Biology subjects

Robson, M.

Publications and source records attributed to Robson, M..

11 recordsLinked to original sources

bioRxiv2023

Saturation genome editing-based functional evaluation and clinical classification of BRCA2 single nucleotide variants

Germline BRCA2 loss-of function (LOF) variants identified by clinical genetic testing predispose to breast, ovarian, prostate and pancreatic cancer. However, variants of uncertain significance (VUS) (n>4000) limit the clinical use of testing results. Thus, there is an urgent need for functional characterization and clinical classification of all BRCA2 variants. Here we report on comprehensive saturation genome editing-based functional characterization of 97% of all possible single nucleotide variants (SNVs) in the BRCA2 DNA Binding Domain hotspot for pathogenic missense variants that is encoded by exons 15 to 26. The assay was based on deep sequence analysis of surviving endogenously targeted haploid cells. A total of 7013 SNVs were characterized as functionally abnormal (n=955), intermediate/uncertain, or functionally normal (n=5224) based on 95% agreement with ClinVar known pathogenic and benign standards. Results were validated relative to batches of nonsense and synonymous variants and variants evaluated using a homology directed repair (HDR) functional assay. Breast cancer case-control association studies showed that pooled SNVs encoding functionally abnormal missense variants were associated with increased risk of breast cancer (odds ratio (OR) 3.89, 95%CI: 2.77-5.51). In addition, 86% of tumors associated with abnormal missense SNVs displayed loss of heterozygosity (LOH), whereas 26% of tumors with normal variants had LOH. The functional data were added to other sources of information in a ClinGen/ACMG/AMP-like model and 700 functionally abnormal SNVs, including 220 missense SNVs, were classified as pathogenic or likely pathogenic, while 4862 functionally normal SNVs, including 3084 missense SNVs, were classified as benign or likely benign. These classified variants can now be used for risk assessment and clinical care of variant carriers and the remaining functional scores can be used directly for clinical classification and interpretation of many additional variants. SummaryGermline BRCA2 loss-of function (LOF) variants identified by clinical genetic testing predispose to several types of cancer. However, variants of uncertain significance (VUS) limit the clinical use of testing results. Thus, there is an urgent need for functional characterization and clinical classification of all BRCA2 variants to facilitate current and future clinical management of individuals with these variants. Here we show the results from a saturation genome editing (SGE) and functional analysis of all possible single nucleotide variants (SNVs) from exons 15 to 26 that encode the BRCA2 DNA Binding Domain hotspot for pathogenic missense variants. The assay was based on deep sequence analysis of surviving endogenously targeted human haploid HAP1 cells. The assay was calibrated relative to ClinVar known pathogenic and benign missense standards and 95% prevalence thresholds for functionally abnormal and normal variants were identified. Thresholds were validated based on nonsense and synonymous variants. SNVs encoding functionally abnormal missense variants were associated with increased risks of breast and ovarian cancer. The functional assay results were integrated into a ClinGen/ACMG/AMP-like model for clinical classification of the majority of BRCA2 SNVs as pathogenic/likely pathogenic or benign/likely benign. The classified variants can be used for improved clinical management of variant carriers.

genomics↗

bioRxiv2023

Model matters: Differential outcomes in traumatic optic neuropathy pathophysiology between blunt and blast-wave mediated head injuries.

