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1.  Overlap between differentially methylated DNA regions in blood B lymphocytes and genetic at-risk loci in primary Sjögren's syndrome 
Annals of the Rheumatic Diseases  2015;75(5):933-940.
Beyond genetics, epigenetics alterations and especially those related to DNA methylation, play key roles in the pathogenesis of autoimmune diseases such as primary Sjögren's syndrome (pSS) and systemic lupus erythematosus. This study aimed to assess the role of methylation deregulation in pSS pathogeny through a genome-wide methylation approach.
Patients and methods
26 female patients with pSS and 22 age-matched controls were included in this study. CD4+ T cells and CD19+ B cells were isolated from peripheral blood mononuclear cells by magnetic microbeads and their genome-wide DNA methylation profiles were analysed using Infinium Human Methylation 450 K BeadChips. Probes with a median DNA methylation difference of at least 7% and p<0.01 between patients and controls were considered significantly differentially methylated.
Methylation alterations were mainly present in B cells compared with T cells. In B cells, an enrichment of genes with differentially methylated probes in genetic at-risk loci was observed, suggesting involvement of both genetic and epigenetic abnormalities in the same genes. Methylation alterations in B cells were more frequent in some specific pathways including Interferon Regulated Genes, mainly among patients who were autoantibody positive. Moreover, genes with differentially methylated probes were over-represented in B cells from patients with active disease.
This study demonstrated more important deregulation of DNA methylation patterns in B cells compared with T cells, emphasising the importance of B cells in the pathogenesis of the disease. Overlap between genes with differentially methylated probes in B lymphocytes and genetic at-risk loci is a new finding highlighting their importance in pSS.
PMCID: PMC4853580  PMID: 26183421
Sjøgren's Syndrome; T Cells; B cells; Autoimmune Diseases; Autoantibodies
2.  Consequences of combining siRNA-mediated DNA methyltransferase 1 depletion with 5-aza-2′-deoxycytidine in human leukemic KG1 cells 
Oncotarget  2015;6(17):15265-15282.
5-azacytidine and 5-aza-2′-deoxycytidine are clinically used to treat patients with blood neoplasia. Their antileukemic property is mediated by the trapping and the subsequent degradation of a family of proteins, the DNA methyltransferases (DNMT1, DNMT3A, and DNMT3B) leading to DNA demethylation, tumor suppressor gene re-expression and DNA damage. Here we studied the respective role of each DNMT in the human leukemia KG1 cell line using a RNA interference approach. In addition we addressed the role of DNA damage formation in DNA demethylation by 5-aza-2′-deoxycytidine. Our data show that DNMT1 is the main DNMT involved in DNA methylation maintenance in KG1 cells and in mediating DNA damage formation upon exposure to 5-aza-2′-deoxycytidine. Moreover, KG1 cells express the DNMT1 protein at a level above the one required to ensure DNA methylation maintenance, and we identified a threshold for DNMT1 depletion that needs to be exceeded to achieve DNA demethylation. Most interestingly, by combining DNMT1 siRNA and treatment with low dose of 5-aza-2′-deoxycytidine, it is possible to uncouple DNA damage formation from DNA demethylation. This work strongly suggests that a direct pharmacological inhibition of DNMT1, unlike the use of 5-aza-2′-deoxycytidine, should lead to tumor suppressor gene hypomethylation and re-expression without inducing major DNA damage in leukemia.
PMCID: PMC4558150  PMID: 25948775
leukemia; DNA methylation; DNMT; 5-aza-2′-deoxycytidine; DNA damage
3.  Identification of Susceptibility Genes for Peritoneal, Ovarian, and Deep Infiltrating Endometriosis Using a Pooled Sample-Based Genome-Wide Association Study 
BioMed Research International  2015;2015:461024.
