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Sex differences and age-related changes in the human heart at the tissue, cell, and molecular level have been well-documented and many may be relevant for cardiovascular disease. However, how molecular programs within individual cell types vary across individuals by age and sex remains poorly characterized. To better understand this variation, we performed single-nucleus combinatorial indexing (sci) ATAC- and RNA-Seq in human heart samples from nine donors. We identify hundreds of differentially expressed genes by age and sex and find epigenetic signatures of variation in ATAC-Seq data in this discovery cohort. We then scale up our single-cell RNA-Seq analysis by combining our data with five recently published single nucleus RNA-Seq datasets of healthy adult hearts. We find variation such as metabolic alterations by sex and immune changes by age in differential expression tests, as well as alterations in abundance of cardiomyocytes by sex and neurons with age. In addition, we compare our adult-derived ATAC-Seq profiles to analogous fetal cell types to identify putative developmental-stage-specific regulatory factors. Finally, we train predictive models of cell-type-specific RNA expression levels utilizing ATAC-Seq profiles to link distal regulatory sequences to promoters, quantifying the predictive value of a simple TF-to-expression regulatory grammar and identifying cell-type-specific TFs. Our analysis represents the largest single-cell analysis of cardiac variation by age and sex to date and provides a resource for further study of healthy cardiac variation and transcriptional regulation at single-cell resolution.
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Cromatina , Análisis de la Célula Individual , Humanos , Análisis de la Célula Individual/métodos , Femenino , Masculino , Adulto , Cromatina/metabolismo , Cromatina/genética , Persona de Mediana Edad , Miocardio/metabolismo , Miocardio/citología , Caracteres Sexuales , Anciano , Factores de Edad , Envejecimiento/genética , Factores Sexuales , Adulto Joven , Miocitos Cardíacos/metabolismo , Corazón/crecimiento & desarrolloRESUMEN
The pathophysiology of silicosis is poorly understood, limiting development of therapies for those who have been exposed to the respirable particle. We explored mechanisms of silica-induced pulmonary fibrosis in human lung samples collected from patients with occupational exposure to silica and in a longitudinal mouse model of silicosis using multiple modalities including whole-lung single-cell RNA sequencing and histological, biochemical, and physiologic assessments. In addition to pulmonary inflammation and fibrosis, intratracheal silica challenge induced osteoclast-like differentiation of alveolar macrophages and recruited monocytes, driven by induction of the osteoclastogenic cytokine, receptor activator of nuclear factor κΒ ligand (RANKL) in pulmonary lymphocytes, and alveolar type II cells. Anti-RANKL monoclonal antibody treatment suppressed silica-induced osteoclast-like differentiation in the lung and attenuated pulmonary fibrosis. We conclude that silica induces differentiation of pulmonary osteoclast-like cells leading to progressive lung injury, likely due to sustained elaboration of bone-resorbing proteases and hydrochloric acid. Interrupting osteoclast-like differentiation may therefore constitute a promising avenue for moderating lung damage in silicosis.
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Diferenciación Celular , Osteoclastos , Fibrosis Pulmonar , Dióxido de Silicio , Silicosis , Dióxido de Silicio/toxicidad , Animales , Humanos , Osteoclastos/metabolismo , Osteoclastos/efectos de los fármacos , Osteoclastos/patología , Fibrosis Pulmonar/inducido químicamente , Fibrosis Pulmonar/patología , Fibrosis Pulmonar/metabolismo , Ratones , Silicosis/patología , Silicosis/metabolismo , Silicosis/etiología , Diferenciación Celular/efectos de los fármacos , Ligando RANK/metabolismo , Modelos Animales de Enfermedad , Masculino , Pulmón/patología , Pulmón/metabolismo , Pulmón/efectos de los fármacos , Macrófagos Alveolares/metabolismo , Macrófagos Alveolares/patología , Macrófagos Alveolares/efectos de los fármacos , FemeninoRESUMEN
The pathophysiology of silicosis is poorly understood, limiting development of therapies for those who have been exposed to the respirable particle. We explored the mechanisms of silica-induced pulmonary fibrosis in a mouse model using multiple modalities including whole-lung single-nucleus RNA sequencing. These analyses revealed that in addition to pulmonary inflammation and fibrosis, intratracheal silica challenge induced osteoclast-like differentiation of alveolar macrophages and recruited monocytes, driven by induction of the osteoclastogenic cytokine, receptor activator of nuclear factor-κB ligand (RANKL) in pulmonary lymphocytes and alveolar type II cells. Furthermore, anti-RANKL monoclonal antibody treatment suppressed silica-induced osteoclast-like differentiation in the lung and attenuated silica-induced pulmonary fibrosis. We conclude that silica induces osteoclast-like differentiation of distinct recruited and tissue resident monocyte populations, leading to progressive lung injury, likely due to sustained elaboration of bone resorbing proteases and hydrochloric acid. Interrupting osteoclast-like differentiation may therefore constitute a promising avenue for moderating lung damage in silicosis.
