Human leukocyte antigen alleles and susceptibility to psoriatic arthritis

Human Immunology 74 (2013) 1333–1338

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Human leukocyte antigen alleles and susceptibility to psoriatic arthritis Vinod Chandran a, Shelley B. Bull b,c, Fawnda J. Pellett a, Renise Ayearst a, Proton Rahman d, Dafna D. Gladman a,⇑ a

Psoriatic Arthritis Program, Centre for Prognosis Studies in The Rheumatic Diseases, Toronto Western Hospital, Toronto, Ontario, Canada Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Ontario, Canada c Dalla Lana School of Public Health, University of Toronto, Toronto, Ontario, Canada d Memorial University of Newfoundland, St. John’s, Newfoundland, Canada b

a r t i c l e

i n f o

Article history: Received 23 February 2013 Accepted 19 July 2013 Available online 2 August 2013

a b s t r a c t Objective: Our purpose was to determine associations between HLA alleles and psoriatic arthritis (PsA). Methods: 678 PsA cases and 688 healthy controls were analyzed in a case–control design. The difference in the proportion of cases and controls with at least 1 copy of HLA alleles were tested for significance using v2 test and Fisher’s exact test. Association analyses of haplotypes inferred by the Expectation–Maximization algorithm were performed. In the family-based association study, data from 283 families were analyzed. Results: Univariate analysis revealed that cases were more likely to be carriers of HLA-C⁄01, -C⁄02, -C⁄06, -C⁄12, -B⁄27, -B⁄38 and -B⁄57, whereas controls were more likely to be carriers of HLA-C⁄03, -C⁄07, -B⁄07, -B⁄51, -DRB1⁄15 and -DQB1⁄0602. In haplotype analyses, PsA cases were more likely to be carriers of the HLA haplotypes -C⁄01/-B⁄27, -C⁄02/-B⁄27, -C⁄12/-B⁄38, and -C⁄06/-B⁄57, while controls were more likely to be carriers of the haplotypes -C⁄07/-B⁄07 and -C⁄15/-B⁄51. In the family-based association analysis, the HLA alleles -A⁄02, -B⁄27 and -DRB1⁄07 were preferentially transmitted to cases, whereas the alleles -A⁄03, -A⁄28, -B⁄51, -DRB1⁄11 and -DQB1⁄0301 were under transmitted. Conclusion: This large case–control and family based association study shows that HLA-C⁄12/B⁄38, HLAB⁄27 and HLA-C⁄06/B⁄57 are haplotypes (alleles) robustly associated with PsA. However, since patients with PsA also have psoriasis it is difficult to determine whether the primary association is with arthritis or psoriasis. Ó 2013 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.

1. Introduction Psoriatic arthritis (PsA) is an inflammatory arthritis associated with psoriasis, a chronic immune-mediated inflammatory skin disease. PsA is classified according to the CASPAR criteria [1]. PsA is a member of a class of inflammatory arthritides called

Abbreviations: AS, ankylosing spondylitis; CASP, Collaborative Association Study of Psoriasis; CASPAR, classification criteria for psoriatic arthritis; CD, cluster of differentiation; DNA, deoxyribonucleic acid; EM, Expectation–Maximization; FBAT, family-based association test; FDR, false discovery rate; GWAS, genome-wide association study; HLA, human leukocyte antigen; JIA, juvenile idiopathic arthritis; KIR, killer-cell immunoglobulin-like receptors; LD, linkage disequilibrium; MHC, major histocompatibility complex; NK, natural killer; OR, odds ratio; PCA, principal component analysis; PCR, polymerase chain reaction; PsA, psoriatic arthritis; SNP, single nucleotide polymorphisms; SpA, spondyloarthritis; SSO, sequence specific oligonucleotide; SSP, sequence specific primers; WHO, World Health Organization. ⇑ Corresponding author. Address: Psoriatic Arthritis Program, Centre for Prognosis Studies in the Rheumatic Diseases, Toronto Western Hospital, 399 Bathurst Street, 1E-410, Toronto, Ontario M5T 2S8, Canada. Fax: +1 416 603 9387. E-mail address: [email protected] (D.D. Gladman).

spondyloarthritis (SpA) that is characterized by peripheral as well as axial arthritis, radiological sacroiliitis, mucosal and skin inflammation, the absence of serum rheumatoid factors and subcutaneous nodules, and a tendency for familial aggregation [2]. Familial aggregation studies have shown that psoriasis, SpA and PsA have a significant genetic component [3,4]. A number of genetic linkage studies have identified the MHC region on chromosome 6p as a region that harbors genes largely contributing to susceptibility to psoriasis and AS (the prototype SpA), two diseases closely related to PsA [5–8]. Recent genomewide association studies in psoriasis and AS are consistent with the results from linkage studies – SNPs in the region of HLA-C are the strongest risk factors for psoriasis, and the SNP tagging HLA-B⁄27 is the strongest risk factor for AS [9–12]. Since PsA is a SpA and most patients with PsA have psoriasis, it is likely that the MHC region also harbors genes for susceptibility to PsA. In fact, in the Collaborative Association Study of Psoriasis (CASP) study, the authors reported that HLA-C provided the strongest association with PsA when compared to normal controls. However, when

