Background/Purpose: DNA methylation represents an important potential mediator of environmental influences on autoimmunity, including rheumatoid arthritis (RA).  Genome-wide methylation in the context of clinical phenotypes is most commonly performed using Illumina Human 450K methylation arrays. These assay only  about 1% of dynamic methylome in the genome and underestimate the impact of methylation differences distal to CpG-rich regions as well as intergenic functional elements. To overcome these limitations, we developed a comprehensive approach to study blood (immune cell) methylomes. We used this to compare DNA methylation in subjects positive vs. negative for anti-cyclic citrullinated peptide (anti-CCP, a key serological marker of RA risk).

Methods:  Using banked serum from a random subset of a general population cohort, we identified 22 subjects who were anti-CCP positive and 24 who were anti-CCP negative. Custom capture of target regions by SeqCap Epi (Roche) allows targeting of desired genomic regions as we described earlier (Allum et al. Nat Comm. 2015) to conduct genome-wide bisulphite sequencing. For this study we have established an “immune-methylome” panel that targets regulatory elements in approximately 20 immune cell subsets along with content of 450K arrays and regions genetically associated with autoimmune/inflammatory diseases. Altogether approximately 120Mb of genomic DNA harboring 4.6M CpG sites were interrogated by our assay.

Results: First-pass data analyses in peripheral blood of 22 anti-CCP positive and 24 anti-CCP negative subjects (sequenced to 10-15x average depth) show the clear advantages of comprehensive methylome assessment. We detected 2600 significant CpGs, of which only about 8% were represented in Illumina 450K arrays. The variation among the groups was depleted at proximal promoters and enriched in gene distal regions. The annotation of immunologic signatures by GREAT for the differentially methylated sites show distinct gene sets impacted by relative hyper- or hypo-methylation in anti-CCP positive vs. negative subjects.

Conclusion: These novel methods represent a comprehensive tool to assess methylation variation and its relation to immunological phenotypes in peripheral blood, with clear differences being shown in anti-CCP positive vs. negative subjects.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/dna-methylation-and-its-relation-to-immunological-phenotypes-in-peripheral-blood-a-study-of-anti-ccp-antibody-positivity-from-a-population-based-pool/