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OBJECTIVE: Among individuals with systemic sclerosis (SSc) randomized to cyclophosphamide (CYC) (n = 34) or hematopoietic stem cell transplantation (HSCT) (n = 33), we examined longitudinal trends of clinical, pulmonary function, and quality of life measures while accounting for the influence of early failures on treatment comparisons. METHODS: Assuming that data were missing at random, mixed-effects regression models were used to estimate longitudinal trends for clinical measures when comparing treatment groups. Results were compared to observed means and to longitudinal trends estimated from shared parameter models, assuming that data were missing not at random. Longitudinal trends for SSc intrinsic molecular subsets defined by baseline gene expression signatures (normal-like, inflammatory, and fibroproliferative signatures) were also studied. RESULTS: Available observed means for pulmonary function tests appeared to improve over time in both arms. However, after accounting for participant loss, forced vital capacity in HSCT recipients increased by 0.77 percentage points/year but worsened by -3.70/year for CYC (P = 0.004). Similar results were found for diffusing capacity for carbon monoxide and quality of life indicators. Results for both analytic models were consistent. HSCT recipients in the inflammatory (n = 20) and fibroproliferative (n = 20) subsets had superior long-term trends compared to CYC for pulmonary and quality of life measures. HSCT was also superior for modified Rodnan skin thickness scores in the fibroproliferative subset. For the normal-like subset (n = 22), superiority of HSCT was less apparent. CONCLUSION: Longitudinal trends estimated from 2 statistical models affirm the efficacy of HSCT over CYC in severe SSc. Failure to account for early loss of participants may distort estimated clinical trends over the long term.
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Trasplante de Células Madre Hematopoyéticas , Esclerodermia Localizada , Esclerodermia Sistémica , Humanos , Trasplante de Células Madre Hematopoyéticas/efectos adversos , Trasplante de Células Madre Hematopoyéticas/métodos , Inmunosupresores/uso terapéutico , Calidad de Vida , Trasplante Autólogo , Ciclofosfamida/uso terapéutico , Esclerodermia Sistémica/diagnóstico , Esclerodermia Sistémica/tratamiento farmacológico , Esclerodermia Localizada/tratamiento farmacológico , Resultado del TratamientoRESUMEN
Here, the efficacy of abatacept in patients with early diffuse systemic sclerosis (dcSSc) was analyzed to test the hypothesis that patients in the inflammatory intrinsic subset would show the most significant clinical improvement. Eighty-four participants with dcSSc were randomized to receive abatacept or placebo for 12 months. RNA-Seq was performed on 233 skin paired biopsies at baseline and at 3 and 6 months. Improvement was defined as a 5-point or more than 20% change in modified Rodnan skin score (mRSS) between baseline and 12 months. Samples were assigned to intrinsic gene expression subsets (inflammatory, fibroproliferative, or normal-like subsets). In the abatacept arm, change in mRSS was most pronounced for the inflammatory and normal-like subsets relative to the placebo subset. Gene expression for participants on placebo remained in the original molecular subset, whereas inflammatory participants treated with abatacept had gene expression that moved toward the normal-like subset. The Costimulation of the CD28 Family Reactome Pathway decreased in patients who improved on abatacept and was specific to the inflammatory subset. Patients in the inflammatory subset had elevation of the Costimulation of the CD28 Family pathway at baseline relative to that of participants in the fibroproliferative and normal-like subsets. There was a correlation between improved ΔmRSS and baseline expression of the Costimulation of the CD28 Family pathway. This study provides an example of precision medicine in systemic sclerosis clinical trials.
