Clinical implementation of RNA sequencing for Mendelian disease diagnostics

Abstract

Lack of functional evidence hampers variant interpretation, leaving a large proportion of cases with a suspected Mendelian disorder without genetic diagnosis after 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. To address these issues, 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. We detected on average 12,500 genes per sample including around 60% 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 one week from sample preparation to result reporting and provided a median of eight disease genes per patient for inspection. A genetic diagnosis was established for 16% of the 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. Together, these results show that streamlined experimental and computational processes can accelerate the implementation of RNA-seq in routine diagnostics.

How Machine Learning and Statistical Models Advance Molecular Diagnostics of Rare Disorders Via Analysis of RNA Sequencing Data

“…Murdock et al (2021) applied OUTRIDER in a cohort of 78 DNA-unsolved patients with diverse disorders and diagnosed five of them with aberrantly expressed genes. OUTRIDER was also applied to a cohort of 303 rare disease patients sequenced in the same center but from different ancestries (mostly European and Asian), which led to the identification of 26 aberrantly expressed disease causal genes (Yépez et al, 2021a).…”

“…The variants can be located in regulatory regions like enhancers, promoters, or suppressors of the corresponding gene, but also in the coding or intronic regions affecting splicing or creating a nonsense codon causing NMD. In Yépez et al (2021a), using WES, the cause of aberrant expression remained elusive in 65% of outliers and without the identification of a causative variant, the case remains undiagnosed. This fraction can be further reduced by using WGS, allowing the discovery of structural variants, which explain up to 25% of expression outliers (Ferraro et al, 2020).…”

“…This fraction can be further reduced by using WGS, allowing the discovery of structural variants, which explain up to 25% of expression outliers (Ferraro et al, 2020). A significant fraction of expression outliers can be explained by NMD affecting a single allele only, showing the sensitivity of RNAseq studies (Li et al, 2017;Ferraro et al, 2020;Yépez et al, 2021a). In the majority of cases solved via aberrant expression, the causal variant affects splicing.…”

“…Without detection of aberrant splicing, they would not have been prioritized. In addition, in some cases, aberrant expression was caused by deletions in the 5'UTR (e.g., NM_004544.3 c.-99_-75del causing 50% depletion in NDUFA10) or promoter regions (e.g., NM_016617.2 c.-273_-271del causing 40% depletion in UFM1) (Yépez et al, 2021a).…”

“…FRASER was also applied to the GEUVADIS multicenter and multi-ancestry cohort and was able to remove sample covariation for all metrics (Yépez et al, 2021b). In addition, FRASER has been used by Murdock et al (2021) leading to the diagnosis of four out of 78 subjects and by Yépez et al (2021a) leading to the diagnosis of 19 (12 in combination with aberrant expression) subjects from various ancestries.…”