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1.  Investigation of polymorphisms in the PADI4 gene in determining severity of inflammatory polyarthritis 
Annals of the Rheumatic Diseases  2005;64(9):1311-1315.
Background: A functional haplotype of the peptidylarginine deiminase 4 (PADI4) gene has recently been identified as a rheumatoid arthritis susceptibility gene in a Japanese but not in a UK population. One possible explanation for this disparity is that the gene determines severity of rather than susceptibility to inflammatory polyarthritis (IP) and that the UK and Japanese cohorts differed in terms of outcome.
Aim: To examine the association between individual PADI4 single nucleotide polymorphisms (SNPs) and haplotypes, with the development and severity of erosions by five years in patients with IP.
Methods: 438 patients from the NOAR inception cohort of patients with IP were x rayed five years after presentation with early IP. Association with four exonic SNPs (padi4_89*G/A, padi4_90*T/C, padi4_92*G/C, and padi4_104*T/C), mapping to the PADI4 gene and defining a haplotype previously reported to be associated with rheumatoid arthritis, was investigated. Patients were compared for the presence, extent, and progression of erosions by five years and the presence of antibodies to citrullinated peptide (anti-CCP antibodies).
Results: There was no association between individual PADI4 SNPs or haplotypes and the development or extent of erosions by five years. Restricting analysis to patients who satisfied ACR criteria for rheumatoid arthritis by five years did not alter the conclusions. No association with presence of anti-CCP antibodies was detected.
Conclusions: No evidence was found for association of the PADI4 gene with severity as assessed by erosive outcome at five years or with presence of anti-CCP antibodies in patients with IP.
doi:10.1136/ard.2004.034173
PMCID: PMC1755648  PMID: 15731287
2.  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.
doi:10.1155/2013/383681
PMCID: PMC3881379  PMID: 24454473
3.  Polymorphisms in peptidylarginine deiminase associate with rheumatoid arthritis in diverse Asian populations: evidence from MyEIRA study and meta-analysis 
Arthritis Research & Therapy  2012;14(6):R250.
Introduction
The majority of our knowledge regarding disease-related mechanisms of uncontrolled citrullination and anti-citrullinated protein antibody development in rheumatoid arthritis (RA) was investigated in Caucasian populations. However, peptidylarginine deiminase (PADI) type 4 gene polymorphisms are associated with RA in East Asian populations and weak or no association was found in Caucasian populations. This study explores the association between the PADI4 polymorphisms and RA risk in a multiethnic population residing in South East Asia with the goal of elucidating generalizability of association in non-Caucasian populations.
Methods
A total of 320 SNPs from the PADI locus (including PADI1, PADI2, PADI3, PADI4 and PADI6 genes) were genotyped in 1,238 RA cases and 1,571 control subjects from the Malaysian Epidemiological Investigation of Rheumatoid Arthritis (MyEIRA) case-control study. Additionally, we conducted meta-analysis of our data together with the previously published studies of RA from East Asian populations.
Results
The overall odds ratio (ORoverall) for the PADI4 (rs2240340) allelic model was 1.11 (95% confidence interval (CI) = 1.00 to 1.23, P = 0.04) and for the genotypic model was 1.20 (95% CI = 1.01 to 1.44, P = 0.04). Haplotype analysis for four selected PADI4 SNPs revealed a significant association of one with susceptibility (P = 0.001) and of another with a protective effect (P = 0.02). The RA susceptibility was further confirmed when combined meta-analysis was performed using these data together with data from five previously published studies from Asia comprising 5,192 RA cases and 4,317 control subjects (ORoverall = 1.23 (95% CI = 1.16 to 1.31, Pheterogeneity = 0.08) and 1.31 (95% CI = 1.20 to 1.44, Pheterogeneity = 0.32) in allele and genotype-based models, respectively). In addition, we also detected a novel association of PADI2 genetic variant rs1005753 with RA (ORoverall = 0.87 (95% CI = 0.77 to 0.99)).
Conclusion
Our study demonstrates an association between PADI4 and RA in the multiethnic population from South East Asia and suggests additional association with a PADI2 gene. The study thus provides further support for the notion that polymorphisms in genes for enzymes responsible for citrullination contribute to RA development in multiple populations of Asian descent.
doi:10.1186/ar4093
PMCID: PMC3674620  PMID: 23164236
4.  PADI4 genotype is not associated with rheumatoid arthritis in a large UK Caucasian population 
Annals of the Rheumatic Diseases  2010;69(4):666-670.
Background
Polymorphisms of the peptidylarginine deiminase type 4 (PADI4) gene confer susceptibility to rheumatoid arthritis (RA) in East Asian people. However, studies in European populations have produced conflicting results. This study explored the association of the PADI4 genotype with RA in a large UK Caucasian population.
Methods
The PADI4_94 (rs2240340) single nucleotide polymorphism (SNP) was directly genotyped in a cohort of unrelated UK Caucasian patients with RA (n=3732) and population controls (n=3039). Imputed data from the Wellcome Trust Case Control Consortium (WTCCC) was used to investigate the association of PADI4_94 with RA in an independent group of RA cases (n=1859) and controls (n=10 599). A further 56 SNPs spanning the PADI4 gene were investigated for association with RA using data from the WTCCC study.
Results
The PADI4_94 genotype was not associated with RA in either the present cohort or the WTCCC cohort. Combined analysis of all the cases of RA (n=5591) and controls (n=13 638) gave an overall OR of 1.01 (95% CI 0.96 to 1.05, p=0.72). No association with anti-CCP antibodies and no interaction with either shared epitope or PTPN22 was detected. No evidence for association with RA was identified for any of the PADI4 SNPs investigated. Meta-analysis of previously published studies and our data confirmed no significant association between the PADI4_94 genotype and RA in people of European descent (OR 1.06, 95% CI 0.99 to 1.13, p=0.12).
