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Purpose Large parts of the Amazon rainforest grow on weathered soils depleted in phosphorus and rock-derived cations. We tested the hypothesis that in this ecosystem, fine roots stimulate decomposition and nutrient release from leaf litter biochemically by releasing enzymes, and by exuding labile carbon stimulating microbial decomposers. Methods We monitored leaf litter decomposition in a Central Amazon tropical rainforest, where fine roots were either present or excluded, over 188 days and added labile carbon substrates (glucose and citric acid) in a fully factorial design. We tracked litter mass loss, remaining carbon, nitrogen, phosphorus and cation concentrations, extracellular enzyme activity and microbial carbon and nutrient concentrations. Results Fine root presence did not affect litter mass loss but significantly increased the loss of phosphorus and cations from leaf litter. In the presence of fine roots, acid phosphatase activity was 43.2% higher, while neither microbial stoichiometry, nor extracellular enzyme activities targeting carbon- and nitrogen-containing compounds changed. Glucose additions increased phosphorus loss from litter when fine roots were present, and enhanced phosphatase activity in root exclusions. Citric acid additions reduced litter mass loss, microbial biomass nitrogen and phosphorus, regardless of fine root presence or exclusion. Conclusions We conclude that plant roots release significant amounts of acid phosphatases into the litter layer and mobilize phosphorus without affecting litter mass loss. Our results further indicate that added labile carbon inputs (i.e. glucose) can stimulate acid phosphatase production by microbial decomposers, highlighting the potential importance of plant-microbial feedbacks in tropical forest ecosystems.

This article belongs to the Special Issue mRNA Metabolism in Health and Disease. Familial hypercholesterolemia (FH) is the most common genetic disorder of lipid metabolism, characterized by increased levels of total and LDL plasma cholesterol, which leads to premature atherosclerosis and coronary heart disease. FH phenotype has considerable genetic heterogeneity and phenotypic variability, depending on LDL receptor activity and lifestyle. To improve diagnosis and patient management, here, we characterized two single nucleotide missense substitutions at Methionine 1 of the human LDLR gene (c.1A>T/p.(Met1Leu) and c.1A>C/p.(Met1Leu)). We used a combination of Western blot, flow cytometry, and luciferase assays to determine the effects of both variants on the expression, activity, and synthesis of LDLR. Our data show that both variants can mediate translation initiation, although the expression of variant c.1A>T is very low. Both variants are in the translation initiation codon and codify for the same amino acid p.(Met1Leu), yet they lead to different levels of impairment on LDLR expression and activity, corroborating different efficiencies of the translation initiation at these non-canonical initiation codons. The functional data of these variants allowed for an improved American College of Medical Genetics (ACMG) classification for both variants, which can allow a more personalized choice of the lipid-lowering treatment and dyslipidemia management, ultimately improving patients' prognosis. The authors acknowledge Fundação para a Ciência e a Tecnologia (Ph.D. scholarship no. SFRH/PD/BD/131427/2017 and SFRH/PD/BD/114392/2016). info:eu-repo/semantics/publishedVersion

