RESUMEN
Bryant-Li-Bhoj syndrome (BLBS), which became OMIM-classified in 2022 (OMIM: 619720, 619721), is caused by germline variants in the two genes that encode histone H3.3 (H3-3A/H3F3A and H3-3B/H3F3B) [1-4]. This syndrome is characterized by developmental delay/intellectual disability, craniofacial anomalies, hyper/hypotonia, and abnormal neuroimaging [1, 5]. BLBS was initially categorized as a progressive neurodegenerative syndrome caused by de novo heterozygous variants in either H3-3A or H3-3B [1-4]. Here, we analyze the data of the 58 previously published individuals along 38 unpublished, unrelated individuals. In this larger cohort of 96 people, we identify causative missense, synonymous, and stop-loss variants. We also expand upon the phenotypic characterization by elaborating on the neurodevelopmental component of BLBS. Notably, phenotypic heterogeneity was present even amongst individuals harboring the same variant. To explore the complex phenotypic variation in this expanded cohort, the relationships between syndromic phenotypes with three variables of interest were interrogated: sex, gene containing the causative variant, and variant location in the H3.3 protein. While specific genotype-phenotype correlations have not been conclusively delineated, the results presented here suggest that the location of the variants within the H3.3 protein and the affected gene (H3-3A or H3-3B) contribute more to the severity of distinct phenotypes than sex. Since these variables do not account for all BLBS phenotypic variability, these findings suggest that additional factors may play a role in modifying the phenotypes of affected individuals. Histones are poised at the interface of genetics and epigenetics, highlighting the potential role for gene-environment interactions and the importance of future research.
Asunto(s)
Histonas , Fenotipo , Humanos , Masculino , Femenino , Histonas/genética , Niño , Enfermedades Neurodegenerativas/genética , Enfermedades Neurodegenerativas/patología , Trastornos del Neurodesarrollo/genética , Trastornos del Neurodesarrollo/patología , Preescolar , Adolescente , Adulto , Discapacidad Intelectual/genética , Discapacidad Intelectual/patologíaRESUMEN
Whole exome and genome sequencing, coupled with refined bioinformatic pipelines, have enabled improved diagnostic yields for individuals with Mendelian conditions and have led to the rapid identification of novel syndromes. For many Mendelian neurodevelopmental disorders (NDDs), there is a lack of pre-existing model systems for mechanistic work. Thus, it is critical for translational researchers to have an accessible phenotype- and genotype-informed approach for model system selection. Single-cell RNA sequencing data can be informative in such an approach, as it can indicate which cell types express a gene of interest at the highest levels across time. For Mendelian NDDs, such data for the developing human brain is especially useful. A valuable single-cell RNA sequencing dataset of the second trimester developing human brain was produced by Bhaduri et al in 2021, but access to these data can be limited by computing power and the learning curve of single-cell data analysis. To reduce these barriers for translational research on Mendelian NDDs, we have built the web-based tool, Neurodevelopment in Trimester 2 - VIsualization of Single cell Data Online Tool (NeuroTri2-VISDOT), for exploring this single-cell dataset, and we have employed it in several different settings to demonstrate its utility for the translational research community.
RESUMEN
TBCK syndrome is an autosomal recessive disorder primarily characterized by global developmental delay, hypotonia, abnormal magnetic resonance imaging (MRI), and distinctive craniofacial phenotypes. High variability is observed among affected individuals and their corresponding variants, making clinical diagnosis challenging. Here, we discuss recent breakthroughs in clinical considerations, TBCK function, and therapeutic development.
Asunto(s)
Enfermedades Neurodegenerativas , Proteínas Serina-Treonina Quinasas , Humanos , Proteínas Serina-Treonina Quinasas/genética , Enfermedades Neurodegenerativas/diagnóstico , Enfermedades Neurodegenerativas/etiología , Hipotonía Muscular/genética , Hipotonía Muscular/patología , FenotipoRESUMEN
Spheroid culture systems have allowed in vitro propagation of cells unable to grow in canonical cell culturing conditions, and may capture cellular contexts that model tumor growth better than current model systems. The insights gleaned from genome-wide clustered regularly interspaced short palindromic repeat (CRISPR) screening of thousands of cancer cell lines grown in conventional culture conditions illustrate the value of such CRISPR pooled screens. It is clear that similar genome-wide CRISPR screens of three-dimensional spheroid cultures will be important for future biological discovery. Here, we present a protocol for genome-wide CRISPR screening of three-dimensional neurospheres. While many in-depth protocols and discussions have been published for more typical cell lines, few detailed protocols are currently available in the literature for genome-wide screening in spheroidal cell lines. For those who want to screen such cell lines, and particularly neurospheres, we provide a step-by-step description of assay development tests to be performed before screening, as well as for the screen itself. We highlight considerations of variables that make these screens distinct from, or similar to, typical nonspheroid cell lines throughout. Finally, we illustrate typical outcomes of neurosphere genome-wide screens, and how neurosphere screens typically produce slightly more heterogeneous signal distributions than more canonical cancer cell lines. Completion of this entire protocol will take 8-12 weeks from the initial assay development tests to deconvolution of the sequencing data.
