Pathomechanisms of Monoallelic variants in TTN causing skeletal muscle disease

Abstract

Pathogenic variants in the titin gene (TTN) are known to cause a wide range of cardiac and musculoskeletal disorders, with skeletal myopathy mostly attributed to biallelic variants. We identified monoallelic truncating variants (TTNtv), splice site or internal deletions in TTN in probands with mild, progressive axial and proximal weakness, with dilated cardiomyopathy frequently developing with age. These variants segregated in an autosomal dominant pattern in 7 out of 8 studied families. We investigated the impact of these variants on mRNA, protein levels, and skeletal muscle structure and function. Results reveal that nonsense-mediated decay likely prevents accumulation of harmful truncated protein in skeletal muscle in patients with TTNtvs. Splice variants and an out-of-frame deletion induce aberrant exon skipping, while an in-frame deletion produces shortened titin with intact N- and C-termini, resulting in disrupted sarcomeric structure. All variant types were associated with genome-wide changes in splicing patterns, which represent a hallmark of disease progression. Lastly, RNA-seq studies revealed that GDF11, a member of the TGF-β superfamily, is upregulated in diseased tissue, indicating that it might be a useful therapeutic target in skeletal muscle titinopathies.

Keywords: TTN deletion; TTNtv; Dominant titinopathy; Titin; titinopathy pathomechanisms.

Figure 1

Pedigrees of titinopathy cases included in this study. Studied cases are indicated by arrows. Individuals with known symptoms are shaded blue (for skeletal muscle) or red (for cardiac muscle). Individuals who underwent genetic testing are denoted with a ‘+’ to indicate the TTN variant was found, or a ‘-’ to indicate the TTN variant was not found. Individuals not marked with ‘+’ or ‘-’ were not available for testing. Cases 12 and 19 are not listed because they were included as disease controls.

Figure 2

Titin transcript levels, allelic balance and exon inclusion levels. Titin transcript levels, determined by RNA sequencing analysis and normalized to nebulin levels (A). Truncating variants displayed reduced transcript levels, while splice site variants were similar to controls. Analysis of biallelic sites confirmed a shift in the allelic balance ratio for this group of variants (B). Numbers represent p-values from unpaired t-tests. Exon 117 was skipped in almost half TTN^splice117 transcripts (C), while only low percentages of exon skipping (1.5%–7.5%) were observed in TTN^splice242 (patients 15, 16 & 18, D). In-frame deletion TTN^del346–362 had the affected exons missing in half of the transcripts (E). Two patients with a large out-of-frame deletion (TTN^del326–347) showed low degrees if exon skipping over the deletion site (F). One PEVK-domain is absent in TTN^splice117 due to exon skipping (G) while the last C-zone super-repeat and most of the M-band (except for M10) are deleted in TTN^del346–362 (H).

Figure 3

A subset of titinopathy samples shows differential splicing patterns. A: An initial analysis of differential splicing between three patient samples with an intron 242 splice acceptor variant [&, 15, 16, 18] and controls revealed a total of 1333 differentially spliced exons (p < 0.05 & PSI change > 5%). Together with two other patient samples [19 & 22], they form a distinct group with splicing patterns that are different from all other control & patient samples during column-wide clustering. Shown are inclusion levels (PSI) of differentially spliced exons, as determined during the initial analysis. B: One or more exons of genes known to be important for muscle development and function are differentially spliced in titinopathy patients (red). Controls are plotted in blue and titinopathy patients without genome-wide splicing changes in black. PSI values are plotted against exon numbers.

Figure 4

GDF11 levels are increased Titinopathy patients. Transcript-specific expression revealed increased levels of GDF11 in titinopathy patients (A). Sample grouping based on variant type revealed a high level of significance in truncating and deletion variants versus controls (B). Significance was calculated from transcript abundances and represents multiple-testing corrected p-values. GDF11 levels did not increase with age of the patient population and correlated negatively in controls (C). Average age did not differ between patients and controls (patients:45.45, controls: 45).

