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Klinefelter syndrome (KS), also known as 47, XXY, is characterized by a distinct set of physiological abnormalities, commonly including infertility. The molecular basis for Klinefelter-related infertility is still unclear, largely because of the cellular complexity of the testis and the intricate endocrine and paracrine signaling that regulates spermatogenesis. Here, we demonstrate an analysis framework for dissecting human testis pathology that uses comparative analysis of single-cell RNA-sequencing data from the biopsies of 12 human donors. By comparing donors from a range of ages and forms of infertility, we generate gene expression signatures that characterize normal testicular function and distinguish clinically distinct forms of male infertility. Unexpectedly, we identified a subpopulation of Sertoli cells within multiple individuals with KS that lack transcription from the XIST locus, and the consequence of this is increased X-linked gene expression compared to all other KS cell populations. By systematic assessment of known cell signaling pathways, we identify 72 pathways potentially active in testis, dozens of which appear upregulated in KS. Altogether our data support a model of pathogenic changes in interstitial cells cascading from loss of X inactivation in pubertal Sertoli cells and nominate dosage-sensitive factors secreted by Sertoli cells that may contribute to the process. Our findings demonstrate the value of comparative patient analysis in mapping genetic mechanisms of disease and identify an epigenetic phenomenon in KS Sertoli cells that may prove important for understanding causes of infertility and sex chromosome evolution.
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Infertilidad Masculina/patología , Síndrome de Klinefelter/complicaciones , Células Intersticiales del Testículo/patología , Células de Sertoli/patología , Análisis de la Célula Individual/métodos , Testículo/patología , Transcriptoma , Humanos , Infertilidad Masculina/etiología , Infertilidad Masculina/metabolismo , Síndrome de Klinefelter/cirugía , Células Intersticiales del Testículo/metabolismo , Masculino , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismo , Células de Sertoli/metabolismo , Espermatogénesis , Testículo/metabolismo , Inactivación del Cromosoma XRESUMEN
MOTIVATION: Single-cell RNA sequencing (scRNA-seq) data, annotated by cell type, is useful in a variety of downstream biological applications, such as profiling gene expression at the single-cell level. However, manually assigning these annotations with known marker genes is both time-consuming and subjective. RESULTS: We present a Graph Convolutional Network (GCN)-based approach to automate the annotation process. Our process builds upon existing labeling approaches, using state-of-the-art tools to find cells with highly confident label assignments through consensus and spreading these confident labels with a semi-supervised GCN. Using simulated data and two scRNA-seq datasets from different tissues, we show that our method improves accuracy over a simple consensus algorithm and the average of the underlying tools. We also compare our method to a nonparametric neighbor majority approach, showing comparable results. We then demonstrate that our GCN method allows for feature interpretation, identifying important genes for cell type classification. We present our completed pipeline, written in PyTorch, as an end-to-end tool for automating and interpreting the classification of scRNA-seq data. AVAILABILITY AND IMPLEMENTATION: Our code for conducting the experiments in this paper and using our model is available at https://github.com/lewinsohndp/scSHARP.
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Análisis de la Célula Individual , Programas Informáticos , Consenso , Análisis de la Célula Individual/métodos , Algoritmos , Análisis de Secuencia de ARN , Perfilación de la Expresión Génica , Análisis por ConglomeradosRESUMEN
MOTIVATION: The mammalian testis is a complex organ with a cellular composition that changes smoothly and cyclically in normal adults. While testis histology is already an invaluable tool for identifying and describing developmental differences in evolution and disease, methods for standardized, digital image analysis of testis are needed to expand the utility of this approach. RESULTS: We developed SATINN (Software for Analysis of Testis Images with Neural Networks), a multi-level framework for automated analysis of multiplexed immunofluorescence images from mouse testis. This approach uses residual learning to train convolutional neural networks (CNNs) to classify nuclei from seminiferous tubules into seven distinct cell types with an accuracy of 81.7%. These cell classifications are then used in a second-level tubule CNN, which places seminiferous tubules into one of 12 distinct tubule stages with 57.3% direct accuracy and 94.9% within ±1 stage. We further describe numerous cell- and tubule-level statistics that can be derived from wild-type testis. Finally, we demonstrate how the classifiers and derived statistics can be used to rapidly and precisely describe pathology by applying our methods to image data from two mutant mouse lines. Our results demonstrate the feasibility and potential of using computer-assisted analysis for testis histology, an area poised to evolve rapidly on the back of emerging, spatially resolved genomic and proteomic technologies. AVAILABILITY AND IMPLEMENTATION: The source code to reproduce the results described here and a SATINN standalone application with graphic-user interface are available from http://github.com/conradlab/SATINN. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
