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

Marc Ciosi¹, Sarah A Cumming¹, Afroditi Chatzi¹

¹Institute of Molecular, Cell and Systems Biology, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, UK.

Journal of Huntington'S Disease

|February 13, 2021

Summary

PCR sequencing can analyze Huntington's disease (HD) alleles but struggles with very large repeat expansions. This method is limited for quantifying somatic mosaicism in alleles exceeding 250 CAG repeats.

Keywords:

Huntington disease Somatic mosaicism huntingtin parallel sequencing repeat expansion

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Area of Science:

Neurogenetics
Molecular Biology

Background:

Huntington's disease (HD) is a neurodegenerative disorder caused by expanded CAG repeats in the HTT gene.
Somatic instability of these repeats contributes to HD onset, severity, and progression.
Traditional methods for assessing somatic mosaicism have limitations.

Purpose of the Study:

To evaluate PCR sequencing for genotyping large HTT CAG repeat alleles in HD.
To assess the utility of PCR sequencing in quantifying somatic mosaicism in HD.

Main Methods:

Applied MiSeq and PacBio sequencing to PCR products of HTT CAG repeats in R6/2 mice.
Examined alleles with approximately 55, 110, 255, and 470 CAG repeats.
Compared repeat length distributions across different tissues and ages.

Main Results:

Full-length sequencing of the CAG repeat was successful for all samples.
Repeat length distributions for alleles with ~470 CAGs showed bias towards shorter lengths.

Conclusions:

PCR sequencing can genotype HD alleles but is unsuitable for quantifying somatic mosaicism in alleles with >250 CAGs.
Limitations of PCR sequencing for large alleles are discussed.
Alternative methods for quantifying extreme somatic expansions and contractions are considered.