Evaluating the pathogenicity of a variant is challenging given the plethora of types of genetic evidence that laboratories consider. Deciding how to weigh each type of evidence is difficult, and ...standards have been needed. In 2015, the American College of Medical Genetics and Genomics (ACMG) and the Association for Molecular Pathology (AMP) published guidelines for the assessment of variants in genes associated with Mendelian diseases. Nine molecular diagnostic laboratories involved in the Clinical Sequencing Exploratory Research (CSER) consortium piloted these guidelines on 99 variants spanning all categories (pathogenic, likely pathogenic, uncertain significance, likely benign, and benign). Nine variants were distributed to all laboratories, and the remaining 90 were evaluated by three laboratories. The laboratories classified each variant by using both the laboratory’s own method and the ACMG-AMP criteria. The agreement between the two methods used within laboratories was high (K-alpha = 0.91) with 79% concordance. However, there was only 34% concordance for either classification system across laboratories. After consensus discussions and detailed review of the ACMG-AMP criteria, concordance increased to 71%. Causes of initial discordance in ACMG-AMP classifications were identified, and recommendations on clarification and increased specification of the ACMG-AMP criteria were made. In summary, although an initial pilot of the ACMG-AMP guidelines did not lead to increased concordance in variant interpretation, comparing variant interpretations to identify differences and having a common framework to facilitate resolution of those differences were beneficial for improving agreement, allowing iterative movement toward increased reporting consistency for variants in genes associated with monogenic disease.
Human glioblastomas harbour a subpopulation of glioblastoma stem cells that drive tumorigenesis. However, the origin of intratumoural functional heterogeneity between glioblastoma cells remains ...poorly understood. Here we study the clonal evolution of barcoded glioblastoma cells in an unbiased way following serial xenotransplantation to define their individual fate behaviours. Independent of an evolving mutational signature, we show that the growth of glioblastoma clones in vivo is consistent with a remarkably neutral process involving a conserved proliferative hierarchy rooted in glioblastoma stem cells. In this model, slow-cycling stem-like cells give rise to a more rapidly cycling progenitor population with extensive self-maintenance capacity, which in turn generates non-proliferative cells. We also identify rare 'outlier' clones that deviate from these dynamics, and further show that chemotherapy facilitates the expansion of pre-existing drug-resistant glioblastoma stem cells. Finally, we show that functionally distinct glioblastoma stem cells can be separately targeted using epigenetic compounds, suggesting new avenues for glioblastoma-targeted therapy.
Molecular programs that underlie precursor progression in multiple myeloma are incompletely understood. Here, we report a disease spectrum-spanning, single-cell analysis of the Vκ*MYC myeloma mouse ...model. Using samples obtained from mice with serologically undetectable disease, we identify malignant cells as early as 30 weeks of age and show that these tumours contain subclonal copy number variations that persist throughout progression. We detect intratumoural heterogeneity driven by transcriptional variability during active disease and show that subclonal expression programs are enriched at different times throughout early disease. We then show how one subclonal program related to GCN2 stress response is progressively activated during progression in myeloma patients. Finally, we use chemical and genetic perturbation of GCN2 in vitro to support this pathway as a therapeutic target in myeloma. These findings therefore present a model of precursor progression in Vκ*MYC mice, nominate an adaptive mechanism important for myeloma survival, and highlight the need for single-cell analyses to understand the biological underpinnings of disease progression.
Human cerebral cancers are known to contain cell types resembling the varying stages of neural development. However, the basis of this association remains unclear. Here, we map the development of ...mouse cerebrum across the developmental time-course, from embryonic day 12.5 to postnatal day 365, performing single-cell transcriptomics on >100,000 cells. By comparing this reference atlas to single-cell data from >100 glial tumours of the adult and paediatric human cerebrum, we find that tumour cells have an expression signature that overlaps with temporally restricted, embryonic radial glial precursors (RGPs) and their immediate sublineages. Further, we demonstrate that prenatal transformation of RGPs in a genetic mouse model gives rise to adult cerebral tumours that show an embryonic/juvenile RGP identity. Together, these findings implicate the acquisition of embryonic-like states in the genesis of adult glioma, providing insight into the origins of human glioma, and identifying specific developmental cell types for therapeutic targeting.