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Esclerodermia Difusa , Esclerodermia Sistémica , Humanos , Abatacept/farmacología , Abatacept/uso terapéutico , Antígenos CD28/metabolismo , Esclerodermia Sistémica/tratamiento farmacológico , Esclerodermia Sistémica/genética , Esclerodermia Sistémica/metabolismo , Esclerodermia Difusa/tratamiento farmacológico , Esclerodermia Difusa/metabolismo , Esclerodermia Difusa/patología , Piel/patologíaRESUMEN
OBJECTIVES: Four intrinsic molecular subsets (inflammatory, fibroproliferative, limited, normal-like) have previously been identified in SSc and are characterized by unique gene expression signatures and pathways. The intrinsic subsets have been linked to improvement with specific therapies. Here, we investigated associations between baseline demographics and intrinsic molecular subsets in a meta-analysis of published datasets. METHODS: Publicly available gene expression data from skin biopsies of 311 SSc patients measured by DNA microarray were classified into the intrinsic molecular subsets. RNA-sequencing data from 84 participants from the ASSET trial were used as a validation cohort. Baseline clinical demographics and intrinsic molecular subsets were tested for statistically significant associations. RESULTS: Males were more likely to be classified in the fibroproliferative subset (P = 0.0046). SSc patients who identified as African American/Black were 2.5 times more likely to be classified as fibroproliferative compared with White/Caucasian patients (P = 0.0378). ASSET participants sera positive for anti-RNA pol I and RNA pol III autoantibodies were enriched in the inflammatory subset (P = 5.8 × 10-5, P = 9.3 × 10-5, respectively), while anti-Scl-70 was enriched in the fibroproliferative subset. Mean modified Rodnan Skin Score (mRSS) was statistically higher in the inflammatory and fibroproliferative subsets compared with normal-like (P = 0.0027). The average disease duration for inflammatory subset was less than fibroproliferative and normal-like intrinsic subsets (P = 8.8 × 10-4). CONCLUSIONS: We identified multiple statistically significant differences in baseline demographics between the intrinsic subsets that may represent underlying features of disease pathogenesis (e.g. chronological stages of fibrosis) and have implications for treatments that are more likely to work in certain SSc populations.
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Esclerodermia Sistémica , Masculino , Humanos , Esclerodermia Sistémica/patología , Genómica , Transcriptoma , Análisis de Secuencia por Matrices de Oligonucleótidos , Piel/patología , ARNRESUMEN
Spatial patterns of gene expression manifest at scales ranging from local (e.g., cell-cell interactions) to global (e.g., body axis patterning). However, current spatial transcriptomics methods either average local contexts or are restricted to limited fields of view. Here, we introduce sci-Space, which retains single-cell resolution while resolving spatial heterogeneity at larger scales. Applying sci-Space to developing mouse embryos, we captured approximate spatial coordinates and whole transcriptomes of about 120,000 nuclei. We identify thousands of genes exhibiting anatomically patterned expression, leverage spatial information to annotate cellular subtypes, show that cell types vary substantially in their extent of spatial patterning, and reveal correlations between pseudotime and the migratory patterns of differentiating neurons. Looking forward, we anticipate that sci-Space will facilitate the construction of spatially resolved single-cell atlases of mammalian development.
