gistfile1.txt

A Functional Variant in ERAP1 Predisposes to Multiple
Sclerosis
Franca Rosa Guerini1*., Rachele Cagliani2., Diego Forni2, Cristina Agliardi1, Domenico Caputo1, Andrea
Cassinotti3, Daniela Galimberti4, Chiara Fenoglio4, Mara Biasin5, Rosanna Asselta6, Elio Scarpini4,
Giacomo P. Comi4, Nereo Bresolin2,4, Mario Clerici1,7, Manuela Sironi2
1 Don C. Gnocchi Foundation ONLUS, Milano, Italy, 2 Bioinformatic Laboratory, Scientific Institute IRCCS E. Medea, Bosisio Parini, Italy, 3 Department of Clinical Sciences,
Chair of Gastroenterology, Luigi Sacco University Hospital, Milano, Italy, 4 Dino Ferrari Centre, Department of Neurological Sciences, University of Milano, Fondazione Ca’
Granda IRCCS Ospedale Maggiore Policlinico, Milano, Italy, 5 DISP LITA Vialba, University of Milano, Milano, Italy, 6 Department of Biology and Genetic for Medical Science,
University of Milan, Milan, Italy, 7 Department of Biomedical Sciences and Technologies LITA Segrate, University of Milan, Milan, Italy

Abstract
The ERAP1 gene encodes an aminopeptidase involved in antigen processing. A functional polymorphism in the gene
(rs30187, Arg528Lys) associates with susceptibility to ankylosying spondylitis (AS), whereas a SNP in the interacting ERAP2
gene increases susceptibility to another inflammatory autoimmune disorder, Crohn’s disease (CD). We analysed rs30187 in
572 Italian patients with CD and in 517 subjects suffering from multiple sclerosis (MS); for each cohort, an independent sexand age-matched control group was genotyped. The frequency of the 528Arg allele was significantly higher in both disease
cohorts compared to the respective control population (for CD, OR = 1.20 95%CI: 1.01–1.43, p = 0.036; for RRMS, OR = 1.26;
95%CI: 1.04–1.51, p = 0.01). Meta-analysis with the Wellcome Trust Cases Control Consortium GWAS data confirmed the
association with MS (pmeta = 0.005), but not with CD. In AS, the rs30187 variant has a predisposing effect only in an HLA-B27
allelic background. It remains to be evaluated whether interaction between ERAP1 and distinct HLA class I alleles also affects
the predisposition to MS, and explains the failure to provide definitive evidence for a role of rs30187 in CD. Results herein
support the emerging concept that a subset of master-regulatory genes underlay the pathogenesis of autoimmunity.
Citation: Guerini FR, Cagliani R, Forni D, Agliardi C, Caputo D, et al. (2012) A Functional Variant in ERAP1 Predisposes to Multiple Sclerosis. PLoS ONE 7(1): e29931.
doi:10.1371/journal.pone.0029931
Editor: Pablo Villoslada, Institute Biomedical Research August Pi Sunyer (IDIBAPS) - Hospital Clinic of Barcelona, Spain
Received September 14, 2011; Accepted December 7, 2011; Published January 12, 2012
Copyright: ß 2012 Guerini et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by the Broad Medical Research Program of The Broad Foundation (grant IBD-0294), by 2010 Ricerca Corrente [Italian Ministry
of Health], and Fondazione CARIPLO. The funding sources had no involvement in study design, collection, analysis and interpretation of data, in the writing of the
report as well as in the decision to submit the paper for publication. No additional external funding received for this study.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: fguerini@dongnocchi
. These authors contributed equally to this work.

