In plants, genomic DNA methylation which contributes to development and stress responses can be actively removed by DEMETER-like DNA demethylases (DMLs). Indeed, in Arabidopsis DMLs are important for maternal imprinting and endosperm demethylation, but only a few studies demonstrate the developmental roles of active DNA demethylation conclusively in this plant. Here, we show a direct cause and effect relationship between active DNA demethylation mainly mediated by the tomato DML, SlDML2, and fruit ripeningan important developmental process unique to plants. RNAi SlDML2 knockdown results in ripening inhibition via hypermethylation and repression of the expression of genes encoding ripening transcription factors and rate-limiting enzymes of key biochemical processes such as carotenoid synthesis. Our data demonstrate that active DNA demethylation is central to the control of ripening in tomato.active DNA demethylation | DNA glycosylase lyase | epigenetic | tomato | fruit ripening G enomic DNA methylation is a major epigenetic mark that is instrumental to many aspects of chromatin function, including gene expression, transposon silencing, or DNA recombination (1-4). In plants, DNA methylation can occur at cytosine both in symmetrical (CG or CHG) and nonsymmetrical (CHH) contexts and is controlled by three classes of DNA methyltransferases, namely, the DNA Methyltransferase 1, Chromomethylases, and the Domain Rearranged Methyltransferases (5-7). Indeed, in all organisms, cytosine methylation can be passively lost after DNA replication in the absence of methyltransferase activity (1). However, plants can also actively demethylate DNA via the action of DNA GlycosylaseLyases, the so-called DEMETER-Like DNA demethylases (DMLs), that remove methylated cytosine, which is then replaced by a nonmethylated cytosine (8

The incidence of renal cell carcinoma (RCC) is increasing worldwide, and its prevalence is particularly high in some parts of Central Europe. Here we undertake whole-genome and transcriptome sequencing of clear cell RCC (ccRCC), the most common form of the disease, in patients from four different European countries with contrasting disease incidence to explore the underlying genomic architecture of RCC. Our findings support previous reports on frequent aberrations in the epigenetic machinery and PI3K/mTOR signalling, and uncover novel pathways and genes affected by recurrent mutations and abnormal transcriptome patterns including focal adhesion, components of extracellular matrix (ECM) and genes encoding FAT cadherins. Furthermore, a large majority of patients from Romania have an unexpected high frequency of A:T4T:A transversions, consistent with exposure to aristolochic acid (AA). These results show that the processes underlying ccRCC tumorigenesis may vary in different populations and suggest that AA may be an important ccRCC carcinogen in Romania, a finding with major public health implications.

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.

DNA methylation has been identified as the most promising molecular biomarker for the prediction of age. Several DNA methylation-based models have been proposed for age prediction based on blood samples, using mainly pyrosequencing. These methods present different performances for age prediction and have rarely, if ever, been evaluated and intercompared in an independent validation study. Here, for the first time, we evaluate and compare six blood-based age prediction models (Bekaert 1 , Park 2 , Thong 3 , Weidner 4 , and the Zbiec-Piekarska 1 5 and Zbiec-Piekarska 2 6 ), using DNA methylation analysis by pyrosequencing on 100 blood samples from French individuals aged between 19–65 years. For each model, we perform correlation analysis and evaluate age-prediction performance (mean absolute deviation (MAD) and standard error of the estimate (SEE)). The best age-prediction performances were found with the Bekaert and Thong models (MAD of 4.5–5.2, SEE of 6.8–7.2), followed by the Zbiec-Piekarska 1 model (MAD of 6.8 and SEE of 9.2), while the Park, Weidner and Zbiec-Piekarska 2 models presented lower performances (MAD of 7.2–8.7 and SEE of 9.2–10.3). Given these results, we recommend performing systematic, independent evaluation of all age prediction models on a same cohort to validate the different models and compare their performance.

Microsatellite instability (MSI) is a genomic alteration in which microsatellites, usually of one to four nucleotide repeats, accumulate mutations corresponding to deletions/insertions of a few nucleotides. The MSI phenotype has been extensively characterized in colorectal cancer and is due to a deficiency of the DNA mismatch repair system. MSI has recently been shown to be present in most types of cancer with variable frequencies (from <1 to 30%). It correlates positively to survival outcome and predicts the response to immune checkpoint blockade therapy. The different methods developed for MSI detection in cancer require taking into consideration two critical parameters which influence method performance. First, the microsatellite markers used should be chosen carefully to ensure they are highly sensitive and specific for MSI detection. Second, the analytical method used should be highly resolute to allow clear identification of MSI and of the mutant allele genotype, and should present the lowest limit of detection possible for application in samples with low mutant allele frequency. In this review, we describe all the different molecular and computational methods developed to date for the detection of MSI in cancer, how they have evolved and improved over the years, and their advantages and drawbacks.

