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Results: In splenocytes, a significant hypermethylation in GATA-3 CpG islands was induced by EPIT versus Sham, starting from the 4 of treatment (p

As the sequence of the human genome is now nearly finished, genome research turns to elucidate gene function and regulation. DNA methylation is of particular importance for gene regulation and is strongly implicated in the pathogenesis of various diseases. The real-time luminometric detection of pyrophosphate release upon nucleotide incorporation in the Pyrosequencing technology is ideally suited for the simultaneous analysis and quantification of the methylation degree of several CpG positions in close proximity. We developed and improved this analysis to obtain reproducible results for as many as 10 successive CpGs in a single sequencing reaction spanning up to 80 nt. Advantages of the Pyrosequencing technology are the ease of its implementation, the high quality and the quantitative nature of the results, and its ability to identify differentially methylated positions in close proximity, which may be used as DNA methylation markers.

Preeclampsia is the major pregnancy-induced hypertensive disorder. It modifies the expression profile of placental genes, including several serine protease inhibitors ( SERPINs ). The objective of this study was to perform a systematic expression analysis of these genes in normal and pathological placentas and to pinpoint epigenetic alterations inside their promoter regions. Expression of 18 placental SERPINs was analyzed by quantitative RT-PCR on placentas from pregnancies complicated by preeclampsia, intrauterine growth restriction, or both and was compared with normal controls. SERPINA3, A5, A8, B2, B5 , and B7 presented significant differences in expression in ≥1 pathological situation. In parallel, the methylation status of the CpG islands located in their promoter regions was studied on a sample of control and preeclamptic placentas. Ten SERPIN promoters were either totally methylated or totally unmethylated, whereas SERPINA3, A5 , and A8 presented complex methylation profiles. For SERPINA3 , the analysis was extended to 81 samples and performed by pyrosequencing. For the SERPINA3 CpG island, the average methylation level was significantly diminished in preeclampsia and growth restriction. The hypomethylated CpGs were situated at putative binding sites for developmental and stress response (hypoxia and inflammation) factors. Our results provide one of the first observations of a specific epigenetic alteration in human placental diseases and provide new potential markers for an early diagnosis.

Expression of imprinted genes is classically asso-ciated with differential methylation of specific CpG-rich DNA regions (DMRs). The H19/IGF2 locus isconsidered a paradigm for epigenetic regulation. Inmice, as in humans, the essential H19 DMR—targetof the CTCF insulator—is located between the twogenes. Here, we performed a pyrosequencing-basedquantitative analysis of its CpG methylation innormal human tissues. The quantitative analysis ofthe methylation level in the H19 DMR revealed threeunexpected discrete, individual-specific methylationstates. This epigenetic polymorphism was confinedto the sixth CTCF binding site while a uniquemedian-methylated profile was found at the thirdCTCF binding site as well as in the H19 promoter.Monoallelic expression of H19 and IGF2 was main-tained independently of the methylation status atthe sixth CTCF binding site and the IGF2 DMR2displayed a median-methylated profile in all indi-viduals and tissues analyzed. Interestingly, themethylation profile was genetically transmitted.Transgenerational inheritance of the H19 methyla-tion profile was compatible with a simple modelinvolving one gene with three alleles. The existenceof three individual-specific epigenotypes in theH19 DMR in a non-pathological situation means itis important to reconsider the diagnostic valueand functional importance of the sixth CTCFbinding site.INTRODUCTIONImprinted genes are expressed from only one of the parentalchromosomes (1,2). Generally they are located in clusters andepigenetically marked by DNA methylation, histoneacetylation/deacetylation and histone methylation and oftenassociated with antisense RNAs (3,4). In mice, extensivestudies of the paradigmatic imprinted H19/Igf2 regionrevealed that the physical contacts between differentiallymethylated regions (DMRs), containing insulators, silencersand activators, lead to transcriptional regulation of bothH19 and Igf2 genes (5,6). Methylation of the paternallyderived allele at an imprinting control region (ICR) located2 kb upstream of H19 (H19 DMR) is required to silenceH19 and to activate Igf2 on the chromosome of paternal ori-gin. Reciprocally, absence of methylation on the maternalallele in the H19 DMR leads to expression of the maternalH19 allele and the silencing of Igf2 throughout development(7). Mechanistically, the H19 DMR is the biological target for

