Interpreting Genetic Variants in Titin in Patients With Muscle Disorders

Abstract

Importance: Mutations in the titin gene (TTN) cause a wide spectrum of genetic diseases. The interpretation of the numerous rare variants identified in TTN is a difficult challenge given its large size.

Objective: To identify genetic variants in titin in a cohort of patients with muscle disorders.

Design, setting, and participants: In this case series, 9 patients with titinopathy and 4 other patients with possibly disease-causing variants in TTN were identified. Titin mutations were detected through targeted resequencing performed on DNA from 504 patients with muscular dystrophy, congenital myopathy, or other skeletal muscle disorders. Patients were enrolled from 10 clinical centers in April 2012 to December 2013. All of them had not received a diagnosis after undergoing an extensive investigation, including Sanger sequencing of candidate genes. The data analysis was performed between September 2013 and January 2017. Sequencing data were analyzed using an internal custom bioinformatics pipeline.

Main outcomes and measures: The identification of novel mutations in the TTN gene and novel patients with titinopathy. We performed an evaluation of putative causative variants in the TTN gene, combining genetic, clinical, and imaging data with messenger RNA and/or protein studies.

Results: Of the 9 novel patients with titinopathy, 5 (55.5%) were men and the mean (SD) age at onset was 25 (15.8) years (range, 0-46 years). Of the 4 other patients (3 men and 1 woman) with possibly disease-causing TTN variants, 2 (50%) had a congenital myopathy and 2 (50%) had a slowly progressive distal myopathy with onset in the second decade. Most of the identified mutations were previously unreported. However, all the variants, even the already described mutations, require careful clinical and molecular evaluation of probands and relatives. Heterozygous truncating variants or unique missense changes are not sufficient to make a diagnosis of titinopathy.

Conclusions and relevance: The interpretation of TTN variants often requires further analyses, including a comprehensive evaluation of the clinical phenotype (deep phenotyping) as well as messenger RNA and protein studies. We propose a specific workflow for the clinical interpretation of genetic findings in titin.

Figure 1.. Western Blot for C-Terminal Titin Fragments

Western blotting using 2 different antibodies (M10-1 and 11-4-3) against the titin C-terminal M10 domain. A, Patient VIII with a single identified protein truncating variant shows a severe reduction of titin C-terminal fractions of all sizes; patient IV presents a reduced amount of the small (<20 kDa) titin fragments, and additionally the presence of a truncated fragment (arrowheads) resulting from the aberrant splicing due to the splice site mutation in intron 362. B, Patient Xa with missense mutations showed a normal titin C-terminal pattern, while patient IXa with a single protein truncating variant and 2 missense variants showed a reduction of the small (<20 kDa) titin C-terminal fragments in particular. The myosin heavy chain (MyHC) serves as the loading control. Ctrl indicates control; LGMD2J, limb-girdle muscular dystrophy 2J; TMD, tibial muscular dystrophy.

Figure 2.. Positions of TTN Missense Variants Identified in Families IX and X on a Structural Model

All images were made in DeepView/Swiss-PdbViewer, version 4.1.0 (GlaxoSmithKline R&D and Swiss Institute of Bioinformatics). The computed molecular surface is semitransparent gray and the secondary structure is shown with yellow β strands and red α helices. The mutated residue is shown as CPK. A, Position of p.Thr6324Pro using the most similar structure available in the Protein Data Bank (3B43). The mutated residue is located in a β strand. The mutation to proline will induce steric restrictions most probably causing a reduced stability and a structural disruption. B, p.Thr31339Ala modeled using the structure 2NZI of titin domains A168-A170. The change from threonine to alanine is predicted in a loop and will probably not interfere with the structure. However, the hydroxyl group on the sidechain of threonine allows for hydrogen bonding with other molecules. The amino acid substitution may alter interactions with TTN ligands in this specific region. C, Position of p.Asn32797Ser using the structure 2NZI. The mutated amino acid, one of the first residues in the domain, is on the surface of the model and it seems not to cause any important structural change. It will probably affect the binding to the interactors of this domain. D, Position of p.Trp33529Arg using the structure 2JBO. The tryptophan residue p.Trp33529 is almost totally buried in the hydrophobic core of the protein. The change to a positively charged arginine will probably be detrimental for the structural stability and will lead to an unfolding of this domain.

Figure 3.. Schematic Representation of Mutations Identified and Algorithm for the Clinical Interpretation of Genetic Findings in Titin

A, Schematic visualization of truncating (circle) and missense (triangle) variants identified in TTN gene in this study. B, Workflow for interpreting titin variants. Previously reported, disease-causing mutations in the TTN gene easily address the diagnosis toward a titinopathy. Identifying 2 truncating variants on both the alleles results in a diagnosis of titinopathy. A single heterozygous protein truncating variant is not sufficient for a diagnosis of titinopathy. Missense variants can lead to a diagnosis of titinopathy only when sufficient evidence supporting their pathogenicity is obtained.