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Background SCN1A is one of the most important epilepsy‐related genes, with pathogenic variants leading to a range of phenotypes with varying disease severity. Different modifying factors have been hypothesized to influence SCN1A‐related phenotypes. We investigate the presence of rare and more common variants in epilepsy‐related genes as potential modifiers of SCN1A‐related disease severity. Methods 87 patients with SCN1A‐related epilepsy were investigated. Whole‐exome sequencing was performed by the Beijing Genomics Institute (BGI). Functional variants in 422 genes associated with epilepsy and/or neuronal excitability were investigated. Differences in proportions of variants between the epilepsy genes and four control gene sets were calculated, and compared to the proportions of variants in the same genes in the ExAC database. Results Statistically significant excesses of variants in epilepsy genes were observed in the complete cohort and in the combined group of mildly and severely affected patients, particularly for variants with minor allele frequencies of <0.05. Patients with extreme phenotypes showed much greater excesses of epilepsy gene variants than patients with intermediate phenotypes. Conclusion Our results indicate that relatively common variants in epilepsy genes, which would not necessarily be classified as pathogenic, may play a large role in modulating SCN1A phenotypes. They may modify the phenotypes of both severely and mildly affected patients. Our results may be a first step toward meaningful testing of modifier gene variants in regular diagnostics for individual patients, to provide a better estimation of disease severity for newly diagnosed patients. Relatively common variants in epilepsy genes may play a large role in modulating SCN1A‐related phenotypes. They may modify the phenotypes of both severely and mildly affected patients.

Background Dilated cardiomyopathy (DCM) is a life-threatening heart disease and a common cause of heart failure due to systolic dysfunction and subsequent left or biventricular dilatation. A significant number of cases have a genetic etiology; however, as a complex disease, the exact genetic risk factors are largely unknown, and many patients remain without a molecular diagnosis. Methods We performed GWAS followed by whole-genome, transcriptome, and immunohistochemical analyses in a spontaneously occurring canine model of DCM. Canine gene discovery was followed up in three human DCM cohorts. Results Our results revealed two independent additive loci associated with the typical DCM phenotype comprising left ventricular systolic dysfunction and dilatation. We highlight two novel candidate genes, RNF207 and PRKAA2 , known for their involvement in cardiac action potentials, energy homeostasis, and morphology. We further illustrate the distinct genetic etiologies underlying the typical DCM phenotype and ventricular premature contractions. Finally, we followed up on the canine discoveries in human DCM patients and discovered candidate variants in our two novel genes. Conclusions Collectively, our study yields insight into the molecular pathophysiology of DCM and provides a large animal model for preclinical studies.

Inherited forms of arrhythmogenic and dilated cardiomyopathy (ACM and DCM) are characterized by variable disease expression and age-related penetrance. Calcium (Ca2+) is crucially important for proper cardiac function, and dysregulation of Ca2+ homeostasis seems to underly cardiomyopathy etiology. A polymorphism, c.286T>G p.(Ser96Ala), in the gene encoding the histidine-rich Ca2+ binding (HRC) protein, relevant for sarcoplasmic reticulum Ca2+ cycling, has previously been associated with a marked increased risk of life-threatening arrhythmias among idiopathic DCM patients. Following this finding, we investigated whether p.(Ser96Ala) affects major cardiac disease manifestations in carriers of the phospholamban (PLN) c.40_42delAGA; p.(Arg14del) pathogenic variant (cohort 1); patients diagnosed with, or predisposed to, ACM (cohort 2); and DCM patients (cohort 3). We found that the allele frequency of the p.(Ser96Ala) polymorphism was similar across the general European–American population (control cohort, 40.3–42.2%) and the different cardiomyopathy cohorts (cohorts 1–3, 40.9–43.9%). Furthermore, the p.(Ser96Ala) polymorphism was not associated with life-threatening arrhythmias or heart failure-related events across various patient cohorts. We therefore conclude that there is a lack of evidence supporting the important role of the HRC p.(Ser96Ala) polymorphism as a modifier in cardiomyopathy, refuting previous findings. Further research is required to identify bona fide genomic predictors for the stratification of cardiomyopathy patients and their risk for life-threatening outcomes.

Dutch genome diagnostic centers (GDC) use next-generation sequencing (NGS)-based diagnostic applications for the diagnosis of primary immunodeficiencies (PIDs). The interpretation of genetic variants in many PIDs is complicated because of the phenotypic and genetic heterogeneity. To analyze uniformity of variant filtering, interpretation, and reporting in NGS-based diagnostics for PID, an external quality assessment was performed. Four main Dutch GDCs participated in the quality assessment. Unannotated variant call format (VCF) files of two PID patient analyses per laboratory were distributed among the four GDCs, analyzed, and interpreted (eight analyses in total). Variants that would be reported to the clinician and/or advised for further investigation were compared between the centers. A survey measuring the experiences of clinical laboratory geneticists was part of the study. Analysis of samples with confirmed diagnoses showed that all centers reported at least the variants classified as likely pathogenic (LP) or pathogenic (P) variants in all samples, except for variants in two genes (PSTPIP1 and BTK). The absence of clinical information complicated correct classification of variants. In this external quality assessment, the final interpretation and conclusions of the genetic analyses were uniform among the four participating genetic centers. Clinical and immunological data provided by a medical specialist are required to be able to draw proper conclusions from genetic data.

