RESUMO
Non-nutritive sweeteners (NNS) are commonly integrated into human diet and presumed to be inert; however, animal studies suggest that they may impact the microbiome and downstream glycemic responses. We causally assessed NNS impacts in humans and their microbiomes in a randomized-controlled trial encompassing 120 healthy adults, administered saccharin, sucralose, aspartame, and stevia sachets for 2 weeks in doses lower than the acceptable daily intake, compared with controls receiving sachet-contained vehicle glucose or no supplement. As groups, each administered NNS distinctly altered stool and oral microbiome and plasma metabolome, whereas saccharin and sucralose significantly impaired glycemic responses. Importantly, gnotobiotic mice conventionalized with microbiomes from multiple top and bottom responders of each of the four NNS-supplemented groups featured glycemic responses largely reflecting those noted in respective human donors, which were preempted by distinct microbial signals, as exemplified by sucralose. Collectively, human NNS consumption may induce person-specific, microbiome-dependent glycemic alterations, necessitating future assessment of clinical implications.
Assuntos
Microbiota , Adoçantes não Calóricos , Adulto , Animais , Aspartame/farmacologia , Glicemia , Humanos , Camundongos , Adoçantes não Calóricos/análise , Adoçantes não Calóricos/farmacologia , Sacarina/farmacologiaRESUMO
Human gut commensals are increasingly suggested to impact non-communicable diseases, such as inflammatory bowel diseases (IBD), yet their targeted suppression remains a daunting unmet challenge. In four geographically distinct IBD cohorts (n = 537), we identify a clade of Klebsiella pneumoniae (Kp) strains, featuring a unique antibiotics resistance and mobilome signature, to be strongly associated with disease exacerbation and severity. Transfer of clinical IBD-associated Kp strains into colitis-prone, germ-free, and colonized mice enhances intestinal inflammation. Stepwise generation of a lytic five-phage combination, targeting sensitive and resistant IBD-associated Kp clade members through distinct mechanisms, enables effective Kp suppression in colitis-prone mice, driving an attenuated inflammation and disease severity. Proof-of-concept assessment of Kp-targeting phages in an artificial human gut and in healthy volunteers demonstrates gastric acid-dependent phage resilience, safety, and viability in the lower gut. Collectively, we demonstrate the feasibility of orally administered combination phage therapy in avoiding resistance, while effectively inhibiting non-communicable disease-contributing pathobionts.
Assuntos
Bacteriófagos , Colite , Microbioma Gastrointestinal , Doenças Inflamatórias Intestinais , Animais , Colite/terapia , Humanos , Inflamação/terapia , Doenças Inflamatórias Intestinais/terapia , Klebsiella pneumoniae , CamundongosRESUMO
Unlike other nucleotide oligomerization domain-like receptors, Nlrp10 lacks a canonical leucine-rich repeat domain, suggesting that it is incapable of signal sensing and inflammasome formation. Here we show that mouse Nlrp10 is expressed in distal colonic intestinal epithelial cells (IECs) and modulated by the intestinal microbiome. In vitro, Nlrp10 forms an Apoptosis-associated speck-like protein containing a caspase-recruitment domain (ASC)-dependent, m-3M3FBS-activated, polyinosinic:polycytidylic acid-modulated inflammasome driving interleukin-1ß and interleukin-18 secretion. In vivo, Nlrp10 signaling is dispensable during steady state but becomes functional during autoinflammation in antagonizing mucosal damage. Importantly, whole-body or conditional IEC Nlrp10 depletion leads to reduced IEC caspase-1 activation, coupled with enhanced susceptibility to dextran sodium sulfate-induced colitis, mediated by altered inflammatory and healing programs. Collectively, understanding Nlrp10 inflammasome-dependent and independent activity, regulation and possible human relevance might facilitate the development of new innate immune anti-inflammatory interventions.
