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Recognizing axial spondyloarthritis
de Boer, J.J.H.
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2018
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de Boer, J. J. H. (2018). Recognizing axial spondyloarthritis.
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Absence of HLA-C*07 is associated with susceptibility
to axial spondyloarthritis data from the GErman
SPondyloarthris: Inception Cohort (GESPIC) and the
SPondyloArthritis Caught Early (SPACE) cohort
Janneke J. de Winter1, Iris C. Blijdorp1,2, Henriëtte M. de Jong1, Jürgen Sauter3,
Alexander H. Schmidt3, Floris A. van Gaalen4, Désirée van der Heijde4, Denis
Podubbnyy5, Nataliya G. Yeremenko1,2, Marleen G. van de Sande1, Dominique L.
Baeten1
1Department of Clinical Immunology and Rheumatology, Amsterdam Rheumatology and
immunology Center, Academic Medical Center/University of Amsterdam, Amsterdam, The Netherlands 2Laboratory of Experimental Immunology, Academic Medical Center/
University of Amsterdam, Amsterdam, The Netherlands 3DKMS German Bone Marrow
Donor Center, Tübingen, Germany 4Department of Rheumatology, Leiden University
Medical Center, Leiden, The Netherlands 5Department of Rheumatology, Charité
Universitätsmedizin Berlin, Berlin, Germany Submitted for publication
ABSTRACT
Objectives
We aimed to assess mRNA expression of MHC class 1 related molecules in ankylosing spondylitis (AS) versus healthy controls (HC) and subsequently if absence of HLA-C*07 is associated with genetic susceptibility to axial spondyloarthritis (axSpA).
Methods
HLA-C*07 was assessed in a) an exploratory cohort of 24 AS patients versus 40 HC, b) a confirmatory cohort of 113 AS patients and 83 non-radiographic axSpA patients from the GErman SPondyloarthritis Inception Cohort (GESPIC) versus 134,528 German potential stem cell donors, and c) an early back pain cohort with 94 early axSpA patients versus 216 chronic back pain (CBP) patients from the SPondyloArthritis Caught Early (SPACE) cohort.
Results
In the exploratory cohort, 79% of the AS patients were HLA-C*07 negative compared to 35% of the HC (p<0.001). This difference was confirmed in GESPIC with 73% of AS patients being HLA-C*07 negative compared to 50% of the controls (p<0.0001); 59% of the nr-axSpA patients were HLA-C*07 negative. In the SPACE cohort, 70% of axSpA patients were HLA-C*07 negative compared to 44% of CBP patients (p<0.0001); the association between HLA-C*07 negativity and a diagnosis of axSpA was independent from HLA-B*27. Exploratory analyses in GESPIC and SPACE suggest that HLA-C*07 negativity in axSpA is associated with increased inflammatory serum markers, radiographic damage, and absence of psoriasis.
Conclusion
The absence of HLA-C*07 is associated with genetic susceptibility to axSpA, independently of HLA-B*27. HLA-C*07 status is possibly linked to an axSpA phenotype with high inflammation and radiographic damage and with a low psoriasis prevalence.
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INTRODUCTION
Axial spondyloarthritis (axSpA) is a common form of chronic inflammatory arthritis affecting the axial skeleton, including the sacroiliac joints and the spine, as well as peripheral joints and extra-articular sites. The disease has a strong genetic predisposition with ankylosing spondylitis (AS), the most homogenous and well-studied clinical phenotype within the axSpA disease spectrum, having a recurrence risk of 8% in first-degree relatives (1). Human leukocyte antigen (HLA)-B*27, part of the major histocompatibility complex (MHC), is the major susceptibility gene for AS. However, it accounts for only 25% of the overall AS heritability (2). Moreover, the concordance rate of AS in monozygotic twins (63%) is higher than in dizygotic twins (12.5%), even when dizygotic twins are concordant for HLA-B*27 (27%) (3,4). These and other observations suggest that AS is a multigenic disease and that other susceptibility genes should contribute beyond HLA-B*27. This concept has been well validated by large Genome Wide Association Studies (GWAS), identifying a whole series of additional genes in AS (5). Albeit the GWAS analyses have contributed significantly to the identification of disease-relevant pathways, the relative contribution of individual susceptibility genes remains modest, indicating that many other genes contributing to the disease may be difficult to identify by this approach. This may include rare variants as well as non-HLA-B*27 genes in the MHC region on chromosome 6p (6). Although most of the genetic association of this locus is driven by the association of AS with HLA-B*27, there is strong evidence for the involvement of other MHC genes.
