Methods for Assessing DNA Repair and Repeat Expansion in Huntington’s Disease

Massey, Thomas; McAllister, Branduff; Jones, Lesley

doi:10.1007/978-1-4939-7825-0_22

Cited by 8 publications

(5 citation statements)

References 71 publications

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“…Somatic CAG tract instability is an exciting and validated course of investigation, and has been reviewed elsewhere [21,22]. The purpose of this review is to discuss alternate hypotheses, as well as the importance of neuronal metabolism in the context of defective DNA damage repair.…”

Section: Hd Gwas Brings Unbiased Hypotheses Back To Huntington's Diseasementioning

confidence: 99%

DNA Repair in Huntington’s Disease and Spinocerebellar Ataxias: Somatic Instability and Alternative Hypotheses

Maiuri

Hung

Suart

et al. 2021

JHD

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The use of genome wide association studies (GWAS) in Huntington’s disease (HD) research, driven by unbiased human data analysis, has transformed the focus of new targets that could affect age at onset. While there is a significant depth of information on DNA damage repair, with many drugs and drug targets, most of this development has taken place in the context of cancer therapy. DNA damage repair in neurons does not rely on DNA replication correction mechanisms. However, there is a strong connection between DNA repair and neuronal metabolism, mediated by nucleotide salvaging and the poly ADP-ribose (PAR) response, and this connection has been implicated in other age-onset neurodegenerative diseases. Validation of leads including the mismatch repair protein MSH3, and interstrand cross-link repair protein FAN1, suggest the mechanism is driven by somatic CAG instability, which is supported by the protective effect of CAA substitutions in the CAG tract. We currently do not understand: how somatic instability is triggered; the state of DNA damage within expanding alleles in the brain; whether this damage induces mismatch repair and interstrand cross-link pathways; whether instability mediates toxicity, and how this relates to human ageing. We discuss DNA damage pathways uncovered by HD GWAS, known roles of other polyglutamine disease proteins in DNA damage repair, and a panel of hypotheses for pathogenic mechanisms.

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Section: Hd Gwas Brings Unbiased Hypotheses Back To Huntington's Diseasementioning

confidence: 99%

DNA Repair in Huntington’s Disease and Spinocerebellar Ataxias: Somatic Instability and Alternative Hypotheses

Maiuri

Hung

Suart

et al. 2021

JHD

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“…In this context, precise and accurate quantification of HTT CAG somatic mosaicism in patient samples and model systems is warranted, since it may provide a suitable phenotype for genetic association studies aimed at identifying genetic variants that act as modifiers of repeat instability [7,15]. Somatic mosaicism in HD patients and model organisms is traditionally assessed by gel or capillary electrophoresis of single-molecule, small pool (SP) or bulk-PCR (PCR using thousands of DNA molecules as a template) products [26]. However, using electrophoresis to estimate the number of repeats in the PCR fragments detected does not provide any information about genetic variants within and around the HTT CAG repeat, which can lead to erroneous electrophoresis estimates of the number of pure CAG repeats [7].…”

Section: Introductionmentioning

confidence: 99%

Approaches to Sequence the HTT CAG Repeat Expansion and Quantify Repeat Length Variation

Ciosi

Cumming

Chatzi

et al. 2021

JHD

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Background: Huntington’s disease (HD) is an autosomal dominant neurodegenerative disorder caused by the expansion of the HTT CAG repeat. Affected individuals inherit ≥36 repeats and longer alleles cause earlier onset, greater disease severity and faster disease progression. The HTT CAG repeat is genetically unstable in the soma in a process that preferentially generates somatic expansions, the proportion of which is associated with disease onset, severity and progression. Somatic mosaicism of the HTT CAG repeat has traditionally been assessed by semi-quantitative PCR-electrophoresis approaches that have limitations (e.g., no information about sequence variants). Genotyping-by-sequencing could allow for some of these limitations to be overcome. Objective: To investigate the utility of PCR sequencing to genotype large (>50 CAGs) HD alleles and to quantify the associated somatic mosaicism. Methods: We have applied MiSeq and PacBio sequencing to PCR products of the HTT CAG repeat in transgenic R6/2 mice carrying ∼55, ∼110, ∼255 and ∼470 CAGs. For each of these alleles, we compared the repeat length distributions generated for different tissues at two ages. Results: We were able to sequence the CAG repeat full length in all samples. However, the repeat length distributions for samples with ∼470 CAGs were biased towards shorter repeat lengths. Conclusion: PCR sequencing can be used to sequence all the HD alleles considered, but this approach cannot be used to estimate modal allele size or quantify somatic expansions for alleles ⪢250 CAGs. We review the limitations of PCR sequencing and alternative approaches that may allow the quantification of somatic contractions and very large somatic expansions.

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“…Nevertheless, given this is likely a stochastic process there might be surviving cells at different points in the pathological trajectory that could be used in single cell experiments to define the pathogenic CAG tract length threshold. There are methods to sequence and size the HTT CAG tract accurately [264,265], which could potentially be applied to single cells, but these would have to be tied to the single cell RNA gene expression data-achievable, but technically challenging.…”

Section: What Evidence Do We Need To Refine Our Definition Of the Intmentioning

confidence: 99%

What is the Pathogenic CAG Expansion Length in Huntington’s Disease?

Donaldson

Powell

Rickards

et al. 2021

JHD

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Huntington’s disease (HD) (OMIM 143100) is caused by an expanded CAG repeat tract in the HTT gene. The inherited CAG length is known to expand further in somatic and germline cells in HD subjects. Age at onset of the disease is inversely correlated with the inherited CAG length, but is further modulated by a series of genetic modifiers which are most likely to act on the CAG repeat in HTT that permit it to further expand. Longer repeats are more prone to expansions, and this expansion is age dependent and tissue-specific. Given that the inherited tract expands through life and most subjects develop disease in mid-life, this implies that in cells that degenerate, the CAG length is likely to be longer than the inherited length. These findings suggest two thresholds— the inherited CAG length which permits further expansion, and the intracellular pathogenic threshold, above which cells become dysfunctional and die. This two-step mechanism has been previously proposed and modelled mathematically to give an intracellular pathogenic threshold at a tract length of 115 CAG (95% confidence intervals 70– 165 CAG). Empirically, the intracellular pathogenic threshold is difficult to determine. Clues from studies of people and models of HD, and from other diseases caused by expanded repeat tracts, place this threshold between 60– 100 CAG, most likely towards the upper part of that range. We assess this evidence and discuss how the intracellular pathogenic threshold in manifest disease might be better determined. Knowing the cellular pathogenic threshold would be informative for both understanding the mechanism in HD and deploying treatments.

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Methods for Assessing DNA Repair and Repeat Expansion in Huntington’s Disease

Cited by 8 publications

References 71 publications

DNA Repair in Huntington’s Disease and Spinocerebellar Ataxias: Somatic Instability and Alternative Hypotheses

DNA Repair in Huntington’s Disease and Spinocerebellar Ataxias: Somatic Instability and Alternative Hypotheses

Approaches to Sequence the HTT CAG Repeat Expansion and Quantify Repeat Length Variation

What is the Pathogenic CAG Expansion Length in Huntington’s Disease?

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