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EZH2, a component of the polycomb repressive complex 2, catalyses the trimethylation of histone H3 lysine 27, a chromatin mark associated with transcriptional repression. EZH2 loss-of-function mutations are seen in myeloid neoplasms and are associated with an adverse prognosis. Missense mutations in the SET/CXC domain abrogate catalytic activity as assessed by in vitro histone methylation assays, but missense mutations clustering in the conserved DI and DII regions retain activity. To understand the role of DI and DII mutations, we initially developed a cell-based histone methylation assay to test activity in a cellular context. Murine induced pluripotent stem cells lacking EZH2 were transiently transfected with wild type or mutant EZH2 (n = 15) and any resulting histone methylation was measured by flow cytometry. All DI mutations (n = 5) resulted in complete or partial loss of methylation activity whilst 5/6 DII mutations retained activity. Next, we assessed the possibility of splicing abnormalities induced by exon 8 mutations (encoding DII) using RT-PCR from primary patient samples and mini-gene assays. Exon 8 mutations resulted in skipping of exon 8 and an out-of-frame transcript. We have therefore shown that mutations within regions encoding EZH2 domains DI and DII are pathogenic by loss of function and exon skipping, respectively.

Ultra-processed foods (UPFs) consumption could affect gut microbiota diversity and profile. We aimed to evaluate the effects of UPFs on microbiota, considering the role of sex. The consumption of UPFs (using NOVA criteria) was assessed with a validated 137-item food-frequency questionnaire. Participants (n = 359) were classified into less than three servings per day (n = 96) of UPFs and more than five (n = 90). Women and men were subclassified following the same criteria. 16S rRNA sequencing was performed from DNA fecal samples, and differences in microbiota were analyzed using EdgeR. The relationship between UPFs and bacteria was assessed by Spearman correlation and comparison of tertiles of consumption. Women who consumed more than five servings/day of UPFs presented an increase in Acidaminococcus, Butyrivibrio, Gemmiger, Shigella, Anaerofilum, Parabacteroides, Bifidobacterium, Enterobacteriales, Bifidobacteriales and Actinobacteria and a decrease in Melainabacter and Lachnospira. Bifidobacterium, Bifidobacteriales and Actinobacteria was positively associated with pizza and Actinobacteria with industrially processed dairy in women. Men who consumed more than five servings/day presented an increase of Granulicatella, Blautia, Carnobacteriaceae, Bacteroidaceae, Peptostreptococcaceae, Bacteroidia and Bacteroidetes and a decrease of Anaerostipes and Clostridiaceae. Bacteroidia and Bacteroidetes correlated positively with industrially processed meat. This study suggests that UPFs may affect microbiota composition differently in women and men.

Fecal microbiome disturbances are linked to different human diseases. In the case of obesity, gut microbiota seems to play a role in the development of low-grade inflammation. The purpose of the present study was to identify specific bacterial families and genera associated with an increased obesity-related inflammatory status, which would allow to build a regression model for the prediction of the inflammatory status of obese and overweight subjects based on fecal microorganisms.A total of 361 volunteers from the Obekit trial (65 normal-weight, 110 overweight, and 186 obese) were classified according to four variables: waist/hip ratio (≥0.86 for women and ≥1.00 for men), leptin/adiponectin ratio (LAR, ≥3.0 for women and ≥1.4 for men), and plasma C-reactive protein (≥2 mg/L) and TNF levels (≥0.85 pg/mL). An inflammation score was designed to classify individuals in low (those subjects who did exceed the threshold value in 0 or 1 variable) or high inflammatory index (those subjects who did exceed the threshold value in 2 or more variables). Fecal 16 S rRNA sequencing was performed for all participants, and differential abundance analyses for family and genera were performed using the MicrobiomeAnalyst web-based platform.Methanobacteriaceae, Christensenellaceae, Coriobacteriaceae, Bifidobacteriaceae, Catabacteriaceae, and Dehalobacteriaceae families, and Methanobrevibacter, Eggerthella, Gemmiger, Anaerostipes, and Collinsella genera were significantly overrepresented in subjects with low inflammatory index. Conversely, Carnobacteriaceae, Veillonellaceae, Pasteurellaceae, Prevotellaceae and Enterobacteriaceae families, and Granulicatella, Veillonella, Haemophilus, Dialister Parabacteroides, Prevotella, Shigella, and Allisonella genera were more abundant in subjects with a high inflammatory index. A regression model adjusted by BMI, sex, and age and including the families Coriobacteriaceae and Prevotellaceae and the genus Veillonella was developed.A microbiota-based regression model was able to predict the obesity-related inflammatory status (area under the ROC curve = 0.8570 ± 0.0092 Harrell's optimism-correction) and could be useful in the precision management of inflammobesity.