An alternative approach to identify disease-relevant pathways in axSpA is to perform large-scale expression analysis using microarray or RNA sequencing technology. We have previously performed unbiased pan-genomic microarray analysis of inflamed synovial tissue (7); this approach, however, can also be used to focus on potential pathways of interest. As the expression of beta-2 microglobulin has been demonstrated to modify the disease phenotype induced by HLA-B*27 in transgenic rats (8), firstly we aimed to assess the level of mRNA expression of MHC and related genes in AS versus peripheral blood mononuclear cells of healthy controls. Since this screening experiment revealed low mRNA expression of a particular HLA-C gene product in AS, secondly the study aimed to assess if this particular MHC gene, HLA-C*07, is associated with susceptibility to AS and axSpA.
METHODS Cohorts
HLA-C*07 was assessed in a) an exploratory cohort of 24 AS patients versus 40 healthy controls, b) a confirmatory cohort of 113 AS patients and 83 non-radiographic axspa patients from the GErman SPondyloarthritis Inception Cohort
(GESPIC) versus 135,168 healthy German potential stem cell donors, and c) an early back pain cohort with 94 early axSpA patients versus 216 chronic back pain (CBP) patients from the SPondyloArthritis Caught Early (SPACE) cohort. All patients and controls gave their written informed consent prior to any study procedures. All studies were approved by the local ethics committees of the Academic Medical Center/University of Amsterdam (exploratory cohort), the Medical University of Berlin (exploratory cohort/GESPIC) and the Leiden University Medical Center (the SPACE cohort).
Exploratory cohort
DNA from patients with AS was collected in the Academic Medical Center/ University of Amsterdam (n=9) and the Medical University of Berlin (n=15). All AS patients were diagnosed by a rheumatologist and fulfilled the modified New York (mNY) criteria (9). DNA from healthy controls was collected in the Academic Medical Center/University of Amsterdam (n=10) and the Medical University of Berlin (n=30).
GESPIC
GESPIC is described in detail elsewhere (10). In short, GESPIC is an ongoing, prospective, longitudinal cohort study that started in 2000. Patients with definite axSpA diagnosed by their treating rheumatologist were included. Patients were further classified as AS or nr-axSpA based on the fulfillment of the radiographic criterion of the modified New York criteria according to the judgement of the central readers (11). The cervical and lumbar spine was scored according to the modified Stoke AS Spine Score (mSASSS) (12). AS patients fulfilled the modified New York criteria (9), the duration of symptoms was restricted to ≤10 years at the time of inclusion. Nr-axSpA patients fulfilled the European Spondylarthropathy Study Group (ESSG) criteria (13), the duration of symptoms was restricted to ≤5 years at the time of inclusion.
We included 196 patients of GESPIC for which DNA for HLA typing was available, including 113 patients with AS (57.7%) and 83 patients with nr-axSpA (42.3%). Of the 196 axSpA patients, 158 were HLA-B*27 positive (80.6%) and 100 were male (51.0%). Data were complete, except for ‘duration of back pain (missing in n=16) and HLA-B*27 (missing in n=1). The results of GESPIC were compared with 135,160 randomly selected potential stem cell donors (in short: donors) from the German Bone Marrow Donor Center (DKMS). Donors were matched with GESPIC patients according to their place of residence. Of the 135,160 donors, 13,120 were HLA-B*27 positive (9.7%) and 50,454 were male (37.3%). Donors were not allowed to have a diagnosis of axSpA.
