RESUMEN
Major histocompatibility complex (MHC)-bound peptides that originate from tumor-specific genetic alterations, known as neoantigens, are an important class of anticancer therapeutic targets. Accurately predicting peptide presentation by MHC complexes is a key aspect of discovering therapeutically relevant neoantigens. Technological improvements in mass-spectrometry-based immunopeptidomics and advanced modeling techniques have vastly improved MHC presentation prediction over the past two decades. However, improvement in the sensitivity and specificity of prediction algorithms is needed for clinical applications such as the development of personalized cancer vaccines, the discovery of biomarkers for response to checkpoint blockade, and the quantification of autoimmune risk in gene therapies. Toward this end, we generated allele-specific immunopeptidomics data using 25 monoallelic cell lines and created Systematic HLA Epitope Ranking Pan Algorithm (SHERPA), a pan-allelic MHC-peptide algorithm for predicting MHC-peptide binding and presentation. In contrast to previously published large-scale monoallelic data, we used an HLA-null K562 parental cell line and a stable transfection of HLA alleles to better emulate native presentation. Our dataset includes five previously unprofiled alleles that expand MHC-binding pocket diversity in the training data and extend allelic coverage in under profiled populations. To improve generalizability, SHERPA systematically integrates 128 monoallelic and 384 multiallelic samples with publicly available immunoproteomics data and binding assay data. Using this dataset, we developed two features that empirically estimate the propensities of genes and specific regions within gene bodies to engender immunopeptides to represent antigen processing. Using a composite model constructed with gradient boosting decision trees, multiallelic deconvolution, and 2.15 million peptides encompassing 167 alleles, we achieved a 1.44-fold improvement of positive predictive value compared with existing tools when evaluated on independent monoallelic datasets and a 1.15-fold improvement when evaluating on tumor samples. With a high degree of accuracy, SHERPA has the potential to enable precision neoantigen discovery for future clinical applications.
Asunto(s)
Antígenos de Neoplasias , Complejo Mayor de Histocompatibilidad , Modelos Teóricos , Péptidos , Algoritmos , Presentación de Antígeno , Línea Celular , Humanos , Proteoma , TranscriptomaRESUMEN
Essential tremor (ET) is the most common adult-onset movement disorder. In the present study, we performed whole exome sequencing of a large ET-affected family (10 affected and 6 un-affected family members) and identified a TUB p.V431I variant (rs75594955) segregating in a manner consistent with autosomal-dominant inheritance. Subsequent targeted re-sequencing of TUB in 820 unrelated individuals with sporadic ET and 630 controls revealed significant enrichment of rare nonsynonymous TUB variants (e.g. rs75594955: p.V431I, rs1241709665: p.Ile20Phe, rs55648406: p.Arg49Gln) in the ET cohort (SKAT-O test p-value = 6.20e-08). TUB encodes a transcription factor predominantly expressed in neuronal cells and has been previously implicated in obesity. ChIP-seq analyses of the TUB transcription factor across different regions of the mouse brain revealed that TUB regulates the pathways responsible for neurotransmitter production as well thyroid hormone signaling. Together, these results support the association of rare variants in TUB with ET.
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Proteínas Adaptadoras Transductoras de Señales/genética , Temblor Esencial/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Anciano , Anciano de 80 o más Años , Animales , Secuenciación de Inmunoprecipitación de Cromatina/métodos , Estudios de Cohortes , Exoma/genética , Familia , Femenino , Humanos , Masculino , Ratones Endogámicos C57BL , Persona de Mediana Edad , Linaje , Polimorfismo de Nucleótido Simple/genética , Factores de Transcripción/genética , Secuenciación del Exoma/métodosRESUMEN
Fetal Alcohol Spectrum Disorders, or FASD, represent a range of adverse developmental conditions caused by prenatal ethanol exposure (PrEE) from maternal consumption of alcohol. PrEE induces neurobiological damage in the developing brain leading to cognitive-perceptual and behavioral deficits in the offspring. Alcohol-mediated alterations to epigenetic function may underlie PrEE-related brain dysfunction, with these changes potentially carried across generations to unexposed offspring. To determine the transgenerational impact of PrEE on neocortical development, we generated a mouse model of FASD and identified numerous stable phenotypes transmitted via the male germline to the unexposed third generation. These include alterations in ectopic intraneocortical connectivity, upregulation of neocortical Rzrß and Id2 expression accompanied by both promoter hypomethylation of these genes and decreased global DNA methylation levels. DNMT expression was also suppressed in newborn PrEE cortex, providing further insight into how ethanol perturbs DNA methylation leading to altered regulation of gene transcription. These PrEE-induced, transgenerational phenotypes may be responsible for cognitive, sensorimotor, and behavioral deficits seen in humans with FASD. Thus, understanding the possible epigenetic mechanisms by which these phenotypes are generated may reveal novel targets for therapeutic intervention of FASD and lead to advances in human health.
