Genetic study confirms association of HLA-DPA1∗01:03 subtype with ankylosing spondylitis in HLA-B27-positive populations

Human Immunology 74 (2013) 764–767

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Genetic study confirms association of HLA-DPA1⁄01:03 subtype with ankylosing spondylitis in HLA-B27-positive populations Roberto Díaz-Peña a,b, Patricia Castro-Santos b, Ana M. Aransay c, Jacome Brüges-Armas d, Fernando M. Pimentel-Santos e,f, Carlos López-Larrea a,g,⇑ a

Department of Immunology, Hospital Universitario Central de Asturias, Oviedo, Spain Faculty of Health Sciences, Universidad Autónoma de Chile, Talca, Chile Genome Analysis Platform, CIC bioGUNE, Bizkaia Technology Park, Derio, Spain d Institute for Molecular and Cell Biology, University of Porto, Porto, Portugal e CEDOC, Faculdade de Ciências Médicas da Universidade Nova de Lisboa, Lisboa, Portugal f CHLO, Department of Rheumatology, Hospital de Egas Moniz, Lisboa, Portugal g Fundación Renal ‘‘Iñigo Alvarez de Toledo’’, Madrid, Spain b c

a r t i c l e

i n f o

Article history: Received 17 August 2012 Accepted 19 February 2013 Available online 28 February 2013

a b s t r a c t The association of human leukocyte antigen (HLA)-B27 with ankylosing spondylitis (AS) has been known for over 38 years. However, it is not the only gene associated with AS. The aim of this study was to confirm the association of HLA markers around HLA-DPA1/DPB1 region with AS in HLA-B27 positive populations. Five SNPs (rs422544, rs6914849, rs92777535, rs3128968 and rs2295119) from the HLA-DPA1/DPB1 region were genotyped in 340 individuals HLA-B27-positive from Portugal (137 AS patients and 203 healthy controls). Characterizations of HLA-DPA1/DPB1 alleles were also performed. rs422544 revealed a significant association with AS (P < 0.05) and sliding windows (SW) analysis showed association of some groups of adjacent SNPs within HLA-DPA1/DPB1 region with AS (P < 0.05). We also found association of the HLA-DPA1⁄01:03 allele with AS (P < 0.05). This is the first study that confirms the association of HLA markers and haplotypes around HLA-DPA1 and HLA-DPB1 with AS. Ó 2013 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.

1. Introduction Ankylosing spondylitis (AS) is an inflammatory rheumatic disease affecting predominantly axial skeleton and sacroiliac joints, but also affect peripheral joints and entheses or extraarticular sites such uvea and tendon insertions. Disease susceptibility is clearly attributable to genetic factors, estimating that disease heritability exceeds 90% [1]. This heritability is also significant with respect to the clinical manifestations of AS [2]. Heritabilities of 40% and 62% have been estimated for radiographic disease severity and age of symptom onset, respectively. Even for disease activity measured by BASDAI and BASFI questionnaires, respective values of Abbreviations: AS, ankylosing spondylitis; HLA, human leukocyte antigen; SNPs, single nucleotide polymorphisms; GWAS, genome-wide association study; MHC, major histocompatibility complex; LD, linkage disequilibrium; PCR, polymerase chain reaction; SSO, sequence-specific oligonucleotides; HWE, Hardy–Weinberg equilibrium; OR, odds ratios; CI, confidence intervals; SW, sliding window; ASP, affected sibling pair. ⇑ Corresponding author. Address: Department of Immunology, Hospital Universitario Central de Asturias, C/ Celestino Villamil s/n, 33006 Oviedo, Spain. Fax: +34 985106195. E-mail address: [email protected] (C. López-Larrea).

51% and 76% have been estimated. The link between human leukocyte antigen (HLA)-B27 and AS remains the strongest association between an HLA class I molecule and a disease [3]. While more than 90% of Caucasian with AS are HLA-B27-positive, only 1–5% of HLA-B27-positive individuals develop the disease, suggesting that other genes, both within and outside HLA, are involved in disease susceptibility. In fact, HLA-B27 accounted only for 16% of the genetic load in AS and indeed its pathogenic role has not yet been resolved [4]. Research in the field of AS genetics and genomics is advancing rapidly, mainly through single nucleotide polymorphisms (SNPs) genotyping (genome-wide association studies, GWAS) and next genome sequencing, two techniques that are improving our understanding of the etiopathogenesis of the disease. The first GWAS for AS has provided an unprecedented view of the genetic markers of AS susceptibility and clinical manifestations [5], increasing the number of genetic loci convincingly associated with AS to nine, seven genes and two dessert regions (ERAP1, IL23R, HLA-B, KIF21B, RUNX3, IL12B and LTBR-TNFRSF1A; 2p15 and 21q22). In spite of this, MHC containing the HLA locus is strongly linked and associated with AS [5].

