June 2022
Volume 63, Issue 7
Open Access
ARVO Annual Meeting Abstract  |   June 2022
Functional investigation of candidate mis-splicing variants in retinal dystrophy genes using nanopore sequencing from patient blood samples
Author Affiliations & Notes
  • Neringa Jurkute
    1. Moorfields Eye Hospital NHS Foundation Trust, London, United Kingdom
    2. Institute of Ophthalmology, University College London, London, United Kingdom
  • Jim Bellingham
    2. Institute of Ophthalmology, University College London, London, United Kingdom
  • Luca Varga
    2. Institute of Ophthalmology, University College London, London, United Kingdom
  • Michel Michaelides
    1. Moorfields Eye Hospital NHS Foundation Trust, London, United Kingdom
    2. Institute of Ophthalmology, University College London, London, United Kingdom
  • Omar Abdul Rahman Mahroo
    1. Moorfields Eye Hospital NHS Foundation Trust, London, United Kingdom
    2. Institute of Ophthalmology, University College London, London, United Kingdom
  • Susanne Roosing
    3. Donders Institute for Brain, Cognition and Behaviour, Radboud University Medical Center, Nijmegen, Netherlands
    5. Department of Human Genetics, Radboud University Medical Center, Nijmegen, Netherlands
  • Andrew R Webster
    1. Moorfields Eye Hospital NHS Foundation Trust, London, United Kingdom
    2. Institute of Ophthalmology, University College London, London, United Kingdom
  • Gavin Arno
    1. Moorfields Eye Hospital NHS Foundation Trust, London, United Kingdom
    2. Institute of Ophthalmology, University College London, London, United Kingdom
  • Footnotes
    Commercial Relationships   Neringa Jurkute None; Jim Bellingham None; Luca Varga None; Michel Michaelides None; Omar Mahroo None; Susanne Roosing None; Andrew Webster None; Gavin Arno None
  • Footnotes
    Support  None
Investigative Ophthalmology & Visual Science June 2022, Vol.63, 515 – A0092. doi:
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    • Get Citation

      Neringa Jurkute, Jim Bellingham, Luca Varga, Michel Michaelides, Omar Abdul Rahman Mahroo, Susanne Roosing, Andrew R Webster, Gavin Arno; Functional investigation of candidate mis-splicing variants in retinal dystrophy genes using nanopore sequencing from patient blood samples. Invest. Ophthalmol. Vis. Sci. 2022;63(7):515 – A0092.

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

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Abstract

Purpose : To functionally investigate candidate splice altering variants in inherited retinal dystrophy (IRD) genes including non-coding variants in genes with restricted expression using RT-PCR and nanopore single-molecule sequencing for transcripts from patient blood derived total RNA samples.

Methods : Patients were recruited from the inherited retinal disease clinics at Moorfields Eye Hospital (London, UK) as part of the UK 100,000 genomes project with an additional individual identified at Radboud University Medical Center (Nijmegen, the Netherlands). Ten probands who were identified to carry a candidate pathogenic non-coding or synonymous variant were selected for investigation. RT-PCR was performed on total RNA derived from affected individuals and control blood to amplify fragments flanking the affected intron or whole transcripts where possible. Nanopore sequencing was performed using the Oxford Nanopore Technologies Genomic DNA by Ligation (SQK-LSK109) protocol and sequencing on a flongle flow-cell.

Results : Ten affected individuals diagnosed with IRD were identified to carry candidate variants that were otherwise only classed as variants of uncertain significance. Variants were identified in CDH23, COQ5, NMNAT1, PDSS1, PROM1, PRPF31 and TULP1 following interrogation of whole genome sequencing data and multidisciplinary team investigation. This study demonstrated mis-splicing for nine intronic variants in genes CDH23, COQ5, PDSS1 (N=2), PROM1, PRPF31 (N=3) and TULP1 and a reduced transcript level for an upstream variant in NMNAT1. It was possible to detect transcript alteration for genes that are not expressed in blood (CDH23, PROM1, TULP1) and where standard techniques failed to demonstrate mis-splicing.

Conclusions : This study shows that nanopore sequencing is effective for detecting transcript alterations from blood derived RNA for retinal disease genes, allowing functional characterisation of non-canonical splice and upstream variants, where no mis-splicing was apparent by agarose gel electrophoresis precluding Sanger sequencing analysis. Thus, simple blood samples can be collected for investigation without the need for complicated cloning or induced pluripotent stem cell derived retinal progenitor or organoid experiments. The functional evidence provides the crucial support for interpretation of pathogenicity of these candidate variants.

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

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