Protein Citrullination by Peptidyl Arginine Deiminase/Arginine Deiminase Homologs in Members of the Human Microbiota and Its Recognition by Anti-Citrullinated Protein Antibodies.

The human microbiome exists throughout the body, and it is essential for maintaining various physiological processes, including immunity, and dysbiotic events, which are associated with autoimmunity. Peptidylarginine deiminase (PAD) enzymes can citrullinate self-proteins related to rheumatoid arthritis (RA) that induce the production of anti-citrullinated protein antibodies (ACPAs) and lead to inflammation and joint damage. The present investigation was carried out to demonstrate the expression of homologs of PADs or arginine deiminases (ADs) and citrullinated proteins in members of the human microbiota. To achieve the objective, we used 17 microbial strains and specific polyclonal antibodies (pAbs) of the synthetic peptide derived from residues 100-200 of human PAD2 (anti-PAD2 pAb), and the recombinant fragment of amino acids 326 and 611 of human PAD4 (anti-PAD4 pAb), a human anti-citrulline pAb, and affinity ACPAs of an RA patient. Western blot (WB), enzyme-linked immunosorbent assay (ELISA), elution, and a test with Griess reagent were used. This is a cross-sectional case-control study on patients diagnosed with RA and control subjects. Inferential statistics were applied using the non-parametric Kruskal-Wallis test and Mann-Whitney U test generated in the SPSS program. Some members of phyla Firmicutes and Proteobacteria harbor homologs of PADs/ADs and citrullinated antigens that are reactive to the ACPAs of RA patients. Microbial citrullinome and homolog enzymes of PADs/ADs are extensive in the human microbiome and are involved in the production of ACPAs. Our findings suggest a molecular link between microorganisms of a dysbiotic microbiota and RA pathogenesis.

Intrinsically Disordered Chromatin Protein NUPR1 Binds to the Enzyme PADI4.

The nuclear protein 1 (NUPR1) is an intrinsically disordered protein involved in stress-mediated cellular conditions. Its paralogue nuclear protein 1-like (NUPR1L) is p53-regulated, and its expression down-regulates that of the NUPR1 gene. Peptidyl-arginine deiminase 4 (PADI4) is an isoform of a family of enzymes catalyzing arginine to citrulline conversion; it is also involved in stress-mediated cellular conditions. We characterized the interaction between NUPR1 and PADI4 in vitro, in silico, and in cellulo. The interaction of NUPR1 and PADI4 occurred with a dissociation constant of 18 ± 6 µM. The binding region of NUPR1, mapped by NMR, was a hydrophobic polypeptide patch surrounding the key residue Ala33, as pinpointed by: (i) computational results; and, (ii) site-directed mutagenesis of residues of NUPR1. The association between PADI4 and wild-type NUPR1 was also assessed in cellulo by using proximity ligation assays (PLAs) and immunofluorescence (IF), and it occurred mainly in the nucleus. Moreover, binding between NUPR1L and PADI4 also occurred in vitro with an affinity similar to that of NUPR1. Molecular modelling provided information on the binding hot spot for PADI4. This is an example of a disordered partner of PADI4, whereas its other known interacting proteins are well-folded. Altogether, our results suggest that the NUPR1/PADI4 complex could have crucial functions in modulating DNA-repair, favoring metastasis, or facilitating citrullination of other proteins.

A Screening of Epigenetic Therapeutic Targets for Non-Small Cell Lung Cancer Reveals PADI4 and KDM6B as Promising Candidates.

Despite advances in diagnostic and therapeutic approaches for lung cancer, new therapies targeting metastasis by the specific regulation of cancer genes are needed. In this study, we screened a small library of epigenetic inhibitors in non-small-cell lung cancer (NSCLC) cell lines and evaluated 38 epigenetic targets for their potential role in metastatic NSCLC. The potential candidates were ranked by a streamlined approach using in silico and in vitro experiments based on publicly available databases and evaluated by real-time qPCR target gene expression, cell viability and invasion assays, and transcriptomic analysis. The survival rate of patients with lung adenocarcinoma is inversely correlated with the gene expression of eight epigenetic targets, and a systematic review of the literature confirmed that four of them have already been identified as targets for the treatment of NSCLC. Using nontoxic doses of the remaining inhibitors, KDM6B and PADI4 were identified as potential targets affecting the invasion and migration of metastatic lung cancer cell lines. Transcriptomic analysis of KDM6B and PADI4 treated cells showed altered expression of important genes related to the metastatic process. In conclusion, we showed that KDM6B and PADI4 are promising targets for inhibiting the metastasis of lung adenocarcinoma cancer cells.