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R. Díaz-Peña et al. / Human Immunology 74 (2013) 764–767

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There is evidence for the implication in AS susceptibility of other non-B27 loci within the major histocompatibility complex (MHC) such as HLA-B alleles, HLA-DRB alleles, Tumour Necrosis Factor a (TNFa) and other genes involved in the antigenic presentation by HLA class I molecules (LMP2, LMP7, and TAP) [6,7]. However, none of these studies took in account the strong linkage of B27 haplotypes with AS. In a previous study reported by us, we investigated the association of SNPs markers within HLA and AS in a Spanish population HLA-B27-positive (AS patients and controls) [8]. We showed that HLA markers and linkage disequilibrium (LD) blocks were associated with AS. In the present study, we further analyze the contribution of HLA markers, different from B27 and located around HLA-DPA1 and HLA-DPB1 regions, to the susceptibility to develop AS in a Portuguese population.

frequencies were compared between AS patients and controls by Chi-Square statistics, and odds ratios (OR) and 95% confidence intervals (95% CI) were calculated within PLINK software [11]; (ii) sliding windows of 2 to 5 SNPs each were tested for association analyses by Chi-Square test within PLINK software [11]. The ‘‘Sliding Windows’’ analyses assess the frequency of composite genotypes of a fixed number of contiguous SNPs (shifting 1 SNP at a time) instead of examining haplotypes on the basis of their linkage disequilibrium (LD). We used SPSS v.13 statistical software to assess the association of each HLA-DP allele on AS susceptibility. We used Fisher’s exact test on two-by-two contingency tables with or without each allele.

2. Materials and methods

Table 1 shows the single marker allelic association results of the 5 SNPs successfully characterized in the studied population. Among all analyzed markers, only rs422544, located downstream of HLA-DPA1, showed significant association with AS after Bonferroni correction (P = 0.029, Odds Ratio (OR) = 1.56). The association of the remaining SNPs was not significant. In addition to single marker association, sliding window (SW) test revealed association between groups of adjacent SNPs and AS (Table 2) [11]. The P-values obtained for the SW test of a region that includes all studied SNPs (Chr6: 33,134,088 to 33,134,088) ranged from P = 0.0025–0.05, supporting the association of this region, located around HLA-DPA1 and HLA-DPB1 loci, with AS. We next tested the possible influence of HLA-DPA1 and HLA-DPB1 alleles in the susceptibility to AS in 128 AS patients and 193 healthy controls. We found association of the HLA-DPA1⁄01:03 allele with AS (P = 0.027) (Table 3). We also combined the results presented in this study (Portuguese population) with the data previously reported in the Spanish population [7]. The results confirm the association of HLA-DPA1⁄01:03 allele with AS in Caucasian populations, being its allele frequency decreased in AS patients compared to B27-positive healthy controls (combined population, P = 1.5  104) (Table 3). No association was seen with other HLA-DP alleles or DPA1-DPB1 haplotypes in the joint analysis. Due to the dissimilarities observed in the allele frequencies of rs422544 in the studied populations, we also evaluated the LD [12] between rs422544 polymorphism and HLA-DPA1⁄01:03 allele. The resulting LD values were D0 = 0.162 and D0 = 0.07 for the Portuguese and Spanish populations, respectively.

2.1. Patients and controls A total of 137 AS patients and 203 healthy controls were enrolled in this study, being both, cases and controls, HLA-B27positive. The patients with AS were diagnosed at the Rheumatology Unit of Hospital de Egas Moniz, Lisboa, Portugal, and at the Rheumatic Diseases Clinic of the Angra do Heroismo Hospital, Azores, Portugal, following New York criteria [9]. Radiographs of the pelvis and lumbar spine were obtained from all AS patients, and they revealed the presence of sacroiliitis bilateral grade II or greater. All individuals gave written informed consent prior to enrolling in the study. The protocol was approved by the ethics committees of the two mentioned hospitals and conducted according to the Declaration of Helsinki. 2.2. SNP genotyping SNPs rs422544, rs9277535, rs3128968 and rs2295119 were genotyped using iPLEX technology (MassARRAY, Sequenom) and rs6914849 was genotyped using pyrosequencing assay. We designed PCR and pyrosequencing primers for rs6914849 for the Pyrosequencing platform (Biotage, Uppsala, Sweden). The primer sequences to genotype rs6914849 were as follows: forward, 50 -biotin-AGC CCT AGG GTC CTA GAA GAG A-30 ; reverse, 50 -GGA ACT CCG AGC TAT GGA CTC T-30 ; sequencing primer, 50 -AAA GAG TGT TTC TGT ATC CT-30 . The pyrosequencing reaction was performed on a PyroMark Q24 instrument (Qiagen) using the Pyro Gold Q24 Reagents (Qiagen,) and Streptavidin Sepharose HP (Amersham Biosciences, Uppsala, Sweden). Purification and subsequent processing of the biotinylated single-stranded DNA was performed according to the manufacturer’s recommendations. Resulting data were analyzed with the PyroMark Q24 software version 2.0.6 (Qiagen). 2.3. Characterization of HLA-DPA1/DPB1 alleles HLA-DPA1 and HLA-DPB1 alleles were genotyped in 128 AS patients and 193 healthy controls. HLA-DPA1 alleles were typed by polymerase chain reaction (PCR) with sequence-specific primers (SSP) [10]. The HLA-DPB1 alleles were typed using HLA-SSO typing kit (Tepnel Lifecodes Corporation) according to the manufacturer’s instructions. 2.4. Statistical test Allele frequencies obtained for each SNP were tested for deviations from Hardy–Weinberg Equilibrium (HWE) expectations. Type II error of 0.5 i.e. a statistical power of 0.5. The statistical analysis of SNPs genotyping data was performed as follows: (i) allele

