RESUMO
In digenic inheritance, pathogenic variants in two genes must be inherited together to cause disease. Only very few examples of digenic inheritance have been described in the neuromuscular disease field. Here we show that predicted deleterious variants in SRPK3, encoding the X-linked serine/argenine protein kinase 3, lead to a progressive early onset skeletal muscle myopathy only when in combination with heterozygous variants in the TTN gene. The co-occurrence of predicted deleterious SRPK3/TTN variants was not seen among 76,702 healthy male individuals, and statistical modeling strongly supported digenic inheritance as the best-fitting model. Furthermore, double-mutant zebrafish (srpk3-/-; ttn.1+/-) replicated the myopathic phenotype and showed myofibrillar disorganization. Transcriptome data suggest that the interaction of srpk3 and ttn.1 in zebrafish occurs at a post-transcriptional level. We propose that digenic inheritance of deleterious changes impacting both the protein kinase SRPK3 and the giant muscle protein titin causes a skeletal myopathy and might serve as a model for other genetic diseases.
Assuntos
Doenças Musculares , Peixe-Zebra , Animais , Humanos , Masculino , Conectina/genética , Conectina/metabolismo , Músculo Esquelético , Doenças Musculares/genética , Doenças Musculares/metabolismo , Doenças Musculares/patologia , Mutação , Peixe-Zebra/genéticaRESUMO
Shigella flexneri is a human-adapted pathovar of Escherichia coli that can invade the intestinal epithelium, causing inflammation and bacillary dysentery. Although an important human pathogen, the host response to S. flexneri has not been fully described. Zebrafish larvae represent a valuable model for studying human infections in vivo. Here, we use a Shigella-zebrafish infection model to generate mRNA expression profiles of host response to Shigella infection at the whole-animal level. Immune response-related processes dominate the signature of early Shigella infection (6â h post-infection). Consistent with its clearance from the host, the signature of late Shigella infection (24â h post-infection) is significantly changed, and only a small set of immune-related genes remain differentially expressed, including acod1 and gpr84. Using mutant lines generated by ENU, CRISPR mutagenesis and F0 crispants, we show that acod1- and gpr84-deficient larvae are more susceptible to Shigella infection. Together, these results highlight the power of zebrafish to model infection by bacterial pathogens and reveal the mRNA expression of the early (acutely infected) and late (clearing) host response to Shigella infection.
Assuntos
Disenteria Bacilar , Animais , Humanos , Disenteria Bacilar/genética , Shigella flexneri/genética , Shigella flexneri/metabolismo , Peixe-Zebra/genética , Peixe-Zebra/microbiologia , Inflamação/microbiologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
Antiphospholipid syndrome (APS) is an uncommon autoantibody-mediated condition characterised by acquired thrombophilia resulting in recurrent arterial and venous thrombosis. An inciting factor allows for the exposure of endothelial phospholipids, causing antigen formation and subsequent creation of antibodies. A woman in her 70s presented after vehicular trauma, suffering broken ribs, pneumothorax and incidentally discovered left adrenal haemorrhage. Two weeks later she presented with acute-onset abdominal pain and was found to have a right adrenal gland haemorrhage on CT imaging without interval trauma occurring. The patient had antiphospholipid antibody laboratory studies drawn and was given intravenous heparin with a bridge to warfarin at discharge. Laboratory results returned positive for lupus anticoagulant, beta-2 glycoprotein and anticardiolipin antibodies indicating triple positivity, with repeated laboratory tests positive in 12 weeks' time, confirming the diagnosis. Bilateral adrenal haemorrhage, rather than traditional venous thromboembolism, was the presenting pathology in this patient's diagnosis of APS.
