Journal of Clinical and Cellular Immunology

Journal of Clinical and Cellular Immunology
Open Access

ISSN: 2155-9899

Research Article - (2014) Volume 5, Issue 3

Methylation Profile of CD4+ T Cells in Chronic Fatigue Syndrome/Myalgic Encephalomyelitis

Ekua W Brenu1,2*, Donald R Staines2 and Sonya M Marshall-Gradisnik1,2
1School of Medical Science, Griffith Health Centre, Griffith University, Gold Coast, QLD, Australia
2The National Centre for Neuroimmunology and Emerging Diseases, Griffith University, Australia
*Corresponding Author: Ekua W Brenu, Parklands Drive Griffith Health Centre National Centre for Neuroimmunology and Emerging Diseases, Griffith University, Southport, QLD, 4215, Australia, Tel: 61756789282 Email:

Abstract

Objective: Methylation is known to regulate biological processes and alterations in methylation patterns have been associated with a variety of diseases. Chronic Fatigue Syndrome/Myalgic Encephalomyelitis (CFS/ME) is an unexplained disorder associated with immunological and molecular changes. CD4+T cells specifically, regulatory T cells (Tregs) have been implicated in CFS/ME patients where significant increases in Tregs have been observed in these patients. Therefore the objective of this study was to examine methylation in CD4+T cells from CFS/ME patients.

Methods: The study comprised twenty-five CFS/ME participants and eighteen controls aged between 25-60 years. A volume of 20 ml whole blood was collected from each participant and peripheral blood mononuclear cells were isolated via density gradient centrifugation. A negative isolation kit was used to isolate the CD4+T cells from the peripheral blood samples. Genome wide methylation studies were performed on isolated CD4+T cells using the Illumina Infinium 450 K Human methylation array system. Data analysis was performed using Genome studio and Partek Enrichment software.

Results: 120 CpGs were observed to be differentially methylated in the CFS/ME patients in comparison to the controls. Of these 70 were associated with known genes. The majority of the differential methylated regions in the CFS/ME patients were hypomethylated. Additionally, most of the genes with differentially methylated regions in the CFS/ME patients were responsible for apoptosis, cell development, cell function and metabolic activity.

Conclusion: The present study demonstrates that epigenetic changes in CD4+T cells may have a potential role in the immunological changes observed in CFS/ME patients.

Keywords: Chronic Fatigue Syndrome, CD4+T cells, Methylation, miRNA

Introduction

Chronic Fatigue Syndrome/Myalgic Encephalomyelitis (CFS/ME) is an inexplicable disorder that affects 1–4% of individuals worldwide [1,2]. CFS/ME patients are subject to severe incapacitating fatigue, post-exertional sickness, discrepancies in cognition, painful lymph nodes, muscle aches and unbalanced sleep patterns [3]. CFS/ME involves disruption to immunological processes including reduced cytotoxic activity and elevated levels of regulatory T cells [4-6]. Furthermore, CFS/ME patients may exhibit differential expression in genes that regulate various physiological processes known to be abnormal in CFS/ME [4,7-12]. To date a succinct pathomechanism for CFS/ME and concrete diagnostic biomarkers have not been identified.

DNA methylation is an epigenetic modification process where DNA methyltransferases adds a methyl group to the 5’ position of cytosines of CpG dinucleotides [13]. The process of methylation has many consequences on gene expression as the extent and pattern of 5-methylcytosines dictates the rate of gene expression in a particular region [14]. In particular, DNA methylation recruits transcriptional co-repressors of the methyl-DNA binding domain family which repress the transcription of certain genes [13]. Regulation of DNA methylation is important, especially, during embryonic development, chromatin condensation and genomic imprinting [15]. Epigenetic modifications may have great value in various diseases including inflammatory and autoimmune diseases. Importantly, epigenetic modifications have been suggested to account for certain immune related diseases [16]. Increases and decreases in DNA methylation has been shown to regulate the expression of T cell related cytokines genes such as IL-2 and IL-6 [17].

In CFS/ME changes in gene expression has been reported for a number of mRNA and microRNA genes involved in various immune processes. However, to date, the role of cell specific methylation in CFS/ME has not been explored. This study performed a differential genome wide methylation analysis on CD4+T cells in CFS/ME patients and non-fatigued controls.

Materials and Methods

Ethical approval

Approval for the study was granted by the Institutional Ethics Review Board at Griffith University. Written informed consent was obtained from all participants prior to the study.

CFS/ME patients and controls

Twenty-five patients with CFS/ME (21 Females and 4 males; age= 50.31 ± 2.27) were enrolled into the study. These patients were assessed using the 1994 Centre for Disease Control and Prevention case definition (1994 CDC) Criteria for CFS/ME [3]. Majority of the CFS/ME cases were women, which is reflective of the high female to male ratio observed in CFS/ME cases. All patients reported active CFS/ME with low to moderate levels of physical activity and full time employment. A non-fatigued control group comprising 18 healthy participants with no incidence of CFS/ME or other medical conditions was also included in the study (10 Females and 8 males; age= 47.44 ± 2.16). Participants with autoimmune diseases, psychosis or smoking were excluded from the study.

CD4+ T cell isolation

A total volume of 20 ml of venous peripheral blood was collected from each participant in to EDTA containing tubes. Ficoll-hypaque density gradient centrifugation was used to isolate the peripheral blood mononuclear cells (PBMCs) from whole blood samples. Isolation of the CD4+T cells was performed according to the manufacture’s directive, using a negative selection protocol which required the use of magnetic beads labelled with markers that exclude the CD4+T cell population (Miltenyi Biotec, Bergisch Gladbach, Germany). Isolated PBMCs were resuspended in a buffer solution of PBS, stained and incubated with a biotin solution at 4°C for 10 minutes. Following incubation samples were resuspended in PBS and stained with microbeads at 4°C for 15 minutes. The samples were washed by adding PBS solution and centrifuging at 300 x g for 5 minutes. The supernatant generated was discarded and samples were resuspended in a buffer. The cells in suspension were applied to columns attached to a magnetic stand, the flow through fluid containing the cells of interest were collected and snap frozen in liquid nitrogen and stored at -80°C until required for further processing.

DNA extraction and methylation

Genomic DNA was extracted using the QIAamp DNA extraction kit according to the manufacturer’s instructions. Genome wide methylation was performed at the Australian Genome Research Facility (AGRF; The Walter and Eliza Hall Institute of Medical Research, Sydney). Assessment of integrity, quality and quantity was determined by the Nanodrop Spectrophotometer and electrophoresis on a 0.8% agarose gel. The EZ DNA Methylaiton kit (Zymo Research, Irvine, CA) was used in the Bisulfite conversion of DNA. This was then hybridized onto a beadchip. The Illumina HumanMethylation450K BeadChip assay (Illumina Inc., San Diego, CA) was the method of choice for performing the whole genome methylation. This assay contains 485,577 CpG targets. Enzymatic end-point fragmentation, precipitation and re-suspension were used to amplify all the DNA samples. These samples were then hybridized for 16 hours at 48°C on to BeadChips. Dioxy nucleotide extension was used to achieve single nucleotide extensions. This was followed by a series of staining steps to differentiate biotin and DNP. The Illumina Human Methylation 450K Bead Chip assay (Illumina Inc., San Diego, CA) also covers miRNA promoter regions therefore we also examined methylation of the miRNA promoter regions with 200bp proximity.

