Milton Wexler Biennial Symposium, 2022

Development of novel methods to quantify somatic CAG repeat expansions in Huntington’s Disease

Shota Shibata1,2, Muzhou Wu1, Sean Koebley3, Andrey Mikheikin3,4, T. Christian Boles5, Jason Reed3, Ricardo Mouro Pinto1,2 

[1 ]Center for Genomic Medicine, Massachusetts General Hospital; [2] Department of Neurology, Harvard Medical School; [3] Department of Physics
and Massey Cancer Center, Virginia Commonwealth University; [4] Evizia, Inc., [5] Sage Science, Inc.

Acknowledgement: Berman/Topper HD Career Development Fellowship, Huntington’s Disease Society of America, National Institute of Neurological Disorders and Stroke

Abstract

Current methodology used to quantify HTT CAG repeat expansions suffers from reduced sensitivity and/or inability to detect large expansions. Most methods require PCR amplification of the repeat which is biased towards amplification of shorter alleles. This can result in an underestimation of the actual extent of CAG expansions in a patient’s sample.

While small-pool PCR followed by Southern blot detection can overcome some of these limitations, this method lacks size resolution, takes days to perform, and has a very small throughput. Next generation short-read sequencing methods can measure a large number of alleles and samples in a single run, as well as provide valuable information in terms of repeat structure (eg. CAA interruptions), but they are unable to generate information on longer alleles (max ~150 CAGs). Capillary-based electrophoresis is the most commonly used assay for its improved resolution, sensitivity and relatively low cost.

However, it is also sensitive to PCR bias and has a maximum detection range of ~200 CAGs. Since somatic expansions >1,000 CAGs have been reported in the striatum of HD mutation carriers, as determined by small-pool PCR and Southern blot analysis, this represents a significant shortcoming of existing methods.

In attempt to address this need, we hereby present preliminary data on the development of two novel methods for HTT CAG repeat quantification:

1. Single-molecule long-read sequencing: This method combines long-read single-molecule next generation sequencing (Pacbio) with the incorporation of unique molecular barcodes, allowing for deduplication of PCR amplicons and therefore circumvent PCR bias for amplification of shorter alleles.

In addition, due to incorporation of unique sample barcodes, this method facilitates the simultaneous quantification of long trinucleotide repeats in multiple samples, from multiple patients at the same time, therefore making for better patient-to-patient or tissue-to-tissue comparisons and substantially reducing costs.

Finally, since this is a sequencing-based method, it provides important information on repeat composition and variants such as CAA interruptions, which have been reported as modifiers of HD onset.

2. Digital PCR and high-speed atomic force microscopy (HSAFM): this is a single-molecule imaging method that accurately measures the length of amplicons from individual digital PCR reactions, thereby avoiding the PCR bias for shorter alleles.

In addition, through automation of the HSAFM measurement process, the method can be scaled to rapidly measure thousands of positive digital PCR reactions.

Huntington’s disease and somatic instability

The CAG repeat expansion is the causative mutation of HD.
The length of the expanded CAG is a significant determinant of the onset age of HD.

The somatic instability of the expanded CAG repeats is itself a significant predictor of the onset age of HD.
This necessitate the evaluation of the expanded repeats at the single molecule resolution.

There is an unmet need for efficient methods of somatic repeat instability characterization.
Ideally, the method needs: (1) single-molecule resolution, (2) high sizing accuracy, (3) ability to detect large repeat expansions, (4) high throughput, and (5) cost efficiency.

Single-molecule long-read sequencing

The schematic workflow of dual-tagging strategy with single-molecule real-time (SMRT) sequencing

The dual-tagging strategy utilize the molecular-tags and the sample-tags.
The molecular consensus sequences are obtained among the reads with the same molecular-tags. This process corrects the PCR biases. The sample-tags enable the multiplexing of samples.
The one-to-one correspondence of the template dsDNA and the circular consensus sequence (CCS) is preserved in the process of SMRT sequencing.
Combining the tagging-strategy and SMRT sequencing, this strategy regain the single-molecule resolution in starting bulk material of multiple samples.

Digital PCR & high-speed atomic force microscopy

The schematic workflow of dPCR combined with HSAFM

Single-molecule resolution and rare variant detection sensitivity can be achieved by limited dilution digital PCR (dPCR).
The speed and comparatively low cost can be achieved by high-throughput HSAFM technology.

Single-molecule long-read sequencing

Proof of concept: (example from non-CAG repeat amplicons)

The dual-tagging strategy was applied to triplet repeat-containing PCR amplicons from multiple samples.
The reads assigned to each sample were identified.
The de-tagging process correcting the PCR bias narrowed down the distributions of the read lengths and the repeat units, resulting in successful calling of repeat lengths at single-molecule resolution.

Digital PCR & high-speed atomic force microscopy

Proof of concept (examples from different datasets)

The distribution of fragment sizes in each reaction is obtained by automated analysis of HSAFM images. The representative fragment sizes for each reaction are determined (A).
The repeat units were deduced from the fragment sizes (B).
The spiked-in rare somatic repeat expansions were successfully detected, proving the high sensitivity of this strategy (C).

Conclusion

Technological limitations reside in the evaluation of somatic repeat instability despite its striking importance.
None of the single applications have sufficient performance for this purpose. Therefore, combination of multiple approaches is necessary to achieve the affordable balance of cost and efficiency.
The two novel methods, 1) the longread sequencing with dual-tagging strategy and 2) the dPCR with HSAFM, were both demonstrated here to be the potential candidates.
Efficient quantification measures of somatic mosaicism would contribute to the elucidation of the pathological mechanism and to the development of feasible biomarkers of Huntington’s Disease.