23P Establishing the genetic diagnosis in patients with suspected recessive titinopathy

• Published in: Neuromuscular disorders : NMD 2024-10, Vol.43, p.104441, Article 104441.221
• Main Authors: Mueller, J., Savarese, M., Lillback, V., Perry, L., Zaharieva, I., Pini, V., Sagath, L., Steyaert, W., Hoischen, A., Yepez, V., Esteve-Codina, A., Neveling, K., Topf, A., Muntoni, F., Sarkozy, A.
• Format: Article
• Language: English
• ISSN: 0960-8966
• DOI: 10.1016/j.nmd.2024.07.230

Pathogenic variants in the Titin gene (TTN) cause a wide spectrum of muscle disorders and cardiomyopathies, with either dominant or recessive inheritance. Early, paediatric onset, recessive Titinopathies are emerging as a common form of congenital myopathy (TTN-CM). Due to the genetic complexity of the TTN gene, molecular diagnosis of TTN-CM relies on a complex, multidisciplinary approach. However, molecular diagnosis may often remain elusive, due to inability to identify and/or interpret TTN variants. In this work, we present results of complementary, clinical and multi-omics investigations on 15 patients, referred to the Highly Specialised Service at the Dubowitz Neuromuscular Centre in London. All patients presented with clinical, pathological and MRI features compatible with TTN-CM, and carried a monoallelic, pathogenic TTN variant. We hypothesised that a second pathogenic variant (either a deep intronic, splicing or structural variant) has so far eluded detection through routine sequencing or was wrongly interpreted. We thus employed complementary technologies including RT-PCR analysis, PacBio HIFI Long Read Whole Genome Sequencing (LR-WGS), Optical Genome Mapping (OGM, Bionano) and RNA Sequencing (RNASeq) to help to achieve a molecular diagnosis for these patients. Direct cDNA analysis was performed in a patient with a homozygous c.29134+5G>A variant. The analysis confirmed aberrant splicing of exon 102 in the TTN gene. We applied LR-WGS in a patient with two pathogenic variants, one maternally inherited (c.105854dupC) and one de novo (c.40652delC) and were able to establish these variants were in trans and thus confirm the diagnosis. To identify second elusive variants, we completed mRNASeq on RNA extracted from muscle in 5 patients. Due to the size of the TTN gene, this analysis was not successful as some areas of the gene were not well covered. Total RNASeq was thus performed in these and additional 7 patients, and results are pending. In addition, we also collected peripheral blood for OGM in 5 patients to investigate possible structural variants, and establish the role of this technique in the diagnostic process for TTN-CM. The presented work indicates that RNA based analysis and LR-WGS can support diagnostic process for the TTN gene and will help to establish a possible role for Total RNASeq and OGM.