By transforming the ACMG/AMP guidelines into a Bayesian framework, we provide a mathematical foundation for what was a qualitative heuristic. Only 2 of the 18 existing ACMG/AMP evidence combinations were mathematically inconsistent with the overall framework. Mixed combinations of pathogenic and benign evidence could yield a likely pathogenic, likely benign, or VUS result. This quantitative framework validates the approach adopted by the ACMG/AMP, provides opportunities to further refine evidence categories and combining rules, and supports efforts to automate components of variant pathogenicity assessments.

Diffuse intrinsic pontine glioma (DIPG) and other H3K27M-mutated diffuse midline gliomas (DMGs) are universally lethal paediatric tumours of the central nervous system1. We have previously shown that the disialoganglioside GD2 is highly expressed on H3K27M-mutated glioma cells and have demonstrated promising preclinical efficacy of GD2-directed chimeric antigen receptor (CAR) T cells2, providing the rationale for a first-in-human phase I clinical trial (NCT04196413). Because CAR T cell-induced brainstem inflammation can result in obstructive hydrocephalus, increased intracranial pressure and dangerous tissue shifts, neurocritical care precautions were incorporated. Here we present the clinical experience from the first four patients with H3K27M-mutated DIPG or spinal cord DMG treated with GD2-CAR T cells at dose level 1 (1 × 106 GD2-CAR T cells per kg administered intravenously). Patients who exhibited clinical benefit were eligible for subsequent GD2-CAR T cell infusions administered intracerebroventricularly3. Toxicity was largely related to the location of the tumour and was reversible with intensive supportive care. On-target, off-tumour toxicity was not observed. Three of four patients exhibited clinical and radiographic improvement. Pro-inflammatory cytokine levels were increased in the plasma and cerebrospinal fluid. Transcriptomic analyses of 65,598 single cells from CAR T cell products and cerebrospinal fluid elucidate heterogeneity in response between participants and administration routes. These early results underscore the promise of this therapeutic approach for patients with H3K27M-mutated DIPG or spinal cord DMG.

T cell exhaustion limits immune responses against cancer and is a major cause of resistance to chimeric antigen receptor (CAR)–T cell therapeutics. Using murine xenograft models and an in vitro model wherein tonic CAR signaling induces hallmark features of exhaustion, we tested the effect of transient cessation of receptor signaling, or rest, on the development and maintenance of exhaustion. Induction of rest through enforced down-regulation of the CAR protein using a drug-regulatable system or treatment with the multikinase inhibitor dasatinib resulted in the acquisition of a memory-like phenotype, global transcriptional and epigenetic reprogramming, and restored antitumor functionality in exhausted CAR-T cells. This work demonstrates that rest can enhance CAR-T cell efficacy by preventing or reversing exhaustion, and it challenges the notion that exhaustion is an epigenetically fixed state.

Identification of the molecular lesion in Caenorhabditis elegans mutants isolated through forward genetic screens usually involves time-consuming genetic mapping. We used Illumina deep sequencing technology to sequence a complete, mutant C. elegans genome and thus pinpointed a single-nucleotide mutation in the genome that affects a neuronal cell fate decision. This constitutes a proof-of-principle for using whole-genome sequencing to analyze C. elegans mutants.C. elegans is used extensively to identify genes involved in various aspects of animal development, behavior and physiology 1 (http://www.wormbook.org/). The traditional forward genetic approach involves random mutagenesis and subsequent isolation of mutants defective in a given process 1 . The ensuing characterization of the molecular lesion in a mutant strain is a painstaking process that involves mapping with genetic and/or single-nucleotide polymorphism (SNP) markers. The relative gene density in C. elegans and limited recombinant frequencies can make traditional mapping a very time-consuming process. This issue becomes even more apparent when the scoring of the mutant phenotype is cumbersome and recombinants are therefore tedious to identify. Another problem with traditional mapping approaches is that genetic-background effects on a given phenotype prohibit the use of many genetic markers and mapping strains.We considered whole-genome sequencing as an approach to identify the molecular lesion in a specific ethyl methanesulfonate (EMS)-induced mutant C. elegans strain. We had previously described a genetic locus, lsy-12, in which a neuronal fate decision is aberrantly executed 2 . Instead of generating two left/right asymmetric, distinct chemosensory neurons, ASEL and ASER, lsy-12 mutants generate two ASER neurons 2 . To determine the molecular identity of lsy-12, we undertook a single mapping cross of the recessive lsy-12 allele ot177 (lsy-12 (ot177)) with a Hawaiian mapping strain 3 , analyzed 200 F 2 progeny for SNPs by PCR and thereby mapped lsy-12(ot177) to a 4-Mb interval on chromosome V. This interval represents 4% of the genome and contains 1,142 predicted genes (5% of total genes).We prepared genomic DNA from lsy-12(ot177) worms and sequenced the DNA using pairedend Illumina (formerly Solexa) sequencing technology 4 . We generated 4. mer sequence reads in a 1-week sequencing run. Then we mapped the sequence data to the wild-type N2 reference genome using ELAND (efficient large-scale alignment of nucleotide databases) and the Maq alignment tools (Supplementary Methods online); 3.1 Gb of reads were mapped exactly onto the genome with an average coverage of ∼28×. To label differences between our sequence data and the N2 reference genome as 'variants', we filtered for those reads that mapped uniquely with high-quality scores on both strands and were read at least ten times, thus eliminating the vast majority of ambiguous calls (Supplementary Methods). The filtering left 80 variants between lsy-12(ot177) genomic DNA and the published N2 wil...

Summary Divergent adaptation can be associated with reproductive isolation in the process of speciation [1]. We recently demonstrated the link between divergent adaptation and the onset of reproductive isolation in experimental populations of the yeast Saccharomyces cerevisiae evolved from a single progenitor in either a high-salt or a low-glucose environment [2]. Here, we used whole-genome re-sequencing of representatives of three populations to identify 17 candidate mutations, six of which explained the adaptive increases in mitotic fitness in the two environments. In two populations evolved in high salt, two different mutations occurred in the proton efflux pump gene PMA1 and the global transcriptional repressor gene CYC8; the ENA genes encoding sodium efflux pumps were over-expressed once through expansion of this gene cluster and once due to mutation in the regulator CYC8. In the population from low glucose, one mutation occurred in MDS3, which modulates growth at high pH, and one in MKT1, a global regulator of mRNAs encoding mitochondrial proteins, the latter recapitulating a naturally-occurring variant. A Dobzhansky-Muller (DM) incompatibility between the evolved alleles of PMA1 and MKT1 strongly depressed fitness in the low-glucose environment. This DM interaction is the first reported between experimentally evolved alleles of known genes and shows how reproductive isolation can arise rapidly when divergent selection is strong.