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patients with PsA were compared to those with psoriasis alone, HLA-C was more strongly associated with psoriasis alone, suggesting that the primary association was with psoriasis and not PsA [9]. The MHC is a gene-rich region that encodes genes involved in the immune response. The classical HLA class I and class II genes in the MHC are highly polymorphic and the proteins they encode play an essential role in self/non-self immune recognition. HLA variation plays a major role in determining transplant rejection and susceptibility to a large number of infectious and autoimmune diseases [13]. However, identification of functional variants is problematic due to linkage disequilibrium (LD) that extends across multiple HLA and non-HLA genes in the MHC [14,15]. In addition to SNP typing, molecular HLA typing may help better delineate the genetic architecture of this region. Studies comparing HLA antigens between patients with psoriasis and PsA as well as non-disease controls have shown that while HLA-B13, -B16 (a broad antigen serotype that recognizes the HLAB38 and -B39 split antigen serotypes), HLA-B17 and HLA-Cw6 are associated with psoriasis, with or without arthritis, HLA-B27 and HLA-B7 are specifically associated with PsA [16,17]. In PsA there is a stronger association with HLA-B than with HLA-C alleles. The association of HLA-C, particularly HLA-C⁄06, with PsA was observed to be specific to association with early onset psoriasis, since no association was found in patients with PsA and late onset psoriasis [18]. Since almost all patients with PsA have psoriasis, it is unclear whether the HLA associations described above are with psoriasis or with PsA, or both. The HLA antigens reported as specific to PsA are HLA-B27 and possibly -B7, -B38 and -B39. The results from HLA association studies in PsA published in the last 25 years on subjects of European descent are summarized in Supplementary Table 1 [16,19–23]. HLA allele association studies conducted to date have been limited by lack of a clear case definition and limited genotyping. Moreover, most studies have used HLA antigen testing and have generally been underpowered. We therefore aimed to undertake a large case–control association study using well defined cases and appropriately matched controls and utilize molecular HLA allele typing methods to determine associations between HLA class I (HLA-A, -C, -B) and class II (HLA-DQ, -DR) alleles and PsA. HLA allele associations were also further investigated using family-based association tests to determine whether the results from the population-based study were robust to bias from population stratification. 2. Methods 2.1. Subjects The University of Toronto PsA Program recruits patients with PsA for genetic and prognostic studies. Healthy controls are also recruited from blood donors, hospital employees and general public through flyers. As of March 31, 2010 the database consisted of 816 patients with PsA and 774 controls. From the database 684 patients with PsA satisfying CASPAR classification criteria and 706 healthy controls of self-reported European ethnicity were selected [1]. Ethics approval for this study was obtained from the University Health Network Research Ethics Board as per REB# 08-0360-AE. All participants in this study have signed the appropriate consent form(s) for their participation. 2.2. HLA typing DNA was extracted from peripheral blood using a modified salting out procedure (GentraÒ PuregeneÒ Blood Kit). Extracted genomic DNA was amplified by PCR using locus specific primers