The year 2020 was interrupted by the coronavirus pandemic, when abruptly universities, schools, commerce and non-essential services were paralyzed. In this adverse context, the project “COVID 19 and the social determination of health” was created. A qualitative research, focusing on the strategy of participatory diagnosis, which sought to discuss with the community of a federal public school, located in Niterói / RJ, about the health conditions and social vulnerability impacted by the pandemic, causing reflections and actions on health inequities. In this article, we describe the actions developed by the fellows of this project, analyzing the development of the actions in the light of the theory of social determination of health and popular education in health. Despite the limitations imposed by social distance, we managed to dialogue with the community virtually through the actions of the project, promoting important discussions and reflections for mutual strengthening, against any type of attack that insists on deepening social inequalities. El año 2020 fue interrumpido por la pandemia de coronavirus, cuando de forma abrupta se paralizaron universidades, escuelas, comercio y servicios no esenciales. En este contexto adverso, se creó el proyecto “COVID 19 y la determinación social de la salud”. Una investigación cualitativa, enfocada en la estrategia de diagnóstico participativo, que buscó discutir con la comunidad de una escuela pública federal, ubicada en Niterói / RJ, sobre las condiciones de salud y vulnerabilidad social impactadas por la pandemia, provocando reflexiones y acciones sobre inequidades en salud. . En este artículo se describen las acciones desarrolladas por los becarios de este proyecto, analizando el desarrollo de las acciones a la luz de la teoría de la determinación social de la salud y la educación popular en salud. A pesar de las limitaciones que impone la distancia social, logramos dialogar con la comunidad virtualmente a través de las acciones del proyecto, promoviendo importantes discusiones y reflexiones para el fortalecimiento mutuo, frente a cualquier tipo de ataque que insista en profundizar las desigualdades sociales. O ano de 2020 foi interrompido pela pandemia do coronavírus, quando abruptamente universidades, escolas, comércio e serviços não essenciais foram paralisados. Neste contexto adverso foi criado o projeto “A COVID 19 e a determinação social da saúde”. Uma pesquisa qualitativa, com enfoque na estratégia do diagnóstico participativo, que buscou discutir com a comunidade de uma escola pública federal, localizada em Niterói/RJ, sobre as condições de saúde e vulnerabilidade social impactadas pela pandemia, provocando reflexões e ações sobre as iniquidades em saúde. Neste artigo descrevemos as ações desenvolvidas pelos bolsistas deste projeto, analisando seus desdobramentos sob a luz da teoria da determinação social da saúde e da educação popular em saúde. Apesar das limitações impostas pelo distanciamento social, conseguimos dialogar virtualmente com a comunidade através das ações do projeto, promovendo discussões e reflexões importantes para o fortalecimento mútuo, contra todo tipo de ataque que insiste em aprofundar as desigualdades sociais.&nbsp

O ano de 2020 foi interrompido pela pandemia do coronavírus, quando abruptamente universidades, escolas, comércio e serviços não essenciais foram paralisados. Neste contexto adverso foi criado o projeto “A COVID 19 e a determinação social da saúde”. Uma pesquisa qualitativa, com enfoque na estratégia do diagnóstico participativo, que buscou discutir com a comunidade de uma escola pública federal, localizada em Niterói/RJ, sobre as condições de saúde e vulnerabilidade social impactadas pela pandemia, provocando reflexões e ações sobre as iniquidades em saúde. Neste artigo descrevemos as ações desenvolvidas pelos bolsistas deste projeto, analisando seus desdobramentos sob a luz da teoria da determinação social da saúde e da educação popular em saúde. Apesar das limitações impostas pelo distanciamento social, conseguimos dialogar virtualmente com a comunidade através das ações do projeto, promovendo discussões e reflexões importantes para o fortalecimento mútuo, contra todo tipo de ataque que insiste em aprofundar as desigualdades sociais.

Innate behaviors consist of a succession of genetically-hardwired motor and physiological subprograms that can be coupled to drastic morphogenetic changes. How these integrative responses are orchestrated is not completely understood. Here, we provide insight into these mechanisms by studying pupariation, a multi-step innate behavior of Drosophila larvae that is critical for survival during metamorphosis. We find that the steroid-hormone ecdysone triggers parallel pupariation neuromotor and morphogenetic subprograms, which include the induction of the relaxin-peptide hormone, Dilp8, in the epidermis. Dilp8 acts on six Lgr3-positive thoracic interneurons to couple both subprograms in time and to instruct neuromotor subprogram switching during behavior. Our work reveals that interorgan feedback gates progression between subunits of an innate behavior and points to an ancestral neuromodulatory function of relaxin signaling., Pupariation in Drosophila is triggered by the steroid-hormone ecdysone and requires coordination between associated behavioral and body-reshaping motor subprograms. The authors show that coordination requires ecdysone-dependent Dilp8-Lgr3 signaling between the cuticle epidermis and interneurons.

