Biology subjects

Aston, A. N.

Publications and source records attributed to Aston, A. N..

4 recordsLinked to original sources

bioRxiv2026

Targeted sequencing of expanded tandem repeats: Identifying interruptions and errors

Expanded repeat disorders remain without a disease modifying treatment. Some of the most important modifiers of disease severity include inherited repeat size, their somatic mosaicism, and whether they contain interruptions. Targeted sequencing approaches are becoming the gold standard on how these parameters are measured despite repeats being notoriously challenging to sequence. Here we developed CHARLIE (Comprehensive High-throughput Analysis of Repeat Length, Interruptions and Expansions) to identify and position interruptions within expanded repeats. We used samples from Huntingtons disease and myotonic dystrophy type 1 sequenced with Illumina MiSeq and PacBios Single-Molecule Real-Time Sequencing. We validated the pipeline against previous methods for identifying germline-inherited interruptions. One challenge that can potentially mask the identification of true interruptions and sequence variation is the accuracy of sequencing platforms, which, when applied to expanded repeats, is largely unknown. Our results suggest that each sequencing platform produces a distinct error profile and we show that PCR-free library preparation for SMRT sequencing improves sequencing accuracy. CHARLIE, therefore, provides a method that can help differentiate between trivial sequencing errors and those interruptions that modify disease presentation, which can be inherited or somatic in origin.

bioinformatics↗

bioRxiv2026

Cell-specific transcription dysregulation in human Huntington's disease-positive developing striatum

Huntingtons disease (HD) is an autosomal dominant neurodegenerative disorder caused by a CAG-repeat expansion in the HTT gene. Progressive loss of striatal projection neurons leads to cognitive, psychiatric, and motor impairments that typically manifest in midlife, despite the presence of the expansion from conception. Increasing evidence supports a neurodevelopmental component to HD; however, authentic human developing HD striatal tissue has not previously been characterised. We analysed an HD positive human fetal striatal sample alongside an age- and sex-matched control. CAG-repeat length was determined, and single-cell RNA sequencing was used to investigate gene expression. We compared the fetal HD transcriptional signature with publicly available datasets from postmortem adult HD brain tissue. We identified 2,032 differentially expressed genes and defined nine cellular clusters, each exhibiting distinct transcriptional profiles. Gene enrichment analysis revealed disruption of key biological processes across the developing HD striatum, with pathway-level dysregulation varying between clusters. There was overlap in gene expression changes between fetal and adult HD striatal tissues. Together, these findings demonstrate that molecular features of HD pathology are present during early human striatal development, supporting the concept that disease mechanisms are established decades prior to clinical onset.

neuroscience↗

bioRxiv2026

SCIA: A fast and widely applicable pipeline for measuring expanded repeat instability

The expansion of short tandem repeats is a feature of over 60 different human diseases. Ongoing somatic instability throughout a patients lifetime can influence disease progression and has emerged as a therapeutic target. Understanding its mechanism is essential for the identification of both drug targets and therapeutic interventions. A major obstacle towards this translational goal has been to measure changes in repeat size distribution given that these are complex datasets. To address this, here we provide a new analysis method, and accompanying software, that generates delta plots, extracts the instability frequency from targeted long-read sequencing data, the bias towards expansion or contraction, and the average size of the changes. It further provides statistical analysis for comparison between treatments. We show its applicability to non-dividing cells, and in vivo datasets. Moreover, we have developed a streamlined experimental design for dividing cells, Single Clone-based Instability Assay (SCIA), that saves weeks in assessing the effect of a gene knockout on repeat instability and is ideal for an initial screen. We have validated the approach using FAN1, PMS1, and MLH1 knockouts. Using SCIA, we find that although FAN1 knockout clones showed increased frequency of expansions, the size of the expansions were smaller. This highlights the wealth of information that can be extracted and the potential for novel insights into the mechanism of repeat instability.

neuroscience↗

bioRxiv2024

Cas9 nickase-mediated contraction of CAG/CTG repeatsat multiple disease loci

Expanded CAG/CTG repeats cause over 15 different diseases that all remain without a disease-modifying treatment. Because repeat length accounts for most of the variation in disease severity, contracting them presents an attractive therapeutic avenue. Here, we show that the CRISPR-Cas9 nickase targeted to CAG/CTG repeats leads to efficient contractions in Huntingtons disease patient-derived neurons and astrocytes, and in myotonic dystrophy type 1 patient-derived neurons. The approach is allele-selective and free of detectable off-target mutations. Striatal injection of the Cas9 nickase in a mouse model for Huntingtons disease using adeno-associated viral vectors led to contractions in over half the infected cells. Upon injection, we observed a reduction in the number of inclusion bodies, improved transcriptome, and ameliorated locomotion. The effects were greater than expected from the contractions induced and suggest that non-cell autonomous mechanisms may be involved. Our results provide the proof-of-concept that correction of CAG/CTG repeats can improve Huntingtons disease phenotypes in vivo. One sentence summaryThe Cas9 nickase contracts CAG/CTG repeats at multiple disease loci in patient-derived cells and improves molecular and behavioral phenotypes in HD.

neuroscience↗