Histoplasma capsulatum-induced extracellular DNA trap release in human neutrophils.

Neutrophils are leukocytes that are capable of eliminating both intra- and extracellular pathogens by mechanisms such as phagocytosis, degranulation, and release of neutrophil extracellular traps (NETs). Histoplasma capsulatum var. capsulatum (H. capsulatum) is a dimorphic fungus with a global distribution that causes histoplasmosis, a disease that is endemic in different geographic areas and is spreading worldwide. The release of NETs has been described as an important host defense mechanism against different fungi; however, there are no reports demonstrating that this process is implicated in neutrophil response to H. capsulatum infection. Therefore, the aim of this work is to investigate whether isolated human neutrophils release NETs in response to H. capsulatum and the potential mechanisms involved, as well as delineate the NETs antifungal activity. Using both confocal fluorescence and scanning electron microscopy techniques, we determined that NETs are released in vitro in response to H. capsulatum via an oxidative mechanism that is downstream of activation of the Syk and Src kinase pathways and is also dependent on CD18. NETs released in response to H. capsulatum yeasts involve the loss of neutrophil viability and are associated with elastase and citrullinated histones, however also can occur in a PAD4 histone citrullination independent pathway. This NETs also presented fungicidal activity against H. capsulatum yeasts. Our findings may contribute to the understanding of how neutrophils recognize and respond as immune effector cells to H. capsulatum, which may lead to better knowledge of histoplasmosis pathophysiology and treatment.

Mac-1 triggers neutrophil DNA extracellular trap formation to Aspergillus fumigatus independently of PAD4 histone citrullination.

Aspergillus fumigatus (A. fumigatus) is an environmental fungus and a human pathogen. Neutrophils are critical effector cells during the fungal infections, and neutropenia is a risk factor for the development of pulmonary aspergillosis. Neutrophil extracellular traps (NETs) are released by neutrophils in response to A. fumigatus and inhibit the conidial germination. In this work, we observed that the receptors TLR2, TLR4, and Dectin-1 were dispensable for the A. fumigatus induced NET release. In contrast CD11b/CD18 was critical for the NET release in response to A. fumigatus conidia, and this required the CD11b I-domain-mediated recognition, whereas the blockade of the CD11b lectin domain did not affect the A. fumigatus induced NET release. A. fumigatus induced NET release relied on the activity of spleen tyrosine kinase (Syk), Src family kinase(s), and class IA PI3 kinase δ. Although A. fumigatus promoted histone citrullination, this process was dispensable for the NET release in response to A. fumigatus conidia. The A. fumigatus induced NET release required the reactive oxygen species generation by the NOX2 complex, in a downstream pathway requiring CD11b/CD18, Src kinase family activity, Syk and PI3K class IA δ. Our findings thus reveal the signaling pathways involved in the formation of NETs in response to A. fumigatus.

Clinical and pathophysiologic relevance of autoantibodies in rheumatoid arthritis.

Rheumatoid arthritis (RA) is an autoimmune/inflammatory disease affecting 0.5 to 1% of adults worldwide and frequently leads to joint destruction and disability. Early diagnosis and early and effective therapy may prevent joint damage and lead to better long-term results. Therefore, reliable biomarkers and outcome measures are needed. Refinement of the understanding of molecular pathways involved in disease pathogenesis have been achieved by combining knowledge on RA-associated genes, environmental factors and the presence of serological elements. The presence of autoantibodies is a distinctive feature of RA. Rheumatoid Factor and Anti-Citrullinated Protein Antibodies are the two most remarkable autoantibodies in RA and provide different clinical and pathophysiological information. They precede the onset of disease symptoms and predict a more severe disease course, indicating a pathogenetic role in RA. Therefore, they promote a more accurate prognosis and contribute for a better disease management. Several RA-associated autoantibody systems have been identified: Anti-Carbamylated Antibodies, Anti-BRAF, Anti-Acetylated, Anti-PAD4 antibodies and others. Hopefully, the characterization of a comprehensive array of novel autoantibody systems in RA will provide unique pathogenic insights of relevance for the development of diagnostic and prognostic approaches compatible with an effective personalized medicine.