Over 3 million people in the United States live with long-term disability as a result of a traumatic brain injury (TBI). The purpose of this study was to characterize and compare two different animal models of TBI (blunt head trauma and blast TBI) to determine common and divergent characteristics of these models. With recent literature reviews noting the prevalence of visual system injury in animal models of TBI, coupled with clinical estimates of 50-75% of all TBI cases, we decided to assess commonalities, if they existed, through visual system injury. Blast, repeat blast, and blunt injury were induced in adult male mice to observe and quantify visual deficits. Retinal ganglion cell loss and axonal degeneration in the optic tract, superior colliculus, and lateral geniculate nuclei were examined to trace injury outcomes throughout major vision-associated areas. Optokinetic response, immunohistochemistry, and western blots were analyzed. Where a single blunt injury produces significant visual deficits a single blast injury appears to have less severe visual consequences. Visual deficits after repeat blasts are similar to a single blast. Single blast injury induces contralateral damage to right optic chiasm and tract whereas bilateral injury follows a single blunt injury. Repeat blast injuries are required to see degeneration patterns in downstream regions similar to the damage seen in a single blunt injury. This finding is further supported by Amyloid Precursor Protein (APP) staining in injured cohorts. Blunt injured groups present with staining 1.2 mm of the optic nerve, indicating axonal breakage closer to the optic chiasm. In blast groups, APP was identifiable in a bilateral pattern only in the geniculate nucleus. Evidence for unilateral neuronal degeneration in brain tissue with bilateral axonal ruptures are pivotal discoveries in this model differentiation. Analysis of the two injury models suggest there is a significant difference in the histological outcomes dependent on injury type, though visual system injury is likely present in more cases than are currently diagnosed clinically.

neuroscience↗

bioRxiv2023

Exploration of T-cell immune responses by expression of a dominant-negative SHP1 and SHP2

SHP1 and SHP2 are SH2 domain-containing proteins which have inhibitory phosphatase activity when recruited to phosphorylated ITIMs and ITSMs on inhibitory immune receptors. Consequently, SHP1 and SHP2 are key proteins in the transmission of inhibitory signals within T cells, constituting an important point of convergence for diverse inhibitory receptors. Therefore, SHP1 and SHP2 inhibition may represent a strategy for preventing immunosuppression of T cells mediated by cancers hence improving immunotherapies directed against these malignancies. Both SHP1 and SHP2 contain dual SH2 domains responsible for localization to the endodomain of inhibitory receptors and a protein tyrosine phosphatase domain which dephosphorylates and thus inhibits key mediators of T cell activation. We explored the interaction of the isolated SH2 domains of SHP1 and SHP2 to inhibitory motifs from PD1 and identified strong binding of both SH2 domains from SHP2 and more moderate binding in the case of SHP1. We next explored whether these a truncated form of SHP1/2 comprising only of SH2 domains (dSHP1/2) could act in a dominant negative fashion by preventing docking of the wild type proteins. When co-expressed with CARs we found that dSHP2 but not dSHP1 could alleviate immunosuppression mediated by PD1. We next explored the capacity of dSHP2 to bind with other inhibitory receptors and observed several potential interactions. In vivo we observed that the expression of PDL1 on tumor cells impaired the ability of CAR T cells to mediate tumor rejection and this effect was partially reversed by the co-expression of dSHP2 albeit at the cost of reduced CAR T cell proliferation. Modulation of SHP1 and SHP2 activity in engineered T cells through the expression of these truncated variants may enhance T cell activity and hence efficacy in the context of cancer immunotherapy.

immunology↗

bioRxiv2023

Enhancing CAR T cell therapy using Fab-Based Constitutively Heterodimeric Cytokine Receptors

Adoptive T cell therapy aims to achieve lasting tumour clearance, requiring enhanced engraftment and survival of the immune cells. Cytokines are paramount modulators of T cell survival and proliferation. Cytokine receptors signal via ligand-induced dimerization, and this principle has been hijacked utilising non-native dimerization domains. A major limitation of current technologies resides in the absence of a module that recapitulates the natural cytokine receptor heterodimeric pairing. To circumvent this, we created a new engineered cytokine receptor able to constitutively recreate receptor-heterodimer utilising the heterodimerization domain derived from the IgG1 antibody (dFab_CCR). We found that the signal delivered by the dFab_CCR-IL2 proficiently mimics the cytokine receptor heterodimerization, with transcriptomic signatures similar to that obtained by the activation of IL2 native receptor. Importantly, we found that this dimerization structure is agnostic, efficiently activating signaling through four cytokine receptor families. Using a combination of in vivo and in vitro screening approaches, we characterized a library of 18 dFab_CCRs co-expressed with a clinically relevant solid tumor-specific GD2 CAR. Based on this characterization we suggest that the co-expression of either the common {beta}-chain GMCSF or the IL18 dFab_CCRs is optimal to improve CAR T cell expansion, engraftment, and efficacy. Our results demonstrate how the Fab dimerization is efficient and versatile in recapitulating a cytokine receptor heterodimerization signal. This module could be applied for the enhancement of adoptive T cell therapies, and therapies based on other immune cell types. Furthermore, these results provide a choice of cytokine signal to incorporate with adoptive T cells therapies.