Assuntos
Doenças das Glândulas Suprarrenais , Síndrome Antifosfolipídica , Doenças das Glândulas Suprarrenais/diagnóstico por imagem , Doenças das Glândulas Suprarrenais/etiologia , Anticorpos Anticardiolipina , Anticorpos Antifosfolipídeos , Síndrome Antifosfolipídica/complicações , Síndrome Antifosfolipídica/diagnóstico , Feminino , Glicoproteínas , Hemorragia/diagnóstico por imagem , Hemorragia/etiologia , Heparina , Humanos , Inibidor de Coagulação do Lúpus , Fosfolipídeos , Varfarina/uso terapêuticoRESUMO
Manganese neurotoxicity is a hallmark of hypermanganesemia with dystonia 2, an inherited manganese transporter defect caused by mutations in SLC39A14. To identify novel potential targets of manganese neurotoxicity, we performed transcriptome analysis of slc39a14-/- mutant zebrafish that were exposed to MnCl2. Differentially expressed genes mapped to the central nervous system and eye, and pathway analysis suggested that Ca2+ dyshomeostasis and activation of the unfolded protein response are key features of manganese neurotoxicity. Consistent with this interpretation, MnCl2 exposure led to decreased whole-animal Ca2+ levels, locomotor defects and changes in neuronal activity within the telencephalon and optic tectum. In accordance with reduced tectal activity, slc39a14-/- zebrafish showed changes in visual phototransduction gene expression, absence of visual background adaptation and a diminished optokinetic reflex. Finally, numerous differentially expressed genes in mutant larvae normalised upon MnCl2 treatment indicating that, in addition to neurotoxicity, manganese deficiency is present either subcellularly or in specific cells or tissues. Overall, we assembled a comprehensive set of genes that mediate manganese-systemic responses and found a highly correlated and modulated network associated with Ca2+ dyshomeostasis and cellular stress. This article has an associated First Person interview with the first author of the paper.
Assuntos
Proteínas de Transporte de Cátions , Distonia , Animais , Cálcio/metabolismo , Proteínas de Transporte de Cátions/genética , Proteínas de Transporte de Cátions/metabolismo , Distonia/genética , Íons/metabolismo , Manganês/metabolismo , Manganês/toxicidade , Peixe-Zebra/genética , Peixe-Zebra/metabolismoRESUMO
In model organisms, RNA-sequencing (RNA-seq) is frequently used to assess the effect of genetic mutations on cellular and developmental processes. Typically, animals heterozygous for a mutation are crossed to produce offspring with different genotypes. Resultant embryos are grouped by genotype to compare homozygous mutant embryos to heterozygous and wild-type siblings. Genes that are differentially expressed between the groups are assumed to reveal insights into the pathways affected by the mutation. Here we show that in zebrafish, differentially expressed genes are often over-represented on the same chromosome as the mutation due to different levels of expression of alleles from different genetic backgrounds. Using an incross of haplotype-resolved wild-type fish, we found evidence of widespread allele-specific expression, which appears as differential expression when comparing embryos homozygous for a region of the genome to their siblings. When analysing mutant transcriptomes, this means that the differential expression of genes on the same chromosome as a mutation of interest may not be caused by that mutation. Typically, the genomic location of a differentially expressed gene is not considered when interpreting its importance with respect to the phenotype. This could lead to pathways being erroneously implicated or overlooked due to the noise of spurious differentially expressed genes on the same chromosome as the mutation. These observations have implications for the interpretation of RNA-seq experiments involving outbred animals and non-inbred model organisms.
Assuntos
Proteínas de Peixe-Zebra , Peixe-Zebra , Alelos , Animais , Expressão Gênica , Heterozigoto , Mutação , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genéticaRESUMO
RNA sequencing allows for the quantification of the transcriptome of embryos to investigate transcriptional responses to various perturbations (e.g., mutations, infections, drug treatments). Previous protocols either lack the option to genotype individual samples, or are laborious and therefore difficult to do at a large scale. We have developed a protocol to extract total nucleic acid from individual zebrafish embryos. Individual embryos are lysed in 96-well plates and nucleic acid is extracted using SPRI beads. The total nucleic acid can be genotyped and then DNase I treated to produce RNA samples for sequencing. This protocol allows for processing large numbers of individual samples, with the ability to genotype each sample, which makes it possible to undertake transcriptomic analysis on mutants at timepoints before the phenotype is visible. Graphic abstract: Extraction of total nucleic acid from individual zebrafish embryos for genotyping and RNA-seq.