for each of the HLA-A, -C, -B, -DR and DQ loci. PCR amplicons were identified by sequence specific oligonucleotide (SSO) probes using the reverse line blot technique (RELI™ SSO HLA typing kits, Invitrogen, Burlington, Ontario, Canada). Ambiguous results were resolved using sequence specific primers (PCR-SSP). The molecular reagents are updated and standardized as new sequences are identified and accepted by the WHO nomenclature committee (http:// www.ihwg.org). HLA allele 2 digit resolution (A–C, DRB1) and 4 digit resolution (DQB1) was used for analysis of 105 alleles over the 5 loci being investigated. 94 alleles were detected in at least one case or one control subject. In case–control genetic association studies, population stratification is of potential concern and comparability of cases and controls is important. In order to exclude subjects who are ethnic outliers, principal component analysis (PCA) using information on SNPs genotyped in the majority of subjects for a study on psoriasis genetics was used. Four hundred and fifty-seven PsA cases and 486 controls with available DNA were genotyped for 7978 SNPs using a custom made Illumina iSelect HD marker set. SNPs included in this marker set were chosen for an association study on psoriasis and included SNPs in all autosomes. For the purpose of identifying ethnic outliers in this sub-sample, SNPs in regions associated with psoriasis and AS, those in the MHC, as well as SNPs in regions of long range LD in chromosomes 1–3, 5–8, 10–12 and 20 were omitted [24]. Two SNPs with insufficient cardinality and 81 that were monomorphic were excluded. Thus, 5405 polymorphic SNPs were used as input to the PCA using the ‘‘SNP and Variation Suite™ 7’’ software, Golden Helix, Bozeman, MT, USA. Assuming an additive genetic model and theoretical sigma at Hardy–Weinberg Equilibrium for marker normalization, up to 10 top components were identified. Components were recomputed up to 5 times after removing outliers (>6 standard deviations) from 5 of the components. PCA produced 10 components; 24 outliers (6 PsA, 18 controls) were found from 4 iterations (see Supplementary Table 2). Supplementary Figs. 1 and 2 plot Eigenvalues 1 against 2 and 2 against 3, respectively, and suggest that after outlier removal, cases and controls were reasonably well matched genetically. Thus, 678 PsA cases and 688 controls were used for all further analyses. 2.3. Statistical analysis The difference in the proportion of cases and controls with at least 1 copy of HLA-A, -C, -B, -DR and -DQ alleles (implying a dominant model) was tested for significance by asymptotic v2 test and Fisher’s exact test. Odds ratios and the corresponding asymptotic 95% confidence intervals were obtained for the PsA case–control comparison. To account for multiple testing, the false discovery rate (FDR) approach was employed with each HLA locus being assessed separately [25]. The threshold for statistical significance after correction for multiple testing using FDR was set at p < 0.01 (FDR p = 0.05 divided by 5, the number of tested HLA loci). Analyses were performed using SASÓ 9.2, SAS Institute Inc., Cary, NC, USA. After single marker association analysis, haplotype association analysis was initially performed using the ‘‘SNP and Variation Suite™ 7’’ software using HLA-C and -B alleles as a single block. Subsequently, haplotype association analysis was performed similarly for -C, -B, -DRB1 alleles as well as for -A, -C, -B, -DRB1 and -DQB1 alleles. Haplotype information was inferred among cases and controls separately using the Expectation–Maximization (EM) algorithm since cases were more frequent in the sample compared to the general population. The EM algorithm generates maximum likelihood estimates given a multi-locus sample of HLA alleles [26]. A maximum of 50 EM iterations was conducted and EM convergence tolerance was set at 0.0001. Posterior prob-

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abilities of all possible haplotypes for an individual, conditional on the observed genotypes, were estimated. Use of the resulting posterior probabilities as dosage made it possible to test the haplotype-disease association while accounting for haplotype estimation. Because of the imprecision involved in estimating the effects of low-frequency haplotypes, only those with an estimated frequency of more than 0.5% in the study population were included in the analysis. Odds ratios comparing haplotype frequency in PsA cases versus controls were calculated with the corresponding asymptotic 95% confidence intervals (CI). To account for multiple testing the false discovery rate (FDR) approach was employed and the threshold for significance was set at p < 0.01 (FDR p = 0.05 divided by 5, the number of tested HLA loci) [25]. Subsequently, multivariate logistic regression analysis using backward elimination was conducted to identify HLA alleles independently associated with PsA compared to controls while accounting for LD between loci and dependence among allele frequencies within a locus. HLA alleles for inclusion in the multivariate analysis were selected based on the results of univariate analyses, according to the criteria: alleles significant at 5% FDR level at each locus and/or those alleles found to be significant in univariate chi-squared test at 5% level if reported to be associated in previous HLA association studies in psoriasis or PsA. Allelic associations were retained in the multivariate regression and considered statistically significant if the p-value from the 2-sided Wald-test was <0.05. Maximum likelihood odds ratios and the corresponding asymptotic 95% CI were obtained from the Wald test in the final model. Statistical analysis was performed using SAS 9.2Ó SAS Institute Inc., Cary, NC, USA. Family-based designs have unique advantages over population-based designs, as they are robust against population admixture and stratification and allow both linkage and association to be tested [27]. Although in the population based case–control association study attempts were made to address population stratification, this might still be an issue. Therefore, a familybased association study using families from Ontario and Newfoundland was conducted to complement the case–control study. A total of 283 families were recruited (202 from Ontario, 81 from Newfoundland, with 263 nuclear and 20 extended families, including 1000 persons in total). The families included probands with PsA and their first-degree relatives. Overall there were 125 trios, 16 families with 1 parent and at least 2 siblings, 10 with at least 3 siblings but no parents and 132 with other complex family structure. Alleles identified as independently associated with PsA following multivariate case–control analysis were further examined for association with PsA using the family-based association test implemented in the FBAT version 2.0.3 [28]. Initially, association analyses were carried out using each individual allele. Alleles with less than 10 informative families were excluded and the per-allele significance level was set at p < 0.05. Since the family-based analyses had less power than the case–control analyses, trends for association (p 6 0.1) were also examined. For example, the case–control study had >80% power to detect an OR of 1.6 for an allele frequency of 0.25 at a significance level of p < 0.001, whereas the family-based study had power of only 55% even when the significance was set at 5%. Subsequently, haplotype based association tests using all 5 markers, using only HLA-C, -B, -DRB1, as well as HLA-C, -B alleles were also conducted using FBAT (hbat command). Although, ideally the cases recruited in the case–control study should not overlap with those in the family based study, the relatively low number of families available demanded that the probands from Ontario be a sub-group of the cases recruited under the case–control study.