SPACE
The SPACE cohort is an ongoing, prospective, multicenter, longitudinal cohort that started in 2009 and is described in detail earlier (14). In short, the SPACE
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cohort is including patients aged ≥16 years with chronic back pain (≥3 months, but ≤2 years) with the onset before the age of 45 years. The local medical ethical committees of the participating sites approved the study and all participants gave their written informed consent. For the present analyses, we used data and DNA of the baseline visit of patients included in the Leiden Universitary Medical Center and the Academic Medical Center Amsterdam. We used the central reading results of radiographs and MRI of the sacroiliac joints (14). We compared patients with axSpA (diagnosed by a rheumatologist and fulfilling the ASAS axSpA criteria) with non-SpA CBP patients (diagnosed by a rheumatologist as not having axSpA). We included 310 patients of the SPACE cohort, including 94 axSpA patients (30.3%) and 216 CBP controls (69.7%). In short, 42 of the 94 axSpA patients were male vs. 61 of the 216 CBP patients (44.7% vs. 28.2%, p=0.005). Of the 94 axSpA patients, 81 were HLA-B*27 positive vs. 32 of the 216 CBP controls (86.2% vs. 14.8%, p<0.0001). The mean age at inclusion of the axSpA patients was 30.5±8.8 years and 31.1±8.2 years of the CBP patients (p=0.57). The mean back pain duration was 14.5±8.1 months in the axSpA patients and 13.6±9.0 months in the CBP patients (p=0.30). Twenty-three of the 94 axSpA patients and 2 of the 216 CBP patients fulfilled the radiographic criterion of the modified New York (mNY) criteria (24.7% vs. 1.0%, p<0.0001). Missing values were ≤ n=1 except for sacroiliitis on MRI (missing n=24), radiographic sacroiliitis (n=13), BASDAI (n=26).
HLA typing
AxSpA patients and controls underwent low-resolution HLA-C typing by a DNA-based technique using sequence-specific oligonucleotides which involved polymerase chain reaction (PCR) amplification of the HLA-C region (Invitrogen, Washington, DC, USA). For the donors enrolled with DKMS, high resolution typing results were available for HLA-B and -C based on Sanger Sequencing or NGS (15). For the SPACE participants enrolled in Leiden, LIFECODES HLA SSO Typing kits were used (Imucor Norcross GA, USA).
HLA-C expression analysis
For the analysis of HLA-C expression, we included patients of the exploratory cohort (24 AS patients and 40 healthy controls), supplemented by 27 AS patients from GESPIC. Peripheral blood mononuclear cells (PBMCs) were isolated from heparinized peripheral blood of 51 patients with AS and 40 healthy controls through density gradient centrifugation using Lymphoprep Ficoll-Isopaque (Axis-Shield, Oslo, NO). RNeasy Micro Kit (Qiagen, Venlo, Netherlands) was used to extract RNA from PBMCs according to the manufacturer’s instructions. RNA concentration was determined with the Nanodrop (Nanodrop Technologies, MA, USA) and reversely transcribed into cDNA (Fermentas, MA, USA). Quantitative real-time PCR was performed on StepOnePlus™ Real-Time PCR System (Applied Biosystems, MA, USA) using TaqMan gene expression assays
for HLA-B (Hs00818803_g1), HLA-C (Hs00740298_g1 and Hs03044135_m1), beta-2 microglobulin (Hs00187842_m1) and GAPDH (4310884E) according to the manufacturer’s protocol (ThermoFisher, MA, USA). mRNA data were missing for n=2 healthy donors because of unreliable results.
Data analyses
Categorical data are presented as numbers (%), continuous data are presented as the mean (SD) or as median (interquartile range, IQR) as appropriate. We used a χ2 test for categorical data and an unpaired T test or Mann-Whitney U for continuous data when data were or were not normally distributed, respectively. Statistical tests were 2-sided, and p-values less than 0.05 were considered significant.
