Biology subjects

Hewitt, A.

Publications and source records attributed to Hewitt, A..

12 recordsLinked to original sources

bioRxiv2024

Genetic variants associated with cell-type-specific intra-individual gene expression variability reveal new mechanisms of genome regulation

Gene expression levels can vary substantially across cells, even in a seemingly homogeneous cell population. Identifying the relationships between genetic variation and gene expression is critical for understanding the mechanisms of genome regulation. However, the genetic control of gene expression variability among the cells within individuals has yet to be extensively examined. This is primarily due to the statistical challenges, such as the need for sufficiently powered cohorts and adjusting mean-variance dependence. Here, we introduce MEOTIVE (Mapping genetic Effects On inTra-Individual Variability of gene Expression), a novel statistical framework to identify genetic effects on the gene expression variability (sc-veQTL) accounting for the mean-variance dependence. Using single-cell RNA-seq data of 1.2 million peripheral blood mononuclear cells from 980 human donors, we identified 14 - 3,488 genes with significant sc-veQTLs (study-wide q-value < 0.05) across different blood cell types, 2,103 of which were shared across more than one cell type. We further detected 55 SNP-gene pairs (in 34 unique genes) by directly linking genetic variations with gene expression dispersion (sc-deQTL) regardless of mean-variance dependence, and these genes were enriched in biological processes relevant to immune response and viral infection. An example is rs1131017 (p<9.08x10-52), a sc-veQTL in the 5UTR of RPS26, which shows a ubiquitous dispersion effect across cell types, with higher dispersion levels associated with lower auto-immune disease risk, including rheumatoid arthritis and type 1 diabetes. Another example is LYZ, which is associated with antibacterial activity against bacterial species and was only detected with a monocyte-specific deQTL (rs1384) located at the 3 UTR region (p=1.48x10-11) and replicated in an independent cohort. Our results demonstrate an efficient and robust statistical method to identify genetic effects on gene expression variability and how these associations and their involved pathways confer auto-immune disease risk. This analytical framework provides a new approach to unravelling the genetic regulation of gene expression at the single-cell resolution, advancing our understanding of complex biological processes.

genomics↗

bioRxiv2024

Sci-comm "behind the scenes": Gendered narratives of scientific outreach activities in the life sciences

Science communication (sci-comm) encompasses activities that promote scientific literacy, inform policy, and inspire future scientists. Despite its value, sci-comm is often considered less pretigious than research and other internal academic activities. In Australia, scientific organisations (e.g., learned societies) rely on members to perform a range of sci-comm activities, typically unpaid. In this pilot study, we surveyed 88 Australian life sciences organisations to examine who performs sci-comm and why. Most respondents were early-career women employed in university research positions. Participants largely agreed that organisational sci-comm brought limited career benefits and was often viewed as feminised or "care work" relative to their research roles. Yet, respondents also cited personal and professional gains and most wished to continue in such roles. Our findings suggest "invisible" sci-comm performed for scientific organisations is disproportionately undertaken by women at early career stages, with implications for career progression and gender equity in STEM.

scientific communication and education↗

bioRxiv2023

The landscape of genomic structural variation in Indigenous Australians

Indigenous Australians harbour rich and unique genomic diversity. However, Aboriginal and Torres Strait Islander ancestries are historically under-represented in genomics research and almost completely missing from reference databases. Addressing this representation gap is critical, both to advance our understanding of global human genomic diversity and as a prerequisite for ensuring equitable outcomes in genomic medicine. Here, we apply population-scale whole genome long-read sequencing to profile genomic structural variation across four remote Indigenous communities. We uncover an abundance of large indels (20-49bp; n=136,797) and structural variants (SVs; [≥]50bp; n=159,912), the majority of which are composed of tandem repeat or interspersed mobile element sequences (90%) and have not been previously annotated (73%). A large fraction of SVs appear to be exclusive to Indigenous Australians (>30%) and the majority of these are found in only a single community, underscoring the need for broad and deep sampling to achieve a comprehensive catalogue of genomic structural variation across the Australian continent. Finally, we explore short-tandem repeats (STRs) throughout the genome to characterise allelic diversity at 50 known disease loci, uncover hundreds of novel repeat expansion sites within protein-coding genes, and identify unique patterns of diversity and constraint among STR sequences. Our study sheds new light on the dimensions, diversity and evolutionary trajectories of genomic structural variation within and beyond Australia.

