A total of 3-6% human genome is composed of microsatellite sequences, which are short DNA elements composed of two to six nucleotide motifs repeated in tandem. Expansion of a subset of these microsatellites is the leading cause of >60 diseases. However, most of these mutations are located in sequences annotated as noncoding, which raises questions about their pathogenicity. Here we found that GGC repeat expansions causing oculopharyngodistal myopathy with or without oculopharyngeal myopathy leukoencephalopathy are located within previously unrecognized open reading frames (ORFs), resulting in their translation into new polyglycine-containing proteins. Antibodies developed against these proteins stain the p62-positive inclusions typical of these diseases. Moreover, expression of these polyglycine proteins causes locomotor and skeletal muscle alterations associated with neurodegeneration in cell, fly and mouse models. Finally, we identified a compound, the cationic porphyrin TMPyP4, targeting the expression of these polyglycine proteins, raising hope to develop a therapy for these disorders. Overall, this work highlights the complexity and richness of the human genome and the importance of mutations in yet-unrecognized small ORFs.

Oculopharyngodistal myopathy (OPDM) is characterized by ptosis, ophthalmoparesis, dysphagia, and distal weakness. Myopathological features include rimmed vacuoles and intranuclear inclusions. OPDM is associated with a pathogenic CGG repeat expansions in the 5'UTR of LRP12, NOTCH2NLC, GIPC1, RILPL1 and ABCD3. Translation of the repeat in the glycine reading frame has been demonstrated for expansions in FMR1, NOTCH2NLC and GIPC1. To assess for a similar phenomenon with LRP12, we expressed normal or expanded CGG repeats in the context of the 5'UTR of LRP12, upstream of a green fluorescent protein (GFP) in the three repeat reading frames. Repeat dependent translation occurs exclusively in the glycine reading frame. However, unlike other CGG repeat disorders, there is no proximal AUG, or near-AUG cognate initiated polyglycine (polyG) open reading frame in LRP12. Instead, our results support a model in which repeat-associated non-AUG (RAN) mediated polyG translation may initiate within the arginine reading frame and then undergo a + 1 translational frameshift into the glycine reading frame. LRP12-associated polyG products form intranuclear SQSTM1/ubiquitin positive inclusions that are cytotoxic and alter the nuclear lamina architecture in transfected cells. While FMR1-associated polyG inclusions are cytosolic, LRP12-associated polyG inclusions are nuclear in transfected skeletal muscle. LRP12 expansion carrier iPSC derived myotubes exhibit SQSTM1 positive intra- and peri- nuclear inclusions when compared with control patient myotubes, suggesting that polyG expression can occur in patients. Together, these findings provide evidence of RAN translation and polyG-toxicity in LRP12-associated OPDM pathology.

GGC repeat expansions in the 5' untranslated region (UTR) of the GIPC1 gene have been implicated in the pathogenesis of oculopharyngodistal myopathy type 2 (OPDM2).To investigate the underlying mechanism, we generated a series of reporter constructs to confirm he translation product of GIPC1 expanded GGC repeats. We further developed a specific antibody targeting the predicted N-terminus of the predominant translation product. Its expression and toxicity were validated in patient-derived induced pluripotent stem cell (iPSC)-derived myotubes and zebrafish model. Here, we demonstrate that the expanded GGC repeats undergo repeat-associated non-AUG (RAN) translation in multiple reading frames, predominantly generating a polyglycine-containing protein (uGIPC1polyG) initiated at an upstream CTG codon. These polyG-containing proteins aggregate and form intranuclear and cytoplasmic p62/ubiquitin-positive inclusions, which are pathogenic hallmarks of OPDM2. The translation of GGC repeats into a polyG protein further causes mitochondrial dysfunction and disrupts nuclear lamina architecture, thereby inducing cytotoxicity and apoptosis in cell lines, including HEK293T cells, fibroblasts, and iPSC-derived myotubes from OPDM2 patients. Additionally, the zebrafish model exhibits developmental malformation and compromised locomotor function, demonstrating the in vivo toxicity of uGIPC1polyG. These findings suggest that the translation of expanded GGC repeats into a toxic polyG protein might play a crucial role in the pathogenesis of OPDM2, highlighting uGIPC1polyG as a potential biomarker and therapeutic target.

CGG expansions in NOTCH2NLC and LRP12 were recently identified as a cause of Charcot-Marie-Tooth disease (CMT) in 1.2%-10.6% of genetically undiagnosed patients in China, Taiwan and Japan. However, their relevance in CMT patients of different ethnic origin is still unknown. Here, we leveraged short-read whole genome sequencing data from the 100 000 Genomes Project to investigate the presence and frequency of CGG expansions in NOTCH2NLC, LRP12 and additional genes associated with oculopharyngodistal myopathy (OPDM), in 560 genetically unsolved patients diagnosed with CMT and 32 509 non-neurological controls. Repeat expansions in NOTCH2NLC, LRP12, RILPL1, NUTM2B-AS1 and ABCD3 were absent from 560 genetically unsolved patients with CMT, mostly of Northern European ancestry. One patient of African ancestry carried an expanded allele in GIPC1, below the reported pathogenic threshold. However, rare expansions in this gene, as well as in NOTCH2NLC, NUTM2B-AS1 and ABCD3, were also detected in controls (≤0.05%). The distribution of repeat size at these loci varied significantly across different ethnicities, with larger non-pathogenic intermediate alleles of NOTCH2NLC and LRP12 typically observed in East Asians. CGG expansions in NOTCH2NLC, LRP12 and other OPDM-associated genes do not appear to be a relevant cause of CMT in the UK. The larger size of non-pathogenic intermediate alleles of NOTCH2NLC and LRP12 in East Asians could explain their ancestry-specific propensity to further expand into the full pathogenic range.

Here, we summarize the current knowledge about the genetics and proposed mechanisms of disease underlying skeletal muscle short tandem repeat (STR) expansion disorders. The human genome contains up to 2 million STRs (also known as microsatellites), which are highly variable repetitions of two to six nucleotide-long DNA motifs. These elements, present in both coding and noncoding sequences, are highly instable, and their polymorphic variations have important roles in genes regulation and human phenotypic trait diversity. Importantly, expansion over a threshold size of a subset of these STR is the cause of approximately 60 neurological diseases, including some major muscle disorders such as myotonic dystrophy, oculopharyngodistal myopathy (OPDM) and oculopharyngeal muscular dystrophy. The discovery and characterisation of a number of these STR expansion disorders, in particular for OPDM, has been enabled in recent years by advanced genomic technologies. Many recently described STR expansion disorders are now recognized and genetic testing of patients is possible on a research basis, clinical testing for these newly described repeat loci is not yet readily available and is complicated by the reduced penetrance seen in some families, rendering clinical interpretation more difficult. The phenotypic spectrums associated with these STR expansion disorders are also evolving as unbiased sequencing approaches identified expansions at known loci in individuals with phenotypes that are quite different to those in which the STR expansions were first characterized. The pathomechanisms associated with these newer STR expansion disorders is still poorly understood, however there is evidence of both RNA toxicity and polyGly toxicity. Additional STR expansions underlying skeletal muscle diseases are likely to be identified in coming years and may shed further light onto the complex genetics, epigenetics and disease mechanisms underlying these disorders.