Laboratory for Functional Genomics

Paris, France

Laboratory for Functional Genomics

Paris, France
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PubMed | Laboratory for Functional Genomics, University of Oslo and French Atomic Energy Commission
Type: Journal Article | Journal: Molecular oncology | Year: 2016

DNA methylation alterations are early events in tumorigenesis and important in the regulation of gene expression in cancer cells. Lung cancer patients have in general a poor prognosis, and a deeper insight into the epigenetic landscape in lung adenocarcinoma tumors and its prognostic implications is needed.We determined whole-genome DNA methylation profiles of 164 fresh frozen lung adenocarcinoma samples and 19 samples of matched normal lung tissue using the Illumina Infinium 450K array. A large number of differentially methylated CpGs in lung adenocarcinoma tissue were identified, and specific methylation profiles were observed in tumors with mutations in the EGFR-, KRAS- or TP53 genes and according to the patients smoking status. The methylation levels were correlated with gene expression and both positive and negative correlations were seen. Methylation profiles of the tumor samples identified subtypes of tumors with distinct prognosis, including one subtype enriched for TP53 mutant tumors. A prognostic index based on the methylation levels of 33 CpGs was established, and was significantly associated with prognosis in the univariate analysis using an independent cohort of lung adenocarcinoma patients from The Cancer Genome Atlas project. CpGs in the HOX B and HOX C gene clusters were represented in the prognostic signature.Methylation differences mirror biologically important features in the etiology of lung adenocarcinomas and influence prognosis.

Gestermann N.,French Institute of Health and Medical Research | Koutero M.,French Atomic Energy Commission | Belkhir R.,French Institute of Health and Medical Research | Tost J.,French Atomic Energy Commission | And 3 more authors.
European Cytokine Network | Year: 2012

The transcription factor interferon regulatory factor 5 (IRF5), in the type I interferon pathway is involved in the genetic susceptibility to various autoimmune diseases. A 5-bp insertion/deletion (CGGGG indel) polymorphism in the promoter region of IRF5 associated with primary Sjögren's syndrome (pSS) could be epigenetically deregulated in this condition. Therefore, we investigated DNA methylation patterns of the promoter region of IRF5 to determine whether its epigenetic deregulation could explain the increased expression of IRF5 mRNA in pSS patients, along with the risk of pSS induced by the genetic polymorphism. DNA extracted from total peripheral blood mononuclear cells, isolated CD4+ T cells, B lymphocytes and monocytes from 19 pSS patients and 24 healthy controls underwent methylation analysis by pyrosequencing. Salivary gland epithelial cells (SGECs) were cultured from minor salivary glands. Regions of interest in the CGGGG repeat and ATG initiation codon region were amplified by PCR and analysed by pyrosequencing. The effect of the demethylating agent 5-AzaC on IRF5 mRNA expression in controls was quantified by RT-PCR. Among the healthy controls, the mean methylation of the nine CpG pairs of the CGGGG repeat region and the 18 CpG pairs of the ATG region was < 15% in CD4+T cells, B lymphocytes, monocytes and SGECs. Patients and controls did not differ in methylation profiles as regards CD4+T cells and B lymphocytes. IRF5 mRNA expression did not differ with or without 5-AzaC in controls. The absence of aberrant DNA methylation profiles for the putative regulatory regions of IRF5 in CD4+T cells, B lymphocytes, and monocytes from patients with pSS, does not support the hypothesis that epigenetic deregulation in combination with the genetic polymorphism explains the increase in IRF5 mRNA levels in pSS patients.

