June 2022
Volume 63, Issue 7
Open Access
ARVO Annual Meeting Abstract  |   June 2022
X-linked retinitis pigmentosa caused by a non-canonical splice variant in RPGR
Author Affiliations & Notes
  • Samuel Koller
    Medical Molecular Genetics, Universitat Zurich Medizinische Fakultat, Zurich, ZH, Switzerland
  • Tim Beltramelli
    Ophthalmology, Ospedale Regionale di Lugano, Lugano, Ticino, Switzerland
  • Agnès Wlodarczyk
    Medical Molecular Genetics, Universitat Zurich Medizinische Fakultat, Zurich, ZH, Switzerland
  • Silke Feil
    Medical Molecular Genetics, Universitat Zurich Medizinische Fakultat, Zurich, ZH, Switzerland
  • Lucy Bähr
    Medical Molecular Genetics, Universitat Zurich Medizinische Fakultat, Zurich, ZH, Switzerland
  • Christina Gerth-Kahlert
    Ophthalmology, UniversitatsSpital Zurich, Zurich, Switzerland
  • Moreno Menghini
    Ophthalmology, Ospedale Regionale di Lugano, Lugano, Ticino, Switzerland
  • Wolfgang Berger
    Medical Molecular Genetics, Universitat Zurich Medizinische Fakultat, Zurich, ZH, Switzerland
    Universitat Zurich Zurich Center for Integrative Human Physiology, Zurich, Switzerland
  • Footnotes
    Commercial Relationships   Samuel Koller None; Tim Beltramelli None; Agnès Wlodarczyk None; Silke Feil None; Lucy Bähr None; Christina Gerth-Kahlert None; Moreno Menghini None; Wolfgang Berger None
  • Footnotes
    Support  None
Investigative Ophthalmology & Visual Science June 2022, Vol.63, 504 – A0081. doi:
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      Samuel Koller, Tim Beltramelli, Agnès Wlodarczyk, Silke Feil, Lucy Bähr, Christina Gerth-Kahlert, Moreno Menghini, Wolfgang Berger; X-linked retinitis pigmentosa caused by a non-canonical splice variant in RPGR. Invest. Ophthalmol. Vis. Sci. 2022;63(7):504 – A0081.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose : Variants in RPGR are associated with severe forms of retinitis pigmentosa (RP) and usually found within the purine rich exon ORF15. Pathogenic variants can also be found in non-canonical splice sites (NCSS), but may be classified as VUS (variant of unknown significance) by ACMG (American College of Medical Genetics) guidelines. We investigated the effect of an NCCS variant 9 nucleotides in front of RPGR exon 12 on splicing by mini-gene assay and whole-blood RNA analysis.

Methods : Clinical diagnosis was established by comprehensive ophthalmic exam and multi-modal imaging of the patient and his mother.
Patient DNA was analyzed by whole exome sequencing. Segregation analysis was performed by Sanger sequencing. Effects of a potential splice variant was analyzed by cDNA derived from a mini-gene assay and a whole-blood sample from the patient and his mother.

Results : A rare NCSS variant was found in a patient with X-linked retinitis pigmentosa (RP) in front of RPGR exon 12, which was predicted to create a novel splice acceptor site 8 nucleotides upstream of exon 12.
Sanger sequencing confirmed the carrier status of the patient’s mother, who showed a tapetal-like fundus reflex as assessed by multi-modal imaging.
Analysis of cDNA form a mini gene assay, designed to study this variant, confirmed the predicted novel acceptor site. This specific acceptor site was not active in the control mini-gene construct which contained the reference sequence. The novel acceptor site was confirmed by the analysis of cDNA generated from a whole blood samples of the proband and his mother, who is a confirmed carrier of the variant.

Conclusions : Care has to be taken by the interpretation of non-canonical splice site variants by ACMG guidelines, as they could be pathogenic. Only a functional assay can classify NCSS as likely pathogenic. It has been suggested that contribution of NCSS variants is underestimated by 35%-40%.
If no blood sample for RNA extraction is available, a mini-gene experiment/approach may be a reliable and a fast assay to characterize NCSS variants in RPGR.
We have shown that NCSS variants can contribute to the diagnostic yield in (RPGR) X-linked RP. These variants should be analyzed on transcript level in order to identify a splicing effect but also for designing therapeutic approaches in the future.

This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.

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