👤 Dianne Matthews

Formalin-fixed paraffin-embedded (FFPE) tissue specimens represent precious resources for clinical genomic profiling studies, especially when coupled with comprehensive medical records. Even though next-generation sequencing (NGS) is an effective tool to detect somatic mutations and somatic copy number alterations (sCNA), the biggest challenges in unlocking clinically translatable genomic information from FFPE tissue are low DNA yields and degraded DNA, affected by variable formalin fixation. Another issue is that the proportion of carcinoma and other noncarcinoma cells is variable and can be confounded by intratumoral heterogeneity. To explore these challenges, we isolated pure carcinoma and stromal cells using the DEPArray™ NxT system, a microchip-based digital sorter that allows isolation of pure, homogeneous subpopulations of cells from FFPE samples. We isolated pure carcinoma and stromal cell populations from 12 FFPE tissues, including tissues from nine primary and metastatic breast cancer and three primary ovarian high-grade serous carcinomas. This was followed by downstream shallow whole-genome sequencing (WGS) for copy number landscape profiling (10 samples) and/or a targeted panel for somatic mutation and sCNA analysis (seven samples), subject to cell availability. Seven out of 10 samples (even some with low tumour content or of old age) produced good-quality genomic data, detecting sCNA in all carcinoma population samples but not in the stromal populations. Mutation analysis was performed successfully in 6/7 samples and somatic mutations were detected in all of them. Our workflow enabled the identification of clinically actionable targets, including PIK3CA, ERBB2, FGFR1/2, CDK6, CCNE1, KRAS amplifications and RB, BRCA1/2 losses in patients that would direct therapy. © 2025 The Author(s). The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland. Show less

Hypertrophic cardiomyopathy (HCM) is an important cause of morbidity and mortality with both monogenic and polygenic components. Here, we report results from a large genome-wide association study and multitrait analysis including 5,900 HCM cases, 68,359 controls and 36,083 UK Biobank participants with cardiac magnetic resonance imaging. We identified 70 loci (50 novel) associated with HCM and 62 loci (20 novel) associated with relevant left ventricular traits. Among the prioritized genes in the HCM loci, we identify a novel HCM disease gene, SVIL, which encodes the actin-binding protein supervillin, showing that rare truncating SVIL variants confer a roughly tenfold increased risk of HCM. Mendelian randomization analyses support a causal role of increased left ventricular contractility in both obstructive and nonobstructive forms of HCM, suggesting common disease mechanisms and anticipating shared response to therapy. Taken together, these findings increase our understanding of the genetic basis of HCM, with potential implications for disease management. Show less

Chronic kidney disease (CKD) is associated with systemic phosphate elevations, called hyperphosphatemia. Translational studies have shown that hyperphosphatemia contributes to CKD-associated inflammation and injury in various tissues, including the kidney, heart, liver, and parathyroid gland. Mechanisms underlying pathologic actions of elevated phosphate on cells are not well understood but seem to involve uptake of phosphate through sodium phosphate cotransporters and phosphate-induced signaling via FGFR1 (fibroblast growth factor receptor 1). Clinical studies indicate patients with CKD are more likely to develop inflammatory and restrictive lung diseases, such as fibrotic interstitial lung diseases, and here we aimed to determine whether hyperphosphatemia can cause lung injury. We found that a mouse model of CKD and hyperphosphatemia, induced by an adenine-rich diet, develops lung fibrosis and inflammation. Elevation of systemic phosphate concentration by administration of a high-phosphate diet in a mouse model of primary lung inflammation and fibrosis, induced by bleomycin, exacerbated lung injury in the absence of kidney damage. Our Show less

