Exonic splice variant discovery using in vitro models of inherited retinal disease

Abstract

ABSTRACTCorrect identification of the molecular consequences of pathogenic genetic variants is essential to the development of allele-specific therapies. However, in some cases, such molecular effects may be ambiguous following genetic sequence analysis alone. One such case is exonic, codon-altering variants that are also predicted to disrupt normal RNA splicing. Here, we identify such cases in the context of inherited retinal disease.NR2E3c.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 ofNR2E3c.932G>A when expressed endogenously during human rod photoreceptor cell development. Retinal organoids carrying theNR2E3c.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 ofNR2E3showed sufficiency of the c.932G>A variant to cause the observed splicing defect. These results support the hypothesis that the pathogenicNR2E3c.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 functionin vitro. We also usedin silicoprediction tools to show that similar changes are likely to affect other inherited retinal disease variants in genes such asCEP290, ABCA4, andBEST1.