The contribution of mast cells in the microenvironment of solid malignancies remains controversial. Here we functionally assess the impact of tumor-adjacent, submucosal mast cell accumulation in murine and human intestinal-type gastric cancer. We find that genetic ablation or therapeutic inactivation of mast cells suppresses accumulation of tumor-associated macrophages, reduces tumor cell proliferation and angiogenesis, and diminishes tumor burden. Mast cells are activated by interleukin (IL)-33, an alarmin produced by the tumor epithelium in response to the inflammatory cytokine IL-11, which is required for the growth of gastric cancers in mice. Accordingly, ablation of the cognate IL-33 receptor St2 limits tumor growth, and reduces mast cell-dependent production and release of the macrophage-attracting factors Csf2, Ccl3, and Il6. Conversely, genetic or therapeutic macrophage depletion reduces tumor burden without affecting mast cell abundance. Therefore, tumor-derived IL-33 sustains a mast cell and macrophage-dependent signaling cascade that is amenable for the treatment of gastric cancer. Mast cells within the tumor microenvironment have controversial roles. Here, the authors show, using genetic mouse models, that in gastric cancer, mast cells at the periphery of the tumors are activated via cancer cell produced-IL33 and promote tumorigenesis by recruiting macrophages within the tumors.

Recent human decedent model studies 1 , 2 and compassionate xenograft use 3 have explored the promise of porcine organs for human transplantation. To proceed to human studies, a clinically ready porcine donor must be engineered and its xenograft successfully tested in nonhuman primates. Here we describe the design, creation and long-term life-supporting function of kidney grafts from a genetically engineered porcine donor transplanted into a cynomolgus monkey model. The porcine donor was engineered to carry 69 genomic edits, eliminating glycan antigens, overexpressing human transgenes and inactivating porcine endogenous retroviruses. In vitro functional analyses showed that the edited kidney endothelial cells modulated inflammation to an extent that was indistinguishable from that of human endothelial cells, suggesting that these edited cells acquired a high level of human immune compatibility. When transplanted into cynomolgus monkeys, the kidneys with three glycan antigen knockouts alone experienced poor graft survival, whereas those with glycan antigen knockouts and human transgene expression demonstrated significantly longer survival time, suggesting the benefit of human transgene expression in vivo. These results show that preclinical studies of renal xenotransplantation could be successfully conducted in nonhuman primates and bring us closer to clinical trials of genetically engineered porcine renal grafts. Using kidneys from a genetically engineered porcine donor transplanted into a cynomolgus monkey model, the design, creation and long-term function of kidney grafts supporting life are explored.

Ample evidence indicates that the gut microbiome is a tumor-extrinsic factor associated with antitumor response to anti-programmed cell death protein-1 (PD-1) therapy, but inconsistencies exist between published microbial signatures associated with clinical outcomes. To resolve this, we evaluated a new melanoma cohort, along with four published datasets. Time-to-event analysis showed that baseline microbiota composition was optimally associated with clinical outcome at approximately 1 year after initiation of treatment. Meta-analysis and other bioinformatic analyses of the combined data show that bacteria associated with favorable response are confined within the Actinobacteria phylum and the Lachnospiraceae/Ruminococcaceae families of Firmicutes. Conversely, Gram-negative bacteria were associated with an inflammatory host intestinal gene signature, increased blood neutrophil-to-lymphocyte ratio, and unfavorable outcome. Two microbial signatures, enriched for Lachnospiraceae spp. and Streptococcaceae spp., were associated with favorable and unfavorable clinical response, respectively, and with distinct immune-related adverse effects. Despite between-cohort heterogeneity, optimized all-minus-one supervised learning algorithms trained on batch-corrected microbiome data consistently predicted outcomes to programmed cell death protein-1 therapy in all cohorts. Gut microbial communities (microbiotypes) with nonuniform geographical distribution were associated with favorable and unfavorable outcomes, contributing to discrepancies between cohorts. Our findings shed new light on the complex interaction between the gut microbiome and response to cancer immunotherapy, providing a roadmap for future studies. Integrated analysis of microbiome and host cell transcriptional data on clinically annotated cohorts of patients with melanoma who were treated with anti-programmed cell death protein-1, uncovers new associations of streptococcus species with immune-related adverse effects and finds consistent microbiome associations with clinical outcomes.

