Rheumatology Advance Access originally published online on November 29, 2006
Rheumatology 2007 46(4):617-621; doi:10.1093/rheumatology/kel381
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The 620W allele is the PTPN22 genetic variant conferring susceptibility to RA in a Dutch population
Department of Rheumatology, Leiden University Medical Center, Leiden, The Netherlands, 1Celera Diagnostics, Alameda, CA, USA and 2Department of Medical Statistics, Leiden University Medical Center, Leiden, The Netherlands
Correspondence to: T. W. J. Huizinga, Department of Rheumatology, Leiden University Medical Center, Leiden, The Netherlands. E-mail: T.W.J.Huizinga{at}lumc.nl
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Abstract |
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Objectives. A missense SNP, C1858T, in PTPN22 has been identified as a genetic risk factor for rheumatoid arthritis (RA). Subsequent work has suggested that other variants in this gene, in particular a haplotype marked by the minor allele of rs3789604, are associated with RA in white North Americans independent of C1858T. We tested this hypothesis in an independent white Dutch study.
Methods. A total of 667 RA patients and 286 controls were genotyped for 13 PTPN22 single nucleotide polymorphisms (SNPs) by allele-specific kinetic polymerase chain reaction. rs3789604 was genotyped in an additional 410 RA and 270 UA patients participating in the Leiden early arthritis inception cohort. We conducted single-marker and haplotype association tests.
Results. The sole haplotype strongly associated with RA in our Dutch population carries the PTPN22 1858T allele. A second haplotype identical at all other SNPs tested except 1858 was not associated with disease. No significant association of the haplotype tagged by the 3' PTPN22 SNP, rs3789604, with RA susceptibility (P = 0.134) was observed in our sample set.
Conclusion. We conclude that C1858T is the sole PTPN22 variant predisposing to RA in our white Dutch sample set.
KEY WORDS: RA, Association study, Haplotype, PTPN22
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Introduction |
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A missense single nucleotide polymorphism (SNP), rs2476601, in the hematopoietic-specific protein tyrosine phosphatase gene, PTPN22, is the first non-HLA (non-human leucocyte antigen) genetic variant consistently associated with susceptibility to a number of autoimmune diseases including rheumatoid arthritis (RA), juvenile idiopathic arthritis (JIA), type 1 diabetes (TID) and Graves disease (reviewed in [1]). The disease-associated C1858T SNP, encoding an arginine to tryptophan substitution at residue 620 (R620W), is located in the P1 proline-rich motif of PTPN22, which binds with high affinity to the Src homology 3 (SH3) domain of the tyrosine kinase, Csk. The minor 1858T (W620) allele disrupts the interaction of PTPN22 with Csk [2] and also encodes a significantly more active phosphatase, which suppresses TCR signalling more efficiently than the wild-type protein [3].
RA is the most common systemic autoimmune disease affecting up to 1% of general population, with a higher prevalence in women [4]. Persistent inflammations that may lead to bone destruction as well as the presence of autoantibodies are hallmarks of RA. Consequently, it is interesting that a prominent feature of PTPN22-associated autoimmune diseases appears to be the presence of autoantibodies. Two autoantibodies are commonly used as diagnostic indicators of RA: IgM rheumatoid factor (RF) directed to the Fc fragment of IgG and anti-citrullinated peptide antibodies (ACPAs) directed against peptides in which arginine residues have been post-translationally modified to citrulline [5]. However, not all RA patients have autoantibodies, and while some studies show exclusive association of the PTPN22 missense SNP with RF-positive RA, others do not [6–12].
Recently Carlton et al. [13] resequenced the PTPN22 gene in 48 RA patients and identified a dense map of SNPs that were characterized in two independent white North American sample sets. Using the resulting genotype data, 10 distinct haplotypes with a frequency of 
1% were predicted, only one of which carried the 1858T risk allele. A second haplotype identical at all SNPs tested, except for 1858, was not associated with disease. This, together with the aforemetioned biological data, provides strong evidence that 1858T is a causative variant. In addition, minor alleles of two SNPs, rs3811021 in the 3' UTR of PTPN22 and rs3789604 in a putative transcription factor binding site 3' of the gene, that are in strong linkage disequilibrium (LD) with one another (R2 > 0.98) tagged a second haplotype that appeared to contribute to RA predisposition independent of 1858T in both sample sets.
