De Novo Variants in CNOT1, a Central Component of the CCR4-NOT Complex Involved in Gene Expression and RNA and Protein Stability, Cause Neurodevelopmental Delay.

CNOT1 is a member of the CCR4-NOT complex, which is a master regulator, orchestrating gene expression, RNA deadenylation, and protein ubiquitination. We report on 39 individuals with heterozygous de novo CNOT1 variants, including missense, splice site, and nonsense variants, who present with a clinical spectrum of intellectual disability, motor delay, speech delay, seizures, hypotonia, and behavioral problems. To link CNOT1 dysfunction to the neurodevelopmental phenotype observed, we generated variant-specific Drosophila models, which showed learning and memory defects upon CNOT1 knockdown. Introduction of human wild-type CNOT1 was able to rescue this phenotype, whereas mutants could not or only partially, supporting our hypothesis that CNOT1 impairment results in neurodevelopmental delay. Furthermore, the genetic interaction with autism-spectrum genes, such as ASH1L, DYRK1A, MED13, and SHANK3, was impaired in our Drosophila models. Molecular characterization of CNOT1 variants revealed normal CNOT1 expression levels, with both mutant and wild-type alleles expressed at similar levels. Analysis of protein-protein interactions with other members indicated that the CCR4-NOT complex remained intact. An integrated omics approach of patient-derived genomics and transcriptomics data suggested only minimal effects on endonucleolytic nonsense-mediated mRNA decay components, suggesting that de novo CNOT1 variants are likely haploinsufficient hypomorph or neomorph, rather than dominant negative. In summary, we provide strong evidence that de novo CNOT1 variants cause neurodevelopmental delay with a wide range of additional co-morbidities. Whereas the underlying pathophysiological mechanism warrants further analysis, our data demonstrate an essential and central role of the CCR4-NOT complex in human brain development.

Dynamic hydrogen peroxide levels reveal a rate-dependent sensitivity in B-cell lymphoma signaling.

Although in vivo extracellular microenvironments are dynamic, most in vitro studies are conducted under static conditions. Here, we exposed diffuse large B-cell lymphoma (DLBCL) cells to gradient increases in the concentration of hydrogen peroxide (H2O2), thereby capturing some of the dynamics of the tumour microenvironment. Subsequently, we measured the phosphorylation response of B-cell receptor (BCR) signalling proteins CD79a, SYK and PLCγ2 at a high temporal resolution via single-cell phospho-specific flow cytometry. We demonstrated that the cells respond bimodally to static extracellular H2O2, where the percentage of cells that respond is mainly determined by the concentration. Computational analysis revealed that the bimodality results from a combination of a steep dose-response relationship and cell-to-cell variability in the response threshold. Dynamic gradient inputs of varying durations indicated that the H2O2 concentration is not the only determinant of the signalling response, as cells exposed to more shallow gradients respond at lower H2O2 levels. A minimal model of the proximal BCR network qualitatively reproduced the experimental findings and uncovered a rate-dependent sensitivity to H2O2, where a lower rate of increase correlates to a higher sensitivity. These findings will bring us closer to understanding how cells process information from their complex and dynamic in vivo environments.