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There is growing recognition that earliest signs of autism need not clearly manifest in the first three years of life. To what extent is this variation in developmental trajectories associated with age at autism diagnosis? Does the genetic profile of autism vary with age at autism diagnosis? Using longitudinal data from four birth cohorts, we demonstrate that two different trajectories of socio-emotional behaviours are associated with age at diagnosis. We further demonstrate that the age at autism diagnosis is partly heritable (h2 SNP = 0.12, s.e.m = 0.01), and is associated with two moderately correlated (rg = 0.38, s.e.m = 0.07) autism polygenic factors. One of these factors is associated with earlier diagnosis of autism, lower social and communication abilities in early childhood. The second factor is associated with later autism diagnosis, increased socio-emotional difficulties in adolescence, and has moderate to high positive genetic correlations with Attention-Deficit/Hyperactivity Disorder, mental health conditions, and trauma. Overall, our research identifies an axis of heterogeneity in autism, indexed by age at diagnosis, which partly explains heterogeneity in autism and the profiles of co-occurring neurodevelopmental and mental health profiles. Our findings have important implications for how we conceptualise autism and provide one model to explain some of the diversity within autism.
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We show that inactivating AMPK in a genetic medulloblastoma model depletes tumor stem cells and slows progression. In medulloblastoma, the most common malignant pediatric brain tumor, drug-resistant stem cells co-exist with transit-amplifying cells and terminally differentiated neuronal progeny. Prior studies show that Hk2-dependent glycolysis promotes medulloblastoma progression by suppressing neural differentiation. To determine how the metabolic regulator AMPK affects medulloblastoma growth and differentiation, we inactivated AMPK genetically in medulloblastomas. We bred conditional Prkaa1 and Prkaa2 deletions into medulloblastoma-prone SmoM2 mice and compared SmoM2-driven medulloblastomas with intact or inactivated AMPK. AMPK-inactivation increased event-free survival (EFS) and altered cellular heterogeneity, increasing differentiation and decreasing tumor stem cell populations. Surprisingly, AMPK-inactivation decreased mTORC1 activity and decreased Hk2 expression. Hk2 deletion similarly depleted medulloblastoma stem cells, implicating reduced glycolysis in the AMPK-inactivated phenotype. Our results show that AMPK inactivation disproportionately impairs medulloblastoma stem cell populations typically refractory to conventional therapies.
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Autozygosity is associated with rare Mendelian disorders and clinically relevant quantitative traits. We investigated associations between the fraction of the genome in runs of homozygosity (FROH) and common diseases in Genes & Health (n = 23,978 British South Asians), UK Biobank (n = 397,184), and 23andMe. We show that restricting analysis to offspring of first cousins is an effective way of reducing confounding due to social/environmental correlates of FROH. Within this group in G&H+UK Biobank, we found experiment-wide significant associations between FROH and twelve common diseases. We replicated associations with type 2 diabetes (T2D) and post-traumatic stress disorder via within-sibling analysis in 23andMe (median n = 480,282). We estimated that autozygosity due to consanguinity accounts for 5%-18% of T2D cases among British Pakistanis. Our work highlights the possibility of widespread non-additive genetic effects on common diseases and has important implications for global populations with high rates of consanguinity.
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Consanguinidad , Diabetes Mellitus Tipo 2 , Humanos , Diabetes Mellitus Tipo 2/genética , Homocigoto , Fenotipo , Polimorfismo de Nucleótido Simple , Bancos de Muestras Biológicas , Genoma Humano , Predisposición Genética a la Enfermedad , Reino UnidoRESUMEN
Recurrence is the primary life-threatening complication for medulloblastoma (MB). In Sonic Hedgehog (SHH)-subgroup MB, OLIG2-expressing tumor stem cells drive recurrence. We investigated the anti-tumor potential of the small-molecule OLIG2 inhibitor CT-179, using SHH-MB patient-derived organoids, patient-derived xenograft (PDX) tumors and mice genetically-engineered to develop SHH-MB. CT-179 disrupted OLIG2 dimerization, DNA binding and phosphorylation and altered tumor cell cycle kinetics in vitro and in vivo, increasing differentiation and apoptosis. CT-179 increased survival time in GEMM and PDX models of SHH-MB, and potentiated radiotherapy in both organoid and mouse models, delaying post-radiation recurrence. Single cell transcriptomic studies (scRNA-seq) confirmed that CT-179 increased differentiation and showed that tumors up-regulated Cdk4 post-treatment. Consistent with increased CDK4 mediating CT-179 resistance, CT-179 combined with CDK4/6 inhibitor palbociclib delayed recurrence compared to either single-agent. These data show that targeting treatment-resistant MB stem cell populations by adding the OLIG2 inhibitor CT-179 to initial MB treatment can reduce recurrence.