The rapid rise in atmospheric CO2 concentration over the past century is unprecedented. It has unambiguously influenced Earth’s climate system and terrestrial ecosystems. Elevated atmospheric CO2 concentrations (eCO2) have induced an increase in biomass and thus, a carbon sink in forests worldwide. It is assumed that eCO2 stimulates photosynthesis and plant productivity and enhances water-use efficiency – the so-called CO2-fertilization effect, which may provide an important buffering effect for plants during adverse climate conditions. For these reasons, current global climate simulations consistently predict that tropical forests will continue to sequester more carbon in aboveground biomass, partially compensating human emissions and decelerating climate change by acting as a carbon sink. In contrast to model simulations, several lines of evidence point towards a decreasing carbon sink strength of the Amazon rainforest. Reliable predictions of eCO2 effects in the Amazon rainforest are hindered by a lack of process-based information gained from ecosystem scale eCO2 experiments. Here we report on baseline measurements from the Amazon Free Air CO2 Enrichment (AmazonFACE) experiment and preliminary results from open-top chamber (OTC) experiments with eCO2. After three months of eCO2, we find that understory saplings increased carbon assimilation by 17% (under light saturated conditions) and water use efficiency by 39% in the OTC experiment. We present our main hypotheses for the FACE experiment, and discuss our expectations on the potential driving processes for limiting or stimulating the Amazon rainforest carbon sink under eCO2. We focus on possible effects of eCO2 on carbon uptake and allocation, nutrient cycling, water-use and plant-herbivore interactions, which need to be implemented in dynamic vegetation models to estimate future changes of the Amazon carbon sink.

Protein S (PS) is a widely studied protein with an important function in the downregulation of thrombin formation. Since its discovery in 1976, more than 400 variants have been described in PS gene (PROS1) associated with PS deficiency and as a risk factor for venous thromboembolism (VTE). We describe a novel variant, c.1871-14T>G, in intron 14 of PROS1 gene identified in two patients with PS deficiency from two unrelated families with a history of thrombotic disease. This alteration leads to a PROS1 mRNA expression reduction, probably due to nonsense-mediated mRNA decay. Our results suggest that c.1871-14T>G is causative of type I PS deficiency in these patients, highlighting the importance of screening not only the coding and the most conserved intron–exon junctions, but also perform mRNA-based studies. We call attention to the potential increased risk of VTE in hereditary type I PS deficiency associated with this cryptic splice-site variant. Key Clinical Message: Our results prove that c.1871-14T>G is causative of type I PS deficiency, highlighting the importance of performing mRNA-based studies in order to evaluate variants pathogenicity. We evidence the increased risk of venous thromboembolism associated with this cryptic splice-site variant if present in patients with PS deficiency. Juliane Menezes is supported by a fellowship from Fundação para a Ciência e a Tecnologia (SFRH/BPD/98360/2013), and this work was partially funded by the Centre for Toxicogenomics and Human Health—ToxOmics, Genetics, Oncology and Human Toxicology from Fundação para a Ciência e a Tecnologia (UID/BIM/00009/2013). info:eu-repo/semantics/publishedVersion

In this article, we present supportive data related to the research article “A role for DIS3L2 over natural nonsense-mediated mRNA decay targets in human cells” [1], where interpretation of the data presented here is available. Indeed, here we analyze the impact of the DIS3L2 exoribonuclease over nonsense-mediated mRNA decay (NMD)-targets. Specifically, we present data on: a) the expression of various reporter human β-globin mRNAs, monitored by Northern blot and RT-qPCR, before and after altering DIS3L2 levels in HeLa cells, and b) the gene expression levels of deregulated transcripts generated by re-analyzing publicly available data from UPF1-depleted HeLa cells that were further cross-referenced with a dataset of transcripts upregulated in DIS3L2-depleted cells. These analyses revealed that DIS3L2 regulates the levels of a subset of NMD-targets. These data can be valuable for researchers interested in the NMD mechanism. This work was partially supported by Fundação para a Ciencia e a Tecnologia (FCT) (PTFC/BIM-MEC/3749/2014 to LR and UID/MULTI/04046/2013 to BioISI). PJdC, HAS and JFG-M are recipients of a fellowship from BioSys PhD programme (SFRH/BD/52495/2014, SFRH/BD/52492/2014, and PD/BD/ 142898/2018, respectively) and JM is a postdoctoral fellow (SFRH/BPD/98360/2013) from FCT. Work at ITQB-NOVA was financially supported by: Project LISBOA-01-0145-FEDER-007660 funded by the European Regional Development Fund (FEDER) through COMPETE2020 - Programa Operacional Competitividade e Internacionalização (POCI) and by FCT funds: PTDC/BIA-MIC/1399/2014 to CMA and PTFC/BIM-MEC/3749/2014 to SCV. SCV was financed by program IF of FCT (IF/00217/2015). MS was financed by an FCT contract according to DL57/2016 [SFRH/BPD/109464/2015] info:eu-repo/semantics/publishedVersion