immunology↗

bioRxiv2023

Novel Fas-TNFR chimeras that prevents Fas ligand-mediated kill and signals synergistically to enhance CAR T-cell efficacy

The hostile tumour microenvironment limits the efficacy of adoptive cell therapies. Activation of the Fas death receptor initiates apoptosis and disrupting these receptors could be key to increase CAR T-cell efficacy. We screened a library of Fas-TNFR proteins identifying several novel chimeras that not only prevented Fas ligand-mediated kill, but also enhanced CAR T-cell efficacy by signalling synergistically with the CAR. Upon binding Fas ligand, Fas-CD40 activated the NF-{kappa}B pathway, inducing greatest proliferation and IFN{gamma} release out of all Fas-TNFRs tested. Fas-CD40 induced profound transcriptional modifications, particularly genes relating to the cell cycle, metabolism, and chemokine signalling. Co-expression of Fas-CD40 with either 4-1BB- or CD28-containing CARs increased in vitro efficacy by eliciting maximal CAR T-cell proliferation and cancer target cytotoxicity, and enhanced tumour killing and overall mouse survival in vivo. Functional activity of the Fas-TNFRs were dependent on the co-stimulatory domain within the CAR, highlighting crosstalk between signalling pathways. Furthermore, we show that a major source for Fas-TNFR activation derives from CAR T cells themselves via activation-induced Fas ligand upregulation, highlighting a universal role of Fas-TNFRs in augmenting CAR T-cell responses. We have identified Fas-CD40 as the optimal chimera for overcoming Fas ligand-mediated kill and enhancing CAR T-cell efficacy.

synthetic biology↗

bioRxiv2022

Single cell, whole embryo phenotyping of pleiotropic disorders of mammalian development

Mouse models are a critical tool for studying human diseases, particularly developmental disorders, as well as for advancing our general understanding of mammalian biology. However, it has long been suspected that conventional approaches for phenotyping are insufficiently sensitive to detect subtle defects throughout the developing mouse. Here we set out to establish single cell RNA sequencing (sc-RNA-seq) of the whole embryo as a scalable platform for the systematic molecular and cellular phenotyping of mouse genetic models. We applied combinatorial indexing-based sc-RNA-seq to profile 101 embryos of 26 genotypes at embryonic stage E13.5, altogether profiling gene expression in over 1.6M nuclei. The 26 genotypes include 22 mouse mutants representing a range of anticipated severities, from established multisystem disorders to deletions of individual enhancers, as well as the 4 wildtype backgrounds on which these mutants reside. We developed and applied several analytical frameworks for detecting differences in composition and/or gene expression across 52 cell types or trajectories. Some mutants exhibited changes in dozens of trajectories (e.g., the pleiotropic consequences of altering the Sox9 regulatory landscape) whereas others showed phenotypes affecting specific subsets of cells. We also identify differences between widely used wildtype strains, compare phenotyping of gain vs. loss of function mutants, and characterise deletions of topological associating domain (TAD) boundaries. Intriguingly, even among these 22 mutants, some changes are shared by heretofore unrelated models, suggesting that developmental pleiotropy might be "decomposable" through further scaling of this approach. Overall, our findings show how single cell profiling of whole embryos can enable the systematic molecular and cellular phenotypic characterization of mouse mutants with unprecedented breadth and resolution.