We investigated the association between HLA-C*07 status and disease characteristics in axSpA patients by logistic regression. Variables were first selected by backward elimination using a p-value of <.20 for inclusion and the model was secondly tested by forward selection and included sex, HLA-B*27 status, the presence of peripheral arthritis, enthesitis, dactylitis, inflammatory bowel disease (IBD), psoriasis and sacroiliitis on MRI or X-ray. Since the SPACE cohort includes both axSpA patients and CBP controls, the SPACE cohort enabled us to investigate the association of HLA-C*07 and HLA-B*27 with a diagnosis of axSpA in a multivariate regression analysis. For that purpose, we used the same logistic regression approach as described earlier: firstly, using backwards elimination and secondly testing the model by forward selection. As a sensitivity model we included all axSpA patients in the analysis, irrespective of fulfilling the ASAS criteria.
RESULTS
mRNA expression of HLA-C*07 is decreased in PBMCs of AS patients versus healthy controls
Analysis of mRNA of MHC class 1 molecules by qPCR demonstrated that the expression levels of HLA-B and Beta-2 microglobulin was not different in peripheral blood of patients with AS and healthy controls (Figure 1, panel A and B). On the contrary, expression of HLA-C was absent in 35 out of 51 AS patients, compared to 14 out of 40 healthy controls (68.7% versus 35.0%, p=0.004) (Figure 1C). Further examination revealed that this absence of expression is explained by the binding of the primers and probe of Hs00740298_g1 Taqman assay within the region transcribed from HLA-C serotype group Cw7 (HLA-C*07). qPCR analysis of the same cohort with Hs03044135_m1 HLA-C Taqman assay revealed similar expression levels of HLA-C between AS patients and healthy controls (Figure 1D).
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Figure 1. mRNA expression of A) HLA-B, B) Beta-2 microglobulin, C) HLA-C using the Hs00740298_g1 assay and D) HLA-C using the Hs03044135_m1 assay
Lower frequency of HLA-C*07 in AS
As one potential explanation for the complete absence of HLA-C*07 mRNA in a majority of the analyzed AS samples is the absence of the HLA-C*07 genotype, we hypothesized that (the absence of) HLA-C*07 is associated with genetic susceptibility for AS and axSpA. Therefore, we first explored the frequency of HLA-C*07 in a relatively small exploratory cohort of 24 AS patients versus 40 healthy controls. Nineteen out of 24 AS patients were HLA-C*07 negative compared to 14 of the 40 healthy controls (79.2% vs. 35.0%, p<0.001, Figure 2). These results correlated perfectly with our hypotheses: all individuals with mRNA expression of HLA-C using probe Hs00740298_g1 had at least 1 HLA-C*07 allele.
Figure 2. Prevalence of HLA-C*07 negativity in
A) patients with AS (n=24) and healthy controls (n=40)
B) patients with AS (n=113) from GESPIC and healthy potential donors (n=135,160) C) patients with axSpA (n=196) and chronic back pain controls (n=216) from the
SPACE Cohort
Confirmation of the association between the absence of HLA-C*07 and AS/axSpA
To confirm this finding, we tested the prevalence of HLA-C*07 in AS patients of GESPIC. Of the 113 AS patients, 82 were HLA-C*07 negative compared to 67,043 of the 135,168 donor controls (72.6% vs. 49.6%, p<0.0001, Figure 2). As the axSpA disease spectrum consists not only of AS but also of non-radiographic axSpA
(nr-axSpA), we additionally tested HLA-C*07 in 83 nr-axSpA patients of GESPIC: HLA-C*07 was negative in 59.0% (p=0.09). HLA-B*27 positivity was equal in HLA-C*07 positive and negative axSpA patients: 49 of the 65 HLA-C*07 positive axSpA patients were HLA-B*27 positive, compared to 109 of the 131 HLA-C*07 negative ax-SpA patients (75.4% vs. 83.8%, p=0.16).