Because ERAP1 also contributes to shedding the membranebound receptor for inflammatory cytokines including IL1R2,
TNFR1, and IL6R [4], ERAP1 is likely to play a pivotal role in
protection from infectious diseases, in maintaining immunotolerance, and in controlling inflammation. A single nucleotide
polymorphism (SNP) in ERAP1 (rs30187), which changes a highly
conserved residue (Arg528Lys), is maintained at intermediate
frequency in human populations by natural selection [5] and
affects the enzyme catalytic activity [6]. This SNP has been
associated with susceptibility to ankylosying spondylitis (AS) [7],
and variants in linkage disequilibrium (LD) with it increase
predisposition to psoriasis [8]. This observation is in line with an
emerging concept whereby a portion of susceptibility alleles is
shared among two or more autoimmune conditions (reviewed in
[9]), suggesting that a subset of master-regulatory genes underlay
the pathogenesis of autoimmunity, although the clinical outcomes
and end-organ targets differ across diseases. For example, variants
in IL23R have been associated with psoriasis, AS, and Crohn’s
Disease (CD). Additional shared variants between CD and AS
have recently been described [10,11], and provide genetic
evidence to the clinical observation that the two diseases have
frequent co-occurrence and co-symptomatology [12].

Introduction
Antigen processing and presentation by MHC class I molecules
is essential for assuring immune surveillance and for establishing
immunodominance. The process initiates with the transport of
proteasome-generated antigenic peptides to the endoplasmic
reticulum (ER), where they are customized to optimal size for
MHC class I loading by resident enzymes. In humans, two ERaminopeptidases, encoded by ERAP1 and ERAP2, trim imported
peptides at their N-terminus and contribute to the shaping of the
antigenic repertoire presented by class I MHC molecules [1].
Studies in humans and mice have shown that, depending on
peptide length and sequence composition, ERAP1 has the
ability to both destroy and create peptide cargos for MHC class
I [2]. Therefore, in mice lacking the enzyme the presentation of
some peptides is dramatically reduced, whereas other peptides
are much more abundant than what is observed in wild-type
animals [3]. This applies to both proteolytic fragments of
pathogen-derived proteins and to endogenous peptides. As a
consequence, immunodominance is disrupted in Erap12/2 mice
and these animals display a distinct repertoire of antigenic
peptides [3].
PLoS ONE | www.plosone.org

1

January 2012 | Volume 7 | Issue 1 | e29931

ERAP1 Allele Predisposing to Multiple Sclerosis

does not reach the GWAS statistical threshold, suggesting the need
to further replicate this association in independent studies.
AS and CD are known to have a close clinical relationship:
about 10% of AS patients also suffer from inflammatory bowel
disease, and most AS cases display evidence of chronic intestinal
inflammation [12]. Arthropathyes are common among CD
patients as well [17]. Consistently, risk alleles that predispose to
both conditions have been recently identified [10,11]. A nonsynonymous variant in ERAP2 (rs2549794), which acts in concert
with ERAP1 in the ER, has been associated with the risk of CD in
a GWAS [10]. Although the two aminopeptidase genes are located
in a cluster on chromosome 5, rs30187 and rs2549794 segregate
independently, as the two SNPs display extremely limited LD both
in Italians [5] and in HapMap populations of European ancestry
(r2 = 0.18, http://hapmap.ncbi.nlm.nih.gov/). These observations
make ERAP1 a good candidate as a susceptibility gene for CD.
Our analysis in the Italian population supported the role of the A
allele of rs31078, which predisposes to AS, in susceptibility to
Crohn’s disease; nonetheless, this finding was not supported when
data from a second study were used for meta-analysis. One
possibility is that the ERAP1 variant genetically interacts with
specific HLA class I alleles. Indeed, in the case of AS, rs30187 was
shown to display a strong genetic interaction with HLA-B27, which
is extremely common in spondylitis patients [7]. This observation
suggests that the co-occurrence of the 528Arg allele at ERAP1 and
HLA-B27 results in the presentation of antigenic species that
prompt disease pathogenesis. Similar observations have been
reported for psoriasis, as variants in ERAP1 have a predisposing
effect only when combined with specific HLA-C allelic backgrounds. No specific MHC allele/haplotype has been reported in
CD, although several significant associations have been described
for SNP alleles within the MHC [18]. Therefore, the role of ERAP1
alleles in the pathogenesis of CD remains to be evaluated, as well
as the presence of possible epistatic effects of HLA alleles.
Recent findings have indicated that a portion of susceptibility
alleles for autoimmune disease is shared among two or more
conditions (reviewed in [9]). Our data indicate that the AS
susceptibility allele in ERAP1 also confers increased risk to develop
MS, and imply a role for antigen presentation and class I MHC
molecules in the pathogenesis of MS. The strongest genetic risk
factor for MS is the HLA DRB1*1501-DQB1*0602 haplotype (also
known as DR15 haplotype) in the HLA-class II region. In Italians,
as well as in other European populations [19–21], DR15 confers
an OR of about 3. Yet, in recent years, it has been suggested that
the HLA-class I region does indeed exert an additional influence
on the risk of MS, analogous to that reported for other
autoimmune diseases [22–24], and with an effect independent
from HLA-DRB1 [25]. Again, further analyses will be required to

