Genetic Association Between Juvenile Rheumatoid Arthritis and Polymorphism in the SH2D2A Gene

BRIEF COMMUNICATION Genetic association between juvenile rheumatoid arthritis and polymorphism in the SH2D2A gene

A Smerdel1, K-Z Dai2, AR Lorentzen1, B Flatø3, S Maslinski4, E Thorsby1, Ø Førre3 and A Spurkland2 1Institute of Immunology, Rikshospitalet University Hospital, Oslo, Norway; 2Department of Anatomy, Institute of Basal Medical Sciences, University of Oslo, Norway; 3The Department of Rheumatology, Rikshospitalet University Hospital, Oslo, Norway; 4Institute of Rheumatology, Warsaw, Poland

T-cell-specific adapter protein (TSAd) involved in the negative control of T-cell activation is encoded by the SH2D2A gene. Our recent studies indicate that homozygosity for short (ie GA13 and GA16) alleles of the SH2D2A gene promoter is associated with development of multiple sclerosis. To study whether the same SH2D2A promoter polymorphism also contributes to the genetic susceptibility to develop juvenile rheumatoid arthritis (JRA), we examined 210 JRA patients and 558 healthy unrelated controls from Norway. The frequency of the short allele GA13 was increased among the JRA patients compared to control (0.098 vs

0.05; Pn ¼ 8 ¼ 0.042). There was a significant increased frequency of HLA-DRB1*08-positive patients carrying two copies of

‘short’ alleles GA13 and/or GA16 compared to healthy controls (16% vs 6%; Pn ¼ 4 ¼ 0.016). Our data indicate that the ‘short’ alleles of the SH2D2A promoter could contribute to the genetic susceptibility to JRA. Genes and Immunity (2004) 5, 310–312. doi:10.1038/sj.gene.6364093 Published online 6 May 2004

Keywords: juvenile rheumatoid arthritis; T-cell-specific adapter protein; SH2D2A gene

T-cell-specific adapter protein (TSAd) is an SH2 reduced expression of TSAd in activated T cells.7 More- domain-containing intracellular adapter molecule that over, there is an increased frequency of ‘short’ alleles 1 is expressed in thymocytes and activated mature T cells. (GA13 and GA16) among patients with multiple sclerosis The exact role played by TSAd in T-cell signal transduc- (MS), suggesting that the SH2D2A gene may contribute tion is unknown. TSAd may be involved in inhibiting to the genetic susceptibility to develop this disease.7 early T-cell signaling events.2 There are also indications This study was aimed to assess whether polymorphism that TSAd may be involved in transcriptional regulation of the SH2D2A gene promoter is also associated with in the cell nucleus.3 Recently, it was reported that mice juvenile rheumatoid arthritis (JRA). JRA is a chronic lacking the SH2D2A gene develop spontaneous systemic inflammation of joints that affects children. The pathogen- lupus-like autoimmune disease when they get older, esis is not known, but an antigen-driven autoimmune probably due to a defect in activation-induced T-cell process is proposed, in which auto-reactive T cells play a death.4 The SH2D2A gene encoding TSAd contains a central role in joint inflammation and destruction.8,9 Both variable number (n ¼ 13–33) of GA repeats in its susceptibility to and severity of this disease are largely promoter region located at position À340 upstream of genetically determined,10,11 and the genes responsible are the first coding ATG.5 Initial analysis of the SH2D2A mostly unknown. The only genetic factors that have been promoter identified the minimal promoter to be located firmly shown to contribute to JRA are genes within the HLA proximal to position À236 upstream of the first coding complex.12 Considering the putative major role of T cells in ATG, whereas a putative silencer element was identified the pathogenesis of JRA, the T-cell regulatory SH2D2A gene in the region encompassed by position À500 to À310, is an aprioricandidate susceptibility gene in JRA. thus including the GA repeat.6 We previously showed We analyzed the distribution of polymorphic GA that ‘short’ alleles of the GA repeat in the SH2D2A alleles of the SH2D2A gene in 210 JRA patients and 558 promoter conferred lower expression of TSAd in healthy unrelated individuals as controls (shown in activated CD4 T cells compared to long alleles. Hence, Figure 1). The frequency of the short GA13 allele was individuals being homozygous for ‘short’ alleles of this significantly increased among JRA patients compared to

GA repeat in the SH2D2A gene promoter display the controls (0.098 vs 0.05; OR ¼ 1.8; Pnc ¼ 0.0053;