We next used the SPACE cohort to test if HLA-C*07 prevalence is lower in early axSpA patients than in CBP controls. Sixty-six of the 94 axSpA patients were HLA-C*07 negative compared to 96 of the 216 CBP patients (70.2% vs. 44.4%, p<0.0001, Figure 2). Of the 23 early axSpA patients fulfilling the mNY criteria, 78.3% was HLA-C*07 negative. Of the 71 axSpA patients with nr-axSpA, 65.8% was HLA-C*07 negative. Also here, absence of HLA-C*07 was similar in HLA*B27 positive and negative patients: 22 of the 28 HLA-C*07 positive axSpA patients were HLA-B*27 positive compared to 59 of the 66 HLA-C*07 negative axSpA patients (78.6% vs. 89.4%, p=0.17). Next, we performed multivariate logistic regression in the complete SPACE population to further test whether absence of HLA-C*07 is associated with an axSpA diagnosis independently from HLA-B*27. The final model, having patients with an axSpA diagnosis and fulfilling the ASAS criteria as outcome, included inflammatory back pain, CRP level, HLA-C*07, HLA-B*27, psoriasis, uveitis and arthritis occurrence ever. This analysis showed that HLA-C*07 negativity was associated with a diagnosis of axSpA (OR 2.2, p=0.038) independently from HLA-B*27 (OR 32.8, p<0.0001). Collectively, these data in two independent cohorts demonstrate that HLA-C*07 prevalence is lower in axSpA patients than in controls, independently from HLA-B*27 prevalence.
Association between HLA-C*07 negativity and disease phenotype in axSpA
We explored if HLA-C*07 negativity was associated with a specific sub-population of axSpA patients. Table 1 shows characteristics of HLA-C*07 positive and negative axSpA patients in GESPIC. Of the 65 HLA-C*07-positive axSpA patients, 26 were male compared to 74 of the HLA-C*07 negative axSpA patients (40.0% vs. 56.5%, p=0.030). The HLA-C*07 positive axSpA patients had a mean back pain duration of 42.4±27.5 months compared to a mean back pain duration of 53.7±24.4 months in the HLA-C*07-negative axSpA patients (p=0.022). Of the 65 C*07-positive axSpA patients, 11 had psoriasis compared to 9 of 131 HLA-C*07-negative axSpA patients (16.9% vs. 6.9%, p=0.029). The mean CRP level of C*07-positive axSpA patients was 7.8±13.9 compared to 12.6±20.5 in HLA-C*07-negative axSpA patients (p=0.02). Thirty-one of the 65 HLA-C*07-positive axSpA patients fulfilled the mNY criteria compared to 82 of the 131 HLA-C*07-negative axSpA patients (47.7% vs. 62.6%, p=0.047). There were no differences in mean age, the prevalence of a positive family history, peripheral arthritis, enthesitis, IBD, uveitis, mean BASDAI or mean baseline mSASSS scores between HLA-C*07-positive and negative axSpA patients (Table 1). Both a univariate and multivariate logistic regression analysis within the axSpA patients showed that psoriasis was conversely correlated with HLA-C*07 negativity (OR 0.21, p=0.004),
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as well as with disease duration (OR 1.02, p=0.009). Other patient characteristics or SpA features were not associated with HLA-C*07 negativity in the GESPIC axSpA population in the multivariate model.
We next assessed if HLA-C*07 absence was associated with similar phenotypic features in SPACE. Table 2 shows characteristics of axSpA patients positive and negative for HLA-C*07. Of the 28 HLA-C*07-positive patients, 9 were male, compared to 33 of the 66 HLA-C*07-negative axSpA patients (32.1% vs. 50.0%, p=0.11). Eight of the 28 HLA-C*07-positive axSpA patients had psoriasis compared to 5 of the 66 HLA-C*07 negative axSpA patients (28.6% vs. 7.6%, p=0.007). The mean ESR of HLA-C*07 positive patients was 12.0 (SD 12.8) and 21.0 (SD 21.9) in HLA-C*07 negative patients (p=0.02). There were no differences in the prevalence of a positive family history, the duration of back pain, the prevalence of inflammatory back pain (IBP), peripheral arthritis, enthesitis, dactylitis, IBD, uveitis or sacroiliitis on MRI or X-ray between HLA-C*07 positive and negative patients (Table 2). Both a univariate and multivariate logistic regression analysis within the axSpA patients confirmed that psoriasis was conversely correlated with HLA-C*07 negativity (OR 0.20, p=0.013). Other patient characteristics or SpA features were not associated with HLA-C*07 absence or presence in the SPACE axSpA population in the multivariate analysis.