How Kit A.,Laboratory for Functional Genomics | Nielsen H.M.,Laboratory for Functional Genomics | Nielsen H.M.,University of Aarhus | Tost J.,Laboratory for Functional Genomics | Tost J.,French Atomic Energy Commission
Biochimie | Year: 2012

A biomarker is a molecular target analyzed in a qualitative or quantitative manner to detect and diagnose the presence of a disease, to predict the outcome and the response to a specific treatment allowing personalized tailoring of patient management. Biomarkers can belong to different types of biochemical molecules such as proteins, DNA, RNA or lipids, whereby protein biomarkers have been the most extensively studied and used, notably in blood-based protein quantification tests or immunohistochemistry. The rise of interest in epigenetic mechanisms has allowed the identification of a new type of biomarker, DNA methylation, which is of great potential for many applications. This stable and heritable covalent modification mostly affects cytosines in the context of a CpG dinucleotide in humans. It can be detected and quantified by a number of technologies including genome-wide screening methods as well as locus- or gene-specific high-resolution analysis in different types of samples such as frozen tissues and FFPE samples, but also in body fluids such as urine, plasma, and serum obtained through non-invasive procedures. In some cases, DNA methylation based biomarkers have proven to be more specific and sensitive than commonly used protein biomarkers, which could clearly justify their use in clinics. However, very few of them are at the moment used in clinics and even less commercial tests are currently available. The objective of this review is to discuss the advantages of DNA methylation as a biomarker, the practical considerations for their development, and their use in disease detection, prediction of outcome or treatment response, through multiple examples mainly focusing on cancer, but also to evoke their potential for complex diseases and prenatal diagnostics. © 2012 Elsevier Masson SAS. All rights reserved.

Brand S.,University of Marburg | Teich R.,Helmholtz Center for Infection Research | Dicke T.,University of Marburg | Harb H.,University of Marburg | And 10 more authors.
Journal of Allergy and Clinical Immunology | Year: 2011

Background: Bronchial asthma is a chronic inflammatory disease resulting from complex gene-environment interactions. Natural microbial exposure has been identified as an important environmental condition that provides asthma protection in a prenatal window of opportunity. Epigenetic regulation is an important mechanism by which environmental factors might interact with genes involved in allergy and asthma development. Objective: This study was designed to test whether epigenetic mechanisms might contribute to asthma protection conferred by early microbial exposure. Methods: Pregnant maternal mice were exposed to the farm-derived gram-negative bacterium Acinetobacter lwoffii F78. Epigenetic modifications in the offspring were analyzed in TH1- and TH2-relevant genes of CD4+ T cells. Results: Prenatal administration of A lwoffii F78 prevented the development of an asthmatic phenotype in the progeny, and this effect was IFN-γ dependent. Furthermore, the IFNG promoter of CD4+ T cells in the offspring revealed a significant protection against loss of histone 4 (H4) acetylation, which was closely associated with IFN-γ expression. Pharmacologic inhibition of H4 acetylation in the offspring abolished the asthma-protective phenotype. Regarding TH2-relevant genes only at the IL4 promoter, a decrease could be detected for H4 acetylation but not at the IL5 promoter or the intergenic TH2 regulatory region conserved noncoding sequence 1 (CNS1). Conclusion: These data support the hygiene concept and indicate that microbes operate by means of epigenetic mechanisms. This provides a new mechanism in the understanding of gene-environment interactions in the context of allergy protection. © 2011 American Academy of Allergy, Asthma & Immunology.

Nelissen E.C.M.,Maastricht University | Dumoulin J.C.M.,Maastricht University | Daunay A.,Laboratory for Functional Genomics | Evers J.L.H.,Maastricht University | And 3 more authors.
Human Reproduction | Year: 2013

