Dystroglycan (DG) is an extracellular matrix receptor consisting of an α- and a β-DG subunit encoded by the DAG1 gene. The homozygous mutation (c.2006G>T, p.Cys669Phe) in β-DG causes muscle-eye-brain disease with multicystic leukodystrophy in humans. In a mouse model of this primary dystroglycanopathy, approximately two-thirds of homozygous embryos fail to develop to term. Mutant mice that are born undergo a normal postnatal development but show a late-onset myopathy with partially penetrant histopathological changes and an impaired performance on an activity wheel. Their brains and eyes are structurally normal, but the localization of mutant β-DG is altered in the glial perivascular end-feet, resulting in a perturbed protein composition of the blood-brain and blood-retina barrier. In addition, α- and β-DG protein levels are significantly reduced in muscle and brain of mutant mice. Owing to the partially penetrant developmental phenotype of the C669F β-DG mice, they represent a novel and highly valuable mouse model with which to study the molecular effects of β-DG functional alterations both during embryogenesis and in mature muscle, brain and eye, and to gain insight into the pathogenesis of primary dystroglycanopathies.

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 all possible coding single nucleotide variants, which aid in resolving clinically reported variants of uncertain significance. 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.

Dystroglycanopathies are a group of congenital muscular dystrophies (CMDs) that include a broad phenotypic spectrum ranging from late-onset limb-girdle muscular dystrophy to severe muscle-eye-brain disease, Walker-Warburg syndrome, and Fukuyama congenital muscular dystrophy. In addition to clinical heterogeneity, CMDs are characterized by genetic heterogeneity. To date, 18 genes have been associated with CMDs. One of them is B3GALNT2, which encodes the β-1,3-N-acetylgalactosaminyltransferase 2 that glycosylates α-dystroglycan. In this study, using exome sequencing, we identify a homozygous frameshift variant in B3GALNT2 due to a mixed uniparental disomy of chromosome 1 in a 7-year-old girl with global developmental delay, severely delayed active language development, and autism spectrum disorder but without any symptoms of muscular dystrophy. In addition to this case, we also provide an overview of all previously reported cases, further expanding the phenotypic spectrum.

Neuromuscular electrical stimulation (NMES) allows activation of muscle fibers in the absence of voluntary force generation. NMES could have the potential to promote muscle homeostasis in the context of muscle disease, but the impacts of NMES on diseased muscle are not well understood. We used the zebrafish Duchenne muscular dystrophy (dmd) mutant and a longitudinal design to elucidate the consequences of NMES on muscle health. We designed four neuromuscular stimulation paradigms loosely based on weightlifting regimens. Each paradigm differentially affected neuromuscular structure, function, and survival. Only endurance neuromuscular stimulation (eNMES) improved all outcome measures. We found that eNMES improves muscle and neuromuscular junction morphology, swimming, and survival. Heme oxygenase and integrin alpha7 are required for eNMES-mediated improvement. Our data indicate that neuromuscular stimulation can be beneficial, suggesting that the right type of activity may benefit patients with muscle disease.

Fukutin-related protein (FKRP) is a glycosyltransferase involved in glycosylation of alpha-dystroglycan (α-DG). Mutations in FKRP are associated with muscular dystrophies (MD) ranging from limb-girdle LGMDR9 to Walker-Warburg Syndrome (WWS), a severe type of congenital MD. Although hypoglycosylation of α-DG is the main hallmark of this group of diseases, a full understanding of the underlying pathophysiology is still missing. Here, we investigated molecular mechanisms impaired by FKRP mutations in pluripotent stem (PS) cell-derived myotubes. FKRP-deficient myotubes show transcriptome alterations in genes involved in extracellular matrix receptor interactions, calcium signaling, PI3K-Akt pathway, and lysosomal function. Accordingly, using a panel of patient-specific LGMDR9 and WWS induced PS cell-derived myotubes, we found a significant reduction in the autophagy-lysosome pathway for both disease phenotypes. In addition, we show that WWS myotubes display decreased ERK1/2 activity and increased apoptosis, which were restored in gene edited myotubes. Our results suggest the autophagy-lysosome pathway and apoptosis may contribute to the FKRP-associated MD pathogenesis.

ISPD gene mutation-related diseases have high clinical and genetic heterogeneity, and no studies have yet reported any effective treatments. We describe six patients with dystroglycanopathies caused by ISPD gene mutations and analyze their genotypes and phenotypes to explore possible effective treatments. Our results confirm that the phenotype of limb-girdle muscular dystrophies can be easily misdiagnosed as Duchenne muscular dystrophy and that exon deletions of ISPD gene are relatively common. Moreover, low-dose prednisone therapy can improve patients\' exercise ability and prolong survival and may be a promising new avenue for ISPD therapy.

Dystroglycanopathy is a type of congenital muscular dystrophy caused by mutations causing defective glycosylation of a dystrophin-associated glycoprotein, dystroglycan and as such is a very rare disease entity. We are reporting a 1-year-old girl child with dystroglycanopathy who presented with motor predominant developmental delay. She had motor development quotient of 52, mental development quotient of 75, facial dysmorphism, mixed hypotonia with a global decrease in muscle power, and areflexia. Serum CPK level was elevated; magnetic resonance imaging brain revealed multiple intraparenchymal cysts in the cerebellum with disorganized folia. Next-generation sequencing revealed a homozygous missense mutation in exon 3 of the ISPD gene (p.Gln215His; ENST00000407010) consistent with the diagnosis of dystroglycanopathy muscle-eye-brain disease. Genetic counseling and prenatal diagnosis for subsequent pregnancies were advised for the family, apart from appropriate rehabilitation for the child.

