{
  "abstract": "Purpose Pathogenic variants in PRPF31 are a leading cause of autosomal dominant retinitis pigmentosa (RP). Despite advances in next-generation sequencing, many patients remain undiagnosed, due to variants of uncertain significance (VUS). These include non-coding variants affecting splicing, which are challenging to interpret. This study uses reverse-transcription polymerase chain reaction (RT-PCR) combined with Oxford Nanopore Technology (ONT) long-read sequencing to functionally characterise non-coding PRPF31 variants.Methods Six individuals with RP and non-coding PRPF31 variants were investigated, including known pathogenic variants (c.527+3A>G and c.528-39_531del) and VUS (c.856-8T>G, c.946-3C>G, c.239-3C>G, c.855+3G>C). SpliceAI and Pangolin were used for in silico splice prediction. RT-PCR was performed on RNA extracted from peripheral blood mononuclear cells, followed by ONT sequencing of full-length PRPF31 transcripts. Results were compared with computational predictions.Results ONT sequencing enabled full-length transcript analysis in a single assay, demonstrating both dominant and low-level splicing events. This included multiple splicing alterations arising from individual non-coding PRPF31 variants, as well as ubiquitous low-level mis-splicing occurring across the gene, reflecting the inherent complexity of physiological splicing. Notably, two variants, c.946-3C>G and c.527+3A>G, demonstrated clear splicing defects on ONT analysis, despite having insignificant SpliceAI scores, highlighting the limitations of in silico predictive tools.Conclusion This study demonstrates utility of ONT-based transcript analysis for functional validation of non-coding PRPF31 VUS, resolving uncertainties over pathogenicity and enabling molecular diagnoses. Findings provide proof-of-principle for integrating ONT-sequencing into clinical diagnostics to aid variant interpretation, benefiting unsolved inherited retinal disease patient cohorts globally. This work also offers insights into complex splicing alterations driven by non-coding variants in PRPF31, advancing understanding of disease mechanisms and informing future therapies.",
  "authors": [
    {
      "affiliations": [
        "Division of Evolution, Infection and Genomics, School of Biological Sciences, Faculty of Biology, Medicine and Health, University of Manchester, Manchester, UK",
        "Manchester Centre for Genomic Medicine and Manchester Royal Eye Hospital, Manchester University NHS Foundation Trust, Manchester, UK",
        "National Institute of Health Research Biomedical Research Centre at Moorfields Eye Hospital and the UCL Institute of Ophthalmology, London, UK"
      ],
      "name": "Siying Lin"
    },
    {
      "affiliations": [
        "UCL Institute of Ophthalmology, University College London, London, UK"
      ],
      "name": "Aleksandr Jestin"
    },
    {
      "affiliations": [
        "Division of Research, Greenwood Genetic Center, Greenwood, South Carolina, USA"
      ],
      "name": "Andrzej Brunon Poplawski"
    },
    {
      "affiliations": [
        "Division of Research, Greenwood Genetic Center, Greenwood, South Carolina, USA"
      ],
      "name": "Theresa Erlenbach"
    },
    {
      "affiliations": [
        "National Institute of Health Research Biomedical Research Centre at Moorfields Eye Hospital and the UCL Institute of Ophthalmology, London, UK",
        "UCL Institute of Ophthalmology, University College London, London, UK"
      ],
      "name": "Michel Michaelides"
    },
    {
      "affiliations": [
        "National Institute of Health Research Biomedical Research Centre at Moorfields Eye Hospital and the UCL Institute of Ophthalmology, London, UK",
        "UCL Institute of Ophthalmology, University College London, London, UK",
        "Department of Ophthalmology, St Thomas’ Hospital, London, UK"
      ],
      "name": "Omar A Mahroo"
    },
    {
      "affiliations": [
        "National Institute of Health Research Biomedical Research Centre at Moorfields Eye Hospital and the UCL Institute of Ophthalmology, London, UK",
        "UCL Institute of Ophthalmology, University College London, London, UK"
      ],
      "name": "Andrew R Webster"
    },
    {
      "affiliations": [
        "National Institute of Health Research Biomedical Research Centre at Moorfields Eye Hospital and the UCL Institute of Ophthalmology, London, UK",
        "UCL Institute of Ophthalmology, University College London, London, UK",
        "Division of Research, Greenwood Genetic Center, Greenwood, South Carolina, USA"
      ],
      "name": "Gavin Arno"
    }
  ],
  "title": "#ECR-Paper-02 Investigating splice defects in PRPF31 transcripts using targeted long-read sequencing",
  "uid": "2fb0601c-467c-5cd3-905e-f58bebc16743"
}