The number of adults living with cystic fibrosis (CF) has already increased significantly because of drastic improvements in life expectancy attributable to advances in treatment, including the development of highly effective modulator therapy. Chronic airway inflammation in CF contributes to morbidity and mortality, and aging processes like inflammaging and cell senescence influence CF pathology. Our results show that single-cell RNA sequencing data, human primary bronchial epithelial cells from non-CF and CF donors, a CF bronchial epithelial cell line, and Cftr-knockout (Cftr-/-) rats all demonstrated increased cell senescence markers in the CF bronchial epithelium. This was associated with upregulation of fibroblast growth factor receptors (FGFRs) and mitogen-activated protein kinase (MAPK) p38. Inhibition of FGFRs, specifically FGFR4 and to some extent FGFR1, attenuated cell senescence and improved mucociliary clearance, which was associated with MAPK p38 signaling. Mucociliary dysfunction could also be improved using a combination of senolytics in a CF ex vivo model. In summary, FGFR/MAPK p38 signaling contributes to cell senescence in CF airways, which is associated with impaired mucociliary clearance. Therefore, attenuation of cell senescence in the CF airways might be a future therapeutic strategy improving mucociliary dysfunction and lung disease in an aging population with CF. Show less

The β-site Amyloid precursor protein Cleaving Enzyme 1 (BACE1) is an extensively studied therapeutic target for Alzheimer's disease (AD), owing to its role in the production of neurotoxic amyloid beta (Aβ) peptides. However, despite numerous BACE1 inhibitors entering clinical trials, none have successfully improved AD pathogenesis, despite effectively lowering Aβ concentrations. This can, in part, be attributed to an incomplete understanding of BACE1, including its physiological functions and substrate specificity. We propose that BACE1 has additional important physiological functions, mediated through substrates still to be identified. Thus, to address this, we computationally analysed a list of 533 BACE1 dependent proteins, identified from the literature, for potential BACE1 substrates, and compared them against proteins differentially expressed in AD. We identified 15 novel BACE1 substrates that were specifically altered in AD. To confirm our analysis, we validated Protein tyrosine phosphatase receptor type D (PTPRD) and Netrin receptor DCC (DCC) using Western blotting. These findings shed light on the BACE1 inhibitor failings and could enable the design of substrate-specific inhibitors to target alternative BACE1 substrates. Furthermore, it gives us a greater understanding of the roles of BACE1 and its dysfunction in AD. Show less

The heart muscle diseases hypertrophic (HCM) and dilated (DCM) cardiomyopathies are leading causes of sudden death and heart failure in young, otherwise healthy, individuals. We conducted genome-wide association studies and multi-trait analyses in HCM (1,733 cases), DCM (5,521 cases) and nine left ventricular (LV) traits (19,260 UK Biobank participants with structurally normal hearts). We identified 16 loci associated with HCM, 13 with DCM and 23 with LV traits. We show strong genetic correlations between LV traits and cardiomyopathies, with opposing effects in HCM and DCM. Two-sample Mendelian randomization supports a causal association linking increased LV contractility with HCM risk. A polygenic risk score explains a significant portion of phenotypic variability in carriers of HCM-causing rare variants. Our findings thus provide evidence that polygenic risk score may account for variability in Mendelian diseases. More broadly, we provide insights into how genetic pathways may lead to distinct disorders through opposing genetic effects. Show less

Glucose-dependent insulinotropic polypeptide (GIP) conveys information from ingested nutrients to peripheral tissues, signaling energy availability. The GIP Receptor (GIPR) is also expressed in the bone marrow, notably in cells of the myeloid lineage. However, the importance of gain and loss of GIPR signaling for diverse hematopoietic responses remains unclear. We assessed the expression of the Gipr in bone marrow (BM) lineages and examined functional roles for the GIPR in control of hematopoiesis. Bone marrow responses were studied in (i) mice fed regular or energy-rich diets, (ii) mice treated with hematopoietic stressors including acute 5-fluorouracil (5-FU), pamsaccharide (LPS), and Pam3CysSerLys4 (Pam3CSK4), with or without pharmacological administration of a GIPR agonist, and (iii) mice with global (Gipr Gipr is expressed within T cells, myeloid cells, and myeloid precursors; however, these cell populations were not different in peripheral blood, spleen, or BM of Gipr These studies identify a functional gut hormone-BM axis positioned for the transduction of signals linking nutrient availability to the control of TLR and Notch genes regulating hematopoiesis. Nevertheless, stimulation or loss of GIPR signaling has minimal impact on basal hematopoiesis or the physiological response to hematopoietic stress. Show less