3. Results 3.1. Case–control study The demographic and disease characteristics of the PsA patients included in the case–control study are given in Table 1. All controls were of European ethnicity. Their mean age at sample collection was 46 (sd 15.2) years and males comprised 40%. In univariate analyses (Supplementary Table 3) HLA-C⁄01, ⁄ C 02, -C⁄06, -C⁄12, -B⁄27, -B⁄38 and -B⁄57 were significantly more frequent in PsA, compared to controls, whereas HLA-C⁄03, -C⁄07, B⁄07, -B⁄51, -DRB1⁄15 and -DQB1⁄0602 were less frequent in PsA compared to controls. Since HLA-C and -B alleles show the strongest association with PsA in single-locus analysis, haplotype analyses using markers at these 2 loci were conducted. Four haplotypes HLA-C⁄06 B⁄57, HLA- C⁄12 B⁄38, HLA- C⁄02 B⁄27 and HLA-C⁄01 B⁄27 (Table 2) were significantly more frequent in PsA, while each of two haplotypes HLA-C⁄07 B⁄07 and HLA-C⁄15 B⁄51 were significantly less frequent. Because previous studies have shown associations between PsA and alleles at HLA-C, -B and -DRB1 loci, haplotype analyses using markers at these 3 loci only were also conducted (Supplementary Table 4). Three haplotypes (HLA-C⁄12 B⁄38 DRB1⁄04, HLA-C⁄12 B⁄38 DRB1⁄13, and HLA-C⁄02 B⁄27 DRB1⁄04) were significantly more frequent in PsA, while one haplotype HLA-C⁄07 B⁄07 DRB1⁄15 was significantly less frequent. Finally, haplotype analyses were conducted using markers at all 5 loci – HLA-A, -C, -B, DRB1 and -DQB1 (Supplementary Table 5). One extended haplotype HLA-A⁄26 C⁄12 B⁄38 DRB1⁄04 DQB1⁄0302 was significantly more frequent in PsA compared to controls, while another haplotype HLA-A⁄03 C⁄07 B⁄07 DRB1⁄15 DQB1⁄0602 was significantly less frequent in PsA compared to controls. To account for LD between loci and dependence among allele frequencies within a locus multivariate logistic regression analyses were performed to identify HLA alleles independently associated with PsA. Alleles selected for inclusion in multivariate analysis were HLA-A⁄26, -A⁄03, -A⁄23, -C⁄01, -C⁄02, -C⁄06, -C⁄12, -C⁄03, -C⁄07, -C⁄15, -B⁄13, -B⁄27, -B⁄38, -B⁄39, -B⁄53, -B⁄57, -B⁄07, -B⁄49, -B⁄51, -B⁄60, -DRB1⁄01, -DRB1⁄11, -DRB1⁄15, -DQB1⁄0501, -DQB1⁄0602 (FDR p < 0.05 from univariate analyses) and HLA alleles -A⁄02 and -C⁄04 (p < 0.05 in univariate analyses and previously reported to be associated with psoriasis or PsA) [29–32]. Six alleles: HLA-B⁄13, -B⁄27, -B⁄38, -B⁄39, -B⁄57 and -DRB1⁄01 were more frequent in PsA whereas the following six alleles HLA-A⁄03,

Table 1 Demographic and disease characteristics at first clinic visit of cases included in the case–control study.

a

Variable

Psoriatic arthritis (N = 678)a

Sex: males vs. females Age at 1st visit Age at diagnosis of psoriasis Age at diagnosis of PsA Duration of psoriasis Duration of PsA Swollen joint count Active joint countb Damaged joint countb Radiographic damage No. of joints with radiographic damage No. of patients with axial arthritisc PASId

293 (43%)/385 (57%) 43.1 (12.8) 28.0 (14.5) 36.1 (13.0) 15.0 (12.3) 7.0 (8.2) 4.7 (4.5) 11.2 (9.7) 8.5 (10.8) 369 (54%) 4.6 (8.1) 221 (35%) 6.3 (8.3)

Frequency (%) or mean (sd). Damage joint count, active joint count and swollen joint count is based on patients with damage, activity and swollen joints, respectively. c Defined as presence of at least unilateral grade 2 sacroiliitis. d PASI score is based on the first available. b

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Table 2 Results of haplotype analyses to evaluate association between HLA-C, B haplotypes and psoriatic arthritis. The alleles in bold script indicate those statistically significant at FDR p < 0.01. Haplotype C, B

