Fibroblast Transcriptomics in Molecular Diagnostics of a Comprehensive Dystonia Cohort

Genomic sequencing leaves >50% of dystonia-affected individuals without a diagnosis. Where DNA-oriented approaches remain insufficient, integrating multiomics is essential to advance genome interpretation. Herein, we incorporated RNA sequencing (RNA-seq) data from 167 patients with dystonia across a range of ages and presentations.

We leveraged an RNA-seq analysis pipeline, focused on the identification of expression and splicing aberrations, on RNA-seq from skin biopsies. The recruited patients had early-onset dystonia in 85.0%, non-focal dystonia in 92.2%, and coexisting features in 76.0%. Thirty-six patient samples with pre-identified variants (36/167, 21.6%) and 131 samples with no previously prioritized diagnostic candidates from genomic sequencing (131/167, 78.4%) were evaluated.

We found that >80% of dystonia-associated genes were detected by fibroblast RNA-seq. Expression and splicing aberration analyses produced a manageable number of significant RNA defects affecting dystonia-associated genes. The approach was especially successful in validating pathogenic effects of loss-of-function variants, with disease-relevant RNA-underexpression detected for 66.7% (10/15). Studying aberrant expression and splicing in the context of other pre-identified variant types yielded relevant results in 28.6% (6/21 samples). We obtained a 6.9% (9/131) diagnostic uplift for patients without prior candidates, all of whom exhibited combined dystonia with autosomal recessive inheritance. The new diagnoses from RNA-seq and genomic reanalysis were based on previously neglected splice-region (3/9) and deep(er) intronic (6/9) variants. For the observed events, integration of new machine-learning scores predicted corresponding aberrant gene expression in the brain.

Fibroblast-based RNA-seq in our selected cohort improved variant interpretation and offered a modest yield in patients without prior candidate variants. ANN NEUROL 2026;99:1363-1378.