Conclusion
In the largest study performed to date, the PADI4 genotype was not a significant risk factor for RA in people of European ancestry, in contrast to Asian populations.
doi:10.1136/ard.2009.111294
PMCID: PMC2927647  PMID: 19470526
5.  A family based study shows no association between rheumatoid arthritis and the PADI4 gene in a white French population 
Annals of the Rheumatic Diseases  2004;64(4):587-593.
Background: Autoantibodies to citrullinated proteins (ACPA) are considered a specific marker for rheumatoid arthritis. Peptidylarginine deiminase (PAD) is the enzyme that converts arginyl into citrullyl residues; different isoforms of the enzyme are expressed in mammals. It has been suggested that the PADI4 gene may contribute to genetic susceptibility to rheumatoid arthritis, but conflicting results have been obtained in different populations.
Objective: To test the hypothesis that the PADI4 gene may confer susceptibility to rheumatoid arthritis in a white French population, using powerful and highly reliable family based association tests.
Methods: DNA samples were analysed from 100 families where one member was affected by rheumatoid arthritis and both parents were available for sampling. Five single nucleotide polymorphisms, located within the PADI4 gene and in its close proximity, were genotyped by restriction fragment length polymorphism, and haplotypes were constructed. The analysis involved use of the transmission disequilibrium test and genotype relative risk. ACPA were detected by ELISA on cyclic citrullinated peptides and on human deiminated fibrinogen.
Results: No single SNP or haplotype was associated with the disease, or was preferentially transmitted. No association was found when patients were partitioned according to ACPA positivity.
Conclusions: No PADI4 haplotype is associated with rheumatoid arthritis in a white French population. The role of genes encoding the other PAD isoforms, or modulating tissue expression or enzyme activity, remains to be elucidated.
doi:10.1136/ard.2004.026831
PMCID: PMC1755438  PMID: 15485997
6.  Peptidyl arginine deiminase type IV (PADI4) haplotypes interact with shared epitope regardless of anti-cyclic citrullinated peptide antibody or erosive joint status in rheumatoid arthritis: a case control study 
Arthritis Research & Therapy  2010;12(3):R115.
Introduction
Anti-cyclic citrullinated peptide autoantibodies (anti-CCP) are the most specific serologic marker for rheumatoid arthritis (RA). Genetic polymorphisms in a citrullinating (or deiminating) enzyme, peptidyl arginine deiminase type IV (PADI4) have been reproducibly associated with RA susceptibility in several populations. We investigated whether PADI4 polymorphisms contribute to anti-CCP-negative as well as -positive RA, whether they influence disease severity (erosive joint status), and whether they interact with two major risk factors for RA, Human Leukocyte Antigen-DRB1 (HLA-DRB1) shared epitope (SE) alleles and smoking, depending on anti-CCP and erosive joint status.
Methods
All 2,317 unrelated Korean subjects including 1,313 patients with RA and 1,004 unaffected controls were genotyped for three nonsynonymous (padi4_89, padi4_90, and padi4_92) and one synonymous (padi4_104) single-nucleotide polymorphisms (SNPs) in PADI4 and for HLA-DRB1 by direct DNA sequence analysis. Odds ratios (OR) were calculated by multivariate logistic regression. Interaction was evaluated by attributable proportions (AP), with 95% confidence intervals (CI).
Results
A functional haplotype of the three fully correlated nonsynonymous SNPs in PADI4 was significantly associated with susceptibility to not only anti-CCP-positive (adjusted OR 1.73, 95% CI 1.34 to 2.23) but also -negative RA (adjusted OR 1.75, 95% CI 1.15 to 2.68). A strong association with both non-erosive (adjusted OR 1.62, 95% CI 1.29 to 2.05) and erosive RA (adjusted OR 1.62, 95% CI 1.14 to 2.31) was observed for PADI4 haplotype. Gene-gene interactions between the homozygous RA-risk PADI4 haplotype and SE alleles were significant in both anti-CCP-positive (AP 0.45, 95% CI 0.20 to 0.71) and -negative RA (AP 0.61, 95% CI 0.29 to 0.92). Theses interactions were also observed for both non-erosive (AP 0.48, 95% CI 0.25 to 0.72) and erosive RA (AP 0.46, 95% CI 0.14 to 0.78). In contrast, no interaction was observed between smoking and PADI4 polymorphisms.
Conclusions
A haplotype of nonsynonymous SNPs in PADI4 contributes to development of RA regardless of anti-CCP or erosive joint status. The homozygous PADI4 haplotype contribution is affected by gene-gene interactions with HLA-DRB1 SE alleles.
doi:10.1186/ar3051
PMCID: PMC2911908  PMID: 20537173
7.  PADI2 Is Significantly Associated with Rheumatoid Arthritis 
PLoS ONE  2013;8(12):e81259.
Citrullination, a posttranslational modification of peptidyl arginine to citrulline, plays an essential role in rheumatoid arthritis (RA). Citrullination is catalyzed by a group of peptidylarginine deiminases (PADs) including PADI 1, 2, 3, 4 and 6. Many studies have indicated that the gene encoding PADI4 is a factor in susceptibility to RA. Some studies have detected PADI2 expression in RA synovial tissues, suggesting that PADI2 also plays an important role in the disease. This study evaluated the possible association between the PADI2-encoding gene and RA. Seventeen tag SNPs across the PAD locus were genotyped using a custom-designed Illumina 96-SNP VeraCode microarray. Peripheral blood samples were collected from patients with RA (n = 267), ankylosing spondylitis (AS, n = 51) and healthy controls (n = 160). The results of genotyping were verified using Sequenom MassARRAY in an independent cohort of 307 patients with RA, 324 patients with AS and 509 healthy controls. A western blot analysis was performed using synovial tissue from patients with RA (n = 7), osteoarthritis (OA, n = 7) and AS (n = 5) to determine the levels of expression of PADI2. A microarray analysis revealed a significant association between three selected PADI2 SNPs (rs2235926, rs2057094, rs2076616) and the presence of RA. The increased susceptibility to RA associated with rs2235926 (OR = 1.706733, 95% CI = [1.576366–1.866587], p = 0.000839) and rs2057094 (OR = 1.360432, 95% CI = [1.065483–1.869482], p = 0.003291) was further confirmed by the Sequenom MassARRAY. No tag SNPs in the PADI2 locus showed a significant association with AS. Increased expression of PADI2 was detected in RA synovial tissues compared with samples from patients with OA and AS. PADI2 is significantly associated with RA and may be involved in the pathogenesis of the disease.
doi:10.1371/journal.pone.0081259
PMCID: PMC3855321  PMID: 24339914
8.  Functional haplotypes of PADI4: relevance for rheumatoid arthritis specific synovial intracellular citrullinated proteins and anticitrullinated protein antibodies 
Annals of the Rheumatic Diseases  2005;64(9):1316-1320.