HRM analysis

HRM analysis was performed for genes including NINJ2, HSPD1, TEX14, HLA-C, RAD51C, FMN2, DGKQ, LIPT1, GJA9 and MYCBP. HRM analysis was performed on the LightCycler 480 II system (Roche Diagnostics, Mannheim, Germany). Each reaction mixture contained 5 µl of precision melt supermix (Bio-rad), 0.5 µl of each primer (200 nM) and 1-50 ng of DNA. The total reaction volume was 10 µl. The analyses were performed on a 96 well plate in triplicates. An initial pre-incubation step was set at 95°C for 10 minutes, followed by 45 cycles of 95°C for 10 seconds, with annealing at 58°C for 15s and extension at 72°C for 25s. Melting analysis was designed to cover temperatures from 65 to 95°C where temperature was increases at 0.1°C increments. Melt curve data was performed on the LightCycler software.

Statistical analysis

Quality control checks were performed for target removal, staining for non-polymorphic probes, staining for negative controls, target removal, bisulfide conversion efficiency, hybridization efficiency and specificity. Analysis of the methylation data was performed with the GenomeStudio Illumina methylation module (version 1.8). Methylation levels of the different CpG loci were determined by the value of ß (which is the ratio of the intensities between methylated and unmethylated alleles). The detection p value was set to <0.001, to eliminate poorly detected CpGs. Differentially methylated genes were characterized as genes with a fold difference = 2.0. Gene Ontolology and Pathways enrichment analysis was performed using Partek® Genomic SuiteTM software, version 6.6 (Partek Inc., St. Louis, MO) where significant differences in expression were determined at enrichment scores = 3 [18]. ANOVA was used to calculate significance of variation in normalized expression values between sample groups, fold change of gene expressions was calculated as mean ratio.

Results

Participant characteristics

The age of the participants was (CFS/ME: 50.31 ± 2.27 years; non-fatigued controls: 47.44 ± 2.16 years) and full blood counts were performed on whole blood samples from all participants prior to CD4+T cell isolation. There were no significant differences observed in either the age or full blood count parameters examined in the CFS/ME and control groups.

Overall methylation pattern in CD4+T cells

The present study compared and examined DNA methylation subtleties in CD4+T cells from CFS/ME patients and non-fatigued controls. A total of 485 577 methylation sites in the genome were examined. Principle component analysis and hierarchical clustering of differentially methylated genes were used to determine the transcriptome profile and sources of variance in the groups (Figure 1). A predominant trend of genome hypomethylation was observed in the CD4+T cells from the CFS/ME patients compared with the controls. We detected 120 CpGs that were differentially methylated between the two groups and of these 85% were hypomethylated while 14.17% were hypermethylated. 75 of these methylated regions were linked to known genes while the remainder were unknown. These differentially methylated (dmCpG) sites were detected on chromosomes 1-22 with no particular concentration on one chromosome. Structurally these dmCpGs were located on the 3’UTR, 5’UTR and transcription start sites with a large proportion located within the gene. 30% of the CpG islands were associated with gene promoters.

cellular-immunology-DNA-methylation

Figure 1: DNA methylation profiles of CD4+T cells in CFS/ME patients and controls. The heat map and hierarchical clustering results of methylated regions in CD4+T cells from CFS/ME patients and control. The blue regions represent hypomethylation while the red regions represent hypermethylation.

Differential methylated genes

The dmCpGs were found in 75 genes in the CD4+T cells. The gene with the most dmCpG site was NINJ2 where three CpG sites in this region were hypomethylated. Incidentally this gene contained CpG sites that were highly hypomethylated in the CFS/ME patients compared with the controls. This gene has not previously been associated with CFS/ME. Additionally, none of the genes identified in the present study has been previously associated with CFS/ME. miRNA methylation analysis generated 176 miRNAs with significant dmCpGs, of these miRNAs, 82 miRNA genes were hypermethylated while 94 were hypomethylated.

Gene ontology and pathway enrichment profile

To determine whether the dmCpGs were biologically significant, the Partek ontology enrichment tool was applied to all genes with dmCpGs that were significantly altered (p<0.05). Gene enrichment analysis detected 59 different functional terms (p<0.05) that were associated with dmCpG sites and these can be classified in to three groups, biological, cellular and molecular processes (Table 1). The most defining factor to suggest that these genes were detected in CD4+T cells was the observation that some of these genes were related to MHC Class II receptor activity (HLA-DQB1). Genetic pathways specific to these sites were determined using the Kegg pathway analysis. The Kegg pathway analysis identified HLA-C and HLA-DQB1 as genes in the pathway with the highest enrichment score (Table 1).

