Long-read genome sequencing resolves complex structural variants in exome-negative developmental and epileptic encephalopathy

Background and Objectives: Developmental and Epileptic Encephalopathies (DEEs) encompass a clinically and molecularly heterogeneous group of mainly genetic disorders. Despite standard genetic testing, approximately two-thirds of patients remain undiagnosed. Pathogenic noncoding and structural variants (SVs), particularly complex rearrangements that are difficult to detect or resolve using short-read sequencing, may contribute to this diagnostic gap. To evaluate the diagnostic contribution of long-read genome sequencing (lrGS), we recruited a cohort of 124 families with sporadic DEE who remained unsolved by prior ES. Methods: Trio lrGS (Oxford Nanopore or PacBio HiFi) was performed, complemented zby short-read genome sequencing (srGS) and mRNA sequencing (blood or fibroblast) of index cases, where available. Comprehensive variant analysis included small nucleotide variants (SNVs), indels, SVs, and short tandem repeats. To identify the diagnostic yield of each method, we compared lrGS, srGS and ES re-analysis, and evaluated the additional value of RNA sequencing. Results: We established a diagnosis in 21 of 124 families (16.9%). Fourteen variants were retrospectively detectable in prior ES., Two cases with non-coding variants in SCN1A and RNU2-2 were detectable by both srGS and lrGS. Five SVs also required genome sequencing (5.6% of the total cohort), three of which could be detected, but not confidently resolved by srGS due to breakpoint complexity, repeat-mediated rearrangements, and large copy-neutral events: LrGS enabled accurate characterization of Alu-mediated CDKL5 and STXBP1 deletions and a MEF2C deletion with large inversion. A copy-neutral SCN1A inversion was well characterizable by both srGS and lrGS. One balanced translocation t(X;17) disrupting CDKL5 was resolved by lrGS (srGS unavailable). RNA sequencing confirmed pathogenicity of two SVs including t(X;17), the MEF2C deletion with inversion, and two CNVs. In addition, ZNF569 and ZNF570 emerged as candidate epilepsy genes from our cohort. Discussion: In ES-negative DEE, the incremental diagnostic yield of genome sequencing was modest, while lrGS provided particular value for the structural resolution of complex rearrangements, especially those involving repetitive sequences. The finding that two-thirds of diagnoses were retrospectively detectable in prior ES highlights the substantial value of systematic reanalysis. Further gains from genome sequencing may increasingly depend on improved interpretation of non-coding variation and integration of long-read-specific information. Our findings indicate that complex SVs represent the class for which lrGS currently yields the most distinctive diagnostic information.

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medRxiv
Published
2026-10-28
DOI
https://doi.org/10.64898/2026.10.26.26365902
Primary Topic
Genomics and Rare Diseases
Type
preprint

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preprint

Long-read genome sequencing resolves complex structural variants in exome-negative developmental and epileptic encephalopathy

Julien Gagneur, Tejasvi S. Niranjan, Heidi Loeffler, Manuela Pendziwiat et al.
2 links
medRxiv
Genomics and Rare Diseases
preprint

Long-read genome sequencing resolves complex structural variants in exome-negative developmental and epileptic encephalopathy

Julien Gagneur, Tejasvi S. Niranjan, Heidi Loeffler, Manuela Pendziwiat, German M. Demidov, Leon Schütz, Sarah Weckhuysen, Renzo Guerrini, Peter Krieg, Tobias B. Haack, Hiltrud Muhle, Elena Parrini, Anna Esteve‐Codina, Isabelle Sabatier, Francesca Bisulli, Christel Depienne, Noor Smal, Josua Kegele, Michael Alber, Hamidah Ghani, Laura Canafoglia, Dorothee M. Ville, Holger Lerche, Leo Strothmann, Evelina Carapancea, Gianpiero L. Cavalleri, Charissa Millevert, Robert J. Lauerer, Maria Roberta Cilio, Eva Matzker, Gaëtan Lesca, Bernd Axel Neubauer, Miriam Scida, Barbara Castellotti, Davide Mei, Simona Balestrini, Patrick May, Merel Swinnen, Els De Vriendt, Raffaella Minardi, Elena Freri, Janna Kenny, Kornelia Ellwanger, An‐Sofie Schoonjans, Wouter De Coster, Matthias De Wachter, John Coleman, Icíar Sánchez‐Albisua, Christina Vossler-Wolf, Damien Sanlaville, Friedhelm Hildebrandt, Simone Olivieri, Marc Sturm, Laura Licchetta, Hannah Stamberger, Vicente A. Yépez, Stephan Ossowski, Nicolas Chatron, Joohyun Park, Susan Byrne, Amelie T. van der Ven, Carsten Wurst, Johanna Pohl, Zakaria Eddafir, Markus Wolff, Oliver Maier, Anna Jansen, Mandana Gheisari, Christiane Wagner, Flavia Fineschi, Thomas Ott, Andrea Rueegger, Solve-RD DITF Epicare, Anne Schulz
preprint en