Thus, we wished to verify whether the ERAP1 susceptibility
allele for AS also predisposes to CD and MS, this latter also
showing some degree of co-morbidity with Crohn’s disease in
affected individuals and their family members [13–15].

Results and Discussion
Multiple SNPs in ERAP1 have been associated with AS, but the
strongest signal is accounted for by rs31087 (Arg528Lys) [7]. As
mentioned above, the variant was recently shown to be functional
by affecting both peptide trimming and antigen presentation [6,7].
Thus, we focused on this SNP and set out to verify whether it may
affect the predisposition to CD and MS. To this aim, rs30187 was
genotyped in 572 patients with CD and in 517 subjects suffering
from relapsing-remitting MS (RRMS); two independent cohorts of
sex- and age-matched controls were also analysed. All individuals
were Italian of European ancestry and the SNP complied to
Hardy-Weinberg equilibrium in the case and control cohorts.
The genotype and allele distributions of rs30187 are shown in
Table 1 for both CD and RRMS patients compared to two
independent healthy control (HC) cohorts. Statistically significant
associations of rs30187 genotype and allele distributions were
observed both in CD and in RRMS.
In particular, the AA genotype was more frequent both in CD
patients (15.0%, CD vs. 11.5%, HC) and MS subjects (15.7%, MS
vs. 11.6%, HC) compared to their respective control samples, and
a statistically significant association of the rs30187 A allele was
observed both in CD (odds ratio, OR: 1.20; 95% confidence
interval, CI: 1.01–1.43) and in MS patients (OR: 1.26; 95% CI:
1.04–1.51) (Tab. 1). Thus, the minor A allele of rs30187 (528Arg),
previously associated with AS, also confers susceptibility to CD
and MS in these Italian cohorts. In order to perform a metaanalysis, we exploited genome-wide association study (GWAS)
data for MS and CD generated by the Wellcome Trust Cases
Control Consortium (WTCCC1 project data). As estimation of
effect heterogeneity is inaccurate when few studies are included in
the meta-analysis, we applied a random-effects model [16].
rs30187 was not genotyped in the CD GWAS; a search for linked
SNPs identified rs27710, which has been genotyped by the
WTCCC1 and is in full LD with rs30187 in the Italian population
(r2 from the 1000 Genomes Project data for TSI = 1), making
imputation straightforward. As for MS, rs30187 was available in
the GWAS study. Random-effect meta-analysis with these data
supported the association between rs31087 and MS susceptibility
(pmeta = 0.005) (Tab. 1). Conversely, high between-study heterogeneity was observed for CD, resulting in failure to confirm the
association we observed in the Italian sample (Tab. 1). It is worth
mentioning that the p value obtained for MS after meta-analysis

Table 1. Association study and meta-analysis for rs30187 in RRMS and CD.

