The type 1 diabetes susceptibility gene SUMO4 at IDDM5 is not associated with susceptibility to rheumatoid arthritis or juvenile idiopathic arthritis

L. J. Gibbons1, W. Thomson1, E. Zeggini3, J. Worthington1, A. Barton1, S. Eyre1, R. Donn1,2 and A. Hinks1

1 Arthritis Research Campaign Epidemiology Unit and 2 Centre for Molecular Medicine, University of Manchester, Manchester and 3 Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford, UK.

Correspondence to: L. J. Gibbons, Arthritis Research Campaign Epidemiology Unit, University of Manchester, Manchester. M13 9PT, UK. E-mail: lgibbons{at}fs1.ser.man.ac.uk


    Abstract
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 Contributors
 References
 
Objectives. Linkage and association of rheumatoid arthritis (RA) and rheumatoid factor (RF)-negative juvenile idiopathic arthritis (JIA) has previously been demonstrated to the type 1 diabetes (T1D) locus, IDDM5, on chromosome 6q25. An association of a methionine-to-valine polymorphism (rs237025, 163A -> G, M55V) in the SUMO4 gene within IDDM5 has recently been described in T1D. The objective of this study was to test the hypothesis that SUMO4 is a general autoimmune susceptibility gene by investigating whether the SUMO4 polymorphism is associated with RA and/or JIA.

Methods. The SUMO4 SNP was genotyped in 875 RA patients, 668 JIA patients and 484 healthy controls using a TaqMan® allelic discrimination assay. Allele and genotype frequencies were compared between cases and controls using the {chi}2 test. Analyses were also carried out with RA patients stratified by gender, age at onset, RF status, the presence of erosive disease and shared epitope status, while JIA patients were stratified by their International League of Associations for Rheumatology (ILAR) subgroup.

Results. No deviation from Hardy–Weinberg equilibrium was detected in either set of cases or controls. No association was observed between rs237025 and RA ({chi}2=0.17, P=0.93), or with any RA subset. Similarly, there was no association between this SNP and JIA ({chi}2=0.21, P=0.90), or with any ILAR subgroup.

Conclusions. The M55V substitution in the SUMO4 gene is not associated with susceptibility to RA or JIA in the UK population studied. However, other candidate genes mapping within IDDM5 remain to be investigated.

KEY WORDS: Rheumatoid arthritis, Juvenile idiopathic arthritis, SUMO4, Autoimmune


    Introduction
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 Contributors
 References
 
Many autoimmune diseases share similar clinical characteristics that point towards a common biological pathway. Several lines of evidence have emerged that suggest that this may arise due to a shared genetic basis. Firstly, the attack on self tissue by the immune system suggests that autoimmune diseases are brought about by similar mechanisms of immune dysregulation, perhaps due to defects in immune response genes. Secondly, autoimmune diseases cluster in families: relatives of a person with RA are more likely to develop an autoimmune disease than relatives of a healthy person [1]. Different autoimmune diseases may also occur in the same individual, and the majority show a substantial female predominance. Finally, the most convincing evidence of a shared genetic basis in autoimmunity is the overlap of autoimmune disease susceptibility loci. This is typified by the central role of the major histocompatibility complex (MHC) in most autoimmune diseases. In addition, in both murine models of autoimmunity [2] and human autoimmune whole-genome scans [3], non-MHC susceptibility loci cluster non-randomly and colocalize with loci from other autoimmune diseases. It has therefore been proposed that ‘autoimmunity’ genes exist which influence susceptibility to autoimmunity in general, while other disease genes determine the tissues affected.

Examples of genes involved in susceptibility to more than one autoimmune disease include cytotoxic T-lymphocyte-associated antigen 4 (CTLA4), which is associated with type 1 diabetes (T1D), Graves’ disease and autoimmune hypothyroidism [4, 5]. Another is protein tyrosine phosphatase non-receptor type 22 (PTPN22), in which a missense single-nucleotide polymorphism (SNP) is associated with T1D [6], RA [7], juvenile idiopathic arthritis (JIA) [8], systemic lupus erythematosus [9] and autoimmune thyroid disease [10].