Asunto(s)
Discapacidades del Desarrollo/etiología , Discapacidades del Desarrollo/patología , Trastornos del Espectro Alcohólico Fetal/etiología , Trastornos del Espectro Alcohólico Fetal/patología , Neocórtex/patología , Efectos Tardíos de la Exposición Prenatal/fisiopatología , Animales , Animales Recién Nacidos , Peso Corporal/efectos de los fármacos , Depresores del Sistema Nervioso Central/toxicidad , Metilación de ADN/efectos de los fármacos , Modelos Animales de Enfermedad , Epigenómica , Etanol/toxicidad , Femenino , Trastornos del Espectro Alcohólico Fetal/fisiopatología , Masculino , Ratones , Trastornos del Humor/etiología , Actividad Motora/fisiología , Neocórtex/crecimiento & desarrollo , Neuronas/patología , Embarazo , ARN Mensajero/metabolismo , AutoadministraciónRESUMEN
BACKGROUND: In utero alcohol, or ethanol (EtOH), exposure produces developmental abnormalities in the brain of the fetus, which can result in lifelong behavioral abnormalities. Fetal alcohol spectrum disorders (FASD) is a term used to describe a range of adverse developmental conditions caused by EtOH exposure during gestation. Children diagnosed with FASD potentially exhibit a host of phenotypes including growth retardation, facial dysmorphology, central nervous system anomalies, abnormal behavior, and cognitive deficits. Previous research suggests that abnormal gene expression and circuitry in the neocortex may underlie reported disabilities of learning, memory, and behavior resulting from early exposure to alcohol (J Neurosci, 33, 2013, 18893). METHODS: Here, we utilize a mouse model of FASD to examine effects of prenatal EtOH exposure (PrEE), on brain anatomy in newborn (postnatal day [P]0), weanling (P20), and early adult (P50) mice. We correlate abnormal cortical and subcortical anatomy with atypical behavior in adult P50 PrEE mice. In this model, experimental dams self-administered a 25% EtOH solution throughout gestation (gestational days 0 to 19, day of birth), generating the exposure to the offspring. RESULTS: Results from these experiments reveal long-term alterations to cortical anatomy, including atypical developmental cortical thinning, and abnormal subcortical development as a result of in utero EtOH exposure. Furthermore, offspring exposed to EtOH during the prenatal period performed poorly on behavioral tasks measuring sensorimotor integration and anxiety. CONCLUSIONS: Insight from this study will help provide new information on developmental trajectories of PrEE and the biological etiologies of abnormal behavior in people diagnosed with FASD.
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Ansiedad , Conducta Animal/efectos de los fármacos , Encéfalo/efectos de los fármacos , Depresores del Sistema Nervioso Central/farmacología , Etanol/farmacología , Trastornos del Espectro Alcohólico Fetal , Efectos Tardíos de la Exposición Prenatal , Animales , Encéfalo/anomalías , Encéfalo/embriología , Recuento de Células , Corteza Cerebral/anomalías , Corteza Cerebral/efectos de los fármacos , Corteza Cerebral/embriología , Modelos Animales de Enfermedad , Femenino , Ratones , Tamaño de los Órganos , Equilibrio Postural/efectos de los fármacos , EmbarazoRESUMEN
In utero ethanol exposure from a mother's consumption of alcoholic beverages impacts brain and cognitive development, creating a range of deficits in the child (Levitt, 1998; Lebel et al., 2012). Children diagnosed with fetal alcohol spectrum disorders (FASD) are often born with facial dysmorphology and may exhibit cognitive, behavioral, and motor deficits from ethanol-related neurobiological damage in early development. Prenatal ethanol exposure (PrEE) is the number one cause of preventable mental and intellectual dysfunction globally, therefore the neurobiological underpinnings warrant systematic research. We document novel anatomical and gene expression abnormalities in the neocortex of newborn mice exposed to ethanol in utero. This is the first study to demonstrate large-scale changes in intraneocortical connections and disruption of normal patterns of neocortical gene expression in any prenatal ethanol exposure animal model. Neuroanatomical defects and abnormal neocortical RZRß, Id2, and Cadherin8 expression patterns are observed in PrEE newborns, and abnormal behavior is present in 20-d-old PrEE mice. The vast network of neocortical connections is responsible for high-level sensory and motor processing as well as complex cognitive thought and behavior in humans. Disruptions to this network from PrEE-related changes in gene expression may underlie some of the cognitive-behavioral phenotypes observed in children with FASD.