The aim of our study was 2-fold. First, we wanted to investigate association of the additional PTPN22 SNPs with RA in an independent white sample set, and second, we wanted to determine whether the 1858T allele was associated with RF-positive and/or RF-negative disease.
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Materials and methods |
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Sample set 1 consisted of 667 white Dutch individuals with RA, all of whom fulfilled the ACR classification criteria for RA and have been described elsewhere [14, 15]. RF status was determined for 302 of the patients as previously described [16]. Sample set 2 consisted of 410 RA patients and 270 undifferentiated arthritis (UA) patients from a population-based inception cohort, the Leiden Early Arthritis Clinic (EAC) [17]. These RA patients also met the ACR classification criteria for RA. UA patients who had symptoms of arthritis that did not exceed 2 yrs, were aged 18 yrs or older and did not fulfil any classification criteria. Controls (n = 286) were unrelated white Dutch individuals with no history of RA [18]. Patients and control individuals were enrolled in the study with informed written consent obtained according to Declaration of Helsinki. Commissie Medische Ethiek, the Leiden institutional review board, approved all protocols.
A combination of 13 informative PTPN22 functional and tagging SNPs (selected from Carlton et al. [13]) was genotyped in both sample set 1 and the controls (Table 1). rs3789604 was genotyped in sample 2; results for rs2476601 (C1858T) in this sample set have been reported previously [11]. Genotyping was done using allele-specific kinetic polymerase chain reaction PCR [19] and the data hand-curated prior to statistical analysis. Previous analyses suggest a genotyping accuracy of >99% [13]. An exact test of Hardy–Weinberg equilibrium (HWE) on the genotyping data for these 13 SNPs, performed separately for cases and controls, showed all SNPs were in HWE (P > 0.05) except for ss38346942 in the controls (expected AA = 276, AT = 9, TT = 0; observed: AA = 278, AT = 5, TT = 2; P < 0.0001). Consequently, ss38346942 was excluded from further analysis (this SNP uniquely marks one of the 10 North American haplotypes; without it two haplotypes, haplotype 10, which is relatively infrequent, and 6, collapse into one haplotype [13]). A chi-square test was used for association analyses; haplotypes were predicted using the Haplo.stats package [20], and LD analysis was performed using Haploview [21]. The haplotype method of Thomson et al. [22, 23] was used for conditional analyses. Regression analysis was done as described by Schaid et al. [20] and power calculations were carried out according to the method of Purcell et al. [24]. P-values <0.05 were considered significant.
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Single-marker association
Genotype data for the 12 PTPN22 SNPs successfully typed in the Dutch cases (sample set 1) and controls were examined by single-marker analysis (Table 1). The results confirm association of the C1858T missense SNP (rs2476601, SNP 22) with RA in Dutch whites [allelic P = 0.0008, odds ratio (OR) = 1.7] [11]. Four additional markers also showed significant association with RA (SNP reference –2, 35, 36 and 37). SNP37, located downstream of PTPN22 and previously reported to tag a W620-independent risk haplotype, was also genotyped in sample set 2 (Table 1). In both sample sets, the minor allele of rs3789604 was less frequent in patients than in the controls (16.8% in sample set 1, 17.7% in sample set 2, 20.6% in controls); this difference between cases and controls was marginally significant in sample set 1 (allelic P = 0.049, OR = 0.78). The UA patients in sample set 2 were also genotyped for rs3789604; again the minor allele frequency was less in cases (15.7%) than in the controls. This is in contrast to the two North American sample sets where the rs3789604 minor allele was more common in cases than in controls (21 vs 16% and 20.6 vs 18.5%).
Association of SNP37 conditional on R620W
The known association of C1858T with RA in addition to the strong LD across the PTPN22 region can confound our ability to determine whether any other SNPs in this region are independently associated with disease. To address this issue, we assessed the association of SNP 37 conditional on 1858T using the haplotype method developed by Thomson and colleagues [22, 23] and used by Carlton et al. [13] to help identify rs3789604 as a disease-associated SNP. In this analysis, two marker haplotypes were estimated using an expectation–maximization algorithm and the case and control chromosomes divided into two bins, those carrying the 1858T risk allele and those carrying the 1858C allele. Focusing on the 1858C chromosomes, we then determined whether the frequency of the rs3789604 minor allele was significantly different between case and control chromosomes. The results showed that SNP37 was not significantly associated with disease conditional on C1858T (P = 0.82), which was confirmed with a regression analysis for genotypes (OR = 0.826, P = 0.15) adjusting for C1858T genotypes.