Supplementary data to this article can be found online at https://doi.org/10.1016/j.bbrc.2019.08.105. The nonsense-mediated decay (NMD) pathway selectively degrades mRNAs carrying a premature translation-termination codon but also regulates the abundance of a large number of physiological mRNAs that encode full-length proteins. In human cells, NMD-targeted mRNAs are degraded by endonucleolytic cleavage and exonucleolytic degradation from both 5-' and 3'-ends. This is done by a process not yet completely understood that recruits decapping and 5'-to-3' exonuclease activities, as well as deadenylating and 3'-to-5' exonuclease exosome activities. In yeast, DIS3/Rrp44 protein is the catalytic subunit of the exosome, but in humans, there are three known paralogues of this enzyme: DIS3, DIS3L1, and DIS3L2. However, little is known about their role in NMD. Here, we show that some NMD-targets are DIS3L2 substrates in human cells. In addition, we observed that DIS3L2 acts over full-length transcripts, through a process that also involves UPF1. Moreover, DIS3L2-mediated decay is dependent on the activity of the terminal uridylyl transferases Zcchc6/11 (TUT7/4). Together, our findings establish a role for DIS3L2 and uridylation in NMD. Highlights: DIS3L2 functions in the decay of natural NMD-targets in a transcript-specific manner; DIS3L2 acts over full-length NMD-targets, through a process that also involves UPF1; DIS3L2 function in NMD is dependent on the terminal uridylyl transferases Zcchc6/11. This work was partially supported by Fundaçao para a Ci ~ encia e ^ a Tecnologia (FCT) (PTFC/BIM-MEC/3749/2014 to LR and UID/ MULTI/04046/2013 to BioISI). PJdC, HAS and JFG-M are recipients of a fellowship from BioSys PhD programme (SFRH/BD/52495/2014, SFRH/BD/52492/2014, and PD/BD/142898/2018, respectively) and JM is a posdoc fellow (SFRH/BPD/98360/2013) from FCT. Work at ITQB-NOVA was financially supported by: Project LISBOA-01-0145-FEDER-007660 funded by the European Regional Development Fund (FEDER) through COMPETE2020 - Programa Operacional Competitividade e Internacionalizaçao (POCI) and by FCT funds: ~ PTDC/BIA-MIC/1399/2014 to CMA and PTDC/BIM-MEC/3749/2014 to SCV. SCV was financed by program IF of FCT (IF/00217/2015). MS was financed by an FCT contract according to DL57/2016 [SFRH/ BPD/109464/2015]. We thank Dr. V. Narry Kim from Seoul National University, who kindly provided us with the pCK-FLAG-TUT4 and pCK-FLAG-TUT7 vectors. info:eu-repo/semantics/publishedVersion

The nonsense-mediated decay (NMD) pathway selectively degrades mRNAs carrying a premature translation-termination codon but also regulates the abundance of a large number of physiological mRNAs that encode full-length proteins. In human cells, NMD-targeted mRNAs are degraded by endonucleolytic cleavage and exonucleolytic degradation from both 5’ and 3’ ends. This is done by a process not yet completely understood that recruits decapping and 5’-to-3’ exonuclease activities, as well as deadenylating and 3’-to-5’ exonuclease exosome activities. In yeast, DIS3/Rrp44 protein is the catalytic subunit of the exosome, but in humans, there are three known paralogues of this enzyme: DIS3, DIS3L1, and DIS3L2. DIS3L1 and DIS3L2 exoribonucleases localize in the same compartment where NMD occurs, but little is known about their role in this process. In order to unveil the role of DIS3L2 in NMD, here we show that some NMD-targets accumulate in DIS3L2-depleted cells. mRNA half-life analysis further supports that these NMD-targets are in fact DIS3L2 substrates. Besides, we observed that DIS3L2 acts over full-length transcripts, through a process that also involves UPF1. Moreover, DIS3L2-mediated decay is dependent on the activity of the terminal uridylyl transferases Zcchc6/11 (TUT7/4). Together, our findings establish a role for DIS3L2 and uridylation in NMD.