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Proteómica , Testículo , Masculino , Ratones , Animales , Redes Neurales de la Computación , Túbulos Seminíferos , Programas Informáticos , MamíferosRESUMEN
Next-generation sequencing is increasingly used for clinical evaluation of patients presenting with thrombotic microangiopathies because it allows for simultaneous interrogation of multiple complement and coagulation pathway genes known to be associated with disease. However, the diagnostic yield is undefined in routine clinical practice. Historic studies relied on case-control cohorts, did not apply current guidelines for variant pathogenicity assessment, and used targeted gene enrichment combined with next-generation sequencing. A clinically enhanced exome, targeting ~54 Mb, was sequenced for 73 patients. Variant analysis and interpretation were performed on genes with biological relevance in thrombotic microangiopathy (C3,CD46, CFB, CFH, CFI, DGKE, and THBD). CFHR3-CFHR1 deletion status was also assessed using multiplex ligation-dependent probe amplification. Variants were classified using American College of Medical Genetics and Genomics guidelines. We identified 5 unique novel and 14 unique rare variants in 25% (18/73) of patients, including a total of 5 pathogenic, 4 likely pathogenic, and 15 variants of uncertain clinical significance. Nine patients had homozygous deletions in CFHR3-CFHR1. The diagnostic yield, defined as the presence of a pathogenic variant, likely pathogenic variant or homozygous deletion of CFHR3-CFHR1, was 25% for all patients tested. Variants of uncertain clinical significance were identified in 21% (15/73) of patients.These results illustrate the expected diagnositic yield in the setting of thrombotic microangiopathies through the application of standardized variant interpretation, and highlight the utility of such an approach. Sequencing a clinically enhanced exome to enable targeted, disease-specific variant analysis is a viable approach. The moderate rate of variants of uncertain clinical significance highlights the paucity of data surrounding the variants in our cohort and illustrates the need for expanded variant curation resources to aid in thrombotic microangiopathy-related disease variant classification.
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Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Microangiopatías Trombóticas/diagnóstico , Microangiopatías Trombóticas/genética , Adulto , Anciano , Estudios de Casos y Controles , Estudios de Cohortes , Exoma , Femenino , Humanos , Masculino , Persona de Mediana EdadRESUMEN
BACKGROUND: Single-cell RNA-seq (scRNA-Seq) has been widely adopted to study gene expression of the human testis. Several datasets of scRNA-Seq from human testis have been generated from different groups processed with different informatics pipelines. An integrated atlas of scRNA-Seq expression constructed from multiple donors, developmental ages, and fertility states would be widely useful for the testis research community. OBJECTIVE: To describe the generation and use of the human infertility single-cell testis atlas (HISTA), an interactive web tool for understanding human spermatogenesis through scRNA-Seq analysis. METHODS: We obtained scRNA-Seq datasets derived from 12 donors, including healthy adult controls, juveniles, and several infertility cases, and reprocessed these data using methods to remove batch effects. Using Shiny, an open-source environment for data visualization, we created numerous interactive tools for exploring the data, some of which support simple statistical hypothesis testing. We used the resulting HISTA browser and its underlying data to demonstrate HISTA's value for testis researchers. RESULTS: A primary application of HISTA is to search by a single gene or a set of genes; thus, we present various analyses that quantify and visualize gene expression across the testis cells and pathology. HISTA also contains machine-learning-derived gene modules ("components") that capture the entire transcriptional landscape of the testis tissue. We show how the use of these components can simplify the highly complex data in HISTA and assist with the interpretation of genes with unknown functions. Finally, we demonstrate the diverse ways HISTA can be used for new data analysis, including hypothesis testing. DISCUSSION AND CONCLUSIONS: HISTA is a research environment that can help scientists organize and understand the high-dimensional transcriptional landscape of the human testis. HISTA has already contributed to published testis research and can be updated as needed with input from the research community or downloaded and modified for individual needs.