Introduction Deux inhibiteurs de fort enrichissement de la mutation au cours d’une PCR ou alleles mutants et sauvages subissent respectivement une amplification exponentielle et lineaire. Materiel et methodes Deux lignees cellulaires derivees de melanome humain porteuses de differentes mutations heterozygote BRAF V600 ont ete utilisees pour la mise au point de cette methodologie. Des analyses moleculaires ont ete realisees sur 18 echantillons tumoraux de melanome congeles, 17 echantillons de melanome conserves en paraffine et 17 echantillons de plasma de patients atteints de melanome avance. La technique de ice-cold PCR a ete realisee en utilisant un thermocycleur 96 puits en temps reel suivi d’un pyrosequencage, puis identification et quantification automatique de la mutation. Resultats Avec ce dosage, une large gamme de 25 pg a 25 ng d’ADN peut etre utilisee sans aucune reduction de l’efficacite de l’enrichissement de mutation. Cette technique est ultrasensible car elle peut detecter jusqu’a 0,01 % d’alleles mutes dans un fond de type sauvage. Le test a ete valide sur prelevement congeles, conserves en paraffine, et sur du plasma de patients atteints de melanome. Il a permis la detection, l’identification et la quantification de differents types de mutation du gene BRAF presents dans les echantillons qui apparaissaient sauvages en utilisant des techniques moins sensibles comme le pyrosequencage standard, la discrimination allelique ou le sequencage Sanger. Discussion Notre test a un niveau de sensibilite eleve proche de la digital PCR, il est facilement realisable facilement en une etape. Conclusion Ce test de ice-cold PCR BRAF V600 est actuellement un outil de diagnostic moleculaire extremement puissant pour la detection ultrasensible et la quantification de mutations BRAF V600 .

Metastatic melanomas are the deadliest form of skin cancer and are very aggressive tumors showing highly invasive properties and a rapid chemoresistance to standard treatment (Dacarbazine) and to specific BRAF-V600E kinase inhibitors (Vemurafenib). Thus, targeting these tumors remains a major concern for novel therapeutic proposals. Abnormal patterns of DNA methylation, an epigenetic modification that cells use to control gene expression, have been described in these tumors. These epigenetic modifications participate in melanoma formation and maintenance. The aim of our project is to characterize the DNA methylation changes that occur in the most aggressive form of melanoma and to reverse these changes by using clinically active DNA methyltransferase inhibitors (5-aza-2′-deoxycytidine, 5-aza-dC). To date a limited number of datasets have been published depicting the effects of inhibitors of DNA methylation on invasive capacities of metastatic melanoma cells. The work presented here, focuses on the study of the effects of DNA methylation inhibition on metastatic melanoma invasiveness. We have set up an original method to quantify DNA methylation by FACS and shown that non cytotoxic nanomolar 5-aza-dC concentrations were able to demethylate DNA of WM-266-4 metastatic melanoma cells. Then using an in vitro 3D spheroids cell invasion assay and fluorescent microscopy to measure invasion capacities of metastatic cell lines, we showed that 5-aza-dC was able to inhibit invasion of WM-266-4 cells at these non-cytotoxic demethylating concentrations. Citation Format: Chantal Etiévant, Cécile Desjobert, Arnaud Carrier, Audrey Delmas, Jorg Tost, Gilles Favre, Joelle Riond, Paola Arimondo. 5-aza-2′-deoxycytidine, a DNA demethylating agent, inhibits metastatic melanoma invasiveness. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5524. doi:10.1158/1538-7445.AM2014-5524

Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry has become one of the most powerful and widely applied technologies for SNP scoring and determination of allele frequencies in the post-genome sequencing era. Although different strategies for allele discrimination combined with MALDI were devised, in practice only primer extension methods are nowadays routinely used. This combination enables the rapid, quantitative, and direct detection of several genetic markers simultaneously in a broad variety of biological samples. In the field of molecular diagnostics, MALDI has been applied to the discovery of genetic markers, that are associated with a phenotype like a disease susceptibility or drug response, as well as an alternative means for diagnostic testing of a range of diseases for which the responsible mutations are already known. It is one of the first techniques with which whole genome scans based on single nucleotide polymorphisms were carried out. It is equally well suited for pathogen identification and the detection of emerging mutant strains as well as for the characterization of the genetic identity and quantitative trait loci mapping in farm animals. MALDI can also be used as a detection platform for a range of novel applications that are more demanding than standard SNP genotyping such as mutation/polymorphism discovery, molecular haplotyping, analysis of DNA methylation, and expression profiling. This review gives an introduction to the application of mass spectrometry for DNA analysis, and provides an overview of most studies using SNPs as genetic markers and MALDI mass spectrometric detection that are related to clinical applications and molecular diagnostics. Further, it aims to show specialized applications that might lead to diagnostic applications in the future. It does not speculate on whether this methodology will ever reach the diagnostic market.

Current protocols for DNA methylation analysis are either labor intensive or limited to the measurement of only one or two CpG positions. Pyrosequencing is a real-time sequencing technology that can overcome these limitations and be used as an epigenotype-mapping tool. Initial experiments demonstrated reliable quantification of the degree of DNA methylation when 2-6 CpGs were analyzed. We sought to improve the sequencing protocol so as to analyze as many CpGs as possible in a single sequencing run. By using an improved enzyme mix and adding single-stranded DNA-binding protein to the reaction, we obtained reproducible results for as many as 10 successive CpGs in a single sequencing reaction spanning up to 75 nucleotides. A minimum amount of 10 ng of bisulfite-treated DNA is necessary to obtain good reproducibility and avoid preferential amplification. We applied the assay to the analysis of DNA methylation patterns in four CpG islands in the vicinity of IGF2 and H19 genes. This allowed accurate and quantitative de novo sequencing of the methylation state of each CpG, showing reproducible variations of methylation state in contiguous CpGs, and proved to be a useful adjunct to current technologies.

of the technology is the close correlation between the peak heights in a Pyrogram (Pyrosequencing AB), representing the sequence raw data, and the amount of nucleotides incorporated by the DNA polymerase during the Pyrosequencing reaction that allows for precise allele frequency determination for a SNP in pooled DNA samples (10–12). Very recently, an approach to the analysis of the degree of methylation on a single MVP after bisulfite treatment was shown using mixtures of PCR products for calibration (13). Bisulfite treatment converts unmethylated cytosines to uracils, while methylated cytosines remain unchanged under the appropriate reaction conditions. Therefore, after PCR, methylation sites can be treated as C/T SNPs with an allele frequency spectrum spanning the entire range (0%–100%). Here we demonstrate the Pyrosequencing technology to analyze and precisely quantify the degree of DNA methylation. Up to six MVPs were examined simultaneously in a single Pyrosequencing reaction. The method is amenable to the analysis of bisulfite-treated DNA derived from paraffin-embedded tissue samples, highly reproducible, and accurate if calibration is carried out properly (to detect any biased PCR amplification).

Reconstruction of haplotypes, or the allelic phase, of single nucleotide polymorphisms (SNPs) is a key component of studies aimed at the identification and dissection of genetic factors involved in complex genetic traits. In humans, this often involves investigation of SNPs in case/control or other cohorts in which the haplotypes can only be partially inferred from genotypes by statistical approaches with resulting loss of power. Moreover, alternative statistical methodologies can lead to different evaluations of the most probable haplotypes present, and different haplotype frequency estimates when data are ambiguous. Given the cost and complexity of SNP studies, a robust and easy-to-use molecular technique that allows haplotypes to be determined directly from individual DNA samples would have wide applicability. Here, we present a reliable, automated and high-throughput method for molecular haplotyping in 2 kb, and potentially longer, sequence segments that is based on the physical determination of the phase of SNP alleles on either of the individual paternal haploids. We demonstrate that molecular haplotyping with this technique is not more complicated than SNP genotyping when implemented by matrix-assisted laser desorption/ionisation mass spectrometry, and we also show that the method can be applied using other DNA variation detection platforms. Molecular haplotyping is illustrated on the well-described beta(2)-adrenergic receptor gene.

