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Epigenomewide Scan Identifies a Treatmentresponsive Pattern Of ARTHRITIS & RHEUMATOLOGY Vol. 66, No. 6, June 2014, pp 1648–1658 DOI 10.1002/art.38409 © 2014, American College of Rheumatology Epigenome-Wide Scan Identifies a Treatment-Responsive Pattern of Altered DNA Methylation Among Cytoskeletal Remodeling Genes in Monocytes and CD4ϩ T Cells From Patients With Behc¸et’s Disease Travis Hughes,1 Filiz Ture-Ozdemir,2 Fatma Alibaz-Oner,2 Patrick Coit,1 Haner Direskeneli,2 and Amr H. Sawalha1 Objective. The pathogenesis of Behc¸et’s disease the cytoskeleton might contribute to the pathogenesis of (BD), an inflammatory disease with multisystem in- BD. Further, treatment of BD modified the differences volvement, remains poorly understood. This study was in DNA methylation observed in patients compared to undertaken to investigate whether there are DNA methyl- controls; indeed, among CpG sites that were differen- ation abnormalities in BD that might contribute to the tially methylated after disease remission versus before pathogenesis of the disease. treatment, there was widespread reversal of the direc- Methods. We examined genome-wide DNA methyl- tion of aberrant DNA methylation observed between the .ation patterns in monocytes and CD4؉ T cells from 16 patient and control groups male patients with untreated BD and age, sex, and Conclusion. The results of this epigenome-wide ethnicity–matched healthy controls. Additional samples study—the first such study in BD—provide strong evi- were collected from 12 of the same BD patients after dence that epigenetic modification of cytoskeletal dy- treatment and achievement of disease remission. namics underlies the pathogenesis of BD and its re- Genome-wide DNA methylation patterns were assessed sponse to treatment. using the Infinium HumanMethylation450 BeadChip array, which includes >485,000 individual methylation Behc¸et’s disease (BD) is an inflammatory disease sites across the genome. characterized by the presence of recurrent oral and Results. We identified 383 CpG sites in mono- genital ulcers, skin lesions, and uveitis. Mucocutaneous ,cytes, and 125 in CD4؉ T cells, that were differentially lesions in BD typically exhibit leukocyte infiltration methylated between BD patients and controls. Bioinfor- primarily by T cells and monocytes (1). Widespread matic analysis revealed a pattern of aberrant DNA enhancement of leukocyte motility and tissue infiltration methylation among genes that regulate cytoskeletal occurs in BD. Monocytes from patients with BD display dynamics, suggesting that aberrant DNA methylation of increased activity and promote increased neutrophil multiple classes of structural and regulatory proteins of adhesion (2). Further, levels of macrophage inflamma- tory protein 1␣, which promotes lymphocyte chemotaxis, are elevated in BD (3). Activated CD4ϩ T cells play a Supported by the Rheumatology Research Foundation critical role in BD pathogenesis, and an expansion of (Rheumatology Investigator award to Dr. Sawalha) and the NIH Th17 cells via interleukin-21 signaling and increased (grant P30-ES-017885 to Dr. Sawalha). ␥ ␦ 1Travis Hughes, MPH, Patrick Coit, BSc, Amr H. Sawalha, levels of / T cells are observed (4–6). MD: University of Michigan, Ann Arbor; 2Filiz Ture-Ozdemir, PhD, Endothelial cell injury is common in BD. Levels Fatma Alibaz-Oner, MD, Haner Direskeneli, MD: Marmara Univer- of soluble E-selectin molecules have been reported to be sity School of Medicine, Istanbul, Turkey. Address correspondence to Amr H. Sawalha, MD, Division of increased in the serum of patients with active disease (7). Rheumatology, Department of Internal Medicine, University of Mich- Vascular endothelial cell function becomes impaired in igan, Room 5520 MSRB-1, SPC 5680, 1150 West Medical Center response to oxidative damage (8). Disruption of tight Drive, Ann Arbor, MI 48109. E-mail: [email protected]. Submitted for publication October 10, 2013; accepted in junctions between endothelial cells is mediated by revised form February 11, 2014. changes in class II myosin phosphorylation and remod- 1648 DNA METHYLATION CHANGES IN BEHC¸ET’S DISEASE 1649 eling of the F-actin cytoskeleton (9). Cytoskeletal re- PATIENTS AND METHODS modeling in endothelial and immune cells accompanies Patient selection and sample collection. Sixteen male lymphocyte adhesion and tissue infiltration in the nor- BD patients and 16 healthy controls matched for age (Ϯ5 mal inflammatory response (10). Treatments that dis- years), sex, and ethnicity were recruited to participate in this rupt microtubule processing might be effective in limit- study. All patients were recruited from the rheumatology ing the frequency and severity of mucocutaneous clinics at Marmara University School of Medicine. Disease manifestations of the patients are reported