The complexity of titin splicing pattern in human adult skeletal muscles

Abstract

BackgroundMutations in the titin gene (TTN) cause a large spectrum of diseases affecting skeletal and/or cardiac muscle. TTN includes 363 coding exons, a repeated region with a high degree of complexity, isoform-specific elements, and metatranscript-only exons thought to be expressed only during fetal development. Although three main classes of isoforms have been described so far, alternative splicing events (ASEs) in different tissues or in different developmental and physiological states have been reported.MethodsTo achieve a comprehensive view of titin ASEs in adult human skeletal muscles, we performed a RNA-Sequencing experiment on 42 human biopsies collected from 12 anatomically different skeletal muscles of 11 individuals without any skeletal-muscle disorders.ResultsWe confirmed that the skeletal muscle N2A isoforms are highly prevalent, but we found an elevated number of alternative splicing events, some at a very high level. These include previously unknown exon skipping events and alternative 5′ and 3′ splice sites. Our data suggests the partial inclusion in the TTN transcript of some metatranscript-only exons and the partial exclusion of canonical N2A exons.ConclusionsThis study provides an extensive picture of the complex TTN splicing pattern in human adult skeletal muscle, which is crucial for a proper clinical interpretation of TTN variants.Electronic supplementary materialThe online version of this article (10.1186/s13395-018-0156-z) contains supplementary material, which is available to authorized users.

Recurrent TTN metatranscript‐only c.39974–11T>G splice variant associated with autosomal recessive arthrogryposis multiplex congenita and myopathy

“…We further performed RNA‐seq of five fetal muscle specimens and showed 4/5 fetal muscle RNA samples showed predominant inclusion of exons 213‐217 (inclusion rate ≥0.66) with the remaining fetal muscle sample showing an inclusion rate of 0.57. Our data are supported by recent studies, which show that the metatranscript‐only exons 213‐217 are more highly expressed in fetal muscle than the adult muscle (Savarese et al, ).…”

“…Titin is encoded by TTN , arguably one of the most complex human genes; with 364 exons encoding extensively alternatively spliced transcripts that are approximately 100,000 nucleotides in length (Bang et al, ; Freiburg et al, ; Labeit & Kolmerer, ). Further contributing to the complexity, TTN bears a triplicated repeat region that encompasses three, near‐identical replicated blocks of nine exons, which are alternatively spliced (Savarese et al, ), and technically very challenging to sequence. When a variant is identified within the triplicated repeat region, in many cases it is impossible to be certain in which exon it resides.…”

“…While the use of the cryptic 3′ splice‐site inducing a frameshift may readily be interpreted as exerting damaging consequences for the encoded titin protein; it remains difficult to interpret the functional implications attributable to the loss of 28 residues within the differentially‐spliced PEVK region of titin. Exons within the PEVK region are extensively alternatively spliced, regulating passive tension and muscle elasticity (Freiburg et al, ; Ottenheijm et al, ; Savarese et al, ). However, exon 214 skipping is not observed in control muscle (Savarese et al, ), is a very rare/absent event among our muscle RNA‐seq data from approximately 50 disease controls (data not shown) and absent from eight control samples by RT‐PCR (results for C1 and C2 are shown in Figure a‐ii).…”

“…In support of pathogenicity, there are several recent reports of autosomal recessive congenital titinopathies associated with variants within metatranscript‐only exons 163, 172, 181, 201 (Oates et al, ), 197 (Fernández‐Marmiesse et al, ), and 167 (Chervinsky et al, ). Junctional reads bridging exons 181 and 170 are detected in adult skeletal muscle (Savarese et al, ), inferring the novel frameshift variants found in families A and F affect transcripts expressed in skeletal muscle. Of note, exons 213‐217 are not expressed at significant levels in cardiac tissue (Savarese et al, ) and reported individuals with a metatranscript‐only pathogenic TTN variant did not present with a cardiac phenotype (herein and in Chervinsky et al, ; Fernández‐Marmiesse et al, ; Oates et al, ).…”

“…Junctional reads bridging exons 181 and 170 are detected in adult skeletal muscle (Savarese et al, ), inferring the novel frameshift variants found in families A and F affect transcripts expressed in skeletal muscle. Of note, exons 213‐217 are not expressed at significant levels in cardiac tissue (Savarese et al, ) and reported individuals with a metatranscript‐only pathogenic TTN variant did not present with a cardiac phenotype (herein and in Chervinsky et al, ; Fernández‐Marmiesse et al, ; Oates et al, ).…”