Table 1. Demographic and disease characteristics of HLA-C*07 positive and negative axSpA patients of GESPIC
HLA-C*07 in axSpA patients
Positive (n=65) Negative (n=131) p-value
Male (n, %) 26 (40.0%) 74 (56.5%) .03
Age (mean, SD) 37.5 (9.6) 37.5 (11.3) .99
HLA-B*27 positive (n, %) 49 (75.4%) 109 (83.8%) .16 Positive family history of AS (n, %) 13 (20.0%) 20 (15.3%) .40 Duration of back pain in
months (mean, SD) 42.4 (SD 27.5) 53.7 (±24.4) .02 Peripheral arthritis baseline (n, %) 8 (12.3%) 22 (16.8%) .41 Enthesitis current (n, %) 13 (20.0%) 31 (23.7%) .56 IBD (n, %) 1 (1.5%) 3 (2.3%) .73 Psoriasis current (n, %) 11 (16.9%) 9 (6.9%) .03 Uveitis ever (n, %) 11 (16.9%) 29 (22.1%) .39 CRP, mg/L (mean, SD) 7.8 (13.9) 12.6 (20.5) .02 mNY criteria (n, %) 31 (47.7%) 82 (62.6%) .047 mSASSS baseline 3.26 4.99 .03
Sacroiliitis on MRI (n, %) Unknown
BASDAI (mean, SD) 3.69 3.70 .96
AxSpA axial Spondyloarthritis, GESPIC GErman SPondyloarthritis Inception Cohort, HLA human leukocyte antigen, AS ankylosing spondylitis, IBD inflammatory bowel disease, CRP C-reactive protein, mNY modified New York, mSASSS modified Stoke Ankylosing
spondylitis disease activity index
Table 2. Demographic and disease characteristics of HLA-C*07 positive and negative axSpA patients of the SPACE cohort
HLA-C*07 in axSpA patients
Positive (n=28) Negative (n=66) p-value Male (n, %)
Age (mean, SD)
HLA-B*27 positive (n, %) Positive family history (n, %) Duration of back pain in months (mean, SD)
Inflammatory back pain (n, %) Peripheral arthritis (n, %) Enthesitis (n, %) Dactylitis (n, %) IBD (n, %) Psoriasis (n, %) Uveitis (n, %) CRP, mg/mL (mean, SD) ESR, mm/h (mean, SD) MNY criteria (n, %) Sacroiliitis on MRI (n, %) BASDAI (mean, SD) 9 (32.1) 30.3 (8.4) 22 (78.6) 15 (53.8) 13.5 (8.2) 20 (71.4) 4 (14.8) 5 (17.9) 2 (7.1) 2 (7.1) 8 (28.6) 4 (14.3) 8.9 (12.9) 12.0 (12.8) 5 (17.9) 12 (46.2) 4.9 (1.7) 33 (50.0) 30.5 (8.1) 59 (89.4) 34 (51.5) 15.0 (7.9) 49 (74.2) 7 (10.6) 6 (9.1) 3 (4.5) 3 (7.0) 5 (7.6) 11 (16.7) 11.6 (13.1) 21.0 (21.9) 18 (27.7) 31 (48.4) 4.1 (2.1) .11 .92 .17 .86 .40 .78 .57 .23 .61 .37 .01 .77 .37 .02 .31 .84 .10 AxSpA axial Spondyloarthritis, SPACE SPondyloarthritis Caught Early, HLA human leukocyte antigen, IBD inflammatory bowel disease, CRP C-reactive protein, MNY modified New York, MRI magnetic resonance imaging, BASDAI Bath ankylosing spondylitis disease activity index
DISCUSSION
The major finding of the current study is that HLA-C*07 prevalence is decreased in axSpA. Based on original observations at the gene expression level and an initial genetic evaluation in a small exploratory cohort, we confirmed a lower HLA-C*07 prevalence in two different settings, showing a difference in HLA-C*07 prevalence not only in a cohort of patients with established AS, GESPIC, but also in an early chronic back pain cohort, SPACE. This enabled us to a) duplicate our findings in two different cohorts in two different countries, b) show that low HLA-C*07 prevalence is not restricted to full-blown AS but also observed in nr-axSpA, and c) establish that this association is independent of HLA-B*27 status. Furthermore, preliminary analyses in both cohorts suggest that HLA-C*07 negative axSpA patients have a lower prevalence of psoriasis but increased serum inflammatory markers and a higher prevalence of radiographic damage.
