ORIGINAL ARTICLE Interleukin-2 receptor-α proximal promoter hypomethylation is associated with multiple sclerosis J Field 1 , A Fox 1 , MA Jordan 2 , AG Baxter 2 , T Spelman 3 , M Gresle 3 , H Butzkueven 3 , TJ Kilpatrick 4 and JP Rubio 1,5 Genetic studies have demonstrated association between single-nucleotide polymorphisms within the IL2RA (interleukin-2 receptor α-subunit) gene and risk of developing multiple sclerosis (MS); however, these variants do not have obvious functional consequences. DNA methylation is a source of genetic variation that could impact on autoimmune disease risk. We investigated DNA methylation of the IL2RA promoter in genomic DNA obtained from peripheral blood mononuclear cells and neural tissue using matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry. A differential methylation profile of IL2RA was identified, suggesting that IL2RA expression was regulated by DNA methylation. We extended our analysis of DNA methylation to peripheral blood mononuclear cell (PBMC) of MS cases and controls using MALDI-TOF and Illumina HumanMethylation450 arrays. Analyses of CpG sites within the proximal promoter of IL2RA in PBMC showed no differences between MS cases and controls despite an increase in IL2RA expression. In contrast, we inferred significant DNA methylation differences specific to particular leukocyte subtypes in MS cases compared with controls by deconvolution of the array data. The decrease in methylation in patients correlated with an increase in IL2RA expression in T cells from MS cases in comparison with controls. Our data suggest that differential methylation of the IL2RA promoter in T cells could be an important pathogenic mechanism in MS. Genes and Immunity advance online publication, 12 January 2017; doi:10.1038/gene.2016.50 INTRODUCTION The interleukin-2 receptor α-subunit (IL2RA) is one of three transmembrane proteins that make up the high-affinity IL-2 receptor. 1 Also known as CD25, this highly inducible α-chain has also been shown to increase the affinity for IL-2 binding. 1 The IL2RA gene is expressed by thymocytes early in T-cell develop- ment but is downregulated once thymocytes reach the CD4 + CD8 + double-positive stage and is not expressed during subsequent T-cell development. 2 Expression of IL2RA is induced in resting mature T-lymphocytes on stimulation with antigen and costimu- latory signals; 3 however, constitutive expression is seen in the regulatory T-cell population. 4 Expression in humans is modulated by at least six positive regulatory regions (PRRI–VI) that are dispersed over a 12 kb segment of the IL2RA gene, which spans 50 kb on chromosome 10p15.1, and each PRR has a complemen- tary role in regulating the transcription of IL2RA. 5 Genetic studies in type 1 diabetes (T1D), Graves’ disease and multiple sclerosis (MS) have led to the identification of single- nucleotide polymorphisms (SNPs) within the IL2RA gene that are associated with disease susceptibility. 6–13 A role for IL2RA in autoimmune disease is further supported by the development of autoimmunity in individuals who lack functional IL2RA, 14 as well as its involvement in murine models of autoimmune disease, 4,15–17 where the absence of CD25-expressing regulatory T cells results in increased inflammation. Autoimmune disease-associated SNPs located within intron 1 of the IL2RA gene has previously been shown to correlate with altered proportions of IL2RA (CD25)- expressing naïve CD4 + T cells, as well as increased levels of soluble IL2RA (sIL2RA). 18–20 Furthermore, some of these associated SNPs (e.g. rs2104286) create or disrupt a CpG dinucleotide dependent on the allele, suggesting a possible role for epigenetic variation such as alteration in the degree of CpG DNA methylation in disease pathogenesis. Epigenetic control of gene expression via differential CpG methylation is well established in normal biological processes such as tissue differentiation, imprinting, X-chromosome inactiva- tion and suppression of endogenous and exogenous gene expression. In addition, DNA methylation has been shown to be important in regulating cell fate decisions in the development of T-helper (Th) lymphocytes, 21–23 and the induction of Th1 and Th2 lymphocyte commitment via epigenetic regulation of cytokines such as interferon-γ, IL-4 and IL-17. The importance of epigenetic mechanisms in modulation of the immune system is further underscored by their involvement in both humans and mice in the induction of the suppressive regulatory T-cell population, through regulation of the forkhead transcription factor 3 (FOXP3). 24–26 In mice, a conserved region within intron 1 of the FoxP3 gene is differentially methylated in CD4 + CD25 - compared with regulatory T cells (CD4 + CD25 + FOXP3 + ), 27 whereas in humans the FOXP3 promoter is completely demethylated in the regulatory T-cell compartment, allowing identification of bone fide regulatory T cells through DNA methylation analysis. 28,29 Altered DNA methylation has also been shown to have a role in systemic lupus erythematosus and rheumatoid arthritis, 30–32 TID, where SNPs associated with T1D also correlate with DNA methylation changes, 33 and more recently in MS, where purified T-cell populations from MS patients have been assessed, 34–36 but have 1 Multiple Sclerosis Division, The Florey Institute of Neuroscience and Mental Health, The University of Melbourne, Melbourne, VIC, Australia; 2 Comparative Genomics Centre, James Cook University, Townsville, QLD, Australia; 3 Department of Medicine, University of Melbourne, Parkville, VIC, Australia; 4 The Melbourne Neuroscience Institute, University of Melbourne, Melbourne, VIC, Australia and 5 Department of Pathology, University of Melbourne, Parkville, VIC, Australia. Correspondence: Dr J Field, Multiple Sclerosis Division, The Florey Institute of Neuroscience and Mental Health, The University of Melbourne, 30 Royal Parade, Melbourne, VIC 3010, Australia. E-mail: judith.field@florey.edu.au Received 6 September 2016; revised 23 November 2016; accepted 28 November 2016 Genes and Immunity (2017), 1 – 8 © 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved 1466-4879/17 www.nature.com/gene