Congenital Titinopathies Linked to Mutations in TTN Metatranscript-Only Exons

Abstract

Congenital titinopathies reported to date show autosomal recessive inheritance and are caused by a variety of genomic variants, most of them located in metatranscript (MTT)-only exons. The aim of this study was to describe additional patients and establish robust genotype-phenotype associations in titinopathies. This study involved analyzing molecular, clinical, pathological, and muscle imaging features in 20 patients who had at least one pathogenic or likely pathogenic TTN variant in MTT-only exons, with onset occurring antenatally or in the early postnatal stages. The 20 patients with recessive inheritance exhibited a heterogeneous range of phenotypes. These included fetal lethality, progressive weakness, cardiac or respiratory complications, hyper-CKemia, or dystrophic muscle biopsies. MRI revealed variable abnormalities in different muscles. All patients presented severe congenital myopathy at birth, characterized by arthrogryposis (either multiplex or axial-distal) or neonatal hypotonia in most cases. This study provides detailed genotype-phenotype correlations in congenital titinopathies caused by mutations in MTT-only exons. The findings highlight the variability in clinical presentation and the severity of phenotypes associated with these specific genetic alterations. RNA-seq analyses provided valuable insights into the molecular consequences of TTN variants, particularly in relation to splicing defects and nonsense-mediated RNA decay. In conclusion, this study reinforces the genotype-phenotype correlations between congenital myopathies and variants in TTN MTT-only exons, improves their molecular diagnosis, and provides a better understanding of their pathophysiology.

Keywords: arthrogryposis; congenital titinopathies; genetics; myopathy; neurology.

Figure 1

Cohort description and location of variants on the TTN gene. (A) Clinical presentation. (B) Genomic variants mapped on the TTN gene; colors are attributed to each patient. Variants described in the upper panel are metatranscript-only exon variants and, in the lower panel, constitutively expressed exons.

Figure 2

MRI data. (A) Patient P14 MRI analysis, selection of axial T1 weighted sections of a patient with a recessive form of titinopathy. Muscle damage with fat infiltration is disseminated; the most preserved muscles are the supraspinatus, psoas–iliacus, gluteus maximus, great and long adductors, and short head of the biceps femoris. Fat content in muscles is more pronounced in anterior and posterior thighs and legs. (B) Assessment of muscular damage to all four limbs using the Mercuri scale. Fatty replacement varies widely from muscle to muscle and from patient to patient. However, involvement predominates in proximal muscles: in the upper limbs, the deltoids, trapezius, rotator, and scapula fixator muscles are the most affected, whereas in the lower limbs, involvement predominates in the glutei muscles, in the thighs and legs, although the sartorius and gracilis muscles are spared.

Figure 3

Molecular analysis of patient P16 and WBs of all available biopsies. (A) Localization of the variants identified in patient P16 on the TTN gene represented with its domains, Z-disk, I-band containing the PEVK domain, A-band, and M-band. The variants are localized in the I-band, including the variant of exon 164 in the PEVK domain. (B) Transcript analyses in the regions of the mutated exons. Exon inclusion rates in transcripts reported in Savarese et al., 2018 [6] are noted as percentages above DNA exons, and RNA splicing with exon inclusion is described. RNA-seq results are represented by a sashimi plot (right panel). Exon 115-carrying variant 1 is expressed in 100% of the transcripts. Exon 164 carrying variant 2 is skipped in mature N2A transcripts. (C) Schematic representation of the proteins theoretically translated by each allele with the predicted sizes. Allele 1 should produce a protein of about 1.1 MDa, whereas allele 2, with a variant in a metatranscript exon, should not express the variant and should translate a normal-sized protein of 3.8 MDa. (D) Western blot analyses of patients with TTN truncating variants and available muscle biopsies. Anti-TTN antibodies against the N-terminal part (Sigma1400284; left panel) and against the C-terminal part (M10.1; right panel). The analysis was performed in patients P15, P16, P3, P17, and P12. Alleles carrying frameshift variants can produce truncated proteins whose theoretical sizes have been calculated: in green, the theoretical sizes of proteins from variants in exons located in the metatranscript (if they are not skipped from N2A transcripts), and in red, the theoretical sizes of proteins from variants in exons expressed constitutively. The size of the observed titin protein is indicated in black. No titin truncated protein of 1.1 MDa is visible on the blot for P16.