Gene Full name Fold change P-value Methylation Chromosome Function
NINJ2 Ninjurin (for nerve injury induced) -6.01644 0.025353 hypomethylation 12 Neuron adhesion
TXNRD1 Thioredoxinreductase 1 -3.55680 0.000165 hypomethylation 12 Ribonucleotide binding, nucleotide binding, oxidation reduction
BRWD1 Bromodomain and WD repeat domain containing 1 -3.31289 0.000813 hypomethylation 21  
ATP9B ATPase, class II, type 9B -3.26934 0.014517 hypomethylation 18 Nucleotide biosynthetic process, purine biosynthetic process
ASXL2 Additionalsex combs like 2 (Drosophila) -3.11677 0.028253 hypomethylation 2  
HSPE1 Heatshock 10kDa protein 1 -3.07642 0.002192 hypomethylation 2 Purine nucleotide binding, Ribonucleotide binding, nucleotide binding,
HSPD1 Heatshock 60kDa protein 1 (chaperonin) -3.03374 0.002294 hypomethylation 2 Purine nucleotide binding, Ribonucleotide binding, nucleotide binding
KDM2B Lysine(K)-specific demethylase 2B -3.01638 0.002514 hypomethylation 12 Oxidation reduction,
COG3 Component of oligomeric golgi complex 3 -2.96826 0.025353 hypomethylation 13 Protein localization in organelle, intra-Golgi vesicle-mediated transport, cellular macromolecule localization, cellular protein localization, retrograde vesicle-mediated transport, Golgi to ER, Golgi transport complex, protein transporter activity
NR3C1 Nuclear receptor subfamily 3, group C, member 1 (glucocorticoid receptor) -2.66399 0.008423 hypomethylation 5 Intracellular signalling cascade,
ADAMTSL1 ADAMTS (a disintegrin and metalloproteinase with thrombospondin motif) -2.65750 0.005077 hypomethylation 9  
SELT Selenoprotein T -2.57892 0.002665 hypomethylation 3  
MX1 Myxovirus (influenza virus) resistance 1, interferon-inducible protein p78 (mouse) -2.53843 0.045739 hypomethylation 21 Purine ribonucleotide binding,
MACF1 Microtubule-actin crosslinking factor 1 -2.53607 0.001098 hypomethylation 1 Protein localization in organelle, cellular macromolecule localization, cellular protein localization, microtubule cytoskeleton,
FRMD4A FERM domain containing 4Ap -2.47480 0.003371 hypomethylation 10  
DMXL1 Dmx-like 1 -2.43990 0.029074 hypomethylation 5  
PGD Phosphogluconate dehydrogenase -2.43270 0.003595 hypomethylation 1 Ribonucleotide binding
MARK1 MAP/microtubule affinity-regulating kinase 1 -2.42785 0.016744 hypomethylation 1 Nucleotide binding, protein kinase cascade, protein amino acid phosphorylation, intracellular signalling cascade, microtubule cytoskeleton, protein kinase activity, phosphotransferase activity, alcohol group as acceptor
FAM13B Family with sequence similarity 13, member B -2.41743 0.006546 hypomethylation 5 GTPase activator activity
ATM Ataxia telangiectasia mutated -2.41147 0.020951 hypomethylation 11 Purine ribonucleotide binding, ribonucleotide binding, nucleotide binding, protein amino acid phosphorylation, intracellular signalling cascade, microtubule cytoskeleton,
NPAT Nuclear protein, ataxia-telangiectasia locus -2.40925 0.020950 hypomethylation 11  
SDCCAG10 CWC27 spliceosome-associated protein homolog (S. cerevisiae) -2.39996 0.004538 hypomethylation 5  
RSBN1 Round spermatid basic protein 1 -2.38842 0.016944 hypomethylation 1  
MED13 Mediator complex subunit 13 -2.32252 0.009664 hypomethylation 17 VitaminD receptor binding, intracellular signalling cascade,
MATN2 Matriline 2 -2.31173 0.021316 hypomethylation 8  
RAD51 RAD51 recombinase -2.28733 0.025275 hypomethylation 15 Purine ribonucleotide binding, ribonucleotide binding, nucleotide binding,
SLC4A5 Solute carrier family 4 (sodium bicarbonate cotransporter), member 5 -2.27009 0.015108 hypomethylation 2 Anion transmembrane transporter activity,
WBSCR17 Williams-Beuren syndrome chromosome region 17 -2.26845 0.015743 hypomethylation 7  
RBM25 RNA binding motif protein 25 -2.24768 0.046929 hypomethylation 14 Ribonucleotide binding, nucleotide binding,
DOCK4 Dedicator of cytokinesis 4 -2.22303 0.023445 hypomethylation 7 Rho GTPase binding, RacGTPase activator activity, RacGTPase binding,
CCDC148 Coiled-coil domain containing 148 -2.19913 0.014485 hypomethylation 2  
PHF19 PHD finger protein 19 -2.19496 0.010693 hypomethylation 9  
RPS6KA2 Ribosomal protein S6 kinase, 90kDa, polypeptide 2 -2.19420 0.004795 hypomethylation 6 Proteinkinase cascade, protein amino acid phosphorylation, intracellular signalling cascade, proteinserine/threonine kinase activity
CTSO Cathepsin O -2.18726 0.035781 hypomethylation 4  
STK17B Serine/threonine kinase 17b (apoptosis-inducing) -2.18165 0.029008 hypomethylation 2 Proteinserine/threonine kinase activity, protein kinase activity, phosphotransferase activity, alcohol group as acceptor
SGK1 Serum glucocorticoid regulated kinase 1 -2.14645 0.013318 hypomethylation 6 Phosphotransferase activity, alcohol group as acceptor, protein amino acid phosphorylation
HLA-C Major histocompatibility complex, class I, C -2.13388 0.006649 hypomethylation 6 MHC class II receptor activity
C20orf3 Chromosome 3 open reading frame, human -2.13244 0.025289 hypomethylation 20  
HLA-C   -2.12459 0.027134 hypomethylation 6 MHC class II receptor activity
ATP13A3 ATPase type 13A3 -2.12378 0.047204 hypomethylation 3 Nucleotide biosynthetic process, purine nucleotide biosynthetic process,
PHF12 PHD finger protein 12 -2.10335 0.024860 hypomethylation 17  
A2BP1 RNA binding protein, fox-1 homolog (C. elegans) 1 -2.10170 0.000697 hypomethylation 16  
MCC Mutatedin colorectal cancers -2.09595 0.028307 hypomethylation 5  
ANKH ANKH inorganic pyrophosphate transport regulator -2.07819 0.041987 hypomethylation 5 Locomotory behaviour, regulation of bone mineralization, inorganic anion transmembrane transporter activity
ERICH1 Glutamate-rich 1 -2.07436 0.013699 hypomethylation 8  
PHC3 Polyhomeotichomolog 3 (Drosophila) -2.05013 0.016319 hypomethylation 3  
LOC404266   -2.04836 0.018899 hypomethylation 17  
HOXB5 HomeoboxB5 -2.04707 0.018899 hypomethylation 17  
KHDRBS2 KH domain containing, RNA binding, signal transduction associated 2 -2.04293 0.022498 hypomethylation 6  
CRIM1 Cysteine rich transmembrane BMP regulator 1 (chordin-like) -2.04276 0.030130 hypomethylation 2 Proteinkinase activity,
AMACR Alpha-methylacyl-CoA racemase -2.04223 0.015920 hypomethylation 5  
ASNA1 arsAarsenite transporter, ATP-binding, homolog 1 (bacterial) -2.03930 0.010834 hypomethylation 19 Inorganic anion transmembrane transporter activity
BNIP2 BCL2/adenovirus E1B 19kDa interacting protein 2 -2.03728 0.040322 hypomethylation 15 GTPase activator activity,
NDUFA5 NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 5 -2.03695 0.020507 hypomethylation 7 Oxidation reduction, mitochondrial respiratory chain, respiratory chain,
UNC84A Sad1 and UNC84 domain containing 1 -2.03190 0.000411 hypomethylation 7  
BTAF1 BTAF1 RNA polymerase II, B-TFIID transcription factor-associated, 170kDa -2.02822 0.042294 hypomethylation 10 Ribonucleotide binding,
OXA1L Oxidase (cytochrome c) assembly 1-like 2.00760 0.034505 hypomethylation 14 Oxidation reduction, negative regulation of ATPase activity, proton-transporting two-sector ATPase complex assembly, mitochondrial respiratory chain complex assembly, aerobic respiration, negative regulation of hydrolase activity, cellular macromolecule localization, cellular protein localization
GMNN Geminin, DNA replication inhibitor 2.02374 0.012055 hypomethylation 6 Negative regulation of DNA replication, nuclear export, Cajal body
LSG1 Large60S subunit nuclear export GTPase 1 2.07421 0.019916 hypermethylation 3  
NUDT1 Nudix (nucleoside diphosphate linked moiety X)-type motif 1 2.11897 0.002342 hypermethylation 7  
FTSJ2 FtsJ RNA methyltransferase homolog 2 (E. coli) 2.12453 0.002342 hypermethylation 7  
HLA-DQB1 Majorhistocompatibility complex, class II, DQ beta 1 2.29574 0.033972 hypermethylation 6 MHC class II receptor activity
ADAMTS12 ADAM metallopeptidase with thrombospondin type 1 motif, 12 2.32516 0.033508 hypermethylation 5  
MED12L Mediator complex subunit 12-like: 2.40530 0.033766 hypermethylation 3  
GPR171 G protein-coupled receptor 171 2.42853 0.033766 hypermethylation 3  
TEX14 Testis expressed 14 3.09392 0.035419 hypermethylation 17 Protein amino acid phosphorylation, protein kinase activity, transferase activity, transferring phosphorus-containing groups
RAD51C RAD51 paralog C 3.47220 0.035419 hypermethylation 17 Purine ribonucleotide binding
MYCBP MYC binding protein;  3.52884 0.000838 hypermethylation 1  
GJA9 Gap junction protein, alpha 9, 59kDa 3.95041 0.000838 hypermethylation 1  
LIPT1 Lipoyl transferase1 5.93865 0.029695 hypermethylation 2  
TSGA10 Testis specific, 10 -6.01644 0.029695 Hypermethylation 2  
DGKQ Diacylglycerol kinase, theta 110kDa -3.66709 0.013574 Hypermethylation 4 Proteinkinase cascade, intracellular signalling cascade
FMN2 formin 2 -3.55680 0.019258 Hypermethylation 1 Establishment of spindle localization, metaphase, cytokinesis during cell cycle, establishment of chromosome localization, intracellular signalling cascade
C1orf52 Chromosome1 open reading frame 52 -3.34680 0.015531 Hypermethylation 1  
PSMD2 Proteasome (prosome, macropain) 26S subunit, non-ATPase, 2 -3.31289 0.023962 Hypermethylation 3 Proteasome regulatory particle
DGKQ Diacylglycerol kinase, theta 110kDa -3.26934 0.016689 hypermethylation 4 Protein kinase cascade, intracellular signalling cascade

Table 1: A list of genes with differential methylated regions in the patient group.