In a field experiment, annual nitrous oxide (N2O) emissions and grassland yield were measured across different plant communities, comprising systematically varying combinations of monocultures and ...mixtures of three functional groups (FG): grasses (Lolium perenne, Phleum pratense), legumes (Trifolium pratense, Trifolium repens) and herbs (Cichorium intybus, Plantago lanceolata). Plots received 150 kg ha−1 year−1 nitrogen (N) (150 N), except L. perenne monocultures which received two N levels: 150 N and 300 N. The effect of plant diversity on N2O emissions was derived from linear combinations of species performances' in monoculture (species identity) and not from strong interactions between species in mixtures. Increasing from 150 N to 300 N in L. perenne resulted in a highly significant increase in cumulative N2O emissions from 1.39 to 3.18 kg N2O-N ha−1 year−1. Higher N2O emissions were also associated with the legume FG. Emissions intensities (yield-scaled N2O emissions) from multi-species mixture communities around the equi-proportional mixture were lowered due to interactions among species. For N2O emissions scaled by nitrogen yield in forage, the 6-species mixture was significantly lower than L. perenne at both 300 N and 150 N. In comparison to 300 N L. perenne, the same N yield or DM yield could have been produced with the equi-proportional 6-species mixture (150 N) while reducing N2O losses by 63% and 58% respectively. Compared to 150 N L. perenne, the same N yield or DM yield could have been produced with the 6-species mixture while reducing N2O losses by 41% and 24% respectively. Overall, this study found that multi-species grasslands can potentially reduce both N2O emissions and emissions intensities, contributing to the sustainability of grassland production.
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•N2O emissions were driven by strong plant identity effects with no interspecific interactions.•The N yield-scaled N2O emissions of the 6-species mixture were 41% lower than the L. perenne monoculture (both at 150 kg ha−1 year−1 of nitrogen fertiliser).•The DM yield-scaled N2O emissions of the 6-species mixture were 24% lower than the L. perenne monoculture (both at 150 kg ha−1 year−1 of nitrogen fertiliser).
Cancer cells can metabolize glutamine to replenish TCA cycle intermediates, leading to a dependence on glutaminolysis for cell survival. However, a mechanistic understanding of the role that ...glutamine metabolism has on the survival of glioblastoma (GBM) brain tumor stem cells (BTSC) has not yet been elucidated. Here, we report that across a panel of 19 GBM BTSC lines, inhibition of glutaminase (GLS) showed a variable response from complete blockade of cell growth to absolute resistance. Surprisingly, BTSC sensitivity to GLS inhibition was a result of reduced intracellular glutamate triggering the amino acid deprivation response (AADR) and not due to the contribution of glutaminolysis to the TCA cycle. Moreover, BTSC sensitivity to GLS inhibition negatively correlated with expression of the astrocytic glutamate transporters EAAT1 and EAAT2. Blocking glutamate transport in BTSCs with high EAAT1/EAAT2 expression rendered cells susceptible to GLS inhibition, triggering the AADR and limiting cell growth. These findings uncover a unique metabolic vulnerability in BTSCs and support the therapeutic targeting of upstream activators and downstream effectors of the AADR pathway in GBM. Moreover, they demonstrate that gene expression patterns reflecting the cellular hierarchy of the tissue of origin can alter the metabolic requirements of the cancer stem cell population. SIGNIFICANCE: Glioblastoma brain tumor stem cells with low astrocytic glutamate transporter expression are dependent on GLS to maintain intracellular glutamate to prevent the amino acid deprivation response and cell death.
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