Toward transcriptomics as a primary tool for rare disease investigation.

In the past 5 years transcriptome or RNA-sequencing (RNA-seq) has steadily emerged as a complementary assay for rare disease diagnosis and discovery. In this perspective, we summarize several recent developments and challenges in the use of RNA-seq for rare disease investigation. Using an accessible patient sample, such as blood, skin, or muscle, RNA-seq enables the assay of expressed RNA transcripts. Analysis of RNA-seq allows the identification of aberrant or outlier gene expression and alternative splicing as functional evidence to support rare disease study and diagnosis. Further, many types of variant effects can be profiled beyond coding variants, as the consequences of noncoding variants that impact gene expression and splicing can be directly observed. This is particularly apparent for structural variants that disproportionately underlie outlier gene expression and for splicing variants in which RNA-seq can both measure aberrant canonical splicing and detect deep intronic effects. However, a major potential limitation of RNA-seq in rare disease investigation is the developmental and cell type specificity of gene expression as a pathogenic variant's effect may be limited to a specific spatiotemporal context and access to a patient's tissue sample from the relevant tissue and timing of disease expression may not be possible. We speculate that as advances in computational methods and emerging experimental techniques overcome both developmental and cell type specificity, there will be broadening use of RNA sequencing and multiomics in rare disease diagnosis and delivery of precision health.