Identification of mutations in the MYO9A gene in patients with congenital myasthenic syndrome.

Congenital myasthenic syndromes are a group of rare and genetically heterogenous disorders resulting from defects in the structure and function of the neuromuscular junction. Patients with congenital myasthenic syndrome exhibit fatigable muscle weakness with a variety of accompanying phenotypes depending on the protein affected. A cohort of patients with a clinical diagnosis of congenital myasthenic syndrome that lacked a genetic diagnosis underwent whole exome sequencing in order to identify genetic causation. Missense biallelic mutations in the MYO9A gene, encoding an unconventional myosin, were identified in two unrelated families. Depletion of MYO9A in NSC-34 cells revealed a direct effect of MYO9A on neuronal branching and axon guidance. Morpholino-mediated knockdown of the two MYO9A orthologues in zebrafish, myo9aa/ab, demonstrated a requirement for MYO9A in the formation of the neuromuscular junction during development. The morphants displayed shortened and abnormally branched motor axons, lack of movement within the chorion and abnormal swimming in response to tactile stimulation. We therefore conclude that MYO9A deficiency may affect the presynaptic motor axon, manifesting in congenital myasthenic syndrome. These results highlight the involvement of unconventional myosins in motor axon functionality, as well as the need to look outside traditional neuromuscular junction-specific proteins for further congenital myasthenic syndrome candidate genes.

Mutations in the collagen XII gene define a new form of extracellular matrix-related myopathy.

Bethlem myopathy (BM) [MIM 158810] is a slowly progressive muscle disease characterized by contractures and proximal weakness, which can be caused by mutations in one of the collagen VI genes (COL6A1, COL6A2 and COL6A3). However, there may be additional causal genes to identify as in ∼50% of BM cases no mutations in the COL6 genes are identified. In a cohort of -24 patients with a BM-like phenotype, we first sequenced 12 candidate genes based on their function, including genes for known binding partners of collagen VI, and those enzymes involved in its correct post-translational modification, assembly and secretion. Proceeding to whole-exome sequencing (WES), we identified mutations in the COL12A1 gene, a member of the FACIT collagens (fibril-associated collagens with interrupted triple helices) in five individuals from two families. Both families showed dominant inheritance with a clinical phenotype resembling classical BM. Family 1 had a single-base substitution that led to the replacement of one glycine residue in the triple-helical domain, breaking the Gly-X-Y repeating pattern, and Family 2 had a missense mutation, which created a mutant protein with an unpaired cysteine residue. Abnormality at the protein level was confirmed in both families by the intracellular retention of collagen XII in patient dermal fibroblasts. The mutation in Family 2 leads to the up-regulation of genes associated with the unfolded protein response (UPR) pathway and swollen, dysmorphic rough-ER. We conclude that the spectrum of causative genes in extracellular matrix (ECM)-related myopathies be extended to include COL12A1.

Hexosamine biosynthetic pathway mutations cause neuromuscular transmission defect.

Neuromuscular junctions (NMJs) are synapses that transmit impulses from motor neurons to skeletal muscle fibers leading to muscle contraction. Study of hereditary disorders of neuromuscular transmission, termed congenital myasthenic syndromes (CMS), has helped elucidate fundamental processes influencing development and function of the nerve-muscle synapse. Using genetic linkage, we find 18 different biallelic mutations in the gene encoding glutamine-fructose-6-phosphate transaminase 1 (GFPT1) in 13 unrelated families with an autosomal recessive CMS. Consistent with these data, downregulation of the GFPT1 ortholog gfpt1 in zebrafish embryos altered muscle fiber morphology and impaired neuromuscular junction development. GFPT1 is the key enzyme of the hexosamine pathway yielding the amino sugar UDP-N-acetylglucosamine, an essential substrate for protein glycosylation. Our findings provide further impetus to study the glycobiology of NMJ and synapses in general.

Efficient and fast functional screening of microdystrophin constructs in vivo and in vitro for therapy of duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is an X-linked, lethal genetic disorder affecting the skeletal muscle compartment, and is caused by mutation(s) in the dystrophin gene. Gene delivery of microdystrophin constructs using adeno-associated virus (AAV) and antisense-mediated exon skipping restoring the genetic reading frame are two of the most promising therapeutic strategies for DMD. Both approaches use microdystrophin proteins either directly as a desired construct for gene delivery, using the capacity-limited AAV vectors, or as the therapeutic outcome of gene splicing. Although functionality of the resulting artificial dystrophin proteins can be predicted in silico, experimental evidence usually obtained in transgenic mice is required before human trials. However, the enormous number of potential constructs makes screening assays for dystrophin protein function in vitro and in vivo highly desirable. Here we present data showing that functionality of microdystrophins can be assessed using relatively simple and fast techniques.

AHNAK, a novel component of the dysferlin protein complex, redistributes to the cytoplasm with dysferlin during skeletal muscle regeneration.

Mutations in dysferlin cause limb girdle muscular dystrophy 2B, Miyoshi myopathy and distal anterior compartment myopathy. Dysferlin is proposed to play a role in muscle membrane repair. To gain functional insight into the molecular mechanisms of dysferlin, we have searched for dysferlin-interacting proteins in skeletal muscle. By coimmunoprecipitation coupled with mass spectrometry, we demonstrate that AHNAK interacts with dysferlin. We defined the binding sites in dysferlin and AHNAK as the C2A domain in dysferlin and the carboxyterminal domain of AHNAK by glutathione S-transferase (GST)-pull down assays. As expected, the N-terminal domain of myoferlin also interacts with the carboxyterminal domain of AHNAK. In normal skeletal muscle, dysferlin and AHNAK colocalize at the sarcolemmal membrane and T-tubules. In dysferlinopathies, reduction or absence of dysferlin correlates with a secondary muscle-specific loss of AHNAK. Moreover, in regenerating rat muscle, dysferlin and AHNAK showed a marked increase and cytoplasmic localization, consistent with the direct interaction between them. Our data suggest that dysferlin participates in the recruitment and stabilization of AHNAK to the sarcolemma and that AHNAK plays a role in dysferlin membrane repair process. It may also have significant implications for understanding the biology of AHNAK-containing exocytotic vesicles, "enlargosomes," in plasma membrane remodeling and repair.