Genetic and genomic studies of PADI4 in rheumatoid arthritis
S. M. J. Harney,
C. Meisel,
A.-M. Sims,
P. Y. Woon1,
B. P. Wordsworth and
M. A. Brown
Institute of Musculoskeletal Sciences, University of Oxford, Botnar Research Centre and 1 Wellcome Trust Centre for Human Genetics, Oxford, UK.
Correspondence to: M. A. Brown, University of Oxford, Institute of Musculoskeletal Sciences, Botnar Research Centre, Nuffield Orthopaedic Centre, Windmill Road, Oxford OX3 7LD, UK. E-mail: mbrown{at}well.ox.ac.uk
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Abstract
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Objectives. Strong genetic association of rheumatoid arthritis (RA) with PADI4 (peptidyl arginine deiminase) has previously been described in Japanese, although this was not confirmed in a subsequent study in the UK. We therefore undertook a further study of genetic association between PADI4 and RA in UK Caucasians and also studied expression of PADI4 in the peripheral blood of patients with RA.
Methods. Seven single-nucleotide polymorphisms (SNP) were genotyped using polymerase chain reaction (PCR)restriction fragment length polymorphism in 111 RA cases and controls. A marker significantly associated with RA (PADI4_100, rs#2240339) in this first data set (P = 0.03) was then tested for association in a larger group of 439 RA patients and 428 controls. PADI4 transcription was also assessed by real-time quantitative PCR using RNA extracted from peripheral blood mononuclear cells from 13 RA patients and 11 healthy controls.
Results. A single SNP was weakly associated with RA (P = 0.03) in the initial casecontrol study, a single SNP (PADI4_100) and a two marker haplotype of that SNP and the neighbouring SNP (PADI4_104) were significantly associated with RA (P = 0.02 and P = 0.03 respectively). PADI4_100 was not associated with RA in a second sample set. PADI4 expression was four times greater in cases than controls (P = 0.004), but expression levels did not correlate with the levels of markers of inflammation.
Conclusion. PADI4 is significantly overexpressed in the blood of RA patients but genetic variation within PADI4 is not a major risk factor for RA in Caucasians.
KEY WORDS: Real-time PCR, Anti-CCP antibodies, Genetic polymorphism
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Introduction
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RA is a multisystem disorder with a complex aetiology, the most obvious manifestation of which is a destructive inflammatory arthritis. Twin studies have estimated that the concordance rate for RA in monozygotic twins is four times that of dizygotic twins. Furthermore, concordance rate is higher in twins that are HLA-DRB1*04-positive [1]. It is also known that concordance in twins is higher in those with more severe disease and that this may account for at least some of the discrepancy in monozygotic twin concordance rates between the studies of Lawrence (30%) [2] and Aho et al. (12%) [3]. Using variance modelling, MacGregor et al. [4] estimate that up to 55% of the population variance in RA is genetic.
The diagnosis of RA can be difficult, especially in the early stages of disease. Not all patients meet the well-established 1987 ACR criteria [5] and 1020% can go into remission after the first inflammatory episode. Published studies on the utility of genetic markers for predicting persistent and severe arthritis are contradictory [6, 7]. Classic rheumatoid factor (RF) is one of the ACR criteria for RA, but it can be positive in other autoimmune and infectious diseases, and may be positive in up to 13% of healthy blood donors over the age of 60 [8]. Its diagnostic utility is limited because of moderate sensitivity (5488%) and specificity (4892%) [913]. Recently, antibodies to cyclic citrullinated peptides (CCP) have been described that are highly specific for RA and, like RF, can predate the onset of RA by many years [14, 15]. Peptidyl arginine deiminase (PADI) enzymes convert arginine within peptides to citrulline, which, because of its neutral oxygen group, is specifically recognized by anti-CCP antibodies [16]. Two whole-genome screens of RA families [17, 18] have suggested linkage with chromosome 1p36, which includes the PADI4 gene. Further, Suzuki et al. [19] demonstrated a strong association of PADI4 with RA in Japanese. This has subsequently been confirmed in Japanese [20], although no association was found in a study of UK Caucasians with RA [21].
We therefore performed a casecontrol study of PADI4 genotypes in RA in a UK Caucasian population to confirm and refine these associations. We also investigated the expression of PADI4 in the peripheral blood of RA patients and healthy controls.
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Materials and methods
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Study population
A preliminary association study was undertaken in 111 patients with RA and 111 healthy controls. All were British Caucasians living in the south of England. All cases satisfied the 1987 American College of Rheumatology criteria. All subjects gave informed written consent and approval had been granted by the local ethics committee. The controls were healthy British Caucasian blood donors.