Background: Haplotypes of PADI4, encoding for a citrullinating enzyme, were associated with rheumatoid arthritis in a Japanese population. It was suggested they were related to the presence of anticitrullinated protein antibodies (ACPA).
Objective: To explore the relation between PADI4 haplotypes, the presence of rheumatoid arthritis specific intracellular citrullinated proteins in synovial membrane, and serum ACPA titres.
Methods: Synovial biopsies and peripheral blood samples were obtained in 59 patients with rheumatoid arthritis. Synovial intracellular citrullinated proteins were detected by immunohistochemistry. Serum ACPA titres were measured by anti-CCP2 ELISA. PADI4 haplotypes were determined by direct sequencing of the four exonic PADI4 single nucleotide polymorphisms.
Results: PADI4 haplotype frequencies and the presence of synovial intracellular citrullinated proteins and ACPA were comparable with previous studies. There was no significant association between PADI4 haplotype 1 or 2 and the presence of synovial intracellular citrullinated proteins, although these proteins were associated with higher serum ACPA. There was no correlation between PADI4 haplotypes and serum ACPA, either by continuous analysis using the titres or by dichotomous analysis using the diagnostic cut off. Further analyses in homozygotes for haplotype 1 or 2 or in heterozygotes (1/2) also failed to show an association between PADI4 polymorphisms and ACPA. This contrasted with the clear association between ACPA levels and HLA-DR shared epitope.
Conclusions: The link between synovial intracellular citrullinated proteins and ACPA emphasises the role of deimination of synovial proteins in rheumatoid arthritis, but the biological relevance of the PADI4 haplotypes for this autoimmune process is questionable, at least in a European population.
doi:10.1136/ard.2004.033548
PMCID: PMC1755666  PMID: 15760928
9.  Long-Range Enhancer Associated with Chromatin Looping Allows AP-1 Regulation of the Peptidylarginine Deiminase 3 Gene in Differentiated Keratinocyte 
PLoS ONE  2008;3(10):e3408.
Transcription control at a distance is a critical mechanism, particularly for contiguous genes. The peptidylarginine deiminases (PADs) catalyse the conversion of protein-bound arginine into citrulline (deimination), a critical reaction in the pathophysiology of multiple sclerosis, Alzheimer's disease and rheumatoid arthritis, and in the metabolism of the major epidermal barrier protein filaggrin, a strong predisposing factor for atopic dermatitis. PADs are encoded by 5 clustered PADI genes (1p35-6). Unclear are the mechanisms controlling the expression of the gene PADI3 encoding the PAD3 isoform, a strong candidate for the deimination of filaggrin in the terminally differentiating epidermal keratinocyte. We describe the first PAD Intergenic Enhancer (PIE), an evolutionary conserved non coding segment located 86-kb from the PADI3 promoter. PIE is a strong enhancer of the PADI3 promoter in Ca2+-differentiated epidermal keratinocytes, and requires bound AP-1 factors, namely c-Jun and c-Fos. As compared to proliferative keratinocytes, calcium stimulation specifically associates with increased local DNase I hypersensitivity around PIE, and increased physical proximity of PIE and PADI3 as assessed by Chromosome Conformation Capture. The specific AP-1 inhibitor nordihydroguaiaretic acid suppresses the calcium-induced increase of PADI3 mRNA levels in keratinocytes. Our findings pave the way to the exploration of deimination control during tumorigenesis and wound healing, two conditions for which AP-1 factors are critical, and disclose that long-range transcription control has a role in the regulation of the gene PADI3. Since invalidation of distant regulators causes a variety of human diseases, PIE results to be a plausible candidate in association studies on deimination-related disorders or atopic disease.
doi:10.1371/journal.pone.0003408
PMCID: PMC2566589  PMID: 18923650
10.  Phosphorylation-Dependent Interaction of Tyrosine 3-Monooxygenase/Tryptophan 5-Monooxygenase Activation Protein (YWHA) with PADI6 Following Oocyte Maturation in Mice1 
Biology of reproduction  2008;79(2):337-347.
Proteins in the tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein family (YWHA; also known as 14-3-3) are involved in the regulation of many intracellular processes. We have examined the interaction of YWHA with peptidylarginine deiminase type VI (PADI6), an abundant protein in mammalian oocytes, eggs, and early embryos. Peptidylarginine deiminases catalyze the posttranslational modification of peptidylarginine to citrulline. PADI6 is associated with oocyte cytoplasmic sheets, and PADI6-deficient mice are infertile because of disruption of development beyond the two-cell stage. We found that PADI6 undergoes a dramatic developmental change in phosphorylation during oocyte maturation. This change in phosphorylation is linked to an interaction of PADI6 with YWHA in the mature egg. Recombinant glutathione S-transferase YWHA pull-down experiments and transgenic tandem affinity purification with liquid chromatography-mass spectrometry demonstrate a binding interaction between YWHA and PADI6 in mature eggs. YWHA proteins modulate or complement intracellular events involving phosphorylation-dependent switching or protein modification. These results indicate that phosphorylation and/or YWHA binding may serve as a means of intracellular PADI6 regulation.
doi:10.1095/biolreprod.108.069328
PMCID: PMC2575841  PMID: 18463355
egg; gamete biology; gametogenesis; mouse; oocyte; oocyte development; oocyte maturation; ovum; PADI6; PAD6; peptidylarginine deiminase; phosphorylation; YWHA; 14-3-3
11.  Increased PADI4 expression in blood and tissues of patients with malignant tumors 
BMC Cancer  2009;9:40.
