IPSC-Derived Corneal Endothelial-like Cells Act as an Appropriate Model System to Assess the Impact of SLC4A11 Variants on Pre-mRNA Splicing.
Adolescent
Adult
Aged
Anion Transport Proteins
/ biosynthesis
Antiporters
/ biosynthesis
Cell Differentiation
Cells, Cultured
Child
Child, Preschool
Corneal Dystrophies, Hereditary
/ genetics
Endothelium, Corneal
/ metabolism
Female
Gene Expression Regulation
Hearing Loss, Sensorineural
/ genetics
Humans
Induced Pluripotent Stem Cells
/ cytology
Male
Middle Aged
Pedigree
RNA
/ genetics
RNA Precursors
RNA Splicing
Young Adult
Journal
Investigative ophthalmology & visual science
ISSN: 1552-5783
Titre abrégé: Invest Ophthalmol Vis Sci
Pays: United States
ID NLM: 7703701
Informations de publication
Date de publication:
01 07 2019
01 07 2019
Historique:
entrez:
20
7
2019
pubmed:
20
7
2019
medline:
18
12
2019
Statut:
ppublish
Résumé
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. 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. 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. This proof-of-concept study highlights the potential of using CE-like cells to investigate the pathogenic consequences of SLC4A11 disease-associated variants.
Identifiants
pubmed: 31323090
pii: 2738678
doi: 10.1167/iovs.19-26930
pmc: PMC6645617
doi:
Substances chimiques
Anion Transport Proteins
0
Antiporters
0
RNA Precursors
0
SLC4A11 protein, human
0
RNA
63231-63-0
Types de publication
Journal Article
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
3084-3090Subventions
Organisme : Department of Health
Pays : United Kingdom
Organisme : Wellcome Trust
Pays : United Kingdom
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