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.

Biopticka laborator s r o Pilsen Czech Republic

Clinic of Ophthalmology University Hospital Ostrava Ostrava Czech Republic

Department of Craniofacial Surgery University of Ostrava Ostrava Czech Republic

Department of Ophthalmology 1st Faculty of Medicine Charles University and General University Hospital Prague Prague Czech Republic

Great Ormond Street Hospital for Children London United Kingdom

Moorfields Eye Hospital NHS Foundation Trust London United Kingdom

Research Unit for Rare Diseases Department of Pediatrics and Adolescent Medicine 1st Faculty of Medicine Charles University and General University Hospital Prague Czech Republic

UCL Institute of Ophthalmology London United Kingdom

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