MicroRNA methylation pattern in CD4+T cells

MicroRNA methylation patterns were examined in the CD4+T cells from CFS/ME patients and non-fatigued controls. A total of 2291 methylation sites observed. Of these, 133 were differentially methylated between the two groups where 51.9% were hypomethylated while 48.1% were hypermethylated (Table 2).

Chromosome miRNA Start End Methylation Fold Change P-value Target Gene
chr2 hsa-mir-7845 208031124 208031222 Hypomethlation -1.17746 0.000205 KLF7
chr2 hsa-mir-5001 233415184 233415283 Hypomethlation -1.10555 0.000806 EIF4E2;TIGD1
chr14 hsa-mir-376c 101506027 101506092 hypermethylation 1.42619 0.001340  
chr3 hsa-mir-4444-2 75263627 75263700 hypermethylation 1.07197 0.001379  
chr22 hsa-mir-3928 31556048 31556105 hypermethylation 1.14670 0.001707  
chr19 hsa-mir-6795 15290094 15290161 Hypomethlation -1.00656 0.002333  
chr22 hsa-mir-3653 29729147 29729256 hypermethylation 1.01139 0.002345  
chr19 hsa-mir-520b 54204481 54204541 Hypomethlation -1.04360 0.002389  
chr17 hsa-mir-33b 17717150 17717245 hypermethylation 1.01560 0.002862  
chr19 hsa-mir-639 14640355 14640452 Hypomethlation -1.11001 0.003032 TECR;MIR639
chr9 hsa-mir-4674 139440625 139440711 Hypomethlation -1.14622 0.003294 NOTCH1
chr15 hsa-mir-3175 93447629 93447705 Hypomethlation -1.11911 0.003765 CHD2
chr14 hsa-mir-4710 105144031 105144086 Hypomethlation -1.12202 0.003959  
chr8 hsa-mir-124-2 65291706 65291814 Hypomethlation -1.13882 0.004364  
chr6 hsa-mir-6891 31323001 31323093 Hypomethlation -1.10710 0.004376  
chr5 hsa-mir-143 148808481 148808586 hypermethylation 1.05776 0.005115  
chr11 hsa-mir-130a 57408671 57408759 hypermethylation 1.02682 0.005571  
chr11 hsa-mir-34c 111384164 111384240 Hypomethlation -1.14561 0.006109  
chr11 hsa-mir-139 72326107 72326174 Hypomethlation -1.01062 0.008066  
chr16 hsa-mir-1225 2140196 2140285 Hypomethlation -1.00302 0.008162  
chr1 hsa-mir-760 94312388 94312467 hypermethylation 1.52577 0.008974  
chr17 hsa-mir-142 56408593 56408679 hypomethylation -1.1211 0.009029  
chr19 hsa-mir-638 10829080 10829179 hypermethylation 1.23848 0.009762 MIR638;DNM2
chr14 hsa-mir-496 101526910 101527011 hypomethylation -1.01214 0.009849  
chr11 hsa-mir-1908 61582633 61582712 hypomethylation -1.08932 0.010624 MIR1908;FADS1
chr3 hsa-mir-425 49057581 49057667 hypermethylation 1.12229 0.010864 NDUFAF3;DALRD3; MIR425
chr13 hsa-mir-320d-1 41301964 41302011 hypermethylation 1.05393 0.011027  
chr19 hsa-mir-523 54201639 54201725 hypomethylation -1.01830 0.011236  
chr6 hsa-mir-6891 31323001 31323093 hypomethylation -1.34121 0.011631  
chr19 hsa-mir-498 54177451 54177574 hypomethylation -1.01044 0.011972  
chr20 hsa-mir-124-3 61809852 61809938 hypomethylation -1.12224 0.012418  
chr10 hsa-mir-2110 115933864 115933938 hypomethylation -1.09673 0.012533  
chr9 hsa-mir-6853 35732919 35732992 hypomethylation -1.05786 0.012640 CREB3;TLN1
chr3 hsa-mir-944 189547711 189547798 hypermethylation 1.02836 0.012847  
chr11 hsa-mir-192 64658609 64658718 hypermethylation 1.00819 0.013255  
chr1 hsa-mir-6742 228584749 228584810 hypermethylation 1.01184 0.013631  
chr19 hsa-mir-4321 2250638 2250717 hypermethylation 1.06948 0.013643  
chr8 hsa-mir-4664 144815253 144815323 hypermethylation 1.04538 0.014353  
chr17 hsa-mir-3184 28444104 28444178 hypermethylation 1.44860 0.014434 MIR423;CCDC55
chr17 hsa-mir-423 28444097 28444190 hypermethylation 1.44860 0.014434 MIR423;CCDC55
chr5 hsa-mir-340 179442303 179442397 hypermethylation 1.01906 0.014508  
chr8 hsa-mir-596 1765397 1765473 hypomethylation -1.19681 0.014675 MIR596
chr6 hsa-mir-6834 33258022 33258102 hypermethylation 1.07284 0.015375  
chr2 hsa-mir-375 219866367 219866430 hypomethylation -1.56597 0.015549  
chr19 hsa-mir-181c 13985513 13985622 hypermethylation 1.00944 0.015609  
chr19 hsa-mir-181d 13985689 13985825 hypermethylation 1.00944 0.015609  
chr4 hsa-mir-5091 13629489 13629581 hypermethylation 1.35117 0.016711 BOD1L
chr20 hsa-mir-647 62573984 62574079 hypermethylation 1.01182 0.017086  
chr1 hsa-mir-6733 43637323 43637383 hypomethylation -1.04073 0.017516 WDR65;EBNA1BP2
chr19 hsa-mir-330 46142252 46142345 hypomethylation -1.11000 0.018314 MIR330;EML2
chr8 hsa-mir-6876 25202918 25202990 hypermethylation 1.26651 0.018319  
chr19 hsa-mir-4746 4445975 4446045 hypomethylation -1.00382 0.018569  
chr2 hsa-mir-375 219866367 219866430 hypomethylation -1.38810 0.019266  
chr19 hsa-mir-638 10829080 10829179 hypomethylation -1.09026 0.020249 DNM2
chr21 hsa-mir-155 26946292 26946356 hypomethylation -1.67121 0.020383  
chr3 hsa-mir-15b 160122376 160122473 hypermethylation 1.07586 0.020474  
chr3 hsa-mir-16-2 160122533 160122613 hypermethylation 1.07586 0.020474  
chr19 hsa-mir-642a 46178186 46178282 hypermethylation 1.00701 0.020574  
chr19 hsa-mir-642b 46178190 46178266 hypermethylation 1.00701 0.020574  
chr22 hsa-mir-658 38240279 38240378 hypomethylation -1.16552 0.020627 ANKRD54;MIR658
chr3 hsa-let-7g 52302294 52302377 hypomethylation -1.15178 0.021046  
chr15 hsa-mir-4515 83736087 83736167 hypomethylation -1.05581 0.021243 BTBD1
chr17 hsa-mir-4523 27717680 27717748 hypomethylation -1.14820 0.021784  
chr7 hsa-mir-6840 99954274 99954344 hypermethylation 1.01168 0.022112  
chr17 hsa-mir-10a 46657200 46657309 hypermethylation 1.14698 0.022855  
chr20 hsa-mir-124-3 61809852 61809938 hypomethylation -1.10597 0.023198  
chr17 hsa-mir-4523 27717680 27717748 hypermethylation 1.32887 0.023291  
chr2 hsa-mir-3679 134884696 134884763 hypomethylation -1.02140 0.023375  
chr1 hsa-mir-6733 43637323 43637383 hypomethylation -1.47410 0.023379 WDR65;EBNA1BP2
chr3 hsa-mir-4792 24562853 24562926 hypomethylation -1.11756 0.023586  
chr17 hsa-mir-1288 16185328 16185402 hypermethylation 1.03806 0.023703  
chr2 hsa-mir-4444-1 178077454 178077527 hypermethylation 1.10018 0.023789 HNRNPA3
chr17 hsa-mir-378j 35974976 35975084 hypomethylation -1.00674 0.024333  
chr2 hsa-mir-1471 232756952 232757008 hypomethylation -1.00916 0.024522  
chr17 hsa-mir-632 30677128 30677221 hypomethylation -1.17464 0.025691 ZNF207;MIR632
chr11 hsa-mir-1304 93466840 93466930 hypermethylation 1.11806 0.026275  
chr15 hsa-mir-3175 93447629 93447705 hypomethylation -1.14724 0.026841 CHD2
chr20 hsa-mir-663a 26188822 26188914 hypomethylation -1.16412 0.026892 MIR663
chr9 hsa-mir-4672 130631694 130631774 hypomethylation -1.00712 0.027149  
chr3 hsa-mir-885 10436173 10436246 hypomethylation -1.00941 0.027299  
chr7 hsa-mir-183 129414745 129414854 hypermethylation 1.01084 0.027378  
chr7 hsa-mir-96 129414532 129414609 hypermethylation 1.01084 0.027380  
chr12 hsa-mir-141 7073260 7073354 hypermethylation 1.01399 0.027805  
chr7 hsa-mir-590 73605528 73605624 hypomethylation -1.01026 0.028022  
chr12 hsa-mir-1178 120151439 120151529 hypermethylation 1.01277 0.028608  
chr4 hsa-mir-1973 117220881 117220924 hypermethylation 1.08332 0.028757  