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Spermatogenesis is a complex process that can be disrupted by genetic and epigenetic changes, potentially leading to male infertility. Recent research has rapidly increased the number of protein coding mutations causally linked to impaired spermatogenesis in humans and mice. However, the role of non-coding mutations remains largely unexplored. As a case study to evaluate the effects of non-coding mutations on spermatogenesis, we first identified an evolutionarily conserved topologically associated domain (TAD) boundary near two genes with important roles in mammalian testis function: Dmrtb1 and Lrp8 . We then used CRISPR-Cas9 to generate a mouse line where 26kb of the boundary was removed including a strong and evolutionarily conserved CTCF binding site. ChIP-seq and Hi-C experiments confirmed the removal of the CTCF site and a resulting increase in the DNA-DNA interactions across the domain boundary. Mutant mice displayed significant changes in testis gene expression, abnormal testis histology, a 35% drop in the estimated efficiency of spermatogenesis and a 28% decrease in daily sperm production compared to littermate controls. Despite these quantitative changes in testis function, mutant mice show no significant changes in fertility. This suggests that non-coding deletions affecting testis gene regulation may have smaller effects on fertility compared to coding mutations of the same genes. Our results demonstrate that disruption of a TAD boundary can have a negative impact on sperm production and highlight the importance of considering non-coding mutations in the analysis of patients with male infertility.
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Male rhesus monkeys (n = 24) had a biopsy of prefrontal cortical area 46 prior to chronic ethanol self-administration (n = 17) or caloric control (n = 7). Fourteen months of daily self-administration (water vs. 4% alcohol, 22 h access/day termed "open-access") was followed by two cycles of prolonged abstinence (5 weeks) each followed by 3 months of open-access alcohol and a final abstinence followed by necropsy. At necropsy, a biopsy of Area 46, contralateral to the original biopsy, was obtained. Gene expression data (RNA-Seq) were collected comparing biopsy/necropsy samples. Monkeys were categorized by drinking status during the final post-abstinent drinking phase as light (LD), binge (BD), heavy (HD) and very heavy (VHD drinkers). Comparing pre-ethanol to post-abstinent biopsies, four animals that converted from HD to VHD status had significant ontology enrichments in downregulated genes (necropsy minus biopsy n = 286) that included immune response (FDR < 9 × 10-7) and plasma membrane changes (FDR < 1 × 10-7). Genes in the immune response category included IL16 and 18, CCR1, B2M, TLR3, 6 and 7, SP2 and CX3CR1. Upregulated genes (N = 388) were particularly enriched in genes associated with the negative regulation of MAP kinase activity (FDR < 3 × 10-5), including DUSP 1, 4, 5, 6 and 18, SPRY 2, 3, and 4, SPRED2, BMP4 and RGS2. Overall, these data illustrate the power of the NHP model and the within-subject design of genomic changes due to alcohol and suggest new targets for treating severe escalated drinking following repeated alcohol abstinence attempts.
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Postzygotic mutations (PZMs) begin to accrue in the human genome immediately after fertilization, but how and when PZMs affect development and lifetime health remain unclear. To study the origins and functional consequences of PZMs, we generated a multitissue atlas of PZMs spanning 54 tissue and cell types from 948 donors. Nearly half the variation in mutation burden among tissue samples can be explained by measured technical and biological effects, and 9% can be attributed to donor-specific effects. Through phylogenetic reconstruction of PZMs, we found that their type and predicted functional impact vary during prenatal development, across tissues, and through the germ cell life cycle. Thus, methods for interpreting effects across the body and the life span are needed to fully understand the consequences of genetic variants.
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Análisis Mutacional de ADN , Longevidad , Cigoto , Femenino , Humanos , Longevidad/genética , Mutación , Filogenia , RNA-SeqRESUMEN
Topological associating domains (TADs) are self-interacting genomic units crucial for shaping gene regulation patterns. Despite their importance, the extent of their evolutionary conservation and its functional implications remain largely unknown. In this study, we generate Hi-C and ChIP-seq data and compare TAD organization across four primate and four rodent species, and characterize the genetic and epigenetic properties of TAD boundaries in correspondence to their evolutionary conservation. We find that only 14% of all human TAD boundaries are shared among all eight species (ultraconserved), while 15% are human-specific. Ultraconserved TAD boundaries have stronger insulation strength, CTCF binding, and enrichment of older retrotransposons, compared to species-specific boundaries. CRISPR-Cas9 knockouts of two ultraconserved boundaries in mouse models leads to tissue-specific gene expression changes and morphological phenotypes. Deletion of a human-specific boundary near the autism-related AUTS2 gene results in upregulation of this gene in neurons. Overall, our study provides pertinent TAD boundary evolutionary conservation annotations, and showcase the functional importance of TAD evolution.
