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IPSC-Derived Corneal Endothelial-like Cells Act as an Appropriate Model System to Assess the Impact of SLC4A11 Variants on Pre-mRNA Splicing

Brejchova, K; Dudakova, L; Skalicka, P; Dobrovolny, R; Masek, P; Putzova, M; Moosajee, M; ... Liskova, P; + view all (2019) IPSC-Derived Corneal Endothelial-like Cells Act as an Appropriate Model System to Assess the Impact of SLC4A11 Variants on Pre-mRNA Splicing. Investigative Ophthalmology & Visual Science , 60 (8) pp. 3084-3090. 10.1167/iovs.19-26930. Green open access

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Abstract

Purpose: To report molecular genetic findings in six probands with congenital hereditary endothelial dystrophy (CHED) variably associated with hearing loss (also known as Harboyan syndrome). Furthermore, we developed a cellular model to determine if disease-associated variants induce aberrant SLC4A11 pre-mRNA splicing. Methods: Direct sequencing of the entire SLC4A11 coding region was performed in five probands. In one individual, whole genome sequencing was undertaken. The effect of c.2240+5G>A on pre-mRNA splicing was evaluated in a corneal endothelial-like (CE-like) cell model expressing SLC4A11. CE-like cells were derived from autologous induced pluripotent stem cells (iPSCs) via neural crest cells exposed to B27, PDGF-BB, and DKK-2. Total RNA was extracted, and RT-PCR was performed followed by Sanger and a targeted next generation sequencing (NGS) approach to identify and quantify the relative abundance of alternatively spliced transcripts. Results: In total, 11 different mutations in SLC4A11 evaluated as pathogenic were identified; of these, c.1237G>A, c.2003T>C, c.1216+1G>A, and c.2240+5G>A were novel. The c.2240+5G>A variant was demonstrated to result in aberrant pre-mRNA splicing. A targeted NGS approach confirmed that the variant introduces a leaky cryptic splice donor site leading to the production of a transcript containing an insertion of six base pairs with the subsequent introduction of a premature stop codon (p.Thr747*). Furthermore, a subset of transcripts comprising full retention of intron 16 also were observed, leading to the same functionally null allele. Conclusions: This proof-of-concept study highlights the potential of using CE-like cells to investigate the pathogenic consequences of SLC4A11 disease-associated variants.

Type: Article
Title: IPSC-Derived Corneal Endothelial-like Cells Act as an Appropriate Model System to Assess the Impact of SLC4A11 Variants on Pre-mRNA Splicing
Location: United States
Open access status: An open access version is available from UCL Discovery
DOI: 10.1167/iovs.19-26930
Publisher version: https://doi.org/10.1167/iovs.19-26930
Language: English
Additional information: Copyright © 2019 The Authors. This work is licensed under a Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0).
UCL classification: UCL
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Brain Sciences
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Brain Sciences > Institute of Ophthalmology
URI: https://discovery.ucl.ac.uk/id/eprint/10078629
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