STUDY QUESTION: Does IVF/ICSI have an effect on the epigenetic regulation of the human placenta?SUMMARY ANSWERWe found a reduced DNA methylation level at the H19 and MEST differentially methylated regions (DMRs), and an increased RNA expression of H19 in placentas from pregnancies conceived by IVF/ICSI when compared with placentas from spontaneous conception.WHAT IS KNOWN ALREADYChanges in fetal environment are associated with adverse health outcomes. The placenta is pivotal for intrauterine environment. Animal studies show that epigenetic regulation plays an important role in these environment-induced phenotypic effects. Also, the preimplantation embryo environment affects birthweight as well as the risk of chronic adult diseases. Epigenetic processes are sensitive to the environment, especially during the period around conception.STUDY DESIGN AND PARTICIPANTSPlacental tissue was collected from 35 spontaneously conceived pregnancies and 35 IVF/ICSI (5 IVF, 30 ICSI) derived pregnancies. We quantitatively analysed the DNA methylation patterns of a number of consecutive CpGs in the core regions of DMRs and other regulatory regions of imprinted genes, since these are involved in placental and fetal growth and development.METHODSBy using pyrosequencing, the DNA methylation at seven germline-derived primary DMRs was analysed quantitatively. Five of these are maternally methylated (MEST isoform α and β, PEG3, KCNQ1OT1 and SNRPN) and two are paternally methylated [H19 DMR and the intergenic region between DLK1 and MEG3 (IG-DMR)]. The post-fertilization-derived secondary DMRs, IGF2 (DMR0 and 2) and IG-DMR (CG7, also called MEG3 DMR), and the MEG3 promoter region were examined as well. In case of differential methylation between the two groups, the effect on gene expression was assessed by quantitative real-time PCR. MAIN RESULTS AND THE ROLE OF CHANCE: Both the promoter region of MEST isoform α and β and the 6th CTCF binding site within the H19 DMR were significantly hypomethylated in the IVF/ICSI group. The phenomenon was consistently observed over all CpG sites analysed and not restricted to single CpG sites. The other primary and secondary DMRs were not affected. Expression of H19 was increased in the IVF/ICSI group, while that of IGF2 and MEST remained similar. LIMITATIONS, REASONS FOR CAUTION: In the IVF/ICSI group, mostly ICSI pregnancies were investigated. The ICSI technique or male subfertility could be a confounding factor. Therefore, our results are less generalizable to IVF pregnancies. WIDER IMPLICATIONS OF THE FINDINGS: The clinical effects of the observed placental hypomethylations on the developmental programming of the IVF/ICSI progeny, if any, are as yet unknown. Whether the hypomethylation is an adaptation of the placenta to maintain fetal supply and ameliorate the effects of environmental cues, or whether it is a deregulation leading to deranged developmental programming with or without increased vulnerability for disease, consistent with the developmental origins of health and disease hypothesis, needs further investigation. STUDY FUNDING/COMPETING INTEREST(S): Partly funded by an unrestricted research grant by Organon BV (now MSD BV) without any role in study design, data collection and analysis, or preparation of the manuscript. No conflict of interests to declare.TRIAL REGISTRATION NUMBERDutch Trial Registry (NTR) number 1298. © 2013 The Author. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email:

Charbel C.,French Institute of Health and Medical Research | Charbel C.,University Pierre and Marie Curie | Fontaine R.H.,French Institute of Health and Medical Research | Fontaine R.H.,University Pierre and Marie Curie | And 16 more authors.
Journal of Investigative Dermatology | Year: 2014

Congenital melanocytic nevus (CMN) is a particular melanocytic in utero proliferation characterized by an increased risk of melanoma transformation during infancy or adulthood. NRAS and BRAF mutations have consistently been reported in CMN samples, but until recently results have been contradictory. We therefore studied a series of large and giant CMNs and compared them with small and medium CMNs using Sanger sequencing, pyrosequencing, high-resolution melting analysis, and mutation enrichment by an enhanced version of ice-COLD-PCR. Large-giant CMNs displayed NRAS mutations in 94.7% of cases (18/19). At that point, the role of additional mutations in CMN pathogenesis had to be investigated. We therefore performed exome sequencing on five specimens of large-giant nevi. The results showed that NRAS mutation was the sole recurrent somatic event found in such melanocytic proliferations. The genetic profile of small-medium CMNs was significantly different, with 70% of cases bearing NRAS mutations and 30% showing BRAF mutations. These findings strongly suggest that NRAS mutations are sufficient to drive melanocytic benign proliferations in utero. © 2014 The Society for Investigative Dermatology.