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Topological associating domains (TADs) are self-interacting genomic units crucial for shaping gene regulation patterns. Despite their importance, the extent of their evolutionary conservation and its functional implications remain largely unknown. In this study, we generate Hi-C and ChIP-seq data and compare TAD organization across four primate and four rodent species and characterize the genetic and epigenetic properties of TAD boundaries in correspondence to their evolutionary conservation. We find 14% of all human TAD boundaries to be shared among all eight species (ultraconserved), while 15% are human-specific. Ultraconserved TAD boundaries have stronger insulation strength, CTCF binding, and enrichment of older retrotransposons compared to species-specific boundaries. CRISPR-Cas9 knockouts of an ultraconserved boundary in a mouse model lead to tissue-specific gene expression changes and morphological phenotypes. Deletion of a human-specific boundary near the autism-related AUTS2 gene results in the upregulation of this gene in neurons. Overall, our study provides pertinent TAD boundary evolutionary conservation annotations and showcases the functional importance of TAD evolution.
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Genoma , Genómica , Animales , Ratones , Humanos , Regulación de la Expresión Génica , Epigenómica , Secuenciación de Inmunoprecipitación de Cromatina , Cromatina , Mamíferos/genéticaRESUMEN
The activity of nuclear transcription factors is often regulated by specific kinase-signaling pathways. We have previously shown that the organochlorine pesticide dichlorodiphenyltrichloroethane (DDT) stimulates activator protein-1 activity through the p38 mitogen-activated protein kinase (MAPK). Here, we show that DDT and its metabolites also stimulate the transcriptional activity of cyclic adenosine monophosphate response element-binding protein and Elk1 and potentiate gene expression through cyclic adenosine monophosphate and hypoxia response elements. Because DDT stimulates gene expression through various transcription factors and hence multiple response elements, we hypothesized that p38 signaling targets a common shared transcriptional activator. Here, we demonstrate using both pharmacological and molecular techniques, the general coactivator p300 is phosphorylated and potentiated by the p38 MAPK signaling cascade. We further show that p38 directly phosphorylates p300 in its N-terminus. These results, together with our previous work, suggest that p38 stimulates downstream transcription factors in part by targeting the general coactivator p300.
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DDT/farmacología , Factores de Transcripción p300-CBP/efectos de los fármacos , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Western Blotting , Línea Celular , Genes Reporteros , Humanos , Sistema de Señalización de MAP Quinasas , Fosforilación , Regiones Promotoras Genéticas , Factores de Transcripción/metabolismo , Transcripción Genética , Factores de Transcripción p300-CBP/metabolismoRESUMEN
Glioblastoma is the most common primary malignancy of the adult central nervous system. Gliomagenesis involves a complex range of alterations, including sequence changes, copy number variations (CNVs), and epigenetic modifications, that have clinical implications for disease classification and prognosis. Thus, multiple testing modalities are required to support a complete diagnostic workup. The goal of this study was to streamline the multipart workflow by predicting both sequence changes and CNVs (specifically EGFR amplifications) from a single next-generation sequencing (NGS) test. Eighty-six primary and secondary glioblastomas were submitted for clinical NGS to report sequence variants from a concise panel of cancer-relevant genes. Most specimens underwent concomitant testing by methylation-specific polymerase chain reaction, immunohistochemistry, and fluorescence in situ hybridization. Using data generated during the course of clinical testing, we found that NGS-based variant predictions were concordant with immunohistochemistry and fluorescence in situ hybridization for IDH mutation and EGFR amplification status, respectively. We also noted that EGFR amplifications correlated with polysomy of chromosome 7, 19, and 20, and loss of PTEN and CDKN2A. EGFR-unamplified cases had lower rates of chromosome 7 polysomy, and PTEN and CDKN2A loss, but more CNVs overall. TP53, NF1, ATRX, and PDGFRA mutations were nearly exclusive to specimens without EGFR amplification. EGFR amplification was not associated with longer progression-free survival in this cohort, but amplifications were enriched in a group with slightly longer overall survival despite radiographic evidence of disease progression. Further study is needed to explore the mechanisms responsible for noted patterns of co-occurring variants and to correlate them with specific clinical outcomes.