Characterizing genetic contributions to endometriosis might help to shorten the time to diagnosis, especially in the most severe forms, but represents a challenge. Previous genome-wide association studies (GWAS) made no distinction between peritoneal endometriosis (SUP), endometrioma (OMA), and deep infiltrating endometriosis (DIE). We therefore conducted a pooled sample-based GWAS and distinguished histologically confirmed endometriosis subtypes. We performed an initial discovery step on 10-individual pools (two pools per condition). After quality control filtering, a Monte-Carlo simulation was used to rank the significant SNPs according to the ratio of allele frequencies and the coefficient of variation. Then, a replication step of individual genotyping was conducted in an independent cohort of 259 cases and 288 controls. Our approach was very stringent but probably missed a lot of information due to the Monte-Carlo simulation, which likely explained why we did not replicate results from “classic” GWAS. Four variants (rs227849, rs4703908, rs2479037, and rs966674) were significantly associated with an increased risk of OMA. Rs4703908, located close to ZNF366, provided a higher risk of OMA (OR = 2.22; 95% CI: 1.26–3.92) and DIE, especially with bowel involvement (OR = 2.09; 95% CI: 1.12–3.91). ZNF366, involved in estrogen metabolism and progression of breast cancer, is a new biologically plausible candidate for endometriosis.
PMCID: PMC4334615  PMID: 25722978
4.  Individual and combined effects of DNA methylation and copy number alterations on miRNA expression in breast tumors 
Genome Biology  2013;14(11):R126.
The global effect of copy number and epigenetic alterations on miRNA expression in cancer is poorly understood. In the present study, we integrate genome-wide DNA methylation, copy number and miRNA expression and identify genetic mechanisms underlying miRNA dysregulation in breast cancer.
We identify 70 miRNAs whose expression was associated with alterations in copy number or methylation, or both. Among these, five miRNA families are represented. Interestingly, the members of these families are encoded on different chromosomes and are complementarily altered by gain or hypomethylation across the patients. In an independent breast cancer cohort of 123 patients, 41 of the 70 miRNAs were confirmed with respect to aberration pattern and association to expression. In vitro functional experiments were performed in breast cancer cell lines with miRNA mimics to evaluate the phenotype of the replicated miRNAs. let-7e-3p, which in tumors is found associated with hypermethylation, is shown to induce apoptosis and reduce cell viability, and low let-7e-3p expression is associated with poorer prognosis. The overexpression of three other miRNAs associated with copy number gain, miR-21-3p, miR-148b-3p and miR-151a-5p, increases proliferation of breast cancer cell lines. In addition, miR-151a-5p enhances the levels of phosphorylated AKT protein.
Our data provide novel evidence of the mechanisms behind miRNA dysregulation in breast cancer. The study contributes to the understanding of how methylation and copy number alterations influence miRNA expression, emphasizing miRNA functionality through redundant encoding, and suggests novel miRNAs important in breast cancer.
PMCID: PMC4053776  PMID: 24257477
5.  Differences in Transcription Patterns between Induced Pluripotent Stem Cells Produced from the Same Germ Layer Are Erased upon Differentiation 
PLoS ONE  2013;8(1):e53033.
Little is known about differences between induced pluripotent stem cells produced from tissues originating from the same germ layer. We have generated human myoblast-derived iPS cells by retroviral transduction of human primary myoblasts with the OCT3/4, SOX2, KLF4 and MYC coding sequences and compared them to iPS produced from human primary fibroblasts. When cultivated in vitro, these iPS cells proved similar to human embryonic stem cells in terms of morphology, expression of embryonic stemness markers and gene promoter methylation patterns. Embryonic bodies were derived that expressed endodermal, mesodermal as well as ectodermal markers. A comparative analysis of transcription patterns revealed significant differences in the gene expression pattern between myoblast- and fibroblast-derived iPS cells. However, these differences were reduced in the mesenchymal stem cells derived from the two iPS cell types were compared.
PMCID: PMC3541362  PMID: 23326377
6.  Identification of Type 1 Diabetes–Associated DNA Methylation Variable Positions That Precede Disease Diagnosis 
PLoS Genetics  2011;7(9):e1002300.