3. Results

4. Discussion Several studies have suggested the existence of non-HLA-B27 MHC genes that influence in AS susceptibility [7]. However, the linkage disequilibrium of those loci with HLA or with B27 haplotypes has made difficult to discern their true contribution to AS proneness. A previous study reported by us investigated the association of HLA markers with AS enrolling an HLA-B27 neutral group (AS cases and controls) from a Spanish population [8], being the first revision to show that a region located around HLA-DPA1 and HLA-DPB1 genes (non-HLA-B27 region) was associated with the susceptibility to AS. Previous studies have investigated MHC class II region and their association with AS, but little replication has been obtained. Genes involved in the antigenic presentation by HLA class I molecules (LMP2, LMP7 and TAP) have been investigated [13–15], although it is early to assign a role for TAP, LMP2 and LMP7 in AS susceptibility [16]. Several studies have indicated the possible involvement of HLA class II molecules in AS development. It has been reported that HLA-DRB alleles, including HLA-DRB1⁄01 and DRB1⁄04, may contribute to AS susceptibility [17,18]. Moreover, HLA-DRB1⁄08

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Table 1 Single-SNP allelic association analysis (Chi-Square test, PLINK software [11]). SNP

Position

Gene symbol

Location

A1

MAF AS patients

MAF controls

P value

P BONF

OR

L95

U95

rs422544 rs6914849 rs9277535 rs3128968 rs2295119

33134088 33148693 33162839 33164231 33168848

HLA-DPA1 HLA-DPA1 HLA-DPB1 HLA-DPB1 HLA-DPB1

flanking_3UTR intron 3UTR flanking_3UTR flanking_3UTR

C A G A A

0.44 0.33 0.19 0.21 0.2

0.34 0.26 0.23 0.23 0.17

0.006 0.08 0.28 0.44 0.38

0.03 0.41 1 1 1

1.56 1.36 0.8 0.86 1.19

1.14 0.96 0.53 0.59 0.80

2.14 1.93 1.20 1.25 1.77

SNP = single nucleotide polymorphism; A1 = minor allele nucleotide; MAF = minor allele frequency; P BONF = P value using Bonferroni correction; OR = Odds ratio; L95 = lower bound on confidence interval for odds ratio; U95 = upper bound on confidence interval for odds ratio. SNPs were genotyped in 340 individuals HLA-B27-positive from Portugal (137 AS patients and 203 healthy controls).

Table 2 Gene region, marker composition and association values (only P-values 6 0.05 are shown) obtained with the Chi-Square test considering sliding of 2–5 SNPs windows (PLINK software [11]). Gene (S)

Markers at each SW

P value

HLA-DPA1 HLA-DPA1 HLA-DPA1, HLA-DPA1, HLA-DPA1, HLA-DPA1, HLA-DPB1 HLA-DPA1, HLA-DPA1, HLA-DPA1, HLA-DPA1,

rs422544|rs6914849 rs422544|rs6914849 rs422544|rs6914849|rs9277535 rs422544|rs6914849|rs9277535 rs422544|rs6914849|rs9277535 rs422544|rs6914849|rs9277535 rs6914849|rs9277535|rs3128968 rs422544|rs6914849|rs9277535|rs3128968 rs422544|rs6914849|rs9277535|rs3128968 rs422544|rs6914849|rs9277535|rs3128968|rs2295119 rs422544|rs6914849|rs9277535|rs3128968|rs2295119