Assuntos
Proteínas Reguladoras de Apoptose , Inflamassomos , Camundongos , Humanos , Animais , Inflamassomos/metabolismo , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Apoptose , Caspase 1/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Interleucina-1beta/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismoRESUMO
Cigarette smoking constitutes a leading global cause of morbidity and preventable death1, and most active smokers report a desire or recent attempt to quit2. Smoking-cessation-induced weight gain (SCWG; 4.5 kg reported to be gained on average per 6-12 months, >10 kg year-1 in 13% of those who stopped smoking3) constitutes a major obstacle to smoking abstinence4, even under stable5,6 or restricted7 caloric intake. Here we use a mouse model to demonstrate that smoking and cessation induce a dysbiotic state that is driven by an intestinal influx of cigarette-smoke-related metabolites. Microbiome depletion induced by treatment with antibiotics prevents SCWG. Conversely, fecal microbiome transplantation from mice previously exposed to cigarette smoke into germ-free mice naive to smoke exposure induces excessive weight gain across diets and mouse strains. Metabolically, microbiome-induced SCWG involves a concerted host and microbiome shunting of dietary choline to dimethylglycine driving increased gut energy harvest, coupled with the depletion of a cross-regulated weight-lowering metabolite, N-acetylglycine, and possibly by the effects of other differentially abundant cigarette-smoke-related metabolites. Dimethylglycine and N-acetylglycine may also modulate weight and associated adipose-tissue immunity under non-smoking conditions. Preliminary observations in a small cross-sectional human cohort support these findings, which calls for larger human trials to establish the relevance of this mechanism in active smokers. Collectively, we uncover a microbiome-dependent orchestration of SCWG that may be exploitable to improve smoking-cessation success and to correct metabolic perturbations even in non-smoking settings.
Assuntos
Microbioma Gastrointestinal , Abandono do Hábito de Fumar , Aumento de Peso , Animais , Estudos Transversais , Disbiose/etiologia , Disbiose/metabolismo , Disbiose/patologia , Camundongos , Modelos Animais , Fumar/metabolismo , Fumar/patologiaRESUMO
Diabetes mellitus (DM) is more common among people living with human immunodeficiency virus (PLWH) compared with healthy individuals. In a prospective multicenter study (N = 248), we identified normoglycemic (48.7%), prediabetic (44.4%), and diabetic (6.9%) PLWH. Glycosylated hemoglobin (HbA1c) and fasting blood glucose (FBG) sensitivity in defining dysglycemia was 96.8%, while addition of oral glucose tolerance test led to reclassification of only 4 patients. Inclusion of 93 additional PLWH with known DM enabled identification of multiple independent predictors of dysglycemia or diabetes: older age, higher body mass index, Ethiopian origin, HIV duration, lower integrase inhibitor exposure, and advanced disease at diagnosis. Shotgun metagenomic microbiome analysis revealed 4 species that were significantly expanded with hyperglycemia/hyperinsulinemia, and 2 species that were differentially more prevalent in prediabetic/diabetic PLWH. Collectively, we uncover multiple potential host and microbiome predictors of altered glycemic status in PLWH, while demonstrating that FBG and HbA1c likely suffice for diabetes screening. These potential diabetic predictors merit future prospective validation.
Assuntos
Glicemia , Diabetes Mellitus , Hemoglobinas Glicadas , Infecções por HIV , Microbiota , Estado Pré-Diabético , Humanos , Estado Pré-Diabético/diagnóstico , Infecções por HIV/complicações , Feminino , Masculino , Pessoa de Meia-Idade , Fatores de Risco , Estudos Prospectivos , Adulto , Glicemia/análise , Hemoglobinas Glicadas/análise , Hemoglobinas Glicadas/metabolismo , Diabetes Mellitus/epidemiologia , Teste de Tolerância a GlucoseRESUMO
Modernization of society from a rural, hunter-gatherer setting into an urban and industrial habitat, with the associated dietary changes, has led to an increased prevalence of cardiometabolic and additional noncommunicable diseases, such as cancer, inflammatory bowel disease, and neurodegenerative and autoimmune disorders. However, while dietary sciences have been rapidly evolving to meet these challenges, validation and translation of experimental results into clinical practice remain limited for multiple reasons, including inherent ethnic, gender, and cultural interindividual variability, among other methodological, dietary reporting-related, and analytical issues. Recently, large clinical cohorts with artificial intelligence analytics have introduced new precision and personalized nutrition concepts that enable one to successfully bridge these gaps in a real-life setting. In this review, we highlight selected examples of case studies at the intersection between diet-disease research and artificial intelligence. We discuss their potential and challenges and offer an outlook toward the transformation of dietary sciences into individualized clinical translation.