At the type 1 diabetes locus, IDDM5, on chromosome 6q25, a study investigating the shared autoimmunity gene hypothesis has demonstrated linkage and association of the microsatellite markers D6S311 and D6S440 with RA [11] (Supplementary Fig. 1). Association of the D6S440 marker with RA has been replicated in an independent cohort [12]. Nominal evidence of linkage and association has also been demonstrated to this marker in the RF-negative polyarthritis subgroup of JIA (Hinks, unpublished data). The absence of association with other JIA subgroups may be a result of low power due to small sample sizes. For example, the RF-positive polyarthritis subgroup included only 10 simplex families, consisting of one affected individual and parent(s). These results suggest that a gene within the IDDM5 region contributes to susceptibility to RA and JIA, in addition to T1D, and may therefore be a general autoimmunity gene. The IDDM5 region spanned by these two microsatellite markers covers approximately 4 Mb and contains several candidates for this autoimmunity gene.

One candidate within IDDM5 is SUMO4, an intronless gene encoding a member of the small ubiquitin-related modifier (SUMO) family of proteins. Although little is known at present about the role of SUMO4, another family member, SUMO1, is known to be an inhibitor of the NF-{kappa}B signalling pathway. The SUMO proteins therefore have the potential to down-regulate NF-{kappa}B signalling, leading to decreased transcription of genes encoding pro-inflammatory cytokines.

Associations of SNPs mapping to SUMO4 and the neighbouring region have recently been reported in T1D [13, 14]. Functional analyses have implicated an SNP encoding a methionine -> valine substitution of SUMO4 in the aetiology of T1D [14, 15]. However, there is some confusion over which allele is associated. In a study of US and UK families, the A allele (methionine) was associated with disease [13, 15], whilst in another study of US and non-UK families, the G allele (valine) was associated with T1D [14]. Larger studies of European [16] and Canadian [17] data sets have failed to replicate the association of the SUMO4 SNP in T1D, whilst a study of Korean cases and controls detected an association of the G allele with T1D [18].

Thus, in the light of the linkage and association demonstrated at the IDDM5 locus in RA and the RF-negative polyarthritis subgroup of JIA, and the recent association of SUMO4 with T1D, we hypothesized that the SUMO4 gene is a shared autoimmunity gene implicated in susceptibility to RA and/or JIA. The aim of this study was therefore to investigate association of the SUMO4 M55V SNP with RA and JIA.


    Patients and methods
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 Contributors
 References
 
Patients and controls
A case–control study design was used to assess association of the SUMO4 M55V SNP (rs237025, 163A -> G) with RA and JIA.

All patients and controls were of UK Caucasoid ethnic origin, were recruited with ethical approval and provided informed consent [North-West Multi-Centre Research Ethics Committee (MREC 99/8/84) and the University of Manchester Committee on the Ethics of Research on Human Beings].

DNA was available for 875 RA cases from the Arthritis Research Campaign National Repository for RA, and from clinics within the Greater Manchester area of the North of England. Classification of RA was by the 1987 American College of Rheumatology (ACR) criteria modified for genetic studies [19]. A positive RF score was defined as a titre of 1 in 40 or greater, as measured by a particle agglutination test. HLA DRB1 genotypes had previously been determined with a commercially available semi-automated polymerase chain reaction (PCR) sequence-specific oligonucleotide probe typing technique (INNO-LiPA; Abbott Laboratories, Maidenhead, UK).

DNA was available for 668 JIA cases from the National Repository for JIA, recruited by the British Society for Paediatric and Adolescent Rheumatology (BSPAR) from 17 centres around the UK. All cases were classified by the International League of Associations for Rheumatology (ILAR) criteria for JIA [20].

DNA was available for 484 healthy control subjects, of whom 172 were blood donors from the Oxford region; 142 were healthy population controls from the North-West of England; and 170 were from the North-West, recruited as part of a media campaign, with an age range of 61–95 yr. Twenty-eight per cent of the controls had one copy of the shared epitope and 6.6% had two copies. The entire control group had a median age of 67, and 59% were female.

Since RA and JIA are clinically heterogeneous diseases, stratification was performed to create more phenotypically homogeneous subgroups. RA patients were stratified according to gender, age at disease onset, shared epitope positivity, RF status and the presence of erosions. The median age at onset in the RA cohort was 41; therefore this age was chosen as the cut-off for early/late onset. JIA patients were subgrouped according to the ILAR classification system [20].

