June 2021
Volume 62, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2021
Molecular inversion probe sequencing to identify USH2A variants underlying Usher syndrome and non-syndromic retinitis pigmentosa
Author Affiliations & Notes
  • Janine Reurink
    Department of Human Genetics, Radboud university medical center, Nijmegen, Netherlands
    Donders Institute for Brain Cognition and Behaviour, Radboud university medical center, Nijmegen, Netherlands
  • Adrian Dockery
    The School of Genetics & Microbiology, Trinity College Dublin, Dublin, Ireland
  • G. Jane Farrar
    The School of Genetics & Microbiology, Trinity College Dublin, Dublin, Ireland
  • Monika Oldak
    Department of Histology and Embryology, Medical University of Warsaw, Warsaw, Poland
  • Jacoline B. ten Brink
    Department of Clinical Genetics, Amsterdam UMC, Amsterdam, Netherlands
  • Arthur A.B. Bergen
    Department of Clinical Genetics, Amsterdam UMC, Amsterdam, Netherlands
  • Ronald J.E. Pennings
    Department of Otorhinolaryngology, Radboud university medical center, Nijmegen, Netherlands
  • Marco Aben
    Department of Human Genetics, Radboud university medical center, Nijmegen, Netherlands
  • Jaap Oostrik
    Department of Otorhinolaryngology, Radboud university medical center, Nijmegen, Netherlands
  • Erwin van Wijk
    Department of Otorhinolaryngology, Radboud university medical center, Nijmegen, Netherlands
    Donders Institute for Brain Cognition and Behaviour, Radboud university medical center, Nijmegen, Netherlands
  • Frans P.M. Cremers
    Department of Human Genetics, Radboud university medical center, Nijmegen, Netherlands
    Donders Institute for Brain Cognition and Behaviour, Radboud university medical center, Nijmegen, Netherlands
  • Susanne Roosing
    Department of Human Genetics, Radboud university medical center, Nijmegen, Netherlands
    Donders Institute for Brain Cognition and Behaviour, Radboud university medical center, Nijmegen, Netherlands
  • Hannie Kremer
    Department of Human Genetics, Radboud university medical center, Nijmegen, Netherlands
    Department of Otorhinolaryngology, Radboud university medical center, Nijmegen, Netherlands
  • Footnotes
    Commercial Relationships   Janine Reurink, None; Adrian Dockery, None; G. Jane Farrar, None; Monika Oldak, None; Jacoline ten Brink, None; Arthur Bergen, None; Ronald Pennings, None; Marco Aben, None; Jaap Oostrik, None; Erwin van Wijk, None; Frans Cremers, None; Susanne Roosing, None; Hannie Kremer, None
  • Footnotes
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Investigative Ophthalmology & Visual Science June 2021, Vol.62, 1517. doi:
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      Janine Reurink, Adrian Dockery, G. Jane Farrar, Monika Oldak, Jacoline B. ten Brink, Arthur A.B. Bergen, Ronald J.E. Pennings, Marco Aben, Jaap Oostrik, Erwin van Wijk, Frans P.M. Cremers, Susanne Roosing, Hannie Kremer; Molecular inversion probe sequencing to identify USH2A variants underlying Usher syndrome and non-syndromic retinitis pigmentosa. Invest. Ophthalmol. Vis. Sci. 2021;62(8):1517.

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

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Abstract

Purpose : A significant proportion of subjects with autosomal recessively inherited retinitis pigmentosa (arRP) and Usher syndrome type 2 (USH2) lacks a genetic diagnosis, subsequent prognosis and possibilities for future therapy. The USH2A gene is the most frequently mutated gene in USH2 and also prevalent in individuals with arRP. As such, USH2A is an important target for genetic screening. The aim of this study is 1) to screen genetically unexplained USH2 and arRP cases in which already one pathogenic USH2A variant was identified using targeted analyses and 2) to include genetically unexplained cases in our future whole genome sequencing effort.

Methods : Eleven unscreened or partially prescreened arRP cases with one previously detected USH2A variant and 29 USH2 cases were included for Molecular Inversion Probe (MIP)-based sequencing of USH2A coding regions, surrounding regions and published deep-intronic variants. After identification of pathogenic coding variants, data was prioritized based on four splice site prediction tools and the deep learning-based tool SpliceAI (Jaganathan et al. 2019). Wild-type and mutant minigene constructs were generated and transfected in Human Embryonic Kidney cells. Transcriptional analysis was performed to determine the splicing effects.

Results : Forty cases were assessed through MIPs. Four unique copy number variants and 107 rare (<1% in gnomAD) unique single nucleotide variants were identified. In 30 cases two (likely) pathogenic variants were identified and in three cases variants of unknown significance were found. One synonymous splice variant and one cryptic acceptor splice site variant that were absent or very rare (<0.001%) in gnomAD were identified. Subsequent minigene splice assays revealed partial exon skipping or no effect on splicing, respectively.

Conclusions : We identified five novel pathogenic variants and have shown the effect on splicing of one known synonymous USH2A variant. This analysis confirms that this MIPs approach is cost-effective. Through this study, genetic diagnoses were completed for at least 30 patients. Six of the unexplained cases will be included for WGS to explore the noncoding regions for pathogenic variants.

This is a 2021 ARVO Annual Meeting abstract.

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