Background
Peptidylarginine deiminase type 4 (PAD4/PADI4) post-translationally converts peptidylarginine to citrulline. Recent studies suggest that PADI4 represses expression of p53-regulated genes via citrullination of histones at gene promoters.
Methods
Expression of PADI4 was investigated in various tumors and non-tumor tissues (n = 1673) as well as in A549, SKOV3 and U937 tumor cell lines by immunohistochemistry, real-time PCR, and western blot. Levels of PADI4 and citrullinated antithrombin (cAT) were investigated in the blood of patients with various tumors by ELISA (n = 1121).
Results
Immunohistochemistry detected significant PADI4 expression in various malignancies including breast carcinomas, lung adenocarcinomas, hepatocellular carcinomas, esophageal squamous cancer cells, colorectal adenocarcinomas, renal cancer cells, ovarian adenocarcinomas, endometrial carcinomas, uterine adenocarcinomas, bladder carcinomas, chondromas, as well as other metastatic carcinomas. However, PADI4 expression was not observed in benign leiomyomas of stomach, uterine myomas, endometrial hyperplasias, cervical polyps, teratomas, hydatidiform moles, trophoblastic cell hyperplasias, hyroid adenomas, hemangiomas, lymph hyperplasias, schwannomas, neurofibromas, lipomas, and cavernous hemangiomas of the liver. Additionally, PADI4 expression was not detected in non-tumor tissues including cholecystitis, cervicitis and synovitis of osteoarthritis, except in certain acutely inflamed tissues such as in gastritis and appendicitis. Quantitative PCR and western blot analysis showed higher PADI4 expression in gastric adenocarcinomas, lung adenocarcinomas, hepatocellular carcinomas, esophageal squamous cell cancers and breast cancers (n = 5 for each disease) than in the surrounding healthy tissues. Furthermore, western blot analysis detected PADI4 expression in cultured tumor cell lines. ELISA detected increased PADI4 and cAT levels in the blood of patients with various malignant tumors compared to those in patients with chronic inflammation and benign tumors. This was consistent with immunohistochemical results. Additionally, PADI4 and cAT levels were significantly associated with higher levels of known tumor markers.
Conclusion
Our results suggest that PADI4 expression is increased in the blood and tissues of many malignant tumors, a finding useful for further understanding of tumorigenesis.
doi:10.1186/1471-2407-9-40
PMCID: PMC2637889  PMID: 19183436
12.  Expression of Peptidylarginine Deiminase Type 4 in Ovarian Tumors 
Peptidylarginine deiminase type 4 (PADI4) converts arginine residues into citrulline. The current study focused on the expression of PADI4 in various subtypes of ovary cancers, and this study investigated the effects of estrogen on PADI4 expression in SKOV-3 cells that originated from ovary tumors. We utilized immunohistochemistry, real-time PCR and western blotting to analyze the expression of PADI4 in the tumor tissues and in the cell line that were cultured with estrodial-17β. PADI4 was detected in serious cystadenocarcinoma (n=39, positivity=100%), clear cell cancer (n=7, positivity= 100%), mucinous cystadenocarcinoma (n=6, positivity=100%), dysgerminoma (n=6, positivity=100%), squamous cell tumor (n=6, positivity=100%), sibnet-ring cell carcinoma (n=6, positivity=100%), endodermal sinus tumor (n=6, positivity=100%), germ cell tumors (n=6, positivity=100%) and immature teratoma (n=6, positivity=100%). However, PADI4 was either not detected or detected at low levels in granulosa cell tumor (n=6), malignant thecoma (n=6), ovarian cystadenoma (n=5) and normal ovarian tissue (n=11). For serious cystadenocarcinoma, all of the samples with high PADI4 expression belonged to the T1 and T2 stages of pTMN, whereas all of the samples that exhibited weak or moderate PADI4 expression belonged to the T3 and T4 stages. PADI4 was evenly distributed in the cytoplasm of tumor cells of serious cystadenocarcinoma that were classified as being grade II and III by histopathological scoring. However, PADI4 showed granular cellular distribution in the tumor tissues that were isolated from grade I cystadenocarcinoma. In addition, the PADI4 level was positively related with the ages of the patients that presented with serious adenocarcinoma (p=0.029). Real-time PCR and western blot analyses confirmed that PADI4 was expressed at higher levels in ovarian adenocarcinoma (n=8) compared to ovarian cystadenoma (n=5) (p< 0.05). The study also detected an increased level of PADI4 in SKOV-3 cells that were incubated with estrodial-17β in the range of 10-12 to 10-4M. The results suggest an important role for PADI4 in the tumorigenesis of ovary cancers that are under the regulation of estrogen.
PMCID: PMC2935668  PMID: 20827398
Peptidylarginine deiminase type 4 (PADI4/PAD4); ovarian cancer (OCa); estrodial-17β.
