Psoriasis pathophysiology: current concepts of pathogenesis. Ann. Rheum. Dis. 64 (Suppl 2), ii3 ii36 (25). 24. Purcell, S. et al. PLINK: a tool set for whole-genome association and population-based linkage analyses. Am. J. Hum. Genet. 81, (27). 25. Price, A.L. et al. Principal components analysis corrects for stratification in genomewide association studies. Nat. Genet. 38, (26). 26. Mantel, N. & Haenszel, W. Statistical aspects of the analysis of data from retrospective studies of disease. J. Natl. Cancer Inst. 22, (1959). 27. Garcia-Closas, M. et al. Heterogeneity of breast cancer associations with five susceptibility loci by clinical and pathological characteristics. PLoS Genet. 4, e154 (28). 21 VOLUME 41 [ NUMBER 2 [ FEBRUARY 29 NATURE GENETICS