Reduced Anti-Histone Antibodies and Increased Risk of Rheumatoid Arthritis Associated with a Single Nucleotide Polymorphism in PADI4 in North Americans

Abstract

Autoantibodies against citrullinated proteins are a hallmark of rheumatoid arthritis, a destructive inflammatory arthritis. Peptidylarginine deiminase 4 (PAD4) has been hypothesized to contribute to rheumatoid arthritis by citrullinating histones to induce neutrophil extracellular traps (NETs), which display citrullinated proteins that are targeted by autoantibodies to drive inflammation and arthritis. Consistent with this theory, PAD4-deficient mice have reduced NETs, autoantibodies, and arthritis. However, PAD4′s role in human rheumatoid arthritis is less clear. Here, we determine if single nucleotide polymorphism rs2240335 in PADI4, whose G allele is associated with reduced PAD4 in neutrophils, correlates with NETs, anti-histone antibodies, and rheumatoid arthritis susceptibility in North Americans. Control and rheumatoid arthritis subjects, divided into anti-cyclic citrullinated peptide (CCP) antibody positive and negative groups, were genotyped at rs2240335. In homozygotes, in vitro NETosis was quantified in immunofluorescent images and circulating NET and anti-histone antibody levels by enzyme linked immunosorbent assay (ELISA). Results were compared by t-test and correlation of rheumatoid arthritis diagnosis with rs2240335 by Armitage trend test. NET levels did not significantly correlate with genotype. G allele homozygotes in the CCP− rheumatoid arthritis group had reduced anti-native and anti-citrullinated histone antibodies. However, the G allele conferred increased risk for rheumatoid arthritis diagnosis, suggesting a complex role for PAD4 in human rheumatoid arthritis.

PADI4 Polymorphisms Confer Risk of Anti-CCP-Positive Rheumatoid Arthritis in Synergy With HLA-DRB1*04 and Smoking

“…The association was largely driven by the North American cohort, in accordance with a previous study showing association with rs2240340, which is in high LD with the SNP in our study (31). Furthermore, the minor allele (A) of the exon-synonymous SNP rs2240335 was associated with a decreased risk of anti-CCP-positive RA with an OR of 0.82, in agreement with previous results (35).…”

“…Written informed consent was obtained from all study subjects, and the study was approved by the Scientific Ethical Committees for Copenhagen and Frederiksberg (KF 01-039/01), the Danish Data Protection Agency (2001–41–0658), and the Institutional Review Board at Statens Serum Institut (21–00050). The North American cohort included 200 RA patients and 100 age- and sex-matched controls without autoimmune disease selected from the University of Wisconsin (UW) Rheumatology Biorepository described in ( 35 , 41 ) ( Table 1 ). The RA patients were initially identified by having at least two outpatient visits with RA-associated ICD codes within 24 months ( 42 ), or one visit and a positive anti-CCP test.…”

“…The haplotype consists of the minor alleles of rs11203366, rs11203367, and rs874881 encoding the amino acid substitutions Gly55Ser, Val82Ala, and Gly112Ala, respectively, and rs1748033 which is a synonymous SNP ( Figure 1 ). Besides, the minor allele (A) of the synonymous SNP rs2240035 has been shown to decrease the risk of RA in Asians and North Americans ( 26 , 27 , 35 ). Together with rs74058715, which is located in the 5’ untranslated region (UTR) of PADI4 , rs2240035 has been shown to regulate PAD4 expression levels in neutrophils and monocytes ( 36 ).…”

Analysis of Polymorphisms rs7093069-IL-2RA, rs7138803-FAIM2, and rs1748033-PADI4 in the Group of Adolescents With Autoimmune Thyroid Diseases

“…Peptidylarginine deiminase 4 (PADI4) catalyzes the citrullines of histones and thus regulates the maintenance of stem cells. This process results in extracellular neutrophil traps (NETs), which as citrullinated proteins, are the target of autoantibodies in the treatment of inflammation and arthritis (31). PADI4 was found in the differentiation of macrophages and its role in inflammatory reactions (32).…”

Neutrophil Extracellular Traps in Autoimmunity and Allergy: Immune Complexes at Work

“…Citrullination is a physiological process that occurs in inflammation and during NETosis, due to the activation of PAD4 (66–69). PAD4 activity contributes to RA development, since PAD4-deficient mice have reduced autoantibodies and joint damage in arthritis models (70, 71), and a single nucleotide polymorphism of PAD4 is associated to an increased risk to develop RA in humans (72). Neutrophils from RA patients are activated and produce spontaneously more NETs than healthy donors (73).…”