The development of selective ubiquitin-specific protease-7 (USP7) inhibitors GNE-6640 and GNE-6776, which induce tumour cell death and reveal differential kinetics of Lys-48 and Lys-63-linked ubiquitin chain depolymerization by USP7. Interfering inhibitors show toxicity to tumours Deubiquitinating enzymes remove the small modifier protein ubiquitin from target substrates regulating their stability. One such enzyme, USP7, is a potential target for anti-cancer therapy, as its inhibition would result in the degradation of the ubiquitinated oncoprotein MDM2, leading to reactivation of the tumour suppressor protein p53. However, selective inhibitors of USP7 have remained elusive. Here, Ingrid Wertz and team develop two USP7 inhibitors, providing structural insights into the mode of action of these compounds and demonstrating their toxicity towards tumour cells. Elsewhere in this issue, David Komander and colleagues independently report the identification of two small molecules that inhibit USP7 with high affinity and specificity both in vitro and within cells, also demonstrating their ability to inhibit tumour growth. The ubiquitin system regulates essential cellular processes in eukaryotes. Ubiquitin is ligated to substrate proteins as monomers or chains and the topology of ubiquitin modifications regulates substrate interactions with specific proteins. Thus ubiquitination directs a variety of substrate fates including proteasomal degradation 1 . Deubiquitinase enzymes cleave ubiquitin from substrates and are implicated in disease 2 ; for example, ubiquitin-specific protease-7 (USP7) regulates stability of the p53 tumour suppressor and other proteins critical for tumour cell survival 3 . However, developing selective deubiquitinase inhibitors has been challenging 4 and no co-crystal structures have been solved with small-molecule inhibitors. Here, using nuclear magnetic resonance-based screening and structure-based design, we describe the development of selective USP7 inhibitors GNE-6640 and GNE-6776. These compounds induce tumour cell death and enhance cytotoxicity with chemotherapeutic agents and targeted compounds, including PIM kinase inhibitors. Structural studies reveal that GNE-6640 and GNE-6776 non-covalently target USP7 12 Å distant from the catalytic cysteine. The compounds attenuate ubiquitin binding and thus inhibit USP7 deubiquitinase activity. GNE-6640 and GNE-6776 interact with acidic residues that mediate hydrogen-bond interactions with the ubiquitin Lys48 side chain 5 , suggesting that USP7 preferentially interacts with and cleaves ubiquitin moieties that have free Lys48 side chains. We investigated this idea by engineering di-ubiquitin chains containing differential proximal and distal isotopic labels and measuring USP7 binding by nuclear magnetic resonance. This preferential binding protracted the depolymerization kinetics of Lys48-linked ubiquitin chains relative to Lys63-linked chains. In summary, engineering compounds that inhibit USP7 activity by attenuating ubiquitin binding suggests opportunities for developing other deubiquitinase inhibitors and may be a strategy more broadly applicable to inhibiting proteins that require ubiquitin binding for full functional activity.

Bacteria of clinical importance, such as Pseudomonas aeruginosa , can become hypermutators upon loss of DNA mismatch repair (MMR) and are clinically correlated with high rates of multidrug resistance (MDR). Here, we demonstrate that hypermutated MMR-deficient P. aeruginosa has a unique mutational signature and rapidly acquires MDR upon repeated exposure to first-line or last-resort antibiotics. MDR acquisition was irrespective of drug class and instead arose through common resistance mechanisms shared between the initial and secondary drugs. Rational combinations of drugs having distinct resistance mechanisms prevented MDR acquisition in hypermutated MMR-deficient P. aeruginosa . Mutational signature analysis of P. aeruginosa across different human disease contexts identified appreciable quantities of MMR-deficient clinical isolates that were already MDR or prone to future MDR acquisition. Mutational signature analysis of patient samples is a promising diagnostic tool that may predict MDR and guide precision-based medical care. Mutational signature analysis of Pseudomonas aeruginosa identifies hypermutator clinical strains and prognostically predicts multidrug resistance acquisiton, potentially serving as a tool to guide effective personalized treatment.

The biodiversity-productivity relationship (BPR) is foundational to our understanding of the global extinction crisis and its impacts on ecosystem functioning. Understanding BPR is critical for the accurate valuation and effective conservation of biodiversity. Using ground-sourced data from 777,126 permanent plots, spanning 44 countries and most terrestrial biomes, we reveal a globally consistent positive concave-down BPR, showing that continued biodiversity loss would result in an accelerating decline in forest productivity worldwide. The value of biodiversity in maintaining commercial forest productivity alone-US$166 billion to 490 billion per year according to our estimation-is more than twice what it would cost to implement effective global conservation. This highlights the need for a worldwide reassessment of biodiversity values, forest management strategies, and conservation priorities.