Severe skeletal muscle wasting is the most debilitating symptom experienced by individuals with myotonic dystrophy type 1 (DM1). We present a DM1 mouse model with inducible and skeletal muscle-specific expression of large tracts of CTG repeats in the context of DMPK exon 15. These mice recapitulate many findings associated with DM1 skeletal muscle, such as CUG RNA foci with Muscleblind-like 1 (MBNL1) protein colocalization, misregulation of developmentally regulated alternative splicing events, myotonia, characteristic histological abnormalities, and increased CUGBP1 protein levels. Importantly, this DM1 mouse model recapitulates severe muscle wasting, which has not been reported in models in which depletion of MBNL1 is the main feature. Using these mice, we discovered previously undescribed alternative splicing events that are responsive to CUGBP1 and not MBNL, and these events were found to be misregulated in individuals with DM1. Our results indicate that increased CUGBP1 protein levels are associated with DMPK-CUG RNA expression, suggesting a role for CUGBP1-specific splicing or cytoplasmic functions in muscle wasting.alternative splicing ͉ CUG-binding protein 1 ͉ Muscleblind-like 1 ͉ muscle atrophy ͉ microsatellite expansion M yotonic dystrophy type 1 (DM1) is a multisystemic, autosomal dominant disease caused by a CTG repeat expansion in the 3Ј untranslated region (UTR) of the DMPK gene (exon 15). Individuals with the disease have expansions ranging from 50 to Ͼ2,000 repeats. Onset and severity of disease correlate with repeat expansion size. The dominant organ system affected is skeletal muscle, which exhibits myotonia and degeneration. Severe skeletal muscle wasting is the primary cause of morbidity and mortality (1).After transcription of the expanded allele, CUG repeats accumulate within the nucleus in discrete RNA foci. Pathology in DM1 is a result of toxic RNA expression and not alteration of DMPK gene expression. This model was solidified by work from Mankodi et al. (2) using HSA LR transgenic mice that expressed 250 CUG repeats in the human skeletal actin 3Ј UTR specifically in skeletal muscle. HSA LR mice develop DM1-characteristic RNA foci, misregulated alternative splicing, myotonia, and histological abnormalities (2). Additionally, a second form of DM (DM2) is caused by expanded CCTG repeats in intron 1 of an unrelated gene, ZNF9, and yet has similar symptoms to those seen in DM1 (3, 4). Accumulation of toxic CUG or CCUG repeats leads to a transdominant misregulation of RNA homeostasis. In particular, developmentally regulated alternative splicing is affected such that there is a failure to express adult isoforms. Thus, pathology results at least in part from inappropriate expression of embryonic splicing patterns in adult tissues (5).Two proteins identified as interacting with CUG RNA repeats, Muscleblind-like 1 (MBNL1) and CUG-binding protein 1 (CUGBP1), play important roles in DM1 pathogenesis. Both proteins normally regulate alternative splicing of exons that are misregulated in DM1 (7, 24)...

Alternative splicing is the primary source of proteome complexity in metazoans and its regulation shapes the proteome in response to shifting physiological requirements. We developed a bichromatic splicing reporter that uses a peculiar feature of some fluorescent protein coding regions to express two different fluorescent proteins from a single alternative splicing event. The mutually exclusive expression of different fluorescent proteins from a single reporter provides a uniquely sensitive approach for high-throughput screening and analysis of cell-specific splicing events in mixed cell cultures and tissues of transgenic animals. This reporter is applicable to the majority of alternative splicing patterns and can be used to quantify alternative splicing within single cells and to select cells that express specific splicing patterns. The ability to perform quantitative single-cell analysis of alternative splicing and high-throughput screens will enhance progress toward understanding splicing regulatory networks and identifying compounds that reverse pathogenic splicing defects.

Optimal lysosome function requires maintenance of an acidic pH maintained by proton pumps in combination with a counterion transporter such as the Cl À /H þ exchanger, CLCN7 (ClC-7), encoded by CLCN7. The role of ClC-7 in maintaining lysosomal pH has been controversial. In this paper, we performed clinical and genetic evaluations of two children of different ethnicities. Both children had delayed myelination and development, organomegaly, and hypopigmentation, but neither had osteopetrosis. Whole-exome and-genome sequencing revealed a de novo c.2144A>G variant in CLCN7 in both affected children. This p.Tyr715Cys variant, located in the C-terminal domain of ClC-7, resulted in increased outward currents when it was heterologously expressed in Xenopus oocytes. Fibroblasts from probands displayed a lysosomal pH approximately 0.2 units lower than that of control cells, and treatment with chloroquine normalized the pH. Primary fibroblasts from both probands also exhibited markedly enlarged intracellular vacuoles; this finding was recapitulated by the overexpression of human p.Tyr715Cys CLCN7 in control fibroblasts, reflecting the dominant, gain-of-function nature of the variant. A mouse harboring the knock-in Clcn7 variant exhibited hypopigmentation, hepatomegaly resulting from abnormal storage, and enlarged vacuoles in cultured fibroblasts. Our results show that p.Tyr715Cys is a gain-of-function CLCN7 variant associated with developmental delay, organomegaly, and hypopigmentation resulting from lysosomal hyperacidity, abnormal storage, and enlarged intracellular vacuoles. Our data supports the hypothesis that the ClC-7 antiporter plays a critical role in maintaining lysosomal pH.

Objective Misregulation of alternative splicing has become a molecular hallmark of myotonic dystrophy type 1 (DM1) in which neonatal splice variants are expressed in adult skeletal muscle. Splicing misregulation is induced by RNA containing expanded CUG repeats expressed from the expanded mutant allele by sequestration of Muscleblind-like 1 (MBNL1) protein within nuclear RNA foci and increased CUGBP, Elav-like family member 1 (CELF1) protein levels. Misregulated splicing has also been identified in other neuromuscular disorders suggesting either that diseases with different molecular causes share a common pathogenic mechanism or that misregulated splicing can also be a common secondary consequence of muscle degeneration and regeneration. Methods In this study we examined regulation of alternative splicing in four different mouse models of muscular dystrophy including DM1, limb-girdle muscular dystrophy, congenital merosin-deficient muscular dystrophy, Duchenne muscular dystrophy, and two myotoxin (cardiotoxin and notexin) muscle injury models. Results We show that DM1-like alternative splicing misregulation and altered expression of MBNL1 and CELF1 occurs in non-DM1 mouse models of muscular dystrophy and muscle injury, most likely due to recapitulation of neonatal splicing patterns in regenerating fibers. In contrast, CELF1 was elevated in nuclei of mature myofibers of the DM1 model consistent with a primary effect of pathogenic RNA expression. Interpretation Splicing misregulation in DM1 is a primary effect of RNA containing expanded CUG repeats. However, we conclude that splicing changes can also be observed secondary to muscle regeneration and this possibility must be taken into account when evaluating cause-effect relationships between misregulated splicing and disease processes.

Patient now 19 years old has intellectual disability, developmental delay, absent speech, seizures, hypotonia, severe motor disability (non-ambulatory), short stature, relative macrocephaly. Patient uses gastric tube for feeding and has gastroesophageal reflux. Facial dysmorphisms include short palpebral fissures, large incisors, full eyebrows. Fingers are short and trident-shaped.Brain MRI revealed progressive cerebral and cerebellar volume loss, hypodensity in the left basal ganglia, unchanged and consistent with a lacune infarct (remote). There is a less conspicuous area of hypodensity on the contralateral side. There are hypodense white matter changes along the periventricular white matter and bilateral centrum semiovale.