Assuntos
Inteligência Artificial , Doenças Autoimunes , Humanos , Dieta , Estado Nutricional , IndústriasRESUMO
Longitudinal 'omics analytical methods are extensively used in the evolving field of precision medicine, by enabling 'big data' recording and high-resolution interpretation of complex datasets, driven by individual variations in response to perturbations such as disease pathogenesis, medical treatment or changes in lifestyle. However, inherent technical limitations in biomedical studies often result in the generation of feature-rich and sample-limited datasets. Analyzing such data using conventional modalities often proves to be challenging since the repeated, high-dimensional measurements overload the outlook with inconsequential variations that must be filtered from the data in order to find the true, biologically relevant signal. Tensor methods for the analysis and meaningful representation of multiway data may prove useful to the biological research community by their advertised ability to tackle this challenge. In this study, we present tcam-a new unsupervised tensor factorization method for the analysis of multiway data. Building on top of cutting-edge developments in the field of tensor-tensor algebra, we characterize the unique mathematical properties of our method, namely, 1) preservation of geometric and statistical traits of the data, which enable uncovering information beyond the inter-individual variation that often takes over the focus, especially in human studies. 2) Natural and straightforward out-of-sample extension, making tcam amenable for integration in machine learning workflows. A series of re-analyses of real-world, human experimental datasets showcase these theoretical properties, while providing empirical confirmation of tcam's utility in the analysis of longitudinal 'omics data.
Assuntos
Algoritmos , Aprendizado de Máquina , Big Data , Humanos , Fenótipo , Medicina de Precisão/métodosRESUMO
Mantle cell lymphoma (MCL) is a mature B-cell neoplasm initially driven by CCND1 rearrangement with 2 molecular subtypes, conventional MCL (cMCL) and leukemic non-nodal MCL (nnMCL), that differ in their clinicobiological behavior. To identify the genetic and epigenetic alterations determining this diversity, we used whole-genome (n = 61) and exome (n = 21) sequencing (74% cMCL, 26% nnMCL) combined with transcriptome and DNA methylation profiles in the context of 5 MCL reference epigenomes. We identified that open and active chromatin at the major translocation cluster locus might facilitate the t(11;14)(q13;32), which modifies the 3-dimensional structure of the involved regions. This translocation is mainly acquired in precursor B cells mediated by recombination-activating genes in both MCL subtypes, whereas in 8% of cases the translocation occurs in mature B cells mediated by activation-induced cytidine deaminase. We identified novel recurrent MCL drivers, including CDKN1B, SAMHD1, BCOR, SYNE1, HNRNPH1, SMARCB1, and DAZAP1. Complex structural alterations emerge as a relevant early oncogenic mechanism in MCL, targeting key driver genes. Breakage-fusion-bridge cycles and translocations activated oncogenes (BMI1, MIR17HG, TERT, MYC, and MYCN), generating gene amplifications and remodeling regulatory regions. cMCL carried significant higher numbers of structural variants, copy number alterations, and driver changes than nnMCL, with exclusive alterations of ATM in cMCL, whereas TP53 and TERT alterations were slightly enriched in nnMCL. Several drivers had prognostic impact, but only TP53 and MYC aberrations added value independently of genomic complexity. An increasing genomic complexity, together with the presence of breakage-fusion-bridge cycles and high DNA methylation changes related to the proliferative cell history, defines patients with different clinical evolution.
Assuntos
Epigênese Genética , Rearranjo Gênico , Linfoma de Célula do Manto/genética , Mutação , Adulto , Idoso , Idoso de 80 Anos ou mais , Proliferação de Células , Ciclina D1/genética , Metilação de DNA , Feminino , Regulação Neoplásica da Expressão Gênica , Genômica , Humanos , Imunoglobulinas/genética , Linfoma de Célula do Manto/patologia , Masculino , Pessoa de Meia-IdadeRESUMO
Risk of developing myelodysplastic syndrome (MDS) is significantly increased in both multiple myeloma (MM) and monoclonal gammopathy of undetermined significance, suggesting that it is therapy independent. However, the incidence and sequelae of dysplastic hematopoiesis at diagnosis are unknown. Here, we used multidimensional flow cytometry (MFC) to prospectively screen for the presence of MDS-associated phenotypic alterations (MDS-PA) in the bone marrow of 285 patients with MM enrolled in the PETHEMA/GEM2012MENOS65 trial (#NCT01916252). We investigated the clinical significance of monocytic MDS-PA in a larger series of 1252 patients enrolled in 4 PETHEMA/GEM protocols. At diagnosis, 33 (11.6%) of 285 cases displayed MDS-PA. Bulk and single-cell-targeted sequencing of MDS recurrently mutated genes in CD34+ progenitors (and dysplastic lineages) from 67 patients revealed clonal hematopoiesis in 13 (50%) of 26 cases with MDS-PA vs 9 (22%) of 41 without MDS-PA; TET2 and NRAS were the most frequently mutated genes. Dynamics of MDS-PA at diagnosis and after autologous transplant were evaluated in 86 of 285 patients and showed that in most cases (69 of 86 [80%]), MDS-PA either persisted or remained absent in patients with or without MDS-PA at diagnosis, respectively. Noteworthy, MDS-associated mutations infrequently emerged after high-dose therapy. Based on MFC profiling, patients with MDS-PA have altered hematopoiesis and T regulatory cell distribution in the tumor microenvironment. Importantly, the presence of monocytic MDS-PA at diagnosis anticipated greater risk of hematologic toxicity and was independently associated with inferior progression-free survival (hazard ratio, 1.5; P = .02) and overall survival (hazard ratio, 1.7; P = .01). This study reveals the biological and clinical significance of dysplastic hematopoiesis in newly diagnosed MM, which can be screened with moderate sensitivity using cost-effective MFC.
