Hereditary myopathies are a group of neuromuscular diseases primarily caused by defects in the skeletal muscle. These conditions show high clinical and genetic heterogeneity, which complicates reaching a precise diagnosis. Despite the advances with the implementation of Next Generation Sequencing (NGS), half of the patients with hereditary myopathies do not have a definitive genetic diagnosis after clinical genetic testing. Pinpointing the molecular defect is crucial for establishing disease prognosis and access to treatments, as well as receiving accurate genetic and reproductive counselling. Several factors can explain the difficulty in reaching a precise molecular diagnosis, including: many variants of uncertain significance are identified, the molecular defect is not captured in exome sequencing (ES), or the gene associated with the disease has not been described to date, among others.
In this thesis, we applied a stepwise multiomics strategy involving muscle RNA analysis and genetic reanalysis in 79 cases where a definitive genetic diagnosis was not established after the first NGS study. In addition, we performed genome sequencing on selected cases. These studies established the genetic cause in 27 out of 79 patients, representing a diagnostic yield of 34.2%.
The first publication included in this thesis focuses on seven dystrophinopathy cases with clear clinical and anatomopathological indications. However, standard genetic testing, including MLPA and ES, did not identify pathogenic variants in these patients. Muscle RNA analysis of the DMD gene showed an alteration in all studied cases, with pseudoexon inclusion being the most common pathogenic alteration. These results highlighted the need to perform additional transcriptomic studies in undiagnosed dystrophinopathy patients.
In the second publication, muscle RNA sequencing was performed in 70 undiagnosed cases with muscular dystrophies or myopathies. RNAseq established a molecular diagnosis in 10/70 (14.3%) patients, while reanalysis of the first NGS test allowed the diagnosis of 9/70 (12.9%) individuals. Seven cases (10%) are still under study since we have identified candidate variants in neuromuscular genes, an OMIM gene with an expanded phenotype and candidate non-OMIM genes highly expressed in skeletal muscle. In addition, a benchmarking of aberrant splicing detection tools was performed in this cohort, and based on our data, FRASER and FRASER2 outperformed LeafCutterMD and rMATS-turbo.
Finally, publication 3 describes a novel autosomal dominant RYR1 founder variant in the Basque population. We showed that the p.Leu2286Val variant is associated with nonprogressive muscle weakness, high CK levels and myalgia in seven families.
Overall, this work contributes to identifying the molecular mechanisms underlying muscle diseases, highlights the utility of complementary omic technologies in interpreting variants of uncertain significance, and improves the diagnosis of hereditary myopathies. We have shown that muscle RNA analysis is instrumental in two scenarios. First, to detect an in trans variant in cases with a single heterozygous pathogenic variant; and second, in cases with strong candidate gene(s) based on clinical presentation, such as dystrophinopathies or COL6-related dystrophies. We also show that the combination of genomics and transcriptomics increases the success rate and accelerates turnaround times to identify causative variants. Altogether, these analyses have contributed to put an end to the patients’ diagnostic odyssey, and have provided a deeper understanding of the molecular basis of hereditary myopathies through the discovery of novel candidate genes, new disease mechanisms and the phenotypic expansion of known conditions.
Improving genetic diagnosis of hereditary myopathies by integrating transcriptome, genome sequencing and reanalysis
Segarra Casas, A. (Author). 23 Oct 2025
Student thesis: Doctoral thesis
Student thesis: Doctoral thesis