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Clinical implementation of RNA sequencing for Mendelian disease diagnostics

Lookup NU author(s): Dr Charlotte Alston, Rui Ban, Professor Robert Taylor

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This work is licensed under a Creative Commons Attribution 4.0 International License (CC BY 4.0).


Abstract

© 2022, The Author(s). 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.


Publication metadata

Author(s): Yepez VA, Gusic M, Kopajtich R, Mertes C, Smith NH, Alston CL, Ban R, Beblo S, Berutti R, Blessing H, Ciara E, Distelmaier F, Freisinger P, Haberle J, Hayflick SJ, Hempel M, Itkis YS, Kishita Y, Klopstock T, Krylova TD, Lamperti C, Lenz D, Makowski C, Mosegaard S, Muller MF, Munoz-Pujol G, Nadel A, Ohtake A, Okazaki Y, Procopio E, Schwarzmayr T, Smet J, Staufner C, Stenton SL, Strom TM, Terrile C, Tort F, Van Coster R, Vanlander A, Wagner M, Xu M, Fang F, Ghezzi D, Mayr JA, Piekutowska-Abramczuk D, Ribes A, Rotig A, Taylor RW, Wortmann SB, Murayama K, Meitinger T, Gagneur J, Prokisch H

Publication type: Article

Publication status: Published

Journal: Genome Medicine

Year: 2022

Volume: 14

Issue: 1

Online publication date: 05/04/2022

Acceptance date: 03/02/2022

Date deposited: 21/04/2022

ISSN (electronic): 1756-994X

Publisher: BioMed Central Ltd

URL: https://doi.org/10.1186/s13073-022-01019-9

DOI: 10.1186/s13073-022-01019-9

PubMed id: 35379322


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Funding

Funder referenceFunder name
01GM1207
01IS18053F
01KU2016A
203105/Z/16/ZWellcome Trust
DMB-1805-0002
G0800674
JP19ek0109273
JP20ek0109468
mitoNET; 01GM1113C
KAKENHI JP20H05519
PDF-2018-11-ST2-021
MR/S005021/1Medical Research Council (MRC)
Open Access funding enabled and organized by Projekt DEAL
SGR 1428
SLT002/16/00174

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