Functional Analysis of a Novel, Non-Canonical RPGR Splice Variant Causing X-Linked Retinitis Pigmentosa

Samuel Koller; Tim Beltraminelli; Jordi Maggi; Agnès Wlodarczyk; Silke Feil; Luzy Baehr; Christina Gerth-Kahlert; Moreno Menghini; Wolfgang Berger

doi:10.3390/genes14040934

Functional Analysis of a Novel, Non-Canonical RPGR Splice Variant Causing X-Linked Retinitis Pigmentosa

Genes (Basel). 2023 Apr 18;14(4):934. doi: 10.3390/genes14040934.

Authors

Affiliations

¹ Institute of Medical Molecular Genetics, University of Zurich, 8952 Schlieren, Switzerland.
² Department of Ophthalmology, Institute of Clinical Neurosciences of Southern Switzerland, Ente Ospedaliero Cantonale (EOC), 6962 Lugano, Switzerland.
³ Department of Ophthalmology, University Hospital, University of Zurich, 8091 Zurich, Switzerland.
⁴ Zurich Center for Integrative Human Physiology (ZIHP), University of Zurich, 8057 Zurich, Switzerland.
⁵ Neuroscience Center Zurich (ZNZ), University and ETH Zurich, 8057 Zurich, Switzerland.

Abstract

X-linked retinitis pigmentosa (XLRP) caused by mutations in the RPGR gene is one of the most severe forms of RP due to its early onset and intractable progression. Most cases have been associated with genetic variants within the purine-rich exon ORF15 region of this gene. RPGR retinal gene therapy is currently being investigated in several clinical trials. Therefore, it is crucial to report and functionally characterize (all novel) potentially pathogenic DNA sequence variants. Whole-exome sequencing (WES) was performed for the index patient. The splicing effects of a non-canonical splice variant were tested on cDNA from whole blood and a minigene assay. WES revealed a rare, non-canonical splice site variant predicted to disrupt the wildtype splice acceptor and create a novel acceptor site 8 nucleotides upstream of RPGR exon 12. Reverse-transcription PCR analyses confirmed the disruption of the correct splicing pattern, leading to the insertion of eight additional nucleotides in the variant transcript. Transcript analyses with minigene assays and cDNA from peripheral blood are useful tools for the characterization of splicing defects due to variants in the RPGR and may increase the diagnostic yield in RP. The functional analysis of non-canonical splice variants is required to classify those variants as pathogenic according to the ACMG's criteria.

Keywords: RPGR; minigene; non-canonical splice variant; retinitis pigmentosa.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

DNA, Complementary
Eye Proteins* / genetics
Humans
Mutation
Retina
Retinitis Pigmentosa* / diagnosis
Retinitis Pigmentosa* / genetics

Substances

Eye Proteins
DNA, Complementary
RPGR protein, human

Grants and funding

This research was partially funded by Velux Stiftung, grant number 1371 (to W.B.). M.M. received an unrestricted research grant from Bayer A.G. Switzerland.