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We show that Polycomb Repressive Complex-2 (PRC2) components EED and EZH2 maintain neural identity in cerebellar granule neuron progenitors (CGNPs) and SHH-driven medulloblastoma, a cancer of CGNPs. Proliferating CGNPs and medulloblastoma cells inherit neural fate commitment through epigenetic mechanisms. The PRC2 is an epigenetic regulator that has been proposed as a therapeutic target in medulloblastoma. To define PRC2 function in cerebellar development and medulloblastoma, we conditionally deleted PRC2 components Eed or Ezh2 in CGNPs and analyzed medulloblastomas induced in Eed-deleted and Ezh2-deleted CGNPs by expressing SmoM2, an oncogenic allele of Smo. Eed deletion destabilized the PRC2, depleting EED and EZH2 proteins, while Ezh2 deletion did not deplete EED. Eed-deleted cerebella were hypoplastic, with reduced proliferation, increased apoptosis, and inappropriate muscle-like differentiation. Ezh2-deleted cerebella showed similar, milder phenotypes, with fewer muscle-like cells and without reduced growth. Eed-deleted and Ezh2-deleted medulloblastomas both demonstrated myoid differentiation and progressed more rapidly than PRC2-intact controls. The PRC2 thus maintains neural commitment in CGNPs and medulloblastoma, but is not required for SHH medulloblastoma progression. Our data define a role for the PRC2 in preventing inappropriate, non-neural fates during postnatal neurogenesis, and caution that targeting the PRC2 in SHH medulloblastoma may not produce durable therapeutic effects.
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Neoplasias Cerebelosas , Meduloblastoma , Humanos , Meduloblastoma/genética , Meduloblastoma/metabolismo , Complejo Represivo Polycomb 2/genética , Complejo Represivo Polycomb 2/metabolismo , Proliferación Celular , Cerebelo/metabolismo , Diferenciación Celular , Neoplasias Cerebelosas/genética , Neoplasias Cerebelosas/metabolismoRESUMEN
Single-cell transcriptomic analysis (scRNA-seq) can enable researchers to explore the gene expression patterns of thousands of individual cells simultaneously. Processing the complex data generated by scRNA-seq requires specialized computational tools. This chapter focuses on the analytical aspect of scRNA-seq workflow, with a focus on resolving biological signals from large-scale scRNA-seq data produced by the Drop-Seq platform.
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Microcefalia , Humanos , Análisis de la Célula Individual , Secuenciación del Exoma , Investigadores , TranscriptomaRESUMEN
Analysis of single-cell RNA sequencing typically includes the clustering of cells and subsequent determination of the population size of each cluster, relative to the whole. In an experimental setting, two or more conditions are compared to assess changes in cellular composition of the sampled tissue. Cluster populations are frequently normalized to the total number of cells from each replicate in order to facilitate comparisons. After normalization, they become interdependent fractions and therefore cannot be compared using individual t-tests. Here we describe the use of Dirichlet regression to compare changes in cellular composition between two or more conditions when multiple biological replicates (three or more) are sampled under each condition. We provide an example of R code to conduct a similar analysis and interpret the results.