Pn ¼ 8 ¼ 0.042; for correction numbers, see figure legends),

whereas the frequency of the GA16 allele did not differ among patients and controls. Since we previously Correspondence: Dr A Smerdel, Institute of Immunology, Rikshospitalet observed that genotypes homozygous for the short University Hospital, N-0027 Oslo, Norway. E-mail: [email protected] GA13 and GA16 alleles or heterozygous GA13/GA16 were Received 09 December 2003; revised 28 February 2004; accepted 12 associated to MS, we specifically examined these three March 2004; published online 6 May 2004 genotypes in JRA patients. As can be seen from Figure 2, JRA and polymorphism in the SH2D2A gene A Smerdel et al 311 0.30 20 DR8 Positive DR8 Negative 0.25 16 ** JRA Patients Controls 0.20 Controls 12 0.15 * 8 0.10 * Allele Frequency 4 0.05 Genotype Frequency (%) 0 0.00 GA13-13 GA13-16 GA16-16 S-S 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 Genotypes GA 1 GA 1 GA 1 GA 1 GA 1 GA 1 GA 1 GA 2 GA 2 GA 2 GA 2 GA 2 GA 2 GA 2 GA 2 GA 2 GA 2 GA Alleles Figure 3 The genotypic frequencies of ‘short’ alleles in JRA patients stratified for the presence of DRB1*08 compared to healthy Figure 1 The allele frequency distribution of SH2D2A GA repeats controls. The genotypic frequencies of ‘short’ alleles in patients with

(GA13–29) in JRA patients and controls. A total of 210 patients with JRA positive for DRB1*08 (N ¼ 68) vs patients negative for DRB1*08 JRA and 558 healthy unrelated individuals as controls from Norway (N ¼ 142) or random healthy controls (N ¼ 558): GA13–13 (2/68 vs 0/ 5 (of which 277 has previously been studied ) were included. The 142 and 1/558, respectively), GA13–16 (6/68 vs 4/142 and 7/558, 16 13 clinical characterization of patients as well as HLA associations respectively), GA16–16 (3/68 vs 7/142 and 24/558, respectively), S–S has previously been reported. Methods:ASH2D2A gene segment (individuals carrying ‘short’ alleles GA13 and/or GA16) (11/68 vs containing the GA repeats was amplified by polymerase chain 11/142, and 32/558, respectively). When patients were subgrouped reaction (PCR) amplification as described previously.7 The PCR according to HLA-DRB1*08 status, the total number of genotype products were separated on a 4.25% urea-polyacrylamide gel and comparisons performed was n ¼ 8, or when ‘short’ alleles were identified according to size on an ABI 377XL DNA sequencer (PE lumped n ¼ 4. *DR8-positive patients vs healthy controls; OR ¼ 7.6, Applied Biosystems, Foster City, CA, USA). The frequencies of 95% CI ¼ 2.2–26, Pn ¼ 8 ¼ 0.008. **DR8-positive patients vs healthy 2 alleles were compared by means of w statistics or Fisher’s exact test, controls; OR ¼ 3.2, 95% CI ¼ 1.4–6.9; Pn ¼ 4 ¼ 0.016. when appropriate. The strength of association was estimated by odds ratios (OR) with 95% confidence interval (95% CI). P-values were corrected according to the Bonferroni method by multiplying with the number of comparisons performed. For comparisons of all the three genotypes were increased among the JRA SH2D2A GA repeat alleles, this was the number of alleles with the patients, but only the heterozygous GA13–16 genotype frequency higher than 5% plus the remaining alleles lumped into one group (n ¼ 8). The significant association is shown with * (0.1 in reached statistical significance (5% vs 1%; OR ¼ 3.9; 95% ¼ ¼ patients vs 0.05 in controls; OR ¼ 1.8; 95% CI ¼ 1.2–2.7; Pnc ¼ 0.0053; CI (1.4–11.6); Pn 4 0.036). Overall, JRA patients carried

Pn ¼ 8 ¼ 0.042). significantly more frequent two copies of ‘short’ alleles

(GA13 and/or GA16) compared to healthy controls (10%

vs 6%; OR ¼ 1.9; 95% CI (1.1–3.5); Pn ¼ 2 ¼ 0.04). Intrigu- ingly, a higher frequency of individuals homozygous for