Monozygotic (MZ) twin pair discordance for childhood-onset Type 1 Diabetes (T1D) is ∼50%, implicating roles for genetic and non-genetic factors in the aetiology of this complex autoimmune disease. Although significant progress has been made in elucidating the genetics of T1D in recent years, the non-genetic component has remained poorly defined. We hypothesized that epigenetic variation could underlie some of the non-genetic component of T1D aetiology and, thus, performed an epigenome-wide association study (EWAS) for this disease. We generated genome-wide DNA methylation profiles of purified CD14+ monocytes (an immune effector cell type relevant to T1D pathogenesis) from 15 T1D–discordant MZ twin pairs. This identified 132 different CpG sites at which the direction of the intra-MZ pair DNA methylation difference significantly correlated with the diabetic state, i.e. T1D–associated methylation variable positions (T1D–MVPs). We confirmed these T1D–MVPs display statistically significant intra-MZ pair DNA methylation differences in the expected direction in an independent set of T1D–discordant MZ pairs (P = 0.035). Then, to establish the temporal origins of the T1D–MVPs, we generated two further genome-wide datasets and established that, when compared with controls, T1D–MVPs are enriched in singletons both before (P = 0.001) and at (P = 0.015) disease diagnosis, and also in singletons positive for diabetes-associated autoantibodies but disease-free even after 12 years follow-up (P = 0.0023). Combined, these results suggest that T1D–MVPs arise very early in the etiological process that leads to overt T1D. Our EWAS of T1D represents an important contribution toward understanding the etiological role of epigenetic variation in type 1 diabetes, and it is also the first systematic analysis of the temporal origins of disease-associated epigenetic variation for any human complex disease.
Author Summary
Type 1 diabetes (T1D) is a complex autoimmune disease affecting >30 million people worldwide. It is caused by a combination of genetic and non-genetic factors, leading to destruction of insulin-secreting cells. Although significant progress has recently been made in elucidating the genetics of T1D, the non-genetic component has remained poorly defined. Epigenetic modifications, such as methylation of DNA, are indispensable for genomic processes such as transcriptional regulation and are frequently perturbed in human disease. We therefore hypothesized that epigenetic variation could underlie some of the non-genetic component of T1D aetiology, and we performed a genome-wide DNA methylation analysis of a specific subset of immune cells (monocytes) from monozygotic twins discordant for T1D. This revealed the presence of T1D–specific methylation variable positions (T1D–MVPs) in the T1D–affected co-twins. Since these T1D–MVPs were found in MZ twins, they cannot be due to genetic differences. Additional experiments revealed that some of these T1D–MVPs are found in individuals before T1D diagnosis, suggesting they arise very early in the process that leads to overt T1D and are not simply due to post-disease associated factors (e.g. medication or long-term metabolic changes). T1D–MVPs may thus potentially represent a previously unappreciated, and important, component of type 1 diabetes risk.
PMCID: PMC3183089  PMID: 21980303
7.  Identification of regions correlating MGMT promoter methylation and gene expression in glioblastomas 
Neuro-Oncology  2009;11(4):348-356.
The O6-methylguanine-DNA methyltransferase gene (MGMT) is methylated in several cancers, including gliomas. However, the functional role of cysteine-phosphate-guanine (CpG) island (CGI) methylation in MGMT silencing is still controversial. The aim of this study was to investigate whether MGMT CGI methylation correlates inversely with RNA expression of MGMT in glioblastomas and to determine the CpG region whose methylation best reflects the level of expression. The methylation level of CpG sites that are potentially related to expression was investigated in 54 glioblastomas by pyrosequencing, a highly quantitative method, and analyzed with respect to their MGMT mRNA expression status. Three groups of patients were identified according to the methylation pattern of all 52 analyzed CpG sites. Overall, an 85% rate of concordance was observed between methylation and expression (p < 0.0001). When analyzing each CpG separately, six CpG sites were highly correlated with expression (p < 0.0001), and two CpG regions could be used as surrogate markers for RNA expression in 81.5% of the patients. This study indicates that there is good statistical agreement between MGMT methylation and expression, and that some CpG regions better reflect MGMT expression than do others. However, if transcriptional repression is the key mechanism in explaining the higher chemosensitivity of MGMT-methylated tumors, a substantial rate of discordance should lead clinicians to be cautious when deciding on a therapeutic strategy based on MGMT methylation status alone.