Figure 5

Titin protein analysis. Titin protein was separated on agarose gels and detected with antibodies against its N-termini (Z1Z2, (A), top panels) and C-termini (M10, bottom panels). (A) Shortened TTN protein could only be detected in-frame deletion TTN^del346–362 (patient 5). Overlays of Z1Z2 and M10 images (B) demonstrate that no C-terminally truncated proteins are present at expected protein sizes (indicated by white boxes). Domains M8 and M9 are internally deleted in sample 5 and not detected in shortened titin with an M8M9-specific antibody (C) in contrast to domain M10 (A & B). Shortened titin is less abundant in sample 5 than full-length protein when quantified on agarose gels (D, values represent intensity). Protein levels of truncating and deletion variants (E) were not significantly changed from controls when normalized to MHC (F). Five biopsy samples had protein levels that were too low to detect by gels or Western blots (samples 8, 11, 16, 19 & 22).

Figure 6

TTN^del346–362 muscle shows abnormalities in muscle structure. Analysis of tibialis anterior muscle by electron microscopy revealed irregular Z-disks and M-lines from in-frame deletion TTN^del346–362 (case 5, first 4 panels), which produces shortened titin protein. Normal muscle structure was observed for two patients with truncating variants (TTN^stop326–2 and TTN^stop326–3), a patient with a splice donor variant (8,TTN^splice117) and controls [9 and 13].

Figure 7

Passive mechanics in slow fibers from titinopathy patients. (A) Left panel: Ramp-stretch protocol for determining passive tension (force divided by fiber cross-sectional area) in skeletal muscle fibers. Fibers were set at slack sarcomere length and stretched in steps of 10% of fiber length (FL) at a speed of 1% FL/s and held for 20 s until the next step. Right panel: Passive force response in a control fiber from the ramp-stretch protocol. (B) Peak tensions plotted against sarcomere length for each step in the stretch protocol. (C) Peak tension at the sarcomere length range 3.0–3.12 μm (shaded area in D). (D) Cut out gel image of silver-stained 8% SDS-PAGE used to determine fiber types. Std; standard, Ctrl; control fiber, Pat-f#; patient fiber. Individual patient fibers are displayed. Controls are the average of 3–11 fibers per individual. ^*p < 0.05, and ^**P < 0.01 vs controls.

Figure 8

Membrane-permeabilized muscle fiber experiments reveal myofilament dysfunction in slow fibers from some individual titinopathy patients. (A) Left panel: Recording of tension developed by a slow skeletal muscle fiber at incremental [Ca2+]. Right panel: Tension-Ca2 + −curve for the fiber in A with EC50 ([Ca2+] for 50% tension) and Hill coefficient (nH) of curve shown. (B) Active stiffness protocol. Left panel: Relative length change; 0.3% of fiber length (FL) 500 Hz sinusoid for 20 ms was used to determine active stiffness. Right panel: Force trace for a fast and a slow fiber. Stiffness is calculated by dividing the force change with the length change. (C) Rate of force redevelopment protocol. Left panel: Relative length change; the fiber is shortened to 75% of FL in a ~ 1 ms step, held for 20 ms, stretched 103% of FL and immediately returned to 100% of FL. Right panel: Force trace for a fast and a slow fiber fitted with double exponential function. (D) Active tension (force normalized to fiber cross-sectional area). (E) Calcium sensitivity as EC50. (F) Hill coefficient (nH) is slope of the steep rise segment (black line in A, left panel) in tension-Ca²⁺-curve. (G) Active stiffness, an indicator of strongly-bound crossbridges. (H) Tension-stiffness ratio. (I) Rate of force redevelopment (k_tr). Control values are average of 5–21 fibers from 8 subjects without known neuromuscular disease. Individual fibers are shown for patients. One-way ANOVA with Dunnett’s multiple comparisons post hoc test was used for statistical testing. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001 and ^****P < 0.0001 vs control.