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Conducta Animal/efectos de los fármacos , Corteza Cerebral/metabolismo , Trastornos del Espectro Alcohólico Fetal/fisiopatología , Trastornos del Espectro Alcohólico Fetal/psicología , Expresión Génica/efectos de los fármacos , Efectos Tardíos de la Exposición Prenatal/fisiopatología , Efectos Tardíos de la Exposición Prenatal/psicología , Animales , Cadherinas/biosíntesis , Cadherinas/genética , Recuento de Células , Corteza Cerebral/efectos de los fármacos , Corteza Cerebral/patología , Etanol/sangre , Femenino , Trastornos del Espectro Alcohólico Fetal/genética , Técnicas In Vitro , Proteína 2 Inhibidora de la Diferenciación/biosíntesis , Proteína 2 Inhibidora de la Diferenciación/genética , Ratones , Microscopía Fluorescente , Miembro 2 del Grupo F de la Subfamilia 1 de Receptores Nucleares/biosíntesis , Miembro 2 del Grupo F de la Subfamilia 1 de Receptores Nucleares/genética , Concentración Osmolar , Embarazo , Preñez/efectos de los fármacos , Efectos Tardíos de la Exposición Prenatal/genética , Aumento de PesoRESUMEN
Human leukocyte antigen loss of heterozygosity (HLA LOH) allows cancer cells to escape immune recognition by deleting HLA alleles, causing the suppressed presentation of tumor neoantigens. Despite its importance in immunotherapy response, few methods exist to detect HLA LOH, and their accuracy is not well understood. Here, we develop DASH (Deletion of Allele-Specific HLAs), a machine learning-based algorithm to detect HLA LOH from paired tumor-normal sequencing data. With cell line mixtures, we demonstrate increased sensitivity compared to previously published tools. Moreover, our patient-specific digital PCR validation approach provides a sensitive, robust orthogonal approach that could be used for clinical validation. Using DASH on 610 patients across 15 tumor types, we find that 18% of patients have HLA LOH. Moreover, we show inflated HLA LOH rates compared to genome-wide LOH and correlations between CD274 (encodes PD-L1) expression and microsatellite instability status, suggesting the HLA LOH is a key immune resistance strategy.
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Pérdida de Heterocigocidad , Neoplasias , Algoritmos , Antígenos HLA/genética , Antígenos de Histocompatibilidad Clase I/genética , Antígenos de Histocompatibilidad Clase II , Humanos , Pérdida de Heterocigocidad/genética , Aprendizaje Automático , Repeticiones de Microsatélite/genética , Neoplasias/genéticaRESUMEN
PURPOSE: While immune checkpoint blockade (ICB) has become a pillar of cancer treatment, biomarkers that consistently predict patient response remain elusive due to the complex mechanisms driving immune response to tumors. We hypothesized that a multi-dimensional approach modeling both tumor and immune-related molecular mechanisms would better predict ICB response than simpler mutation-focused biomarkers, such as tumor mutational burden (TMB). EXPERIMENTAL DESIGN: Tumors from a cohort of patients with late-stage melanoma (n = 51) were profiled using an immune-enhanced exome and transcriptome platform. We demonstrate increasing predictive power with deeper modeling of neoantigens and immune-related resistance mechanisms to ICB. RESULTS: Our neoantigen burden score, which integrates both exome and transcriptome features, more significantly stratified responders and nonresponders (P = 0.016) than TMB alone (P = 0.049). Extension of this model to include immune-related resistance mechanisms affecting the antigen presentation machinery, such as HLA allele-specific LOH, resulted in a composite neoantigen presentation score (NEOPS) that demonstrated further increased association with therapy response (P = 0.002). CONCLUSIONS: NEOPS proved the statistically strongest biomarker compared with all single-gene biomarkers, expression signatures, and TMB biomarkers evaluated in this cohort. Subsequent confirmation of these findings in an independent cohort of patients (n = 110) suggests that NEOPS is a robust, novel biomarker of ICB response in melanoma.