Pharmacophore and structure-based drug design, molecular dynamics and admet/tox studies to design novel potential pad4 inhibitors.

We have used docking (GLIDE), pharmacophore modeling (Discovery Studio), long trajectory molecular dynamics (Discovery Studio) and ADMET/Tox (QikProp and DEREK) to investigate PAD4 in order to determine potential novel inhibitors and hits. We have carried out virtual screening in the ZINC natural compounds database. Pharmacokinetics and Toxicity of the best hits were assessed using databases implemented in softwares that create models based on chemical structures taking into account consideration about the toxicophoric groups. A wide variety of pharmaceutical relevant properties are determined in order to make decisions about molecular suitability. After screening and analysis, the 6 most promising PAD4 inhibitors are suggested, with strong interactions (pi-stacking, hydrogen bonds, hydrophobic contacts) and suitable pharmacotherapeutic profile as well.

Respiratory Syncytial Virus induces the classical ROS-dependent NETosis through PAD-4 and necroptosis pathways activation.

Respiratory syncytial virus (RSV) is a major cause of diseases of the respiratory tract in young children and babies, being mainly associated with bronchiolitis. RSV infection occurs primarily in pulmonary epithelial cells and, once infection is established, an immune response is triggered and neutrophils are recruited. In this study, we investigated the mechanisms underlying NET production induced by RSV. We show that RSV induced the classical ROS-dependent NETosis in human neutrophils and that RSV was trapped in DNA lattices coated with NE and MPO. NETosis induction by RSV was dependent on signaling by PI3K/AKT, ERK and p38 MAPK and required histone citrullination by PAD-4. In addition, RIPK1, RIPK3 and MLKL were essential to RSV-induced NETosis. MLKL was also necessary to neutrophil necrosis triggered by the virus, likely promoting membrane-disrupting pores, leading to neutrophil lysis and NET extrusion. Finally, we found that RSV infection of alveolar epithelial cells or lung fibroblasts triggers NET-DNA release by neutrophils, indicating that neutrophils can identify RSV-infected cells and respond to them by releasing NETs. The identification of the mechanisms responsible to mediate RSV-induced NETosis may prove valuable to the design of new therapeutic approaches to treat the inflammatory consequences of RSV bronchiolitis in young children.

Peptidylarginine deiminase 4 concentration, but not PADI4 polymorphisms, is associated with ICU mortality in septic shock patients.

The objective of our study was to evaluate the association between peptidylarginine deiminase 4 (PAD4) concentration and its polymorphisms with mortality in patients with septic shock. We prospectively evaluated 175 patients aged over 18 years with septic shock upon intensive care unit (ICU) admission. However, 48 patients were excluded. Thus, 127 patients were enrolled in the study. At the time of the patients' enrollment, demographic information was recorded. Blood samples were taken within the first 24 hours of the patient's admission to determine serum PAD4 concentrations and its polymorphism PADI4_89 [rs11203366], PADI4_94 [rs2240340] and PADI4_104 [rs1748033]. The mean age was 63.3 ± 15.2 years, 56.7% were male, PAD4 concentration was 4.62 (2.48-6.20) ng/mL and the ICU mortality rate was 67.7%. The patients who died in the ICU had higher APACHE II and Sequential Organ Failure Assessment (SOFA) scores. In addition, PAD4 concentration was higher in patients who died during ICU stay. However, there were no differences regarding PADI4 polymorphisms and ICU mortality. In the logistic regression models, PAD4 concentrations were associated with ICU mortality when adjusted for APACHE II score and lactate (OR: 1.477; CI 95%: 1.186-1.839; P < .001), and when adjusted for age, gender and APACHE II score (OR: 1.392; CI 95%: 1.145-1.692; P < .001). In conclusion, PAD4 concentration, but not PADI4_89, PADI4_94 and PADI4_104 polymorphisms, is associated with ICU mortality in septic shock patients.

Entamoeba histolytica Trophozoites Induce a Rapid Non-classical NETosis Mechanism Independent of NOX2-Derived Reactive Oxygen Species and PAD4 Activity.

