Clinical phenotype and loss of the slow skeletal muscle troponin T in three new patients with recessive TNNT1 nemaline myopathy

Figures

• 

  Figure 1. Representative frozen sections (A–E) and electronic microscopy images (F–H). Gomori trichrome: patient 1 (A): marked fibre size irregularity. Rare atrophic fibres with hyaline core-like intracytoplasmic inclusions (white arrows), slight increased endomysial connective tissue. Patient 2 (B): numerous rods and endomysial fibrosis. Patient 3 (C): rods are visualised in disorganised fibres. ATPase pH 4.6: patient 1 (D) and patient 3 (E): type I fibres are dark; type IIA fibres are pale, type IIB are intermediate stained; type I fibres are irregular and smaller than type II. No myopathic grouping. The proportion of type I and type II is preserved. No fibre-type grouping. Ultrastructure: patient 1 (F): the sarcomere structure is disrupted in a large circumscribed central area of a muscle fibre (fibre on the right, white arrows showing the outlines of this area). Rods are visualised within this area (white arrow heads showed some rods). Detail: a typical rod with homogeneous lattice Z disk-like structure in continuity with thin filaments. Patient 2 (G): numerous electron dense rods in a disorganised area. Patient 3 (H): rods are observed throughout the cytoplasm. Detail of a typical rod…

• 

  Figure 2. Homozygous deletion of exons 8–9 of the TNNT1 gene. Integrative Genomics Viewer visualisation of the deletion identified by NGS in case1 (I401). Precise breakpoint determination was further obtained by Sanger sequencing (hg19chr2 : g.55650633_55652981del; NM_003283.5 : c.192+244_388-1191del). NGS, next-generation sequencing; TNNT1, troponin T type 1.

• 

  Figure 3. TNNT1 variants identified in patients. Patient 1 revealed a homozygous deletion of exons 8 and 9 of the TNNT1 gene. These two exons encode part of the Tm1 of slow skeletal muscle troponin T. Patient two carried the heterozygous nonsense variant c.334G>T in exon 9 leading to a predicted stop codon p.(Glu112*) in Tm1. Through muscle transcript analysis, a second heterozygous variant was identified in this patient in intron 4, c.74–67C>A, activating a cryptic splice-accepting site and predicted to lead to intron retention. Patient 3 revealed a very early homozygous stop codon p.(Glu6*). Tm1, tropomyosin binding site 1 domain; TNNT1, troponin T type 1.

• 

  Figure 4. Transcript analyses from muscle biopsies. Visualisation on Integrative Genomics Viewer. In grey are represented the coverage of each sequence. The red and blue crescent represent the splice junction. 3.1. Transcript patterns from exons 7 to 10 from a healthy control (A) and patient 1 (B) samples. The complete skip of exons 8 and 9 observed in patient 1 is represented, compared with the normal splicing of exons 7–10 in the healthy control. 3.2. Transcript patterns from exon 4 to 5 from a healthy control (A) and patient 2 (B) samples. Two transcripts were identified in patient 2: one normal splice pattern between exon 4 and 5 (similar to the healthy control) and another transcript with retention of 65 nucleotides of intron 4.

• 

  Figure 5. Western blot analysis of sTNT. Muscular biopsy homogenates were realised for the three patients (patients 1–3) and an age relative control biopsy (CTRL). An antitubulin antibody was used to control the protein amount loading. An anti sTNT was used to evaluate the quantity of sTNT. Compared with the control, the three patients have nearly null expression of sTNT. The very faint bands detected in patients’ biopsy most probably correspond to non-specific reactivity of the antibodies but could also reflect cross-reactivity with other TNNT isoforms. They represent less than 0.5% of the TNNT1 amount in the control biopsy (TNNT1/tubulin amount fixed to 100% in control). sTNT, skeletal muscle-specific troponin T; TNNT1, troponin T type 1.