Moison C.,Pierre Fabre | Moison C.,French National Center for Scientific Research | Moison C.,University Pierre and Marie Curie | Assemat F.,Pierre Fabre | And 5 more authors.
Epigenetics | Year: 2014

DNA methylation and polycomb proteins are well-known mediators of epigenetic silencing in mammalian cells. Usually described as mutually exclusive, this statement is today controversial and recent in vitro studies suggest the coexistence of both repressor systems. We addressed this issue in the study of Retinoic Acid Receptor β (RARβ), a tumor suppressor gene frequently silenced in prostate cancer. We found that the RARβ promoter is hypermethylated in all studied prostate tumors and methylation levels are positively correlated with H3K27me3 enrichments. Thus, by using bisulfite conversion and pyrosequencing of immunoprecipitated H3K27me3 chromatin, we demonstrated that DNA methylation and polycomb repression co-exist in vivo at this locus. We found this repressive association in 6/6 patient tumor samples of different Gleason score, suggesting a strong interplay of DNA methylation and EZH2 to silence RARβ during prostate tumorigenesis. © 2014 Landes Bioscience.

PubMed | Laboratory for Functional Genomics, French Institute of Health and Medical Research, University of Paris Descartes, Assistance Publique Hopitaux de Paris and University Pierre and Marie Curie
Type: Journal Article | Journal: Experimental dermatology | Year: 2016

Congenital melanocytic nevi (CMN) are benign proliferations that may be associated with various consequences depending on their size. They are characterized by a specific molecular signature, namely a postzygotic somatic NRAS or BRAF mutation. We have recently reported that large CMN (lCMN), which are classically associated with an increased melanoma risk, harbour cell subpopulations with specific clonogenic and tumorigenic potential. We wished to ascertain whether cells displaying similar properties persisted postnatally in medium CMN (mCMN). Eighteen medium M1, nine large and one giant NRAS-mutated CMN were prospectively included in the study. Subpopulations of mCMN cells expressed stem cell/progenitor lineage markers such as Sox10, nestin and Oct4, as was the case in lCMN. Nevertheless, conversely to lCMN, mCMN cells with clonogenic properties were rarer. In vitro, approximatively one in 1500 cells isolated from fresh mCMN formed colonies that could be passaged. In vivo, mCMN seemed to harbour cells with less proliferative potential than the larger lesions as lCMN biopsies displayed a threefold expansion compared to mCMN when xenografted in Rag2(-/-) mice. Thus, our data revealed variations in clonogenicity and tumorigenic properties in NRAS-mutated CMN according to size.

Rakyan V.K.,Queen Mary, University of London | Beyan H.,Queen Mary, University of London | Down T.A.,University of Cambridge | Hawa M.I.,Queen Mary, University of London | And 13 more authors.
PLoS Genetics | Year: 2011

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. © 2011 Rakyan et al.

How Kit A.,Laboratory for Functional Genomics | Mazaleyrat N.,French Atomic Energy Commission | Daunay A.,Laboratory for Functional Genomics | Nielsen H.M.,Laboratory for Functional Genomics | And 4 more authors.
Human Mutation | Year: 2013

A number of methods allowing the detection of low levels of KRAS mutations have been developed in the last years. However, although these methods have become increasingly sensitive, they can rarely identify the mutated base directly without prior knowledge on the mutated base and are often incompatible with a sequencing-based read-out desirable in clinical practice. Here, we present a modified version of the ice-COLD-PCR assay called Enhanced-ice-COLD-PCR (E-ice-COLD-PCR) for KRAS mutation detection and identification, which allows the enrichment of the six most frequent KRAS mutations. The method is based on a nonextendable chemically modified blocker sequence, complementary to the wild-type (WT) sequence leading to the enrichment of mutated sequences. This assay permits the reliable detection of down to 0.1% mutated sequences in a WT background. A single genotyping assay of the amplification product by pyrosequencing directly following the E-ice-COLD-PCR is performed to identify the mutated base. This developed two-step method is rapid and cost-effective, and requires only a small amount of starting material permitting the sensitive detection and sequence identification of KRAS mutations within 3 hr. This method is applied in the current study to clinical colorectal cancer samples and enables detection of mutations in samples, which appear as WT using standard detection technologies. © 2013 WILEY PERIODICALS, INC.

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