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

Abstract

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.

Keywords: MYO9A; congenital myasthenic syndrome; neuromuscular junction; unconventional myosin; whole exome sequencing.

Congenital myasthenic syndromes result from defects in the neuromuscular junction. Using whole exome sequencing, O'Connor et al. identify mutations in a novel candidate gene, MYO9A, which encodes an unconventional myosin. They provide preliminary evidence that MYO9A contributes to formation of the neuromuscular junction via effects on the presynaptic motor axon.

Figure 1

Structure of MYO9A and patient pedigrees. (A) Diagram depicting the structure of MYO9A and the protein position of the variants identified in Patients 1–3. Sequences before and after the Rho-GTPase domain are predicted to adopt a coiled coil structure (Gorman et al., 1999), represented by orange boxes. Accession number: NM_006901, Transcript: ENST00000356056. (B) Pedigree for Patient 1. (C) Pedigree for Patients 2 and 3. (D) Image of Patients 2 and 3 displaying bilateral ptosis and ophthalmoplegia; with asymmetric upward deviation of gaze (left eye) in the male patient.

Figure 2

MYO9A is expressed at the mouse neuromuscular junction. MYO9A (green) co-localizes with Synaptophysin (blue) and acetylcholine receptor (AChR) clusters (α-bungarotoxin, red) in mouse whole lumbrical muscle mounts. Scale bar = 50 µm.

Figure 3

Knockdown of MYO9A in NSC-34 cells causes increased length and branching of neurites. (A) MYO9A (red) co-localizes with F-actin (phalloidin, green) at the growth cones of mouse derived NSC-34 cells. (B) Relative expression ratio of quantitative RT-PCR showing the expression of Myo9a mRNA in control cells and in MYO9A shRNA-mediated knockdown cells (NSC-34). A 6.4-fold decrease in Myo9a expression is observed. (C) Number of primary, secondary and tertiary branches of control and MYO9A knockdown cells (two-sample t-test, *P < 0.05). Graph depicts mean ± standard error of the mean (SEM). (D) Knockdown of MYO9A in NSC-34 cells leads to aberrant neurite length and branching as compared to control cells. Scale bar = 100 μm.

Figure 4

Knockdown of MYO9A in zebrafish. (A) RT-PCR confirming knockdown success of myo9aa and myo9ab in zebrafish injected with morpholino oligonucleotide. There is loss of the myo9aa transcript and an alternatively spliced myo9ab transcript produced due to intron retention. Bands from the same gel have been cropped and put together for comparison, demarcated by the white lines. (B) Images of non-injected and mild/moderate myo9aa/ab morpholino oligonucleotide-injected zebrafish (48 hpf), with the latter displaying varying severities of tail curvature. Scale bar = 100 μm.

Figure 5

Motor axons of morphant zebrafish are shortened and abnormally branched. Neuromuscular junctions in absence of Myo9aa/ab. Non-injected and myo9aa/ab morpholino oligonucleotide-injected embryos were stained for presynaptic motor axons (SV2, green) and postsynaptic AChRs (α-bungarotoxin, red) at 48 hpf. Morphant zebrafish display shortened (arrow) and abnormally branched motor axons, with some innervating adjacent myotomes (arrowhead). Scale bar = 100 µm.