TY  - INPR
AV  - public
A1  - Hafford-Tear, NJ
A1  - Tsai, Y-C
A1  - Sadan, AN
A1  - Sanchez-Pintado, B
A1  - Zarouchlioti, C
A1  - Maher, GJ
A1  - Liskova, P
A1  - Tuft, SJ
A1  - Hardcastle, AJ
A1  - Clark, TA
A1  - Davidson, AE
SN  - 1530-0366
KW  - Fuchs endothelial corneal dystrophy
KW  -  amplification-free sequencing
KW  -  no-amp targeted sequencing
KW  -  somatic mosaicism
KW  -  triplet repeat-mediated disease
N2  - 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 (n?=?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 (?50 repeats) phased CTG18.1 alleles analyzed. Furthermore, higher levels of repeat instability were associated with increased CTG18.1 allele length (mode length ?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.
JF  - Genetics in Medicine
ID  - discovery10067655
Y1  - 2019/02/08/
UR  - https://doi.org/10.1038/s41436-019-0453-x
TI  - CRISPR/Cas9-targeted enrichment and long-read sequencing of the Fuchs endothelial corneal dystrophy?associated TCF4 triplet repeat
N1  - © The Author(s) 2019. This article is licensed under a Creative Commons Attribution 4.0 International License (https://creativecommons.org/licenses/by/4.0/).
ER  -