Obesity has been related to intestinal dysbiosis and the modification of gut microbiota composition by dietary strategies becomes a promising strategy to help manage obesity. The aim of the current study was to evaluate the effect of two weight-loss diets on the composition and functional profile of gut microbiota.55 men and 124 women with BMI 25 kg/mAfter the intervention, MHP-men showed a significant decrease in Negativicutes, Selenomonadales, Dielma and Dielma fastidiosa. LF-men showed a significant increase in Bacilli, Lactobacillales, Christensenellaceae, Peptococcaceae, and Streptococcaceae, Peptococcus, Streptococcus and Christensenella, Duncaniella dubosii_CP039396_93.49%, Roseburia sp_AB744234_98.96% and Alistipes inops_KJ572413_99.57%. MHP-women increased Pasteurellales, Phascolarctobacterium succinatutens, Ruthenibacterium lactatiformans_LR215981_99.55% and decreased in Phascolarctobacterium succinatutens_NR112902_99.56%. Finally, LF-women presented a significant decrease in Bacteroides clarus and Erysipelothrix inopinata_CP060715_84.4%. Surprisingly, no matching bacterial changes were found between these four groups. A total of 42 KO, 10 metabolic pathways and 107 related metabolites related were found implicated in these bacterial changes. Seven metabolites were confirmed in plasma.Weight-loss-related-changes in gut microbiome composition and the functional profile occur in a sex- and diet-related manner, showing that women and men could differentially benefit from the consumption of MHP and LF diets.NCT02737267, 10th March 2016 retrospectively registered.

Cocoa polyphenols exhibit high antioxidant activity and have been proposed as a potential adjuvant for the treatment of metabolic disturbances. Here, we demonstrate that supplementation with low doses (14 and 140 mg per kg per rat) of a complete cocoa extract induces metabolic benefits in a diet-induced obesity (DIO) model of Wistar rats. After 10 weeks, cocoa extract-supplemented animals exhibited significantly lower body weight gain and food efficiency, with no differences in energy intake. Cocoa significantly reduced visceral (epididymal and retroperitoneal) and subcutaneous fat accumulation accompanied by a significant reduction in the adipocyte size, which was mediated by downregulation of the adipocyte-specific genes Cebpa, Fasn and Adipoq. Additionally, cocoa extract supplementation reduced the triacylglycerol/high density lipoprotein (TAG/HDL) ratio, decreased hepatic triglyceride accumulation, improved insulin sensitivity by reducing HOMA-IR, and significantly ameliorated glucose tolerance after an intraperitoneal glucose tolerance test. Finally, no adverse effect was observed in an in vivo toxicity evaluation of our cocoa extract at doses up to 500 mg kg-1 day-1. Our data demonstrate that low doses of cocoa extract supplementation (14 and 140 mg kg-1 day-1) are safe and sufficient to counteract obesity and type-2 diabetes in rats and provide new insights into the potential application of cocoa supplements in the management of the metabolic syndrome.

