Abstract
Spinocerebellar ataxias (SCAs) are caused by nucleotide repeat expansions that are intronic or exonic, the latter being translated as polyglutamine repeats. These translated exonic repeat expansion disorders are SCA1, SCA2, SCA3, SCA6, SCA7, SCA17, and dentatorubral-pallidoluysian atrophy (DRPLA). Other SCAs, i.e., SCA10, SCA31, SCA36, and SCA37, present with intronic repeat expansions and can be much longer than the polyglutamine repeats. It is not uncommon that such intronic repeat expansions comprise of several hundred or even thousand repeats; hence, the expansion can be between 2500 and 22,500 base pairs in length.
Such long repeats present a challenge for standard sequencing and detection techniques in the research and diagnostic setting. Most clinical diagnostics are PCR-based methods and Southern blotting; however, such methods have their shortcomings, and it is known that repeat sizing can vary between labs. In addition, next-generation sequencing technologies with short reads also have their limitations because of the repetitive nature of the repeats resulting in alignment problems.
Clinically, it has also become clear that repeat compositions, repeat interruptions, and mosaic variability can play a disease-modifying role and affect the symptoms, penetrance, and disease progression. Therefore, it is very important in the research and clinical setting to establish methods that provide (1) accurate mutant repeat size, (2) accurate mutant repeat composition at the nucleotide level, and (3) estimate of mosaicism of repeat length as these repeat regions can be highly mutable due to replication errors.
In this chapter, we describe the use of long-read sequencing using single-molecule real-time sequencing (SMRT) combined with clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 targeting of multiple loci, which allowed us to develop a 15-gene panel for repeat expansion disorders of spinocerebellar ataxias. The advantage of SMRT sequencing are the long reads of over 10,000 base pairs from amplification-free genomic DNA. CRISPR/Cas9 allows for targeting specific repeat loci and multiplexing them to develop a sequencing panel of repeat expansion SCAs relevant for both research applications and diagnostic development.
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Tsai, YC. et al. (2022). Multiplex CRISPR/Cas9-Guided No-Amp Targeted Sequencing Panel for Spinocerebellar Ataxia Repeat Expansions. In: Proukakis, C. (eds) Genomic Structural Variants in Nervous System Disorders. Neuromethods, vol 182. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2357-2_6
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DOI: https://doi.org/10.1007/978-1-0716-2357-2_6
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