Disease

Genotype Counts
(GG/AG/AA)
Cases

Controls

RRMS

182/254/81

209/233/58

CD

211/275/86

247/273/68

Pgeno

Allele Counts
(A/G)

Pallelica

OR (95% CI)

Meta-analysis
Het. P-valueb

Cases
0.043

416/618

349/651

0.014

0.094

447/697

409/767

0.036

I2c

Pmetad

ORmeta

1.26 (1.04–1.51)

0.29

8.7

0.005

1.16

1.20 (1.01–1.43)

0.02

81.1

0.58

1.06

Controls

a

P value from Pearson’s Chi-squared test with Yates’ continuity correction.
P value from Cochran Q heterogeneity test.
Heterogeneity index.
d
Random-effects meta-analysis p value.
doi:10.1371/journal.pone.0029931.t001
b
c

PLoS ONE | www.plosone.org

2

January 2012 | Volume 7 | Issue 1 | e29931

ERAP1 Allele Predisposing to Multiple Sclerosis

verify whether the ERAP1 Arg528Lys variant interacts with
specific HLA class I alleles to modulate predisposition to MS.
As mentioned above, in addition to its role as an ERaminopeptidase, ERAP1 also functions as a cleavage enzyme for
IL1R2, TNFR1 (also known as TNFRSF1A), and IL6R. TNFRSF1A
is a susceptibility locus for MS and CD [26,27], and variants in IL1R2
have been associated with AS and ulcerative colitis [7,28], while IL6
is a central mediator of inflammation. Thus, the associations we
detected between ERAP1 and MS might relate to the role of the
enzyme as a receptor sheddase, although it is presently unknown
whether the Arg528Lys also affects this cleavage activity.
In summary, we report that a functional ERAP1 allele previously
associated to AS confers susceptibility to MS in Italian
populations, whereas its role in predisposing to CD remains to
be evaluated. Thus, results herein add further support to the
shared genetic architecture of autoimmune diseases.

For the CD case/control cohorts, 1160 individuals: 572
suffering from CD (301 males, 271 females) and 588 age- and
sex-matched healthy individuals (305 males, 283 females) were
recruited by the IBD Unit of the Luigi Sacco Hospital in Milano, a
third-level centre for the management of IBD patients. The
diagnosis of CD was based on international published criteria,
according to clinical, endoscopic, histological and/or radiological
data [30]. A detailed clinical history, as well as laboratory and
instrumental diagnostic data, were collected. Also in this case, all
patients and controls were Italians of Caucasian ethnicity.
Genotyping of rs30187 was performed by a TaqMan probe
assay (TaqMan SNP genotyping assay, Applied Biosystems, Foster
City, CA, USA) using the allelic discrimination real-time PCR
method.
Genotype data for rs30187 and rs27710 from the WTCCC1 studies
has been retrieved from the European Genome-phenome Archive
(EGA, http://www.ebi.ac.uk/ega/) which is hosted by the EBI, under
accessions EGAS00000000006 (CD) and EGAS00000000022 (MS).
For meta-analysis, we applied a random-effects model as implemented
in PLINK [31].

Materials and Methods
For the MS case/control association study, a total of 1017
individuals were enrolled: 517 patients (343 females and 174
males) suffering from RRMS and 500 age- and sex-matched
healthy controls (325 females and 175 males) were recruited at the
MS Centre of Don Gnocchi Foundation in Milan and at
Department of Neurological Sciences, University of Milan. All
subjects gave informed consent according to protocols approved
by the local Ethic Committees. All patients and controls were
Italians of European origin. Patients underwent a standard battery
of examinations, including medical history, physical and neurological examination, screening laboratory test, and brain Magnetic
Resonance Imaging (MRI). Patients with RRMS fulfilled the
McDonald’s criteria [29]. Median age was 42.1+11.9 and
43.12+18.22 years for RRMS and controls, respectively.

Acknowledgments
This work was supported by the Broad Medical Research Program of The
Broad Foundation (grant IBD-0294), by 2010 Ricerca Corrente [Italian
Ministry of Health], and Fondazione CARIPLO.
We wish to thank the Wellcome Trust Case Control Consortium for
allowing access to genotype data.

Author Contributions
Conceived and designed the experiments: MS FRG MC. Performed the
experiments: RC DF CA CF AC DC RA. Analyzed the data: MS FRG
MB DG GPC RC. Contributed reagents/materials/analysis tools: ES NB.
Wrote the paper: MS FRG MC.