Throughout evolution, eukaryotic cells have devised different mechanisms to cope with stressful environments. When eukaryotic cells are exposed to stress stimuli, they activate adaptive pathways that allow them to restore cellular homeostasis. Most types of stress stimuli have been reported to induce a decrease in overall protein synthesis accompanied by induction of alternative mechanisms of mRNA translation initiation. Here, we present well-studied and recent examples of such stress responses and the alternative translation initiation mechanisms they induce, and discuss the consequences of such regulation for cell homeostasis and oncogenic transformation.

The scanning model for eukaryotic mRNA translation initiation states that the small ribosomal subunit, along with initiation factors, binds at the cap structure at the 5' end of the mRNA and scans the 5' untranslated region (5'UTR) until an initiation codon is found. However, under conditions that impair canonical cap-dependent translation, the synthesis of some proteins is kept by alternative mechanisms that are required for cell survival and stress recovery. Alternative modes of translation initiation include cap- and/or scanning-independent mechanisms of ribosomal recruitment. In most cap-independent translation initiation events there is a direct recruitment of the 40S ribosome into a position upstream, or directly at, the initiation codon via a specific internal ribosome entry site (IRES) element in the 5'UTR. Yet, in some cellular mRNAs, a different translation initiation mechanism that is neither cap- nor IRES-dependent seems to occur through a special RNA structure called cap-independent translational enhancer (CITE). Recent evidence uncovered a distinct mechanism through which mRNAs containing N 6-methyladenosine (m6A) residues in their 5'UTR directly bind eukaryotic initiation factor 3 (eIF3) and the 40S ribosomal subunit in order to initiate translation in the absence of the cap-binding proteins. This review focuses on the important role of cap-independent translation mechanisms in human cells and how these alternative mechanisms can either act individually or cooperate with other cis-acting RNA regulons to orchestrate specific translational responses triggered upon several cellular stress states, and diseases such as cancer. Elucidation of these non-canonical mechanisms reveals the complexity of translational control and points out their potential as prospective novel therapeutic targets. This work was partially supported by Fundação para a Ciência e a Tecnologia (UID/MULTI/04046/2013 to BioISI from FCT/MCTES/PIDDAC). Rafaela Lacerda and Juliane Menezes were supported by fellowships from Fundação para a Ciência e a Tecnologia (SFRH/BD/74778/2010 and SFRH/BPD/98360/2013, respectively). info:eu-repo/semantics/publishedVersion

The eukaryotic initiation factor 3 (eIF3) is one of the most complex translation initiation factors in mammalian cells, consisting of several subunits (eIF3a to eIF3m). It is crucial in translation initiation and termination, and in ribosomal recycling. Accordingly, deregulated eIF3 expression is associated with different pathological conditions, including cancer. In this manuscript, we discuss the interactome and function of each subunit of the human eIF3 complex. Furthermore, we review how altered levels of eIF3 subunits correlate with neurodegenerative disorders and cancer onset and development; in addition, we evaluate how such misregulation may also trigger infection cascades. A deep understanding of the molecular mechanisms underlying eIF3 role in human disease is essential to develop new eIF3-targeted therapeutic approaches and thus, overcome such conditions. This work was partially supported by Fundação para a Ciência e a Tecnologia (UID/MULTI/04046/2013 to BioISI from FCT/MCTES/PIDDAC and PTFC/BIM-MEC/3749/2014 to LR). JM is supported by a fellowship from Fundação para a Ciência e a Tecnologia (SFRH/BPD/98360/2013). RL is a fellow from Fundação para a Ciência e a Tecnologia through the PTDC/BIMONC/4890/2014 project. info:eu-repo/semantics/publishedVersion

The mechanistic/mammalian target of rapamycin (mTOR) is a conserved serine/threonine kinase that integrates cellular signals from the nutrient and energy status to act, namely, on the protein synthesis machinery. While major advances have emerged regarding the regulators and effects of the mTOR signaling pathway, little is known about the regulation of mTOR gene expression. Here, we show that the human mTOR transcript can be translated in a cap-independent manner, and that its 5′ untranslated region (UTR) is a highly folded RNA scaffold capable of binding directly to the 40S ribosomal subunit. We further demonstrate that mTOR is able to bypass the cap requirement for translation both in normal and hypoxic conditions. Moreover, our data reveal that the cap-independent translation of mTOR is necessary for its ability to induce cell-cycle progression into S phase. These results suggest a novel regulatory mechanism for mTOR gene expression that integrates the global protein synthesis changes induced by translational inhibitory conditions.

