Background/Purpose: Rheumatoid arthritis (RA) is an autoimmune disease that results in inflammatory destruction of synovial joints in affected patients. The involvement of genetic and epigenetic factors in RA is recognized, but not well understood. Using proteoglycan (PG)-induced arthritis (PGIA), an autoimmune mouse model of RA, previously we identified a genomic region in mouse chromosome 3 (mChr3) which was associated with PGIA susceptibility independently of MHC. The goals of the present study were (i) to generate MHC-matched PGIA-susceptible and -resistant interval-specific congenic (IVSC) strains in order to narrow this genomic region to a size that can be sequenced by next generation sequencing (NGS) methods, and (ii) to identify disease-associated genomic alterations/mutations (e.g., nucleotide insertions or deletions: “indels”) within this region in PGIA-susceptible and resistant IVSC mice.

Methods: IVSC strains were created by intercrossing MHC-matched PGIA-resistant DBA/2 and PGIA-susceptible BALB/c strains, which resulted in IVSC offspring carrying partially overlapping DBA/2 genomic segments within mChr3 on a full BALB/c background. Heterozygous mice with the same narrow genomic intervals were mated and homozygous offspring were tested for susceptibility to PGIA. On the basis of arthritis incidence and severity in IVSC mice, a 21 Mbp region of Chr3 (99.4-120.4 Mbp) was selected, and sequenced by NGS in parent and IVSC mice (n=16). All sequence alterations were compiled into a database and analyzed using GeneSpring NGS software (Agilent) and Microsoft Visual Studio program scripts.

Results: The sequenced 21 Mbp genomic region of mChr3 contained a total of 164,380 nucleotide changes (mutations) in DBA/2 and BALB/c as compared to the corresponding core (reference) C57BL/6 genomic sequences. Of these mutations, 42% were unique to BALB/c and 21% were unique to DBA/2. However, less than 1% of the mutations were located within protein coding (exonic) regions. All missense exonic mutations were predicted to have negligible or no effect on RNA or protein sequences, and no nonsense mutation was found. A total of 4,429 mutations were located within the region containing the Ptpn22 and Cd2 genes and their flanking sequences, which have been reported to harbor RA-associated SNPs on human Chr1. However, a number of indels found in the intergenic regions Ptpn22 and Cd2loci of mChr3 were predicted to create or eliminate binding sites for potentially important transcription factors.

Conclusion: We have identified arthritis-associated sequence alterations within a genomic region of mChr3 containing the Ptpn22 and Cd2 genes in mice, which region is syntenic with a prominent RA susceptibility locus on human Chr1. Although the majority of the mutations are located within non-coding sequences, they may alter the binding of transcription factors or epigenetic modulators to promoter regions, thus affecting the transcription of the neighboring genes. Our finding warrant further genetic, epigenetic, and functional studies on this arthritis-associated locus in mice and humans.  

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/arthritis-associated-sequence-alterations-within-a-genetic-susceptibility-region-of-mouse-chromosome-3-a-genomic-region-which-is-syntenic-with-a-prominent-non-mhc-locus-in-rheumatoid-arthritis/