1. IPSC-Derived Corneal Endothelial-like Cells Act as an Appropriate Model System to Assess the Impact of SLC4A11 Variants on Pre-mRNA Splicing
- Author
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Kristyna, Brejchova, Lubica, Dudakova, Pavlina, Skalicka, Robert, Dobrovolny, Petr, Masek, Martina, Putzova, Mariya, Moosajee, Stephen J, Tuft, Alice E, Davidson, and Petra, Liskova
- Subjects
Adult ,Male ,Adolescent ,induced pluripotent stem cells ,Hearing Loss, Sensorineural ,RNA Splicing ,Anion Transport Proteins ,Antiporters ,Young Adult ,RNA Precursors ,Genetics ,Humans ,Child ,corneal endothelial-like cells model ,Cells, Cultured ,Aged ,Corneal Dystrophies, Hereditary ,Endothelium, Corneal ,Cell Differentiation ,Middle Aged ,Pedigree ,Gene Expression Regulation ,congenital hereditary endothelial dystrophy ,Child, Preschool ,RNA ,Female ,SLC4A11 - 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.
- Published
- 2019