Introduction Un nombre croissant d’etudes indique que l’exposition maternelle aux polluants atmospheriques est associee a une croissance fœtale plus faible mais les effets a plus long terme et les mecanismes biologiques sous-jacents sont peu decrits. Methodes La cohorte suivi de l’exposition aux polluants atmospheriques durant la grossesse et l’enfance et sante (SEPAGES) repose sur 700 femmes enceintes recrutees en 2014–2015 au premier trimestre de la grossesse dans l’agglomeration grenobloise. Son originalite reside dans un suivi intensif de la grossesse puis de l’enfant qui permettra d’etudier les effets a long terme d’expositions precoces. Afin d’explorer les mecanismes biologiques qui regissent ces effets a long terme, une etude similaire a SEPAGES est conduite sur des lapines gestantes avec un suivi a long terme de la descendance. De plus, une etude sur la cohorte mere-enfant EDEN s’interesse au role de la methylation de l’ADN dans les associations entre pollution de l’air et croissance fœtale. Resultats Une revue des resultats recents obtenus chez l’animal et chez l’homme sera presentee. Chez le lapin, la croissance fœtale tend a etre plus faible chez les meres exposees aux emissions d’un moteur diesel comparees aux non exposees. Chez l’homme, l’ADN de 600 placentas d’EDEN qui etaient stockes a −80 °C a ete extrait et la methylation mesuree sur plus de 480 000 sites CpG (Puce Illumina 450K). Des premiers resultats sur les associations avec l’exposition a la pollution atmospherique et le poids de naissance seront presentes. Discussion Ces resultats preliminaires suggerent de poursuivre les efforts consistant a mener des travaux pluri-disciplinaires afin de repondre aux defis poses par la DOHaD.

9020 Background: Two BRAF inhibitors have been approved for patients with BRAF V600 activating mutation. However, to date the different tools developed for the detection of BRAF V600 mutations lack...

9005 Background: Resistance mechanisms acquired during BRAF inhibitor (BRAFi) treatment were shown to involve multiple signalling pathways including MAPK, PI3K pathways and tumor microenvironment. They may also be complicated by intra-tumor heterogeneity of BRAF mutational status which paradoxically enhances wild-type BRAF cells proliferation. We therefore wanted to evaluate using a quantitative pyrosequencing assay whether the level of BRAFV600 mutation in tumor tissue could predict clinical response and outcome of BRAFV600melanoma patients treated with the BRAFi, vemurafenib. Methods: melanoma specimen from 44 patients with advanced melanoma treated with vemurafenib were available at abseline. The BRAFV600 mutation level was defined as the ratio of the BRAFV600 allele quantification to the percentage of tumor cells in the sample. The main end point were response according to RECIST, progression free survival, overall survival and ratio of the mutated allele to percentage of tumor cells. Results: The BRA...