References
13. Langer-Gould A, Albers KB, Van Den Eeden SK, Nelson LM (2010)
Autoimmune diseases prior to the diagnosis of multiple sclerosis: A population-based case-control study. Mult Scler 16(7): 855–861.
14. Weng X, Liu L, Barcellos LF, Allison JE, Herrinton LJ (2007) Clustering of
inflammatory bowel disease with immune mediated diseases among members of
a northern california-managed care organization. Am J Gastroenterol 102(7):
1429–1435.
15. Nielsen NM, Frisch M, Rostgaard K, Wohlfahrt J, Hjalgrim H, et al. (2008)
Autoimmune diseases in patients with multiple sclerosis and their first-degree
relatives: A nationwide cohort study in denmark. Mult Scler 14(6): 823–829.
16. Kavvoura FK, Ioannidis JP (2008) Methods for meta-analysis in genetic association
studies: A review of their potential and pitfalls. Hum Genet 123(1): 1–14.
17. de Vlam K, Mielants H, Cuvelier C, De Keyser F, Veys EM, et al. (2000)
Spondyloarthropathy is underestimated in inflammatory bowel disease:
Prevalence and HLA association. J Rheumatol 27(12): 2860–2865.
18. Barrett JC, Hansoul S, Nicolae DL, Cho JH, Duerr RH, et al. (2008) Genomewide association defines more than 30 distinct susceptibility loci for crohn’s
disease. Nat Genet 40(8): 955–962. 10.1038/ng.175.
19. Fogdell A, Hillert J, Sachs C, Olerup O (1995) The multiple sclerosis- and
narcolepsy-associated HLA class II haplotype includes the DRB5*0101 allele.
Tissue Antigens 46(4): 333–336.
20. Olerup O, Hillert J (1991) HLA class II-associated genetic susceptibility in
multiple sclerosis: A critical evaluation. Tissue Antigens 38(1): 1–15.
21. Barcellos LF, Sawcer S, Ramsay PP, Baranzini SE, Thomson G, et al. (2006)
Heterogeneity at the HLA-DRB1 locus and risk for multiple sclerosis. Hum Mol
Genet 15(18): 2813–2824.
22. Howson JM, Walker NM, Clayton D, Todd JA, Type 1 Diabetes Genetics
Consortium (2009) Confirmation of HLA class II independent type 1 diabetes
associations in the major histocompatibility complex including HLA-B and
HLA-A. Diabetes Obes Metab 11 Suppl 1: 31–45.
23. Eike MC, Becker T, Humphreys K, Olsson M, Lie BA (2009) Conditional
analyses on the T1DGC MHC dataset: Novel associations with type 1 diabetes
around HLA-G and confirmation of HLA-B. Genes Immun 10(1): 56–67.
24. Bolognesi E, Karell K, Percopo S, Coto I, Greco L, et al. (2003) Additional
factor in some HLA DR3/DQ2 haplotypes confers a fourfold increased genetic
risk of celiac disease. Tissue Antigens 61(4): 308–316.