Genotyping
The PADI4 gene is made up of 16 exons in total and is 17 Mb in length (17 015 79917 071 331 bp). The transcript length is 1992 bp and the translated length is 66 amino acid residues. Seven SNPs (PADI4_92, PADI4_94, PADI4_97, PADI4_99, PADI4_100, PADI4_103 and PADI4_104) were genotyped using polymerase chain reaction (PCR) amplification of 5 ng genomic DNA and restriction fragment length polymorphism in 111 RA cases and 111 controls. The reaction mix per sample was 0.09 µl TaqGold (Applied Biosystems, Foster City, CA, USA), 1 µl 10 x PCR buffer, 1 µl of 200 µM dNTPs, 0.61.0 µl 25 mmol MgCl2, 0.4 µl pooled diluted primers (10 µmol/l each primer), and made up to a final reaction volume of 10 µl with water. The cycling conditions for PADI4_99 and PADI4_104 were 94°C for 12 min then 34 cycles of 94°C for 30 s, annealing at 56°C for 30 s and 72°C for 60 s, then 72°C for 10 min, then holding at 15°C. A similar programme was employed for PADI4_103 but with an annealing temperature of 65°C. A touchdown programme was used for PADI4_92, PADI4_94, PADI4_97 and PADI4_100; using the following genotyping conditions: 95°C for 15 min, 14 cycles of 94°C for 20 s, 65°C for 30 s (0.5°/cycle), 72°C for 60 s, then 19 cycles of 94°C for 20 s, 56°C for 30 s and 72°C for 60 s, ending with 72°C for 5 min. The products were incubated overnight at 37°C with their respective restriction enzymes and then separated by electrophoresis using a 3% agarose gel, stained with ethidium bromide. One SNP (rs#2240339) found to be associated with RA in this initial cohort was then genotyped in a second larger population of patients with erosive RA (n = 439 cases, n = 428 controls) by TaqMan Invader assay (KBiosciences, Hoddesden, UK). All genotypes were read by two independent observers and any inconsistencies were excluded from further analysis.
PADI4 expression
RNA was extracted from peripheral blood mononuclear cells from 13 early, DMARD-naïve RA patients (duration of symptoms <2 yr) and 11 healthy controls, and PADI4 transcription was assessed by reverse transcriptionPCR using an ABI Prism 7700 Sequence Detection System and Assays-on-Demand Gene Expression probes (Hs00202612_m1 PADI4; Hs00355752_m1 HPRT1; Applied Biosystems). All reactions were performed in duplicate under standard TaqMan thermal cycling conditions and data were analysed using Sequence Detection Systems software (Applied Biosystems).
Anti-CCP antibody levels
Serum samples were collected from RA patients and healthy controls and stored at 20°C until analysis. Anti-CCP antibody levels were measured by enzyme-linked immunosorbent assay (ELISA; Axis-Shield DiastatTM Anti-CCP ELISA) and values of <2 U/ml were taken as negative.
Statistical analysis
All case and control genotyping was analysed using the
2 test. Haplotypes were constructed in unrelated cases and controls using the program Phase v2.0 [22, 23]. All cases and controls were permutated and only haplotypes with a posterior probability of >90% were analysed. PADI4 gene expression was studied using the Mann-Whitney U test.
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Results
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A single SNP (marker rs#2240339) was weakly associated with RA (0.03) in the initial casecontrol study; the accuracy of genotyping was confirmed by regenotyping using a different method. However, association was noted with a two-marker haplotype (PADI4_100/PADI4_103) carrying this SNP, although the association was marginal (P = 0.03). When marker PADI4_100 was tested in the larger population (cases n = 439, controls n = 438), no association was seen (P = 0.4). The genotyping results are summarized in Table 1. The seven SNPs were in strong linkage disequilibrium, with D' values between 0.7 and 1. Haplotype frequencies in the UK population were similar to the previously reported Japanese frequencies [19]. Three 7-marker haplotypes represented over 80% of the chromosomes in both the cases and controls (cases, 82%; controls, 81%). PADI4 expression was four-fold greater in RA-patients compared with controls (P = 0.004) (Fig. 1) but expression levels did not significantly correlate with inflammatory markers (CRP, r = 0.23; ESR, r = 0.17) or anti-CCP levels.