Neutrophil extracellular traps (NETs) are DNA fibers decorated with histones and antimicrobial proteins from cytoplasmic granules released into the extracellular space in a process denominated NETosis. The molecular pathways involved in NETosis have not been completely understood. Classical NETosis mechanisms involve the neutrophil elastase (NE) translocation to nucleus due to the generation of reactive oxygen species (ROS) by NADPH oxidase (NOX2) or the peptidyl arginine deiminase 4 (PAD4) activation in response to an increase in extracellular calcium influx; both mechanisms result in DNA decondensation. Previously, we reported that trophozoites and lipopeptidophosphoglycan from Entamoeba histolytica trigger NET release in human neutrophils. Here, we demonstrated in a quantitative manner that NETs were rapidly form upon treatment with amoebic trophozoites and involved both nuclear and mitochondrial DNA (mtDNA). NETs formation depended on amoeba viability as heat-inactivated or paraformaldehyde-fixed amoebas were not able to induce NETs. Interestingly, ROS were not detected in neutrophils during their interaction with amoebas, which could explain why NOX2 inhibition using apocynin did not affect this NETosis. Surprisingly, whereas calcium chelation reduced NET release induced by amoebas, PAD4 inhibition by GSK484 failed to block DNA extrusion but, as expected, abolished NETosis induced by the calcium ionophore A23187. Additionally, NE translocation to the nucleus and serine-protease activity were necessary for NET release caused by amoeba. These data support the idea that E. histolytica trophozoites trigger NETosis by a rapid non-classical mechanism and that different mechanisms of NETs release exist depending on the stimuli used.

Meta-analysis of the association between PADI4 -92C/G polymorphism and rheumatoid arthritis in the Chinese population.

Many studies have evaluated the correlation between peptidylarginine deiminase 4 (PADI4) -92C/G polymorphism and rheumatoid arthritis (RA), but the results remain inconclusive. Therefore, we performed a meta-analysis in the Chinese population to provide comprehensive data on the association between PADI4 -92C/G polymorphism and RA. Eligible studies published before May 2016 were identified in PubMed and Chinese databases. The strengths of these associations were assessed by pooled odds ratios (OR) and 95% confidence interval (CI). Eight studies documenting a total of 1351 RA cases and 1585 controls were included in this meta-analysis. In the overall analysis, a significant association between the PADI4 -92C/G polymorphism and RA was found in the Chinese population (G vs C: OR=1.32, 95%CI=1.02-1.71; GG+CG vs CC: OR=1.75, 95%CI=1.20-2.53). The subgroup analyses stratified by geographic area(s) and source of controls revealed significant results in South China, in hospital-based studies and population-based studies. In summary, this meta-analysis suggested that PADI4 -92C/G polymorphism may be associated with the RA incidence in the Chinese population, especially for South China. Further studies conducted on other ethnic groups are required for definite conclusions.

PADI4 polymorphisms and the functional haplotype are associated with increased rheumatoid arthritis susceptibility: A replication study in a Southern Mexican population.

Rheumatoid arthritis (RA) is a common autoimmune disease with a complex genetic background. The peptidyl arginine deiminase type IV (PADI4) gene has been associated with RA susceptibility in several populations. We addressed the relationship between three exonic PADI4 gene single nucleotide polymorphisms (SNPs) PADI4_89 (rs11203366), PADI4_90 (rs11203367) and PADI4_92 (rs874881) and related haplotypes with RA in a population from Southern México. This study included 200 RA patients and 200 control subjects. The SNPs were evaluated using the polymerase chain reaction-restriction fragment-length polymorphism (PCR-RFLP) technique, and antibodies to cyclic citrullinated peptides (anti-CCP) were measured by enzyme-linked immunosorbent assay (ELISA). In this population, the minor alleles of PADI4_89∗G, PADI4_90∗T and PADI4_92∗G gene polymorphisms were associated with RA susceptibility (OR=1.34, p=0.04; OR=1.35, p=0.03; OR=1.34, p=0.04; respectively). The GTG haplotype was also significantly associated with RA (OR=2.27 95%CI=1.18-4.41; p=0.008), but did not show association with levels of anti-CCP antibodies and clinical parameters. In conclusion, our replication study in a Southern Mexican population suggests that PADI4 individual polymorphisms and the related susceptibility haplotype (GTG) are also genetic risk markers for RA.

Activation of Peptidylarginine Deiminase in the Salivary Glands of Balb/c Mice Drives the Citrullination of Ro and La Ribonucleoproteins.

