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.

because it expresses also the N2A element, a mechanosensory signaling hub. We targeted N2BA titin by generating mice in which titin exons 112-158 are deleted (Ttn D112-158). These exons encode spring region sequences that are specific to the N2BA isoform. The Left ventricle (LV) of Ttn D112-158 shows considerable downregulation of N2BA mRNA and protein. At 6-months of age (6mo), functional analysis reveals LV dilation and systolic dysfunction with normal diastolic function, a phenotype consistent with DCM. The DCM is progressive and at 6-weeks of age (6wks) the LV does not reveal any abnormalities. Our hypothesis is that downregulation of N2BA also downregulates N2Adependent signaling mediators which disrupts cardiomyocyte homeostasis, thus causing the observed DCM. Of the mediators that bind to the N2A element, CARP exhibited significant downregulation at the pre-DCM, 6wks time point. To further examine the contribution of CARP downregulation on we crossbred Ttn D112-158 mice with CARP knockout mice to produce mice that are homozygous for the Ttn D112-158 deletion and Heterozygous for the CARP deletion. We found that even this 50% reduction in CARP allelic representation was sufficient to increase the severity of DCM in 6mo LV. In summary, we generated a novel titin-based DCM mouse model and provided functional evidence that downregulation of CARP is an early pathological modulator in this model.

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cross-linked dimer form of the TDP-43 C-terminal domain and observe large enhancement of helical structure by NMR spectroscopy. Both simulation and experiment point to the role of two conserved glycine residues (G335 and G338) as potent inhibitors of helical extension and helix-helix interaction. Interestingly, one of these positions is substituted in ALS-associated mutation G335D. We find that substitution to helix-enhancing alanine at either of these positions substantially enhances in vitro phase separation and reduces fluidity of in cell phase separated TDP-43 reporter compartments. Furthermore, we find that G335A enhances TDP-43 splicing function in the well-established CFTR mini-gene assay. In conclusion, the conserved region of the TDP-43 C-terminal domain encodes a short but highly tunable alpha-helical module for precise regulation of assembly and cellular function. We suggest that this region and future modified sequences based on it will be important tools to enable new applications of LLPS for membraneless organelle design and synthetic biology.