Detailed analysis of the variability of peptidylarginine deiminase type 4 in German patients with rheumatoid arthritis: a case–control study

Peptidylarginine deiminases (EC 3.5.3.15) are enzymes involved in the post-translational deimination of protein-bound arginine to citrulline [1]. Five different types of peptidylarginine deiminases encoded by the genes PADI1‐PADI4 and PADI6 are currently known [1]. The presence of citrulline-modified target epitopes for autoantibodies is a well-known phenomenon in rheumatoid arthritis (RA) [2, 3]. Peptidylarginine deiminases were recently implicated in the generation of anti-cyclic citrullinated peptide antibodies (anti-CCP) detectable in early stages of the disease [2‐4]. The process resulting in anti-CCP formation is thought to play a pivotal role in early stages of RA evolvement since it is detectable several years before the onset of symptoms [5]. Certain evidence suggests that deimination of arginine at those peptide side-chain positions that interact with the so-called shared epitope of some major histocompatibility complex class II molecules (for example, HLA-DRB1*0401) may result in the generation of high-affinity peptides, thus inducing a strong in-vitro T cell activation [4, 6].

A Japanese research group recently identified a genomic region (1p36) containing the genes PADI1–PADI4, which were suspected to be associated with susceptibility to RA [7]. Peptidylarginine deiminase type 4 (PADI4) was identified as the gene actually responsible for the association with RA. PADI4 has at least five main haplotypes that differ at four exonic single nucleotide polymorphisms (SNPs) and three subsequent amino acid substitutions [7, 8]. While the so-called susceptibility haplotypes 2, 3, and 4 were found to be significantly more frequent in Japanese individuals suffering from RA, the non-susceptibility haplotype 1 predominated in healthy individuals [7]. These results could be confirmed by a further Japanese study [9]. However, studies in different European countries did not reveal significantly different PADI4 haplotype distributions in RA patients and healthy individuals. Moreover, no influence of the PADI4 genotype on disease severity could be detected [10‐14]. Thus, the relevance of PADI4 variability for susceptibility to RA is still unclear.

A recent analysis of our group characterising exons 2–4 of the PADI4 gene identified new variants and haplotypes by a novel haplotype-specific sequencing-based approach [8]. Importantly, three novel coding SNPs in exons 2, 3, and 4 and three SNPs in introns 2 and 3 located near the exon–intron boundaries were found in 11/102 individuals (10.8%). Moreover, a closely related novel haplotype (haplotype 1B) was found in 2.9% of healthy individuals, which differs from haplotype 1 by padi4_92*G/padi4_96*C [8]. Since this additional variability of the PADI4 gene has not been assessed by other studies, the aim of the present case–control study was to investigate the possible influence of PADI4 genotypes including previously unknown PADI4 variants on susceptibility to RA in a German population.

This study provides a hint that variability of the PADI4 gene is related to the susceptibility to RA in the German population, whereas certain differences of hitherto unknown PADI4 variants between patients and controls were not found. The impact of PADI4 genotypes on susceptibility to RA remains controversial [7, 9‐14]. Until now, certain PADI4 genotypes (haplotypes 2, 3, and 4) have been implicated to be involved in the pathogenesis of RA only in Japanese populations [7, 9]. No such association of PADI4 variability with RA prevalence and severity could be demonstrated in various European populations [10‐14]. In our study, also, an influence of PADI4 genotype on disease activity or anti-CCP level could not be demonstrated. The mechanism by which PADI4 variability may influence the break of tolerance is still unknown. Initially, it was argued that detectable differences in mRNA stability could result in higher enzymatic activity in cases where the susceptibility haplotypes (2,3 and 4) of PADI4 are present, leading to the generation of larger amounts of citrullinated peptides [7]. Most recently, a close association of the production of anti-CCP antibodies and HLA-DRB1 has been described [6, 11, 13, 19], indicating the importance of antigen presentation in the induction of autoimmunity. This finding clearly could be confirmed in our study.

With the exception of haplotype 4, the frequencies of all other PADI4 haplotypes in our control individuals were comparable with those reported by other groups [7, 9, 10, 14]. While the frequency of PADI4 haplotype 4 in our study (7.8%) was similar to that reported by groups from the United Kingdom (9.4%, P = 0.51; here termed haplotype 3) [10], Spain (5.9%, P = 0.32; padi4_94*T, padi4_104*C) [14], and Japan (5.5%, P = 0.17) [9], it was statistically significant different from the frequency reported by the large initial Japanese study (4.0%, P = 0.013) [7]. All of our patients and healthy individuals were Caucasian. The fact that the PADI4 haplotype 4 frequency in our control population was significantly higher compared with one of the Japanese studies [7] may therefore be influenced by differences in the ethnic background.

In our study, a statistically significant positive association of PADI4 haplotype 4 with RA was observed (odds ratio = 2.0, 95% confidence interval = 1.1–3.8). The presence of this haplotype did not influence disease activity or the anti-CCP level. We did not found an association of RA and PADI4 haplotypes 2 and 3, which were described as the principal susceptibility haplotypes in the Japanese population [7]. However, we cannot exclude that this difference may be influenced by the size of our study population.

When analysing the distributions of those PADI4 SNPs covered by our genotyping approach, padi4_89A→G, padi4_90C→T, and padi4_94C→T were found to be significantly associated with RA. These SNPs are common with PADI4 haplotype 4 and haplotype 2/3, whereas padi4_104C→T, padi4_95G→C, and padi4_96T→C, which are common with PADI4 haplotype 4 and haplotype 1, exhibited no association with RA.

The present study identified uncommon PADI4 variants that are not typically included among the five main PADI4 haplotypes. Consistent with our previous findings in healthy individuals [8], this study also revealed additional variability in PADI4 exons 2–4 in RA patients. As a result of this study, the frequency of uncommon PADI4 variants as identified earlier [8] was apparently not different quantitatively or qualitatively between patients and controls.

Of note, a statistically significant association between certain PADI4 genotypes and RA was detected in our study, in contrast to reports from other European groups [10‐14]. This puzzling discrepancy may be due to influencing factors, such as a homogeneous Caucasian population, although we cannot definitely exclude other selection biases.

The question of whether PADI4 variability alters the interactions between the enzyme and possible target proteins remains unclear [20]. Further studies are needed to characterise the influence of this variability on the repertoire of deiminated target proteins.

In summary, the PADI4 haplotype 4 and the SNPs padi4_89A→G, padi4_90C→T, and padi4_94C→T were found to be significantly associated with RA in a German population. The genomic region of PADI4 exons 2–4 of RA patients exhibits additional variability, which is apparently not different quantitatively and qualitatively between RA patients and controls. While the PADI4 genotype did not influence disease activity or the anti-CCP level, the presence of the HLA-DRB1 shared epitope was associated with significantly higher anti-CCP levels.