in Supplementary symptoms in BD, but their exact mechanism of action Table 1 (on the Arthritis & Rheumatology web site at http:// remains uncertain. onlinelibrary.wiley.com/doi/10.1002/art.38409/abstract). All BD has a strong genetic component, as evidenced patients studied had either not received treatment for BD or by the geographic concentration of the disease along had not received treatment for at least 3 months prior to the Old Silk Road (11). The most robust genetic asso- enrollment, and samples were collected at the initial study visit. Samples were further collected from 12 of the 16 BD patients ciation observed in BD to date is within the HLA region. following treatment and achievement of disease remission Recent evidence suggests that there are multiple inde- (defined as the absence of any disease-associated symptoms or pendent genetic susceptibility loci for BD in the HLA organ involvement for at least 1 month). The study was class I region, with the strongest effect localized to approved by the ethics committees and institutional review boards at Marmara University School of Medicine and the the genetic region between HLA-B and MICA (12). University of Michigan. All study participants provided written Multiple associations outside of the HLA region have informed consent prior to enrollment. also been reported in BD, including associations with Isolation of monocytes and CD4؉ T cells and DNA IL10, IL23R, UBAC2, STAT4, CCR1, KLRC4, ERAP1, extraction. Peripheral blood mononuclear cells (PBMCs) were isolated, by density-gradient centrifugation (Amersham Biosci- and GIMAP2/GIMAP4 (13). While there is a strong ences), from fresh blood samples obtained from BD patients genetic component to BD, genetic variation alone is not and healthy controls. Monocytes and CD4ϩ T cells were sufficient to explain the heritability and pathogenesis of purified from PBMCs by magnetic bead separation (Miltenyi the disease. Biotec). The purity of isolated cell populations, confirmed by flow cytometry using fluorochrome-conjugated antibodies To date there have been no reported studies of against CD14 (for monocytes) and CD4 (for T helper lympho- the involvement of DNA methylation (the addition of a cytes), was Ͼ90% for both monocytes and CD4ϩ T cells. methyl group to the fifth carbon in cytosine rings within Genomic DNA was obtained from isolated monocytes and ϩ CpG dinucleotides) in BD. However, there is a growing CD4 T cells using a DNeasy Blood and Tissue Kit (Qiagen). Genome-wide DNA methylation profiling. Bisulfite body of evidence that changes in DNA methylation have conversion was performed using an EZ DNA Methylation Kit an important role in multiple immune-mediated diseases (Zymo Research). Whole-genome amplification of bisulfite- (14–17). DNA methylation ex utero is primarily medi- converted DNA was performed prior to array hybridization. ated by DNA methyltransferase 1 and is generally a An Infinium HumanMethylation450 BeadChip array kit was used to assess the methylation status of Ͼ485,000 individual repressive epigenetic mark. Hypermethylation of DNA methylation sites throughout the genome. This array covers results in transcriptional gene repression, while hypo- 99% of RefSeq genes, with an average of 17 CpG sites per methylation is associated with a chromatin configuration gene across the promoter region, 5Ј-untranslated region (5Ј- Ј that is transcriptionally permissive (18). UTR), first exon, gene body, and 3 -UTR. It also covers 96% of CpG islands. Non-CpG methylated sites recently identified Herein we report the results of an epigenome- in human stem cells are also covered, as are microRNA wide study of the methylation status of Ͼ485,000 indi- promoter regions. vidual methylation sites across the genome, among Quality control, identification of differentially methyl- treatment-naive BD patients and healthy matched con- ated CpG sites, and bioinformatic analysis. Comparisons of DNA methylation levels between BD patients and healthy trols. We also evaluated epigenome-wide DNA methyl- controls were performed separately for monocytes and CD4ϩ ation status in individual BD patients before and after T cells. Differences in DNA methylation associated with treatment with achievement of disease remission. We treatment for BD were also assessed separately in monocytes ϩ obtained evidence of widespread DNA methylation and CD4 T cells. Differential methylation analysis was performed using the Genome Studio methylation package. changes in BD across the genome. Our data suggest that Beta values were used to represent the percent methylation at DNA methylation changes in genes involved in cytoskel- each CpG site, and were calculated using the ratio of intensi- etal dynamics contribute to the pathogenesis of BD and ties between methylated and unmethylated
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