The goal of the present study was to determine whether peptidylarginine deiminase PAD2 and PAD4 enzymes are present in Balb/c mouse salivary glands and whether they are able to citrullinate Ro and La ribonucleoproteins. Salivary glands from Balb/c mice were cultured in DMEM and supplemented with one of the following stimulants: ATP, LPS, TNF, IFNγ, or IL-6. A control group without stimulant was also evaluated. PAD2, PAD4, citrullinated peptides, Ro60, and La were detected by immunohistochemistry and double immunofluorescence. PAD2 and PAD4 mRNAs and protein expression were detected by qPCR and Western blot analysis. PAD activity was assessed using an antigen capture enzyme-linked immunosorbent assay. LPS, ATP, and TNF triggered PAD2 and PAD4 expression; in contrast, no expression was detected in the control group (p < 0.001). PAD transcription slightly increased in response to stimulation. Additionally, PAD2/4 activity modified the arginine residues of a reporter protein (fibrinogen) in vitro. PADs citrullinated Ro60 and La ribonucleoproteins in vivo. Molecular stimulants induced apoptosis in ductal cells and the externalization of Ro60 and La ribonucleoproteins onto apoptotic membranes. PAD enzymes citrullinate Ro and La ribonucleoproteins, and this experimental approach may facilitate our understanding of the role of posttranslational modifications in the pathophysiology of Sjögren's syndrome.

Association between peptidyl arginine deiminase 4 (PADI4)-104C/T polymorphism and rheumatoid arthritis: a meta-analysis in the Chinese population.

The correlation between the -104C/T polymorphism in the peptidyl arginine deiminase 4 (PADI4) gene and rheumatoid arthritis (RA) risk has been analyzed in several studies. However, the results are inconclusive and remain to be confirmed in several ethnic groups. The effect of the PADI4-104C/T polymorphism on RA risk in the Chinese population was evaluated in a meta-analysis. Studies with dates of publication up to July 2015 conforming to the inclusion criteria were retrieved from PubMed and Chinese databases. The associations were assessed with pooled odds ratios (ORs) and 95% confidence intervals (CIs). Ten studies, including 2119 RA cases and 1962 controls, that conformed to the study criteria were included in this analysis. The overall analysis indicated a significant association between the PADI4-104C/T polymorphism and RA risk in the Chinese population (T vs C: OR = 1.45, 95%CI = 1.18-1.78; TT vs CC: OR = 1.49, 95%CI = 1.24-1.80; TT vs CC+CT: OR = 1.28, 95%CI = 1.08-1.51; TT+CT vs CC: OR = 1.75, 95%CI = 1.30-2.37). Analysis of data stratified by the geographic area and source of controls revealed that the PADI4-104C/T polymorphism was significantly associated with RA risk in a North Chinese population. In conclusion, the results of this meta-analysis indicated that the PADI4-104C/T variants could influence the risk of RA in the Chinese population; further studies in other ethnic groups are required to draw definite conclusions.

Association between the PADI4 -94G/A polymorphism and rheumatoid arthritis: a meta-analysis in the Chinese population.

Although a number of studies have been conducted on the association between the peptidylarginine deiminase (PADI4) -94G/A polymorphism and rheumatoid arthritis (RA) in the Chinese population, the association remains elusive and controversial. To clarify the impact of the PADI4 -94G/A polymorphism on the risk of RA, a meta-analysis was performed in the Chinese population. Related studies were identified from databases such as, Springer Link, Ovid, Chinese Wanfang Data Knowledge Service Platform, Chinese National Knowledge Infrastructure (CNKI), and Chinese Biology Medicine (CBM) up to May 21, 2015. Pooled odds ratios (ORs) and 95% confidence intervals (CIs) were used to assess the strength of associations. A total of 10 studies with 2783 RA cases and 2887 controls were included in this meta-analysis. Overall, a significantly elevated risk of RA was associated with all variants of PADI4 -94G/A (A vs G: OR = 1.24, 95%CI = 1.15-1.34; AA + GA vs GG: OR = 1.45, 95%CI = 1.29-1.62; AA vs GG: OR = 1.49, 95%CI = 1.28-1.73; AA vs GG + GA: OR = 1.19, 95%CI = 1.04-1.35). Subgroup analyses stratified by geographic areas and source of controls revealed significant results in the population-based studies in North and South China. In conclusion, this meta-analysis showed that the PADI4 -94G/A variants may influence RA risk in the Chinese population. However, further studies with gene-gene and gene-environment interactions are required for definite conclusions.

The amount of citrullinated proteins in synovial tissue is related to serum anti-cyclic citrullinated peptide (anti-CCP) antibody levels.