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Microcefalia , Análisis de la Célula Individual , Humanos , Análisis de Secuencia de ARN/métodos , Análisis de la Célula Individual/métodos , Análisis por Conglomerados , Secuenciación del ExomaRESUMEN
Genetically engineered neural stem cells (NSCs) are a promising therapy for the highly aggressive brain cancer glioblastoma (GBM); however, treatment durability remains a major challenge. We sought to define the events that contribute to dynamic adaptation of GBM during treatment with human skin-derived induced NSCs releasing the pro-apoptotic agent TRAIL (iNSC-TRAIL) and develop strategies that convert initial tumor kill into sustained GBM suppression. In vivo and ex vivo analysis before, during, and after treatment revealed significant shifts in tumor transcriptome and spatial distribution as the tumors adapted to treatment. To address this, we designed iNSC delivery strategies that increased spatiotemporal TRAIL coverage and significantly decreased GBM volume throughout the brain, reducing tumor burden 100-fold as quantified in live ex vivo brain slices. The varying impact of different strategies on treatment durability and median survival of both solid and invasive tumors provides important guidance for optimizing iNSC therapy.
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PURPOSE: Patients with MYC-amplified medulloblastoma (MB) have poor prognosis and frequently develop recurrence, thus new therapeutic approaches to prevent recurrence are needed. EXPERIMENTAL DESIGN: We evaluated OLIG2 expression in a panel of mouse Myc-driven MB tumors, patient MB samples, and patient-derived xenograft (PDX) tumors and analyzed radiation sensitivity in OLIG2-high and OLIG2-low tumors in PDX lines. We assessed the effect of inhibition of OLIG2 by OLIG2-CRISPR or the small molecule inhibitor CT-179 combined with radiotherapy on tumor progression in PDX models. RESULTS: We found that MYC-associated MB can be stratified into OLIG2-high and OLIG2-low tumors based on OLIG2 protein expression. In MYC-amplified MB PDX models, OLIG2-low tumors were sensitive to radiation and rarely relapsed, whereas OLIG2-high tumors were resistant to radiation and consistently developed recurrence. In OLIG2-high tumors, irradiation eliminated the bulk of tumor cells; however, a small number of tumor cells comprising OLIG2- tumor cells and rare OLIG2+ tumor cells remained in the cerebellar tumor bed when examined immediately post-irradiation. All animals harboring residual-resistant tumor cells developed relapse. The relapsed tumors mirrored the cellular composition of the primary tumors with enriched OLIG2 expression. Further studies demonstrated that OLIG2 was essential for recurrence, as OLIG2 disruption with CRISPR-mediated deletion or with the small molecule inhibitor CT-179 prevented recurrence from the residual radioresistant tumor cells. CONCLUSIONS: Our studies reveal that OLIG2 is a biomarker and an effective therapeutic target in a high-risk subset of MYC-amplified MB, and OLIG2 inhibitor combined with radiotherapy represents a novel effective approach for treating this devastating disease.
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Neoplasias Cerebelosas , Meduloblastoma , Animales , Biomarcadores , Línea Celular Tumoral , Neoplasias Cerebelosas/tratamiento farmacológico , Neoplasias Cerebelosas/genética , Modelos Animales de Enfermedad , Humanos , Meduloblastoma/tratamiento farmacológico , Meduloblastoma/genética , Meduloblastoma/radioterapia , Ratones , Recurrencia Local de Neoplasia/genética , Factor de Transcripción 2 de los Oligodendrocitos/genética , Factor de Transcripción 2 de los Oligodendrocitos/uso terapéutico , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Proto-Oncogénicas c-myc/metabolismoRESUMEN
The therapeutic potential of CDK4/6 inhibitors for brain tumors has been limited by recurrence. To address recurrence, we tested a nanoparticle formulation of CDK4/6 inhibitor palbociclib (POx-Palbo) in mice genetically-engineered to develop SHH-driven medulloblastoma, alone or in combination with specific agents suggested by our analysis. Nanoparticle encapsulation reduced palbociclib toxicity, enabled parenteral administration, improved CNS pharmacokinetics, and extended mouse survival, but recurrence persisted. scRNA-seq identified up-regulation of glutamate transporter Slc1a2 and down-regulation of diverse ribosomal genes in proliferating medulloblastoma cells in POx-Palbo-treated mice, suggesting mTORC1 signaling suppression, subsequently confirmed by decreased 4EBP1 phosphorylation. Combining POx-Palbo with the mTORC1 inhibitor sapanisertib produced mutually enhancing effects and prolonged mouse survival compared to either agent alone, contrasting markedly with other tested drug combinations. Our data show the potential of nanoparticle formulation and scRNA-seq analysis of resistance to improve brain tumor treatment and identify POx-Palbo + Sapanisertib as effective combinatorial therapy for SHH medulloblastoma.