1. Jensen PE (2007) Recent advances in antigen processing and presentation. Nat
Immunol 8(10): 1041–1048.
2. Wearsch PA, Cresswell P (2008) The quality control of MHC class I peptide
loading. Curr Opin Cell Biol 20(6): 624–631.
3. York IA, Brehm MA, Zendzian S, Towne CF, Rock KL (2006) Endoplasmic reticulum
aminopeptidase 1 (ERAP1) trims MHC class I-presented peptides in vivo and plays an
important role in immunodominance. Proc Natl Acad Sci U S A 103(24): 9202–9207.
4. Haroon N, Inman RD (2010) Endoplasmic reticulum aminopeptidases: Biology
and pathogenic potential. Nat Rev Rheumatol 6(8): 461–467.
5. Cagliani R, Riva S, Biasin M, Fumagalli M, Pozzoli U, et al. (2010) Genetic
diversity at endoplasmic reticulum aminopeptidases is maintained by balancing
selection and is associated with natural resistance to HIV-1 infection. Hum Mol
Genet 19(23): 4705–4714.
6. Evnouchidou I, Kamal RP, Seregin SS, Goto Y, Tsujimoto M, et al. (2011)
Coding single nucleotide polymorphisms of endoplasmic reticulum aminopeptidase 1 can affect antigenic peptide generation in vitro by influencing basic
enzymatic properties of the enzyme. J Immunol 186(4): 1909–1913.
7. The Australo-Anglo-American Spondyloarthritis Consortium (TASC), the
Wellcome Trust Case Control Consortium 2 (WTCCC2), Evans DM,
Spencer CC, Pointon JJ, et al. (2011) Interaction between ERAP1 and HLAB27 in ankylosing spondylitis implicates peptide handling in the mechanism for
HLA-B27 in disease susceptibility. Nat Genet 43(8): 761–767.
8. Genetic Analysis of Psoriasis Consortium & the Wellcome Trust Case Control
Consortium 2, Strange A, Capon F, Spencer CC, Knight J, et al. (2010) A
genome-wide association study identifies new psoriasis susceptibility loci and an
interaction between HLA-C and ERAP1. Nat Genet 42(11): 985–990.
9. Zenewicz LA, Abraham C, Flavell RA, Cho JH (2010) Unraveling the genetics
of autoimmunity. Cell 140(6): 791–797.
10. Franke A, McGovern DP, Barrett JC, Wang K, Radford-Smith GL, et al. (2010)
Genome-wide meta-analysis increases to 71 the number of confirmed crohn’s
disease susceptibility loci. Nat Genet 42(12): 1118–1125.
11. Danoy P, Pryce K, Hadler J, Bradbury LA, Farrar C, et al. (2010) Association of
variants at 1q32 and STAT3 with ankylosing spondylitis suggests genetic overlap
with crohn’s disease. PLoS Genet 6(12): e1001195.
12. Mielants H, Veys EM, De Vos M, Cuvelier C, Goemaere S, et al. (1995) The
evolution of spondyloarthropathies in relation to gut histology. I. clinical aspects.
J Rheumatol 22(12): 2266–2272.

PLoS ONE | www.plosone.org

3

January 2012 | Volume 7 | Issue 1 | e29931

ERAP1 Allele Predisposing to Multiple Sclerosis

25. Bergamaschi L, Leone MA, Fasano ME, Guerini FR, Ferrante D, et al. (2010)
HLA-class I markers and multiple sclerosis susceptibility in the italian
population. Genes Immun 11(2): 173–180.
26. De Jager PL, Jia X, Wang J, de Bakker PI, Ottoboni L, et al. (2009) Metaanalysis of genome scans and replication identify CD6, IRF8 and TNFRSF1A as
new multiple sclerosis susceptibility loci. Nat Genet 41(7): 776–782.
27. Waschke KA, Villani AC, Vermeire S, Dufresne L, Chen TC, et al. (2005)
Tumor necrosis factor receptor gene polymorphisms in crohn’s disease:
Association with clinical phenotypes. Am J Gastroenterol 100(5): 1126–1133.
28. Anderson CA, Boucher G, Lees CW, Franke A, D’Amato M, et al. (2011) Metaanalysis identifies 29 additional ulcerative colitis risk loci, increasing the number
of confirmed associations to 47. Nat Genet 43(3): 246–252.

PLoS ONE | www.plosone.org

29. McDonald WI, Compston A, Edan G, Goodkin D, Hartung HP, et al. (2001)
Recommended diagnostic criteria for multiple sclerosis: Guidelines from the
international panel on the diagnosis of multiple sclerosis. Ann Neurol 50(1):
121–127.
30. Lennard-Jones JE (1989) Classification of inflammatory bowel disease.
Scand J Gastroenterol Suppl 170: 2–6; discussion 16-9.
31. Purcell S, Neale B, Todd-Brown K, Thomas L, Ferreira MA, et al. (2007)
PLINK: A tool set for whole-genome association and population-based linkage
analyses. Am J Hum Genet 81(3): 559–575.

4

January 2012 | Volume 7 | Issue 1 | e29931