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This study shows a major difference in prevalence of an MHC class I gene in axSpA. In recent years, extensive progress has been made identifying susceptibility alleles in the disease with over 100 additional loci identified (16–21). Using a GWAS approach to identify additional loci in the HLA region remains, however, very challenging because of the huge diversity in this genetic region and the potential linkage disequilibrium with HLA-B*27. In this study we used a ‘reverse’ approach to identify HLA-C*07 as a candidate susceptibility gene: rather than performing large-scale genetic screening and then assessing altered expression and/or function of the gene products of interest, we started by assessing altered expression of a focused set of molecules in a small homogenous sample set in order to select strong signals and avoid false positive data. Subsequently, we analyzed whether altered expression of target molecules was related to genetic variation, allowing us to uncover the association with HLA-C*07.
In sharp contrast to psoriasis, where there is a clear genetic association with HLA-C06 (22,23), indications that HLA-C could play a role in axSpA are sparse. One study in Korean patients showed an increased prevalence of HLA-C15:02 in AS patients, mainly if they were HLA-B*27 negative (24). In PsA, a condition closely related to axSpA, it was reported that patients without HLA-C*0701 had a 42.2% chance of symmetrical sacroiliitis, when compared to 15.8% of PsA patients with an HLA-C*0701 allele (25). This observation is in line with our study, suggesting a link between radiographic damage and HLA-C*07 negativity. In IBD, one Spanish study showed an interaction between a single nucleotide polymorphism (SNP) in endoplasmic reticulum aminopeptidase (ERAP)-1 with the HLA-C*07 antigen (26). Interestingly, ERAP-1 SNPs are also associated with AS (20,21), but the cohorts analyzed in the present study were too small to assess reliably such interaction in axSpA. Nevertheless, these data in PsA and IBD are consistent with our genetic findings in axSpA that HLA-C*07 could play a role in these closely related conditions.
The proposed genetic association between absence of HLA-C*07 and susceptibility to axSpA could potentially be relevant for pathophysiology, for diagnosis, and/or for disease stratification. As to pathophysiology, it is tempting to hypothesize that HLA-C*07 (or its absence) can shape the inflammatory response through its impact on killer cell immunoglobulin-like receptors (KIRs). KIRs regulate natural killer (NK) and T cell function by recognition of HLA class I molecules (27). The regulation of NK and T cell responses appear to depend on KIR genotype, HLA genotype, and the interaction between HLA and KIR products. Depending on the receptor type, KIRs activate or inhibit NK and T cell responses. An imbalance between activating and inhibitory KIRs in the presence of specific HLA genotypes may play a role in susceptibility to or protection against several conditions. Such a mechanism has been shown to play a role in the maintenance of pregnancy (28), in clearance of hepatitis C viral infection (29), as well as in inflammatory diseases such as PsA (30) and psoriasis (22). Interestingly, HLA-B*27 homodimers have been suggested to contribute to axSpA by triggering
IL-17 production through KIR ligation (31,32). Whereas further research is needed to assess similar functions for HLA-C*07, it is important to note that we observed that HLA-C*07 contributed to axSpA independent from HLA-B*27 in the GESPIC and SPACE cohorts.