Cases (%) Controls (%) v2 p value v2 FDR OR p value

⁄

07, ⁄08 11.87 07, ⁄07 8.48 ⁄ 12, ⁄38 7.23 ⁄ 06, ⁄57 6.86 ⁄ 04, ⁄35 6.49 ⁄ 05, ⁄44 6.4 ⁄ ⁄ 02, 27 4.97 ⁄ ⁄ 06, 13 4.13 ⁄ 01, ⁄27 3.86 ⁄ 08, ⁄14 3.83 ⁄ 03, ⁄40 (⁄4001) 3.17 ⁄ 03, ⁄15 (⁄1501) 3.08 ⁄ 16, ⁄44 2.8 ⁄ 07, ⁄18 1.88 ⁄ ⁄ 12, 39 1.74 ⁄ 07, ⁄39 1.35 ⁄ 06, ⁄50 1.25 ⁄ 06, ⁄37 1.18 ⁄ 05, ⁄18 1.12 ⁄ 03, ⁄55 1.1 ⁄ ⁄ ⁄ 02, 40 ( 4002) 1.03 ⁄ ⁄ 12, 52 1.03 ⁄ 04, ⁄44 1.02 ⁄ 17, ⁄41 0.81 ⁄ 07, ⁄15 (⁄1517) 0.81 ⁄ 14, ⁄51 0.74 ⁄ 12, ⁄18 0.69 ⁄ ⁄ 07, 44 0.68 ⁄ ⁄ 15, 51 0.59 ⁄ ⁄ 07, 49 0.52 ⁄ 15, ⁄07 0.44 ⁄

13 13.13 1.02 4 8.17 6.51 1.74 2.25 1.52 4.36 5.14 4.4 3.95 2.05 0.94 0.59 0.58 1.53 0.73 1.29 1.02 0.87 1.41 0.58 0.36 1.31 1.31 1.6 2.31 1.6 0.65

0.301 <0.0001 <0.0001 0.0013 0.0722 0.8324 <0.0001 0.0063 0.0002 0.4419 0.0079 0.0596 0.0824 0.7131 0.0748 0.0448 0.0699 0.409 0.3058 0.6325 0.9982 0.6903 0.332 0.4885 0.1322 0.1282 0.096 0.0211 0.0001 0.0051 0.4291

0.4666 0.0005 <0.0001 0.0065 0.1599 0.8601 0.0001 0.0243 0.0011 0.5479 0.0271 0.1541 0.1597 0.7623 0.1547 0.1263 0.1667 0.5513 0.4514 0.7262 0.9982 0.7643 0.4677 0.5824 0.2157 0.2208 0.175 0.0653 0.0011 0.0227 0.5543

0.89 0.6 7.52 1.75 0.77 0.97 2.92 1.85 2.58 0.86 0.59 0.68 0.69 0.9 1.84 2.29 2.14 0.76 1.52 0.84 1 1.17 0.71 1.38 2.21 0.55 0.51 0.41 0.25 0.31 0.66

95% CI 0.70, 1.11 0.47, 0.77 4.27, 13.25 1.24, 2.46 0.57, 1.03 0.71, 1.31 1.82, 4.69 1.18, 2.88 1.54, 4.31 0.59, 1.26 0.40, 0.88 0.46, 1.02 0.45, 1.05 0.53, 1.55 0.93, 3.65 1.0, 5.24 0.92, 4.99 0.39, 1.46 0.68, 3.38 0.42, 1.69 0.48, 2.11 0.54, 2.54 0.35, 1.42 0.55, 3.44 0.77, 6.38 0.25, 1.20 0.23, 1.14 0.19, 0.90 0.11, 0.54 0.13, 0.74 0.23, 1.86

-C⁄03, -B⁄49, -B⁄51, -DRB1⁄11 and -DQB1⁄0602 were less frequent (Table 3) 3.2. Family-based association study The demographics and disease characteristics of the probands from these families are given in Supplementary Table 6. Although probands from Newfoundland were older than those from Ontario, there was no significant difference in the age at onset of psoriasis or PsA. The results of the univariate analysis with individual HLA alleles are given in Supplementary Table 7. Z values greater than 0 indicate over-transmission of the allele, whereas values less than 0 indicate under-transmission of allele. In univariate analyses, HLA alleles -A⁄02, -B⁄27 and -DRB1⁄07 were more frequently transmitted in PsA, whereas five other HLA alleles -A⁄03, -A⁄28, -B⁄51, -DRB1⁄11 and -DQB1⁄0301 were less frequently transmitted. A trend towards higher transmission was found with HLA-C⁄12,

Table 3 Results of multivariate logistic regression analysis to determine independent association between HLA alleles and PsA case–control status. HLA allele

Odds ratio

95% Confidence interval

p value

A⁄03 C⁄03 B⁄13 B⁄27 B⁄38 B⁄39 B⁄57 B⁄49 B⁄51 DRB1⁄01 DRB1⁄11 DQB1⁄0602

0.68 0.74 2.23 3.39 9.32 2.39 2.41 0.37 0.56 1.40 0.62 0.71

0.52, 0.55, 1.40, 2.35, 5.16, 1.39, 1.66, 0.15, 0.36, 1.05, 0.44, 0.53,

0.0061 0.0397 0.0008 <0.0001 <0.0001 0.0016 <0.0001 0.0348 0.0105 0.0214 0.0060 0.0214

0.90 0.99 3.56 4.89 16.85 4.10 3.49 0.93 0.87 1.86 0.87 0.95

-B⁄39, -B⁄57, -DRB1⁄16 & -DQB1⁄0303 while a trend towards lesser transmission was found with the HLA alleles -C⁄15, -B⁄07 and DQB1⁄0503. Haplotype transmission analysis showed that A⁄02 C⁄06 B⁄57 DRB1⁄07 DQB1⁄0303 formed an extended haplotype with excess transmission to PsA. HLA-B⁄27 was independently associated with risk. When comparing the results from the complementary univariate case–control and family-based analyses, similar trends in both analyses were noted at all loci except for the DQB1 locus (Table 4) It was concluded that that HLA-C⁄12/B⁄38, HLA-B⁄27 and HLAC⁄06/B⁄57 are haplotypes (alleles) robustly associated with PsA.