chr2 hsa-mir-375 219866367 219866430 hypomethylation -1.29192 0.029281  
chr10 hsa-mir-2110 115933864 115933938 hypomethylation -1.09997 0.029535 MIR2110;C10orf118
chr10 hsa-mir-1296 65132717 65132808 hypermethylation 1.01367 0.029866  
chr19 hsa-mir-23a 13947401 13947473 hypermethylation 1.06274 0.029990 MIR27A;MIR24-2
chr19 hsa-mir-24-2 13947101 13947173 hypermethylation 1.06274 0.029990  
chr19 hsa-mir-27a 13947254 13947331 hypermethylation 1.06274 0.029990 MIR27A;MIR24-2
chr17 hsa-mir-152 46114527 46114613 hypomethylation -1.30883 0.030070 MIR152;COPZ2
chr17 hsa-mir-6516 75085499 75085579 hypermethylation 1.14486 0.030396 SCARNA16;  C17orf86
chr8 hsa-mir-320a 22102475 22102556 hypermethylation 1.08542 0.030987 POLR3D;MIR320A
chr1 hsa-mir-320b-2 224444706 224444843 hypomethylation -1.00847 0.031140  
chr1 hsa-mir-5087 147806603 147806678 hypomethylation -1.47707 0.031743  
chr17 hsa-mir-4521 8090263 8090322 hypomethylation -1.10388 0.032254  
chr14 hsa-mir-496 101526910 101527011 hypomethylation -1.01232 0.033237  
chr1 hsa-mir-320b-1 117214371 117214449 hypermethylation 1.01642 0.033722  
chr2 hsa-mir-933 176032361 176032437 hypermethylation 1.15198 0.033760 ATF2;MIR933
chr12 hsa-mir-7107 121882076 121882155 hypermethylation 1.01076 0.033806 KDM2B
chr14 hsa-mir-6717 21491473 21491545 hypermethylation 1.03920 0.033889  
chr7 hsa-mir-4648 2566708 2566779 hypermethylation 1.00823 0.034164  
chr10 hsa-mir-938 29891193 29891275 hypomethylation -1.00845 0.034456  
chr6 hsa-mir-6832 31601564 31601635 hypermethylation 1.00795 0.034535  
chr15 hsa-mir-627 42491768 42491864 hypomethylation -1.01772 0.035449  
chr3 hsa-mir-128-2 35785968 35786051 hypermethylation 1.01399 0.035564  
chr3 hsa-mir-4444-2 75263627 75263700 hypermethylation 1.06916 0.035786  
chr14 hsa-mir-300 101507700 101507782 hypermethylation 1.40643 0.036924  
chr6 hsa-mir-6891 31323001 31323093 hypomethylation -1.13122 0.037499  
chr17 hsa-mir-142 56408593 56408679 hypomethylation -1.10981 0.038468  
chr14 hsa-mir-409 101531637 101531715 hypomethylation -1.01538 0.038623  
chr14 hsa-mir-412 101531784 101531874 hypomethylation -1.01538 0.038623  
chr15 hsa-mir-628 55665138 55665232 hypermethylation 1.06044 0.038658  
chr13 hsa-mir-8073 110993305 110993376 hypomethylation -1.12176 0.039730  
chr4 hsa-mir-572 11370451 11370545 hypermethylation 1.13340 0.040330  
chr22 hsa-mir-3928 31556048 31556105 hypermethylation 1.20598 0.040468  
chr14 hsa-mir-4505 74225450 74225522 hypomethylation -1.10764 0.040469  
chr17 hsa-mir-365b 29902430 29902540 hypomethylation -1.00811 0.040829  
chr17 hsa-mir-4725 29902288 29902377 hypomethylation -1.00811 0.040829  
chr19 hsa-mir-4754 58898137 58898225 hypomethylation -1.12062 0.040839 RPS5
chr5 hsa-mir-1229 179225278 179225346 hypomethylation -1.00370 0.041022  
chr15 hsa-mir-7706 85923827 85923893 hypomethylation -1.10880 0.041546 AKAP13
chr11 hsa-mir-4492 118781417 118781496 hypomethylation -1.13724 0.042569 BCL9L
chr18 hsa-mir-4741 20513312 20513401 hypomethylation -1.13235 0.042592 RBBP8
chr13 hsa-mir-19a 92003145 92003226 hypermethylation 1.03689 0.043500  
chr13 hsa-mir-19b-1 92003446 92003532 hypermethylation 1.03689 0.043500  
chr13 hsa-mir-20a 92003319 92003389 hypermethylation 1.03689 0.043500  
chr13 hsa-mir-92a-1 92003568 92003645 hypermethylation 1.03689 0.043500  
chr12 hsa-mir-7107 121882076 121882155 hypomethylation -1.00568 0.044221 KDM2B
chr8 hsa-mir-661 145019359 145019447 hypermethylation 1.01970 0.044401  
chr14 hsa-mir-4308 55344831 55344911 hypermethylation 1.00911 0.044475  
chr12 hsa-mir-1251 97885687 97885756 hypermethylation 1.02643 0.045113  
chr5 hsa-mir-4638 180649566 180649633 hypermethylation 1.03734 0.045950 TRIM41
chr11 hsa-mir-4687 3877292 3877371 hypomethylation -1.06416 0.046474 STIM1
chr19 hsa-mir-1909 1816158 1816237 hypermethylation 1.01084 0.046474  
chr10 hsa-mir-1307 105154010 105154158 hypermethylation 1.01492 0.046703  
chr19 hsa-mir-769 46522190 46522307 hypermethylation 1.02118 0.046976  
chr3 hsa-mir-6828 170140891 170140950 hypomethylation -1.00978 0.047242  
chr7 hsa-mir-339 1062569 1062662 hypermethylation 1.02186 0.047576  
chr1 hsa-mir-181a-1 198828173 198828282 hypermethylation 1.02246 0.048357  
chr1 hsa-mir-181b-1 198828002 198828111 hypermethylation 1.02246 0.048356  
chr14 hsa-mir-369 101531935 101532004 hypomethylation -1.00698 0.048589  
chr14 hsa-mir-409 101531637 101531715 hypomethylation -1.00698 0.048589  
chr14 hsa-mir-412 101531784 101531874 hypomethylation -1.00698 0.048589  
chr7 hsa-mir-196b 27209099 27209182 hypomethylation -1.09179 0.048613 MIR196B
chr11 hsa-mir-675 2017989 2018061 hypomethylation -1.01792 0.048989  
chr19 hsa-mir-638 10829080 10829179 hypomethylation -1.15170 0.049180 DNM2;MIR638
chr11 hsa-mir-34c 111384164 111384240 hypomethylation -1.04702 0.049253  
chr2 hsa-mir-5703 228336848 228336903 hypomethylation -1.11947 0.049776 AGFG1
chr8 hsa-mir-6850 146017316 146017376 hypomethylation -1.25334 0.050049 RPL8
chr6 hsa-mir-6891 31323001 31323093 hypomethylation -1.16699 0.050185  
chr2 hsa-mir-5001 233415184 233415283 hypomethylation -1.08397 0.050224 EIF4E2;TIGD1
chr11 hsa-mir-194-2 64658827 64658911 hypomethylation -1.00694 0.051008  
chr17 hsa-mir-33b 17717150 17717245 hypermethylation 1.01486 0.051177  
chr17 hsa-mir-6777 17716794 17716859 hypermethylation 1.01486 0.051177  
chr22 hsa-mir-3653 29729147 29729256 hypermethylation 1.01870 0.051352  
chr14 hsa-mir-127 101349316 101349412 hypomethylation -1.01120 0.051369  
chr19 hsa-mir-519a-2 54265598 54265684 hypermethylation 1.02831 0.051921  
chr14 hsa-mir-369 101531935 101532004 hypomethylation -1.00538 0.052194  
chr14 hsa-mir-409 101531637 101531715 hypomethylation -1.00538 0.052194  
chr14 hsa-mir-412 101531784 101531874 hypomethylation -1.00538 0.052194  
chr19 hsa-mir-638 10829080 10829179 hypomethylation -1.07948 0.052329 DNM2;MIR638
chr7 hsa-mir-550a-2 32772593 32772689 hypomethylation -1.03930 0.052335  
chr7 hsa-mir-550b-2 32772593 32772689 hypomethylation -1.03930 0.052335  
chr14 hsa-mir-1247 102026624 102026759 hypomethylation -1.09325 0.052954 DIO3
chr8 hsa-mir-1208 129162362 129162434 hypermethylation 1.02168 0.053031  
chr22 hsa-mir-1286 20236657 20236734 hypermethylation 1.01313 0.053109  
chr1 hsa-mir-4259 159869769 159869869 hypomethylation -1.06762 0.053249  
chr1 hsa-mir-200b 1102484 1102578 hypermethylation 1.01675 0.053309 MIR200B;MIR200A
chr9 hsa-mir-204 73424891 73425000 hypermethylation 1.00784 0.053460  
chr16 hsa-mir-662 820183 820277 hypomethylation -1.01052 0.053469  
chr4 hsa-mir-572 11370451 11370545 hypermethylation 1.08956 0.054065  
chr2 hsa-mir-933 176032361 176032437 hypermethylation 1.28796 0.054611 ATF2;MIR933
chr16 hsa-mir-762 30905224 30905306 hypomethylation -1.08783 0.054681 BCL7C;MIR762

