Improvements in NGS have led to significant DNA sequencing information output used for diagnosing rare conditions. Nevertheless, the interpretation of this data remains limited. Exome sequencing (ES) and whole genome sequencing (WGS) provide modest diagnostic yields, with WGS ranging between 19.1% and 68.3%. RNA sequencing is currently being utilized to complement DNA sequencing, enabling the identification of erroneous splicing and gene expression. Unlike RT-PCR, RNA sequencing (RNA-seq) offers a broader, agnostic approach to detecting abnormalities, improving the diagnosis of rare diseases where DNA analysis has failed.
This study included 86 individuals with rare diseases. RNA sequencing was performed using total cell RNA. Erroneous splicing events were assessed in patients with VUS variants and those with no known genetic cause. Various bioinformatics tools were used to detect splicing abnormalities, including exon skipping and intron retention. Validation was conducted using RT-PCR and Sanger sequencing. Expression outliers were detected using the OUTRIDER pipeline, while splicing events were analyzed using several methods, including FRASER2 and rMATS-turbo.
RNA sequencing revealed a mean of 76.6 million reads per sample, with 80% mapping uniquely. Spearman’s relationship among median TPM values and GTEx was 0.79. Disease genes from OMIM and the UK PanelApp showed 67% and 75% expression in blood, respectively. Out of 48 cases with Variant of unknown siognificance (VUSs), RNA-seq identified erroneous splicing in 14 patients, confirmed by RT-PCR. SpliceAI accurately predicted splicing in most cases. For cases without candidate variants, splicing tools and RNA-seq revealed new candidate variants linked to developmental impairment.
This study demonstrates that RNA-seq can be a valuable tool in diagnosing rare diseases, even in patients without candidate VUSs. By combining RNA-seq and RT-PCR, the researchers identified new splicing abnormalities and enhanced the diagnostic yield in 35% of cases with a VUS. In patients without a candidate VUS, RNA-seq detected three pertinent splicing variations and one instance of skewed X-inactivation, providing a 7% diagnostic uplift. The study recommends using RT-PCR for genes with low expression and RNA-seq for comprehensive splicing analysis, suggesting that RNA-seq should be integrated into genetic testing for rare diseases.
[Press release] Geneyx and Ocean Genomics Announce SVDuo™ Release, Delivering Integrated DNA and RNA Analysis to Enhance Genomic Variant Detection for Research and Clinical Genomics