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Resistencia a Antineoplásicos/inmunología , Melanoma/tratamiento farmacológico , Melanoma/inmunología , Modelos Inmunológicos , Predicción , Humanos , Resultado del TratamientoRESUMEN
Maternal consumption of alcohol during pregnancy can generate a multitude of deficits in the offspring. Fetal Alcohol Spectrum Disorders, or FASD, describe a palette of potentially life-long phenotypes that result from exposure to ethanol during human gestation. There is no cure for FASD and cognitive-behavioral therapies typically have low success rates, especially in severe cases. The neocortex, responsible for complex cognitive and behavioral function, is altered by prenatal ethanol exposure (PrEE). Supplementation with choline, an essential nutrient, during the prenatal ethanol insult has been associated with a reduction of negative outcomes associated with PrEE. However, choline's ability to prevent deficits within the developing neocortex, as well as the underlying mechanisms, remain unclear. Here, we exposed pregnant mice to 25% ethanol in addition to a 642 mg/L choline chloride supplement throughout gestation to determine the impact of choline supplementation on neocortical and behavioral development in ethanol-exposed offspring. We found that concurrent choline supplementation prevented gross developmental abnormalities associated with PrEE including reduced body weight, brain weight, and cortical length as well as partially ameliorated PrEE-induced abnormalities in intraneocortical circuitry. Additionally, choline supplementation prevented altered expression of RZRß and Id2, two genes implicated in postmitotic patterning of neocortex, and global DNA hypomethylation within developing neocortex. Lastly, choline supplementation prevented sensorimotor behavioral dysfunction and partially ameliorated increased anxiety-like behavior observed in PrEE mice, as assessed by the Suok and Ledge tests. Our results suggest that choline supplementation may represent a potent preventative measure for the adverse outcomes associated with PrEE.
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Colina/administración & dosificación , Suplementos Dietéticos , Etanol/toxicidad , Trastornos del Espectro Alcohólico Fetal/tratamiento farmacológico , Neocórtex/efectos de los fármacos , Fenotipo , Animales , Animales Recién Nacidos , Ansiedad/inducido químicamente , Ansiedad/tratamiento farmacológico , Ansiedad/patología , Etanol/administración & dosificación , Femenino , Trastornos del Espectro Alcohólico Fetal/patología , Masculino , Ratones , Neocórtex/metabolismo , Neocórtex/patología , EmbarazoRESUMEN
Preterm birth (PTB), or the delivery prior to 37 weeks of gestation, is a significant cause of infant morbidity and mortality. Although twin studies estimate that maternal genetic contributions account for approximately 30% of the incidence of PTB, and other studies reported fetal gene polymorphism association, to date no consistent associations have been identified. In this study, we performed the largest reported genome-wide association study analysis on 1,349 cases of PTB and 12,595 ancestry-matched controls from the focusing on genomic fetal signals. We tested over 2 million single nucleotide polymorphisms (SNPs) for associations with PTB across five subpopulations: African (AFR), the Americas (AMR), European, South Asian, and East Asian. We identified only two intergenic loci associated with PTB at a genome-wide level of significance: rs17591250 (P = 4.55E-09) on chromosome 1 in the AFR population and rs1979081 (P = 3.72E-08) on chromosome 8 in the AMR group. We have queried several existing replication cohorts and found no support of these associations. We conclude that the fetal genetic contribution to PTB is unlikely due to single common genetic variant, but could be explained by interactions of multiple common variants, or of rare variants affected by environmental influences, all not detectable using a GWAS alone.
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Polimorfismo de Nucleótido Simple , Nacimiento Prematuro/genética , Grupos Raciales/genética , Adulto , Cromosomas Humanos Par 1/genética , Cromosomas Humanos Par 8/genética , Femenino , Humanos , Recién Nacido , Masculino , Persona de Mediana Edad , Nacimiento Prematuro/etnologíaRESUMEN
Mammalian neocortical development is regulated by neural patterning mechanisms, with distinct sensory and motor areas arising through the process of arealization. This development occurs alongside developing central or peripheral sensory systems. Specifically, the parcellation of neocortex into specific areas of distinct cytoarchitecture, connectivity and function during development is reliant upon both cortically intrinsic mechanisms, such as gene expression, and extrinsic processes, such as input from the sensory receptors. This developmental program shifts from patterning to maintenance as the animal ages and is believed to be active throughout life, where the brain's organization is stable yet plastic. In this study, we characterize the long-term effects of early removal of visual input via bilateral enucleation at birth. To understand the long-term effects of early blindness we conducted anatomical and molecular assays 18 months after enucleation, near the end of lifespan in the mouse. Bilateral enucleation early in life leads to long-term, stable size reductions of the thalamic lateral geniculate nucleus (LGN) and the primary visual cortex (V1) alongside a increase in individual whisker barrel size. Neocortical gene expression in the aging brain has not been previously identified; we document cortical expression of multiple regionalization genes. Expression patterns of Ephrin A5, COUP-TFI, and RZRß and patterns of intraneocortical connectivity (INC) are altered in the neocortices of aging blind mice. Sensory inputs from different modalities during development likely play a major role in the development of cortical areal and thalamic nuclear boundaries. We suggest that early patterning by prenatal retinal activity combined with persistent gene expression within the thalamus and cortex is sufficient to establish and preserve a small but present LGN and V1 into late adulthood.