genomics↗

bioRxiv2022

Water-Only Look-Locker Inversion recovery (WOLLI) T1 mapping

PurposeModified Look-Locker Inversion recovery (MOLLI) and Shortened MOLLI (ShMOLLI) T1 values can deviate substantially from the water T1 in voxels containing large amounts of water and fat. We introduce Water-Only Look-Locker Inversion recovery (WOLLI) to map water T1 in tissue containing fat. TheoryWOLLI comprises adiabatic water-selective inversion and several balanced steady-state free precession (bSSFP) readouts, followed immediately by adiabatic fat-selective inversion and bSSFP readout(s). Data are fitted pixel-wise and an adapted Deichmann-Haase saturation-correction gives the water T1. MethodsWe compared two WOLLI protocols (WOLLI-w7f1 and WOLLI-w7f2f1), ShMOLLI and single-voxel spectroscopy at 3T in: simulations, fat/water phantoms, and 12 subjects livers. ResultsIn simulations with 0-40% fat fraction, T1 varied by: <0.5% (WOLLI-w7f1), <12% (WOLLI-w7f2f1), and >100% (MOLLI). In phantoms, the accuracy of the methods (i.e. worst-case T1w difference vs spectroscopy) was: WOLLI-w7f1 (best, maximum difference of 10.2%), WOLLI-w7f2f1 (max. 10.5%), and ShMOLLI (max. 81.5%). In the liver, the root-mean-squared deviations vs spectroscopy were: 374ms (WOLLI-w7f1), 645ms (WOLLI-w7f2f1), and 700ms (ShMOLLI). ConclusionCompared to ShMOLLI, WOLLI is less influenced by fat, but more by B0-inhomogeneity. WOLLI was a clear improvement in phantoms but less so in vivo. WOLLI may be suited to scan organs with high fat content such as the liver.

biophysics↗

bioRxiv2021

Distal and proximal cis-regulatory elements sense X-chromosomal dosage and developmental state at the Xist locus

Developmental genes such as Xist, the master regulator of X-chromosome inactivation (XCI), are controlled by complex cis-regulatory landscapes, which decode multiple signals to establish specific spatio-temporal expression patterns. Xist integrates information on X-chromosomal dosage and developmental stage to trigger XCI at the primed pluripotent state in females only. Through a pooled CRISPR interference screen in differentiating mouse embryonic stem cells, we identify functional enhancer elements of Xist during the onset of random XCI. By quantifying how enhancer activity is modulated by X-dosage and differentiation, we find that X-dosage controls the promoter-proximal region in a binary switch-like manner. By contrast, differentiation cues activate a series of distal elements and bring them into closer spatial proximity of the Xist promoter. The strongest distal element is part of an enhancer cluster [~]200 kb upstream of the Xist gene which is associated with a previously unannotated Xist-enhancing regulatory transcript, we named Xert. Developmental cues and X-dosage are thus decoded by distinct regulatory regions, which cooperate to ensure female-specific Xist upregulation at the correct developmental time. Our study is the first step to disentangle how multiple, functionally distinct regulatory regions interact to generate complex expression patterns in mammals.

genomics↗

bioRxiv2019

Enhancer hijacking determines intra- and extrachromosomal circular MYCN amplicon architecture in neuroblastoma

MYCN amplification drives one in six cases of neuroblastoma. The supernumerary gene copies are commonly found on highly rearranged, extrachromosomal circular DNA. The exact amplicon structure has not been described thus far and the functional relevance of its rearrangements is unknown. Here, we analyzed the MYCN amplicon structure and its chromatin landscape. This revealed two distinct classes of amplicons which explain the regulatory requirements for MYCN overexpression. The first class always co-amplified a proximal enhancer driven by the noradrenergic core regulatory circuit (CRC). The second class of MYCN amplicons was characterized by high structural complexity, lacked key local enhancers, and instead contained distal chromosomal fragments, which harbored CRC-driven enhancers. Thus, ectopic enhancer hijacking can compensate for the loss of local gene regulatory elements and explains a large component of the structural diversity observed in MYCN amplification.