genomics↗

bioRxiv2023

High throughput functional profiling of genes at intraocular pressure loci reveals distinct networks for glaucoma

INTRODUCTIONPrimary open angle glaucoma (POAG) is a leading cause of blindness globally. Characterised by progressive retinal ganglion cell degeneration, the precise pathogenesis remains unknown. Genome-wide association studies (GWAS) have uncovered many genetic variants associated with elevated intraocular pressure (IOP), one of the key risk factors for POAG. This study sought to investigate the morphological and transcriptional consequences of perturbation of key genes at IOP loci in trabecular meshwork cell (TMC); the cellular regulators of IOP. We aimed to identify genetic and morphological variation that can be attributed to TMC dysfunction and raised IOP in POAG. METHODS62 genes across 55 loci were knocked-out in a primary human TMC line. Each knockout group, including five non-targeting control groups, underwent single-cell RNA-sequencing (scRNA-seq) for differentially-expressed gene (DEG) analysis. Multiplexed fluorescent staining of key organelles, was coupled with high-throughput microscopy for single-cell morphological analysis using CellProfiler image analysis. RESULTSAcross many of the individual gene knockouts scRNA-seq highlighted genes relating to matrix metalloproteinases and interferon-induced proteins. Our work has prioritised genes at four loci of interest to identify gene knockouts that may contribute to the pathogenesis of POAG, including ANGPTL2, LMX1B, CAV1, and KREMEN1. Three genetic networks of gene knockouts with similar transcriptomic profiles were identified (ABO / CAV1 / MYOC, ANGPT2 / PKHD1 / TNS1 / TXNRD2, and CAPZA1 / KALRN / LMO7 / PLEKHA7 / GNB1L / TEX41), suggesting a synergistic function in trabecular meshwork cell physiology. TEK knockout caused significant upregulation of nuclear granularity on morphological analysis, whilst knockout of TRIOBP, TMCO1 and PLEKHA7 increased granularity and intensity of actin and the cell-membrane. CONCLUSIONHigh throughput analysis of cellular structure and function through multiplex fluorescent single-cell analysis and scRNA-seq assays enabled the direct study of genetic perturbations at the single-cell resolution. This work provides a framework for investigating the role of genes in the pathogenesis of glaucoma and heterogenous diseases with a strong genetic basis.

genetics↗

bioRxiv2020

Investigating the role of APOE: limitations of human pluripotent stem cell-derived cerebral organoids

Apolipoprotein E (APOE) is the most important susceptibility gene for late onset of Alzheimers disease, with the presence of APOE-{varepsilon}4 associated with increased risk of developing Alzheimers disease. Here, we reprogrammed human fibroblasts from individuals with different APOE-{varepsilon} genotypes into induced pluripotent stem cells, and generated isogenic lines with different APOE profiles. We then differentiated these into cerebral organoids for six months and assessed the suitability of this in vitro system to measure APOE, {beta} amyloid, and Tau phosphorylation levels. We identified intra- and inter-variabilities in the organoids cell composition. Using the CRISPR-edited APOE isogenic lines, we observed more homogenous cerebral organoids, and similar levels of APOE, {beta} amyloid, and Tau between the isogenic lines, with the exception of one site of Tau phosphorylation which was higher in the APOE-{varepsilon}4/{varepsilon}4 organoids. These data describe that pathological hallmarks of AD are observed in cerebral organoids, and that their variation is mainly independent of the APOE-{varepsilon} status of the cells, but associated with the high variability of cerebral organoid differentiation. It demonstrates that the batch-to-batch and cell-line-to-cell-line variabilities need to be considered when using cerebral organoids.

neuroscience↗

bioRxiv2019

Comparative performance of the BGI and Illumina sequencing technology for single-cell RNA-sequencing

The libraries generated by high-throughput single cell RNA-sequencing platforms such as the Chromium from 10X Genomics require considerable amounts of sequencing, typically due to the large number of cells. The ability to use this data to address biological questions is directly impacted by the quality of the sequence data. Here we have compared the performance of the Illumina NextSeq 500 and NovaSeq 6000 against the BGI MGISEQ-2000 platform using identical Single Cell 3 libraries consisting of over 70,000 cells. Our results demonstrate a highly comparable performance between the NovaSeq 6000 and MGISEQ-2000 in sequencing quality, and cell, UMI, and gene detection. However, compared with the NextSeq 500, the MGISEQ-2000 platform performs consistently better, identifying more cells, genes, and UMIs at equalised read depth. We were able to call an additional 1,065,659 SNPs from sequence data generated by the BGI platform, enabling an additional 14% of cells to be assigned to the correct donor from a multiplexed library. However, both the NextSeq 500 and MGISEQ-2000 detected similar frequencies of gRNAs from a pooled CRISPR single cell screen. Our study provides a benchmark for high capacity sequencing platforms applied to high-throughput single cell RNA-seq libraries.