Background Autoimmune result from the interaction between genetic and environmental factors. Previous studies have demonstrated the implication of DNA methylation alterations in many autoimmune diseases. 1 Sjogren’s Syndrome (SS) is a prototypic systemic autoimmune disease that can be primary or associated with other systemic connective tissue diseases. DNA methylation is almost exclusively found in the context of the dinucleotide sequence CpG. Methylation of regulatory sequences can lead to gene silencing and the interest in DNA methylation has been raised through multiple studies demonstrating its potential as biomarker containing valuable information for diagnosis, classification and prognosis of disease. Until now, very few data are available in pSS. Objectives To date, no treatment has proven effective in SS. The identification of differentially methylated regions could provide information on novel key players involved in the pathogenesis of pSS and new targets for therapeutic intervention in the future. Methods We analyzed genome-wide DNA methylation patterns in FACS sorted B and T-lymphocytes from 12 SS patients and 12 controls using the Illumina 450K Infinium Human Methylation 450K BeadChip monitoring quantitatively more than 480,000 CpG positions. Data was analyzed using a newly developed preprocessing pipeline for 450K data using an original subset quantile normalization approach that performs both sample normalization and efficient Infinium I/Infinium II shift correction. 2 Differentially methylated regions of interest are validated in an additional set of 12 SS patients and 12 controls as well as salivary gland biopsies using pyrosequencing. Results 1537 probes associated with 993 genes were differentially methylated between patients and controls in B lymphocytes, and 1129 probes associated with 723 genes were differentially methylated in T lymphocytes including genes from the MHC and genes associated with other autoimmune diseases. Pathway analysis showed a highly significant overlap of the genes identified in B lymphocytes with rheumatoid arthritis (p -8 ) and lupus associated genes (p -6 ) including SLC15A4 and IKFZ1, lymphomagenesis and regulation of apoptosis. Replication is currently ongoing, but the first genes replicated well in an independent biological samples. Further, comparing our data to genes found differentially methylated in synviocytes from rheumatoid arthritis (RA) patients we show that the RA-signature classified reasonably well the B cell samples into pSS patients and controls. Although the separation was not perfect (as expected as different tissues were analyzed), these results raise the hypothesis of a common DNA methylation signature of autoimmune diseases. Conclusions Genome-wide DNA methylation profiling identified widespread epigenetic deregulation which provide novel insight in the disease pathology and raises the possibility of a common epigenetic deregulation in AIDs. References Myrtue Nielsen, H. and J. Tost (2012) Epigenetic changes in inflammatory and autoimmune diseases, Subcellular Biochemistry, 61 , 455-78. Touleimat, N., and J. Tost (2012) A complete pipeline for Infinium Methylation 450K BeadChip data processing using subset quantile normalization for accurate DNA methylation estimation, Epigenomics, 4 , 325-41. Acknowledgements This work was supported by the Agence Nationale pour la Recherche (BLAN 2010 R11035LL). Disclosure of Interest None Declared

Background The pathophysiology of lymphomas in auto immune disease (AID) involves persistent inflammation and activation of autoimmune B cells leading to NF-kB activation. The TNFAIP3 gene encodes the A20 protein, a central gatekeeper of NF-kB activation. Germinal abnormalities in TNFAIP3 have been associated with different AID and somatic mutations of the gene have been observed in several lymphoma subtypes, particularly MALT lymphoma, the lymphoma subtype most frequently associated with pSS. Objectives To investigate whether TNFAIP3 abnormalities are involved in the lymphomagenesis process in pSS. Methods The discovery set was constituted by 584 pSS patients including 25 patients with lymphoma and 451 controls of Caucasian ancestry, addressed by 48 Ancestry Informative Markers. Three SNPs encompassing the TNFAIP3 locus located on 6q23 (rs13192841, rs2230926 and rs6922466) and known to be associated with SLE and RA were genotyped. 19 additional patients with pSS and lymphoma were used for extension and replication. We sequenced all TNFAIP3 exons in germinal and lymphoma DNA from 20 pSS patients with lymphoma. Functional abnormalities of A20 were assessed by gene reporter assays. Results The 3 TNFAIP3 SNPs were not significantly associated with risk of pSS. But multivariate analysis demonstrated a significant association between the rs2230926 SNP (coding for an amino acid substitution in exon 3) and pSS complicated by lymphoma: OR vs controls = 3.36 (95%CI 1.34 - 8.42) p= 0.0097, OR vs pSS without lymphoma = 3.26 [95%CI 1.31 – 8.12], p=0.011. TNFAIP3 gene sequencing of germinal DNA from 43 patients with pSS and lymphoma confirmed the more frequent presence of the rs2230926G risk variant in 11/43 patients (25.6%, versus 11% in controls, p=0.018). Twelve of the 20 (60%) patients with paired germinal and lymphoma TNFAIP3 sequence data had functional abnormalities of A20. The frequency was even higher (77%) among pSS patients with MALT lymphoma (n=28). Mutated A20 variants (rs2230926G and GG insertion) were both less effective than the wild type A20 in inhibiting NF-kB-dependent activation (p Conclusions This study demonstrates that A20 inactivation plays a key role in lymphomagenesis in the context of autoimmunity. It supports a scenario in which the presence of germinal and/or somatic abnormalities of genes leading to impaired control of NF-kB activation in B cells continuously stimulated by autoimmunity enhances the risk of lymphoma. Disclosure of Interest None Declared

