CRISPR/Cas9-targeted enrichment and long-read sequencing of the Fuchs endothelial corneal dystrophy-associated TCF4 triplet repeat

Abstract

Purpose : To demonstrate the utility of an amplification-free long-read sequencing method to characterise the Fuchs endothelial corneal dystrophy (FECD)-associated intronic TCF4 triplet repeat (termed CTG18.1) at the nucleotide level.

Methods : We applied an amplification-free method, utilising the CRISPR/Cas9 system, in combination with PacBio single molecule real time (SMRT) long-read sequencing, to study CTG18.1. FECD patient samples displaying a diverse range of CTG18.1 allele lengths and zygosity status (n=11) were analysed. A robust data analysis pipeline was developed to effectively filter, align and interrogate CTG18.1 specific reads. We compared these results with a conventional PCR-based short tandem repeat (STR) assay.

Results : CRISPR-guided SMRT sequencing of CTG18.1 provided accurate genotyping information for all samples and phasing was possible for 18/22 alleles sequenced. Repeat length instability was observed for all expanded (≥50 repeats) phased CTG18.1 alleles analysed. Furthermore, higher levels of repeat instability were observed with increased CTG18.1 allele lengths (mode length ≥91 repeats) indicating that expanded alleles behave dynamically.

Conclusions : CRISPR-guided SMRT sequencing of CTG18.1 has revealed novel insights into CTG18.1 length instability and highlights that size estimates provided by conventional genotyping assays (e.g. STR analysis and Southern blot) do not provide a full representation of the dynamic nature of this repeat element in its expanded state. Furthermore, this study provides a framework to improve the molecular diagnostic accuracy for CTG18.1-mediated FECD, which we anticipate will become increasingly important as gene-directed therapies are developed for this common age-related disease.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.