Abstract
PurposeTo 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.MethodsDirect 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.ResultsIn 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.ConclusionsThis proof-of-concept study highlights the potential of using CE-like cells to investigate the pathogenic consequences of SLC4A11 disease–associated variants.
Highlights
To report molecular genetic findings in six probands with congenital hereditary endothelial dystrophy (CHED) variably associated with hearing loss
A targeted next generation sequencing (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*)
This proof-of-concept study highlights the potential of using corneal endothelial-like (CE-like) cells to investigate the pathogenic consequences of SLC4A11 disease–associated variants
Summary
Direct sequencing of the entire SLC4A11 coding region was performed in five probands. Total RNA was extracted, and RT-PCR was performed followed by Sanger and a targeted generation sequencing (NGS) approach to identify and quantify the relative abundance of alternatively spliced transcripts. The study adhered to the tenets set out in the Declaration of Helsinki and was approved by the ethics committee of the General University Hospital in Prague (151/11 S-IV) and Moorfields Eye Hospital (13/LO/1084 and 09/H0724/25). All participants or their legal representatives signed informed consent before inclusion in the study. The frequency of the detected SLC4A11 variants was established from the Genome Aggregation Database (gnomAD; http://gnomad.broadinstitute.org/)[16] providing data on more than 120,000 individuals and in 4528 Czech chromosomes available through the NGS projects of the National Centre for Medical Genomics (https://ncmg.cz/en)
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