NEWS & VIEWS GENETICS

Insights into RA pathogenesis from DNA methylome analysis Esteban Ballestar Refers to Ai, R. et al. DNA methylome signature in early rheumatoid arthritis synoviocytes compared with longstanding rheumatoid arthritis synoviocytes. Arthritis Rheumatol. http://dx.doi.org/10.1002/art.39123

Could temporal changes in DNA methylation patterns of rheumatoid arthritis (RA) synoviocytes provide insights into the progression of the disease? A study comparing synoviocyte DNA methylation profiles in early and long-standing RA suggests this might be the case, with potential implications for understanding and perhaps modulating RA progression. Compelling evidence has shown that the pathogenesis of autoimmune rheumatic diseases such as rheumatoid arthritis (RA) involves a combination of genetic predisposition, resulting from the combined effect of multiple genetic variants, and epigenetic alterations, which can emerge as a consequence of environmental factors or the activity of gene products. In contrast to genetic factors, which generally remain stable over time, epigenetic modifications are cell-type specific, can evolve over time and have an effect on gene expression patterns, which ultimately determine cell function. Epigenetic changes, such as gain or loss of DNA methy­ lation, are heritable during cell divisions and influence disease development in a manner complementary to mutations or polymorphisms. Analysis of DNA methylation in samples from patients with RA, as done in a new study by Ai et al.,1 is interesting not only because of the relevance of epigenetic changes to the pathogenesis and progression of the disease, but also because of the stability and convenience of DNA for conducting quick, reliable tests in clinical practice. Previous studies by different groups have shown that fibroblast-like synoviocytes (FLSs) from patients with RA have altered DNA methylation patterns. The study by Ai et al.1 shows that the DNA methylation patterns of FLSs from individuals with early RA, juvenile idiopathic arthritis (JIA) and longstanding RA cluster together and are distinct from those of FLSs obtained from patients with osteoarthritis (OA). However, substantial differences in DNA methylation between early and late RA suggest the possibility that

the transition to chronic RA involves acquisition of additional DNA methylation changes, and could therefore serve as a marker to monitor disease progression. Differences in DNA methylation of FLSs between early and late RA could help us to understand or even modulate the progression of the disease, at least at the level of the contribution of this cell type. Healthy/pre-RA

Ai et al.1 used bead arrays to compare the DNA methylation profiles of FLSs obtained from four patients with early RA, three with JIA, 11 with late RA and 11 with OA.1 Despite the small number of samples, DNA methylation patterns of early RA, JIA and late RA samples clustered together and separated from another branch comprising OA samples. Interestingly, these researchers’ findings that the DNA methylation profiles associated with early RA, JIA and late RA stand apart from each other, and that a number of loci are differentially methylated, suggest that variations in DNA methylation among these clinical entities are also relevant. Moreover, the functional pathways associated with the subset of genes differentially methylated in early RA and late RA relate to cell migration, differentiation and adhesion, changes that could perhaps be associated with the increasingly aggressive behaviour of synoviocytes as RA progresses (Figure 1). FLSs (sometimes termed RA synovial fibroblasts) are a major focus of DNA methy­ lation studies in RA.2 Initially, such studies Early RA

Hyperplasia and hypertrophy

Late RA

Bone erosion

Synovial membrane Cartilage Bone

Gene expression in synoviocytes

CDH11, VCAM-1, UDPGDH

MMP-1, MMP-13, ADAMTS4, ADAMTS5

IL-6, IL-1, PDGF, CCL2

Accumulation of DNA methylation changes?

Stage-specific DNA methylation changes?

Early and late RA common pattern Early-RA-specific pattern Late-RA-specific pattern

Figure 1 | RA synoviocytes and their role in the destruction of joints. In a healthy knee, the synovial membrane is 1–2 cells thick. In early RA, synovial lining cells undergo hyperplasia and Nature Reviews | Rheumatology hypertrophy and begin to invade the cartilage. In late RA, the synovial membrane becomes inflammatory tissue, the pannus, which invades and destroys adjacent cartilage and bone. Changes in gene expression are associated with the progressive transformation of the phenotype of synoviocytes. Identified differences between early and late RA epigenetic profiles could represent either the gradual accumulation of DNA methylation changes during RA progression or stage-specific patterns of differential methylation (purple) with respect to the normal/physiological levels of methylation (blue). Abbreviation: RA, rheumatoid arthritis.

