Biology subjects

Monckton, D. G.

Publications and source records attributed to Monckton, D. G..

7 recordsLinked to original sources

bioRxiv2026

Unscheduled DNA synthesis reveals a DNA repair hotspot and biomarker of somatic instability at the expanded-CAG repeat tract in the huntingtin gene

Somatic instability (SI) of expanded DNA repeats is a hallmark of repeat expansion disorder (REDs) and drives onset and progression in Huntingtons disease (HD) yet the absence of target engagement (TE) biomarkers for SI-modulating therapies represents a critical gap to clinical development. Here, we describe the development of the unscheduled repair synthesis assay (URSA)--combining 5-ethynyl-2-deoxyuridine (EdU) pulse-labeling with digital PCR or sequencing--and show that the CAG-expanded huntingtin (HTT) exon 1 allele is a highly active DNA repair hotspot in cells from people with HD (PwHD). Repair activity increases with repeat length, is allele-specific, and depends strongly on MSH3, a central driver of somatic expansion. URSA robustly quantifies MSH3 modulation in preclinical models within days compared to weeks or months required by conventional repeat-length measurements. Critically, substantial repair activity is detectable in peripheral blood mononuclear cells (PBMCs) from PwHD, where signal correlates with CAG length and improves predictive models of somatic expansion (SE) beyond age and CAG length alone. Unlike repeat-length changes, which require years to accumulate in blood, URSA signal is measurable within days. These findings establish DNA repair activity at the mutant HTT locus as a mechanistically grounded pharmacodynamic biomarker, enabling TE monitoring on a clinically actionable timescale, and with broad applicability to REDs and other diseases where modulation of the DNA damage response is therapeutically targeted.

neuroscience↗

bioRxiv2024

The TRX assay for triplet repeat expansions

Expansion mutations of triplet repeat sequences cause numerous inherited neurological diseases. In some diseases, affected individuals display somatic expansions in affected tissues which have been linked to accelerated disease onset and progression. There is currently considerable interest in developing therapies to slow somatic repeat expansions to delay or block disease onset. In vitro assays are particularly important to evaluate potential therapeutic interventions. Current assays typically use physical methods to monitor triplet repeat lengths within a population of cells. While useful, most of these assays are relatively slow ([~]six weeks) and are somewhat limited in sensitivity to rare events. Here, a new assay, called TRX, is described to monitor CAG*CTG triplet repeat expansions more rapidly and with better sensitivity. TRX uses human tissue culture cells expressing two fluorescent proteins. Red fluorescent protein TagRFP658 is constitutively expressed and serves as an internal control. GFP is expressed in a CAG*CTG repeat length-dependent manner, with longer repeat lengths predicted to give higher green fluorescence intensity. Standard flow cytometry allows quantification of changes in fluorescent signal as a simple readout with <2% sensitivity. Two independently derived cell lines with 63 or 59 CAG repeats yielded similar rates of TRX activity. Cells with increased green fluorescence were observed within one to two weeks of culture, with longer times leading to additional signal. The appearance of green fluorescence was partly dependent on MutS{beta}, the DNA MSH2-MSH3 complex, based on siRNA knockdown of MSH3. However, physical analysis of the CAG*CTG repeat tracts by MiSeq deep sequencing or capillary electrophoresis showed limited changes in the length of the repeat tracts. We conclude that the TRX assay is a promising new tool for monitoring CAG*CTG repeat expansions but that further development of the assay is needed to make it fully useful.

genetics↗

bioRxiv2024

Genetic modifiers of somatic expansion and clinical phenotypes in Huntington's disease reveal shared and tissue-specific effects

Huntingtons disease (HD), due to expansion of a CAG repeat in HTT, is representative of a growing number of disorders involving somatically unstable short tandem repeats. We find that overlapping and distinct genetic modifiers of clinical landmarks and somatic expansion in blood DNA reveal an underlying complexity and cell-type specificity to the mismatch repair-related processes that influence disease timing. Differential capture of non-DNA-repair gene modifiers by multiple measures of cognitive and motor dysfunction argues additionally for cell-type specificity of pathogenic processes. Beyond trans modifiers, differential effects are also illustrated at HTT by a 5-UTR variant that promotes somatic expansion in blood without influencing clinical HD, while, even after correcting for uninterrupted CAG length, a synonymous sequence change at the end of the CAG repeat dramatically hastens onset of motor signs without increasing somatic expansion. Our findings are directly relevant to therapeutic suppression of somatic expansion in HD and related disorders and provide a route to define the individual neuronal cell types that contribute to different HD clinical phenotypes.