Genotyping
Genotyping of the SUMO4 SNP was carried out using a Custom TaqMan® 5' allelic discrimination assay (ABI, Warrington, UK). Each PCR reaction was carried out using 15 ng DNA in a 5 µl reaction mixture containing 2.5 µl TaqMan ready reaction mix, 0.06 µl 40x assay and 2.44 µl dH2O per sample. Samples were placed in an ABI 9700 thermocycler at 95°C for 10 min, followed by 15 s at 92°C, then 60 s at 60°C for 40 cycles. Plates were read on a TaqMan® 7700 platform using the allelic discrimination option.

Statistical analysis
The {chi}2 test was used to test for any association between alleles or genotypes at the SUMO4 M55V SNP and RA or JIA, or any subgroup of these diseases. {chi}2 tests for carriage of either allele were also performed.

Carriage of the G allele of the SUMO4 SNP has been reported to confer an odds ratio (OR) of 1.98 in Finnish T1D cases [14]. The sample size used in our study provided 100% power to detect a risk allele of the same frequency and effect size at the 0.05 significance level.


    Results
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 Contributors
 References
 
Genotypes at the M55V SNP were in Hardy–Weinberg equilibrium in both sets of cases (RA, P = 1.00; JIA, P = 0.22) and in controls (P = 0.72). Furthermore, allele frequencies were almost identical to those reported previously (the A allele had a frequency of 48%, compared with 49% in a Caucasian control population [15]).

No association was detected between the M55V SNP and RA (df = 2, {chi}2 = 0.17, P = 0.93) (Table 1) or JIA (df = 2, {chi}2 = 0.21, P = 0.90) (Table 2). Similarly, no association was detected when RA patients were stratified according to RF status, erosive disease, shared epitope status, gender and age at onset (Table 1). No association was detected between the M55V SNP and any ILAR subgroup of JIA patients (Table 2).


View this table:
[in this window]
[in a new window]
 
TABLE 1. Genotype frequencies at the SUMO4 M55V SNP in RA and controls, and in RA subgroups

 

View this table:
[in this window]
[in a new window]
 
TABLE 2. Genotype frequencies at the SUMO4 M55V SNP in JIA and controls, and in ILAR subgroups

 

    Discussion
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 Contributors
 References
 
In order to provide more evidence upon which to assess the hypothesis of a shared genetic basis to diverse autoimmune diseases, we chose the SUMO4 gene at the type 1 diabetes locus, IDDM5, as the focus to test association with RA and JIA. The IDDM5 region maps to 6q25 and is of interest since, not only has the region been linked and associated with T1D, but two microsatellite markers, D6S440 and D6S311, have also shown linkage and association with RA. Furthermore, D6S440 has been linked and associated with the RF-negative polyarthritis subgroup of JIA. The IDDM5 gene locus is large and contains several candidate genes that could contribute to susceptibility to these diseases. One such gene is the recently identified SUMO4 [14, 15], which lies ~1 Mb 3' to D6S311 and ~2.7 Mb 5' from D6S440 (Supplementary Fig. 1).

An association of the A allele (methionine) of the M55V SNP in SUMO4 with increased risk of disease has been described in US and UK T1D families [15]. Luciferase reporter assays demonstrated that SUMO4-V brought about lower activation of expression from heat shock elements than did SUMO4-M. The authors proposed that the increased heat shock factor activation by SUMO4-M alters regulation of the 60 kDa heat shock proteins (Hsp60), against which autoantibodies have been identified in RA and T1D [15].

A second study described an association of the alternative G allele (valine) with T1D susceptibility in the European American population, whilst in the UK population the A allele showed a trend towards overtransmission [14]. In TNF-{alpha}-stimulated NF-{kappa}B luciferase reporter assays, SUMO4-V caused 5.5 times greater activity than SUMO4-M. This suggests that the valine substitution reduces the ability of SUMO4 to inhibit NF-{kappa}B activation, resulting in transcription of downstream pro-inflammatory genes [14].

Therefore, despite the discrepancy in the polarity of the association of the M55V SNP with T1D, functional work in both studies suggests a plausible role for the SNP in the immune response, and highlight SUMO4 as an appropriate candidate as a susceptibility gene in RA and JIA. The SUMO4-V variant not only appears to lower the activation of heat shock factors, but also to provide less effective inhibition of NF-{kappa}B signalling, both of which are pathways implicated in autoimmune diseases.

The findings in T1D prompted us to investigate association of the SUMO4 M55V SNP in a large sample of RA and JIA patients. However, we failed to detect an association with either disease or with any of their clinical subgroups.