13.  Investigating the Pathogenic Role of PADI4 in Oesophageal Cancer 
PADI4 post-translationally converts peptidylarginine to citrulline. PADI4 can disrupt the apoptotic process via the citrullination of histone H3 in the promoter of p53-target genes. The current study focused on PADI4 expression in various subtypes of oesophageal carcinoma (EC) by immunohistochemistry, western blotting and real time PCR. The study also investigated the effect of bile acid deoxycholate (DCA) on PADI4 expression in Eca-109 cells that originated from EC. Apoptosis and DCA-induced toxicity were analyzed by TUNEL, MTT assay and flow cytometry. Additionally, the present study investigated the correlation between single nucleotide polymorphism (SNP) in PADI4 gene and EC risk in Chinese population using Illumina GoldenGate assay. Compared with paraneoplastic tissues, the transcriptional and translational levels of PADI4 were significantly elevated in oesophageal squamous cell carcinoma (ESCC, n=9) and oesophageal adenocarcinoma (EAC, n=5) tissues. Immunolabeling detected expression of PADI4 in ESCC tissues (98.56%, n=139), EAC samples (87.5%, n=16) and oesophageal small cell undifferentiated carcinoma (91.7%, n=12) but not in normal tissues (0%, n=16). Furthermore, PADI4 levels is positively correlated with the pathological classification of ESCC (p=0.009). PADI4 expression levels were consistent with the number of apoptotic cells in the induced Eca-109 cells. rs10437048 [OR= 0.012831; 95% CI, 0.001746~0.094278; p=1.556×10-12] were significantly associated with decreased risk of EC, whereas rs41265997 [OR=12.7; 95% CI, 0.857077~33.207214; p=3.896×10-8] were significantly associated with increased risk of EC. rs41265997 in exon 3 of PADI4 gene is non-synonymous and converts ACG to ATG resulting in a threonine /methionine conversion at position 274 of the protein. Haplotypes GC that carries the variant alleles for rs2501796 and rs2477134 was significantly associated with increased risk of EC (frequency=0.085, p=0.0256, OR=2.7). The results suggest that PADI4 expression is related to the tumorigenic process of EC and the DCA-induced apoptosis. The PADI4 gene may be a valid EC susceptibility gene.
PMCID: PMC3119849  PMID: 21698003
Peptidyl arginine deaminase type 4 (PADI4/PAD4); oesophageal squamous cell carcinoma (ESCC); oesophageal adenocarcinoma (EAC); deoxycholate (DCA); apoptosis; genotyping; susceptibility; haplotype.
14.  Genome-Wide Analysis Reveals PADI4 Cooperates with Elk-1 to Activate c-Fos Expression in Breast Cancer Cells 
PLoS Genetics  2011;7(6):e1002112.
Peptidylarginine deiminase IV (PADI4) catalyzes the conversion of positively charged arginine and methylarginine residues to neutrally charged citrulline, and this activity has been linked to the repression of a limited number of target genes. To broaden our knowledge of the regulatory potential of PADI4, we utilized chromatin immunoprecipitation coupled with promoter tiling array (ChIP-chip) analysis to more comprehensively investigate the range of PADI4 target genes across the genome in MCF-7 breast cancer cells. Results showed that PADI4 is enriched in gene promoter regions near transcription start sites (TSSs); and, surprisingly, this pattern of binding is primarily associated with actively transcribed genes. Computational analysis found potential binding sites for Elk-1, a member of the ETS oncogene family, to be highly enriched around PADI4 binding sites; and coimmunoprecipitation analysis then confirmed that Elk-1 physically associates with PADI4. To better understand how PADI4 may facilitate gene transactivation, we then show that PADI4 interacts with Elk-1 at the c-Fos promoter and that, following Epidermal Growth Factor (EGF) stimulation, PADI4 catalytic activity facilitates Elk-1 phosphorylation, histone H4 acetylation, and c-Fos transcriptional activation. These results define a novel role for PADI4 as a transcription factor co-activator.
Author Summary
Peptidylarginine deiminase 4 (PADI4) converts positively charged arginine and methylarginine residues on histones to the neutrally charged non-standard amino acid citrulline. We and others have previously shown that citrullination of a small subset of gene promoters, such as the estrogen receptor target, TFF1, appears to downregulate gene expression. In this study, we looked across the human genome using ChIP-chip to better define the full repertoire of genes that are regulated by PADI4 in breast cancer cells. Surprisingly, we found that PADI4 appears to primarily be involved in gene activation, as opposed to gene repression. Further, we found that PADI4 is likely recruited as a co-activator to these target genes by a range of well-defined transcription factors, such as Elk-1. With respect to how PADI4 activates gene transcription, we show that PADI4 directly interacts with Elk-1 at its well-defined target, the c-Fos oncogene. Additionally, we found that, following stimulation with epidermal growth factor, PADI4 appears to directly target Elk-1 for citrullination, which in turn leads to increased histone acetylation and gene transcription. These novel genome-wide and gene-specific findings suggest that PADI4 plays a much broader role in gene activation than previously thought.
doi:10.1371/journal.pgen.1002112
PMCID: PMC3107201  PMID: 21655091
15.  Functional Connection between Deimination and Deacetylation of Histones▿  
Molecular and Cellular Biology  2009;29(18):4982-4993.
Histone methylation plays key roles in regulating chromatin structure and function. The recent identification of enzymes that antagonize or remove histone methylation offers new opportunities to appreciate histone methylation plasticity in the regulation of epigenetic pathways. Peptidylarginine deiminase 4 (PADI4; also known as PAD4) was the first enzyme shown to antagonize histone methylation. PADI4 functions as a histone deiminase converting a methylarginine residue to citrulline at specific sites on the tails of histones H3 and H4. This activity is linked to repression of the estrogen-regulated pS2 promoter. Very little is known as to how PADI4 silences gene expression. We show here that PADI4 associates with the histone deacetylase 1 (HDAC1). Kinetic chromatin immunoprecipitation assays revealed that PADI4 and HDAC1, and the corresponding activities, associate cyclically and coordinately with the pS2 promoter during repression phases. Knockdown of HDAC1 led to decreased H3 citrullination, concomitantly with increased histone arginine methylation. In cells with a reduced HDAC1 and a slightly decreased PADI4 level, these effects were more pronounced. Our data thus suggest that PADI4 and HDAC1 collaborate to generate a repressive chromatin environment on the pS2 promoter. These findings further substantiate the “transcriptional clock” concept, highlighting the dynamic connection between deimination and deacetylation of histones.
doi:10.1128/MCB.00285-09
PMCID: PMC2738279  PMID: 19581286
16.  Identification of PADI2 as a potential breast cancer biomarker and therapeutic target 
BMC Cancer  2012;12:500.