RESUMO
Developmental consequences of prenatal drug exposure have been reported in many human cohorts and animal studies. The long-lasting impact on the offspring-including motor and cognitive impairments, cranial and cardiac anomalies and increased prevalence of ADHD-is a socioeconomic burden worldwide. Identifying the molecular changes leading to developmental consequences could help ameliorate the deficits and limit the impact. In this study, we have used zebrafish, a well-established behavioral and genetic model with conserved drug response and reward pathways, to identify changes in behavior and cellular pathways in response to developmental exposure to amphetamine, nicotine or oxycodone. In the presence of the drug, exposed animals showed altered behavior, consistent with effects seen in mammalian systems, including impaired locomotion and altered habituation to acoustic startle. Differences in responses seen following acute and chronic exposure suggest adaptation to the presence of the drug. Transcriptomic analysis of exposed larvae revealed differential expression of numerous genes and alterations in many pathways, including those related to cell death, immunity and circadian rhythm regulation. Differential expression of circadian rhythm genes did not correlate with behavioral changes in the larvae, however, two of the circadian genes, arntl2 and per2, were also differentially expressed at later stages of development, suggesting a long-lasting impact of developmental exposures on circadian gene expression. The immediate-early genes, egr1, egr4, fosab, and junbb, which are associated with synaptic plasticity, were downregulated by all three drugs and in situ hybridization showed that the expression for all four genes was reduced across all neuroanatomical regions, including brain regions implicated in reward processing, addiction and other psychiatric conditions. We anticipate that these early changes in gene expression in response to drug exposure are likely to contribute to the consequences of prenatal exposure and their discovery might pave the way to therapeutic intervention to ameliorate the long-lasting deficits.
RESUMO
Apolipoprotein B-containing lipoproteins (B-lps) are essential for the transport of hydrophobic dietary and endogenous lipids through the circulation in vertebrates. Zebrafish embryos produce large numbers of B-lps in the yolk syncytial layer (YSL) to move lipids from yolk to growing tissues. Disruptions in B-lp production perturb yolk morphology, readily allowing for visual identification of mutants with altered B-lp metabolism. Here we report the discovery of a missense mutation in microsomal triglyceride transfer protein (Mtp), a protein that is essential for B-lp production. This mutation of a conserved glycine residue to valine (zebrafish G863V, human G865V) reduces B-lp production and results in yolk opacity due to aberrant accumulation of cytoplasmic lipid droplets in the YSL. However, this phenotype is milder than that of the previously reported L475P stalactite (stl) mutation. MTP transfers lipids, including triglycerides and phospholipids, to apolipoprotein B in the ER for B-lp assembly. In vitro lipid transfer assays reveal that while both MTP mutations eliminate triglyceride transfer activity, the G863V mutant protein unexpectedly retains ~80% of phospholipid transfer activity. This residual phospholipid transfer activity of the G863V mttp mutant protein is sufficient to support the secretion of small B-lps, which prevents intestinal fat malabsorption and growth defects observed in the mttpstl/stl mutant zebrafish. Modeling based on the recent crystal structure of the heterodimeric human MTP complex suggests the G865V mutation may block triglyceride entry into the lipid-binding cavity. Together, these data argue that selective inhibition of MTP triglyceride transfer activity may be a feasible therapeutic approach to treat dyslipidemia and provide structural insight for drug design. These data also highlight the power of yolk transport studies to identify proteins critical for B-lp biology.
Assuntos
Proteínas de Transporte/genética , Lipídeos/genética , Lipoproteínas/genética , Triglicerídeos/genética , Animais , Fígado Gorduroso/genética , Fígado Gorduroso/patologia , Trato Gastrointestinal/metabolismo , Humanos , Imunoprecipitação , Gotículas Lipídicas/metabolismo , Lipoproteínas/metabolismo , Mutação de Sentido Incorreto/genética , Mutação Puntual/genética , Transporte Proteico/genética , Triglicerídeos/metabolismo , Peixe-Zebra/genéticaRESUMO
PURPOSE: Immunotherapy has demonstrated efficacy in treatment of intracranial metastasis from melanoma, calling into question the role of intracranial radiotherapy (RT). Herein, we assessed the utilization patterns of intracranial RT in patients with melanoma brain metastasis and compared outcomes in patients treated with immunotherapy alone versus immunotherapy in addition to intracranial RT. METHODS: We queried the National Cancer Database (NCDB) for patients with melanoma brain metastases treated with immunotherapy and intracranial RT or immunotherapy alone. Multivariable logistic regression identified variables associated with increased likelihood of receiving immunotherapy alone. Multivariable Cox regression was used to identify co-variates predictive of overall survival (OS). Propensity matching was used to account for indication bias. RESULTS: We identified 528 and 142 patients that were treated with combination therapy and immunotherapy alone, respectively. Patients with lower comorbidity score were more likely to receive immunotherapy alone. Extracranial disease and treatment at a non-academic center were associated with worse OS. Median OS for all patients was 11.0 months. Treatment with stereotactic radiosurgery (SRS) in addition to immunotherapy was superior to immunotherapy alone, median OS of 19.0 versus 11.5 months (p = 0.006). Whole brain radiation therapy (WBRT) in combination with immunotherapy performed worse than immunotherapy alone, median OS of 7.7 versus 11.5 months (p = 0.0255). CONCLUSIONS: For melanoma patients requiring WBRT, immunotherapy alone may be reasonable in asymptomatic patients. For those eligible for SRS, combination therapy may provide better outcomes. Results of ongoing prospective studies will help provide guidance regarding the use of radioimmunotherapy in this population.