As to potential use of HLA-C*07 as a biomarker, the diagnostic value of HLA-C*07 alone appeared to be limited in the SPACE cohort, with a positive predictive value (PPV) of 40.7% (95% CI 36.0%-45.6%) and a negative predictive value of 81.1% (95% CI 75.5%-85.7%). HLA-B*27 has a PPV of 87.9% (95% CI 82.7-91.6%) and a NPV of 91.5% (95% CI 97.7-94.2%). HLA-C*07 negativity in combination with HLA-B*27 positivity increases the PPV to 97.88% (95% CI 95.8-98.9%), the NPV declines to 58.1% (95% CI 50.1-65.8%). Beyond diagnosis, HLA-C*07 genotyping could help to stratify axSpA sub-populations. Although preliminary, our data suggest that HLA-C*07 negativity is linked to a phenotype with higher CRP levels, more radiographic damage and less psoriasis prevalence. Confirmation within other big cohort studies is essential to test for these associations.
This study has several limitations. First, our study was not powered to fully correct for possible confounding caused by linkage disequilibrium (LD) of HLA-C with HLA-B*27 (16). However, HLA-B*27 prevalence did not differ significantly between HLA-C*07 negative and positive axSpA patients, both in GESPIC and in the SPACE cohort. And, although the gathering of axSpA and CBP patients in one cohort in the SPACE cohort enabled us to perform multivariate regression, showing that HLA-C*07 was linked to axSpA independently from HLA-B*27, larger cohorts including both axSpA and non-axSpA individuals will have to answer that question. Second, participants of the DKMS healthy donor cohort self-reported the absence of diseases, including axSpA, and one can thus not formally exclude that some of the healthy donors might have had axSpA or axSpA symptoms at the time of registration. It is however very unlikely that the prevalence of axSpA in the DKMS population is higher than in the general population and, if any selection bias would occur, the true HLA-C*07 prevalence difference would be larger than we measured. Third, GESPIC could not be corrected in detail for ethnicity with the DKMS donor cohort. Different ethnicity might result in different HLA-C allotype frequencies. However, HLA-C allotype frequency of all DKMS donors (n=141,049) did not differ from HLA-C allotype frequencies selected in our population (n=135,160) (data not shown), showing that matching for ethnicity does not influence HLA-C*07 prevalence in this cohort.
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Conclusions
In conclusion, the absence of HLA-C*07 is strongly associated with susceptibility to axSpA, including both AS and non-radiographic axSpA. Future research needs to 1) study HLA-C*07 prevalence in other, larger axSpA populations, thereby controlling for a possible LD between HLA-B*27 and HLA-C*07, 2) study the role of HLA-C*07 in axSpA pathogenesis, focusing on new bone formation and 3) examine HLA-C*07 as a biological marker for axSpA diagnosis, prognosis, or classification.
Acknowledgements: We acknowledge Prof. Martin Rudwaleit and Prof. Joachim
Sieper for their role in establishing GESPIC. We thank Junior Lardy, PhD for his help with the HLA-typing. GESPIC has been financially supported by the German Federal Ministry of Education and Research (Bundesministerium für Bildung und Forschung – BMBF). As funding by BMBF was reduced according to schedule in 2005 and stopped in 2007, complementary financial support has been obtained also from Abbott / Abbvie, Amgen, Centocor, Schering-Plough, and Wyeth. Since 2010 GESPIC is supported by Abbvie. Additional support has being obtained also from ANCYLOSS (grant number FKZ 01EC1002D), ArthroMark (grants numbers FKZ 01EC1009A and FKZ 01EC1401A) and METARTHROS (grant number FKZ 01EC1407A) projects funded by BMBF. DB is supported by a VICI grant from the Netherlands Scientific Organization (NWO), a Consolidator grant from the European Research Council (ERC) and a grant from the Dutch Arthritis Foundation (Reumafonds).
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