Assuntos
Hematopoiese Clonal , Mieloma Múltiplo/patologia , Síndromes Mielodisplásicas/etiologia , Idoso , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Ensaios Clínicos Fase III como Assunto , Terapia Combinada , Feminino , Citometria de Fluxo/métodos , Transplante de Células-Tronco Hematopoéticas , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Hibridização in Situ Fluorescente , Estimativa de Kaplan-Meier , Masculino , Pessoa de Meia-Idade , Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/mortalidade , Mieloma Múltiplo/terapia , Mutação , Prognóstico , Intervalo Livre de Progressão , Estudos Prospectivos , Ensaios Clínicos Controlados Aleatórios como Assunto , Transplante Autólogo , Microambiente TumoralRESUMO
BACKGROUND & AIMS: The intestinal barrier protects intestinal cells from microbes and antigens in the lumen-breaches can alter the composition of the intestinal microbiota, the enteric immune system, and metabolism. We performed a screen to identify molecules that disrupt and support the intestinal epithelial barrier and tested their effects in mice. METHODS: We performed an imaging-based, quantitative, high-throughput screen (using CaCo-2 and T84 cells incubated with lipopolysaccharide; tumor necrosis factor; histamine; receptor antagonists; and libraries of secreted proteins, microbial metabolites, and drugs) to identify molecules that altered epithelial tight junction (TJ) and focal adhesion morphology. We then tested the effects of TJ stabilizers on these changes. Molecules we found to disrupt or stabilize TJs were administered mice with dextran sodium sulfate-induced colitis or Citrobacter rodentium-induced intestinal inflammation. Colon tissues were collected and analyzed by histology, fluorescence microscopy, and RNA sequencing. RESULTS: The screen identified numerous compounds that disrupted or stabilized (after disruption) TJs and monolayers of epithelial cells. We associated distinct morphologic alterations with changes in barrier function, and identified a variety of cytokines, metabolites, and drugs (including inhibitors of actomyosin contractility) that prevent disruption of TJs and restore TJ integrity. One of these disruptors (putrescine) disrupted TJ integrity in ex vivo mouse colon tissues; administration to mice exacerbated colon inflammation, increased gut permeability, reduced colon transepithelial electrical resistance, increased pattern recognition receptor ligands in mesenteric lymph nodes, and decreased colon length and survival times. Putrescine also increased intestine levels and fecal shedding of viable C rodentium, increased bacterial attachment to the colonic epithelium, and increased levels of inflammatory cytokines in colon tissues. Colonic epithelial cells from mice given putrescine increased expression of genes that regulate metal binding, oxidative stress, and cytoskeletal organization and contractility. Co-administration of taurine with putrescine blocked disruption of TJs and the exacerbated inflammation. CONCLUSIONS: We identified molecules that disrupt and stabilize intestinal epithelial TJs and barrier function and affect development of colon inflammation in mice. These agents might be developed for treatment of barrier intestinal impairment-associated and inflammatory disorders in patients, or avoided to prevent inflammation.