Assuntos
Neoplasias Encefálicas/mortalidade , Irradiação Craniana/mortalidade , Imunoterapia/mortalidade , Melanoma/mortalidade , Adulto , Idoso , Idoso de 80 Anos ou mais , Neoplasias Encefálicas/secundário , Neoplasias Encefálicas/terapia , Terapia Combinada , Feminino , Seguimentos , Humanos , Masculino , Melanoma/patologia , Melanoma/terapia , Pessoa de Meia-Idade , Prognóstico , Estudos Retrospectivos , Taxa de Sobrevida , Adulto JovemRESUMO
BACKGROUND: Squamous cell carcinoma (SCC) of larynx is a common head and neck cancer. For cases that are node negative, the role of definitive concurrent chemoradiation is unclear and not supported by guidelines but used at provider discretion. To address this knowledge gap, we examined the oncological outcomes with additional chemotherapy and factors correlated with the chemotherapy administration. METHODS: We queried the National Cancer Database for patients with early stage (T2N0M0) laryngeal SCC treated nonsurgically. Multivariable logistic regression identified predictors of chemotherapy. Multivariable Cox regression evaluated predictors of survival. Propensity matching accounted for indication biases. RESULTS: We identified 7181 patients meeting the eligibility criteria, of which 1568 (22%) patients received chemotherapy in addition to radiation. Predictors of chemotherapy use included younger age, Caucasian race, more remote year of treatment, higher grade, sites other than glottis, treatment at a community cancer center, and use of intensity-modulated radiation therapy. Median overall survival was not significantly different in the two arms analyzed-65 months (95% confidence interval [CI] 60, 72months) with chemotherapy compared to 70 months without chemotherapy (95% CI 66, 75 months, P<.37). Predictors for survival on propensity-matched multivariable analysis were increased age, male sex, less education, lower income, higher comorbidity score, receipt of treatment at a community center, and nonglottic sites. CONCLUSIONS: This study shows no clear survival benefit with chemotherapy in early stage disease. Although this implies that chemotherapy should not be routinely delivered, individualized judgment and prospective studies are recommended as the biology behind this interesting finding is undefined. LEVEL OF EVIDENCE: 2C (Outcomes Research).
RESUMO
OBJECTIVES: The appropriate timing of chemotherapy following surgery for resectable pancreatic adenocarcinoma is controversial. Using the National Cancer Database we evaluated time to initiation of chemotherapy postresection and correlated with outcome. METHODS: We identified stage I-III pancreatic adenocarcinoma treated surgically with adjuvant chemoradiotherapy. Receiver operator curve analysis identified an interval of 66 days as the a priori value for largest discrepancy in outcome. Multivariable logistic regression analysis identified variables associated with increased time to chemotherapy postoperatively (>66 days). Propensity matching was performed to account for indication bias. RESULTS: In total, 6873 and 3348 patients received chemotherapy before and after the 66-day cutoff, respectively. Predictors of expedited chemotherapy included lower comorbidity, treatment outside a community program in an urban location, having insurance, white race, and treatment after 2009. Propensity-matched median survival was 21.8 months for all patients, and of these, 6462 were stage 1. Five-year survival was 20% in patients receiving chemotherapy within 66 days and 18% in those not (P = 0.0266). In stage 1 patients, 5-year survival was 23% versus 21% (P = 0.0116) in favor of expedited chemotherapy. CONCLUSIONS: The present propensity-matched analysis showed a significant association with survival for earlier delivery of chemotherapy in the adjuvant setting.
