Biology subjects

Washer, S.

Publications and source records attributed to Washer, S..

3 recordsLinked to original sources

bioRxiv2025

Integrated QTL mapping and CRISPR screening in pooled iPSC-derived microglia reveals genetic drivers of neurodegenerative risk

Mounting evidence implicates microglia in neurodegeneration, but linking disease-associated genetic variants to target genes and cellular phenotypes is hindered by the inaccessibility of these cells. We differentiated 261 human iPSC lines into microglia-like cells (iMGL) in pools with phenotypic (differentiation, phagocytosis and migration) and single-cell transcriptomic readouts. Burden analysis of deleterious variants detected 36 genes influencing microglial phenotypes. Expression quantitative trait locus (eQTL) analysis found 7,121 eGenes, and 79 colocalizations across four neurodegenerative disease GWAS, half of which had limited prior evidence of causality. Integration of eQTL and phenotypic associations highlighted the role of disease-relevant variants including LRRK2 and TREM2 acting via microglial phagocytosis. A coupled CRISPR screen identified a role of TREM2 in phagocytosis and highlighted the importance of cellular state in directionality of phenotype. By contextualizing variant effects within disease-relevant microglial states, we provide a comprehensive framework for interpreting the function of risk loci in neurodegenerative disorders.

genomics↗

bioRxiv2022

Evaluation of nanopore sequencing for epigenetic epidemiology: a comparison with DNA methylation microarrays

Most epigenetic epidemiology to date has utilized microarrays to identify positions in the genome where variation in DNA methylation is associated with environmental exposures or disease. However, these profile less than 3% of DNA methylation sites in the human genome, potentially missing affected loci and preventing the discovery of disrupted biological pathways. Third generation sequencing technologies, including Nanopore sequencing, have the potential to revolutionise the generation of epigenetic data, not only by providing genuine genome-wide coverage but profiling epigenetic modifications direct from native DNA. Here we assess the viability of using Nanopore sequencing for epidemiology by performing a comparison with DNA methylation quantified using the most comprehensive microarray available, the Illumina EPIC array. We implemented a CRISPR-Cas9 targeted sequencing approach in concert with Nanopore sequencing to profile DNA methylation in three genomic regions to attempt to rediscover genomic positions that existing technologies have shown are differentially methylated in tobacco smokers. Using Nanopore sequencing reads, DNA methylation was quantified at 1,779 CpGs across three regions, providing a finer resolution of DNA methylation patterns compared to the EPIC array. The correlation of estimated levels of DNA methylation between platforms was high. Furthermore, we identified 12 CpGs where hypomethylation was significantly associated with smoking status, including 10 within the AHRR gene. In summary, Nanopore sequencing is a valid option for identifying genomic loci where large differences in DNAm are associated with a phenotype and has the potential to advance our understanding of the role differential methylation plays in the aetiology of complex disease.

neuroscience↗

bioRxiv2020

Analysis of the transcriptome and DNA methylome in response to acute and recurrent low glucose in human primary astrocytes

Aims/hypothesisRecurrent hypoglycaemia (RH) is a major side-effect of intensive insulin therapy for people with diabetes. Changes in hypoglycaemia sensing by the brain contribute to the development of impaired counterregulatory responses to and awareness of hypoglycaemia. Little is known about the intrinsic changes in human astrocytes in response to acute and recurrent low glucose (RLG) exposure. MethodsHuman primary astrocytes (HPA) were exposed to zero, one, three or four bouts of low glucose (0.1 mmol/l) for three hours per day for four days to mimic RH. On the fourth day, DNA and RNA were collected. Differential gene expression and ontology analyses were performed using DESeq2 and GOseq respectively. DNA methylation was assessed using the Infinium MethylationEPIC BeadChip platform. Results24 differentially expressed genes (DEGs) were detected (after correction for multiple comparisons). One bout of low glucose exposure had the largest effect on gene expression. Pathway analyses revealed that endoplasmic-reticulum (ER) stress-related genes such as HSPA5, XBP1, and MANF, involved in the unfolded protein response (UPR), were all significantly increased following LG exposure, which was diminished following RLG. There was little correlation between differentially methylated positions and changes in gene expression yet the number of bouts of LG exposure produced distinct methylation signatures. Conclusions/interpretationThese data suggest that exposure of human astrocytes to transient LG triggers activation of genes involved in the UPR linked to endoplasmic reticulum (ER) stress. Following RLG, the activation of UPR related genes was diminished, suggesting attenuated ER stress. This may be mediated by metabolic adaptations to better preserve intracellular and/or ER ATP levels, but this requires further investigation.

neuroscience↗