Clinical implementation of RNA sequencing for Mendelian disease diagnostics

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.

A multinational team led by researchers at the Technical University of Munich implemented an automated RNA-seq protocol and a computational workflow with which they analyzed skin fibroblasts of 303 individuals with a suspected mitochondrial disease that previously underwent WES. They also assessed through simulations how aberrant expression and mono-allelic expression tests depend on RNA-seq coverage.

The researchers 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. They 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.

RNA-seq-based diagnostic flow chart

Fig. 2

Flow diagram showing the diagnostic decision guideline after detecting a gene with an aberrant event in RNA-seq data. Identification of an aberrant event can lead to genetic diagnosis (diagnostic setting), lead to the discovery of a candidate new disease gene (research setting), or alternatively be of unlikely diagnostic significance, after which the next aberrant event is analyzed following the same path

Together, these results show that streamlined experimental and computational processes can accelerate the implementation of RNA-seq in routine diagnostics.

Yépez 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, Häberle J, Hayflick SJ, Hempel M, Itkis YS, Kishita Y, Klopstock T, Krylova TD, Lamperti C, Lenz D, Makowski C, Mosegaard S, Müller MF, Muñoz-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, Rötig A, Taylor RW, Wortmann SB, Murayama K, Meitinger T, Gagneur J, Prokisch H. (2022) Clinical implementation of RNA sequencing for Mendelian disease diagnostics. Genome Med 14(1):38. [article]

Leave a Reply

Your email address will not be published. Required fields are marked *


Time limit is exhausted. Please reload CAPTCHA.