Flavones provide resistance to DUX4-induced toxicity via an mTor-independent mechanism.

Facioscapulohumeral muscular dystrophy (FSHD) is among the most common of the muscular dystrophies, affecting nearly 1 in 8000 individuals, and is a cause of profound disability. Genetically, FSHD is linked to the contraction and/or epigenetic de-repression of the D4Z4 repeat array on chromosome 4, thereby allowing expression of the DUX4 gene in skeletal muscle. If the DUX4 transcript incorporates a stabilizing polyadenylation site the myotoxic DUX4 protein will be synthesized, resulting in muscle wasting. The mechanism of toxicity remains unclear, as many DUX4-induced cytopathologies have been described, however cell death does primarily occur through caspase 3/7-dependent apoptosis. To date, most FSHD therapeutic development has focused on molecular methods targeting DUX4 expression or the DUX4 transcript, while therapies targeting processes downstream of DUX4 activity have received less attention. Several studies have demonstrated that inhibition of multiple signal transduction pathways can ameliorate DUX4-induced toxicity, and thus compounds targeting these pathways have the potential to be developed into FSHD therapeutics. To this end, we have screened a group of small molecules curated based on their reported activity in relevant pathways and/or structural relationships with known toxicity-modulating molecules. We have identified a panel of five compounds that function downstream of DUX4 activity to inhibit DUX4-induced toxicity. Unexpectedly, this effect was mediated through an mTor-independent mechanism that preserved expression of ULK1 and correlated with an increase in a marker of active cellular autophagy. This identifies these flavones as compounds of interest for therapeutic development, and potentially identifies the autophagy pathway as a target for therapeutics.

Deep Mutational Scanning in Disease-related Genes with Saturation Mutagenesis-Reinforced Functional Assays (SMuRF).

Interpretation of disease-causing genetic variants remains a challenge in human genetics. Current costs and complexity of deep mutational scanning methods hamper crowd-sourcing approaches toward genome-wide resolution of variants in disease-related genes. Our framework, Saturation Mutagenesis-Reinforced Functional assays (SMuRF), addresses these issues by offering simple and cost-effective saturation mutagenesis, as well as streamlining functional assays to enhance the interpretation of unresolved variants. Applying SMuRF to neuromuscular disease genes FKRP and LARGE1, we generated functional scores for over 99.8% of all possible coding single nucleotide variants and resolved 310 clinically reported variants of uncertain significance with high confidence, enhancing clinical variant interpretation in dystroglycanopathies. SMuRF also demonstrates utility in predicting disease severity, resolving critical structural regions, and providing training datasets for the development of computational predictors. Our approach opens new directions for enabling variant-to-function insights for disease genes in a manner that is broadly useful for crowd-sourcing implementation across standard research laboratories.

Development of Assays to Measure GNE Gene Potency and Gene Replacement in Skeletal Muscle.

BACKGROUND: GNE myopathy (GNEM) is a severe muscle disease caused by mutations in the UDP-GlcNAc-2-epimerase/ManNAc-6-kinase (GNE) gene, which encodes a bifunctional enzyme required for sialic acid (Sia) biosynthesis. OBJECTIVE: To develop assays to demonstrate the potency of AAV gene therapy vectors in making Sia and to define the dose required for replacement of endogenous mouse Gne gene expression with human GNE in skeletal muscles. METHODS: A MyoD-inducible Gne-deficient cell line, Lec3MyoDI, and a GNE-deficient human muscle cell line, were made and tested to define the potency of various AAV vectors to increase binding of Sia-specific lectins, including MAA and SNA. qPCR and qRT-PCR methods were used to quantify AAV biodistribution and GNE gene expression after intravenous delivery of AAV vectors designed with different promoters in wild-type mice. RESULTS: Lec3 cells showed a strong deficit in MAA binding, while GNE-/-MB135 cells did not. Overexpressing GNE in Lec3 and Lec3MyoDI cells by AAV infection stimulated MAA binding in a dose-dependent manner. Use of a constitutive promoter, CMV, showed higher induction of MAA binding than use of muscle-specific promoters (MCK, MHCK7). rAAVrh74.CMV.GNE stimulated human GNE expression in muscles at levels equivalent to endogenous mouse Gne at a dose of 1×1013vg/kg, while AAVs with muscle-specific promoters required higher doses. AAV biodistribution in skeletal muscles trended higher when CMV was used as the promoter, and this correlated with increased sialylation of its viral capsid. CONCLUSIONS: Lec3 and Lec3MyoDI cells work well to assay the potency of AAV vectors in making Sia. Systemic delivery of rAAVrh74.CMV.GNE can deliver GNE gene replacement to skeletal muscles at doses that do not overwhelm non-muscle tissues, suggesting that AAV vectors that drive constitutive organ expression could be used to treat GNEM.

Flavones provide resistance to DUX4-induced toxicity via an mTor-independent mechanism.

Facioscapulohumeral muscular dystrophy (FSHD) is among the most common of the muscular dystrophies, affecting nearly 1 in 8000 individuals, and is a cause of profound disability. Genetically, FSHD is linked to the contraction and/or epigenetic de-repression of the D4Z4 repeat array on chromosome 4, thereby allowing expression of the DUX4 gene in skeletal muscle. If the DUX4 transcript incorporates a stabilizing polyadenylation site the myotoxic DUX4 protein will be synthesized, resulting in muscle wasting. The mechanism of toxicity remains unclear, as many DUX4-induced cytopathologies have been described, however cell death does primarily occur through caspase 3/7-dependent apoptosis. To date, most FSHD therapeutic development has focused on molecular methods targeting DUX4 expression or the DUX4 transcript, while therapies targeting processes downstream of DUX4 activity have received less attention. Several studies have demonstrated that inhibition of multiple signal transduction pathways can ameliorate DUX4-induced toxicity, and thus compounds targeting these pathways have the potential to be developed into FSHD therapeutics. To this end, we have screened a group of small molecules curated based on their reported activity in relevant pathways and/or structural relationships with known toxicity-modulating molecules. We have identified a panel of five compounds that function downstream of DUX4 activity to inhibit DUX4-induced toxicity. Unexpectedly, this effect was mediated through an mTor-independent mechanism that preserved expression of ULK1 and correlated with an increase in a marker of active cellular autophagy. This identifies these flavones as compounds of interest for therapeutic development, and potentially identifies the autophagy pathway as a target for therapeutics.

Neuromuscular disorders: finding the missing genetic diagnoses.

Neuromuscular disorders (NMDs) are a wide-ranging group of diseases that seriously affect the quality of life of affected individuals. The development of next-generation sequencing revolutionized the diagnosis of NMD, enabling the discovery of hundreds of NMD genes and many more pathogenic variants. However, the diagnostic yield of genetic testing in NMD cohorts remains incomplete, indicating a large number of genetic diagnoses are not identified through current methods. Fortunately, recent advancements in sequencing technologies, analytical tools, and high-throughput functional screening provide an opportunity to circumvent current challenges. Here, we discuss reasons for missing genetic diagnoses in NMD, how emerging technologies and tools can overcome these hurdles, and examine future approaches to improving diagnostic yields in NMD.