Mutations in DPAGT1 cause a limb-girdle congenital myasthenic syndrome with tubular aggregates

Abstract

Congenital myasthenic syndromes are a heterogeneous group of inherited disorders that arise from impaired signal transmission at the neuromuscular synapse. They are characterized by fatigable muscle weakness. We performed whole-exome sequencing to determine the underlying defect in a group of individuals with an inherited limb-girdle pattern of myasthenic weakness. We identify DPAGT1 as a gene in which mutations cause a congenital myasthenic syndrome. We describe seven different mutations found in five individuals with DPAGT1 mutations. The affected individuals share a number of common clinical features, including involvement of proximal limb muscles, response to treatment with cholinesterase inhibitors and 3,4-diaminopyridine, and the presence of tubular aggregates in muscle biopsies. Analyses of motor endplates from two of the individuals demonstrate a severe reduction of endplate acetylcholine receptors. DPAGT1 is an essential enzyme catalyzing the first committed step of N-linked protein glycosylation. Our findings underscore the importance of N-linked protein glycosylation for proper functioning of the neuromuscular junction. Using the DPAGT1-specific inhibitor tunicamycin, we show that DPAGT1 is required for efficient glycosylation of acetylcholine-receptor subunits and for efficient export of acetylcholine receptors to the cell surface. We suggest that the primary pathogenic mechanism of DPAGT1 mutations is reduced levels of acetylcholine receptors at the endplate region. These individuals share clinical features similar to those of congenital myasthenic syndrome due to GFPT1 mutations, and their disorder might be part of a larger subgroup comprising the congenital myasthenic syndromes that result from defects in the N-linked glycosylation pathway and that manifest through impaired neuromuscular transmission.

Figure 1

Segregation of DPAGT1 Mutations Pedigrees of the families of all affected individuals are shown together with Sanger-sequencing verification of variants for each family member. For case 1, alignment of several representative reads generated by next-generation sequencing is shown to demonstrate that two mutations are always present in different reads and never occur in the same read.

Figure 2

DPAGT1 Structure and Conservation Predicted transmembrane regions are shown in green. Protein alignment was performed in ClustalW2 (see Web Resources). Positions of amino acid substitutions are shown with arrows.

Figure 3

DPAGT1 Is Required for AChR-Subunit Glycosylation and Receptor Export (A) The electron micrograph from the muscle biopsy from case 1 shows the presence of multiple tubular aggregates. (B) The electron micrograph from motor-endplate regions in case 1 shows a severe reduction of postsynaptic folding. Various magnifications are shown. (C) Assay for the level of α-BuTx binding to the surface of HEK 293 cells. Cells were transfected with 1.5 μg of expression plasmids with AChR subunits (2α, 1β, 1δ, and 1ε) and with 4.5 μg of an empty pcDNA vector, a pcDNA-DPAGT1-WT plasmid, or a pcDNA-DPAGT1-c.699dup plasmid. Sixteen hours after transfection, cells were incubated with or without 1 μg/ml tunicamycin. Binding of α-BuTx was normalized to surface expression in the absence of tunicamycin (100%). Error bars represent the standard error of the mean. (D) Immunoblot of cell extracts from the α-BuTx cell-surface-binding experiment (above). As described, cells were transfected with 1.5 μg of plasmids with AChR subunits (2α, 1β, 1δ, and 1ε) and with 4.5 μg of an empty pcDNA vector (−), a pcDNA-DPAGT1-WT (“WT”) plasmid, or a pcDNA-DPAGT1-c.699dup (“dup”) plasmid. Sixteen hours after transfection, cells were incubated with ± 1 μg/ml tunicamycin. Cell extracts were subject to immunoblotting and probed with antibodies against the AChR δ subunit, DPAGT1 (raised to amino acids 27–57 of DPAGT1), or α-tubulin as a loading control. The levels of endogenous DPAGT1 were too low to be detected at the exposure conditions shown.