A stepwise short- and long-read whole genome sequencing strategy resolves previous genetically unresolved inherited retinal disease cases
Abstract
Purpose A significant proportion of cases with rare inherited retinal disease (IRD) remain genetically unresolved following short-read whole genome sequencing (WGS). This study aimed to increase the diagnostic yield of two cohorts in a research setting. One cohort was previously screened by short-read WGS (n=120 cases), while the second cohort (n=28 cases) was not included in any previous short-read WGS studies. In contrast to the larger cohort, these cases only underwent either exome sequencing (ES) or single-molecule molecular inversion probe (smMIPs) sequencing as pre-screening in earlier studies. For all cases, we performed a stepwise, case-by-case reanalysis in which short-read WGS was generated (n=28 cases) and (re)analyzed short-read WGS data using an optimized approach. For a subset of cases (n=20) that remained unresolved, this was followed by long-read WGS. Together, these steps aimed to improve the diagnostic yield in these cohorts. Methods Short-read WGS data of 148 IRD-cases were (re)analyzed using updated allele frequency databases, new variant caller and variant predictor tools, as well as updated and extended gene-panels to identify causal single nucleotide variants (SNVs) and structural variants (SVs). For 20 genetically unresolved cases with sufficient high-quality DNA available, long-read WGS was performed. Results The (re)analysis of short-read WGS data in the 120 previously WGS-screened unresolved cases resulted in a diagnostic yield of 15% (18/120). For the 28 individuals without prior WGS, performing WGS analysis within this study provided an additional yield of 32% (9/28). Furthermore, long-read WGS contributed two genetic diagnoses among the 20 long-read WGS-sequenced probands. Amongst other reasons, new genetic diagnoses were attributed to pathogenic variants in genes newly associated with IRDs and in genes that were not included in the gene panels applied in previous WGS studies. The Mobile Element Locator Tool facilitated the identification of two pathogenic Alu insertions in short-read WGS. Long-read WGS identified two additional pathogenic variants. Conclusion These results support periodic reanalysis of existing short-read WGS data as a key component of IRD genetic diagnostics, with selective long-read WGS representing a valuable additional approach for unresolved cases. Together, these complementary strategies offer a practical framework for improving molecular diagnosis and narrowing the remaining diagnostic gap in IRDs.
Authors 30
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Radboud University Nijmegen · Radboud University Medical Center
Affiliation as printed
Department of Human Genetics, Radboud University Medical Center, Geert Grooteplein Zuid 10, Nijmegen, The Netherlands
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Radboud University Nijmegen · Radboud University Medical Center
Affiliation as printed
Department of Human Genetics, Radboud University Medical Center, Geert Grooteplein Zuid 10, Nijmegen, The Netherlands
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Radboud University Nijmegen · Radboud University Medical Center
Affiliation as printed
Department of Human Genetics, Radboud University Medical Center, Geert Grooteplein Zuid 10, Nijmegen, The Netherlands
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Radboud University Nijmegen · Maastricht University Medical Centre · Radboud University Medical Center · Maastricht University
Affiliation as printed
Department of Clinical Genetics, Maastricht University Medical Center, Maastricht, The Netherlands
Department of Human Genetics, Radboud University Medical Center, Geert Grooteplein Zuid 10, Nijmegen, The Netherlands
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Radboud University Nijmegen · Maastricht University Medical Centre · Radboud University Medical Center · Maastricht University
Affiliation as printed
Department of Clinical Genetics, Maastricht University Medical Center, Maastricht, The Netherlands
Department of Human Genetics, Radboud University Medical Center, Geert Grooteplein Zuid 10, Nijmegen, The Netherlands
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Radboud University Nijmegen · Radboud University Medical Center
Affiliation as printed
Department of Human Genetics, Radboud University Medical Center, Geert Grooteplein Zuid 10, Nijmegen, The Netherlands
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Radboud University Nijmegen · Radboud University Medical Center
Affiliation as printed
Department of Human Genetics, Radboud University Medical Center, Geert Grooteplein Zuid 10, Nijmegen, The Netherlands
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University of Zurich · ETH Zurich
Affiliation as printed
Center for Integrative Human Physiology, University of Zurich, Zurich, Switzerland
Institute of Medical Molecular Genetics, University of Zurich, Schlieren, Switzerland
Neuroscience Center Zurich, University and ETH Zurich, Zurich, Switzerland
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Technion – Israel Institute of Technology
Affiliation as printed
Ruth and Bruce Rappaport Faculty of Medicine, Technion-Israel Institute of Technology, Haifa, Israel
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Leiden University Medical Center · Amsterdam UMC Location University of Amsterdam · Amsterdam University Medical Centers
Affiliation as printed