Alternative pre-mRNA splicing (AS) affects gene expression as it generates proteome diversity. Nonsense-mediated mRNA decay (NMD) is a surveillance pathway that recognizes and selectively degrades mRNAs carrying premature translation-termination codons (PTCs), preventing the production of truncated proteins that could result in disease. Several studies have also implicated NMD in the regulation of steady-state levels of physiological mRNAs. In addition, it is known that several regulated AS events do not lead to generation of protein products, as they lead to transcripts that carry PTCs and thus, they are committed to NMD. Indeed, an estimated one-third of naturally occurring, alternatively spliced mRNAs is targeted for NMD, being AS coupled to NMD (AS-NMD) an efficient strategy to regulate gene expression. In this review, we will focus on how AS mechanism operates and how can be coupled to NMD to fine-tune gene expression levels. Furthermore, we will demonstrate the physiological significance of the interplay among AS and NMD in human disease, such as cancer and neurological disorders. The understanding of how AS-NMD orchestrates expression of vital genes is of utmost importance for the advance in diagnosis, prognosis and treatment of many human disorders. This work was partially supported by Fundação para a Ciência e a Tecnologia (PTFC/BIM-MEC/3749/2014 to LR and UID/MULTI/04046/2013 to BioISI from FCT/MCTES/PIDDAC. Paulo Costa and Juliane Menezes were supported by fellowships from Fundação para a Ciência e a Tecnologia (SFRH/BD/52495/2014 and SFRH/BPD/98360/2013, respectively). info:eu-repo/semantics/publishedVersion

Acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS) represent a group of heterogeneous diseases with a considerable diversity in molecular pathogenesis and clinical outcomes. Recurrent...

Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a very rare disease that currently lacks genomic and genetic biomarkers to assist in its clinical management. We performed whole-exome sequencing (WES) of three BPDCN cases. Based on these data, we designed a resequencing approach to identify mutations in 38 selected genes in 25 BPDCN samples. WES revealed 37-99 deleterious gene mutations per exome with no common affected genes between patients, but with clear overlap in terms of molecular and disease pathways (hematological and dermatological disease). We identified for the first time deleterious mutations in IKZF3, HOXB9, UBE2G2 and ZEB2 in human leukemia. Target sequencing identified 29 recurring genes, ranging in prevalence from 36% for previously known genes, such as TET2, to 12-16% for newly identified genes, such as IKZF3 or ZEB2. Half of the tumors had mutations affecting either the DNA methylation or chromatin remodeling pathways. The clinical analysis revealed that patients with mutations in DNA methylation pathway had a significantly reduced overall survival (P=0.047). We provide the first mutational profiling of BPDCN. The data support the current WHO classification of the disease as a myeloid disorder and provide a biological rationale for the incorporation of epigenetic therapies for its treatment.

Genetic aberrations involving the mixed lineage leukemia ( MLL ) gene are frequently diagnosed in infant acute lymphoblastic and acute myeloid leukemia. More than 60 fusion partner genes have been described at the molecular level, 31 of which have been characterized solely in infant leukemia cases. Here we describe a new MLL fusion partner gene, NEBL, which was identified in a case of acute myeloid leukemia in an infant. The chromosomal breakpoints of the MLL–NEBL and NEBL–MLL fusion genes were cloned by long-distance inverse polymerase chain reaction. The chromosomal breakpoints were located at 10p12, approximately 570 kb telomic of the MLLT10 ( AF10 ) gene. AF10 and NEBL are localized in such close vicinity that they cannot be distinguished cytogenetically by G banding. Therefore, the combination of cytogenetic and independent molecular techniques such as long-distance inverse polymerase chain reaction are indispensable for the rapid identification and characterization of rare MLL rearrangements.