Table 2: A list of miRNA genes with differential methylated regions in the CFS/ME patients in comparison to controls.

Validation of methylated genes via HRM

Of the genes that were selected for HRM analysis we observed significant changes in the melting peak temperatures of DGKQ (Figure 2).

cellular-immunology-HRM-Validation

Figure 2: HRM Validation of some methylated genes in the CD4+T cells from the CFS/ME patients and controls. The bar graphs represent the average melting temperatures of the genes examined, where the black bars are results from the CFS/ME patients and the white bars are results from the controls. * denotes significance at pvalue <0.05.

Discussion

This is the first study to report on a genome-wide DNA methylation analysis in CD4+T cells from CFS/ME patients. This is also the first study to demonstrate significant hypo-and hyper- methylation sites in CD4+T cells in CFS/ME patients. A predominant hypomethylation was observed in the CFS/ME patients and these were mostly located in the promoter regions of genes. The genes with dmCpG sites were associated with a number of gene ontology terms and pathways which were significantly enriched in the CFS/ME group.

Pathway enrichment analysis showed that most of the genes with significant dmCpG sites were involved in forty seven different pathways. Among these pathways the most significantly enriched were involved in type I diabetes mellitus, autoimmune thyroid disease, viral myocarditis, antigen processing and presentation and cell adhesion molecules. The genes related to these pathways were HLA-C and HLA-DQB1. HLA-C is a major histocompatibility complex I (MHCI) gene which has been associated with the progression of Human Immunodeficiency Virus (HIV) [19,20]. The HLA-C molecule is recognized by Killer Immunoglobulin-like Receptors (KIR)s, KIR2DL1 and KIR2DL2,3. Amongst the CD4+T cells there is a subgroup of cells characterized by non-MHC restricted cytotoxicity, with Natural Killer (NK)-like activity and HLA-Cw7 dependent inhibition of cytotoxic activity [21-24]. Methylation in HLA-C may therefore affect cytotoxic activity mediated by CD4+T cells and may suggest a role of HLA-C restricted T cell response in CFS/ME. In CFS/ME patients, cytotoxic activity is known to be reduced in both NK and CD8+T cells, a global reduction in NK activity may persist in CFS/ME patients and this may be related to changes in the epigenetic patterns that regulate cytotoxic activity. Additionally, methylation in HLA-C may represent diversity in HLA-C restricted T cells as a consequence of reduced HLA-C expression on dendritic cells in the thymus during T cell thymic development [25]. HLA-DQB1 is a MHCII gene. Polymorphisms in a number of HLA-DQB1 haplotypes have been associated with susceptibility to certain types of cancers [26]. In particular, HLA-DQB1 has been identified as a risk factor for oesophageal cancer [27,28]. HLA-DQ alleles, in particular HLA-DQA1*01 and HLA-DQB1*06 have been observed to be increased in CFS/ME patients although the increase in HLA-DQB1*06 was only minimal [29].

