G.P.17: TTN a challenge for next generation sequencing

Abstract

Tibial muscular dystrophy (TMD) is caused by mutations in the TTN gene. TTN is transcribing a >100 kb long mRNA, coding for the sarcomeric, largest known human protein, titin, spanning one half sarcomere in striated muscle. Titin (TTN) binds many other proteins and has important structural, mechanical and regulatory roles in muscles. The first TMD causing mutation, FINmaj a deletion/insertion of eleven base pairs, in the last 363:rd exon, Mex6, was reported by our group in 2002. Several different TTN mutations in the last two exons causing TMD have since been reported in European families. Due to its giant size and complexity analysis of TTN by conventional Sanger sequencing has been difficult and laborous. Usually only certain selected parts of the gene has been screened. Therefore the general spectrum and incidence of titinopathies has been underestimated. Next generation sequencing (NGS) methods have enabled an extensive increase in identification of mutations, and more than 120 TTN mutations have been reported in patients with at least 10 different conditions. In addition to using exome sequencing we have developed a targeted NGS custom panel, MyoCap, with the coding exons and UTRs of 236 muscular dystrophy related genes, including all exons of TTN. Due to the giant size of the gene, rare and novel TTN variants are now identified in many individuals analysed. Thus we have found more than ten patients with previously un-described phenotypes, with potential disease causing variants. This puts challenges in assessing the relevance and real correlation of these mutations with a certain disease phenotype. Since TTN is very repetitive, some regions are not well covered by NGS techniques. Insertion/deletion mutations may be missed in the variant calling. All this adds challenges to the development of the NGS methods and analysis of the results. Moreover, usage of the NGS for TTN requires large repertoire of RNA and protein techniques for the assessment of new variants.