478P RNA sequencing as a diagnostic tool in a cohort of 54 undiagnosed patients with neuromuscular diseases

• Published in: Neuromuscular disorders : NMD Vol. 43; p. 104441
• Main Authors: Segarra-Casas, A., Domínguez-González, C., Natera-de-Benito, D., Ortez, C., Nascimiento, A., Hernández-Laín, A., Kapetanovic, S., Rodríguez, M., González-Mera, L., Nedkova, V., Fernández-Torrón, R., López-de Munain, A., Jimenez-Mallebrera, C., Rodríguez-Santiago, B., Gallardo, E., Olivé, M., Gallano, P., González-Quereda, L.
• Format: Journal Article
• Language: English
• Published: Elsevier B.V 01.10.2024
• ISSN: 0960-8966
• DOI: 10.1016/j.nmd.2024.07.324

The implementation of Next Generation Sequencing, particularly exome sequencing (ES), into clinical practice has revolutionised the genetic diagnosis of rare diseases. However, it is estimated that 50% of patients with neuromuscular diseases remain undiagnosed after ES. Here, we evaluated total RNA sequencing (RNAseq) from muscle biopsies as a diagnostic tool in genetically unsolved patients. Total RNAseq was extracted from muscle biopsies of 54 undiagnosed patients after ES or gene panel sequencing, 3 positive controls (patients with previously identified RNA defects in the DMD gene) and 17 healthy controls. We evaluated the presence of aberrant splicing, outlier gene expression and allelic imbalance in autosomal recessive genes. In addition, variant calling was performed from the RNAseq data. In 7/54 patients (12.9%) we detected pathogenic alterations at the mRNA level, including two exon skipping events (COL6A1), one intronic retention (TTN), one alternative splicing caused by a synonymous variant (RYR1), one pseudoexon (DMD) and two patients with allelic imbalance in an autosomal recessive gene (GNE). In one of these cases, WGS was required to achieve the genetic diagnosis. Three patients (5.55%) remain under investigation due to the detection of alternative splicing events in new candidate genes. In addition, reanalysis of existing ES data allowed the diagnosis of 8/54 individuals (14.8%). In this study, we show that RNAseq analysis improves the diagnostic rate in patients with neuromuscular diseases after inconclusive results in ES. RNAseq not only enables the identification of molecular events that would not have been detected by other methods, but also allows the reclassification of VUS variants identified at the gDNA level. We also highlight the importance of a periodic reanalysis of existing genomic data and the utility of an integrated genomic and transcriptomic analysis.