Journal article
Exonic splice variant discovery using in vitro models of inherited retinal disease
HGG advances, Vol.6(1), 100357
01/2025
DOI: 10.1016/j.xhgg.2024.100357
PMCID: PMC11550365
PMID: 39354715
Abstract
Correct identification of the molecular consequences of pathogenic genetic variants is essential to the development of allele-specific therapies. However, such molecular effects may remain ambiguous following genetic sequence analysis alone. Here, we identify exonic codon-altering variants that are also predicted to disrupt normal RNA splicing in the context of inherited retinal disease. NR2E3 c.932G>A (p.Arg311Gln) is a variant commonly associated with Enhanced S Cone Syndrome (ESCS). Previous studies using mutagenized cDNA constructs have shown that the arginine to glutamine substitution at position 311 of NR2E3 does not meaningfully diminish function of the rod-specific transcription factor. Using retinal organoids, we explored the molecular consequences of NR2E3 c.932G>A when expressed endogenously during human rod photoreceptor cell development. Retinal organoids carrying the NR2E3 c.932G>A allele expressed a transcript containing a 186-nucleotide deletion of exon 6 within the ligand binding domain. This short transcript was not detected in control organoids or control human donor retina samples. A minigene containing exons 5 and 6 of NR2E3 showed sufficiency of the c.932G>A variant to cause the observed splicing defect. These results support the hypothesis that the pathogenic NR2E3 c.932G>A variant leads to photoreceptor disease by causing a splice defect and not through an amino acid substitution as previously supposed. They also explain the relatively mild effect of Arg311Gln on NR2E3 function in vitro. We also used in silico prediction tools to show that similar changes are likely to affect other inherited retinal disease variants in genes such as CEP290, ABCA4, and BEST1.
Using patient-derived retinal organoids, minigene assays, and in silico prediction, this study shows that nonsynonymous exonic variants can act through splice alteration in inherited retinal disease. The NR2E3 variant c.932G>A (R311Q) causes aberrant splicing when expressed in retinal cells, contrary to previous reports of this variant’s mechanism.
Details
- Title: Subtitle
- Exonic splice variant discovery using in vitro models of inherited retinal disease
- Creators
- Nathaniel K. Mullin - University of IowaLaura R. Bohrer - University of IowaKristin R. Anfinson - University of IowaJeaneen L. Andorf - University of Iowa, The University of Iowa Institute for Vision ResearchRobert F. Mullins - University of IowaBudd A. Tucker - University of IowaEdwin M. Stone - University of Iowa
- Resource Type
- Journal article
- Publication Details
- HGG advances, Vol.6(1), 100357
- DOI
- 10.1016/j.xhgg.2024.100357
- PMID
- 39354715
- PMCID
- PMC11550365
- NLM abbreviation
- HGG Adv
- ISSN
- 2666-2477
- eISSN
- 2666-2477
- Publisher
- Elsevier Inc
- Grant note
- NIH: T32GM139776, F30EY034009 University of Iowa Institute for Vision ResearchUniversity of Iowa Carver College of Medicine
This work was funded in part by NIH grants T32GM139776 and F30EY034009, and by the University of Iowa Institute for Vision Research. We acknowledge the extraordinary generosity of the patients whose donation of material made this study possible. We also thank the members of the Institute for Vision Research for their helpful conversation and advice. Data presented herein were obtained at the Genomics Division of the Iowa Institute of Human Genetics, which is supported, in part, by the University of Iowa Carver College of Medicine. We acknowledge specifically the advice and support of M. Boes, G. Hauser, K. Knudtson, A. Sheehan, and E. Snir.
- Language
- English
- Electronic publication date
- 09/30/2024
- Date published
- 01/2025
- Academic Unit
- The University of Iowa Institute for Vision Research; Iowa Neuroscience Institute; John and Marcia Carver Nonprofit Genetic Testing Laboratory; Neuroscience and Pharmacology; Ophthalmology and Visual Sciences
- Record Identifier
- 9984721248202771
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