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Neoplasias Encefálicas/genética , Variaciones en el Número de Copia de ADN , Glioblastoma/genética , Mutación , Adulto , Anciano , Anciano de 80 o más Años , Neoplasias Encefálicas/patología , Receptores ErbB/genética , Femenino , Amplificación de Genes , Regulación Neoplásica de la Expresión Génica , Glioblastoma/patología , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Masculino , Persona de Mediana Edad , PronósticoRESUMEN
Infertility in men and women is a complex genetic trait with shared biological bases between the sexes. Here, we perform a series of rare variant analyses across 73,185 women and men to identify genes that contribute to primary gonadal dysfunction. We report CSMD1, a complement regulatory protein on chromosome 8p23, as a strong candidate locus in both sexes. We show that CSMD1 is enriched at the germ-cell/somatic-cell interface in both male and female gonads. Csmd1-knockout males show increased rates of infertility with significantly increased complement C3 protein deposition in the testes, accompanied by severe histological degeneration. Knockout females show significant reduction in ovarian quality and breeding success, as well as mammary branching impairment. Double knockout of Csmd1 and C3 causes non-additive reduction in breeding success, suggesting that CSMD1 and the complement pathway play an important role in the normal postnatal development of the gonads in both sexes.
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Infertilidad/genética , Proteínas de la Membrana/genética , Proteínas Supresoras de Tumor/genética , Factores de Edad , Animales , Complemento C3/metabolismo , Femenino , Estudios de Asociación Genética , Genotipo , Humanos , Masculino , Glándulas Mamarias Animales/crecimiento & desarrollo , Glándulas Mamarias Animales/patología , Menopausia/genética , Ratones Noqueados , Mutación , Ovario/patología , Maduración Sexual , Testículo/metabolismoRESUMEN
Vascular anomalies are variably associated with overgrowth, skeletal anomalies, and abnormalities of the brain, leptomeninges, and eye. We assembled a 16-institution network to determine the range of genetic variants associated with a spectrum of vascular anomalies with overgrowth, ranging from mild to severe. Because of the overlap between cancer-associated variants and previously described somatic variants in vascular overgrowth syndromes, we employed tumor genetic profiling via high-depth next-generation sequencing using a panel to assay affected tissue from a diverse cohort of subjects with vascular anomalies with overgrowth. Seventy-five percent (43/57) harbored pathogenic or likely pathogenic variants in 10 genes. We identified two genes (mTOR, PIK3R1) and several variants previously described in the setting of cancer but that, to our knowledge, have not been described in vascular malformations. All were identified at low variant allele frequency consistent with somatic mosaic etiology. By leveraging somatic variant detection technology typically applied to cancer in a cohort inclusive of broad phenotypic severity, we demonstrated that most vascular anomalies with overgrowth harbor postzygotic gain-of-function mutations in oncogenes. Furthermore, continued interrogation of oncogenes in benign developmental disorders could provide insight into fundamental mechanisms regulating cell growth.
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ADN de Neoplasias/genética , Genes Relacionados con las Neoplasias/genética , Genómica/métodos , Mutación , Neoplasias/genética , Malformaciones Vasculares/genética , Adolescente , Adulto , Niño , Preescolar , Femenino , Frecuencia de los Genes , Pruebas Genéticas , Humanos , Lactante , Masculino , Persona de Mediana Edad , Neoplasias/diagnóstico , Neoplasias/etiología , Fenotipo , Malformaciones Vasculares/complicaciones , Malformaciones Vasculares/metabolismo , Adulto JovenRESUMEN
FGFR2 is recurrently amplified in 5% of gastric cancers and 1%-4% of breast cancers; however, this molecular alteration has never been reported in a primary colorectal cancer specimen. Preclinical studies indicate that several FGFR tyrosine-kinase inhibitors (TKIs), such as AZD4547, have in vitro activity against the FGFR2-amplified colorectal cell line, NCI-H716. The efficacy of these inhibitors is currently under investigation in clinical trials for breast and gastric cancer. Thus, better characterizing colorectal tumors for FGFR2 amplification could identify a subset of patients who may benefit from FGFR TKI therapies. Here, we describe a novel FGFR2 amplification identified by clinical next-generation sequencing in a primary colorectal cancer. Further characterization of the tumor by immunohistochemistry showed neuroendocrine differentiation, similar to the reported properties of the NCI-H716 cell line. These findings demonstrate that the spectrum of potentially clinically actionable mutations detected by targeted clinical sequencing panels is not limited to only single-nucleotide polymorphisms and insertions/deletions but also to copy-number alterations.
