July 2018
Volume 59, Issue 9
Open Access
ARVO Annual Meeting Abstract  |   July 2018
Functional study of two canonical splice site variants c.3877+1G>A and c.2992_2992+6delinsTG in the EYS gene associated with recessive retinitis pigmentosa in northern Sweden
Author Affiliations & Notes
  • Ida Maria Westin
    Department of Medical Biosciences, Faculty of Medicine, Umeå, Sweden
    Department of Clinical Sciences, Faculty of Medicine, Umeå, Sweden
  • Frida Jonsson
    Department of Medical Biosciences, Faculty of Medicine, Umeå, Sweden
  • Lennart Österman
    Department of Medical Biosciences, Faculty of Medicine, Umeå, Sweden
  • Marie Burstedt
    Department of Clinical Sciences, Faculty of Medicine, Umeå, Sweden
  • Monica Holmberg
    Department of Medical Biosciences, Faculty of Medicine, Umeå, Sweden
  • Irina Golovleva
    Department of Medical Biosciences, Faculty of Medicine, Umeå, Sweden
  • Footnotes
    Commercial Relationships   Ida Maria Westin, None; Frida Jonsson, None; Lennart Österman, None; Marie Burstedt, None; Monica Holmberg, None; Irina Golovleva, None
  • Footnotes
    Support  None
Investigative Ophthalmology & Visual Science July 2018, Vol.59, 5413. doi:
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      Ida Maria Westin, Frida Jonsson, Lennart Österman, Marie Burstedt, Monica Holmberg, Irina Golovleva; Functional study of two canonical splice site variants c.3877+1G>A and c.2992_2992+6delinsTG in the EYS gene associated with recessive retinitis pigmentosa in northern Sweden
      . Invest. Ophthalmol. Vis. Sci. 2018;59(9):5413.

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

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Abstract

Purpose : The homolog of Drosophila eyes shut/spacemaker (EYS) is a common genetic cause of retinitis pigmentosa (RP) worldwide, with a prevalence of 5-12% among recessive RP patients. Approximately 6% reported pathogenic variants in the EYS gene are predicted to affect splicing, however these variants are usually not tested functionally if they are located at canonical splice sites (GT-AG). Since bioinformatics tools can only predict an outcome of splicing mutations, the impact on molecular level is unknown for each of these private splice variants.
In this study, we investigate how two canonical splice sites intronic variants found in RP patients from northern Sweden affect splicing of the EYS gene.

Methods : Screening of the EYS gene with Sanger sequencing was performed in RP patients from northern Sweden. Prediction of aberrant splicing was done in silico by using bioinformatics tools MaxEnt, NNSplice and HSF included in Alamut Visual software, version 2.9 (Interactive Biosoftware, Rouen, France). Functional effects of the splice variants were studied by using a minigene system based on exon-trapping vector pSPL3, transfected to human embryonic kidney cells (HEK293T) and to human retinal pigment epithelium cells (ARPE-19).

Results : Two canonical splice sites intronic variants EYS c.3877+1G>A and EYS c.2992_2992+6delinsTG were identified in RP patients from northern Sweden. Exon skipping was observed in HEK293T and in ARPE-19 cells when both mutant variants were introduced. Inclusion of intronic sequence at a non-canonical splice site of the EYS gene was observed when mutant c.3877+1A was transfected to HEK293T cells, but not in ARPE-19 cells.

Conclusions : We report on two novel intronic variants in the EYS gene, c.3877+1G>A and c.2992_2992+6delinsTG, both causing exon skipping in HEK293T and ARPE-19 cells and, therefore, interpreted as pathogenic according to ACMG-AMP variant classification criteria. The splicing differences in HEK293T and ARPE-19 cells seen for variant c.3877+1G>A emphasize the importance of cell type chosen for in vitro splice assays, and point towards species-specific and/or cell type specific dissimilarities in splicing machineries.

This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.

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