Genome-wide association studies (GWAS) have identified numerous loci linked to breast cancer susceptibility, but few of these loci have been convincingly linked to causal variants. We identified genes whose mRNA expression is linked to germline variation in normal mammary tissue and breast tumors by performing a genome-wide expression Quantitative Trait Locus (eQTL) analysis in 97 samples of normal mammary tissue and 286 breast adenocarcinomas. In the tumors we found 164 cis-acting loci, which affect expression of nearby genes. Twenty nine of these loci are previously unreported in other eQTL studies, including a cis-acting locus at 5p12 affecting expression of the mitochondrial ribosomal 28S subunit protein MRPS30 in estrogen receptor-positive but not estrogen receptor-negative tumors. This locus has been associated with susceptibility to estrogen receptor-positive breast cancer in several GWAS, with MRPS30 being the suggested candidate gene. One of these studies found that the rs771660 variant was most significantly associated with breast cancer in the 5p12 region, and we found that rs771660 was most strongly associated with MRPS30 gene expression. We provide the first evidence that this breast cancer susceptibility locus affects MRPS30 expression specifically in estrogen receptor-positive tumors and suggest a mechanism for estrogen activation of MRPS30 via proteins in the AP-1 complex. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P5-05-03.

Glutathione-S-transferase P1 (GSTP1) is involved in thiol-mediated detoxification and breakdown of reactive oxygen species created by anticancer drug exposure. GSTP1 is also an inhibitor of c-Jun N-terminal kinase 1, a kinase involved in stress response, apoptosis and cellular proliferation. Hypermethylation of the GSTP1 promoter has been associated with gene silencing in prostate cancer, kidney cancer, and breast cancer, among others. Although frequently described, the mechanism underlying promoter hypermethylation of the GSTP1 gene is poorly understood. It has been reported that an ATAAA repeat of the GSTP1 promoter separates methylated from unmethylated CpGs in normal prostate tissue [1]. These separate methylation domains are lost in prostate cancer, and methylation extends throughout the whole promoter region. It has been proposed that hypermethylation of GSTP1 requires a combination of gene silencing and random seeds of methylation in prostate cancer cells, and that these combinatorial effects lead to histone deacetylation and subsequent chromatin remodeling [2]. To further elucidate the mechanisms underlying the hypermethylation of the GSTP1 promoter, we genotyped the (ATAAA) repeat and the linked SNPs in positions -354, -288, -287 and -282 in the GSTP1 promoter and we performed methylation analysis using mass spectrometry in tumor DNA from 82 breast cancer patients. The role of the different allelic variants on methylation status of the GSTP1 promoter and expression levels was assessed. We quantitatively determined the methylation status of six CpGs spanning the transcription start site of the GSTP1 promoter: -22, +8, +14, +38, +47 and +55. The average percentage methylation for each individual CpG for the 82 tumor samples analyzed was 16.9%, 30.3%, 18.2%, 21.2%, 18.6% and 8.1%, respectively. The average percentage methylation for all CpGs in all tumor samples was 19%. There was a correlation between the degree of methylation of the individual CpGs and their neighboring CpGs (P < 0.001). When correlating the extent of methylation to the mRNA levels previously assessed by whole genome gene-expression profiling of the same tumors, a significant inverse correlation was observed (P < 0.01). The methylation status of the three CpGs closest to the transcriptional start site was more highly associated with the level of GSTP1 mRNA expression than the CpGs further downstream of the +1 site. Furthermore, we observed differences in the degree of GSTP1 promoter methylation between the different tumor subclasses defined by whole-genome microarray analysis [3]. The methylation of the GSTP1 promoter was significantly lower in the basal subtype compared with the luminal subtype, which corresponded to elevated GSTP1 mRNA levels in the basal subtypes [4]. We further analyzed the impact of the most frequent haplotype structure of the GSTP1 promoter in relation to the extent of methylation, and a correlation was observed (P = 0.003) suggesting that haplotype structures can affect de novo methylation of adjacent sequences.