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Tipificación del Cuerpo/genética , Embrión de Mamíferos/embriología , Desarrollo Embrionario/genética , Perfilación de la Expresión Génica/métodos , Análisis de la Célula Individual/métodos , Transcriptoma , Animales , Atlas como Asunto , Encéfalo/embriología , Movimiento Celular , Ratones , Neurogénesis/genética , Neuronas/citologíaRESUMEN
BACKGROUND: Racial disparities in unintended pregnancy and contraceptive use in the United States are not mediated by access to family planning services alone. Rather, a history of medical mistrust underlies Black Americans' adoption of new medical technologies, inclusive of contraception. Efforts to develop hormonal male contraceptives need to incorporate Black Americans' experiences and perspectives so that new contraceptives enable their reproductive goals and promote gender equity. STUDY DESIGN: Working with our community-based partner, Healthy African American Families in Los Angeles, California, we conducted six 60-minute focus group discussions with 39 Black men over age 18, in ongoing heterosexual relationships, to explore attitudes towards and willingness to use hormonal male contraceptives. RESULTS: Just over one-third (35%) of respondents reported willingness to use or rely on hormonal male contraceptives. The majority held negative attitudes about hormonal male contraceptives, citing concerns about side effects and safety. Several respondents expressed mistrust of the medical community and medical research, noting that hormonal male contraceptives could be used against Black communities; several expressed unwillingness to trial hormonal male contraceptives without years of testing. However, all groups described scenarios where they would use them despite stated concerns. CONCLUSIONS: Black men's hypothetical willingness to use hormonal male contraceptives is limited by medical mistrust, which may be overcome by their concerns about the unreliability of current options or the contraceptive behaviors of female partners. Nevertheless, addressing Black Americans' history of medical mistreatment and exploitation will be essential for hormonal male contraceptives to positively contribute to Black men's reproductive options and agency. IMPLICATIONS: While the development of reversible, hormonal male contraception intends to fulfill unmet global needs for contraception, the utility of these hormonal male contraceptive methods among Black men living on low incomes in Los Angeles, California cannot be fully realized until developers address and overcome historical and ongoing medical mistrust.
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Negro o Afroamericano , Confianza , Anticoncepción , Humanos , Los Angeles , Masculino , Hombres , Estados UnidosRESUMEN
OBJECTIVE: We sought to determine histologic and gene expression features of clinical improvement in early diffuse cutaneous systemic sclerosis (dcSSc; scleroderma). METHODS: Fifty-eight forearm biopsies were evaluated from 26 individuals with dcSSc in two clinical trials. Histologic/immunophenotypic assessments of global severity, alpha-smooth muscle actin (aSMA), CD34, collagen, inflammatory infiltrate, follicles and thickness were compared with gene expression and clinical data. Support vector machine learning was performed using scleroderma gene expression subset (normal-like, fibroproliferative, inflammatory) as classifiers and histology scores as inputs. Comparison of w-vector mean absolute weights was used to identify histologic features most predictive of gene expression subset. We then tested for differential gene expression according to histologic severity and compared those with clinical improvement (according to the Combined Response Index in Systemic Sclerosis). RESULTS: aSMA was highest and CD34 lowest in samples with highest local Modified Rodnan Skin Score. CD34 and aSMA changed significantly from baseline to 52 weeks in clinical improvers. CD34 and aSMA were the strongest predictors of gene expression subset, with highest CD34 staining in the normal-like subset (p<0.001) and highest aSMA staining in the inflammatory subset (p=0.016). Analysis of gene expression according to CD34 and aSMA binarised scores identified a 47-gene fibroblast polarisation signature that decreases over time only in improvers (vs non-improvers). Pathway analysis of these genes identified gene expression signatures of inflammatory fibroblasts. CONCLUSION: CD34 and aSMA stains describe distinct fibroblast polarisation states, are associated with gene expression subsets and clinical assessments, and may be useful biomarkers of clinical severity and improvement in dcSSc.