The sample size used in this study provided 100% power to detect a risk allele with a frequency of 0.48, as observed in our control group, and an OR of 1.98, as determined for T1D [14]. The effect size of the M55V SNP in an alternative disease may be considerably lower than in T1D. Our study had ~90% power to detect an effect size as low as 1.2, and we therefore had sufficient power to detect an association with RA or JIA with an effect size above or equal to this. However, due to the small number of subjects within subgroups, the study had low power to detect a subgroup-specific association.

Recently, several groups have sought to independently replicate the association of SUMO4 with T1D [16–18]. In 471 UK and 336 US T1D families, and 3442 UK cases and 3788 controls, no evidence of association between the SUMO4 M55V variant and T1D was detected [16]. In another study of 1188 Canadian families, no association was detected, neither in the data set as a whole nor in a subset of individuals descended from English and Irish settlers [17]. However, a case–control analysis of 386 Korean T1D cases and 553 controls found that carriage of the G allele was significantly more frequent in cases [18].

Several explanations have been proposed for the conflicting allelic associations observed at this SNP in different populations of T1D families [16–18]. Firstly, the association with either allele may be a false positive, arising due to the study of numerous polymorphisms in many candidate genes by hundreds of research groups. Secondly, epistasis between SUMO4 and other loci could influence the direction of the association. Finally, complex gene–environment interactions may exist that could render an allele predisposing in one population yet protective in another.

Our results demonstrate that SUMO4 is not a susceptibility gene for RA or JIA in the UK population studied. However, the IDDM5 locus is a large region containing several other candidates for the role of a general autoimmunity gene. D6S440 is situated within intron 5 of the oestrogen receptor {alpha} gene (ESR1), more than 2.6 Mb 3' from SUMO4. Other candidates are vasoactive intestinal peptide (VIP) and the three UL16-binding protein genes (ULBP1–3). Thus, IDDM5 remains an intriguing locus at which to search for susceptibility genes in RA and JIA.


    Contributors
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 Contributors
 References
 
Contributors to the British Society of Paediatric and Adolescent Rheumatology (BSPAR) study group are M. Abinum, MD, M. Becker, MD, A. Bell, MD, A. Craft, MD, E. Crawley, MD, J. David, MD, H. Foster, MD, J. Gardener-Medwin, MD, J. Griffin, MD, A. Hall, MD, M. Hall, MD, A. Herrick, MD, P. Hollingworth, MD, L. Holt, MD, S. Jones, MD, G. Pountain, MD, C. Ryder, MD, T. Southwood, MD, I. Stewart, MD, H. Venning. L. Wedderburn, MD, P. Woo, MD, and S. Wyatt, MD.
Figure 1


    Acknowledgments
 
This work was funded by the Arthritis Research Campaign. A.B. is in receipt of a Wellcome Trust Advance Fellowship.

The authors have no conflicting interests.

Supplementary data

Formula

    Supplementary data are available at Rheumatology Online.


    References
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 Contributors
 References
 