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Poliposis Adenomatosa del Colon/genética , Receptor Tipo 2 de Factor de Crecimiento de Fibroblastos/genética , Adenocarcinoma/genética , Línea Celular Tumoral , Neoplasias Colorrectales/genética , Variaciones en el Número de Copia de ADN/genética , Femenino , Amplificación de Genes/genética , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Persona de Mediana Edad , Receptor Tipo 2 de Factor de Crecimiento de Fibroblastos/metabolismo , Neoplasias Gástricas/genéticaRESUMEN
The 2007 World Health Organization Classification of Tumours of the Central Nervous System classifies lower-grade gliomas [LGGs (grades II to III diffuse gliomas)] morphologically as astrocytomas or oligodendrogliomas, and tumors with unclear ambiguous morphology as oligoastrocytomas. The World Health Organization's newly released (2016) classification incorporates molecular data. A single, targeted next-generation sequencing (NGS) panel was used for detecting single-nucleotide variation and copy number variation in 50 LGG cases originally classified using the 2007 criteria, including 36 oligoastrocytomas, 11 oligodendrogliomas, 2 astrocytomas, and 1 LGG not otherwise specified. NGS results were compared with those from IHC analysis and fluorescence in situ hybridization to assess concordance and to categorize the tumors according to the 2016 criteria. NGS results were concordant with those from IHC analysis in all cases. In 3 cases, NGS was superior to fluorescence in situ hybridization in distinguishing segmental chromosomal losses from whole-arm deletions. The NGS approach was effective in reclassifying 36 oligoastrocytomas as 30 astrocytomas (20 IDH1/2 mutant and 10 IDH1/2 wild type) and 6 oligodendrogliomas, and 1 oligodendroglioma as an astrocytoma (IDH1/2 mutant). Here we show that a single, targeted NGS assay can serve as the sole testing modality for categorizing LGG according to the World Health Organization's 2016 diagnostic scheme. This modality affords greater accuracy and efficiency while reducing specimen tissue requirements compared with multimodal approaches.
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Biomarcadores de Tumor , Neoplasias Encefálicas/diagnóstico , Neoplasias Encefálicas/genética , Glioma/diagnóstico , Glioma/genética , Secuenciación de Nucleótidos de Alto Rendimiento , Adolescente , Adulto , Anciano , Niño , Preescolar , Biología Computacional/métodos , Variaciones en el Número de Copia de ADN , Manejo de la Enfermedad , Femenino , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Humanos , Masculino , Persona de Mediana Edad , Mutación , Clasificación del Tumor , Polimorfismo de Nucleótido Simple , Reproducibilidad de los Resultados , Flujo de Trabajo , Adulto JovenRESUMEN
BACKGROUND: Current diagnostics for allergies, such as skin prick and radioallergosorbent tests, do not allow for inexpensive, high-throughput screening of patients. Additionally, extracts used in these methods are made from washed pollen that lacks pollen surface materials that may contain allergens. METHODOLOGY/PRINCIPAL FINDINGS: We sought to develop a high-throughput assay to rapidly measure allergen-specific IgE in sera and to explore the relative allergenicity of different pollen fractions (i.e. surface, cytoplasmic, commercial extracts). To do this, we generated a protein microarray containing surface, cytoplasmic, and commercial extracts from 22 pollen species, commercial extracts from nine non-pollen allergens, and five recombinant allergenic proteins. Pollen surface and cytoplasmic fractions were prepared by extraction into organic solvents and aqueous buffers, respectively. Arrays were incubated with <25 uL of serum from 176 individuals and bound IgE was detected by indirect immunofluorescence, providing a high-throughput measurement of IgE. We demonstrated that the allergen microarray is a reproducible method to measure allergen-specific IgE in small amounts of sera. Using this tool, we demonstrated that specific IgE clusters according to the phylogeny of the allergen source. We also showed that the pollen surface, which has been largely overlooked in the past, contained potent allergens. Although, as a class, cytoplasmic fractions obtained by our pulverization/precipitation method were comparable to commercial extracts, many individual allergens showed significant differences. CONCLUSIONS/SIGNIFICANCE: These results support the hypothesis that protein microarray technology is a useful tool for both research and in the clinic. It could provide a more efficient and less painful alternative to traditionally used skin prick tests, making it economically feasible to compare allergen sensitivity of different populations, monitor individual responses over time, and facilitate genetic studies on pollen allergy.