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

Invest Ophthalmol Vis Sci. 2019 Jul 1;60(8):3084-3090. doi: 10.1167/iovs.19-26930.


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adolescent
  • Adult
  • Aged
  • Anion Transport Proteins / biosynthesis
  • Anion Transport Proteins / genetics*
  • Antiporters / biosynthesis
  • Antiporters / genetics*
  • Cell Differentiation
  • Cells, Cultured
  • Child
  • Child, Preschool
  • Corneal Dystrophies, Hereditary / genetics*
  • Corneal Dystrophies, Hereditary / metabolism
  • Corneal Dystrophies, Hereditary / pathology
  • Endothelium, Corneal / metabolism
  • Endothelium, Corneal / pathology*
  • Female
  • Gene Expression Regulation*
  • Hearing Loss, Sensorineural / genetics*
  • Hearing Loss, Sensorineural / metabolism
  • Hearing Loss, Sensorineural / pathology
  • Humans
  • Induced Pluripotent Stem Cells / cytology*
  • Induced Pluripotent Stem Cells / metabolism
  • Male
  • Middle Aged
  • Pedigree
  • RNA / genetics*
  • RNA Precursors
  • RNA Splicing
  • Young Adult


  • Anion Transport Proteins
  • Antiporters
  • RNA Precursors
  • SLC4A11 protein, human
  • RNA

Supplementary concepts

  • Corneal dystrophy and perceptive deafness