genetics↗

bioRxiv2022

Repeat Detector: accurate, efficient, and flexible sizing of expanded CAG/CTG repeats from targeted DNA sequencing

Targeted DNA sequencing approaches will improve how the size of short tandem repeats is measured for diagnostic tests and pre-clinical studies. The expansion of these sequences causes dozens of disorders, with longer tracts generally leading to a more severe disease. Interrupted alleles are sometimes present within repeats and can alter disease manifestation. Determining repeat size mosaicism and identifying interruptions in targeted sequencing datasets remains a major challenge. This is in part because standard alignment tools are ill-suited for repetitive and unstable sequences. To address this, we have developed Repeat Detector (RD), a deterministic profile weighting algorithm for counting repeats in targeted sequencing data. We tested RD using blood-derived DNA samples from Huntingtons disease and Fuchs endothelial corneal dystrophy patients sequenced using either Illumina MiSeq or Pacific Biosciences single-molecule, real-time sequencing platforms. RD was highly accurate in determining repeat sizes of 609 blood-derived samples from Huntingtons disease individuals and did not require prior knowledge of the flanking sequences. Furthermore, RD can be used to identify alleles with interruptions and provide a measure of repeat instability within an individual. RD is therefore highly versatile and may find applications in the diagnosis of expanded repeat disorders and the development of novel therapies.

neuroscience↗

bioRxiv2021

FAN1 nuclease activity affects CAG expansion and age at onset of Huntington's disease

The age at onset of motor symptoms in Huntingtons disease (HD) is driven by HTT CAG repeat length but modified by other genes. We used exome sequencing of 683 HD patients with extremes of onset or phenotype relative to CAG length to identify rare variants associated with clinical effect. We identified damaging coding variants in candidate modifier genes from prior genome-wide association studies associated with altered HD onset or severity. Variants in FAN1 clustered in its DNA-binding and nuclease domains and were associated predominantly with earlier onset HD. Nuclease activities of these variants correlated with residual age at motor onset of HD. Mutating endogenous FAN1 to a nuclease-inactive form in an induced pluripotent stem cell model of HD led to rates of CAG expansion comparable to those observed with complete FAN1 knock out. Together, these data implicate FAN1 nuclease activity in slowing somatic repeat expansion and hence onset of HD.

neuroscience↗

bioRxiv2019

Towards development of a statistical framework to evaluate myotonic dystrophy type 1 mRNA biomarkers in the context of a clinical trial

Myotonic dystrophy type 1 (DM1) is a rare genetic disorder, characterised by muscular dystrophy, myotonia, and other symptoms. DM1 is caused by the expansion of a CTG repeat in the 3-untranslated region of DMPK. Longer CTG expansions are associated with greater symptom severity and earlier age at onset. The primary mechanism of pathogenesis is thought to be mediated by a gain of function of the CUG-containing RNA, that leads to trans-dysregulation of RNA metabolism of many other genes. Specifically, the alternative splicing (AS) and alternative polyadenylation (APA) of many genes is known to be disrupted. In the context of clinical trials of emerging DM1 treatments, it is important to be able to objectively quantify treatment efficacy at the level of molecular biomarkers. We show how previously described candidate mRNA biomarkers can be used to model an effective reduction in CTG length, using modern high-dimensional statistics (machine learning), and a blood and muscle mRNA microarray dataset. We show how this model could be used to detect treatment effects in the context of a clinical trial.

bioinformatics↗

bioRxiv2019

Huntington's disease onset is determined by length of uninterrupted CAG, not encoded polyglutamine, and is modified by DNA maintenance mechanisms

The effects of variable, glutamine-encoding, CAA interruptions indicate that a property of the uninterrupted HTT CAG repeat sequence, distinct from huntingtins polyglutamine segment, dictates the rate at which HD develops. The timing of onset shows no significant association with HTT cis-eQTLs but is influenced, sometimes in a sex-specific manner, by polymorphic variation at multiple DNA maintenance genes, suggesting that the special onset-determining property of the uninterrupted CAG repeat is a propensity for length instability that leads to its somatic expansion. Additional naturally-occurring genetic modifier loci, defined by GWAS, may influence HD pathogenesis through other mechanisms. These findings have profound implications for the pathogenesis of HD and other repeat diseases and question a fundamental premise of the "polyglutamine disorders".

genetics↗