Approaches to sequence the HTT CAG repeat expansion and quantify repeat length variation

Ciosi, M. et al. (2021) Approaches to sequence the HTT CAG repeat expansion and quantify repeat length variation. Journal of Huntington's Disease, 10(1), pp. 53-74. (doi: 10.3233/jhd-200433) (PMID:33579864) (PMCID:PMC7990409)

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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.

Item Type:Articles
Additional Information:This work was supported by the CHDI Foundation (awards to D.G.M. and A.J.M.) and the National Institutes of Health USA (Grants Nos. R01NS049206 to V.C.W.).
Glasgow Author(s) Enlighten ID:Lomeikaite, Miss Vilija and Hamilton, Dr Graham and Monckton, Professor Darren and Tottey, Dr William and Cumming, Dr Sarah and Chatzi, Ms Afroditi and Larson, Eloise and Ciosi, Dr Marc
Authors: Ciosi, M., Cumming, S. A., Chatzi, A., Larson, E., Tottey, W., Lomeikaite, V., Hamilton, G., Wheeler, V. C., Pinto, R. M., Kwak, S., Morton, A. J., and Monckton, D. G.
College/School:College of Medical Veterinary and Life Sciences > Institute of Molecular Cell and Systems Biology
Journal Name:Journal of Huntington's Disease
Publisher:IOS Press
ISSN (Online):1879-6400
Copyright Holders:Copyright © 2021 – The authors
First Published:First published in Journal of Huntington's Disease 10(1):53-74
Publisher Policy:Reproduced under a Creative Commons Licence

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