Clinical implementation of RNA sequencing for Mendelian disease diagnostics

Research output: Contribution to journal/Conference contribution in journal/Contribution to newspaperJournal articleResearchpeer-review

  • Vicente A. Yépez, Technical University of Munich, Ludwig Maximilian University of Munich
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  • Mirjana Gusic, Technical University of Munich, Helmholtz Zentrum München - German Research Center for Environmental Health, German Centre for Cardiovascular Research
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  • Robert Kopajtich, Technical University of Munich, Helmholtz Zentrum München - German Research Center for Environmental Health
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  • Christian Mertes, Technical University of Munich
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  • Nicholas H. Smith, Technical University of Munich
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  • Charlotte L. Alston, Newcastle University, Newcastle upon Tyne Hospitals NHS Foundation Trust
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  • Rui Ban, Helmholtz Zentrum München - German Research Center for Environmental Health, Capital Medical University
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  • Skadi Beblo, Leipzig University
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  • Riccardo Berutti, Technical University of Munich, Helmholtz Zentrum München - German Research Center for Environmental Health
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  • Holger Blessing, Friedrich-Alexander University Erlangen-Nürnberg
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  • Elżbieta Ciara, Children's Memorial Health Institute
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  • Felix Distelmaier, Heinrich Heine University Düsseldorf
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  • Peter Freisinger, Klinikum Reutlingen
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  • Johannes Häberle, University of Zurich
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  • Susan J. Hayflick, Oregon Health and Science University
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  • Maja Hempel, University of Hamburg
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  • Yulia S. Itkis, Russian Academy of Medical Sciences
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  • Yoshihito Kishita, Juntendo University Graduate School of Medicine, Kindai University
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  • Thomas Klopstock, Ludwig Maximilian University of Munich, German Center for Neurodegenerative Diseases, Munich Cluster for Systems Neurology (SyNergy)
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  • Tatiana D. Krylova, Russian Academy of Medical Sciences
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  • Costanza Lamperti, IRCCS Fondazione Istituto Neurologico Carlo Besta - Milano
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  • Dominic Lenz, Heidelberg University 
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  • Christine Makowski, Technical University of Munich
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  • Signe Mosegaard
  • Michaela F. Müller, Technical University of Munich
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  • Gerard Muñoz-Pujol, University of Barcelona
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  • Agnieszka Nadel, Technical University of Munich, Helmholtz Zentrum München - German Research Center for Environmental Health
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  • Akira Ohtake, Saitama Medical University
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  • Yasushi Okazaki, Juntendo University Graduate School of Medicine
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  • Elena Procopio, Azienda Ospedaliero Universitaria Meyer
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  • Thomas Schwarzmayr, Technical University of Munich, Helmholtz Zentrum München - German Research Center for Environmental Health
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  • Joél Smet, Ghent University
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  • Christian Staufner, Heidelberg University 
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  • Sarah L. Stenton, Technical University of Munich, Helmholtz Zentrum München - German Research Center for Environmental Health
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  • Tim M. Strom, Technical University of Munich, Helmholtz Zentrum München - German Research Center for Environmental Health
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  • Caterina Terrile, Helmholtz Zentrum München - German Research Center for Environmental Health
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  • Frederic Tort, University of Barcelona
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  • Rudy Van Coster, Ghent University
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  • Arnaud Vanlander, Ghent University
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  • Matias Wagner, Technical University of Munich, Helmholtz Zentrum München - German Research Center for Environmental Health
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  • Manting Xu, Helmholtz Zentrum München - German Research Center for Environmental Health, Capital Medical University
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  • Fang Fang, Capital Medical University
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  • Daniele Ghezzi, IRCCS Fondazione Istituto Neurologico Carlo Besta - Milano, University of Milan
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  • Johannes A. Mayr, Paracelsus Private Medical University
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  • Dorota Piekutowska-Abramczuk, Children's Memorial Health Institute
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  • Antonia Ribes, University of Barcelona
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  • Agnès Rötig, Université de Paris
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  • Robert W. Taylor, Newcastle University, Newcastle upon Tyne Hospitals NHS Foundation Trust
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  • Saskia B. Wortmann, Technical University of Munich, Paracelsus Private Medical University, Radboud University Nijmegen
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  • Kei Murayama, Chiba Children's Hospital
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  • Thomas Meitinger, Technical University of Munich
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  • Julien Gagneur, Technical University of Munich, Helmholtz Zentrum München - German Research Center for Environmental Health
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  • Holger Prokisch, Technical University of Munich, Helmholtz Zentrum München - German Research Center for Environmental Health, Capital Medical University

Background: Lack of functional evidence hampers variant interpretation, leaving a large proportion of individuals with a suspected Mendelian disorder without genetic diagnosis after whole genome or whole exome sequencing (WES). Research studies advocate to further sequence transcriptomes to directly and systematically probe gene expression defects. However, collection of additional biopsies and establishment of lab workflows, analytical pipelines, and defined concepts in clinical interpretation of aberrant gene expression are still needed for adopting RNA sequencing (RNA-seq) in routine diagnostics. Methods: We implemented an automated RNA-seq protocol and a computational workflow with which we analyzed skin fibroblasts of 303 individuals with a suspected mitochondrial disease that previously underwent WES. We also assessed through simulations how aberrant expression and mono-allelic expression tests depend on RNA-seq coverage. Results: We detected on average 12,500 genes per sample including around 60% of all disease genes—a coverage substantially higher than with whole blood, supporting the use of skin biopsies. We prioritized genes demonstrating aberrant expression, aberrant splicing, or mono-allelic expression. The pipeline required less than 1 week from sample preparation to result reporting and provided a median of eight disease-associated genes per patient for inspection. A genetic diagnosis was established for 16% of the 205 WES-inconclusive cases. Detection of aberrant expression was a major contributor to diagnosis including instances of 50% reduction, which, together with mono-allelic expression, allowed for the diagnosis of dominant disorders caused by haploinsufficiency. Moreover, calling aberrant splicing and variants from RNA-seq data enabled detecting and validating splice-disrupting variants, of which the majority fell outside WES-covered regions. Conclusion: Together, these results show that streamlined experimental and computational processes can accelerate the implementation of RNA-seq in routine diagnostics.

Original languageEnglish
Article number38
JournalGenome Medicine
Volume14
Issue1
Number of pages26
ISSN1756-994X
DOIs
Publication statusPublished - Apr 2022

    Research areas

  • Genetic diagnostics, Mendelian diseases, RNA-seq

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