Mutations in the fukutin-related protein (FKRP) cause Walker-Warburg syndrome (WWS), a severe form of congenital muscular dystrophy. Here, we established a WWS human induced pluripotent stem cell-derived myogenic model that recapitulates hallmarks of WWS pathology. We used this model to investigate the therapeutic effect of metabolites of the pentose phosphate pathway in human WWS. We show that functional recovery of WWS myotubes is promoted not only by ribitol but also by its precursor ribose. Moreover, we found that the combination of each of these metabolites with NAD+ results in a synergistic effect, as demonstrated by rescue of α-dystroglycan glycosylation and laminin binding capacity. Mechanistically, we found that FKRP residual enzymatic capacity, characteristic of many recessive FKRP mutations, is required for rescue as supported by functional and structural mutational analyses. These findings provide the rationale for testing ribose/ribitol in combination with NAD+ to treat WWS and other diseases associated with FKRP mutations.
Healthy muscles are complex machines that require a myriad of finely tuned molecules to work properly. For instance, a protein called alpha-DG sits at the surface of healthy muscle cells, where it strengthens the tissue by latching onto other proteins in the environment. To perform its role correctly, it first needs to be coated with sugar molecules, a complex process which requires over 20 proteins, including the enzyme FKRP. Faulty forms of FKRP reduce the number of sugars added to alpha-DG, causing the muscle tissue to weaken and waste away, potentially leading to severe forms of diseases known as muscular dystrophies. Drugs that can restore alpha-DG sugar molecules could help to treat these conditions. Previous studies on mice and fish have highlighted two potential candidates, known as ribitol and NAD+, which can help to compensate for reduced FKRP activity and allow sugars to be added to alpha-DG again. Yet no model is available to test these molecules on actual human muscle cells. Here, Ortiz-Cordero et al. developed such a model in the laboratory by growing muscle cells from naïve, undifferentiated cells generated from skin given by a muscular dystrophy patient with a faulty form of FKRP. The resulting muscle fibers are in essence identical to the ones present in the individual. As such, they can help to understand the effect various drugs have on muscular dystrophies. The cells were then put in contact with either NAD+, ribitol, or a precursor of ribitol known as ribose. Ortiz-Cordero et al. found that ribitol and ribose restored the ability of FKRP to add sugars to alpha-DG, reducing muscle damage. Combining NAD+ with ribitol or ribose had an even a bigger impact, further increasing the number of sugars on alpha-DG. The human muscle cell model developed by Ortiz-Cordero et al. could help to identify new compounds that can treat muscular conditions. In particular, the findings point towards NAD+, ribose and ribitol as candidates for treating FKRP-related muscular dystrophies. Further safety studies are now needed to evaluate whether these compounds could be used in patients.

Laminin α2 gene (LAMA2)-related Congenital Muscular Dystrophy (CMD) was distinguished by a defining central nervous system (CNS) abnormality-aberrant white matter signals by MRI-when first described in the 1990s. In the past 25 years, researchers and clinicians have expanded our knowledge of brain involvement in LAMA2-related CMD, also known as Congenital Muscular Dystrophy Type 1A (MDC1A). Neurological changes in MDC1A can be structural, including lissencephaly and agyria, as well as functional, including epilepsy and intellectual disability. Mouse models of MDC1A include both spontaneous and targeted LAMA2 mutations and range from a partial loss of LAMA2 function (e.g., dy2J/dy2J ), to a complete loss of LAMA2 expression (dy 3K/dy 3K). Diverse cellular and molecular changes have been reported in the brains of MDC1A mouse models, including blood-brain barrier dysfunction, altered neuro- and gliogenesis, changes in synaptic plasticity, and decreased myelination, providing mechanistic insight into potential neurological dysfunction in MDC1A. In this review article, we discuss selected studies that illustrate the potential scope and complexity of disturbances in brain development in MDC1A, and as well as highlight mechanistic insights that are emerging from mouse models.

Dystroglycan is a major non-integrin adhesion complex that connects the cytoskeleton to the surrounding basement membranes, thus providing stability to skeletal muscle. In Vertebrates, hypoglycosylation of α-dystroglycan has been strongly linked to muscular dystrophy phenotypes, some of which also show variable degrees of cognitive impairments, collectively termed dystroglycanopathies. Only a small number of mutations in the dystroglycan gene, leading to the so called primary dystroglycanopathies, has been described so far, as opposed to the ever-growing number of identified secondary or tertiary dystroglycanopathies (caused by genetic abnormalities in glycosyltransferases or in enzymes involved in the synthesis of the carbohydrate building blocks). The few mutations found within the autonomous N-terminal domain of α-dystroglycan seem to destabilise it to different degrees, without influencing the overall folding and targeting of the dystroglycan complex. On the contrary other mutations, some located at the α/β interface of the dystroglycan complex, seem to be able to interfere with its maturation, thus compromising its stability and eventually leading to the intracellular engulfment and/or partial or even total degradation of the dystroglycan uncleaved precursor.