Assuntos
Colite/tratamento farmacológico , Colo/efeitos dos fármacos , Infecções por Enterobacteriaceae/tratamento farmacológico , Células Epiteliais/efeitos dos fármacos , Fármacos Gastrointestinais/farmacologia , Ensaios de Triagem em Larga Escala , Absorção Intestinal/efeitos dos fármacos , Mucosa Intestinal/efeitos dos fármacos , Junções Íntimas/efeitos dos fármacos , Animais , Células CACO-2 , Citrobacter rodentium/patogenicidade , Colite/induzido quimicamente , Colite/metabolismo , Colite/microbiologia , Colo/metabolismo , Colo/microbiologia , Colo/patologia , Sulfato de Dextrana , Modelos Animais de Doenças , Infecções por Enterobacteriaceae/metabolismo , Infecções por Enterobacteriaceae/microbiologia , Células Epiteliais/metabolismo , Células Epiteliais/microbiologia , Células Epiteliais/patologia , Microbioma Gastrointestinal , Interações Hospedeiro-Patógeno , Humanos , Mucosa Intestinal/metabolismo , Mucosa Intestinal/microbiologia , Mucosa Intestinal/patologia , Masculino , Camundongos Endogâmicos C57BL , Permeabilidade , Putrescina/farmacologia , Taurina/farmacologia , Junções Íntimas/metabolismo , Junções Íntimas/microbiologia , Junções Íntimas/patologiaRESUMO
Mantle cell lymphoma (MCL) is characterized by the t(11;14)(q13;q32) translocation resulting in overexpression of cyclin D1. However, a small subset of cyclin D1- MCL has been recognized, and approximately one-half of them harbor CCND2 translocations while the primary event in cyclin D1-/D2- MCL remains elusive. To identify other potential mechanisms driving MCL pathogenesis, we investigated 56 cyclin D1-/SOX11+ MCL by fluorescence in situ hybridization (FISH), whole-genome/exome sequencing, and gene-expression and copy-number arrays. FISH with break-apart probes identified CCND2 rearrangements in 39 cases (70%) but not CCND3 rearrangements. We analyzed 3 of these negative cases by whole-genome/exome sequencing and identified IGK (n = 2) and IGL (n = 1) enhancer hijackings near CCND3 that were associated with cyclin D3 overexpression. By specific FISH probes, including the IGK enhancer region, we detected 10 additional cryptic IGK juxtapositions to CCND3 (6 cases) and CCND2 (4 cases) in MCL that overexpressed, respectively, these cyclins. A minor subset of 4 cyclin D1- MCL cases lacked cyclin D rearrangements and showed upregulation of CCNE1 and CCNE2. These cases had blastoid morphology, high genomic complexity, and CDKN2A and RB1 deletions. Both genomic and gene-expression profiles of cyclin D1- MCL cases were indistinguishable from cyclin D1+ MCL. In conclusion, virtually all cyclin D1- MCLs carry CCND2/CCND3 rearrangements with immunoglobulin genes, including a novel IGK/L enhancer hijacking mechanism. A subset of cyclin D1-/D2-/D3- MCL with aggressive features has cyclin E dysregulation. Specific FISH probes may allow the molecular identification and diagnosis of cyclin D1- MCL.
Assuntos
Ciclina D2/genética , Ciclina D3/genética , Elementos Facilitadores Genéticos , Rearranjo Gênico , Cadeias Leves de Imunoglobulina/genética , Linfoma de Célula do Manto/genética , Idoso , Ciclina D1/genética , Ciclina D1/metabolismo , Feminino , Humanos , Linfoma de Célula do Manto/patologia , Masculino , Pessoa de Meia-Idade , Prognóstico , Fatores de Transcrição SOXC/genética , Translocação GenéticaRESUMO
Chronic lymphocytic leukaemia (CLL) is a frequent disease in which the genetic alterations determining the clinicobiological behaviour are not fully understood. Here we describe a comprehensive evaluation of the genomic landscape of 452 CLL cases and 54 patients with monoclonal B-lymphocytosis, a precursor disorder. We extend the number of CLL driver alterations, including changes in ZNF292, ZMYM3, ARID1A and PTPN11. We also identify novel recurrent mutations in non-coding regions, including the 3' region of NOTCH1, which cause aberrant splicing events, increase NOTCH1 activity and result in a more aggressive disease. In addition, mutations in an enhancer located on chromosome 9p13 result in reduced expression of the B-cell-specific transcription factor PAX5. The accumulative number of driver alterations (0 to ≥4) discriminated between patients with differences in clinical behaviour. This study provides an integrated portrait of the CLL genomic landscape, identifies new recurrent driver mutations of the disease, and suggests clinical interventions that may improve the management of this neoplasia.