genomics↗

bioRxiv2018

Generation of human neural retina transcriptome atlas by single cell RNA sequencing

The retina is a highly specialized neural tissue that senses light and initiates image processing. Although the functional organisation of specific cells within the retina has been well-studied, the molecular profile of many cell types remains unclear in humans. To comprehensively profile cell types in the human retina, we performed single cell RNA-sequencing on 20,009 cells obtained post-mortem from three donors and compiled a reference transcriptome atlas. Using unsupervised clustering analysis, we identified 18 transcriptionally distinct clusters representing all known retinal cells: rod photoreceptors, cone photoreceptors, Muller glia cells, bipolar cells, amacrine cells, retinal ganglion cells, horizontal cells, retinal astrocytes and microglia. Notably, our data captured molecular profiles for healthy and early degenerating rod photoreceptors, and revealed a novel role of MALAT1 in putative rod degeneration. We also demonstrated the use of this retina transcriptome atlas to benchmark pluripotent stem cell-derived cone photoreceptors and an adult Muller glia cell line. This work provides an important reference with unprecedented insights into the transcriptional landscape of human retinal cells, which is fundamental to our understanding of retinal biology and disease.

systems biology↗

bioRxiv2018

Congenital glaucoma with anterior segment dysgenesis in individuals with biallelic CPAMD8 variants

PurposeCongenital glaucoma is a significant cause of irreversible blindness. In some instances glaucoma is associated with developmental abnormalities of the ocular anterior segment, which can impair drainage of aqueous humor, leading to an increase in intraocular pressure.\n\nMethodsGenome sequencing was performed on a parent-proband congenital glaucoma trio, with exome sequencing of 79 additional individuals with suspected primary congenital glaucoma.\n\nResultsWe describe a unique ocular anterior segment dysgenesis associated with congenital glaucoma in four individuals from three unrelated families. In each case, disease was associated with compound heterozygous variants in CPAMD8, a gene of unknown function recently associated with ocular anterior segment dysgenesis, myopia, and ectopia lentis. CPAMD8 expression was highest in neural crest-derived tissues of the adult anterior segment, suggesting that CPAMD8 variation may cause malformation of key drainage structures and the development of high intraocular pressure and glaucoma.\n\nConclusionsThis study reveals a unique genetic cause of childhood glaucoma, and expands the phenotypic spectrum of CPAMD8-associated ocular disease.

genetics↗

bioRxiv2018

Longitudinal expression profiling of CD4+ and CD8+ cells in patients with active to quiescent Giant Cell Arteritis

BackgroundGiant cell arteritis (GCA) is the most common form of vasculitis affecting elderly people. It is one of the few true ophthalmic emergencies. GCA is a heterogenous disease, symptoms and signs are variable thereby making it challenging to diagnose and often delaying diagnosis. A temporal artery biopsy is the gold standard to test for GCA, and there are currently no specific biochemical markers to categorize or aid diagnosis of the disease. We aimed to identify a less invasive method to confirm the diagnosis of GCA, as well as to ascertain clinically relevant predictive biomarkers by studying the transcriptome of purified peripheral CD4+ and CD8+ T lymphocytes in patients with GCA.\n\nMethods and FindingsWe recruited 16 patients with histological evidence of GCA at the Royal Victorian Eye and Ear Hospital (RVEEH), Melbourne, Australia, and aimed to collect blood samples at six time points: acute phase, 2-3 weeks, 6-8 weeks, 3 months, 6 months and 12 months after clinical diagnosis. CD4+ and CD8+ T-cells were positively selected at each time point through magnetic-assisted cell sorting (MACS). RNA was extracted from all 195 collected samples for subsequent RNA sequencing. The expression profiles of patients were compared to those of 16 age-matched controls. Over the 12-month study period, polynomial modelling analyses identified 179 and 4 statistically significant transcripts with altered expression profiles (FDR < 0.05) between cases and controls in CD4+ and CD8+ populations, respectively. In CD8+ cells, we identified two transcripts that remained differentially expressed after 12 months, namely SGTB, associated with neuronal apoptosis, and FCGR3A, which has been found in association with Takayasu arteritis (TA), another large vessel vasculitis. We detected genes that correlate with both symptoms and biochemical markers used in the acute setting for predicting long-term prognosis. 15 genes were shared across 3 phenotypes in CD4 and 16 across CD8 cells. In CD8, IL32 was common to 5 phenotypes: a history of Polymyalgia Rheumatica, both visual disturbance and raised neutrophils at the time of presentation, bilateral blindness and death within 12 months. Altered IL32 gene expression could provide risk evaluation of GCA diagnosis at the time of presentation and give an indication of prognosis, which may influence management.\n\nConclusionsThis is the first longitudinal gene expression study undertaken to identify robust transcriptomic biomarkers of GCA. Our results show cell type-specific transcript expression profiles, novel gene-phenotype associations, and uncover important biological pathways for this disease. These data significantly enhance the current knowledge of relevant biomarkers, their association with clinical prognostic markers, as well as potential candidates for detecting disease activity in whole blood samples. In the acute phase, the gene-phenotype relationships we have identified could provide insight to potential disease severity and as such guide us in initiating appropriate patient management.