4. Discussion The MHC region on chromosome 6p is the major contributor to PsA susceptibility. Although extensive LD makes identification of the true disease susceptibility locus/loci difficult, it is believed that the HLA region, especially HLA-B and C, are the most important loci associated with PsA. Using serological assays, it was previously shown that HLA-B13, -B16 (and its splits -B38 and -B39), -B17 and -Cw6 are associated with psoriasis, with or without arthritis, while HLA-B27 and -B7 are specifically associated with PsA [16]. We report results of a large case–control and family-based study of association of PsA defined according to CASPAR criteria with HLA alleles identified using molecular methods, showing that the frequency of HLA alleles HLA-C⁄12, -B⁄27 and -B⁄57 is higher in PsA compared to controls, whereas the HLA alleles -B⁄07, -B⁄51 and -DRB1⁄11 are less frequent. This is the largest study on HLA allele association with PsA to date involving 678 cases and 688 controls as well as 283 families with 1000 individuals. Application of the CASPAR criteria ensured that the cases were well-characterized. The data were examined for population stratification and ethnic outliers were excluded. Both case–control and family-based analyses were conducted to obtain robust results. To increase power, families were recruited from both Ontario and Newfoundland. In spite of a large number of families recruited, the family-based study was less powerful than the case–control study, although associations were generally consistent. Moreover, the results are largely consistent with those reported by smaller studies mostly done using serological assays

Table 4 Summary of comparisons between case–control and family-based association tests for the association between HLA alleles and PsA. HLA allele

Direction of association with PsA

Case–control association analysis

Family-based association analysis

OR

Z statistic

FDR p value

p value

HLA-A A⁄02 A⁄03 A⁄28

Higher Lower Lower

1.28 0.65 0.58

0.0694 0.0104 0.0593

2.78 2.28 3.38

0.0054 0.0226 0.0007

HLA-C C⁄12 C⁄15

Higher Lower

2.58 0.49

<0.0001 0.0169

1.83 1.92

0.0667 0.0551

HLA-B B⁄27 B⁄39 B⁄57 B⁄07 B⁄51

Higher Higher Higher Lower Lower

3.03 2.01 1.96 0.58 0.45

<0.0001 0.0276 0.0009 0.0003 0.0008

3.74 1.60 1.75 1.77 2.54

0.0002 0.1088 0.0797 0.0769 0.0112

HLA-DRB1 DRB1⁄16 Higher DRB1⁄11 Lower

1.80 0.67

0.0945 0.0362

1.71 3.82

0.0881 0.0001

V. Chandran et al. / Human Immunology 74 (2013) 1333–1338

for HLA, and are an important contribution to the literature on HLA allele association with PsA. However, the associations obtained may be due to association with psoriasis and/or PsA. Investigating the differential association between the two traits could be attempted only if an additional control group of patients with psoriasis were recruited from the same population. This analysis was recently conducted and demonstrated that HLA-B⁄27 and -B⁄38 (which is in LD with C⁄12) alleles are more prevalent in PsA compared to PsC, and that the allele -C⁄06 (which is in LD with -B⁄57) is less prevalent [23]. Thus HLA-B⁄27 and -C⁄12 are alleles that specifically increase risk for PsA. Other non-HLA genes within the MHC region may also increase susceptibility to PsA. This was not specifically investigated in this study – only HLA genes were genotyped and analyzed. Confirmation of the results in an independent population was also not done. Because similar results were reported earlier for some of the reported associations it is arguable that this may not be necessary. In a recent study in a homogeneous Irish population, HLA-B⁄27 and -B⁄38 was also found to be specifically associated with PsA when compared to PsC [19]. Alleles found to be less common in PsA compared to unaffected controls need independent verification. However, an independent cohort with complete HLA genotyping was not available to us. Recently, methods for imputing HLA alleles from SNP data have been developed and therefore we plan to replicate the associations reported in this report in large cohorts of psoriasis and PsA that are being SNP genotyped for GWAS in additional Caucasian populations [33]. HLA-C⁄12 is present in 20% of patients with PsA and in this study carries a strong association with PsA. The association between this allele and psoriasis has been controversial. Positive association with psoriasis was shown in some studies [34,35]. However, other studies have shown no such association or even significantly lower frequency in psoriasis patents than in controls [36–38]. A recent genome-wide association study showed that HLA-C⁄12 is associated with psoriasis among Caucasians but not among Chinese, independent of HLA C⁄0602 [39]. HLA-C⁄12 forms a common haplotype with HLA-B⁄38 and this haplotype is strongly associated with PsA in our case–control analysis. HLA-B⁄38 has been shown to be associated with PsA in previous studies, and was also strongly associated with PsA in this case–control study [40–42]. However, there was only a trend for association between PsA and -C⁄12 and -B⁄38 in the family-based association tests. This could reflect lower power in this analysis; alternatively the association in the case–control study may be due to population stratification. Given the previous reports of a positive association, the strong association in the case–control study and a trend for association in the family-based study, we believe it is likely that HLAC⁄12 (or the allele -B⁄38 in strong LD with it) is associated with PsA. Clarification of whether this association is with the skin or joint disease requires comparison of PsA with a control group of patients with psoriasis but without PsA. Such a comparison has been recently reported in studies done by our group, confirming an association between HLA-C⁄12 and PsA [22,23]. Haplotype analyses have shown that the extended haplotype that contains HLAC⁄12 and -B⁄38 (HLA-A⁄26, C⁄12, B⁄38, DR⁄04, DQ⁄0302) is present in a significant proportion of cases and therefore must represent events in the past thousand years (because recent events cannot appear in a large fraction of the population). Positive selection may play a role since alleles predisposing to excessive inflammation may be beneficial to the individual by leading to better control of infection. For example, HLA B⁄27 is a risk allele for ankylosing spondylitis and HLA-C⁄06 for psoriasis, but patients with HIV carrying these alleles have better clearance of HIV virus after infection [33]. Thus this association could indicate a founder effect with subsequent positive selection, and needs further study.