GA13–13 and/or heterozygous for GA13–16 was observed in patients carrying the DRB1*08 allele, the strongest risk factor in pauci- and poly-RF-negative JRA subgroups,13 compared to those patients being negative for the 12 JRA Patients ** DRB1*08 allele (Figure 3); however, these differences 10 Controls did not reach statistical significance due to insufficient number of patients in each group. The SH2D2A gene is 8 located on chromosome 1, and DRB1 is located on chromosome 6; thus alleles at these two loci are inherited 6 * independently of each other. We therefore also compared patients carrying the DRB1*08 allele with the total group 4 of healthy controls, and observed significant differences in the frequency of individuals being heterozygous for 2

Genotype Frequency (%) GA13–16 (9% in patients vs 1% in controls; OR ¼ 7.6, 95% 0 CI (2.2–26), Pn ¼ 8 ¼ 0.008). When considering the three short genotypes as a group, a total of 16% in patients GA13-13 GA13-16 GA16-16 S-S compared to 6% of the controls carried such genotypes;

Genotypes OR ¼ 3.2, 95% CI ¼ 1.4–6.9; Pn ¼ 4 ¼ 0.016) (Figure 3). Figure 2 The genotypic frequencies of ‘short’ alleles in JRA There were no significant differences in the genotype patients and healthy controls. Genotypes of JRA patients (N ¼ 210) frequencies between controls and patients being negative and healthy controls (N ¼ 558) were only compared in individuals for the DRB1*08 allele. having inherited at least one copy of the short allele (eg GA13 and JRA comprises a heterogeneous group of rheumatic GA16). Thus, the total number of genotypes considered was n ¼ 4, or disorders that differ in severity, outcome and HLA n ¼ 2, when the short genotypes were lumped together into one association. To examine whether the observed SH2D2A group. GA13–13 (2/210 vs 1/558), GA13–16 (10/210 vs 7/558; OR ¼ 3.9; Ã allelic association is due to a specific subgroup of JRA we 95% CI ¼ 1.4–11.6; Pn¼4 ¼ 0.036), GA16–16 (10/210 vs 24/558), S–S (individuals carrying ‘short’ alleles GA13 and/or GA16) (22/210 vs divided patients into subgroups that include: systemic ÃÃ 32/558; OR ¼ 1.9; 95% CI ¼ 1.1–3.5; Pn¼2 ¼ 0.04). JRA (n ¼ 20), polyarticular rheumatoid factor (RF)-

Genes and Immunity JRA and polymorphism in the SH2D2A gene A Smerdel et al 312 positive JRA (n ¼ 11), polyarticular RF- negative JRA Acknowledgements (n ¼ 59) and pauciarticular JRA (n ¼ 120). An increased

frequency of GA13 was observed in all JRA subgroups I Knutson is thanked for technical support. Financial (0.10 vs 0.05 in controls), however, only in patients with support has been given from the Norwegian Women

pauciarticular arthritis the GA13 allele showed tendency Public Health Organization, Norwegian Research Council, to significant increase in frequency (OR ¼ 1.8; 95% Grethe Harbitz Legacy, a grant from MSD, from Schering-

CI ¼ 1.3–2.8; Pnc ¼ 0.03), probably due to the higher Plough, from Wyeth, and from Abbott Immunology. number of investigated patients.