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NEWS & VIEWS focused on immune cells, which showed moderate or even an absence of DNA methy­ lation changes,3 perhaps because only a small number of genes were analysed or because of the use of whole blood rather than isolated cell types. However, data from e­pigenomewide association analysis of blood-derived DNA samples have implicated DNA methylation as an intermediary of genetic risk in RA.4 Nonetheless, FLSs might be more suitable than blood cells for DNA methylation studies in the context of RA for several reasons. Firstly, FLSs are the effector cells of cartilage and bone destruction, and display an ‘intrinsically’ activated and aggressive phenotype in RA. Secondly, changes in the expression profile of these cells, such as the increased production of matrix-degrading enzymes and adhesion molecules, are likely to be associated with DNA methylation changes. Thirdly, the aberrant DNA methy­ lation profile of these cells is conserved even after a few passages in vitro. A research group led by Steffen Gay was among the first to identify a role for DNA methylation alterations in FLSs.5 Gary Firestein’s group (who also undertook the current study 1) generated high-throughput DNA methylation data for these cells that showed the acquisition of DNA methylation alterations in genes rele­ vant to RA-related functional categories, suggesting that these changes contribute to the development of an aggressive phenotype in FLSs.6 A drawback of the study by Ai and colleagues is the small number of samples, as acknowledged by the authors in their report.1 This limitation is due to the difficulties of obtaining synoviocytes from patients with early RA and is not an easy one to overcome. The small sample size also makes it difficult to draw broad conclusions about the effect of DNA methylation on FLSs, as the diversity of treatments for RA could

affect both the phenotypic progression of these cells and their DNA methylation patterns. These considerations only underscore the need for cooperation in undertaking studies with larger cohorts to obtain more robust data. The inclusion of samples from patients with very early RA would also be interesting; however, obtaining cells from synovial biopsies of such patients would be even more challenging than for patients with more advanced disease. Finally, OA synoviocytes might not be the ideal cell type to use as a control, as suggested by transcriptomic studies that show robust and differential gene expression in OA synoviocytes versus RA and healthy synoviocytes.7 Hypothetically, large studies and the inclusion of samples from patients with very early RA as well as early RA and late RA could enable the exploration of two possibilities regarding the mechanism by which DNA methylation changes are acquired in RA synoviocytes. One possibility is that the progressive accumulation of DNA methylation alterations in synoviocytes generates patterns that strictly reflect progressive changes in the phenotype of these cells in patients with RA. Alternatively, specific DNA methy­lation alterations might characterize each stage of the disease, not necessarily in a progressive manner (Figure 1), despite the existence of common trends between early RA and late RA. In summary, the study by Ai et al.1 provides new clues about the relevance of studying DNA methylation of FLSs to investigate RA development and progression, and highlights the need for large comprehensive epi­ genomic studies to fully develop the potential of this approach. The hope is that such strategies will identify markers that could be used in the clinical setting, for molecular stratification of patients and to predict their response to therapies.

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Chromatin and Disease Group, Cancer Epigenetics and Biology Programme (PEBC), Bellvitge Biomedical Research Institute (IDIBELL), Avenida Gran Via 199–203, 08908 L’Hospitalet de Llobregat, Barcelona, Spain. [email protected] doi:10.1038/nrrheum.2015.66 Published online 19 May 2015 Acknowledgements E.B. thanks C. Gómez-Vaquero for insightful comments on rheumatoid arthritis synoviocytes. E.B. is supported by Fundación Ramón Areces (grant CIVP16A1834) and European Union/ European Federation of Pharmaceutical Industries and Associations Innovative Medicines Initiative Joint Undertaking PRECISESADS (grant 115565). Competing interests The author declares no competing interests. 1.

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Ai, R. et al. DNA methylome signature in early rheumatoid arthritis synoviocytes compared with longstanding rheumatoid arthritis synoviocytes. Arthritis Rheumatol. http:// dx.doi.org/10.1002/art.39123. Karouzakis, E., Gay, R. E., Gay, S. & Neidhart, M. Epigenetic control in rheumatoid arthritis synovial fibroblasts. Nat. Rev. Rheumatol. 5, 266–272 (2009). Javierre, B. M. et al. Changes in the pattern of DNA methylation associate with twin discordance in systemic lupus erythematosus. Genome Res. 20, 170–179 (2010). Liu, Y. et al. Epigenome-wide association data implicate DNA methylation as an intermediary of genetic risk in rheumatoid arthritis. Nat. Biotechnol. 31, 142–147 (2013). Neidhart, M. et al. Retrotransposable L1 elements expressed in rheumatoid arthritis synovial tissue: association with genomic DNA hypomethylation and influence on gene expression. Arthritis Rheum. 43, 2634–2647 (2000). Whitaker, J. W. et al. An imprinted rheumatoid arthritis methylome signature reflects pathogenic phenotype. Genome Med. 5, 40 (2013). Del Rey, M. J. et al. Transcriptome analysis reveals specific changes in osteoarthritis synovial fibroblasts. Ann. Rheum. Dis. 71, 275–280 (2012).

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Genetics: Insights into RA pathogenesis from DNA methylome analysis.

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