Glucagon-like peptide-1 is a nutrient-sensitive hormone secreted from enteroendocrine L cells within the small and large bowel. Although GLP-1 levels rise rapidly in response to food ingestion, the greatest density of L cells is localized to the distal small bowel and colon. Here, we assessed the importance of the distal gut in the acute L cell response to diverse secretagogues. Circulating levels of glucose and plasma GLP-1 were measured in response to the administration of L cell secretagogues in wild-type mice and in mice with (1) genetic reduction of Gcg expression throughout the small bowel and large bowel (Gcg The acute GLP-1 response to olive oil or arginine administration was markedly diminished in Gcg These findings further establish the importance of the proximal gut for the acute response to nutrient-related GLP-1 secretagogues. In contrast, we identify essential contributions of the distal gut to (i) the rapid induction of circulating GLP-1 levels in response to pharmacological selective agonism of G-protein-coupled receptors, (ii) the increased GLP-1 levels following the activation of Toll-Like Receptors with LPS, and iii) the acute GLP-1 response to metformin. Collectively, these results reveal that distal gut Gcg + endocrine cells are rapid responders to structurally and functionally diverse GLP-1 secretagogues. Show less

The cholesterol-sensing nuclear receptor liver X receptor (LXR) and the glucose-sensing transcription factor carbohydrate responsive element-binding protein (ChREBP) are central players in regulating glucose and lipid metabolism in the liver. More knowledge of their mechanistic interplay is needed to understand their role in pathological conditions like fatty liver disease and insulin resistance. In the current study, LXR and ChREBP co-occupancy was examined by analyzing ChIP-seq datasets from mice livers. LXR and ChREBP interaction was determined by Co-immunoprecipitation (CoIP) and their transactivity was assessed by real-time quantitative polymerase chain reaction (qPCR) of target genes and gene reporter assays. Chromatin binding capacity was determined by ChIP-qPCR assays. Our data show that LXRα and ChREBPα interact physically and show a high co-occupancy at regulatory regions in the mouse genome. LXRα co-activates ChREBPα and regulates ChREBP-specific target genes in vitro and in vivo. This co-activation is dependent on functional recognition elements for ChREBP but not for LXR, indicating that ChREBPα recruits LXRα to chromatin in Show less

The Salmonella enterica effector SteD depletes mature MHC class II (mMHCII) molecules from the surface of infected antigen-presenting cells through ubiquitination of the cytoplasmic tail of the mMHCII β chain. Here, through a genome-wide mutant screen of human antigen-presenting cells, we show that the NEDD4 family HECT E3 ubiquitin ligase WWP2 and a tumor-suppressing transmembrane protein of unknown biochemical function, TMEM127, are required for SteD-dependent ubiquitination of mMHCII. Although evidently not involved in normal regulation of mMHCII, TMEM127 was essential for SteD to suppress both mMHCII antigen presentation in mouse dendritic cells and MHCII-dependent CD4 Show less