Obesity is a medical condition with increasing prevalence, characterized by an accumulation of excess fat that could be improved using some bioactive compounds. However, many of these compounds with in vitro activity fail to respond in vivo, probably due to the sophistication of the physiological energy regulatory networks. In this context, C. elegans has emerged as a plausible model for the identification and characterization of the effect of such compounds on fat storage in a complete organism. However, the results obtained in such a simple model are not easily extrapolated to more complex organisms such as mammals, which hinders its application in the short term. Therefore, it is necessary to obtain new experimental data about the evolutionary conservation of the mechanisms of fat loss between worms and mammals. Previously, we found that some omega-6 fatty acids promote fat loss in C. elegans by up-regulation of peroxisomal fatty acid β-oxidation in an omega-3 independent manner. In this work, we prove that the omega-6 fatty acids' effects on worms are also seen when they are supplemented with a natural omega-6 source (borage seed oil, BSO). Additionally, we explore the anti-obesity effects of two doses of BSO in a diet-induced obesity rat model, validating the up-regulation of peroxisomal fatty acid β-oxidation. The supplementation with BSO significantly reduces body weight gain and energy efficiency and prevents white adipose tissue accumulation without affecting food intake. Moreover, BSO also increases serum HDL-cholesterol levels, improves insulin resistance and promotes the down-regulation of Cebpa, an adipogenesis-related gene. Therefore, we conclude that the effects of omega-6 fatty acids are highly conserved between worms and obesity-induced mammals, so these compounds could be considered to treat or prevent obesity-related disorders.

Bioinformatic analyses of CALR mutations in myeloproliferative neoplasms support a role in signaling

Summary: Whole exome sequencing was performed in a patient with myelodysplastic syndrome before and after progression to acute myeloid leukaemia. Mutations in several genes, including SETBP1, were identified following leukaemic transformation. Screening of 328 patients with myeloid disorders revealed SETBP1 mutations in 14 patients (4·3%), 7 of whom had -7/del(7q) and 3 had i(17)(q10), cytogenetic markers associated with shortened overall survival and increased risk of leukaemic evolution. SETBP1 mutations were frequently acquired at the time of leukaemic evolution, coinciding with increase of leukaemic blasts. These data suggest that SETBP1 mutations may play a role in MDS and chronic myelomonocytic leukaemia disease progression. © 2013 John Wiley and Sons Ltd.

Recurrent loss of heterozygosity (LOH) in 4q24 in patients with myeloid malignancies has led to the identifi cation of mutations in TET2 in diff erent myeloid neoplasms including myeloproliferative neoplasm (MPN), myelodysplastic syndrome (MDS), mixed myelodysplastic/myeloproliferative neoplasm (MDS/MPN) and acute myeloid leukemia (AML) [1 – 13]. Th eir impact on prognosis remains unresolved, and the mechanisms by which TET2 leads to transformation remain unclear. Based on its homology to TET1, it is possible that TET2 may play a role in epigenetic regulation [14] via the presence of catalytic domains in the two highly conserved regions of the gene. Mutations in these two regions could, by this mechanism, lead to inactivation of a specifi c tumor suppressor gene and/or activation of pro-proliferative pathways, although mutations in other areas of the gene could also result in a modifi cation of the protein. Studies using murine models of Tet2 inactivation show that inactivation of this gene establishes important roles for Tet2 in hematopoiesis and the development of myeloproliferative disease. Beacuse TET2 alterations in patients are commonly heterozygous, TET2 haploinsuffi ciency could be enough to promote defects in a hematopoietic progenitor and myeloproliferation [15]. We have collected data on all TET2 mutations published to date [1 – 13] (a total of 725 mutations in 3274 patients) (Table I) in order to analyze whether: (a) they are evenly distributed throughout the gene, (b) diff erent mutation types (missense, nonsense or frameshift) are more prevalent in diff erent regions of the gene and (c) distinct mutational patterns are found in diff erent types of myeloid neoplasms. To this end, we divided the sequence of the TET2 gene into four regions. Region B1 ( Box 1 ) and region B2 ( Box 2 ) are highly conserved in several species and in all three proteins of the TET family. Th e other two regions, which we denominated F1 and F2, are non-conserved regions (Figure 1). We calculated the frequency of mutations per amino acid in each of these regions (number of mutations divided by the number of amino acids