  1. Lin JP, Cash JM, Doyle SZ et al. Familial clustering of rheumatoid arthritis with other autoimmune diseases. Hum Genet 1998;103:475–82.[CrossRef][ISI][Medline]
  2. Vyse TJ, Todd JA. Genetic analysis of autoimmune disease. Cell 1996;85:311–8.[CrossRef][ISI][Medline]
  3. Becker KG, Simon RM, Bailey-Wilson JE et al. Clustering of non-major histocompatibility complex susceptibility candidate loci in human autoimmune diseases. Proc Natl Acad Sci USA 1998;95:9979–84.[Abstract/Free Full Text]
  4. Marron MP, Raffel LJ, Garchon HJ et al. Insulin-dependent diabetes mellitus (IDDM) is associated with CTLA4 polymorphisms in multiple ethnic groups. Hum Mol Genet 1997;6:1275–82.[Abstract/Free Full Text]
  5. Ueda H, Howson JM, Esposito L et al. Association of the T-cell regulatory gene CTLA4 with susceptibility to autoimmune disease. Nature 2003;423:506–11.[CrossRef][ISI][Medline]
  6. Bottini N, Musumeci L, Alonso A et al. A functional variant of lymphoid tyrosine phosphatase is associated with type I diabetes. Nat Genet 2004;36:337–8.[CrossRef][ISI][Medline]
  7. Begovich AB, Carlton VE, Honigberg LA et al. A missense single-nucleotide polymorphism in a gene encoding a protein tyrosine phosphatase (PTPN22) is associated with rheumatoid arthritis. Am J Hum Genet 2004;75:330–7.[CrossRef][ISI][Medline]
  8. Hinks A, Barton A, John S et al. Association between the PTPN22 gene and rheumatoid arthritis and juvenile idiopathic arthritis in a UK population: further support that PTPN22 is an autoimmunity gene. Arthritis Rheum 2005;52:1694–9.[CrossRef][ISI][Medline]
  9. Kyogoku C, Langefeld CD, Ortmann WA et al. Genetic association of the R620W polymorphism of protein tyrosine phosphatase PTPN22 with human SLE. Am J Hum Genet 2004;75:504–7.[CrossRef][ISI][Medline]
  10. Velaga MR, Wilson V, Jennings CE et al. The codon 620 tryptophan allele of the lymphoid tyrosine phosphatase (LYP) gene is a major determinant of Graves’ disease. J Clin Endocrinol Metab 2004;89:5862–5.[Abstract/Free Full Text]
  11. Myerscough A, John S, Barrett JH, Ollier WE, Worthington J. Linkage of rheumatoid arthritis to insulin-dependent diabetes mellitus loci: evidence supporting a hypothesis for the existence of common autoimmune susceptibility loci. Arthritis Rheum 2000;43:2771–5.[CrossRef][ISI][Medline]
  12. Cheng SF, Lum RF, Peden E, Li H, Seldin MF, Criswell LA. Additional support for an autoimmune suceptibility gene(s) at 6q24-27. Arthritis Rheum 2001;44(Suppl.):S104.
  13. Owerbach D, Pina L, Gabbay KH. A 212-kb region on chromosome 6q25 containing the TAB2 gene is associated with susceptibility to type 1 diabetes. Diabetes 2004;53:1890–3.[Abstract/Free Full Text]
  14. Guo D, Li M, Zhang Y et al. A functional variant of SUMO4, a new IkappaBalpha modifier, is associated with type 1 diabetes. Nat Genet 2004;36:837–41.[CrossRef][ISI][Medline]
  15. Bohren KM, Nadkarni V, Song JH, Gabbay KH, Owerbach D. A M55V polymorphism in a novel SUMO gene (SUMO-4) differentially activates heat shock transcription factors and is associated with susceptibility to type I diabetes mellitus. J Biol Chem 2004;279:27233–8.[Abstract/Free Full Text]
  16. Smyth DJ, Howson JM, Lowe CE et al. Assessing the validity of the association between the SUMO4 M55V variant and risk of type 1 diabetes. Nat Genet 2005;37:110–1.[CrossRef][ISI][Medline]
  17. Qu H, Bharaj B, Liu XQ et al. Assessing the validity of the association between the SUMO4 M55V variant and risk of type 1 diabetes. Nat Genet 2005;37:111–2.[CrossRef][ISI][Medline]
  18. Park Y, Park S, Kang J, Yang S, Kim D. Assessing the validity of the association between the SUMO4 M55V variant and risk of type 1 diabetes. Nat Genet 2005;37:112.[Medline]
  19. MacGregor AJ, Bamber S, Silman AJ. A comparison of the performance of different methods of disease classification for rheumatoid arthritis. Results of an analysis from a nationwide twin study. J Rheumatol 1994;21:1420–6.[ISI][Medline]
  20. Petty RE, Southwood TR, Baum J et al. Revision of the proposed classification criteria for juvenile idiopathic arthritis: Durban, 1997. J Rheumatol 1998;25:1991–4.[Medline]
Submitted 25 April 2005; revised version accepted 30 June 2005.



This Article
Abstract
Full Text (PDF)
Supplementary data
All Versions of this Article:
44/11/1390    most recent
kei041v1
Alert me when this article is cited
Alert me if a correction is posted
Services
Email this article to a friend
Similar articles in this journal
Similar articles in PubMed
Alert me to new issues of the journal
Add to My Personal Archive
Download to citation manager
Disclaimer
Request Permissions
Google Scholar
Articles by Gibbons, L. J.
Articles by Hinks, A.
PubMed
PubMed Citation
Articles by Gibbons, L. J.
Articles by Hinks, A.
Related Collections
Rheumatoid Arthritis