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BACKGROUND: Medulloblastoma (MB) is a heterogeneous disease in which neoplastic cells and associated immune cells contribute to disease progression. We aimed to determine the influence of neoplastic and immune cell diversity on MB biology in patient samples and animal models. METHODS: To better characterize cellular heterogeneity in MB we used single-cell RNA sequencing, immunohistochemistry, and deconvolution of transcriptomic data to profile neoplastic and immune populations in patient samples and animal models across childhood MB subgroups. RESULTS: Neoplastic cells cluster primarily according to individual sample of origin which is influenced by chromosomal copy number variance. Harmony alignment reveals novel MB subgroup/subtype-associated subpopulations that recapitulate neurodevelopmental processes, including photoreceptor and glutamatergic neuron-like cells in molecular subgroups GP3 and GP4, and a specific nodule-associated neuronally differentiated subpopulation in the sonic hedgehog subgroup. We definitively chart the spectrum of MB immune cell infiltrates, which include subpopulations that recapitulate developmentally related neuron-pruning and antigen-presenting myeloid cells. MB cellular diversity matching human samples is mirrored in subgroup-specific mouse models of MB. CONCLUSIONS: These findings provide a clearer understanding of the diverse neoplastic and immune cell subpopulations that constitute the MB microenvironment.
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Neoplasias Cerebelosas , Meduloblastoma , Animales , Neoplasias Cerebelosas/genética , Regulación Neoplásica de la Expresión Génica , Proteínas Hedgehog/genética , Humanos , Meduloblastoma/genética , Ratones , Transcriptoma , Microambiente Tumoral/genéticaRESUMEN
Previous genetic and public health research in the Pakistani population has focused on the role of consanguinity in increasing recessive disease risk, but little is known about its recent population history or the effects of endogamy. Here, we investigate fine-scale population structure, history and consanguinity patterns using genotype chip data from 2,200 British Pakistanis. We reveal strong recent population structure driven by the biraderi social stratification system. We find that all subgroups have had low recent effective population sizes (Ne), with some showing a decrease 15â20 generations ago that has resulted in extensive identity-by-descent sharing and homozygosity, increasing the risk of recessive disorders. Our results from two orthogonal methods (one using machine learning and the other coalescent-based) suggest that the detailed reporting of parental relatedness for mothers in the cohort under-represents the true levels of consanguinity. These results demonstrate the impact of cultural practices on population structure and genomic diversity in Pakistanis, and have important implications for medical genetic studies.
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Pueblo Asiatico/genética , Consanguinidad , Genética de Población , Población Blanca/genética , Estudios de Cohortes , Demografía , Genotipo , Homocigoto , Humanos , Matrimonio , Modelos Genéticos , Pakistán , Padres , Densidad de Población , Estatus SocialRESUMEN
It is unclear why medulloblastoma patients receiving similar treatments experience different outcomes. Transcriptomic profiling identified subgroups with different prognoses, but in each subgroup, individuals remain at risk of incurable recurrence. To investigate why similar-appearing tumors produce variable outcomes, we analyzed medulloblastomas triggered in transgenic mice by a common driver mutation expressed at different points in brain development. We genetically engineered mice to express oncogenic SmoM2, starting in multipotent glio-neuronal stem cells, or committed neural progenitors. Both groups developed medulloblastomas with similar transcriptomic profiles. We compared medulloblastoma progression, radiosensitivity, and cellular heterogeneity, determined by single-cell transcriptomic analysis (scRNA-seq). Stem cell-triggered medulloblastomas progressed faster, contained more OLIG2-expressing stem-like cells, and consistently showed radioresistance. In contrast, progenitor-triggered MBs progressed slower, down-regulated stem-like cells and were curable with radiation. Progenitor-triggered medulloblastomas also contained more diverse stromal populations, with more Ccr2+ macrophages and fewer Igf1+ microglia, indicating that developmental events affected the subsequent tumor microenvironment. Reduced mTORC1 activity in M-Smo tumors suggests that differential Igf1 contributed to differences in phenotype. Developmental events in tumorigenesis that were obscure in transcriptomic profiles thus remained cryptic determinants of tumor composition and outcome. Precise understanding of medulloblastoma pathogenesis and prognosis requires supplementing transcriptomic/methylomic studies with analyses that resolve cellular heterogeneity.
