RNA sequencing provides functional insights and diagnostic resolution in previously unsolved rare disease cases
Recommended Citation
Lewis RG, O'Shea JM, Pizzo L, Wen T, Fulmer ML, Zhao J, Verheijen J, Zhang C, Velinder M, Nicholas TJ, Boyden SE, Ward A, Baldwin EE, Andrews A, Ruiz JH, Marchetti M, Viskochil D, Carey JC, Bleyl SB, Butterfield RJ, Taliercio V, Botto LD, Mao R, and Bayrak-Toydemir P. RNA sequencing provides functional insights and diagnostic resolution in previously unsolved rare disease cases. BMC Med Genomics 2025;18(1):182.
Document Type
Article
Publication Date
11-14-2025
Publication Title
BMC Med Genomics
Keywords
Humans, Rare Diseases, Male, Female, Sequence Analysis, RNA, Retrospective Studies, RNA Splicing
Abstract
Exome and genome sequencing have greatly improved the diagnosis of rare genetic disorders but remain limited in their ability to identify and classify non-coding variants, including intronic variants, cryptic splice-site alterations, and disruptions in regulatory regions. RNA sequencing (RNA-seq) has emerged as a powerful tool to bridge this gap by providing functional insights into genomic variants that disrupt splicing or gene expression, thereby aiding in variant interpretation and classification. We retrospectively reviewed 30 cases from the Utah Penelope Program and the Undiagnosed Diseases Network over a three-year period, in which RNA-seq was performed on whole blood and/or fibroblasts following either negative DNA sequencing or the identification of candidate variants requiring functional assessment. In these cases, RNA-seq identified exon skipping, cryptic splice-site activation, and intron retention, leading to transcript disruption. Additionally, positional enrichment analysis clarified X-inactivation patterns and dosage effects, confirming the pathogenicity of copy number variants. By detecting these transcript-level alterations, RNA-seq provided functional evidence supporting the reclassification of multiple variants of uncertain significance, contributing to diagnostic resolution in selected cases. This study underscores the clinical utility of RNA-seq in detecting splicing and regulatory defects that DNA sequencing and predictive tools alone cannot resolve. Integrating RNA-seq into clinical workflows can support variant classification, aid in diagnostic resolution for selected cases, and provide mechanistic insights into genetic disorders, contributing to patient care and genetic counseling.
Medical Subject Headings
Humans; Rare Diseases; Male; Female; Sequence Analysis, RNA; Retrospective Studies; RNA Splicing
PubMed ID
41239373
Volume
18
Issue
1
First Page
182
Last Page
182