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.

Background: There is a substantial genetic component for birthweight variation, and although there are known associations between fetal genotype and birthweight, the role of common epigenetic variation in influencing the risk for small for gestational age (SGA) is unknown. The two imprinting control regions (ICRs) located on chromosome 11p15.5, involved in the overgrowth disorder Beckwith-Wiedemann syndrome (BWS) and the growth restriction disorder Silver-Russell syndrome (SRS), are prime epigenetic candidates for regulating fetal growth. We investigated whether common variation in copy number in the BWS/SRS 11p15 region or altered methylation levels at IGF2/H19 ICR or KCNQ10T1 ICR was associated with SGA. Methods: We used a methylation-specific multiplex-ligation-dependent probe amplification assay to analyse copy number variation in the 11p15 region and methylation of IGF2/H19 and KCNQ10T1 ICRs in blood samples from 153 children (including 80 SGA), as well as bisulfite pyrosequencing to measure methylation at IGF2 differentially methylated region (DMR)0 and H19 DMR. Results: No copy number variants were detected in the analyzed cohort. Children born SGA had 2.7% lower methylation at the IGF2 DMR0. No methylation differences were detected at the H19 or KCNQ10T1 DMRs. Conclusions: We confirm that a small hypomethylation of the IGF2 DMR0 is detected in peripheral blood leucocytes of children born SGA at term. Copy number variation within the 11p15 BWS/SRS region is not an important cause of non-syndromic SGA at term.

As the DNA sequence of the human genome is now nearly finished, the main task of genome research is to elucidate gene function and regulation. DNA methylation is of particular importance for gene regulation and is strongly implicated in the development of cancer. Even minor changes in the degree of methylation can have severe consequences. An accurate quantification of the methylation status at any given position of the genome is a powerful diagnostic indicator. Here we present the first assay for the analysis and precise quantification of methylation on CpG positions in simplex and multiplex reactions based on matrix-assisted laser desorption/ ionisation mass spectrometry detection. Calibration curves for CpGs in two genes were established and an algorithm was developed to account for systematic fluctuations. Regression analysis gave R(2) >or= 0.99 and standard deviation around 2% for the different positions. The limit of detection was approximately 5% for the minor isomer. Calibrations showed no significant differences when carried out as simplex or multiplex analyses. All variable parameters were thoroughly investigated, several paraffin-embedded tissue biopsies were analysed and results were verified by established methods like analysis of cloned material. Mass spectrometric results were also compared to chip hybridisation.

International audience; The International Cancer Genome Consortium (ICGC) was launched to coordinate large-scale cancer genome studies in tumors from 50 different cancer types and/or subtypes that are of clinical and societal importance across the globe. Systematic studies of over 25,000 cancer genomes at the genomic, epigenomic, and transcriptomic levels will reveal the repertoire of oncogenic mutations, uncover traces of the mutagenic influences, define clinically-relevant subtypes for prognosis and therapeutic management, and enable the development of new cancer therapies.

Background Breast cancer is a disease caused by uncontrolled cell division of epithelial cells in the ducts or the lobules of the breast. The ducts and lobules are enclosed by a basement membrane, and during progression of the disease the invading cells will breach the membrane and invade adjacent tissue. A tumor that is still enclosed in the basement membrane is called a carcinoma in situ, while a tumor that has breached the basement membrane is called an invasive carcinoma. DNA methylation is a DNA modification where methyl groups are added to CpG dinucleotides and thought to regulate gene expression through blocking of transcription factor binding or through chromatin remodeling. The aim of the study was to determine what genes get differentially methylated when the cancer progresses from a less to a more aggressive carcinoma. In addition, by applying integrated analysis of other molecular data such as gene expression and copy number, we could investigate how more elaborate biological processes change during progression. Being able to determine the processes that take place during progression of breast cancer may give valuable insight into cancer biology, as well as identification of early markers of disease.