Chronic lymphocytic leukemia (CLL) is a heterogeneous disease without a well-defined genetic alteration responsible for the onset of the disease. Several lines of evidence coincide in identifying stimulatory and growth signals delivered by B-cell receptor (BCR), and co-receptors together with NFkB pathway, as being the driving force in B-cell survival in CLL. However, the molecular mechanism responsible for this activation has not been identified. Based on the hypothesis that BCR activation may depend on somatic mutations of the BCR and related pathways we have performed a complete mutational screening of 301 selected genes associated with BCR signaling and related pathways using massive parallel sequencing technology in 10 CLL cases. Four mutated genes in coding regions (KRAS, SMARCA2, NFKBIE and PRKD3) have been confirmed by capillary sequencing. In conclusion, this study identifies new genes mutated in CLL, all of them in cases with progressive disease, and demonstrates that next-generation sequencing technologies applied to selected genes or pathways of interest are powerful tools for identifying novel mutational changes.

Infant leukemia (IL) is characterised by the presence of MLL rearrangements and a poor outcome. FLT3 gene is consistently highly expressed in MLL+ patients. To correlate the clinical aspects of IL with FLT3 sequence alterations, we have analysed 159 children included in the Brazilian Collaborative Study Group of Infant Acute Leukemia. FLT3-D835 mutations and FLT3-ITD were detected by PCR-RFLP assay and standard PCR amplification, respectively. Mean age at diagnosis was 11.3 months. Overall, 7.5% (ITDs n=6 and D835 n=6) of patients contained FLT3 mutations. FLT3 mutated cases exhibited significantly higher white blood cells (WBC) than wild-type patients (p=0.013). Median overall survival time was 9.2 months (SE 3.3, 95% CI 2.8-15.6). Variables with significant poorer outcomes were age

Although acute leukaemia is rare in pregnancy its importance lies in its life-threatening potential, both to the child and the mother. The possibility of vertical transmission of leukemic cells increases the attention devoted to these patients and their offspring. Three cases of pregnant young women (15-17 years of age) with AML are presented. This series of cases is the first report where gene abnormalities such as ITD mutations of the FLT3 gene and AML1/ETO fusion genes were screened in pregnant AML patients and their babies, so far. Unfortunately, very poor outcomes have been associated to similar cases described in literature, and the same was true to the patients described herein. Although very speculative, we think that the timing and possible similar exposures would be involved in all cases.

Chronic neutrophilic leukemia (CNL) is a rare myeloproliferative neoplasm (MPN) that includes only 150 patients described to date meeting the latest World Health Organization (WHO) criteria and the recently reported CSF3R mutations. The diagnosis is based on morphological criteria of granulocytic cells and the exclusion of genetic drivers that are known to occur in others MPNs, such as BCR-ABL1, PDGFRA/B, or FGFR1 rearrangements. However, this scenario changed with the identification of oncogenic mutations in the CSF3R gene in approximately 83% of WHO-defined and no monoclonal gammopathy-associated CNL patients. CSF3R T618I is a highly specific molecular marker for CNL that is sensitive to inhibition in vitro and in vivo by currently approved protein kinase inhibitors. In addition to CSF3R mutations, other genetic alterations have been found, notably mutations in SETBP1, which may be used as prognostic markers to guide therapeutic decisions. These findings will help to understand the pathogenesis of CNL and greatly impact the clinical management of this disease. In this review, we discuss the new genetic alterations recently found in CNL and the clinical perspectives in its diagnosis and treatment. Fortunately, since the diagnosis of CNL is not based on exclusion anymore, the molecular characterization of the CSF3R gene must be included in the WHO criteria for CNL diagnosis.