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Fibroblastos/fisiología , Aprendizaje Automático , Esclerodermia Difusa/genética , Índice de Severidad de la Enfermedad , Actinas/metabolismo , Adulto , Antígenos CD34/metabolismo , Ensayos Clínicos como Asunto , Colágeno/metabolismo , Femenino , Antebrazo , Expresión Génica , Humanos , Masculino , Persona de Mediana Edad , Piel/metabolismoRESUMEN
OBJECTIVE: The Scleroderma: Cyclophosphamide or Transplantation (SCOT) trial demonstrated clinical benefit of haematopoietic stem cell transplant (HSCT) compared with cyclophosphamide (CYC). We mapped PBC (peripheral blood cell) samples from the SCOT clinical trial to scleroderma intrinsic subsets and tested the hypothesis that they predict long-term response to HSCT. METHODS: We analysed gene expression from PBCs of SCOT participants to identify differential treatment response. PBC gene expression data were generated from 63 SCOT participants at baseline and follow-up timepoints. Participants who completed treatment protocol were stratified by intrinsic gene expression subsets at baseline, evaluated for event-free survival (EFS) and analysed for differentially expressed genes (DEGs). RESULTS: Participants from the fibroproliferative subset on HSCT experienced significant improvement in EFS compared with fibroproliferative participants on CYC (p=0.0091). In contrast, EFS did not significantly differ between CYC and HSCT arms for the participants from the normal-like subset (p=0.77) or the inflammatory subset (p=0.1). At each timepoint, we observed considerably more DEGs in HSCT arm compared with CYC arm with HSCT arm showing significant changes in immune response pathways. CONCLUSIONS: Participants from the fibroproliferative subset showed the most significant long-term benefit from HSCT compared with CYC. This study suggests that intrinsic subset stratification of patients may be used to identify patients with SSc who receive significant benefit from HSCT.
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Perfilación de la Expresión Génica/métodos , Trasplante de Células Madre Hematopoyéticas/métodos , Aprendizaje Automático , Esclerodermia Difusa/clasificación , Esclerodermia Difusa/terapia , Adulto , Ciclofosfamida/uso terapéutico , Femenino , Trasplante de Células Madre Hematopoyéticas/mortalidad , Humanos , Inmunosupresores/uso terapéutico , Masculino , Persona de Mediana Edad , Esclerodermia Difusa/patología , Transcriptoma , Resultado del TratamientoRESUMEN
Systemic sclerosis (SSc) is a heterogeneous autoimmune disorder that results in skin fibrosis, autoantibody production, and internal organ dysfunction. We previously identified 4 "intrinsic" subsets of SSc based upon skin gene expression that are found across organ systems. Gene expression regulators that underlie the SSc-intrinsic subsets, or are associated with clinical covariates, have not been systematically characterized. Here, we present a computational framework to calculate the activity scores of gene expression regulators and identify their associations with SSc clinical outcomes. We found that regulator activity scores can reproduce the intrinsic molecular subsets, with distinct sets of regulators identified for inflammatory, fibroproliferative, limited, and normal-like samples. Regulators most highly correlated with modified Rodnan skin score (MRSS) also varied by intrinsic subset. We identified subgroups of patients with fibroproliferative and inflammatory SSc with more severe pathophenotypes, such as higher MRSS and increased likelihood of interstitial lung disease (ILD). Using an independent cohort, we show that the group with more severe ILD was more likely to show forced vital capacity decline over a period of 36-54 months. Our results demonstrate an association among the activation of regulators, gene expression subsets, and clinical variables that can identify patients with SSc with more severe disease.
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Biología Computacional/métodos , Redes Reguladoras de Genes , Fibrosis Pulmonar/genética , Esclerodermia Sistémica/genética , Algoritmos , Biomarcadores/metabolismo , Humanos , Fibrosis Pulmonar/complicaciones , Fibrosis Pulmonar/patología , Esclerodermia Sistémica/complicaciones , Esclerodermia Sistémica/patología , TranscriptomaRESUMEN
OBJECTIVES: Determine global skin transcriptome patterns of early diffuse systemic sclerosis (SSc) and how they differ from later disease. METHODS: Skin biopsy RNA from 48 patients in the Prospective Registry for Early Systemic Sclerosis (PRESS) cohort (mean disease duration 1.3 years) and 33 matched healthy controls was examined by next-generation RNA sequencing. Data were analysed for cell type-specific signatures and compared with similarly obtained data from 55 previously biopsied patients in Genetics versus Environment in Scleroderma Outcomes Study cohort with longer disease duration (mean 7.4 years) and their matched controls. Correlations with histological features and clinical course were also evaluated. RESULTS: SSc patients in PRESS had a high prevalence of M2 (96%) and M1 (94%) macrophage and CD8 T cell (65%), CD4 T cell (60%) and B cell (69%) signatures. Immunohistochemical staining of immune cell markers correlated with the gene expression-based immune cell signatures. The prevalence of immune cell signatures in early diffuse SSc patients was higher than in patients with longer disease duration. In the multivariable model, adaptive immune cell signatures were significantly associated with shorter disease duration, while fibroblast and macrophage cell type signatures were associated with higher modified Rodnan Skin Score (mRSS). Immune cell signatures also correlated with skin thickness progression rate prior to biopsy, but did not predict subsequent mRSS progression. CONCLUSIONS: Skin in early diffuse SSc has prominent innate and adaptive immune cell signatures. As a prominently affected end organ, these signatures reflect the preceding rate of disease progression. These findings could have implications in understanding SSc pathogenesis and clinical trial design.