Power analysis
Given that the effect of SNP 37 was relatively modest compared with the missense SNP (C1858T) in the original report [13], we calculated the power of our study to detect a similar effect using the method of Purcell et al. [24]. Since the original association of SNP37 with RA was observed in patients and controls negative for 1858T, the sample size of our study for this calculation is 256 controls and 559 cases. Assuming an RA disease prevalence of 1%, a risk allele frequency of 0.19 for SNP 37 (derived from Dutch controls used in this study, Table 2), genotypic relative risks for AA and Aa of 1.78 and 1.15, respectively (derived from Carlton et al. [13]), and a type 1 error rate of 0.05, we estimate we have 80.8% power to detect association in this sample set.
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LD structure of the PTPN22 gene and haplotype analysis
Pairwise LD for the 12 PTPN22 SNPs was defined using the genotype data from sample set 1 cases and the controls separately, and the summary statistics D' and R2 calculated using the HaploView program [21]. The LD structure for the PTPN22 region in the Dutch population was similar to the structure reported by the International HapMap project (data not shown). SNPs 36 and 37 were in nearly complete LD (R2 = 0.98 for cases and R2 = 1 for controls), as previously reported in the North American samples [13].
Haplotypes were predicted for these 12 markers using the EM algorithm in the Haplo.stats package (Table 2). Nine haplotypes, each with a frequency >1%, were predicted in both cases and controls accounting for >96% of all the haplotypes. Overall, association between the PTPN22 haplotypes and disease status was highly significant (global P-value = 0.00848; df = 9). The most common haplotype, haplotype 5, had a frequency of approximately 26% in both cases and controls. This was also the most common haplotype in the controls from the two white North American sample sets (frequencies of 29.8 and 35%) [13]. As expected from the single marker data, the sole haplotype carrying the 1858T risk allele, haplotype 2, was increased in patients compared with controls (P = 0.00078). Haplotype 1, identical at every SNP tested except 1858, is not associated with disease. Additionally, we observed a marginally significant increase in the frequency of haplotype 7 in the RA cases (P = 0.048). Haplotype 4, reported to be significantly increased in RA patients relative to controls independent of R620W [13], was actually more common in controls than cases in the Dutch sample set (19 and 16%, respectively); however, these frequencies were not significantly different (P = 0.134). The relative effect of each haplotype was also analysed using regression analysis with the most common haplotype, haplotype 5, serving as the reference group (haplo.stats). Relative to haplotype 5, the 620W-associated haplotype (haplotype 2) showed strong association with disease (P = 0.006, OR = 1.68), whereas haplotype 4 was not significant (P = 0.4, OR = 0.88).
Meta-analysis of PTPN22 3'UT0R polymorphism rs3789604
We summarized the four available studies and performed a meta-analysis on the rs3789604 polymorphism. Using a random effects model, the common OR for the minor allele of SNP 37 was calculated as 1.08 (95% CI: 0.8–1.4) with Pcommon = 0.6, showing no overall association of the minor allele with RA (Cochran Q = 5.91, df = 2, P = 0.0522).
Association of the PTPN22 1858T allele with RF-positive and/or RF-negative disease
It has been postulated that the PTPN22 1858T allele is associated with autoimmunity characterized by the presence of autoantibodies [6]. We previously reported association of the PTPN22 risk allele with both RF-positive and ACPA-positive disease in a Dutch population of RA patients participating in our EAC—in this article referred to as sample set 2 [11]. Here we investigated whether the 1858T polymorphism was associated with RF-positive and/or RF-negative disease in 302 RA patients from sample set 1 (Table 3). ACPA-status was not known for this sample set. In line with our previous report [11] we observed a significant increase in the frequency of 1858T in 197 RF-positive RA patients compared with controls (0.142 in cases vs 0.097 in controls, P = 0.03, OR = 1.55). The frequency of the minor allele was also increased in the 105 RF-negative patients (0.133 in cases vs 0.097 in controls) although this difference was not statistically significant (P = 0.14; OR = 1.43). Comparison of allelle frequencies of RF-positive and RF-negative revealed no significant differences between the two groups with OR = 1.03 and 95% CI: 0.87–1.21.
