Background/Purpose: Anti-citrullinated protein antibodies (ACPA) are frequently detected in Rheumatoid Arthritis (RA) patients and used as diagnostic biomarkers at a very early stage of the disease. ACPAs are raised against various peptide epitopes carrying citrullinated arginines generated by peptidyl arginine deiminase (PADI) enzyme family members. PADI4 gene is highly expressed in neutrophils in peripheral blood and also known to citrullinate histone proteins. RA-associated PADI4 single nucleotide polymorphisms (SNPs) do not alter amino acid composition; therefore, it is challenging to explore how they can contribute to RA pathology. We investigated the effect of SNP rs2240335 on transcriptional regulation of PADI4 gene.

Methods: We have investigated the epigenetic landscape of the human chromosome region harboring the PADI4 gene and focused on histone modifications that predict active promoters. Using 5’RACE method we explored alternative isoform-encoding transcript of PADI4 gene. Quantitative reverse transcription PCR was used to explore isoform expression in human tissues. Cellular distribution of a novel short PADI4 isoform was studied by confocal fluorescence microscopy. Short PADI4 isoform’s potential catalytic function was tested using in vitro transcription and translation system. The disease-associated SNP’s effect on transcription was assessed in transient expression studies.

Results: Epigenetic profile analysis around the PADI4 gene revealed a new promoter region, which proved to be active on bone marrow. We cloned and sequenced the corresponding transcript and designated PADI4-ΔN. PADI4-ΔN is a truncated isoform that only encodes the C-terminal catalytic domain of the enzyme, which is highly expressed in bone marrow sample and neutrophils. PADI4-ΔN was fused to red fluorescent protein (RFP) and overexpressed in cells. Confocal microscopic studies detected the RFP:PADI4-ΔN in cytoplasmic region and nuclei of the transfected cells. Studies addressed to reveal catalytic activity of PADI4-ΔN could not detect any enzymatic activity in vitro assays. Transient expression studies revealed that the two allele variants (i.e., G or T at rs2240335 position) can contribute differently to the novel PADI4 promoter activity, which is ultimately reflected by Luciferase activity in cell lysates.

Conclusion: The PADI4 gene encodes at least three isoforms: (i) the well-characterized full-length isoform, (ii) a C-terminus deleted (PADI4-ΔC) isoform and (iii) the novel N-terminus truncated (PADI4-ΔN) isoform. The newly discovered isoform is not able to catalyze citrullination of vimentin (a well-characterized PADI4 target) and does not affect the enzymatic activity of the full-length isoform in in vitro competitive assays. These data suggest that allele variants (at the RA-associated SNP position) primarily alter the physiological ratio of the full-length and truncated PADI4 isoforms, which might lead to RA pathogenesis by an unknown mechanism.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/rheumatoid-arthritis-associated-genetic-alteration-defines-a-new-promoter-for-peptydil-arginine-deiminase-4-gene/