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HLA-B⁄27 has been found to be associated with PsA, but not with psoriasis [16,37,43]. Our study confirms this previously reported association in a well-defined PsA population using both case–control and family-based designs. HLA-B⁄27 was present in 20% of the cases and in multiple regression analysis the association was independent of HLA-C⁄12/B⁄38. HLA-B⁄27 forms haplotypes with HLA-C⁄01 and HLA-C⁄02; both haplotypes are associated with PsA. We also found that HLA-B⁄57 is strongly associated with PsA. HLA-B⁄57 was present in 15% of the cases and in multiple regression analysis the association was independent of HLAC⁄12/B⁄38 and HLA-B⁄27. HLA-B⁄57 is in tight LD with HLAC⁄06. Therefore, the association between PsA/psoriasis and HLAB⁄57 could be indirectly due to association between HLA-C⁄06 and psoriasis [44]. In fact, HLA-C⁄06 is strongly associated with PsA in univariate analysis in the case–control study, but not in the family-based study, whereas HLA-B⁄57 was associated with PsA in the univariate & multivariate case–control study and there was a trend for association in the family-based study (p = 0.08). A recent genome-wide study demonstrated that HLA-B⁄57 was associated with psoriasis independent of HLA-C⁄0602, but association with PsA was not evaluated [39]. HLA-B⁄57 could therefore represent an additional risk allele for psoriasis and/or PsA. When comparing PsA to PsC, HLA-B⁄57 as well as C⁄06 are less prevalent in PsA [23]. HLA-B⁄07, -B⁄51 and DRB1⁄11 were less prevalent in PsA cases compared to controls and thus may have a protective role. There have been no previous reports of a protective role for these alleles for psoriasis/PsA although HLA-B⁄07 was reported to be more common in juvenile idiopathic arthritis (JIA) as was HLA-B⁄51 for PsA in the Japanese [45,46]. In addition, HLA-B7 antigen was associated with milder disease in patients with PsA [16]. HLA-DRB1⁄11 was shown to differentiate juvenile PsA from oligoarticular and polyarticular subtypes of JIA [47]. Instead of comparing results obtained from case–controls and family-based association analyses, it would have been ideal to combine the two strategies in a hybrid design [48]. This was however not done since most of the Ontario probands were also included in the case–control analyses and therefore the two were not completely independent. Moreover, the families included had many with more complex structure than simple trios. The association between alleles of HLA Class I molecules and PsA indicates a potential role for both the adaptive (CD8+ T cells) as well as the innate immune system in PsA pathogenesis. HLA Class I restricted CD8+ T cells influencing the response to an as yet unidentified peptide could lead to an immune response in the skin and musculoskeletal structures. However, in ankylosing spondylitis, a disease with features in common to PsA, evidence for such a response has been hard to find [49]. One important innate immune mechanism involving HLA class I recognition is through activation of NK cells upon engagement of NK cell receptors, chiefly the killer-cell immunoglobulin-like receptors (KIR), the locus of which is on chromosome 19q13.4 within the 1 Mb leukocyte receptor complex [50]. Studies on the interaction between HLA and KIR polymorphisms and susceptibility to PsA may provide clues to the pathogenesis of PsA. Thus, to conclude, this large case–control and family based association study using molecular HLA typing and well phenotyped cases (satisfying CASPAR criteria) and controls, demonstrates that HLA-C⁄12/B⁄38, HLA-B⁄27 and HLA-C⁄06/B⁄57 are alleles/haplotypes robustly associated with PsA. However, since patients with PsA also have psoriasis it is difficult to determine whether the primary association is with arthritis or psoriasis. Further studies to distinguish differential genetic associations with cutaneous and arthritic traits of PsA as well as the associations with varied arthritic manifestations of PsA are warranted. Population genetic studies to