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Discussion |
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PTPN22 has been now unanimously accepted as a RA susceptibility gene due to robust replication of association with disease in multiple independent case-control and family based sample sets. However it is still a matter of debate whether the R620W polymorphism is the only PTPN22 disease-predisposing variant. In general, identification of independent genetic effects in a region of strong LD is difficult. A clear example is the HLA locus—the most significant RA-susceptibility region estimated to account for up to one-third of the total genetic effect. After nearly 30 yrs of research there is still no clear consensus on which genes, let alone alleles, in this region play a role in RA susceptibility/protection and/or disease severity.
We confirmed the PTPN22 haplotype structure reported by Carlton et al. [13] for their two North American sample sets identifying 9 of the 10 reported haplotypes. We failed to detect the tenth haplotype for technical reasons; the SNP that uniquely marks this haplotype was removed from this analysis because control genotypes were not in HWE. In addition, we confirmed that the 1858T risk allele was found on a single haplotype significantly associated with disease. As in the North American study, this haplotype was identical to a second haplotype (haplotype 1) at all positions genotyped except that it carried 1858C. Haplotype 1 was not associated with disease, confirming that 1858T is a disease-associated SNP. Although the same haplotypes were found in both sample sets, there were notable frequency differences between the two populations. In particular, haplotype 5, the most common haplotype in both populations, was more frequent in the North American controls while haplotype 8 was more frequent in the Dutch controls. We also found a marginally significant association of haplotype 7 with RA, marked by the minor allele of rs38346944 (SNP 23) (P = 0.048), which was not seen in the North American sample sets (P = 0.23 and P = 0.3).
The most striking difference, however, was that haplotype 4, tagged by SNP 37 and found to be associated with RA independent of the C1858T missense SNP in the North American study, was not associated with disease in the Dutch sample set. In fact, unlike the North American results, this particular SNP was actually more frequent in Dutch controls than cases. This discrepancy may be due to clinical heterogeneity of the groups studied, the relatively small size of the Dutch control population (although our calculations suggest 80% power to detect a similar effect), or population-based genetic diversity. Given the observation that the frequency of the 1858T risk allele increases in frequency as one travels north through Europe from Italy/Sardinia (2.3%) to Finland (15.5%) [1] and that North Americans are a composite of various European subpopulations, it is also possible that the original finding could be the result of population admixture that was consistent across both North American data sets.
The PTPN22 1858T risk allele has been associated with a number of autoimmune diseases characterized by the presence of autoantibodies: SLE, TID, Graves's disease and Wegener's Granulomatosis [1]. Given the well-defined role of T-cells in the production of autoantibodies and modulation of B-cell activity, it is plausible that subtle changes in T-cell function could affect the breakdown of tolerance leading to autoantibody production. However, association of the minor PTPN22 allele with autoantibody-positive RA remains controversial. Four studies report association of the PTPN22 minor allele exclusively with RF-positive disease; however, in three studies the minor allele is associated with both RF-positive and RF-negative disease [6–12]. Although the power of the sample set reported here is limited, we see significant association of the 1858T risk allele with RF-positive disease (P = 0.03, OR = 1.55). Clinical heterogeneity could be the cause of the discrepancies between studies as misclassification of RF status can occur when different detection methods are used. In addition, fluctuations of RF titres can occur. A large adequately powered study is clearly necessary to unambiguously define the association of PTPN22 with autoantibody negative RA. There are additional indications that other PTPN22- independent mechanisms of antibody expression and regulation may be involved in autoantibody-mediated autoimmune disease. Vandiedonck et al. [25] found association of the PTPN22 1858T allele with autoantibody-negative form of Myasthenia Gravis. Although this finding requires replication, it suggests that although autoimmune diseases share general features, such as the presence of autoantibodies, PTPN22 status may influence different pathways in different diseases.
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We are grateful to the RA patients and control individuals for participating in these studies and to members of the Celera high throughput genotyping team, in particular D. Leong and J. Catanese, for invaluable help. This project was partially founded by European Community FPs funding, project 018661 AutoCure.
A.B.B. is an employee of Celera Diagnostics and, as such, owns stock and has stock options in Applena Corporation (made up of Celera Genomics Group, Applied Biosystems Group and Celena Diagnostics). X.H. was an employee of Celera Diagnostics and holds CRA stocks. The other authors have declared no conflicts of interest.
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Present address: *Institute of Biology & Biotechnology, Adam Mickiewicz University, Poznan, Poland;
Genentech, South San Francisco, CA, USA. 
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