Assuntos
Adenocarcinoma/terapia , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Pancreatectomia/métodos , Neoplasias Pancreáticas/terapia , Adenocarcinoma/patologia , Adulto , Idoso , Quimioterapia Adjuvante/métodos , Estudos de Coortes , Terapia Combinada , Feminino , Humanos , Estimativa de Kaplan-Meier , Masculino , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Neoplasias Pancreáticas/patologia , Pontuação de Propensão , Fatores de TempoRESUMO
The neural crest (NC) is a vertebrate-specific cell type that contributes to a wide range of different tissues across all three germ layers. The gene regulatory network (GRN) responsible for the formation of neural crest is conserved across vertebrates. Central to the induction of the NC GRN are AP-2 and SoxE transcription factors. NC induction robustness is ensured through the ability of some of these transcription factors to compensate loss of function of gene family members. However the gene regulatory events underlying compensation are poorly understood. We have used gene knockout and RNA sequencing strategies to dissect NC induction and compensation in zebrafish. We genetically ablate the NC using double mutants of tfap2a;tfap2c or remove specific subsets of the NC with sox10 and mitfa knockouts and characterise genome-wide gene expression levels across multiple time points. We find that compensation through a single wild-type allele of tfap2c is capable of maintaining early NC induction and differentiation in the absence of tfap2a function, but many target genes have abnormal expression levels and therefore show sensitivity to the reduced tfap2 dosage. This separation of morphological and molecular phenotypes identifies a core set of genes required for early NC development. We also identify the 15 somites stage as the peak of the molecular phenotype which strongly diminishes at 24 hpf even as the morphological phenotype becomes more apparent. Using gene knockouts, we associate previously uncharacterised genes with pigment cell development and establish a role for maternal Hippo signalling in melanocyte differentiation. This work extends and refines the NC GRN while also uncovering the transcriptional basis of genetic compensation via paralogues.
Assuntos
Desenvolvimento Embrionário/genética , Crista Neural/crescimento & desenvolvimento , Fatores de Transcrição SOXE/genética , Fator de Transcrição AP-2/genética , Proteínas de Peixe-Zebra/genética , Animais , Diferenciação Celular/genética , Linhagem da Célula/genética , Embrião não Mamífero , Regulação da Expressão Gênica no Desenvolvimento/genética , Redes Reguladoras de Genes/genética , Melanócitos/metabolismo , Fator de Transcrição Associado à Microftalmia/genética , Crista Neural/metabolismo , Pigmentação/genética , Proteínas Serina-Treonina Quinases/genética , Serina-Treonina Quinase 3 , Peixe-Zebra/genética , Peixe-Zebra/crescimento & desenvolvimentoRESUMO
The Deciphering the Mechanisms of Developmental Disorders programme has analysed the morphological and molecular phenotypes of embryonic and perinatal lethal mouse mutant lines in order to investigate the causes of embryonic lethality. Here we show that individual whole-embryo RNA-seq of 73 mouse mutant lines (>1000 transcriptomes) identifies transcriptional events underlying embryonic lethality and associates previously uncharacterised genes with specific pathways and tissues. For example, our data suggest that Hmgxb3 is involved in DNA-damage repair and cell-cycle regulation. Further, we separate embryonic delay signatures from mutant line-specific transcriptional changes by developing a baseline mRNA expression catalogue of wild-type mice during early embryogenesis (4-36 somites). Analysis of transcription outside coding sequence identifies deregulation of repetitive elements in Morc2a mutants and a gene involved in gene-specific splicing. Collectively, this work provides a large scale resource to further our understanding of early embryonic developmental disorders.