RESUMEN
Functional sensory and motor areas in the developing mammalian neocortex are formed through a complex interaction of cortically intrinsic mechanisms, such as gene expression, and cortically extrinsic mechanisms such as those mediated by thalamic input from the senses. Both intrinsic and extrinsic mechanisms are believed to be involved in cortical patterning and the establishment of areal boundaries in early development; however, the nature of the interaction between intrinsic and extrinsic processes is not well understood. In a previous study, we used a perinatal bilateral enucleation mouse model to test some aspects of this interaction by reweighting sensory input to the developing cortex. Visual deprivation at birth resulted in a shift of intraneocortical connections (INCs) that aligned with ectopic ephrin A5 expression in the same location ten days later at postnatal day (P) 10. A prevailing question remained: Does visual deprivation first induce a change in gene expression, followed by a shift in INCs, or vice versa? In the present study, we address this question by investigating the neuroanatomy and patterns of gene expression in post-natal day (P) 1 and 4 mice following bilateral enucleation at birth. Our results demonstrate a rapid reduction in dorsal lateral geniculate nucleus (dLGN) size and ephrin A5 gene expression 24-hours post-enucleation, with more profound effects apparent at P4. The reduced nuclear size and diminished gene expression mirrors subtle changes in ephrin A5 expression evident in P1 and P4 enucleated neocortex, 11 and 8 days prior to natural eye opening, respectively. Somatosensory and visual INCs were indistinguishable between P1 and P4 mice bilaterally enucleated at birth, indicating that perinatal bilateral enucleation initiates a rapid change in gene expression (within one day) followed by an alteration of sensory INCs later on (second postnatal week). With these results, we gain a deeper understanding of how gene expression and sensory input together regulate cortical arealization and plasticity during early development.
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Desarrollo Embrionario/genética , Efrina-A5/genética , Neocórtex/metabolismo , Tálamo/metabolismo , Animales , Embrión de Mamíferos , Efrina-A5/biosíntesis , Enucleación del Ojo , Femenino , Regulación del Desarrollo de la Expresión Génica , Cuerpos Geniculados/metabolismo , Ratones , Neocórtex/crecimiento & desarrollo , Embarazo , Corteza Somatosensorial/crecimiento & desarrollo , Corteza Somatosensorial/metabolismo , Tálamo/crecimiento & desarrollo , Visión Ocular/fisiología , Corteza Visual/metabolismo , Corteza Visual/fisiologíaRESUMEN
BACKGROUND: Anatomically and functionally distinct sensory and motor neocortical areas form during mammalian development through a process called arealization. This process is believed to be reliant on both activity-dependent and activity-independent mechanisms. Although both mechanisms are thought to function concurrently during arealization, the nature of their interaction is not understood. To examine the potential interplay of extrinsic activity-dependent mechanisms, such as sensory input, and intrinsic activity-independent mechanisms, including gene expression in mouse neocortical development, we performed bilateral enucleations in newborn mice and conducted anatomical and molecular analyses 10 days later. In this study, by surgically removing the eyes of the newborn mouse, we examined whether early enucleation would impact normal gene expression and the development of basic anatomical features such as intraneocortical connections and cortical area boundaries in the first 10 days of life, before natural eye opening. We examined the acute effects of bilateral enucleation on the lateral geniculate nucleus of the thalamus and the neocortical somatosensory-visual area boundary through detailed analyses of intraneocortical connections and gene expression of six developmentally regulated genes at postnatal day 10. RESULTS: Our results demonstrate short-term plasticity on postnatal day 10 resulting from the removal of the eyes at birth, with changes in nuclear size and gene expression within the lateral geniculate nucleus as well as a shift in intraneocortical connections and ephrin A5 expression at the somatosensory-visual boundary. In this report, we highlight the correlation between positional shifts in ephrin A5 expression and improper refinement of intraneocortical connections observed at the somatosensory-visual boundary in enucleates on postnatal day 10. CONCLUSIONS: Bilateral enucleation induces a positional shift of both ephrin A5 expression and intraneocortical projections at the somatosensory-visual border in only 10 days. These changes occur prior to natural eye opening, suggesting a possible role of spontaneous retinal activity in area border formation within the neocortex. Through these analyses, we gain a deeper understanding of how extrinsic activity-dependent mechanisms, particularly input from sensory organs, are integrated with intrinsic activity-independent mechanisms to regulate neocortical arealization and plasticity.