Making sense of missense variants in TTN-related congenital myopathies

Abstract

Mutations in the sarcomeric protein titin, encoded by TTN, are emerging as a common cause of myopathies. The diagnosis of a TTN-related myopathy is, however, often not straightforward due to clinico-pathological overlap with other myopathies and the prevalence of TTN variants in control populations. Here, we present a combined clinico-pathological, genetic and biophysical approach to the diagnosis of TTN-related myopathies and the pathogenicity ascertainment of TTN missense variants. We identified 30 patients with a primary TTN-related congenital myopathy (CM) and two truncating variants, or one truncating and one missense TTN variant, or homozygous for one TTN missense variant. We found that TTN-related myopathies show considerable overlap with other myopathies but are strongly suggested by a combination of certain clinico-pathological features. Presentation was typically at birth with the clinical course characterized by variable progression of weakness, contractures, scoliosis and respiratory symptoms but sparing of extraocular muscles. Cardiac involvement depended on the variant position. Our biophysical analyses demonstrated that missense mutations associated with CMs are strongly destabilizing and exert their effect when expressed on a truncating background or in homozygosity. We hypothesise that destabilizing TTN missense mutations phenocopy truncating variants and are a key pathogenic feature of recessive titinopathies that might be amenable to therapeutic intervention.

Fig. 1

Typical features in TTN-related myopathies. This 38-year-old patient presented with hypotonia and ventricular non-compaction from birth. Subsequently, his motor development was delayed. His stature was short, he had a myopathic face with pronounced ptosis (a), multiple contractures prominently involving the elbows and shoulders (b, c), and spinal rigidity (d). He developed a dilated cardiomyopathy from his teens. The most prominent feature on muscle biopsy are numerous centralized nuclei (e) leading to an initial diagnosis of CNM, but there were additional cores (f, g) and few nemaline rods (h) on EM. Scale bars 40 µm (e, f), 2 µm (g) and 1 µm (h). Lower extremity muscle MRI from another patient showing prominent hamstring involvement in the thigh (i). Scale bar 5 cm, L indicates left side

Fig. 2

Localisation and type of variants along Titin in our patient cohort. N2A, N2B and IC transcripts detailed, with exons absent in N2B highlighted on plot in light grey and exons absent in both N2A and N2B in dark grey. Positioning of antibodies used in immunofluorescence studies also indicated (green diamonds). DNA and protein numbered according to the IC transcript NM_001267550

Fig. 3

Immunofluorescence analysis demonstrates the sarcomeric integration of truncating and missense titin variants in patients 22, 24 and 29. Cryosections of heart tissue (patients 22 and 24) and skeletal muscle (patient 29) were stained for N-terminal Z-disk titin (red; Z1Z2 antibody) and C-terminal M-band titin (green; M2, Mis6 and M8-M9 antibodies). Note that only the C-terminal epitope Mis6 is absent for patient 29, carrying two truncating mutations before the Mis6 epitope. Scale bar: 10 µm

Fig. 4

Crystal structures or homology models highlighting atomic environment of residue mutated in patient missense variants. The side-chains of residues (and main chain for Fn3-90) mutated in patients are represented as labelled green sticks, with surrounding side-chains of interest also represented as sticks. Polar contacts are shown via yellow dashed lines. Ig-1 structure is from the crystal structure of first two titin domains, PDB code 2a38. All other models were generated from TitinDB. Images generated using PyMol 2.1.1

Fig. 5

Western blot assessing soluble expression of WT and missense variant-containing titin domains. Bacterial cultures expressing His-tagged titin domains were lysed, separated into total and soluble fractions, run on Western blot and probed with anti-His tag antibody

Fig. 6

Biophysical characterisation of WT and missense variant-containing titin domains. a 1D NMR of Fn3-49 WT (black) and Val22232Glu (lilac). b Differential scanning fluorimetry of Fn3-119 WT (black) and Pro31732Leu (lilac). c thermal denaturation measured by circular dichroism of Fn3-120 WT (left) and Arg31847Pro (right)

Fig. 7

Confocal immunofluorescence microscopy of neonatal rat cardiomyocytes expressing GFP-tagged titin Ig-125-126 WT and Val22232Glu, and Ig-141-142 WT and Gly27849Val. Cells were counterstained with antibodies against myosin heavy chain (red) and Obscurin O59 (blue, marking the M-band); GFP-titin in green. Small arrows mark the A-band doublets labelled by the A4.1025 monoclonal antibody against myosin heads [9] and large arrows mark the intercalated disk. Scale bar: 10 µm

Fig. 8

Confocal microscopic images showing expression of titin fragments in C2C12 myocytes. GFP-tagged titin fragments Ig-125-126 WT and Val22232Glu, and Ig-141-142 WT and Gly27849Val, were transfected into C2C12 myoblasts and seven days post-transfection were fixed and stained with antibodies against p62/SQSTM1 and conjugated ubiquitin (c-Ub). 4–8 transfected cells per titin fragment were imaged, with representative cells shown here. Only the GFP and p62/SQSTM1 channels were used for the merged image. Arrows indicate examples of colocalisation of mutant titin, p62/SQSTM1 and conjugated ubiquitin