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Inmunidad Adaptativa/genética , Inmunidad Innata/genética , Esclerodermia Difusa/genética , Esclerodermia Difusa/inmunología , Adulto , Biomarcadores/análisis , Biopsia , Femenino , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Masculino , Persona de Mediana Edad , Análisis Multivariante , Estudios Prospectivos , Sistema de Registros , Análisis de Regresión , Esclerodermia Difusa/patología , Análisis de Secuencia de ARN , Índice de Severidad de la Enfermedad , Piel/inmunología , Piel/patología , TranscriptomaRESUMEN
OBJECTIVE: High-throughput gene expression profiling of tissue samples from patients with systemic sclerosis (SSc) has identified 4 "intrinsic" gene expression subsets: inflammatory, fibroproliferative, normal-like, and limited. Prior methods required agglomerative clustering of many samples. In order to classify individual patients in clinical trials or for diagnostic purposes, supervised methods that can assign single samples to molecular subsets are required. We undertook this study to introduce a novel machine learning classifier as a robust accurate intrinsic subset predictor. METHODS: Three independent gene expression cohorts were curated and merged to create a data set covering 297 skin biopsy samples from 102 unique patients and controls, which was used to train a machine learning algorithm. We performed external validation using 3 independent SSc cohorts, including a gene expression data set generated by an independent laboratory on a different microarray platform. In total, 413 skin biopsy samples from 213 individuals were analyzed in the training and testing cohorts. RESULTS: Repeated cross-fold validation identified consistent and discriminative markers using multinomial elastic net, performing with an average classification accuracy of 87.1% with high sensitivity and specificity. In external validation, the classifier achieved an average accuracy of 85.4%. Reanalyzing data from a previous study, we identified subsets of patients that represent the canonical inflammatory, fibroproliferative, and normal-like subsets. CONCLUSION: We developed a highly accurate classifier for SSc molecular subsets for individual patient samples. The method can be used in SSc clinical trials to identify an intrinsic subset on individual samples. Our method provides a robust data-driven approach to aid clinical decision-making and interpretation of heterogeneous molecular information in SSc patients.