Department of Ophthalmology, Amsterdam University Medical Center, Amsterdam, The Netherlands
Department of Ophthalmology, Leiden University Medical Center, Leiden, The Netherlands
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Radboud University Nijmegen · Radboud University Medical Center
Affiliation as printed
Department of Human Genetics, Radboud University Medical Center, Geert Grooteplein Zuid 10, Nijmegen, The Netherlands
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Trinity College · Trinity College Dublin
Affiliation as printed
Institute of Genetics, School of Genetics and Microbiology, Trinity College Dublin, The University of Dublin
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Radboud University Nijmegen · Radboud University Medical Center
Affiliation as printed
Department of Human Genetics, Radboud University Medical Center, Geert Grooteplein Zuid 10, Nijmegen, The Netherlands
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Affiliation as printed
Department of Ophthalmology and Vision Sciences, Ocular Genetics Program, The Hospital for Sick Children, 555 University Avenue, Toronto, Ontario, Canada
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Radboud University Nijmegen · Radboud University Medical Center
Affiliation as printed
Department of Ophthalmology, Radboud University Medical Center, Geert Grooteplein Zuid 10, Nijmegen, The Netherlands
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Radboud University Nijmegen · Erasmus MC · Radboud University Medical Center
Affiliation as printed
Department of Ophthalmology, Erasmus MC University Medical Center, Rotterdam, The Netherlands
Department of Ophthalmology, Radboud University Medical Center, Geert Grooteplein Zuid 10, Nijmegen, The Netherlands
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Affiliation as printed
Division Laboratories, Pharmacy and Biomedical Genetics, Department of Genetics, University Medical Center of Utrecht, Utrecht, The Netherlands
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Charles University · General University Hospital in Prague
Affiliation as printed
Department of Ophthalmology, First Faculty of Medicine, Charles University and General University Hospital in Prague, Prague, Czech Republic
Department of Paediatrics and Inherited Metabolic Disorders, First Faculty of Medicine, Charles University and General University Hospital in Prague, Prague, Czech Republic
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Medical University of Warsaw · Institute of Physiology and Pathology of Hearing
Affiliation as printed
Department of Genetics, Institute of Physiology and Pathology of Hearing, M. Mochnackiego 10, Warsaw, Poland
Department of Histology and Embryology, Medical University of Warsaw, T. Chałubińskiego 5, Warsaw, Poland
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Institute of Physiology and Pathology of Hearing
Affiliation as printed
Department of Genetics, Institute of Physiology and Pathology of Hearing, M. Mochnackiego 10, Warsaw, Poland
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Amsterdam University Medical Centers · University of Amsterdam
Affiliation as printed
Department of Human Genetics, Amsterdam University Medical Center, University of Amsterdam, Amsterdam, The Netherlands
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Amsterdam University Medical Centers · University of Amsterdam
Affiliation as printed
Department of Human Genetics, Amsterdam University Medical Center, University of Amsterdam, Amsterdam, The Netherlands
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Affiliation as printed
Department of Ophthalmology, Erasmus MC University Medical Center, Rotterdam, The Netherlands
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University Medical Center Groningen · University of Groningen
Affiliation as printed
Department of Medical Genetics, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands
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Affiliation as printed
CRO Oftalmic, Moscow, Russia
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STZ eyetrial · University of Tübingen
Affiliation as printed
Institute for Ophthalmic Research, Centre for Ophthalmology, University Hospital Tübingen, Elfriede-Aulhorn-Str. 7, Tübingen, Germany
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Radboud University Nijmegen · Radboud University Medical Center
Affiliation as printed
Department of Human Genetics, Radboud University Medical Center, Geert Grooteplein Zuid 10, Nijmegen, The Netherlands
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Maastricht University Medical Centre · Maastricht University
Affiliation as printed
Department of Clinical Genetics, Maastricht University Medical Center, Maastricht, The Netherlands
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Radboud University Nijmegen · Radboud University Medical Center
Affiliation as printed
Department of Human Genetics, Radboud University Medical Center, Geert Grooteplein Zuid 10, Nijmegen, The Netherlands
Department of Otorhinolaryngology, Hearing & Genes, Radboud University Medical Center, Geert Grooteplein Zuid 10, Nijmegen, The Netherlands
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Susanne Roosing corresponding
Radboud University Nijmegen · Radboud University Medical Center
Affiliation as printed
Department of Human Genetics, Radboud University Medical Center, Geert Grooteplein Zuid 10, Nijmegen, The Netherlands
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