Background/Purpose: Somatic mutations caused by reactive oxygen and nitrogen species in inflamed joints, have been implicated in the destructive nature of RA. Mutation analysis also helps identify mechanisms contributing to the aggressive behavior of RA FLS. However, previous analyses focused on candidate gene approaches. This work represents the first whole genome sequencing study of RA FLS.

Methods: Next generation sequencing of the whole genome was carried out for 10 RA and 10 osteoarthritis (OA) human FLS cell lines at 100X. Reads were aligned, sorted according to chromosome and position, duplicates and those with a MAQ score < 30 removed. Base quality score recalibration of aligned reads was then performed with GATK 3.8. Variant calling was performed using VarScan2 with low quality base calls of < Q20 filtered out. Finally, we removed low coverage and low confidence sites. To account for a lack of a germline reference for each line and potential culture effects, we used a novel approach by filtering for sequences with mutation frequencies (MF) in >2% and < 5-8% of reads and discarding those found in at least >92% of the reads to remove putative germline polymorphsms and identify somatic mutations. For each set of mutations, single nucleotide variants (SNV) and insertion/deletion (Indel) locations were overlapped with gene bodies and promoters to identify the associated genes or with gene exons only. Genes specific to RA were analyzed for enriched functions and pathways.

Results: Indel and Missense SNV somatic mutations in the range 2%< MF< 5% were overlapped with gene bodies and promoters. Mutated genes unique to RA were identified and ranked by the number of RA cell lines with the mutant gene. Only 43 genes had mutations in > 5/10 RA lines and 0/10 OA lines. The top ranking genes, which had 7/10 RA lines with intronic mutations included MTMR3, which plays a role in autophagosome formation and CLNK which plays a role in immunoreceptor signaling. Other RA-specific mutated genes included multiple E3 ubiquitin ligases and DNA repair genes. Using the same filter and overlapping somatic mutations only with exons yielded additional RA-specific genes. Of the 33 exon-only mutations in 3/10 or more RA lines and 0/10 OA lines, 5 are also unexpectedly involved ubiquitination. We then extended the mutation range to 2%< MF< 6% and only considered missense somatic mutations in exons. 260 and 207 genes unique to RA or OA, respectively, were identified. Pathway analysis on these gene sets identified 11 significant pathways (p< 1 x10^-5 ) for the RA genes but no significant pathways for OA suggesting that the latter were not functionally relevant. 3 of the mutant RA pathways involved antigen processing including “Antigen Presentation: Folding, assembly and peptide loading of class 1 MHC” (p< 1 x10^-16).

Conclusion: Whole genome sequencing of RA and OA human FLS lines identified somatic mutations unique to RA in genes consistent with immune deregulation. Our novel computational approach corrected for potential tissue culture effects and showed a high mutation rate in RA. The unbiased analysis showed significant mutant pathways enriched in RA and suggests that the mutations play a role in disease pathogenesis.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/novel-somatic-mutations-identified-by-whole-genome-sequencing-of-rheumatoid-arthritis-ra-fibroblast-like-synoviocytes-fls/