The objective of this study was to determine the relationship between citrullinated proteins in synovial tissue with peripheral anti-citrullinated peptides autoantibodies (ACPA) and peptidylarginine deiminase (PADI) PADI2, PADI3, and PADI4 messenger RNA (mRNA) expressions in synovial tissue and fibroblast-like synoviocytes in rheumatoid arthritis (RA) patients. Eleven RA and 12 osteoarthritis (OA) patients who underwent knee replacement surgery were studied. We detected citrullinated proteins in synovial tissue homogenates by western blot and serum ACPA by ELISA to anti-cyclic citrullinated peptide (anti-CCP) antibodies, and PADI2, PADI3, and PADI4 mRNA expressions in synovial tissue and in fibroblast-like synoviocytes. Patients with high amount of citrullinated proteins in synovial tissue (3 out of 7) have high levels of anti-CCP in serum. However, in the remaining 4 patients, the amount of synovial citrullinated proteins was minimal and their sera showed low levels of anti-CCP antibodies. Furthermore, we observed an increase in PADI2 mRNA expression in RA synovial tissue compared with OA patients (p = 0.02). We detected PADI3 mRNA in the synovial tissue of RA patients, but not in the tissue of OA patients. Even though fibroblast-type synoviocytes in RA are not the main source of PADs in the synovial tissue, they express PADI2 mRNA moderately, PADI4 mRNA weakly, while there is no detectable expression of PADI3 mRNA. In conclusion, we found a variety of citrullinated proteins in the synovial tissue of RA patients and the amount of such proteins is related to serum concentration of anti-CCP antibodies. We identified the presence of PADI3 mRNA expression in synovial tissue and PADI2 and PADI4 mRNA expressions in fibroblast-like synoviocytes from patients with RA.

Polymorphisms and functional haplotype in PADI4: further evidence for contribution on rheumatoid arthritis susceptibility and anti-cyclic citrullinated peptide antibodies in a western Mexican population.

Peptidyl arginine deiminase IV (PADI4) enzyme catalyzes the citrullination of proteins, which are recognized by anti-cyclic citrullinated peptide antibodies (anti-CCP) in rheumatoid arthritis (RA) patients. Here, we determined the association between PADI4 gene polymorphisms and haplotypes with RA susceptibility and clinical characteristics in a western Mexican population. The relationship of PADI4 polymorphisms with anti-CCP and PADI4 mRNA expression was also evaluated. PADI4_89, PADI4_90 and PADI4_92 polymorphisms were individually associated with RA susceptibility. The GTG haplotype was significantly associated with: RA susceptibility; disease onset at ≤ 40 years and anti-CCP antibodies. PADI4 expression was three fold higher in RA patients carrying the susceptibility haplotype (GTG) than in non-susceptibility haplotype carriers (ACC). In conclusion, polymorphisms and functional haplotype (GTG) in PADI4 are associated with RA susceptibility as well as anti-CCP antibodies in a Mexican population. This supports the role of PADI4 early in RA pathogenesis by promoting the generation of citrullinated autoantigens.

PADI4 haplotypes in association with RA Mexican patients, a new prospect for antigen modulation.

Peptidyl arginine deiminase IV (PAD 4) is the responsible enzyme for a posttranslational modification called citrullination, originating the antigenic determinant recognized by anti-cyclic citrullinated peptide antibodies (ACPA). Four SNPs (single nucleotide polymorphisms) have been described in PADI4 gene to form a susceptibility haplotype for rheumatoid arthritis (RA); nevertheless, results in association studies appear contradictory in different populations. The aim of the study was to analyze if the presence of three SNPs in PADI4 gene susceptibility haplotype (GTG) is associated with ACPA positivity in patients with RA. This was a cross-sectional study that included 86 RA patients and 98 healthy controls. Polymorphisms PADI4_89, PADI4_90, and PADI4_92 in the PADI4 gene were genotyped. The susceptibility haplotype (GTG) was more frequent in RA patients; interestingly, we found a new haplotype associated with RA with a higher frequency (GTC). There were no associations between polymorphisms and high scores in Spanish HAQ-DI and DAS-28, but we did find an association between RARBIS index and PADI4_89, PADI4_90 polymorphisms. We could not confirm an association between susceptibility haplotype presence and ACPA positivity. Further evidence about proteomic expression of this gene will determine its participation in antigenic generation and autoimmunity.