International audience; Immunostaining is widely used in cell biology for the in situ detection of proteins in fixed cells. The method is based on the specificity of antibodies for recognizing and binding to a selected target, combined with immunolabeling techniques for microscopic imaging. Antibodies with high specificities for modified nucleotides have also been widely developed, and among those, antibodies that recognize modified cytosine: 5-methylcytosine (5mC), and more recently, its derivates 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC). To allow for their detection, primary antibody signals can be amplified using secondary antibodies coupled to fluorophores for immunofluorescence, or other molecules for immunocytochemistry.Immunostaining can be used to gain information on the spatial distribution and levels of DNA methylation states within the nucleus. Although the resolution remains quite low in genomic terms, advanced microscopy techniques and image analysis can obtain detailed spatial information content from immunostained sites. The technique complements genomic approaches that permit the assessment of DNA methylation on specific sequences, but that cannot provide global nuclear spatial context. Immunostaining is an accessible method of great benefit in several cases: when working with limited material (such as embryos or primary cells), to quickly assess at the level of individual cells the effect of siRNA, drugs, or biological processes that promote or inhibit DNA methylation or demethylation, or to study the 3D nuclear organization of regions with high DNA methylation, such as constitutive heterochromatin.Here, we review and outline protocols for the fluorescent and enzymatic immunodetection of DNA methylation in the nuclei of cells, tissue sections, and mammalian embryos.

The analysis of DNA methylation patterns has become of great interest as methylome alterations have been found in many diseases. MeDIP (Methylated DNA ImmunoPrecipitation) immunoprecipitates genome-wide methylated sequences many of which are located in the repetitive sequences. Such sequences are difficult to align unambiguously after sequencing (MeDIP-Seq) leading to a large number of sequences that are currently not used for further analysis. We present an innovative method called MeDIP-dep-Seq which depletes a significant part of several classes of these highly repetitive sequences (up to 300-fold decrease), while unique sequences of interest are not affected. After sequencing on a second generation sequencer (GAIIx, Illumina) the alignment rate substantially enhanced increasing thus the amount of usable sequences. We have further developed a pipeline for the analysi of MeDIP-Seq datasets. Potential candidate regions identified in this genome-wide assay can then be validated by the use of selector probes that specifically capture genomic regions of interest. We introduced a bisulfite treatment in the selection protocol and developed a novel multiplex assay. 98 gene loci were enriched in 6 samples and were then sequenced in parallel on a bench sequencer (GS Junior, Roche). The combination of these technologies will permit the establishment of methylome maps and the identification of novel epigenetic biomarkers for cancer and complex diseases diagnostics and prognosis., L'analyse des profils de méthylation présente un grand intérêt car des altérations du méthylome sont impliquées dans de nombreuses pathologies. Le MeDIP (Methylated DNA ImmunoPrecipitation) immunoprécipite les séquences méthylées sur le génome entier, la plupart étant localisées dans les séquences répétées. De telles séquences sont difficiles à aligner après séquençage (MeDIP-Seq) et bon nombre d'entre elles ne peuvent donc être utilisées pour la suite des analyses. Nous présentons une méthode innovante appelée MeDIP-dep-Seq permettant de supprimer une quantité significative de plusieurs familles de ces éléments répétés (diminution d'un facteur de 300 au maximum) tandis que les séquences uniques d'intérêt ne sont pas affectées. Après séquençage sur un séquenceur de seconde génération (GAIIx, Illumina), le taux d'alignement est amélioré de façon conséquente permettant ainsi d'augmenter la quantité de séquences analysables. Nous avons également développé une plateforme d'analyse des données issues du MeDIP-Seq. De potentielles régions candidates identifiées par cette technique sur le génome entier peuvent ensuite être validées en utilisant des sélectors, sondes permettant la capture de régions génomiques d'intérêt. Nous avons introduit un traitement au bisulphite dans le protocole de sélection afin de développer un nouvel outil pour une analyse multiplexe. 98 loci ont été enrichis dans 6 échantillons puis séquencés en parallèle sur un séquenceur de paillasse (GS Junior, Roche). La combinaison de ces technologies permettra d'établir des cartes du méthylome et d'identifier des nouveaux biomarqueurs épigénétiques pour diagnostiquer et pronostiquer les cancers et maladies complexes.