Assuntos
Embrião de Mamíferos/metabolismo , Análise de Sequência de RNA , Transcrição Gênica , Animais , Regulação da Expressão Gênica no Desenvolvimento , Camundongos , Mutação , TranscriptomaRESUMO
IMPORTANCE: Primary Adenocarcinoma of the anus is a rare disease with a poor prognosis and thus tends to have a more aggressive treatment algorithm, typically involving a surgical approach. Prior to 2001, a few retrospective studies outlined improved outcomes with the incorporation of surgery with chemoradiation. However, since the publication of these studies, advancement in radiotherapy modalities and imaging have left the question of improved outcomes while reserving surgery for salvage. OBJECTIVE: We conducted this National Cancer Database (NCDB)-driven retrospective study to analyze treatment trends and outcomes in the current time from 2004 to 2015 with respect to chemoradiation and surgery. DESIGN: Retrospective NCDB tumor registry data review-using propensity score-adjusted multivariable analyses for survival. SETTING: Database review. PARTICIPANTS: We selected for patients listed in the NCDB with AJCC stage 1-3 anal adenocarcinoma diagnosed between 2004 and 2015 and selected out patients with undocumented/stage 4 disease, those with radiation outside the pelvis, not treated with systemic therapy and patients lost to follow-up. EXPOSURE(S): None. MAIN OUTCOMES AND MEASURES: Overall survival and use of surgery in the up-front management of anal adenocarcinoma. RESULTS: Of the 1729 patients eligible in this study, 1028 were treated with surgery as up-front management and 701 had definitive chemoradiation. Median overall survival for all patients was 55 months with a 5-year survival rate of 55%. Patients treated without surgery had worse overall survival, median survival of 45 months compared to 87 months (P < 0.0001) with 5-year survival rates of 42% and 55% in favor of incorporation of surgery. Analysis across patients treated with surgery alone, surgery followed by adjuvant chemoradiation, neoadjuvant chemoradiation followed by surgery, and chemoradiation alone had median survival rates of 78, 83, 92, and 46 months, respectively. Propensity score-adjusted multivariable analysis identified older age, grade 3, high comorbidity score, and lack of surgery as predictive of worse outcome. CONCLUSIONS AND RELEVANCE: The results of the NCDB analysis indicate improved overall survival with the incorporation of surgery into the initial management of anal adenocarcinoma when compared to chemoradiation alone, despite the omission of surgery in up to 50% of the cases logged. Our results corroborate earlier studies published prior to the year 2000 for surgery to be included in the definitive management of anal adenocarcinoma.
Assuntos
Adenocarcinoma/epidemiologia , Neoplasias do Ânus/epidemiologia , Adenocarcinoma/diagnóstico , Adenocarcinoma/mortalidade , Adenocarcinoma/terapia , Neoplasias do Ânus/diagnóstico , Neoplasias do Ânus/mortalidade , Neoplasias do Ânus/terapia , Terapia Combinada , Comorbidade , Bases de Dados Factuais , Gerenciamento Clínico , Feminino , Pesquisas sobre Atenção à Saúde , Humanos , Estimativa de Kaplan-Meier , Masculino , Estadiamento de Neoplasias , Razão de Chances , Prognóstico , Doenças Raras , Sistema de Registros , Estudos Retrospectivos , Estados Unidos/epidemiologiaRESUMO
BACKGROUND: Anal canal squamous cell carcinoma (SCC) is managed definitively with chemoradiation, reserving surgery for salvage. The dosage of radiation has varied from 30 Gy to in excess of 60 Gy. RTOG 0529 established intensity modulated radiation therapy (IMRT) as standard of care for anal canal SCC with doses of 50.4 to 54 Gy. We sought to use the National Cancer Database to examine trends in dose selection and radiation technique over time. METHODS: We queried the National Cancer Database from 2004 to 2015 for cases of anal cancer stage groups 1 to 3, treated with definitive doses of radiation with chemotherapy. Dose escalation was defined as >54 Gy. Univariable and multivariable analyses were performed to identify factors predictive of dose, IMRT, and overall survival. Propensity-adjusted Cox proportional hazard ratios for survival were used to account for indication bias. RESULTS: We identified 7792 patients meeting the eligibility criteria, with 4269 treated to doses of 45 to 54 Gy and 3163 treated to doses >54 Gy. Patients who were older, had government or private insurance, IMRT treatment, treatment at an academic center, or more recent years were less likely to get dose escalation. The use of dose escalation decreased over time, from 50% in 2005 to 30% in 2015. IMRT use increased over time from 2% to 63%. On multivariable analysis with propensity score included it was found that increased age, higher comorbidity score, lower income, shorter distance to facility, and male sex were predictive of decreased overall survival. In addition, escalated dose was associated with a lower survival (hazard ratio: 1.10, 95% confidence interval: 1.01-1.20, P=0.03). CONCLUSIONS: The results of this analysis show a steady increase in the use of IMRT, with corresponding decrease in dose escalation. These findings correlate with the results of RTOG 0529 establishing IMRT as standard of care for anal SCC, using doses of 50.4 to 54 Gy.