Assuntos
Leucemia Linfocítica Crônica de Células B/genética , Mutação/genética , Regiões 3' não Traduzidas/genética , Processamento Alternativo/genética , Linfócitos B/metabolismo , Proteínas de Transporte/genética , Cromossomos Humanos Par 9/genética , Análise Mutacional de DNA , DNA de Neoplasias/genética , Proteínas de Ligação a DNA , Elementos Facilitadores Genéticos/genética , Genômica , Humanos , Leucemia Linfocítica Crônica de Células B/metabolismo , Leucemia Linfocítica Crônica de Células B/patologia , Proteínas do Tecido Nervoso/genética , Proteínas Nucleares/genética , Fator de Transcrição PAX5/biossíntese , Fator de Transcrição PAX5/genética , Proteína Tirosina Fosfatase não Receptora Tipo 11/genética , Receptor Notch1/genética , Receptor Notch1/metabolismo , Fatores de Transcrição/genéticaRESUMO
Genomic studies have recently identified RPS15 as a new driver gene in aggressive and chemorefractory cases of chronic lymphocytic leukemia (CLL). RPS15 encodes a ribosomal protein whose conserved C-terminal domain extends into the decoding center of the ribosome. We demonstrate that mutations in highly conserved residues of this domain affect protein stability, by increasing its ubiquitin-mediated degradation, and cell-proliferation rates. On the other hand, we show that mutated RPS15 can be loaded into the ribosomes, directly impacting on global protein synthesis and/or translational fidelity in a mutation-specific manner. Quantitative mass spectrometry analyses suggest that RPS15 variants may induce additional alterations in the translational machinery, as well as a metabolic shift at the proteome level in HEK293T and MEC-1 cells. These results indicate that CLL-related RPS15 mutations might act following patterns known for other ribosomal diseases, likely switching from a hypo- to a hyperproliferative phenotype driven by mutated ribosomes. In this scenario, loss of translational fidelity causing altered cell proteostasis can be proposed as a new molecular mechanism involved in CLL pathobiology.
Assuntos
Leucemia Linfocítica Crônica de Células B/genética , Mutação , Proteínas Ribossômicas/genética , Ribossomos/genética , Linhagem Celular Tumoral , Estudos de Coortes , Células HEK293 , Humanos , Leucemia Linfocítica Crônica de Células B/patologia , Taxa de Mutação , Mutação Puntual , Biossíntese de Proteínas , Domínios Proteicos , Proteínas Ribossômicas/química , Ribossomos/patologiaRESUMO
BACKGROUND & AIMS: Although there is a genetic predisposition to colorectal cancer (CRC), few of the genes that affect risk have been identified. We performed whole-exome sequence analysis of individuals in a high-risk family without mutations in genes previously associated with CRC risk to identify variants associated with inherited CRC. METHODS: We collected blood samples from 3 relatives with CRC in Spain (65, 62, and 40 years old at diagnosis) and performed whole-exome sequence analyses. Rare missense, truncating or splice-site variants shared by the 3 relatives were selected. We used targeted pooled DNA amplification followed by next generation sequencing to screen for mutations in candidate genes in 547 additional hereditary and/or early-onset CRC cases (502 additional families). We carried out protein-dependent yeast growth assays and transfection studies in the HT29 human CRC cell line to test the effects of the identified variants. RESULTS: A total of 42 unique or rare (population minor allele frequency below 1%) nonsynonymous genetic variants in 38 genes were shared by all 3 relatives. We selected the BRF1 gene, which encodes an RNA polymerase III transcription initiation factor subunit for further analysis, based on the predicted effect of the identified variant and previous association of BRF1 with cancer. Previously unreported or rare germline variants in BRF1 were identified in 11 of 503 CRC families, a significantly greater proportion than in the control population (34 of 4300). Seven of the identified variants (1 detected in 2 families) affected BRF1 mRNA splicing, protein stability, or expression and/or function. CONCLUSIONS: In an analysis of families with a history of CRC, we associated germline mutations in BRF1 with predisposition to CRC. We associated deleterious BRF1 variants with 1.4% of familial CRC cases, in individuals without mutations in high-penetrance genes previously associated with CRC. Our findings add additional evidence to the link between defects in genes that regulate ribosome synthesis and risk of CRC.