Members of the poly-ADP-ribose polymerase (PARP) family catalyse the ADP-ribosylation of target proteins and are known to play important roles in many cellular processes, including DNA repair, differentiation and transcription. The majority of PARPs exhibit mono-ADP-ribosyltransferase activity rather than PARP activity; however, little is known about their biological activity. In the present study, we report that 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-inducible poly-ADP-ribose polymerase (TIPARP), mono-ADP-ribosylates and positively regulates liver X receptor α (LXRα) and LXRβ activity. Overexpression of TIPARP enhanced LXR-reporter gene activity. TIPARP knockdown or deletion reduced LXR regulated target gene expression levels in HepG2 cells and in Tiparp(-/-)mouse embryonic fibroblasts (MEFs) respectively. Deletion and mutagenesis studies showed that TIPARP's zinc-finger and catalytic domains were required to enhance LXR activity. Protein interaction studies using TIPARP and LXRα/β peptide arrays revealed that LXRs interacted with an N-terminal sequence (a.a. 209-236) of TIPARP, which also overlapped with a putative co-activator domain of TIPARP (a.a. 200-225). Immunofluorescence studies showed that TIPARP and LXRα or LXRβ co-localized in the nucleus.In vitroribosylation assays provided evidence that TIPARP mono-ADP-ribosylated both LXRα and LXRβ. Co-immunoprecipitation (co-IP) studies revealed that ADP-ribosylase macrodomain 1 (MACROD1), but not MACROD2, interacted with LXRs in a TIPARP-dependent manner. This was complemented by reporter gene studies showing that MACROD1, but not MACROD2, prevented the TIPARP-dependent increase in LXR activity. GW3965-dependent increases in hepatic Srebp1 mRNA and protein expression levels were reduced in Tiparp(-/-)mice compared with Tiparp(+/+)mice. Taken together, these data identify a new mechanism of LXR regulation that involves TIPARP, ADP-ribosylation and MACROD1. Show less

Liver X receptor (LXR)α and LXRβ play key roles in hepatic de novo lipogenesis through their regulation of lipogenic genes, including sterol regulatory element-binding protein (SREBP)-1c and carbohydrate responsive element-binding protein (ChREBP). LXRs activate lipogenic gene transcription in response to feeding, which is believed to be mediated by insulin. We have previously shown that LXRs are targets for glucose-hexosamine-derived O-linked β-N-acetylglucosamine (O-GlcNAc) modification enhancing their ability to regulate SREBP-1c promoter activity in vitro. To elucidate insulin-independent effects of feeding on LXR-mediated lipogenic gene expression in vivo, we subjected control and streptozotocin-treated LXRα/β(+/+) and LXRα/β(-/-) mice to a fasting-refeeding regime. We show that under hyperglycemic and hypoinsulinemic conditions, LXRs maintain their ability to upregulate the expression of glycolytic and lipogenic enzymes, including glucokinase (GK), SREBP-1c, ChREBPα, and the newly identified shorter isoform ChREBPβ. Furthermore, glucose-dependent increases in LXR/retinoid X receptor-regulated luciferase activity driven by the ChREBPα promoter was mediated, at least in part, by O-GlcNAc transferase (OGT) signaling in Huh7 cells. Moreover, we show that LXR and OGT interact and colocalize in the nucleus and that loss of LXRs profoundly reduced nuclear O-GlcNAc signaling and ChREBPα promoter binding activity in vivo. In summary, our study provides evidence that LXRs act as nutrient and glucose metabolic sensors upstream of ChREBP by modulating GK expression, nuclear O-GlcNAc signaling, and ChREBP expression and activity. Show less

Glucose-dependent insulinotropic polypeptide (GIP) is an endogenous hormonal factor (incretin) that, upon binding to its receptor (GIPr; a class B G-protein-coupled receptor), stimulates insulin secretion by beta cells in the pancreas. There has been a lack of potent inhibitors of the GIPr with prolonged in vivo exposure to support studies on GIP biology. Here we describe the generation of an antagonizing antibody to the GIPr, using phage and ribosome display libraries. Gipg013 is a specific competitive antagonist with equally high potencies to mouse, rat, dog, and human GIP receptors with a Ki of 7 nm for the human GIPr. Gipg013 antagonizes the GIP receptor and inhibits GIP-induced insulin secretion in vitro and in vivo. A crystal structure of Gipg013 Fab in complex with the human GIPr extracellular domain (ECD) shows that the antibody binds through a series of hydrogen bonds from the complementarity-determining regions of Gipg013 Fab to the N-terminal α-helix of GIPr ECD as well as to residues around its highly conserved glucagon receptor subfamily recognition fold. The antibody epitope overlaps with the GIP binding site on the GIPr ECD, ensuring competitive antagonism of the receptor. This well characterized antagonizing antibody to the GIPr will be useful as a tool to further understand the biological roles of GIP. Show less