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

Abstract

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.

Repeat Detector: versatile sizing of expanded tandem repeats and identification of interrupted alleles from targeted DNA sequencing

“…was a bias against longer repeats during PCR, loading of the SMRT flowcell, the sequencing reaction, and/or the generation of CCSs. These results are in line with recent findings suggesting that up to at least 550 CAG repeats can be sequenced using SMRT sequencing[30,31].…”

“…2). However, in PCR-based library preparation methods there may be slippage errors and other PCR artefacts that may contribute to size heterogeneity, and the distribution of repeat size may not be limited to biological variation [9,31]. Up to 80% of Fuchs endothelial corneal dystrophy (FECD) patients have an expansion of 50 or more CTGs in the third intron of TCF4 (termed CTG18.1) [52] Here, we analysed a high-quality amplification-free library generated from FECD patient-derived whole blood genomic DNA samples (n=11) displaying a diverse range of CTG18.1 allele lengths and zygosity status (Fig.…”

Modifiers of CAG/CTG Repeat Instability: Insights from Mammalian Models

“…Fragment analysis, e.g., using GeneMapper [111] is quicker and easier than the small-pool PCR, is not as sensitive to rare events but is also quantitative [87]. Methods for studying repeat instability are discussed in detail in [146]. A shuttle vector assay in which repeat length changes are detected directly has been used for mapping cis-acting elements, including origin of replication distance and direction as well as DNA methylation [129,147].…”

The Contribution of Somatic Expansion of the CAG Repeat to Symptomatic Development in Huntington’s Disease: A Historical Perspective

“…The high-throughput HTT sequencing assay developed by Ciosi et al also allows for the quantification of the relative ratio of somatic expansions in blood DNA (see Ciosi et al, this issue for comparison of approaches to quantifying somatic mosaicism in HD) [282], that after correcting for age and CAG length effects, results in an individual-specific somatic expansion score [262]. As expected, assuming that somatic expansion profiles in blood DNA at least broadly parallel those in the brain, the somatic expansion score was inversely associated with variation in age at onset and positively associated with individual-specific disease progression scores (i.e., individuals with a faster rate of somatic expansion have an earlier age at onset than expected and more rapid disease course).…”