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Esclerodermia Sistémica/clasificación , Aprendizaje Automático Supervisado , Transcriptoma , Adulto , Anciano , Algoritmos , Conjuntos de Datos como Asunto , Femenino , Expresión Génica , Perfilación de la Expresión Génica , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Aprendizaje Automático , Masculino , Persona de Mediana Edad , Esclerodermia Sistémica/genética , Adulto JovenRESUMEN
MOTIVATION: The accumulation of publicly available DNA methylation datasets has resulted in the need for tools to interpret the specific cellular phenotypes in bulk tissue data. Current approaches use either single differentially methylated CpG sites or differentially methylated regions that map to genes. However, these approaches may introduce biases in downstream analyses of biological interpretation, because of the variability in gene length. There is a lack of approaches to interpret DNA methylation effectively. Therefore, we have developed computational models to provide biological interpretation of relevant gene sets using DNA methylation data in the context of The Cancer Genome Atlas. RESULTS: We illustrate that Biological interpretation of DNA Methylation (BioMethyl) utilizes the complete DNA methylation data for a given cancer type to reflect corresponding gene expression profiles and performs pathway enrichment analyses, providing unique biological insight. Using breast cancer as an example, BioMethyl shows high consistency in the identification of enriched biological pathways from DNA methylation data compared to the results calculated from RNA sequencing data. We find that 12 out of 14 pathways identified by BioMethyl are shared with those by using RNA-seq data, with a Jaccard score 0.8 for estrogen receptor (ER) positive samples. For ER negative samples, three pathways are shared in the two enrichments with a slight lower similarity (Jaccard score = 0.6). Using BioMethyl, we can successfully identify those hidden biological pathways in DNA methylation data when gene expression profile is lacking. AVAILABILITY AND IMPLEMENTATION: BioMethyl R package is freely available in the GitHub repository (https://github.com/yuewangpanda/BioMethyl). SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
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Metilación de ADN , Genoma , Algoritmos , Secuencia de Bases , Islas de CpGRESUMEN
BACKGROUND: Infectious agents have long been postulated to be disease triggers for systemic sclerosis (SSc), but a definitive link has not been found. Metagenomic analyses of high-throughput data allows for the unbiased identification of potential microbiome pathogens in skin biopsies of SSc patients and allows insight into the relationship with host gene expression. METHODS: We examined skin biopsies from a diverse cohort of 23 SSc patients (including lesional forearm and non-lesional back samples) by RNA-seq. Metagenomic filtering and annotation was performed using the Integrated Metagenomic Sequencing Analysis (IMSA). Associations between microbiome composition and gene expression were analyzed using single-sample gene set enrichment analysis (ssGSEA). RESULTS: We find the skin of SSc patients exhibits substantial changes in microbial composition relative to controls, characterized by sharp decreases in lipophilic taxa, such as Propionibacterium, combined with increases in a wide range of gram-negative taxa, including Burkholderia, Citrobacter, and Vibrio. CONCLUSIONS: Microbiome dysbiosis is associated with disease duration and increased inflammatory gene expression. These data provide a comprehensive portrait of the SSc skin microbiome and its association with local gene expression, which mirrors the molecular changes in lesional skin.
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Disbiosis/genética , Inflamación/genética , Microbiota/genética , Esclerodermia Sistémica/genética , Piel/metabolismo , Adulto , Anciano , Bacterias/clasificación , Bacterias/genética , Biopsia , Estudios de Cohortes , Femenino , Perfilación de la Expresión Génica , Humanos , Inflamación/microbiología , Inflamación/patología , Masculino , Metagenómica/métodos , Persona de Mediana Edad , Dinámica Poblacional , Esclerodermia Sistémica/microbiología , Esclerodermia Sistémica/patología , Piel/microbiología , Piel/patología , Factores de TiempoRESUMEN
Fewer than half of patients with systemic sclerosis demonstrate modified Rodnan skin score improvement during mycophenolate mofetil (MMF) treatment. To understand the molecular basis for this observation, we extended our prior studies and characterized molecular and cellular changes in skin biopsies from subjects with systemic sclerosis treated with MMF. Eleven subjects completed ≥24 months of MMF therapy. Two distinct skin gene expression trajectories were observed across six of these subjects. Three of the six subjects showed attenuation of the inflammatory signature by 24 months, paralleling reductions in CCL2 mRNA expression in skin and reduced numbers of macrophages and myeloid dendritic cells in skin biopsies. MMF cessation at 24 months resulted in an increased inflammatory score, increased CCL2 mRNA and protein levels, modified Rodnan skin score rebound, and increased numbers of skin myeloid cells in these subjects. In contrast, three other subjects remained on MMF >24 months and showed a persistent decrease in inflammatory score, decreasing or stable modified Rodnan skin score, CCL2 mRNA reductions, sera CCL2 protein levels trending downward, reduction in monocyte migration, and no increase in skin myeloid cell numbers. These data summarize molecular changes during MMF therapy that suggest reduction of innate immune cell numbers, possibly by attenuating expression of chemokines, including CCL2.