genomics↗

bioRxiv2018

Efficacy and dynamics of self-targeting CRISPR/Cas constructs for gene editing in the retina.

Safe delivery of CRISPR/Cas endonucleases remains one of the major barriers to the widespread application of in vivo genome editing including the anticipatory treatment of monogenic retinal diseases. We previously reported the utility of adeno-associated virus (AAV)-mediated CRISPR/Cas genome editing in the retina; however, with this type of viral delivery system, active endonucleases will remain in the retina for an extended period, making genotoxicity a significant consideration in clinical applications. To address this issue, we have designed a self-destructing \"kamikaze\" CRISPR/Cas system that disrupts the Cas enzyme itself following expression. Four guide RNAs (sgRNAs) were designed to target Streptococcus pyogenes Cas9 (SpCas9), after in situ validation, the selected sgRNAs were cloned into a dual AAV vector. One construct was used to deliver SpCas9 and the other delivered sgRNAs directed against SpCas9 and the target locus (yellow fluorescent protein, YFP), in the presence of mCherry. Both constructs were packaged into AAV2 vector and intravitreally administered in C57BL/6 and Thy1-YFP transgenic mice. After 8 weeks the expression of SpCas9, the efficacy of YFP gene disruption was quantified. A reduction of SpCas9 mRNA was found in retinas treated with AAV2-mediated-YFP/SpCas9 targeting CRISPR/Cas compared to those treated with YFP targeting CRISPR/Cas alone. We also show that AAV2-mediated delivery of YFP/SpCas9 targeting CRISPR/Cas significantly reduced the number of YFP fluorescent cells among mCherry-expressing cells (~85.5% reduction compared to LacZ/SpCas9 targeting CRISPR/Cas) in transfected retina of Thy1-YFP transgenic mice. In conclusion, our data suggest that a self-destructive \"kamikaze\" CRISPR/Cas system can be used as a robust tool for refined genome editing in the retina, without compromising on-target efficiency.

genetics↗

bioRxiv2017

Single Cell RNA Sequencing of stem cell-derived retinal ganglion cells.

We used human embryonic stem cell-derived retinal ganglion cells (RGCs) to characterize the transcriptome of 1,174 cells at the single cell level. The human embryonic stem cell line BRN3B-mCherry A81-H7 was differentiated to RGCs using a guided differentiation approach. Cells were harvested at day 36 and subsequently prepared for single cell RNA sequencing. Our data indicates the presence of three distinct subpopulations of cells, with various degrees of maturity. One cluster of 288 cells upregulated genes involved in axon guidance together with semaphorin interactions, cell-extracellular matrix interactions and ECM proteoglycans, suggestive of a more mature phenotype.

genomics↗

bioRxiv2017

Mitochondrial replacement in an iPSC model of Leber Hereditary Optic Neuropathy.

Cybrid technology was used to replace Leber hereditary optic neuropathy (LHON) causing mitochondrial DNA (mtDNA) mutations from patient-specific fibroblasts with wildtype mtDNA, and mutation-free induced pluripotent stem cells (iPSCs) were generated subsequently. Retinal ganglion cell (RGC) differentiation demonstrates increased cell death in LHON-RGCs and can be rescued in cybrid corrected RGCs.

cell biology↗