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investigate founder effect and selection are also required to further understand the genetic architecture of psoriasis and PsA. Acknowledgments Vinod Chandran was supported by a Canadian Institutes of Health Research (CIHR) – Clinical Research Initiative Fellowship and by the Krembil Foundation. This work was supported in part by Grants from The Arthritis Society, the CIHR Institute of Musculoskeletal Health and Arthritis and the Krembil Foundation. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.humimm.2013. 07.014. References [1] Taylor W, Gladman D, Helliwell P, et al. Classification criteria for psoriatic arthritis: development of new criteria from a large international study. Arthritis Rheum 2006;54:2665–73. [2] Zeidler H, Amor B. The assessment in Spondyloarthritis International Society (ASAS) classification criteria for peripheral spondyloarthritis and for spondyloarthritis in general: the spondyloarthritis concept in progress. Ann Rheum Dis 2011;70:1–3. [3] Rahman P, Elder JT. Genetic epidemiology of psoriasis and psoriatic arthritis. Ann Rheum Dis 2005;64(Suppl. 2):ii37–9. discussion ii40–1. [4] Chandran V, Schentag CT, Brockbank JE, et al. Familial aggregation of psoriatic arthritis. Ann Rheum Dis 2009;68:664–7. [5] Nair RP, Henseler T, Jenisch S, et al. Evidence for two psoriasis susceptibility loci (HLA and 17q) and two novel candidate regions (16q and 20p) by genomewide scan. Hum Mol Genet 1997;6:1349–56. [6] Trembath RC, Clough RL, Rosbotham JL, et al. 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;6:813–20. [7] Leder RO, Mansbridge JN, Hallmayer J, et al. Familial psoriasis and HLA-B: unambiguous support for linkage in 97 published families. Hum Hered 1998;48:198–211. [8] Carter KW, Pluzhnikov A, Timms AE, et al. Combined analysis of three whole genome linkage scans for ankylosing spondylitis. Rheumatology (Oxford) 2007;46:763–71. [9] Nair RP, Duffin KC, Helms C, et al. Genome-wide scan reveals association of psoriasis with IL-23 and NF-kappaB pathways. Nat Genet 2009;41:199–204. [10] Hüffmeier U, Uebe S, Ekici AB, et al. Common variants at TRAF3IP2 are associated with susceptibility to psoriatic arthritis and psoriasis. Nat Genet 2010;42:996–9. [11] Reveille JD, Sims AM, et al. Australo-Anglo-American Spondyloarthritis Consortium (TASC). Genome-wide association study of ankylosing spondylitis identifies non-MHC susceptibility loci. Nat Genet 2010;42:123–7. [12] Evans DM et al. The Australo-Anglo-American Spondyloarthritis Consortium (TASC); the Wellcome Trust Case–Control Consortium 2 (WTCCC2). Interaction between ERAP1 and HLA-B27 in ankylosing spondylitis implicates peptide handling in the mechanism for HLA-B27 in disease susceptibility. Nat Genet 2011;43:761–7. [13] de Bakker PI, McVean G, Sabeti PC, et al. A high-resolution HLA and SNP haplotype map for disease association studies in the extended human MHC. Nat Genet 2006;38:1166–72. [14] Miretti MM, Walsh EC, Ke X, et al. A high-resolution linkage-disequilibrium map of the human major histocompatibility complex and first generation of tag single-nucleotide polymorphisms. Am J Hum Genet 2005;76:634–46. [15] Walsh EC, Mather KA, Schaffner SF, et al. An integrated haplotype map of the human major histocompatibility complex. Am J Hum Genet 2003;73:580–90. [16] Gladman DD, Anhorn KA, Schachter RK, et al. HLA antigens in psoriatic arthritis. J Rheumatol 1986;13:586–92. [17] Gladman DD, Cheung C, Ng CM, et al. HLA C-locus alleles in psoriatic arthritis. Hum Immunol 1999;60:259–61. [18] Ho PY, Barton A, Worthington J, et al. HLA-Cw6 and HLA-DRB1⁄07 together are associated with less severe joint disease in psoriatic arthritis. Ann Rheum Dis 2007;66:807–11. [19] Winchester R, Minevich G, Steshenko V, et al. HLA associations reveal genetic heterogeneity in psoriatic arthritis and in the psoriasis phenotype. Arthritis Rheum 2012;64:1134–44. [20] Popa OM, Popa L, Dutescu MI, et al. HLA-C locus and genetic susceptibility to psoriatic arthritis in Romanian population. Tissue Antigens 2011;77:325–8.

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