PMCID: PMC2743215  PMID: 19224763
CpG island methylation; expression; glioblastomas; MGMT
8.  Frequent aberrant DNA methylation of ABCB1, FOXC1, PPP2R2B and PTEN in ductal carcinoma in situ and early invasive breast cancer 
Ductal carcinoma in situ (DCIS) is a non-invasive lesion of the breast that is frequently detected by mammography and subsequently removed by surgery. However, it is estimated that about half of the detected lesions would never have progressed into invasive cancer. Identifying DCIS and invasive cancer specific epigenetic lesions and understanding how these epigenetic changes are involved in triggering tumour progression is important for a better understanding of which lesions are at risk of becoming invasive.
Quantitative DNA methylation analysis of ABCB1, CDKN2A/p16INK4a, ESR1, FOXC1, GSTP1, IGF2, MGMT, MLH1, PPP2R2B, PTEN and RASSF1A was performed by pyrosequencing in a series of 27 pure DCIS, 28 small invasive ductal carcinomas (IDCs), 34 IDCs with a DCIS component and 5 normal breast tissue samples. FOXC1, ABCB1, PPP2R2B and PTEN were analyzed in 23 additional normal breast tissue samples. Real-Time PCR expression analysis was performed for FOXC1.
Aberrant DNA methylation was observed in all three diagnosis groups for the following genes: ABCB1, FOXC1, GSTP1, MGMT, MLH1, PPP2R2B, PTEN and RASSF1A. For most of these genes, methylation was already present at the DCIS level with the same frequency as within IDCs. For FOXC1 significant differences in methylation levels were observed between normal breast tissue and invasive tumours (P < 0.001). The average DNA methylation levels were significantly higher in the pure IDCs and IDCs with DCIS compared to pure DCIS (P = 0.007 and P = 0.001, respectively). Real-time PCR analysis of FOXC1 expression from 25 DCIS, 23 IDCs and 28 normal tissue samples showed lower gene expression levels of FOXC1 in both methylated and unmethylated tumours compared to normal tissue (P < 0.001). DNA methylation levels of FOXC1, GSTP1, ABCB1 and RASSF1A were higher in oestrogen receptor (ER) positive vs. ER negative tumours; whereas methylation levels of FOXC1, ABCB1, PPP2R2B and PTEN were lower in tumours with a TP53 mutation.
Quantitative methylation analysis identified ABCB1, FOXC1, PPP2R2B and PTEN as novel genes to be methylated in DCIS. In particular, FOXC1 showed a significant increase in the methylation frequency in invasive tumours. Low FOXC1 gene expression in both methylated and unmethylated DCIS and IDCs indicates that the loss of its expression is an early event during breast cancer progression.
PMCID: PMC2880421  PMID: 20056007
9.  Differential DNA methylation analysis of breast cancer reveals the impact of immune signaling in radiation therapy 
Radiotherapy (RT) is a central treatment modality for breast cancer patients. The purpose of our study was to investigate the DNA methylation changes in tumors following RT, and to identify epigenetic markers predicting treatment outcome. Paired biopsies from patients with inoperable breast cancer were collected both before irradiation (n = 20) and after receiving 10–24 Gray (Gy) (n = 19). DNA methylation analysis was performed by using Illumina Infinium 27K arrays. Fourteen genes were selected for technical validation by pyrosequencing. Eighty-two differentially methylated genes were identified in irradiated (n = 11) versus nonirradiated (n = 19) samples (false discovery rate, FDR = 1.1%). Methylation levels in pathways belonging to the immune system were most altered after RT. Based on methylation levels before irradiation, a panel of five genes (H2AFY, CTSA, LTC4S, IL5RA and RB1) were significantly associated with clinical response (p = 0.041). Furthermore, the degree of methylation changes for 2,516 probes correlated with the given radiation dose. Within the 2,516 probes, an enrichment for pathways involved in cellular immune response, proliferation and apoptosis was identified (FDR < 5%). Here, we observed clear differences in methylation levels induced by radiation, some associated with response to treatment. Our study adds knowledge on the molecular mechanisms behind radiation response.
PMCID: PMC4298788  PMID: 24658971
irradiation; immune response; methylation; breast cancer; dose dependent

Results 1-9 (9)