2.54  103 0.02 0.03 0.05 0.02 0.05 0.03 8.79  103 0.05 0.02 0.04

HLA-DPB1 HLA-DPB1 HLA-DPB1 HLA-DPB1 HLA-DPB1 HLA-DPB1 HLA-DPB1 HLA-DPB1

has also been implicated both in susceptibility to uveitis, in the setting of AS and to juvenile-onset AS [7]. A further report examined non-B27 MHC association with AS in B27-matched MHC haplotypes in AS cases and controls and found an association with DRB1 irrespective of the presence of HLA-B27 in the haplotype [19]. In order to control the effect of HLA-B27, the cited study genotyped nineteen SNPs within the 1-Mb region between TNF and DRB1 with an average inter-marker distance of 35 Kb in 229 affected sibling pair (ASP) families, whose members were typed for B27 status and DRB1 subtypes. The conclusion from this research was that a second disease gene is probably located in the HLA class II region. Our research support the possible involvement of HLA class II (DP) molecules in the development of AS. A significant association was detected between SNP rs422544 and AS susceptibility, and sliding window omnibus test also revealed several haplotype associations with AS in a region located around the HLA-DPA1 and HLADPB1 genes. We are aware that the significance of rs422544 alone could increase the statistical significance of the blocks that contain

this SNP and that the frequency of rs422544-C allele differs between the Spanish and Portuguese AS populations, being 22.8% in Spanish cases and 44.4% in Portuguese cases. However, the HLA-DPAI⁄01:03 allele was present in a more similar way in both AS populations and was significantly associated with a protective influence in AS. In fact, we evaluated the LD between the rs422544 and HLA-DPA1⁄01:03 allele in both populations and concluded that the observed differences are influenced by the genetic background of each population. HLA-DP molecules present processed peptides, derived predominantly from membrane and extracellular proteins, to CD4+ T cells, triggering subsequent immunological responses. In this way, HLA molecules capable of binding a particular epitope can restrict T cell induced-immune responses, leading to an association between particular HLA types and immune-related diseases. HLADP allelic and haplotypic diversity as well as SNPs located in the HLA-DP locus have been associated with several diseases, such as diabetes mellitus [20,21], juvenile rheumatoid arthritis [22], sarcoidosis [23], chronic berylliasis [24], and systemic sclerosis

Table 3 Frequencies of HLA-DPA1 alleles genotyped in patients with AS and healthy controls from a Portuguese and a Spanish populations and, the significance (P-value) of the allele frequencies differences (shown in %) found between the cases and controls of each population separately as well as in an artificial joint-population (the combination of the two previous ones). Portuguese population HLA-DPA1 alleles

Controls

AS patients

Total number of alleles (2n) DPA1⁄01:03 DPA1⁄01:04 DPA1⁄01:05 DPA1⁄01:10 DPA1⁄02:01 DPA1⁄02:02 DPA1⁄02:03 DPA1⁄03:01 DPA1⁄04:01

386 73.8 0 0 0 23.1 1.8 0.3 0 0

256 65.6 0.8 2.7 0.4 23.4 3.1 1.6 0.8 1.6

AS = ankylosing spondylitis; NS = not significant.

Spanish population [6] P value

Controls

AS patients

0.03 NS NS NS NS NS NS NS NS

624 79.3 0 0 0.2 20.3 0 0 0 0.2

522 71.4 0 0 0 28.5 0 0 0 0

Joint population P value

Controls

AS patients

P value

0.002 – – NS 0.001 – – – NS

1010 77.2 0 0.4 0.1 21.4 0.7 0.1 0 0.1

778 69.5 0.3 0.9 0.1 26.9 1 0.5 0.3 0.5

1.5  104 NS NS NS NS NS NS NS NS

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[25,26]. Here, we found evidence for supporting previously reported association of HLA-DPA1 allele with AS susceptibility. Regarding the mechanism, CD4+ T cells can recognize normal and abnormal forms of HLA-B27 and self-peptides from HLA-B27 might be recognized as autoantigenic by CD4+ T cells [27], raising the question of whether this might explain the importance of these cells for spondyloarthropaty-like disease in B27 transgenic rats and also about the function of HLA-B27 in human disease development. HLA-B and HLA-DP could contribute to AS in an additive manner, but further studies with different ethnic backgrounds (different from Caucasian), involving even more samples, will be required to confirm these findings. The challenge in the next years will be to identify genetic variants involved in the different clinical manifestations and AS-associated features in order to predict the evolution of the disease. All these studies can provide predictive information about AS patients, taking into account both environmental and unknown genetic factors. Acknowledgments This work was supported by Fundación para el Fomento en Asturias de la Investigación Científica (FICYT) PC10-70 - Fondos FEDER European Union. CIC bioGUNE support was provided from The Department of Industry, Tourism and Trade of the Government of the Autonomous Community of the Basque Country (Etortek Research Programs 2009–2011) and from the Innovation Technology Department of the Bizkaia County.

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