Assuntos
Neoplasias do Ânus/radioterapia , Carcinoma de Células Escamosas/radioterapia , Radioterapia de Intensidade Modulada/tendências , Neoplasias do Ânus/patologia , Carcinoma de Células Escamosas/patologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Prognóstico , Dosagem Radioterapêutica , Radioterapia de Intensidade Modulada/métodos , Estudos Retrospectivos , Taxa de SobrevidaRESUMO
PURPOSE: Traditionally, three-dimensional conformal radiation therapy (3D-CRT) is used for neoadjuvant chemoradiation in locally advanced rectal cancer. Intensity-modulated radiation therapy (IMRT) was later developed for more conformal dose distribution, with the potential for reduced toxicity across many disease sites. We sought to use the National Cancer Database (NCDB) to examine trends and predictors for IMRT use in rectal cancer. MATERIALS AND METHODS: We queried the NCDB from 2004 to 2015 for patients with rectal adenocarcinoma treated with neoadjuvant concurrent chemoradiation to standard doses followed by surgical resection. Odds ratios were used to determine predictors of IMRT use. Univariable and multivariable Cox regressions were used to determine potential predictors of overall survival (OS). Propensity matching was used to account for any indication bias. RESULTS: Among 21,490 eligible patients, 3,131 were treated with IMRT. IMRT use increased from 1% in 2004 to 22% in 2014. Predictors for IMRT use included increased N stage, higher comorbidity score, more recent year, treatment at an academic facility, increased income, and higher educational level. On propensity-adjusted, multivariable analysis, male gender, increased distance to facility, higher comorbidity score, IMRT technique, government insurance, African-American race, and non-metro location were predictive of worse OS. Of note, the complete response rate at time of surgery was 28% with non-IMRT and 21% with IMRT. CONCLUSION: IMRT use has steadily increased in the treatment of rectal cancer, but still remains only a fraction of overall treatment technique, more often reserved for higher disease burden.
RESUMO
We have produced an mRNA expression time course of zebrafish development across 18 time points from 1 cell to 5 days post-fertilisation sampling individual and pools of embryos. Using poly(A) pulldown stranded RNA-seq and a 3' end transcript counting method we characterise temporal expression profiles of 23,642 genes. We identify temporal and functional transcript co-variance that associates 5024 unnamed genes with distinct developmental time points. Specifically, a class of over 100 previously uncharacterised zinc finger domain containing genes, located on the long arm of chromosome 4, is expressed in a sharp peak during zygotic genome activation. In addition, the data reveal new genes and transcripts, differential use of exons and previously unidentified 3' ends across development, new primary microRNAs and temporal divergence of gene paralogues generated in the teleost genome duplication. To make this dataset a useful baseline reference, the data can be browsed and downloaded at Expression Atlas and Ensembl.
Assuntos
Desenvolvimento Embrionário , Regulação da Expressão Gênica no Desenvolvimento , RNA Mensageiro/biossíntese , Peixe-Zebra/embriologia , Animais , Perfilação da Expressão Gênica , Análise de Sequência de RNA , Fatores de TempoRESUMO
KDM2A is a histone demethylase associated with transcriptional silencing, however very little is known about its in vivo role in development and disease. Here we demonstrate that loss of the orthologue kdm2aa in zebrafish causes widespread transcriptional disruption and leads to spontaneous melanomas at a high frequency. Fish homozygous for two independent premature stop codon alleles show reduced growth and survival, a strong male sex bias, and homozygous females exhibit a progressive oogenesis defect. kdm2aa mutant fish also develop melanomas from early adulthood onwards which are independent from mutations in braf and other common oncogenes and tumour suppressors as revealed by deep whole exome sequencing. In addition to effects on translation and DNA replication gene expression, high-replicate RNA-seq in morphologically normal individuals demonstrates a stable regulatory response of epigenetic modifiers and the specific de-repression of a group of zinc finger genes residing in constitutive heterochromatin. Together our data reveal a complex role for Kdm2aa in regulating normal mRNA levels and carcinogenesis. These findings establish kdm2aa mutants as the first single gene knockout model of melanoma biology.