Background
We have recently reported that the expression of peptidylarginine deiminase 2 (PADI2) is regulated by EGF in mammary cancer cells and appears to play a role in the proliferation of normal mammary epithelium; however, the role of PADI2 in the pathogenesis of human breast cancer has yet to be investigated. Thus, the goals of this study were to examine whether PADI2 plays a role in mammary tumor progression, and whether the inhibition of PADI activity has anti-tumor effects.
Methods
RNA-seq data from a collection of 57 breast cancer cell lines was queried for PADI2 levels, and correlations with known subtype and HER2/ERBB2 status were evaluated. To examine PADI2 expression levels during breast cancer progression, the cell lines from the MCF10AT model were used. The efficacy of the PADI inhibitor, Cl-amidine, was tested in vitro using MCF10DCIS cells grown in 2D-monolayers and 3D-spheroids, and in vivo using MCF10DCIS tumor xenografts. Treated MCF10DCIS cells were examined by flow-cytometry to determine the extent of apoptosis and by RT2 Profiler PCR Cell Cycle Array to detect alterations in cell cycle associated genes.
Results
We show by RNA-seq that PADI2 mRNA expression is highly correlated with HER2/ERBB2 (p = 2.2 × 106) in luminal breast cancer cell lines. Using the MCF10AT model of breast cancer progression, we then demonstrate that PADI2 expression increases during the transition of normal mammary epithelium to fully malignant breast carcinomas, with a strong peak of PADI2 expression and activity being observed in the MCF10DCIS cell line, which models human comedo-DCIS lesions. Next, we show that a PADI inhibitor, Cl-amidine, strongly suppresses the growth of MCF10DCIS monolayers and tumor spheroids in culture. We then carried out preclinical studies in nude (nu/nu) mice and found that Cl-amidine also suppressed the growth of xenografted MCF10DCIS tumors by more than 3-fold. Lastly, we performed cell cycle array analysis of Cl-amidine treated and control MCF10DCIS cells, and found that the PADI inhibitor strongly affects the expression of several cell cycle genes implicated in tumor progression, including p21, GADD45α, and Ki67.
Conclusion
Together, these results suggest that PADI2 may function as an important new biomarker for HER2/ERBB2+ tumors and that Cl-amidine represents a new candidate for breast cancer therapy.
doi:10.1186/1471-2407-12-500
PMCID: PMC3571905  PMID: 23110523
Peptidylarginine deiminase; PAD2/PADI2; HER2/ERBB2; Breast cancer; Luminal; Cl-amidine; Citrullination
17.  FCRL3 promoter 169 CC homozygosity is associated with susceptibility to rheumatoid arthritis in Dutch Caucasians 
Annals of the Rheumatic Diseases  2006;66(6):803-806.
Background
Human leucocyte antigen is the only genetic risk factor for rheumatoid arthritis (RA) that has been consistently observed in different populations. A number of other genes such as PTPN22 and PADI4 showed population‐specific association with RA susceptibility. Recently, Fc receptor‐like 3 (FCRL3) gene was found to be associated with RA susceptibility in Japanese, but with conflicting results in other populations.
Objective
To investigate the association of FCRL3 polymorphism with RA susceptibility and severity in Dutch Caucasian patients with RA, as well as to perform a meta‐analysis to reveal the contribution of this gene to RA susceptibility.
Methods
A total of 931 Dutch RA cases and 570 unrelated Dutch controls were genotyped for four FCRL3 single‐nucleotide polymorphisms (SNPs). Genotyping was performed using the MassArray matrix‐assisted laser desorption ionisation‐time‐of‐flight mass spectrometry. Association of the FCRL3 SNPs with susceptibility to RA was examined by single‐marker, carrier and haplotype analysis.
Results
Carrier analysis of the SNP (rs7528684) revealed the association of CC genotype with a higher risk of developing RA as compared with TT and TC carriers (p = 0.039 and OR = 1.31). There was no significant difference in the genotype and allele frequencies of all investigated SNPs between cases and controls. Meta‐analysis of all studies comparing 9467 individuals showed that the OR for the CC genotype to develop RA was 1.2 and the p value <0.001.
Conclusion
A promoter polymorphism of FCRL3 (rs7528684) is associated with an increased risk of developing RA in Dutch Caucasians, suggesting that this association is relevant for RA in both Japanese and Caucasian populations.
doi:10.1136/ard.2006.064949
PMCID: PMC1954681  PMID: 17179172
18.  PADI4 and tumourigenesis 
PADI4 post-translationally converts peptidylarginine to citrulline, a process called citrullination. Studies have demonstrated the high expression of PADI4 in various malignant tumour tissues. PADI4 is also expressed at high levels in the blood of patients with some malignant tumours. Thus far, citrullination of histone, cytokeratin, antithrombin and fibronectin have been confirmed to be involved in abnormal apoptosis, high coagulation, and disordered cell proliferation and differentiation, all of which are main features of malignant tumours. PADI4 is expressed in CD34+ stem cells in normal tissues, and many more CD34+ cells expressing PADI4 are present in tumour tissues. These findings suggest that PADI4 may play an important role in tumourigenesis.
doi:10.1186/1475-2867-10-7
PMCID: PMC2845578  PMID: 20222985
19.  Potential role for PADI-mediated histone citrullination in preimplantation development 
Background
The peptidylarginine deiminases (PADIs) convert positively charged arginine residues to neutrally charged citrulline on protein substrates in a process that is known as citrullination or deimination. Previous reports have documented roles for histone citrullination in chromatin remodeling and gene regulation in several tissue types, however, a potential role for histone citrullination in chromatin-based activities during early embryogenesis has not been investigated.