Asunto(s)
Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Neoplasias , Humanos , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética , Sistemas CRISPR-Cas , Genoma , Línea CelularRESUMEN
Forkhead box R2 (FOXR2) is a forkhead transcription factor located on the X chromosome whose expression is normally restricted to the testis. In this study, we performed a pan-cancer analysis of FOXR2 activation across more than 10,000 adult and pediatric cancer samples and found FOXR2 to be aberrantly upregulated in 70% of all cancer types and 8% of all individual tumors. The majority of tumors (78%) aberrantly expressed FOXR2 through a previously undescribed epigenetic mechanism that involves hypomethylation of a novel promoter, which was functionally validated as necessary for FOXR2 expression and proliferation in FOXR2-expressing cancer cells. FOXR2 promoted tumor growth across multiple cancer lineages and co-opted ETS family transcription circuits across cancers. Taken together, this study identifies FOXR2 as a potent and ubiquitous oncogene that is epigenetically activated across the majority of human cancers. The identification of hijacking of ETS transcription circuits by FOXR2 extends the mechanisms known to active ETS transcription factors and highlights how transcription factor families cooperate to enhance tumorigenesis. SIGNIFICANCE: This work identifies a novel promoter that drives aberrant FOXR2 expression and delineates FOXR2 as a pan-cancer oncogene that specifically activates ETS transcriptional circuits across human cancers. See related commentary by Liu and Northcott, p. 2977.
Asunto(s)
Factores de Transcripción Forkhead , Neoplasias , Adulto , Carcinogénesis/genética , Proliferación Celular , Niño , Epigénesis Genética , Factores de Transcripción Forkhead/genética , Humanos , Masculino , Neoplasias/genética , Oncogenes/genética , Proteínas Proto-Oncogénicas c-ets/genética , Proteínas Proto-Oncogénicas c-ets/metabolismo , Activación TranscripcionalRESUMEN
The role of PPM1D mutations in de novo gliomagenesis has not been systematically explored. Here we analyze whole genome sequences of 170 pediatric high-grade gliomas and find that truncating mutations in PPM1D that increase the stability of its phosphatase are clonal driver events in 11% of Diffuse Midline Gliomas (DMGs) and are enriched in primary pontine tumors. Through the development of DMG mouse models, we show that PPM1D mutations potentiate gliomagenesis and that PPM1D phosphatase activity is required for in vivo oncogenesis. Finally, we apply integrative phosphoproteomic and functional genomics assays and find that oncogenic effects of PPM1D truncation converge on regulators of cell cycle, DNA damage response, and p53 pathways, revealing therapeutic vulnerabilities including MDM2 inhibition.
Asunto(s)
Glioma/genética , Mutación , Oncogenes/genética , Proteína Fosfatasa 2C/genética , Adolescente , Adulto , Animales , Neoplasias del Tronco Encefálico/genética , Carcinogénesis/genética , Ciclo Celular , Niño , Preescolar , Daño del ADN , Modelos Animales de Enfermedad , Femenino , Células HEK293 , Humanos , Lactante , Masculino , Ratones , Proteínas Proto-Oncogénicas c-mdm2 , Transcriptoma , Proteína p53 Supresora de Tumor/genética , Adulto JovenRESUMEN
BET-bromodomain inhibition (BETi) has shown pre-clinical promise for MYC-amplified medulloblastoma. However, the mechanisms for its action, and ultimately for resistance, have not been fully defined. Here, using a combination of expression profiling, genome-scale CRISPR/Cas9-mediated loss of function and ORF/cDNA driven rescue screens, and cell-based models of spontaneous resistance, we identify bHLH/homeobox transcription factors and cell-cycle regulators as key genes mediating BETi's response and resistance. Cells that acquire drug tolerance exhibit a more neuronally differentiated cell-state and expression of lineage-specific bHLH/homeobox transcription factors. However, they do not terminally differentiate, maintain expression of CCND2, and continue to cycle through S-phase. Moreover, CDK4/CDK6 inhibition delays acquisition of resistance. Therefore, our data provide insights about the mechanisms underlying BETi effects and the appearance of resistance and support the therapeutic use of combined cell-cycle inhibitors with BETi in MYC-amplified medulloblastoma.