Assuntos
Histona Desmetilases com o Domínio Jumonji/genética , Melanoma/genética , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas de Peixe-Zebra/genética , Peixe-Zebra/genética , Animais , Replicação do DNA , Modelos Animais de Doenças , Epigênese Genética , Exoma , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Técnicas de Inativação de Genes , Masculino , Mutação , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Análise de Sequência de RNA , Peixe-Zebra/embriologiaRESUMO
Functional genomic elements are marked by characteristic DNA and histone modification signatures. How combinatorial chromatin modification states are recognized by epigenetic reader proteins and how this is linked to their biological function is largely unknown. Here we provide a detailed molecular analysis of chromatin recognition by the lysine demethylase KDM2A. Using biochemical approaches we identify a nucleosome interaction module within KDM2A consisting of a CXXC type zinc finger, a PHD domain and a newly identified Heterochromatin Protein 1 (HP1) interaction motif that mediates direct binding between KDM2A and HP1. This nucleosome interaction module enables KDM2A to decode nucleosomal H3K9me3 modification in addition to CpG methylation signals. The multivalent engagement with DNA and HP1 results in a nucleosome binding circuit in which KDM2A can be recruited to H3K9me3-modified chromatin through HP1, and HP1 can be recruited to unmodified chromatin by KDM2A. A KDM2A mutant deficient in HP1-binding is inactive in an in vivo overexpression assay in zebrafish embryos demonstrating that the HP1 interaction is essential for KDM2A function. Our results reveal a complex regulation of chromatin binding for both KDM2A and HP1 that is modulated by DNA- and H3K9-methylation, and suggest a direct role for KDM2A in chromatin silencing.
Assuntos
Proteínas Cromossômicas não Histona/química , Proteínas Cromossômicas não Histona/metabolismo , Proteínas F-Box/química , Proteínas F-Box/metabolismo , Histona Desmetilases com o Domínio Jumonji/química , Histona Desmetilases com o Domínio Jumonji/metabolismo , Animais , Animais Geneticamente Modificados , Linhagem Celular , Homólogo 5 da Proteína Cromobox , Proteínas Cromossômicas não Histona/genética , Ilhas de CpG , Cricetinae , Metilação de DNA , Proteínas F-Box/genética , Células HEK293 , Células HeLa , Histonas/metabolismo , Humanos , Histona Desmetilases com o Domínio Jumonji/genética , Modelos Genéticos , Mutação , Neoplasias/genética , Neoplasias/metabolismo , Nucleossomos/genética , Nucleossomos/metabolismo , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Técnicas do Sistema de Duplo-Híbrido , Peixe-Zebra , Dedos de ZincoRESUMO
BACKGROUND: Mutations in proteins involved in telomere maintenance lead to a range of human diseases, including dyskeratosis congenita, idiopathic pulmonary fibrosis and cancer. Telomerase functions to add telomeric repeats back onto the ends of chromosomes, however non-canonical roles of components of telomerase have recently been suggested. METHODS: Here we use a zebrafish telomerase mutant which harbours a nonsense mutation in tert to investigate the adult phenotypes of fish derived from heterozygous parents of different ages. Furthermore we use whole genome sequencing data to estimate average telomere lengths. RESULTS: We show that homozygous offspring from older heterozygotes exhibit signs of body wasting at a younger age than those of younger parents, and that offspring of older heterozygous parents weigh less irrespective of genotype. We also demonstrate that tert homozygous mutant fish have a male sex bias, and that clutches from older parents also have a male sex bias in the heterozygous and wild-type populations. Telomere length analysis reveals that the telomeres of younger heterozygous parents are shorter than those of older heterozygous parents. CONCLUSIONS: These data indicate that the phenotypes observed in offspring from older parents cannot be explained by telomere length. Instead we propose that Tert functions outside of telomere length maintenance in an age-dependent manner to influence the adult phenotypes of the next generation.