Results
In the present study, we tested by laser scanning confocal indirect immunofluorescence microscopy whether specific arginine residues on the histone H3 and H4 N-terminal tails (H4R3, H3R2 + 8 + 17, and H3R26) were citrullinated in mouse oocytes and preimplantation embryos. Results showed that all of the tested residues were deiminated with each site showing a unique localization pattern during early development. Given these findings, we next tested whether inhibition of PADI activity using the PADI-specific inhibitor, Cl-amidine, may affect embryonic development. We found that treatment of pronuclear stage zygotes with Cl-amidine reduces both histone H3 and H4 tail citrullination and also potently blocks early cleavage divisions in vitro. Additionally, we found that the Cl-amidine treatment reduces acetylation at histone H3K9, H3K18, and H4K5 while having no apparent effect on the repressive histone H3K9 dimethylation modification. Lastly, we found that treatment of zygotes with trichostatin A (TSA) to induce hyperacetylation also resulted in an increase in histone citrullination at H3R2 + 8 + 17.
Conclusions
Given the observed effects of Cl-amidine on embryonic development and the well documented correlation between histone acetylation and transcriptional activation, our findings suggest that histone citrullination may play an important role in facilitating gene expression in early embryos by creating a chromatin environment that is permissive for histone acetylation.
doi:10.1186/1471-213X-12-19
PMCID: PMC3430579  PMID: 22712504
Peptidylarginine deiminase; Citrullination; Histone modification; Preimplantation development; Cl-amidine
20.  Multiple Marker Detection in Peripheral Blood for NSCLC Diagnosis 
PLoS ONE  2013;8(2):e57401.
Background
Non-invasive early detection of lung cancer could reduce the number of patients diagnosed with advanced disease, which is associated with a poor prognosis. We analyzed the diagnostic accuracy of a panel of peripheral blood markers in detecting non small cell lung cancer (NSCLC).
Methods
100 healthy donors and 100 patients with NSCLC were enrolled onto this study. Free circulating DNA, circulating mRNA expression of peptidylarginine deiminase type 4 (PAD4/PADI4), pro-platelet basic protein (PPBP) and haptoglobin were evaluated using a Real-Time PCR-based method.
Results
Free circulating DNA, PADI4, PPBP and haptoglobin levels were significantly higher in NSCLC patients than in healthy donors (p<0.0001, p<0.0001, p = 0.0002 and p = 0.0001, respectively). The fitted logistic regression model demonstrated a significant direct association between marker expression and lung cancer risk. The odds ratios of individual markers were 6.93 (95% CI 4.15–11.58; p<0.0001) for free DNA, 6.99 (95% CI 3.75–13.03; p<0.0001) for PADI4, 2.85 (95% CI 1.71–4.75; p<0.0001) for PPBP and 1.16 (95% CI 1.01–1.33; p = 0.031) for haptoglobin. Free DNA in combination with PPBP and PADI4 gave an area under the ROC curve of 0.93, 95% CI = 0.90–0.97, with sensitivity and specificity over 90%.
Conclusions
Free circulating DNA analysis combined with PPBP and PADI4 expression determination appears to accurately discriminate between healthy donors and NSCLC patients. This non-invasive multimarker approach warrants further research to assess its potential role in the diagnostic or screening workup of subjects with suspected lung cancer.
doi:10.1371/journal.pone.0057401
PMCID: PMC3582604  PMID: 23468981
21.  Pharmacogenetics of rheumatoid arthritis: Potential targets from susceptibility genes and present therapies 
Rheumatoid arthritis (RA) is a chronic heterogeneous autoimmune disorder of unknown etiology resulting in inflammation in the synovium, cartilage, and bone. Genetic factors play an important role in susceptibility to RA as the heritability of RA is between 50% and 60%, with the human leukocyte antigen (HLA) locus accounting for at least 30% of overall genetic risk. Outside the major histocompatibility complex (MHC) region, six additional risk loci have been identified and validated including PTPN22, STAT4, PADI4, CTLA4, TNFAIP3-OLIG3, and TRAF1/C5. Genetic factors are also important in RA pharmacotherapy due to the gene-dependent activity of enzymes involved in the pharmacokinetics and/or pharmacodynamics of RA medications. Indeed, there is great variability in drug efficacy as well as adverse events associated with any anti-rheumatic therapy and genetics is thought to contribute significantly to this inter-individual variability in response. This review will summarize the genetic factors that have been implicated in the pathogenesis of RA, and how these determinants may factor into the potential pharmacogenetics of this disease. We will also review the therapeutic agents that are currently being utilized or presently being evaluated in the treatment of RA, along with potential pharmacogenetic markers that have been proposed for such medications.
PMCID: PMC3513198  PMID: 23226040
rheumatoid arthritis; susceptibility genes; pharmacogenetics
22.  Role for PADI6 and the CPLs in Ribosomal Storage in Oocytes and Translation in the Early Embryo 
Development (Cambridge, England)  2008;135(15):2627-2636.
The mechanisms mediating the establishment of totipotency during the egg-to-embryo transition in mammals remain poorly understood. The cytoplasmic lattices (CPLs), a structure unique to mammalian oocytes and preimplantation embryos, have long been predicted to function as a storage form for the maternal contribution of ribosomes and mRNA to the early embryo. Here, we demonstrate that oocyte- and embryo-restricted peptidylarginine deiminase 6 (PADI6) is required for incorporation of ribosomal components into the oocyte CPLs. Next, we show that the abundance and localization of ribosomal components is dramatically affected in PADI6-/- two-cell embryos and that de novo protein synthesis is also disregulated in these embryos. Finally, we demonstrate that embryonic genome activation (EGA) is defective in PADI6 -/- two-cell embryos. These results suggest that, in mammals, ribosomal components are stored in the oocyte CPLs and are required for protein translation during early development.
doi:10.1242/dev.016329
PMCID: PMC2708103  PMID: 18599511
23.  Rheumatoid arthritis-associated gene-gene interaction network for rheumatoid arthritis candidate genes 
BMC Proceedings  2009;3(Suppl 7):S75.