The mitogen-activated protein kinase (MAPK) pathway is constantly activated in Langerhans cell histiocytosis (LCH). Mutations of the downstream kinases BRAF and MAP2K1 mediate this activation in a subset of LCH lesions. In this study, we attempted to identify other mutations which may explain the MAPK activation in nonmutated BRAF and MAP2K1 LCH lesions.We analysed 26 pulmonary and 37 nonpulmonary LCH lesions for the presence of BRAF, MAP2K1, NRAS and KRAS mutations. Grossly normal lung tissue from 10 smoker patients was used as control. Patient spontaneous outcomes were concurrently assessed.BRAF(V600E) mutations were observed in 50% and 38% of the pulmonary and nonpulmonary LCH lesions, respectively. 40% of pulmonary LCH lesions harboured NRAS(Q61K) (/R) mutations, whereas no NRAS mutations were identified in nonpulmonary LCH biopsies or in lung tissue control. In seven out of 11 NRAS(Q61K) (/R)-mutated pulmonary LCH lesions, BRAF(V600) (E) mutations were also present. Separately genotyping each CD1a-positive area from the same pulmonary LCH lesion demonstrated that these concurrent BRAF and NRAS mutations were carried by different cell clones. NRAS(Q61K) (/R) mutations activated both the MAPK and AKT (protein kinase B) pathways. In the univariate analysis, the presence of concurrent BRAF(V600E) and NRAS(Q61K) (/R) mutations was significantly associated with patient outcome.These findings highlight the importance of NRAS genotyping of pulmonary LCH lesions because the use of BRAF inhibitors in this context may lead to paradoxical disease progression. These patients might benefit from MAPK kinase inhibitor-based treatments.

The clinical significance of the BRAFV600E mutation in adult Langerhans cell histiocytosis (LCH), including pulmonary Langerhans cell histiocytosis (PLCH), is not well understood. Similarly, the spectrum of molecular alterations involved in adult LCH has not been fully delineated. To address these issues, we genotyped a large number of adult LCH biopsies and searched for an association of identified molecular alterations with clinical presentation and disease outcome.Biopsies from 117 adult LCH patients, 83 with PLCH (median age 36.4 years, 56 females, 38 multisystem disease, 79 single system disease, 65 current smokers) were genotyped for the BRAFV600E mutation. In 69 cases, LCH lesions were also genotyped by whole-exome sequencing (WES) or targeted gene panel next-generation sequencing (NGS). Cox models were used to estimate the association of baseline characteristics with the hazard of LCH progression.MAPK pathway alterations were detected in 59 out of 69 cases (86%) (BRAFV600E mutation: 36%, BRAFN486_P490 deletion: 28%, MAP2K1 mutations: 15%, isolated NRASQ61 mutations: 4%), while KRAS mutations were virtually absent in PLCH lesions. The BRAFV600E mutation was not associated with LCH presentation at diagnosis, including smoking status and lung function, in PLCH patients. BRAFV600E status did not influence the risk of LCH progression over time.Thus, MAPK alterations are present in most lesions from adult LCH patients, particularly in PLCH. Unlike reports in paediatric LCH, BRAFV600E genotyping did not provide additional information on disease outcome. The search for alterations involved in the MAPK pathway, including BRAF deletions, is useful for guiding targeted treatment in selected patients with refractory progressive LCH.

The Enhancer of Zeste Polycomb group proteins, which are encoded by a small gene family in Arabidopsis thaliana, participate to the control of plant development. In the tomato (Solanum lycopersicum), these proteins are encoded by three genes (SlEZ1, SlEZ2 and SlEZ3) that display specific expression profiles. Using a gene specific RNAi strategy, we demonstrate that repression of SlEZ2 correlates with a general reduction of H3K27me3 levels, indicating that SlEZ2 is part of an active PRC2 complex. Reduction of SlEZ2 gene expression impacts the vegetative development of tomato plants, consistent with SlEZ2 having retained at least some of the functions of the Arabidopsis CURLY LEAF (CLF) protein. Notwithstanding, we observed significant differences between transgenic SlEZ2 RNAi tomato plants and Arabidopsis clf mutants. First, we found that reduced SlEZ2 expression has dramatic effects on tomato fruit development and ripening, functions not described in Arabidopsis for the CLF protein. In addition, repression of SlEZ2 has no significant effect on the flowering time or the control of flower organ identity, in contrast to the Arabidopsis clf mutation. Taken together, our results are consistent with a diversification of the function of CLF orthologues in plants, and indicate that although partly conserved amongst plants, the function of EZ proteins need to be newly investigated for non-model plants because they might have been recruited to specific developmental processes.