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Genet Med. 2019 Feb 8. doi: 10.1038/s41436-019-0453-x. [Epub ahead of print]

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

Author information

1
UCL Institute of Ophthalmology, London, UK.
2
Pacific Biosciences, Menlo Park, CA, USA.
3
Clinical Genetics Group, MRC Weatherall Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital, Oxford, UK.
4
Department of Ophthalmology, First Faculty of Medicine, Charles University and General University Hospital in Prague, Prague, Czech Republic.
5
Moorfields Eye Hospital, London, UK.
6
UCL Institute of Ophthalmology, London, UK. alice.davidson@ucl.ac.uk.

Abstract

PURPOSE:

To demonstrate the utility of an amplification-free long-read sequencing method to characterize the Fuchs endothelial corneal dystrophy (FECD)-associated intronic TCF4 triplet repeat (CTG18.1).

METHODS:

We applied an amplification-free method, utilizing 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 (nā€‰=ā€‰11) were analyzed. A robust data analysis pipeline was developed to effectively filter, align, and interrogate CTG18.1-specific reads. All results were compared with conventional polymerase chain reaction (PCR)-based fragment analysis.

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 (ā‰„50 repeats) phased CTG18.1 alleles analyzed. Furthermore, higher levels of repeat instability were associated with increased CTG18.1 allele length (mode length ā‰„91 repeats) indicating that expanded alleles behave dynamically.

CONCLUSION:

CRISPR-guided SMRT sequencing of CTG18.1 has revealed novel insights into CTG18.1 length instability. 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 and sight threatening disease.

KEYWORDS:

Fuchs endothelial corneal dystrophy; amplification-free sequencing; no-amp targeted sequencing; somatic mosaicism; triplet repeat-mediated disease

PMID:
30733599
DOI:
10.1038/s41436-019-0453-x

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