Rheumatoid arthritis (RA, MIM 180300) is a chronic and complex autoimmune disease. Using the North American Rheumatoid Arthritis Consortium (NARAC) data set provided in Genetic Analysis Workshop 16 (GAW16), we used the genotype-trait distortion (GTD) scores and proposed analysis procedures to capture the gene-gene interaction effects of multiple susceptibility gene regions on RA. In this paper, we focused on 27 RA candidate gene regions (531 SNPs) based on a literature search. Statistical significance was evaluated using 1000 permutations. HLADRB1 was found to have strong marginal association with RA. We identified 14 significant interactions (p < 0.01), which were aggregated into an association network among 12 selected candidate genes PADI4, FCGR3, TNFRSF1B, ITGAV, BTLA, SLC22A4, IL3, VEGF, TNF, NFKBIL1, TRAF1-C5, and MIF. Based on our and other contributors' findings during the GAW16 conference, we further studied 24 candidate regions with 336 SNPs. We found 23 significant interactions (p-value < 0.01), nine interactions in addition to our initial findings, and the association network was extended to include candidate genes HLA-A, HLA-B, HLA-C, CTLA4, and IL6. As we will discuss in this paper, the reported possible interactions between genes may suggest potential biological activities of RA.
PMCID: PMC2795977  PMID: 20018070
24.  Citrullination of Histone H3 Interferes with HP1-Mediated Transcriptional Repression 
PLoS Genetics  2012;8(9):e1002934.
Multiple Sclerosis (MS) is an autoimmune disease associated with abnormal expression of a subset of cytokines, resulting in inappropriate T-lymphocyte activation and uncontrolled immune response. A key issue in the field is the need to understand why these cytokines are transcriptionally activated in the patients. Here, we have examined several transcription units subject to pathological reactivation in MS, including the TNFα and IL8 cytokine genes and also several Human Endogenous RetroViruses (HERVs). We find that both the immune genes and the HERVs require the heterochromatin protein HP1α for their transcriptional repression. We further show that the Peptidylarginine Deiminase 4 (PADI4), an enzyme with a suspected role in MS, weakens the binding of HP1α to tri-methylated histone H3 lysine 9 by citrullinating histone H3 arginine 8. The resulting de-repression of both cytokines and HERVs can be reversed with the PADI-inhibitor Cl-amidine. Finally, we show that in peripheral blood mononuclear cells (PBMCs) from MS patients, the promoters of TNFα, and several HERVs share a deficit in HP1α recruitment and an augmented accumulation of histone H3 with a double citrulline 8 tri-methyl lysine 9 modifications. Thus, our study provides compelling evidence that HP1α and PADI4 are regulators of both immune genes and HERVs, and that multiple events of transcriptional reactivation in MS patients can be explained by the deficiency of a single mechanism of gene silencing.
Author Summary
In patients suffering from Multiple Sclerosis (MS), T lymphocytes express abnormally high levels of a subset of cytokines. The same cells also transcribe a series of vestigial retroviral sequences normally silenced by chromatin factors. In this study, we have searched for regulatory mechanisms shared between the cytokines and the retroviral sequences. We find that the repressor protein HP1α is present on the promoter of both types of transcription units in normal cells and that the recruitment of this protein to these promoters is decreased in MS patients. Furthermore, we show that the delocalization of HP1α from these promoters can be caused by citrullination of histone H3, and we provide evidence indicating that levels of this histone modification is augmented in MS patients. Together our data provide a possible explanation for the simultaneously increased transcriptional activity of cytokines and endogenous retroviruses in MS-patient T lymphocytes and suggest that inhibitors of the enzyme responsible for the increased citrullination of histone H3 could help restore normal levels of cytokine activity in the patients.
doi:10.1371/journal.pgen.1002934
PMCID: PMC3441713  PMID: 23028349
25.  Analysis of polymorphisms in 16 genes in type 1 diabetes that have been associated with other immune-mediated diseases 
BMC Medical Genetics  2006;7:20.
Background
The identification of the HLA class II, insulin (INS), CTLA-4 and PTPN22 genes as determinants of type 1 diabetes (T1D) susceptibility indicates that fine tuning of the immune system is centrally involved in disease development. Some genes have been shown to affect several immune-mediated diseases. Therefore, we tested the hypothesis that alleles of susceptibility genes previously associated with other immune-mediated diseases might perturb immune homeostasis, and hence also associate with predisposition to T1D.
Methods
We resequenced and genotyped tag single nucleotide polymorphisms (SNPs) from two genes, CRP and FCER1B, and genotyped 27 disease-associated polymorphisms from thirteen gene regions, namely FCRL3, CFH, SLC9A3R1, PADI4, RUNX1, SPINK5, IL1RN, IL1RA, CARD15, IBD5-locus (including SLC22A4), LAG3, ADAM33 and NFKB1. These genes have been associated previously with susceptibility to a range of immune-mediated diseases including rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), Graves' disease (GD), psoriasis, psoriatic arthritis (PA), atopy, asthma, Crohn disease and multiple sclerosis (MS). Our T1D collections are divided into three sample subsets, consisting of set 1 families (up to 754 families), set 2 families (up to 743 families), and a case-control collection (ranging from 1,500 to 4,400 cases and 1,500 to 4,600 controls). Each SNP was genotyped in one or more of these subsets. Our study typically had approximately 80% statistical power for a minor allele frequency (MAF) >5% and odds ratios (OR) of 1.5 with the type 1 error rate, α = 0.05.
Results
We found no evidence of association with T1D at most of the loci studied 0.02

Conclusion
Polymorphisms in a variety of genes previously associated with immune-mediated disease susceptibility and/or having effects on gene function and the immune system, are unlikely to be affecting T1D susceptibility in a major way, even though some of the genes tested encode proteins of immune pathways that are believed to be central to the development of T1D. We cannot, however, rule out effect sizes smaller than OR 1.5.
doi:10.1186/1471-2350-7-20
PMCID: PMC1420277  PMID: 16519819

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