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Linaje de la Célula , Neoplasias Cerebelosas/patología , Regulación del Desarrollo de la Expresión Génica/efectos de la radiación , Meduloblastoma/patología , Tolerancia a Radiación/genética , Células Madre/patología , Transcriptoma/efectos de la radiación , Animales , Neoplasias Cerebelosas/genética , Neoplasias Cerebelosas/radioterapia , Heterogeneidad Genética , Humanos , Meduloblastoma/genética , Meduloblastoma/radioterapia , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Análisis de la Célula Individual , Células Madre/efectos de la radiación , Microambiente TumoralRESUMEN
Targeting oncogenic pathways holds promise for brain tumor treatment, but inhibition of Sonic Hedgehog (SHH) signaling has failed in SHH-driven medulloblastoma. Cellular diversity within tumors and reduced lineage commitment can undermine targeted therapy by increasing the probability of treatment-resistant populations. Using single-cell RNA-seq and lineage tracing, we analyzed cellular diversity in medulloblastomas in transgenic, medulloblastoma-prone mice, and responses to the SHH-pathway inhibitor vismodegib. In untreated tumors, we find expected stromal cells and tumor-derived cells showing either a spectrum of neural progenitor-differentiation states or glial and stem cell markers. Vismodegib reduces the proliferative population and increases differentiation. However, specific cell types in vismodegib-treated tumors remain proliferative, showing either persistent SHH-pathway activation or stem cell characteristics. Our data show that even in tumors with a single pathway-activating mutation, diverse mechanisms drive tumor growth. This diversity confers early resistance to targeted inhibitor therapy, demonstrating the need to target multiple pathways simultaneously.
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Neoplasias Cerebelosas/genética , Resistencia a Antineoplásicos/genética , Proteínas Hedgehog/antagonistas & inhibidores , Meduloblastoma/genética , Transducción de Señal/genética , Anilidas/farmacología , Anilidas/uso terapéutico , Animales , Proliferación Celular/efectos de los fármacos , Proliferación Celular/genética , Neoplasias Cerebelosas/tratamiento farmacológico , Neoplasias Cerebelosas/patología , Cerebelo/citología , Cerebelo/patología , Femenino , Mutación con Ganancia de Función , Proteínas Hedgehog/genética , Humanos , Masculino , Meduloblastoma/tratamiento farmacológico , Meduloblastoma/patología , Ratones , Ratones Transgénicos , Terapia Molecular Dirigida/métodos , Proteína MioD/genética , Células Madre Neoplásicas/efectos de los fármacos , Piridinas/farmacología , Piridinas/uso terapéutico , RNA-Seq , Transducción de Señal/efectos de los fármacos , Análisis de la Célula Individual , Receptor Smoothened/genética , Factor de Transcripción HES-1/genéticaRESUMEN
Noradrenergic signaling plays a well-established role in promoting the stress response. Here we identify a subpopulation of noradrenergic neurons, defined by developmental expression of Hoxb1, that has a unique role in modulating stress-related behavior. Using an intersectional chemogenetic strategy, in combination with behavioral and physiological analyses, we show that activation of Hoxb1-noradrenergic (Hoxb1-NE) neurons decreases anxiety-like behavior and promotes an active coping strategy in response to acute stressors. In addition, we use cerebral blood volume-weighted functional magnetic resonance imaging to show that chemoactivation of Hoxb1-NE neurons results in reduced activity in stress-related brain regions, including the bed nucleus of the stria terminalis, amygdala, and locus coeruleus. Thus, the actions of Hoxb1-NE neurons are distinct from the well-documented functions of the locus coeruleus in promoting the stress response, demonstrating that the noradrenergic system contains multiple functionally distinct subpopulations.