Our results suggest that the shortest allele (GA13)of the SH2D2A gene promoter could contribute to the genetic susceptibility of JRA, similar to what we have References observed in MS.7 The associations of JRA and MS with 1 Spurkland A, Brinchmann JE, Markussen G et al. Molecular less transcriptionally active short SH2D2A alleles sup- cloning of a Tcell-specific adapter protein (TSAd) containing an port our notion that reduced expression of the immune- Src homology (SH) 2 domain and putative SH3 and phospho- regulatory TSAd protein in activated T cells may increase tyrosine binding sites. J Biol Chem 1998; 273: 4539–4546. the susceptibility to develop autoimmune diseases. The 2 Sundvold V, Torgersen KM, Post NH et al. T cell-specific recent report that mice lacking the murine TSAd protein adapter protein inhibits T cell activation by modulating Lck develop spontaneous autoimmune disease4 lend support activity. J Immunol 2000; 165: 2927–2931. to this view. Interestingly, the SH2D2A gene is located 3 Marti F, Post NH, Chan E et al. A transcription function for the T within a congenic fragment on the murine chromosome cell-specific adapter (TSAd) protein in T cells: critical role of the 3, which influences genetic susceptibility to collagen- TSAd Src homology 2 domain. J Exp Med 2001; 193: 1425–1430. et al. induced arthritis, as well as the chronicity of experi- 4 Drappa J, Kamen LA, Chan E Impaired T cell death and 14 lupus-like autoimmunity in T cell-specific adapter protein- mental allergic encephalomyelitis (Sundvall et al, Jirholt deficient mice. J Exp Med 2003; 198: 809–821. 15 et al and Martina Johannesson, personal communica- 5 Dai KZ, Vergnaud G, Ando A et al. The SH2D2A gene tion). encoding the T-cell-specific adapter protein (TSAd) is loca- The observed association of the short GA repeat to JRA lized centromeric to the CD1 gene cluster on human could primarily be due to other polymorphisms in the chromosome 1. Immunogenetics 2000; 51: 179–185. SH2D2A gene region in linkage disequilibrium (LD) with 6 Dai KZ, Johansen FE, Melkevik-Kolltveit K et al. Transcrip- the GA repeat alleles. We previously identified two tional activation of the SH2D2A gene is dependent on a cyclic single-nucleotide polymorphisms (SNPs) in the 1-kb adenosine 5’ monophosphate responsive element in the fragment of the SH2D2A promoter upstream of the first proximal SH2D2A promoter. J Immunol 2004; 172. 7 Dai KZ, Harbo HF, Celius EG et al. The T cell regulator gene ATG, which were only present on chromosomes carrying 6 5 SH2D2A contributes to the genetic susceptibility of multiple the GA16 allele. Three SNPs in intron 2 were found not sclerosis. Genes Immun 2001; 2: 263–268. to be in LD with the GA repeats examined in this study 8 Moore TL. Immunopathogenesis of juvenile rheumatoid (Dai and Spurkland, unpublished results). A number of arthritis. Curr Opin Rheumatol 1999; 11: 377–383. other SNPs in the SH2D2A gene have now also been 9 Murray K, Thompson SD, Glass DN. Pathogenesis of juvenile identified (http://www.ncbi.nlm.nih.gov/SNP/ chronic arthritis: genetic and environmental factors. Arch Dis snp_ref.cgi?locusId ¼ 9047). Whether these SNPs in the Child 1997; 77: 530–534. promoter or within the gene influence the expression 10 Glass DN, Giannini EH. Juvenile rheumatoid arthritis as a level or function of the SH2D2A gene is at present complex genetic trait. Arthritis Rheum 1999; 42: 2261–2268. unknown. 11 Forre O, Smerdel A. Genetic epidemiology of juvenile idiopathic arthritis. Scand J Rheumatol 2002; 31: 123–128. Taken together, the SH2D2A gene encoding TSAd may 12 Albert E, Ansell BM. Immunogenetics of juvenile chronic represent a novel susceptibility gene in JRA, and may arthritis. Scand J Rheumatol Suppl 1987; 66: 85–91. possibly also represent a general susceptibility gene for 13 Smerdel A, Ploski R, Flato B et al. Juvenile idiopathic arthritis autoimmune diseases. In polygenic diseases, such as (JIA) is primarily associated with HLA-DR8 but not DQ4 on juvenile arthritis and multiple sclerosis, several genes with the DR8-DQ4 haplotype. Ann Rheum Dis 2002; 61: 354–357. only modest effect are thought to determine whether 14 Sundvall M, Jirholt J, Yang HT et al. Identification of murine disease occurs. The observation that DRB1*08-positive loci associated with susceptibility to chronic experimental patients display even higher frequency of the associated autoimmune encephalomyelitis. Nat Genet 1995; 10: 313–317. SH2D2A alleles strengthen the impression that SH2D2A 15 Jirholt J, Cook A, Emahazion T et al. Genetic linkage analysis of collagen-induced arthritis in the mouse. Eur J Immunol 1998; polymorphism contributes to the genetic susceptibility in 28: 3321–3328. already genetically predisposed individuals. Additional 16 Flato B, Smerdel A, Johnston V et al. The influence of patient studies in patients from different populations are thus characteristics, disease variables, and HLA alleles on the necessary to confirm the association of SH2D2A gene to development of radiographically evident sacroiliitis in juve- development of autoimmune disease. nile idiopathic arthritis. Arthritis Rheum 2002; 46: 986–994.

Genes and Immunity