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Myotonic dystrophy type 1 (DM1) is a severe neuromuscular disorder caused by the expression of trinucleotide repeat-containing DMPK transcripts. Abnormally expanded (CUG)n repeats in these transcripts form hairpin-like structures that cause the RNA to accumulate in the cell nucleus by sequestering isoforms of the Muscleblind (MBNL) family, tissue-specific regulators of developmentally programmed, post-transcriptional processes in RNA metabolism. Through this mechanism, the function of MBNL in RNA processing becomes dominantly perturbed, which eventually leads to aberrant alternative splicing and the expression of foetal splice variants of a wide variety of proteins, including the MBNL isoforms themselves. Here, we employ a patient-derived muscle cell model for DM1 to examine in detail the expression of MBNL RNA and protein variants during myogenic differentiation. This DM1 model consists of a panel of isogenic myoblast cell lines that either contain a pathogenic DMPK allele with a congenital mutation of 2600 triplets, or lack this expanded repeat through CRISPR/Cas9-mediated gene editing. We found that the temporal expression levels of MBNL1, MBNL2 and MBNL3 RNAs are not influenced by presence of the (CTG)2600 repeat during myogenesis in vitro. However, throughout myoblast proliferation and differentiation to myotubes a disproportionate inclusion of MBNL1 exon 5 and MBNL2 exons 5 and 8 occurs in cells with the (CTG)2600 repeat. As a consequence, a reduced quantity and imbalanced collection of splice variants of MBNL1 and MBNL2 accumulates in both the cytoplasm and the nucleus of DM1 myoblasts and myotubes. We thus propose that both the quantitative and qualitative changes in the intracellular partitioning of MBNL proteins are a pivotal cause of skeletal muscle problems in DM1, starting already in muscle progenitor cells.

Background Myotonic dystrophy type 1 (DM1) is an incurable multisystem disease caused by a CTG-repeat expansion in the DM1 protein kinase ( DMPK ) gene. The OPTIMISTIC clinical trial demonstrated positive and heterogenous effects of cognitive behavioral therapy (CBT) on the capacity for activity and social participations in DM1 patients. Through a process of reverse engineering, this study aims to identify druggable molecular biomarkers associated with the clinical improvement in the OPTIMISTIC cohort. Methods Based on full blood samples collected during OPTIMISTIC, we performed paired mRNA sequencing for 27 patients before and after the CBT intervention. Linear mixed effect models were used to identify biomarkers associated with the disease-causing CTG expansion and the mean clinical improvement across all clinical outcome measures. Results We identified 608 genes for which their expression was significantly associated with the CTG-repeat expansion, as well as 1176 genes significantly associated with the average clinical response towards the intervention. Remarkably, all 97 genes associated with both returned to more normal levels in patients who benefited the most from CBT. This main finding has been replicated based on an external dataset of mRNA data of DM1 patients and controls, singling these genes out as candidate biomarkers for therapy response. Among these candidate genes were DNAJB12 , HDAC5 , and TRIM8 , each belonging to a protein family that is being studied in the context of neurological disorders or muscular dystrophies. Across the different gene sets, gene pathway enrichment analysis revealed disease-relevant impaired signaling in, among others, insulin-, metabolism-, and immune-related pathways. Furthermore, evidence for shared dysregulations with another neuromuscular disease, Duchenne muscular dystrophy, was found, suggesting a partial overlap in blood-based gene dysregulation. Conclusions DM1-relevant disease signatures can be identified on a molecular level in peripheral blood, opening new avenues for drug discovery and therapy efficacy assessments.

The congenital form of myotonic dystrophy type 1 (cDM) is caused by the large-scale expansion of a (CTG•CAG)n repeat in DMPK and DM1-AS. The production of toxic transcripts with long trinucleotide tracts from these genes results in impairment of the myogenic differentiation capacity as cDM’s most prominent morpho-phenotypic hallmark. In the current in vitro study, we compared the early differentiation programs of isogenic cDM myoblasts with and without a (CTG)2600 repeat obtained by gene editing. We found that excision of the repeat restored the ability of cDM myoblasts to engage in myogenic fusion, preventing the ensuing myotubes from remaining immature. Although the cDM-typical epigenetic status of the DM1 locus and the expression of genes therein were not altered upon removal of the repeat, analyses at the transcriptome and proteome level revealed that early abnormalities in the temporal expression of differentiation regulators, myogenic progression markers, and alternative splicing patterns before and immediately after the onset of differentiation became normalized. Our observation that molecular and cellular features of cDM are reversible in vitro and can be corrected by repeat-directed genome editing in muscle progenitors, when already committed and poised for myogenic differentiation, is important information for the future development of gene therapy for different forms of myotonic dystrophy type 1 (DM1).

Protein tyrosine phosphatases, together with protein tyrosine kinases, control many molecular signaling steps that control life at cellular and organismal levels. Impairing alterations in the genes encoding the involved proteins is expected to profoundly affect the quality of life—if compatible with life at all. Here, we review the current knowledge on the effects of germline variants that have been reported for genes encoding a subset of the protein tyrosine phosphatase superfamily; that of the thirty seven classical members. The conclusion must be that the newest genome research tools produced an avalanche of data that suggest ‘guilt by association’ for individual genes to specific disorders. Future research should face the challenge to investigate these accusations thoroughly and convincingly, to reach a mature genotype-phenotype map for this intriguing protein family.

Abstract In patients with myotonic dystrophy type 1 (DM1), dysregulation of RNA-binding proteins like MBNL and CELF1 leads to alternative splicing of exons and is thought to induce a return to fetal splicing patterns in adult tissues, including the central nervous system (CNS). To comprehensively evaluate this, we created an atlas of developmentally regulated splicing patterns in the frontal cortex of healthy individuals and DM1 patients, by combining RNA-seq data from BrainSpan, GTEx and DM1 patients. Thirty-four splice events displayed an inclusion pattern in DM1 patients that is typical for the fetal situation in healthy individuals. The regulation of DM1-relevant splicing patterns could partly be explained by changes in mRNA expression of the splice regulators MBNL1, MBNL2 and CELF1. On the contrary, interindividual differences in splicing patterns between healthy adults could not be explained by differential expression of these splice regulators. Our findings lend transcriptome-wide evidence to the previously noted shift to fetal splicing patterns in the adult DM1 brain as a consequence of an imbalance in antagonistic MBNL and CELF1 activities. Our atlas serves as a solid foundation for further study and understanding of the cognitive phenotype in patients.

Citrullination is the conversion of peptidylarginine to peptidylcitrulline, which is catalyzed by peptidylarginine deiminases. This conversion is involved in different physiological processes and is associated with several diseases, including cancer and rheumatoid arthritis. A common method to detect citrullinated proteins relies on anti-modified citrulline antibodies directed to a specific chemical modification of the citrulline side chain. Here, we describe a versatile, antibody-independent method for the detection of citrullinated proteins on a membrane, based on the selective reaction of phenylglyoxal with the ureido group of citrulline under highly acidic conditions. The method makes use of 4-azidophenylglyoxal, which, after reaction with citrullinated proteins, can be visualized with alkyne-conjugated probes. The sensitivity of this procedure, using an alkyne-biotin probe, appeared to be comparable to the antibody-based detection method and independent of the sequence surrounding the citrulline.