Assuntos
Neoplasias Colorretais/genética , Mutação em Linhagem Germinativa/genética , Fatores Associados à Proteína de Ligação a TATA/genética , Adulto , Idoso , Neoplasias Colorretais/mortalidade , Neoplasias Colorretais/patologia , Feminino , Predisposição Genética para Doença/genética , Humanos , Masculino , Pessoa de Meia-Idade , Linhagem , EspanhaRESUMO
The t(4;11)(q21;q23) translocation is associated with high-risk infant pro-B-cell acute lymphoblastic leukemia and arises prenatally during embryonic/fetal hematopoiesis. The developmental/pathogenic contribution of the t(4;11)-resulting MLL-AF4 (MA4) and AF4-MLL (A4M) fusions remains unclear; MA4 is always expressed in patients with t(4;11)+ B-cell acute lymphoblastic leukemia, but the reciprocal fusion A4M is expressed in only half of the patients. Because prenatal leukemogenesis manifests as impaired early hematopoietic differentiation, we took advantage of well-established human embryonic stem cell-based hematopoietic differentiation models to study whether the A4M fusion cooperates with MA4 during early human hematopoietic development. Co-expression of A4M and MA4 strongly promoted the emergence of hemato-endothelial precursors, both endothelial- and hemogenic-primed. Double fusion-expressing hemato-endothelial precursors specified into significantly higher numbers of both hematopoietic and endothelial-committed cells, irrespective of the differentiation protocol used and without hijacking survival/proliferation. Functional analysis of differentially expressed genes and differentially enriched H3K79me3 genomic regions by RNA-sequencing and H3K79me3 chromatin immunoprecipitation-sequencing, respectively, confirmed a hematopoietic/endothelial cell differentiation signature in double fusion-expressing hemato-endothelial precursors. Importantly, chromatin immunoprecipitation-sequencing analysis revealed a significant enrichment of H3K79 methylated regions specifically associated with HOX-A cluster genes in double fusion-expressing differentiating hematopoietic cells. Overall, these results establish a functional and molecular cooperation between MA4 and A4M fusions during human hematopoietic development.
Assuntos
Diferenciação Celular/genética , Desenvolvimento Embrionário/genética , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Hematopoese/genética , Proteína de Leucina Linfoide-Mieloide/genética , Proteínas de Fusão Oncogênica/genética , Animais , Apoptose/genética , Ciclo Celular/genética , Técnicas de Cocultura , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Histonas/metabolismo , Células-Tronco Embrionárias Humanas/citologia , Células-Tronco Embrionárias Humanas/metabolismo , Humanos , Metilação , Camundongos , Camundongos KnockoutRESUMO
The causal association of NUDT1 (=MTH1) and OGG1 with hereditary colorectal cancer (CRC) remains unclear. Here, we sought to provide additional evidence for or against the causal contribution of NUDT1 and OGG1 mutations to hereditary CRC and/or polyposis. Mutational screening was performed using pooled DNA amplification and targeted next-generation sequencing in 529 families (441 uncharacterized MMR-proficient familial nonpolyposis CRC and 88 polyposis cases). Cosegregation, in silico analyses, in vitro functional assays, and case-control associations were carried out to characterize the identified variants. Five heterozygous carriers of novel (n = 1) or rare (n = 4) NUDT1 variants were identified. In vitro deleterious effects were demonstrated for c.143G>A p.G48E (catalytic activity and protein stability) and c.403G>T p.G135W (protein stability), although cosegregation data in the carrier families were inconclusive or nonsupportive. The frequency of missense, loss-of-function, and splice-site NUDT1 variants in our familial CRC cohort was similar to the one observed in cancer-free individuals, suggesting lack of association with CRC predisposition. No OGG1 pathogenic mutations were identified. Our results suggest that the contribution of NUDT1 and OGG1 germline mutations to hereditary CRC and to polyposis is inexistent or, at most, negligible. The inclusion of these genes in routine genetic testing is not recommended.
Assuntos
Polipose Adenomatosa do Colo/genética , Neoplasias Colorretais Hereditárias sem Polipose/genética , DNA Glicosilases/genética , Enzimas Reparadoras do DNA/genética , Monoéster Fosfórico Hidrolases/genética , Polipose Adenomatosa do Colo/patologia , Neoplasias Colorretais Hereditárias sem Polipose/patologia , Reparo do DNA/genética , Feminino , Estudos de Associação Genética , Predisposição Genética para Doença , Variação Genética/genética , Genótipo , Mutação em Linhagem Germinativa/genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Mutação com Perda de Função/genética , Masculino , Mutação de Sentido Incorreto/genética , Estresse Oxidativo , Isoformas de Proteínas/genéticaRESUMO
Germline mutations in BUB1 and BUB3 have been reported to increase the risk of developing colorectal cancer (CRC) at young age, in presence of variegated aneuploidy and reminiscent dysmorphic traits of mosaic variegated aneuploidy syndrome. We performed a mutational analysis of BUB1 and BUB3 in 456 uncharacterized mismatch repair-proficient hereditary non-polyposis CRC families and 88 polyposis cases. Four novel or rare germline variants, one splice-site and three missense, were identified in four families. Neither variegated aneuploidy nor dysmorphic traits were observed in carriers. Evident functional effects in the heterozygous form were observed for c.1965-1G>A, but not for c.2296G>A (p.E766K), in spite of the positive co-segregation in the family. BUB1 c.2473C>T (p.P825S) and BUB3 c.77C>T (p.T26I) remained as variants of uncertain significance. As of today, the rarity of functionally relevant mutations identified in familial and/or early onset series does not support the inclusion of BUB1 and BUB3 testing in routine genetic diagnostics of familial CRC.