ASXL1 , TP53 and IKZF3 mutations are present in the chronic phase and blast crisis of chronic myeloid leukemia

Mutations in CSF3R have been recently defined as the common genetic event in patients with myeloid neoplasms, including the rare entity known as chronic neutrophilic leukemia (CNL),1, 2, 3 becoming a potentially useful biomarker for diagnosing and therapy target.4 CSF3R encodes the transmembrane receptor for granulocyte colony-stimulating factor (G-CSF; CSF3), which provides the proliferative and survival signal for granulocytes and also contributes to their differentiation and function.5 Although there are several studies on massive next-generation sequencing of myeloid disorders, not a single comprehensive study has been reported in CNL. Here, we used whole-exome sequencing (WES) and RNA sequencing (RNA-seq) to identify new candidate genes to the disease pathogenesis of an index CNL patient. A 66-year-old man was diagnosed with CNL, according to the 2008 World Health Organization (WHO) classification. At diagnosis, the patient presented peripheral blood leukocytosis (66 × 109/l), segmented neutrophils and band forms were 91.5% of the white blood cells counts (WBCs), immature granulocytes were

Abstract 1415 Background: Chronic myeloid leukemia (CML) is one of the best examples of a disease that can be targeted by molecular therapy however, the success of new designed drugs is largely restricted to the chronic phase of the disease. If not cured at this stage, CML invariably progresses and transforms into an acute-type leukemia undegoing a blast crisis, that is characterized by a rapid expansion of myeloid or lymphoid differentiation-arrested blast cells leading to short median survival. To investigate the genetic changes associated with CML progression under tyrosin-kinase inhibitor treatment, and to determine whether clonal evolution contributes to blast crisis, we performed whole-exome sequencing of an individual patient at three different times of the CML progression: chronic phase (CP), complete hematological remission (CHR) and blast crisis (BC). Methods and Case Description: We collected genomic DNA from bone marrow cells (tumor DNA) at three disease evolution points and from epithelial cells (germline DNA). The sequence capture, enrichment and elution was performed according to manufacturer's instructions and protocols (SureSelect, Agilent). Each eluted-enriched DNA sample was sequenced on an Illumina GAIIX as paired-end 75b reads. The bioinformatics analysis of sequencing data was based on a pipeline which includes alignment, annotation, and filtering of the somatic variants, all linked to a coverage/depth statistical analysis. Validation of somatic mutations was done by capilary sequencing. The patient, a 65 year-old men, showed at diagnosis a classic CML with a 45,XY,t(9;22)(q34;q11.2),rob(13;14)(q10;q10)c [20]. He was treated with Imatinib (400 mg/day) achieving complete hematological and cytogenetic remission after 12 months of treatment. Real-time qRT-PCR demonstrated molecular response: BCR/ABL1 ratio decreased from 53% to 13% within the first year. Unfortunately, the disease progressed at month 14 to a blast crisis with a complex karyotype that did not respond to 2nd line treatment (Dasatinib + Idaurobinice-AraC) and the patient died of the disease 18 months after diagnosis. Results: After discarding the variants present in the matched normal DNA and in the dbSNP132 database, we obteined a total of 3123, 7678 and 3306 single nucleotide substitutions (SNSs) and small insertions and deletions (indels) for CP, CHR and BC, respectively. Next, we selected only those variants within coding regions that, passing depth and quality controls, were predicted to produce non-synonymous amino acid changes. This resulted in 27, 30 and 26 SNSs for CP, CHR and BC, respectively, (Fig. 1). Among those SNSs, we validated mutations in genes known to be involved in CML (such as ASXL1 and TP53) as well as in genes that have not been described so far in the disease (such as UBE2G2, ZEB2 and IKZF3). TP53 mutation (p.E286K) was found in the three phases of CML progression. However, ASXL1 (p.G679*), UBE2G2 (p.D35V), ZEB2 (p.L420R) and IKZF3 (p.E272K) were present only in the CP and BC-CML. On the other hand, only 7%, 42% and 6% of the mutated gene were exclusively found in CP, CHR and BC, respectively (Fig. 1). The evaluation of the number of mutated reads for each gene allowed us to study clonality and clonal evolution patters during CML progression. 93% of the selected SNSs that were present in the CP were also seen in BC (only 46% during CHR). In fact, the percentages of reads of the mutant alleles identified for the most relevant genes were the same (around 50%) both at CP and at BC. Conclusions: Whole-exome sequencing allowed to identify a large number of mutated genes, even at the chonic phase of CML, that harbour clear prognostic and predictive significance (TP53, IKZF3, absence of ABL1 mutations). The study of the mutation profile through the disease progression indicated that, at least in this patient, the number and the type of mutations were rather similar at CP and BC. Disclosures: No relevant conflicts of interest to declare.