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FIG. 1. PADI4 gene expression in RA patients vs controls. The PADI4 gene expression was significantly higher in peripheral blood mononuclear cells from early RA patients compared with healthy controls (P = 0.004, Student's t-test). Solid lines indicate the mean of each group.
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Discussion
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We have shown that PADI4 is significantly overexpressed in the blood of RA patients regardless of their disease activity. No single marker in PADI4 was associated with RA, although a two-marker haplotype was weakly associated with disease. Given the strong replicated evidence of association between PADI4 and RA in Japanese, this is worthy of further investigation [19, 20]. The disease presentation between the two groups has always been similar and the criteria used to define RA is the same. Consequently, we cannot explain our findings by disease onset differences.
Technically, the Japanese study is a lot more comprehensive, with 119 SNPs done in a larger cohort. But in our study the only significant SNP was regenotyped in a much larger group, where it was no longer significant, though a two-marker haplotype showed marginal significance. Our study supports the work of others in the UK and in France. Caponi et al. [25] have shown in 100 French trio families that no single SNP or haplotype was associated with disease. In addition they also showed no correlation with anti-CCP positivity. Barton et al. [21] in a large British study of 839 RA patients and 481 controls also showed no association between PADI4 SNPs and disease. Potential explanation for the differences between the findings in Japanese and Caucasian populations include differences in the genetic variation in PADI4, or in gene-gene interaction or gene-environment in the different ethnic groups. The SNPs we have studied may well be on an extended haplotype that is in linkage disequilibrium with the true disease-causing variant in the Japanese but not in the British population. Consequently, there may be inherent differences in genetic risk between UK and Japanese populations. A recent paper supports this by showing marked variability in the haplotypic patterns in the PADI4 gene between a healthy German population and the Japanese [26]. The findings in the German population are surprising and difficult to reconcile with the findings of the current study and other studies of Caucasians which have generally found similar haplotype patterns to the Japanese population. We have replicated the findings of two other Caucasian groups using a different experimental design. These findings together indicate that genetic variation in PADI4 plays a substantially smaller role in susceptibility to RA in Caucasians than in Japanese.
In the inflamed rheumatoid synovium, there is oxygen disequilibrium. This causes excess reactive oxygen species and simultaneously areas of microinfarction, with resultant production of extravascular citrullinated fibrin [15]. The latter may induce antigen-driven activation of B cells, producing antibodies to such citrullinated proteins (anti-CCP antibodies) [27]. In addition, there is extensive cell death secondary to the oxidative stress in the RA synovium, which may facilitate the leakage of the PADI enzymes. In contrast, PADI4, which catalyses the conversion of arginine to citrulline, resulting in the production of anti-CCP antibodies, seems to be very important. There is no doubt that the presence of anti-CCP antibodies is a very useful adjunct to the diagnosis of RA and may also influence the prognosis.
Citrullination of peptides is likely to be important in the generation of this antibody response and PADI4 transcription was increased in our patients with RA. A recent paper by Yamada et al. [28] has shown that the expression of the PADI enzyme, citrullination of proteins and production of anti-citrullinated protein antibodies occurs in the synovium of RA patients, thus highlighting its importance in disease pathogenesis. However, since the genetic association is apparently quite variable between different ethnic groups with RA it remains to be established whether in situ deiminization of arginine to citrulline is a primary influence in susceptibility to RA or represents a pathway triggered by other aetiological factors. In UK Caucasians, in contrast to Japanese, the contribution of genetic variation in PADI4 to the aetiology of RA is either lacking or very weak. This might reflect the differing HLA background of the disease in these two populations. RA is strongly associated with the HLA-DRB1 shared epitope in both populations [29] and it has been shown previously that citrullinated peptides bind preferentially to shared epitope alleles, particularly HLA-DRB1*0401 [30]. PADI4 may preferentially citrullinate peptides presented by HLA-DRB1 alleles which play a greater role in susceptibility to RA in Japanese, such as HLA-DRB1*0405 and -DRB1*0410, which are uncommon DRB1 alleles in Caucasions. It is also conceivable that the differential association of RA with PADI4 reflects the differing contribution of DRB1 alleles in populations throughout the world. Much is still unknown about the important role PADI4 plays in RA pathogenesis.
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Acknowledgments
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S.H. is an Arthritis Research Campaign Clinical Research Fellow. C.M. is a European Union Marie Curie Fellow and M.A.B. is an Arthritis Research Campaign Senior Research Fellow.
The authors have declared no conflicts of interest.
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Submitted 28 October 2004;
revised version accepted 22 February 2005.