Methylation in genes related to molecular processes such as membrane transport, kinase activity, nucleotide and ribonucleotide binding, GTPase activity and transferase activity, may suggest breakdown in molecular processes specific to CD4+T cells in these patients. For example STK17B is involved in calcium and ROS signalling in CD4+T cells [30] and hypomethylation in this gene may alter this process. Additionally, methylation in TXNRD1 may affect the antioxidant capacity of T cells [31]. RPS6KA2 and SGK1 are associated with the regulation of the mechanistic target of rapamycin (mTOR) signalling which is important in lymphocyte survival, growth, differentiation and proliferation of T cells ensuring efficient metabolism, cytoskeletal organization and apoptosis [32-34]. Importantly, RPS6KA2 is also observed to be associated with the MAPK signalling pathway and these are important in T cell mediated responses. During T cell activation HSPE1 and HSPD1 form a complex which regulates the activity of pro-caspase 3 [35]. Differential methylation in these genes may therefore be detrimental to caspase activity. Importantly, mitochondrial dysfunction has been proposed to be involved in the CFS/ME disease presentation [36-38], this may be associated with changes in NDUFA and OXA1L. Similarly, genes responsible for DNA repair, including ATM, RAD51 and RAD51C, were also differentially methylated in the CFS/ME patients.

Although, the precise function of a number of these genes in T cells is unknown (DOCK4, BRWD1, ASXL2, MED13, NPAT, C20ORF3 and PHF19) the observation that most of these genes are related to intracellular processes suggest potential abnormalities within the cell that may present in the form of increased cell numbers and changes in cytokine levels.

DGKζ (Diacylglycerol kinase, theta) is expressed in T cells and is known to regulate the magnitude of the TCR response by inhibiting MAPK activation and the expression of co-stimulatory molecules such as CD69 and CD25 [39]. In CFS/ME little is known about the status of the TCR. However, modulations in the expression of DGKζ may be important in the mechanism of the disorder.

Of the 176 miRNAs with significant dmCpGs, eight were CD4+T cell specific miRNA genes including miR-124, miR-155, miR-181a, miR-142, miR-27a, miR-339, miR-340 and miR-425. MiR-155 is necessary for the differentiation and proliferation of CD4+T cells in to the four main subsets (Th1, Th2, Th17 and Tregs) [40-42]. In FOXP3 specific Tregs, miR-155 is upregulated and a decrease in miR-155 decreases the number of these cells [40]. Changes in miR-155 regulation may account for the increases in Tregs observed in CFS/ME patients and the shifts in Th1/Th2 immune related responses [43]. Activation of the T cell receptor (TCR) involves a number of signalling pathways whose genes were methylated in the present study. Importantly, TCR signalling is modulated by miR-181a, as it inhibits autoreactivity by promoting central tolerance and enhancing TCR responsiveness [44,45]. In the absence of miR-181a, autoreactive immune responses occur resulting in autoimmunity. miR-124 and miR-27a are over expressed in central memory and effector memory CD4+T cells respectively during differentiation [46]. Among the CD4+ helper T cells, miR-425 is reduced in Th1 cells while miR-142 is increased in expression in Tregs [46].

In conclusion, the present study has identified for the first time potential disruption in epigenetic pathways in CD4+T cells and this may contribute to the pathogenesis of CFS/ME. The most important finding in the present study is the dmCpG in the DGKζ gene and CD4+T cell specific miRNAs. As previous studies have observed changes in these molecules in CFS/ME patients it is possible to posit that these molecules may be important in deciphering a mechanism for CFS/ME. Epigenetic changes in immune cells may be an important component of CFS/ME.

Acknowledgement

This project was funded by grant contributions from the Alison Hunter Memorial Foundation, Mason Foundation and Queensland Government Co-Investment Funding Grant Program.