Abstract

Background and Objectives: Developmental and Epileptic Encephalopathies (DEEs) encompass a clinically and molecularly heterogeneous group of mainly genetic disorders. Despite standard genetic testing, approximately two-thirds of patients remain undiagnosed. Pathogenic noncoding and structural variants (SVs), particularly complex rearrangements that are difficult to detect or resolve using short-read sequencing, may contribute to this diagnostic gap. To evaluate the diagnostic contribution of long-read genome sequencing (lrGS), we recruited a cohort of 124 families with sporadic DEE who remained unsolved by prior ES. Methods: Trio lrGS (Oxford Nanopore or PacBio HiFi) was performed, complemented zby short-read genome sequencing (srGS) and mRNA sequencing (blood or fibroblast) of index cases, where available. Comprehensive variant analysis included small nucleotide variants (SNVs), indels, SVs, and short tandem repeats. To identify the diagnostic yield of each method, we compared lrGS, srGS and ES re-analysis, and evaluated the additional value of RNA sequencing. Results: We established a diagnosis in 21 of 124 families (16.9%). Fourteen variants were retrospectively detectable in prior ES., Two cases with non-coding variants in SCN1A and RNU2-2 were detectable by both srGS and lrGS. Five SVs also required genome sequencing (5.6% of the total cohort), three of which could be detected, but not confidently resolved by srGS due to breakpoint complexity, repeat-mediated rearrangements, and large copy-neutral events: LrGS enabled accurate characterization of Alu-mediated CDKL5 and STXBP1 deletions and a MEF2C deletion with large inversion. A copy-neutral SCN1A inversion was well characterizable by both srGS and lrGS. One balanced translocation t(X;17) disrupting CDKL5 was resolved by lrGS (srGS unavailable). RNA sequencing confirmed pathogenicity of two SVs including t(X;17), the MEF2C deletion with inversion, and two CNVs. In addition, ZNF569 and ZNF570 emerged as candidate epilepsy genes from our cohort. Discussion: In ES-negative DEE, the incremental diagnostic yield of genome sequencing was modest, while lrGS provided particular value for the structural resolution of complex rearrangements, especially those involving repetitive sequences. The finding that two-thirds of diagnoses were retrospectively detectable in prior ES highlights the substantial value of systematic reanalysis. Further gains from genome sequencing may increasingly depend on improved interpretation of non-coding variation and integration of long-read-specific information. Our findings indicate that complex SVs represent the class for which lrGS currently yields the most distinctive diagnostic information.

medRxiv
Université Claude Bernard Lyon 1 (FR), Centre National de la Recherche Scientifique (FR), Cliniques Universitaires Saint-Luc (BE), Royal College of Surgeons in Ireland (IE), University of Antwerp (BE), Inserm (FR), University of Luxembourg (LU), Université de Lyon (FR), Universitätsklinikum Gießen und Marburg (DE), University Hospital of Bern (CH), Children's Health Ireland at Crumlin (IE), Christian-Albrechts-Universität zu Kiel (DE), Sana Klinikum Lichtenberg (DE), Städtisches Klinikum Karlsruhe (DE), Antwerp University Hospital (BE), Meyer Children's Hospital (IL), University Medical Center Hamburg-Eppendorf (DE), University Hospital Schleswig-Holstein (DE), Max Planck Institute for Biological Cybernetics (DE), Meyer Children's Hospital (IT), Essen University Hospital (DE), Istituto delle Scienze Neurologiche di Bologna (IT), University Children's Hospital Tübingen (DE), University Children's Hospital Zurich (CH), VIB-UAntwerp Center for Molecular Neurology (BE), Fraunhofer Project Centre Wolfsburg (DE), Hertie Institute for Clinical Brain Research (DE), Centro Nacional de Análisis Genómico (ES), Medizinische Universität Lausitz - Carl Thiem (DE), Klinikum Kassel (DE), Fondazione IRCCS Istituto Neurologico Carlo Besta (IT), Ostschweizer Kinderspital (CH), ERN EpiCARE (FR), Laboratoire Physiopathologie et Génétique du Neurone et du Muscle (FR), Luxembourg Centre for Systems Biomedicine (LU), University of Duisburg-Essen (DE), Technical University of Munich (DE), Universitat de Barcelona (ES), University of Tübingen (DE), University of Bologna (IT), UCLouvain (BE), KU Leuven (BE)
European Commission, Deutsche Forschungsgemeinschaft, Eberhard Karls Universität Tübingen, Else Kröner-Fresenius-Stiftung, Fonds Wetenschappelijk Onderzoek, Ministero della Salute, GIS-Institut des Maladies Rares, Universiteit Antwerpen, Vlaamse regering, Epilepsy Society, Fondation Médicale Reine Elisabeth, European Regional Development Fund, NextGenerationEU, Research Ireland
Good health and well-being
Genomics and Rare Diseases
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