Integrated genome and RNA sequencing analysis as a powerful technical framework to diagnose primary ciliary dyskinesia

Primary ciliary dyskinesia (PCD) is a rare, genetically heterogeneous respiratory disorder. Exome sequencing results unsuccessful in almost 30% of patients, likely due to variants in noncoding regions or structural variants. We applied RNA sequencing (RNA-seq) on epithelial nasal tissue in combination with Genome Sequencing (GS) as a second-tier strategy. Total RNA was extracted from 29 genetically undiagnosed PCD cases (study group), 8 genetically solved cases and 15 healthy participants. Ciliary enrichment was assessed before analysis of aberrant expression, aberrant splicing and monoallelic expression. GS was also performed in the study group. Ciliary transcriptome was robustly captured in 88% of the samples. Causative RNA alterations were identified in all samples from the positive control group. In the study group, causative transcriptomic outliers were detected in 22% of families, and the integration of GS increased the molecular diagnostic yield to 30% of families. A range of transcriptomic alterations was observed in association to their underlying causative variants, including single-nucleotide variants, copy number variants, and an insertion of a LINE element, most of them being novel. The affected genes included FOXJ1 , CCDC40 , RSPH1 , CFAP54 , SPAG1 , HYDIN and AK8 . In conclusion, the combination of GS and RNA-seq constitute a valuable diagnostic strategy for PCD.