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Inmunosupresores/uso terapéutico , Ácido Micofenólico/uso terapéutico , Células Mieloides/efectos de los fármacos , Esclerodermia Sistémica/tratamiento farmacológico , Adulto , Biopsia , Estudios de Casos y Controles , Recuento de Células , Quimiocina CCL2/inmunología , Quimiocina CCL2/metabolismo , Femenino , Perfilación de la Expresión Génica , Humanos , Inmunosupresores/farmacología , Estudios Longitudinales , Masculino , Persona de Mediana Edad , Ácido Micofenólico/farmacología , Células Mieloides/inmunología , Estudios Prospectivos , Esclerodermia Sistémica/inmunología , Esclerodermia Sistémica/patología , Piel/citología , Piel/efectos de los fármacos , Piel/inmunología , Piel/patología , Transcriptoma/efectos de los fármacos , Transcriptoma/inmunología , Resultado del TratamientoRESUMEN
Motivation: Molecular subtypes of cancers and autoimmune disease, defined by transcriptomic profiling, have provided insight into disease pathogenesis, molecular heterogeneity and therapeutic responses. However, technical biases inherent to different gene expression profiling platforms present a unique problem when analyzing data generated from different studies. Currently, there is a lack of effective methods designed to eliminate platform-based bias. We present a method to normalize and classify RNA-seq data using machine learning classifiers trained on DNA microarray data and molecular subtypes in two datasets: breast invasive carcinoma (BRCA) and colorectal cancer (CRC). Results: Multiple analyses show that feature specific quantile normalization (FSQN) successfully removes platform-based bias from RNA-seq data, regardless of feature scaling or machine learning algorithm. We achieve up to 98% accuracy for BRCA data and 97% accuracy for CRC data in assigning molecular subtypes to RNA-seq data normalized using FSQN and a support vector machine trained exclusively on DNA microarray data. We find that maximum accuracy was achieved when normalizing RNA-seq datasets that contain at least 25 samples. FSQN allows comparison of RNA-seq data to existing DNA microarray datasets. Using these techniques, we can successfully leverage information from existing gene expression data in new analyses despite different platforms used for gene expression profiling. Availability and implementation: FSQN has been submitted as an R package to CRAN. All code used for this study is available on Github (https://github.com/jenniferfranks/FSQN). Contact: michael.l.whitfield@dartmouth.edu. Supplementary information: Supplementary data are available at Bioinformatics online.
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Perfilación de la Expresión Génica/métodos , Neoplasias/genética , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Máquina de Vectores de Soporte , Neoplasias de la Mama/genética , Neoplasias del Colon/genética , Neoplasias Colorrectales/genética , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Análisis de Secuencia de ARN/métodos , Programas InformáticosRESUMEN
OBJECTIVE: To assess the safety and efficacy of treatment with belimumab in patients with early diffuse cutaneous systemic sclerosis (dcSSc) treated with background mycophenolate mofetil (MMF). METHODS: In this 52-week, investigator-initiated, single-center, double-blind, placebo-controlled, pilot study, 20 patients with dcSSc recently started on MMF were randomized 1:1 to additionally receive belimumab at 10 mg/kg intravenously or placebo. We assessed safety, efficacy, and differential gene expression. RESULTS: In the belimumab group, the median modified Rodnan skin thickness score (MRSS) decreased from 27 (interquartile range [IQR] 26.5, 31) to 18 (IQR 11, 23) (P = 0.039). In the placebo group, the median MRSS decreased from 28 (IQR 22, 28) to 21 (IQR 14, 25) (P = 0.023). The median change in MRSS was -10 (IQR -13, -9) in the belimumab group and -3.0 (IQR -15, -1) in the placebo group (P = 0.411). There were no significant differences between the groups in the number of adverse events (AEs). A significant decrease in expression of B cell signaling and profibrotic genes and pathways was observed in patients with improved MRSS in the belimumab group but not in the placebo group. CONCLUSION: Patients in both treatment groups experienced significant improvements in MRSS. The median difference was greater in the belimumab group but did not achieve statistical significance in this small pilot study. AEs were similar between the groups. Changes in gene expression were consistent with mechanism of action and showed that clinical response to treatment with belimumab is associated with a significant decrease in profibrotic genes and pathways. Additional studies are needed to determine the role of belimumab in the treatment of dcSSc.