Biology subjects

Hawkins, V.

Publications and source records attributed to Hawkins, V..

2 recordsLinked to original sources

bioRxiv2023

Evidence for glutamate excitotoxicity that occurs before the onset of cell loss and motor symptoms in an ovine Huntington's Disease model.

BackgroundHuntingtons disease (HD) is a neurodegenerative genetic disorder caused by an expansion in the CAG repeat tract of the huntingtin (HTT) gene resulting in a triad of behavioural, cognitive, and motor defects. Current knowledge of disease pathogenesis remains incomplete, and no disease course-modifying interventions are in clinical use. We have previously reported the development and characterisation of the OVT73 transgenic sheep model of HD. OVT73 captures an early prodromal phase of the disease with an absence of motor symptomatology even at 5-years of age and no detectable striatal cell loss. MethodsTo better understand the disease-initiating events we have undertaken a single nuclei transcriptome study of the striatum of an extensively studied cohort of 5-year-old OVT73 HD sheep and age matched wild-type controls. ResultsWe have identified transcriptional upregulation of genes encoding N-methyl-D-aspartate (NMDA), -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and kainate receptors in OVT73 medium spiny neurons, the cell type preferentially lost early in HD. This observation supports the glutamate excitotoxicity hypothesis as an early neurodegeneration cascade-initiating process. Moreover, we also observed the downstream consequences of excitotoxic stress, including a downregulation of transcription of components for the oxidative phosphorylation complexes. We also found that pathways whose activity has been proposed to reduce excitotoxicity, including the CREB family of transcription factors (CREB1, ATF2, ATF4 and ATF7) were transcriptionally downregulated. ConclusionsTo our knowledge, the OVT73 model is the first large mammalian HD model that exhibits transcriptomic signatures of an excitotoxic process in the absence of neuronal loss. Our results suggest that glutamate excitotoxicity is a disease-initiating process. Addressing this biochemical defect early may prevent neuronal loss and avoid the more complex secondary consequences precipitated by cell death.

neuroscience↗

bioRxiv2021

Optimised multiplex amplicon sequencing for mutation identification using the MinION nanopore sequencer

ObjectiveRapid, cost-effective identification of genetic variants in small candiate genomic regions remains a challenge, particularly for less well equipped or lower throughput laboratories. Application of Oxford Nanopore Technologies MinION sequencer has the potential to fulfil this requirement. We have developed a multiplexing assay which pools PCR amplicons for MinION sequencing to enable sequencing of multiple templates from multiple individuals which could be applied to gene-targeted diagnostics. MethodsA combined strategy of barcoding and sample pooling was developed for simultaneous multiplex MinION sequencing of 100 PCR amplicons, spanning 30 loci in DNA isolated from 82 neurodevelopmental cases and family members. The target regions were chosen for further interegation because a potentially disease-causative variants had been identified in affected individuals by Illumina exome sequencing. The pooled MinION sequences were deconvoluted by aligning to custom references using the guppy aligner software. ResultsOur multiplexing approach produced interpretable and expected sequence from 29 of the 30 targeted genetic loci. The sequence variant which was not correctly resolved in the MinION sequence was adjacent to a five nucleotide homopolymer. It is already known that homopolymers present a resolution problem with the MinION approach. Interstingly despite equimolar quantities of PCR amplicon pooled for sequencing, significant variation in the depth of coverage (139x - 21,499x; mean = 9,050, std err = 538.21) was observed. We observed independent relationships between depth of coverage and target length, and depth of coverage and GC content. These relationships demonstrate biases of the MinION sequencer for longer templates and those with lower GC content. ConclusionWe demonstrate an efficient approach for variant discovery or confirmation from short DNA templates using the MinION sequencing device. With less than 140x depth of coverage required for accurate genotyping, the methodology described here allows for rapid highly multiplexed targeted sequencing of large numbers of samples in a minimally equipped laboratory.

bioinformatics↗