References

  1. Prins JB, van der Meer JW, Bleijenberg G (2006) Chronic fatigue syndrome. Lancet 367: 346-355.
  2. Johnston S, Brenu EW, Staines D, Marshall-Gradisnik S (2013) The prevalence of chronic fatigue syndrome/ myalgic encephalomyelitis: a meta-analysis. Clin Epidemiol 5: 105-110.
  3. Fukuda K, Straus SE, Hickie I, Sharpe MC, Dobbins JG, et al. (1994) The chronic fatigue syndrome: a comprehensive approach to its definition and study. International Chronic Fatigue Syndrome Study Group. Ann Intern Med 121: 953-959.
  4. Brenu EW, Ashton KJ, van Driel M, Staines DR, Peterson D, et al. (2012) Cytotoxic lymphocyte microRNAs as prospective biomarkers for Chronic Fatigue Syndrome/Myalgic Encephalomyelitis. J Affect Disord 141: 261-269.
  5. Brenu EW, van Driel ML, Staines DR, Ashton KJ, Hardcastle SL, et al. (2012) Longitudinal investigation of natural killer cells and cytokines in chronic fatigue syndrome/myalgic encephalomyelitis. J Transl Med 10: 88.
  6. Brenu EW, Staines DR, Baskurt OK, Ashton KJ, Ramos SB, et al. (2010) Immune and hemorheological changes in chronic fatigue syndrome. J Transl Med 8: 1.
  7. Kaushik N, Fear D, Richards SC, McDermott CR, Nuwaysir EF, et al. (2005) Gene expression in peripheral blood mononuclear cells from patients with chronic fatigue syndrome. J Clin Pathol 58: 826-832.
  8. Frampton D, Kerr J, Harrison TJ, Kellam P (2011) Assessment of a 44 gene classifier for the evaluation of chronic fatigue syndrome from peripheral blood mononuclear cell gene expression. PLoS One 6: e16872.
  9. Kerr JR (2008) Gene profiling of patients with chronic fatigue syndrome/myalgic encephalomyelitis. Curr Rheumatol Rep 10: 482-491.
  10. Kerr JR, Petty R, Burke B, Gough J, Fear D, et al. (2008) Gene expression subtypes in patients with chronic fatigue syndrome/myalgic encephalomyelitis. J Infect Dis 197: 1171-1184.
  11. Kerr JR, Burke B, Petty R, Gough J, Fear D, et al. (2008) Seven genomic subtypes of chronic fatigue syndrome/myalgic encephalomyelitis: a detailed analysis of gene networks and clinical phenotypes. J Clin Pathol 61: 730-739.
  12. Brenu EW, van Driel ML, Staines DR, Ashton KJ, Ramos SB, et al. (2011) Immunological abnormalities as potential biomarkers in Chronic Fatigue Syndrome/Myalgic Encephalomyelitis. J Transl Med 9: 81.
  13. Szyf M (2012) The early-life social environment and DNA methylation. Clin Genet 81: 341-349.
  14. Lewandowska J, Bartoszek A (2011) DNA methylation in cancer development, diagnosis and therapy--multiple opportunities for genotoxic agents to act as methylome disruptors or remediators. Mutagenesis 26: 475-487.
  15. Dolinoy DC, Weidman JR, Jirtle RL (2007) Epigenetic gene regulation: linking early developmental environment to adult disease. Reprod Toxicol 23: 297-307.
  16. Suarez-Alvarez B, Rodriguez RM, Fraga MF, López-Larrea C (2012) DNA methylation: a promising landscape for immune system-related diseases. Trends Genet 28: 506-514.
  17. Agarwal S, Rao A (1998) Modulation of chromatin structure regulates cytokine gene expression during T cell differentiation. Immunity 9: 765-775.
  18. Grayson BL, Aune TM (2011) A comparison of genomic copy number calls by Partek Genomics Suite, Genotyping Console and Birdsuite algorithms to quantitative PCR. BioData Min 4: 8.
  19. Kulkarni S, Qi Y, O'hUigin C, Pereyra F, Ramsuran V, et al. (2013) Genetic interplay between HLA-C and MIR148A in HIV control and Crohn disease. Proc Natl Acad Sci U S A 110: 20705-20710.
  20. Apps R, Qi Y, Carlson JM, Chen H, Gao X, et al. (2013) Influence of HLA-C expression level on HIV control. Science 340: 87-91.
  21. Falk CS, Nössner E, Frankenberger B, Schendel DJ (2000) Non-MHC-restricted CD4+ T lymphocytes are regulated by HLA-Cw7-mediated inhibition. Hum Immunol 61: 1219-1232.
  22. Falk CS, Schendel DJ (1997) HLA-C revisited. Ten years of change. Immunol Res 16: 203-214.
  23. Nössner E, Falk CS, Jantzer P, Reinhardt C, Steinle A, et al. (1996) The HLA likes and dislikes of allospecific and non-MHC-restricted cytotoxic T lymphocytes. Immunol Rev 154: 105-135.
  24. Falk CS, Steinle A, Schendel DJ (1995) Expression of HLA-C molecules confers target cell resistance to some non-major histocompatibility complex-restricted T cells in a manner analogous to allospecific natural killer cells. J Exp Med 182: 1005-1018.
  25. Blais ME, Dong T, Rowland-Jones S (2011) HLA-C as a mediator of natural killer and T-cell activation: spectator or key player? Immunology 133: 1-7.
  26. Feng B, Awuti I, Deng Y, Li D, Niyazi M, et al. (2014) Human papillomavirus promotes esophageal squamous cell carcinoma by regulating DNA methylation and expression of HLA-DQB1. Asia Pac J Clin Oncol 10: 66-74.
  27. Hu J, Li L, Pang L, Chen Y, Yang L, et al. (2012) HLA-DRB1*1501 and HLA-DQB1*0301 alleles are positively associated with HPV16 infection-related Kazakh esophageal squamous cell carcinoma in Xinjiang China. Cancer Immunol Immunother 61: 2135-2141.
  28. Chen YZ, Hu JM, Liu CX, Yang L, Qi Y, et al. (2009) [Correlation between HLA-DRB1*0901 and 1501, HLA-DQB1*0301 and 0602 alleles and esophageal squamous cell carcinoma of Kazakh in Xinjiang, China]. Zhonghua Bing Li Xue Za Zhi 38: 816-819.
  29. Smith J, Fritz EL, Kerr JR, Cleare AJ, Wessely S, et al. (2005) Association of chronic fatigue syndrome with human leucocyte antigen class II alleles. J Clin Pathol 58: 860-863.
  30. Fracchia KM, Pai CY, Walsh CM (2013) Modulation of T Cell Metabolism and Function through Calcium Signaling. Front Immunol 4: 324.
  31. Kaur N, Naga OS, Norell H, Al-Khami AA, Scheffel MJ, et al. (2011) T cells expanded in presence of IL-15 exhibit increased antioxidant capacity and innate effector molecules. Cytokine 55: 307-317.
  32. Zeng H, Chi H (2013) mTOR and lymphocyte metabolism. Curr Opin Immunol 25: 347-355.
  33. Junta CM, Sandrin-Garcia P, Fachin-Saltoratto AL, Mello SS, Oliveira RD, et al. (2009) Differential gene expression of peripheral blood mononuclear cells from rheumatoid arthritis patients may discriminate immunogenetic, pathogenic and treatment features. Immunology 127: 365-372.
  34. Powell JD, Pollizzi KN, Heikamp EB, Horton MR (2012) Regulation of immune responses by mTOR. Annu Rev Immunol 30: 39-68.
  35. Wang M, Windgassen D, Papoutsakis ET (2008) A global transcriptional view of apoptosis in human T-cell activation. BMC Med Genomics 1: 53.
  36. Booth NE, Myhill S, McLaren-Howard J (2012) Mitochondrial dysfunction and the pathophysiology of Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS). Int J Clin Exp Med 5: 208-220.
  37. Castro-Marrero J, Cordero MD, Saez-Francas N, Jimenez-Gutierrez C, Aguilar-Montilla FJ, et al. (2013) Could mitochondrial dysfunction be a differentiating marker between chronic fatigue syndrome and fibromyalgia? Antioxid Redox Signal 19: 1855-1860.
  38. Myhill S, Booth NE, McLaren-Howard J (2009) Chronic fatigue syndrome and mitochondrial dysfunction. Int J Clin Exp Med 2: 1-16.
  39. Mérida I1, Avila-Flores A, Merino E (2008) Diacylglycerol kinases: at the hub of cell signalling. Biochem J 409: 1-18.
  40. Lu LF, Thai TH, Calado DP, Chaudhry A, Kubo M, et al. (2009) Foxp3-dependent microRNA155 confers competitive fitness to regulatory T cells by targeting SOCS1 protein. Immunity 30: 80-91.
  41. O'Connell RM, Kahn D, Gibson WS, Round JL, Scholz RL, et al. (2010) MicroRNA-155 promotes autoimmune inflammation by enhancing inflammatory T cell development. Immunity 33: 607-619.
  42. Rodriguez A, Vigorito E, Clare S, Warren MV, Couttet P, et al. (2007) Requirement of bic/microRNA-155 for normal immune function. Science 316: 608-611.
  43. Brenu EW, Huth TK, Hardcastle SL, Fuller K, Kaur M, et al. (2014) Role of adaptive and innate immune cells in chronic fatigue syndrome/myalgic encephalomyelitis. Int Immunol 26: 233-242.
  44. Li G, Yu M, Lee WW, Tsang M, Krishnan E, et al. (2012) Decline in miR-181a expression with age impairs T cell receptor sensitivity by increasing DUSP6 activity. Nat Med 18: 1518-1524.
  45. Li QJ, Chau J, Ebert PJ, Sylvester G, Min H, et al. (2007) miR-181a is an intrinsic modulator of T cell sensitivity and selection. Cell 129: 147-161.
  46. Sethi A, Kulkarni N, Sonar S, Lal G (2013) Role of miRNAs in CD4 T cell plasticity during inflammation and tolerance. Front Genet 4: 8.
Citation: Brenu EW, Staines DR, Marshall-Gradisnik SM (2014) Methylation Profile of CD4+ T Cells in Chronic Fatigue Syndrome/Myalgic Encephalomyelitis. J Clin Cell Immunol 5:228.

Copyright: © 2014 Brenu EW, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
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