cancer biology↗

bioRxiv2019

Range-wide variation in local adaptation and phenotypic plasticity of fitness-related traits in Fagus sylvatica and their implications under climate change

AimTo better understand and more realistically predict future species distribution ranges, it is critical to account for local adaptation and phenotypic plasticity in populations responses to climate. This is challenging because local adaptation and phenotypic plasticity are trait-dependent and traits co-vary along climatic gradients, with differential consequences for fitness. Our aim is to quantify local adaptation and phenotypic plasticity of vertical and radial growth, leaf flushing and survival across Fagus sylvatica range and to estimate each trait contribution to explain the species occurrence.\n\nLocationEurope\n\nTime period1995 - 2014; 2070\n\nMajor taxa studiedFagus sylvatica L.\n\nMethodsWe used vertical and radial growth, flushing phenology and mortality of Fagus sylvatica L. recorded in BeechCOSTe52 (>150,000 trees). Firstly, we performed linear mixed-effect models that related trait variation and co-variation to local adaptation (related to the planted populations climatic origin) and phenotypic plasticity (accounting for the climate of the plantation), and we made spatial predictions under current and RCP 8.5 climates. Secondly, we combined spatial trait predictions in a linear model to explain the occurrence of the species.\n\nResultsThe contribution of plasticity to intra-specific trait variation is always higher than that of local adaptation, suggesting that the species is less sensitive to climate change than expected; different traits constrain beechs distribution in different parts of its range: the northernmost edge is mainly delimited by flushing phenology (mostly driven by photoperiod and temperature), the southern edge by mortality (mainly driven by intolerance to drought), and the eastern edge is characterised by decreasing radial growth (mainly shaped by precipitation-related variables in our model); considering trait co-variation improved single-trait predictions.\n\nMain conclusionsPopulation responses to climate across large geographical gradients are trait-dependent, indicating that multi-trait combinations are needed to understand species sensitivity to climate change and its variation across distribution ranges.

ecology↗

bioRxiv2018

Towards automation of germline variant curation inclinical cancer genetics

Cancer care professionals are confronted with interpreting results from multiplexed gene sequencing of patients at hereditary risk for cancer. Assessments for variant classification now require orthogonal data searches, requiring aggregation of multiple lines of evidence from diverse resources. The burden of evidence for each variant to meet thresholds for pathogenicity or actionability now poses a growing challenge for those seeking to counsel patients and families following germline genetic testing. A computational algorithm that automates, provides uniformity and significantly accelerates this interpretive process is needed. The tool described here, Pathogenicity of Mutation Analyzer (PathoMAN) automates germline genomic variant curation from clinical sequencing based on ACMG guidelines. PathoMAN aggregates multiple tracks of genomic, protein and disease specific information from public sources. We compared expert manually curated variant data from studies on (i) prostate cancer (ii) breast cancer and (iii) ClinVar to assess performance. PathoMAN achieves high concordance (83.1% pathogenic, 75.5% benign) and negligible discordance (0.04% pathogenic, 0.9% benign) when contrasted against expert curation. Some loss of resolution (8.6% pathogenic, 23.64% benign) and gain of resolution (6.6% pathogenic, 1.6% benign) was also observed. We highlight the advantages and weaknesses related to the programmable automation of variant classification. We also propose a new nosology for the five ACMG classes to facilitate more accurate reporting to ClinVar. The proposed refinements will enhance utility of ClinVar to allow further automation in cancer genetics. PathoMAN will reduce the manual workload of domain level experts. It provides a substantial advance in rapid classification of genetic variants by generating robust models using a knowledge-base of diverse genetic data https://pathoman.mskcc.org.

genetics↗