Hematological malignancies with eosinophilia are often associated with fusions in PDGFRA, PDGFRB, or FGFR1 genes. RT-PCR has proved to be useful for finding new PDGFRA gene fusions, but some studies have shown overexpression of the TK domain which cannot be explained by the existence of such aberrations. This fact could be related to the expression of alternative PDGFRA transcripts. We show that quantification of the expression of three different PDGFRA fragments discriminates between PDGFRA alternative transcripts and fusion genes, and we have tested this novel methodological approach in a group of eosinophilia cases. Our data show that alternative PDGFRA transcripts should be taken into account when screening for PDGFRA aberrations, such as gene fusions, by RT-PCR. Expression from an internal PDGFRA promoter seems to be a frequent event, in both normal and leukemic samples, and is probably related to physiological conditions, but it could have a role in other tumors. Even so, we show that our RQ-PCR methodology can discriminate expression of alternative transcripts from the presence of X-PDGFRA fusion genes.

BCR/ABL1-negative chronic myeloproliferative neoplasms (CMPNs) are a heterogeneous group of clonal hematological malignancies. Over recent years, some genetic events in tyrosine kinase (TK) genes have been described as causal events of these diseases. To identify new genetic aberrations underlying these diseases, we used denaturing high performance liquid chromatography and fluorescence in situ hybridization (FISH) to analyze 17 genes from two receptor-TK families (III and IV) and from three cytoplasmic-TK families (Syk, Abl, and Jak) on samples from 44 BCR/ABL1-negative and JAK2(V617F)-negative CMPN patients with different clinical phenotypes. Although screening by FISH did not reveal novel chromosomal aberrations, several sequence changes were detected. None of them were frequent events, but we identified a new potential activating mutation in the FERM domain of JAK2(R340Q). None of the germline JAK2(V617F) single-nucleotide polymorphisms detected differed in distribution between patients and control subjects. In summary, data presented here show that these genes are not frequently mutated or rearranged in CMPNs, suggesting that molecular events causing these disorders must be located in other genes.

The BCR-ABL negative chronic myeloproliferative disorders (CMPDs) are a group of stem cell clonal haematological malignancies characterised by abnormal proliferation and survival of one or more myeloid lineage cells. It has been proposed that they are caused by abnormalities in some signal transduction pathways mainly due to acquired somatic mutations, some of them in tyrosine kinase genes. One of the most prevalent mutations is V617F in the pseudokinase domain (JH2) coded by JAK2 exon 14. This mutation, reported in 2005, has been associated with nearly 95% of patients diagnosed of polycythaemia vera (PV) and near a half of the patients with essential thrombocythaemia (ET) and primary myelofibrosis (MF). However, the frequency of this mutation is below 20% for the remaining chronic myeloproliferative disorders and is absent in lymphoid neoplasms. JAK2V617F has been an important milestone in the knowledge of the molecular mechanisms leading to classic myeloproliferative disorders. However, there are still some patients with PV, ET and MF lacking V617F whose molecular defect is unknown. The disease in these cases could be due to other abnormalities in JAK2 or other related genes. In fact, in a few cases new point mutations have been reported, affecting also the JH2 domain, and one mutation affecting the JH1 domain [1] and reviewed in [2], some of them in other haematological malignancies. In addition, 10% of patients diagnosed of MF and some with ET show somatic activating mutations in the thrombopoietin receptor gene (MPL) inducing constitutive cytokine-independent activation of the JAK-STAT pathway.