Assuntos
Polipose Adenomatosa do Colo/genética , Proteínas de Ciclo Celular/genética , Neoplasias Colorretais Hereditárias sem Polipose/genética , Mutação em Linhagem Germinativa , Proteínas de Ligação a Poli-ADP-Ribose/genética , Proteínas Serina-Treonina Quinases/genética , Fuso Acromático/genética , Proteínas de Ciclo Celular/química , Humanos , Modelos Moleculares , Linhagem , Proteínas de Ligação a Poli-ADP-Ribose/química , Conformação Proteica , Proteínas Serina-Treonina Quinases/químicaRESUMO
Identification of genes associated with hereditary cancers facilitates management of patients with family histories of cancer. We performed exome sequencing of DNA from 3 individuals from a family with colorectal cancer who met the Amsterdam criteria for risk of hereditary nonpolyposis colorectal cancer. These individuals had mismatch repair-proficient tumors and each carried nonsense variant in the FANCD2/FANCI-associated nuclease 1 gene (FAN1), which encodes a nuclease involved in DNA inter-strand cross-link repair. We sequenced FAN1 in 176 additional families with histories of colorectal cancer and performed in vitro functional analyses of the mutant forms of FAN1 identified. We detected FAN1 mutations in approximately 3% of families who met the Amsterdam criteria and had mismatch repair-proficient cancers with no previously associated mutations. These findings link colorectal cancer predisposition to the Fanconi anemia DNA repair pathway, supporting the connection between genome integrity and cancer risk.
Assuntos
Neoplasias Colorretais Hereditárias sem Polipose/genética , Reparo do DNA/genética , Exodesoxirribonucleases/genética , Mutação em Linhagem Germinativa , Adulto , Idoso , Idoso de 80 Anos ou mais , Linhagem Celular Tumoral , Pré-Escolar , Neoplasias Colorretais Hereditárias sem Polipose/enzimologia , Neoplasias Colorretais Hereditárias sem Polipose/patologia , Endodesoxirribonucleases , Exodesoxirribonucleases/metabolismo , Feminino , Predisposição Genética para Doença , Células HEK293 , Hereditariedade , Humanos , Masculino , Pessoa de Meia-Idade , Enzimas Multifuncionais , Linhagem , Fenótipo , Adulto JovemRESUMO
PURPOSE: Germ-line mutations in the exonuclease domains of POLE and POLD1 have been recently associated with polyposis and colorectal cancer (CRC) predisposition. Here, we aimed to gain a better understanding of the phenotypic characteristics of this syndrome to establish specific criteria for POLE and POLD1 mutation screening and to help define the clinical management of mutation carriers. METHODS: The exonuclease domains of POLE and POLD1 were studied in 529 kindred, 441 with familial nonpolyposis CRC and 88 with polyposis, by using pooled DNA amplification and massively parallel sequencing. RESULTS: Seven novel or rare genetic variants were identified. In addition to the POLE p.L424V recurrent mutation in a patient with polyposis, CRC and oligodendroglioma, six novel or rare POLD1 variants (four of them, p.D316H, p.D316G, p.R409W, and p.L474P, with strong evidence for pathogenicity) were identified in nonpolyposis CRC families. Phenotypic data from these and previously reported POLE/POLD1 carriers point to an associated phenotype characterized by attenuated or oligo-adenomatous colorectal polyposis, CRC, and probably brain tumors. In addition, POLD1 mutations predispose to endometrial and breast tumors. CONCLUSION: Our results widen the phenotypic spectrum of the POLE/POLD1-associated syndrome and identify novel pathogenic variants. We propose guidelines for genetic testing and surveillance recommendations.Genet Med 18 4, 325-332.