Two megalencephaly (MEG) syndromes, megalencephaly-capillary malformation (MCAP) and megalencephaly-polymicrogyriapolydactyly-hydrocephalus (MPPH), have recently been defined on the basis of physical and neuroimaging features. Subsequently, exome sequencing of ten MEG cases identified de-novo postzygotic mutations in PIK3CA which cause MCAP and de-novo mutations in AKT and PIK3R2 which cause MPPH. Here we present findings from exome sequencing three unrelated megalencephaly patients which identified a causal PIK3CA mutation in two cases and a causal PIK3R2 mutation in the third case. However, our patient with the PIK3R2 mutation which is considered to cause MPPH has a marked bifrontal band heterotopia which is a feature of MCAP. Furthermore, one of our patients with a PIK3CA mutation lacks syndactyly/polydactyly which is a characteristic of MCAP. These findings suggest that the overlap between MCAP and MPPH may be greater than the available studies suggest. In addition, the PIK3CA mutation in one of our patients could not be detected using standard exome analysis because the mutation was observed at a low frequency consistent with somatic mosaicism. We have therefore investigated several alternative methods of exome analysis and demonstrate that alteration of the initial allele frequency spectrum (AFS), used as a prior for variant calling in samtools, had the greatest power to detect variants with low mutant allele frequencies in our 3 MEG exomes and in simulated data. We therefore recommend non-default settings of the AFS in combination with stringent quality control when searching for causal mutation(s) that could have low levels of mutant reads due to post-zygotic mutation.

When studying mutations in DNA samples, determining whether novel sequence changes are somatic mutations or germline polymorphisms can be difficult. Here we describe a novel and very simple approach for identification of somatic mutations and loss of heterozygosity (LoH) events in DNA samples where no matched tissue sample is available. Our method makes use of heterozygous polymorphisms that are located near the putative mutation to trace both germinal alleles.

Core-binding factor acute myeloid leukemia (CBF-AML) is an acute myeloid neoplasm characterized by the presence of t(8;21)(q22;q22) [abbreviated as t(8;21)] or inv(16)(p13q22)/t(16;16)(p13;q22) [in...

Background Despite the discovery of the p.V617F in JAK2 , the molecular pathogenesis of some chronic myeloproliferative neoplasms remains unclear. Although very rare, different studies have identified CBL (Cas-Br-Murine ecotropic retroviral transforming sequence) mutations in V617F JAK2 -negative patients, mainly located in the RING finger domain. In order to determine the frequency of CBL mutations in these diseases, we studied different regions of all CBL family genes ( CBL , CBLB and CBLC ) in a selected group of patients with myeloproliferative neoplasms. We also included V617F JAK2- positive patients to check whether mutations in CBL and JAK2 are mutually exclusive events. Design and Methods Using denaturing high performance liquid chromatography, we screened for mutations in CBL , CBLB and CBLC in a group of 172 V617F JAK2 -negative and 232 V617F JAK2 -positive patients with myeloproliferative neoplasms not selected for loss of heterozygosity. The effect on cell proliferation of the mutations detected was analyzed on a 32D(FLT3) cell model. Results An initial screening of all coding exons of CBL , CBLB and CBLC in 44 V617F JAK2 -negative samples revealed two new CBL mutations (p.C416W in the RING finger domain and p.A678V in the proline-rich domain). Analyses performed on 128 additional V617F JAK2 -negative and 232 V617F JAK2 -positive samples detected three CBL changes (p.T402HfsX29, p.P417R and p.S675C in two cases) in four V617F JAK2 -positive patients. None of these mutations was found in 200 control samples. Cell proliferation assays showed that all of the mutations promoted hypersensitivity to interleukin-3 in 32D(FLT3) cells. Conclusions Although mutations described to date have been found in the RING finger domain and in the linker region of CBL, we found a similar frequency of mutations in the proline-rich domain. In addition, we found CBL mutations in both V617F JAK2 -positive (4/232; 1.7%) and negative (2/172; 1.2%) patients and all of them promoted hypersensitivity to interleukin-3.