GFPT1-myasthenia: clinical, structural, and electrophysiologic heterogeneity

Abstract

Objective: To identify patients with GFPT1-related limb-girdle myasthenia and analyze phenotypic consequences of the mutations.

Methods: We performed genetic analysis, histochemical, immunoblot, and ultrastructural studies and in vitro electrophysiologic analysis of neuromuscular transmission.

Results: We identified 16 recessive mutations in GFPT1 in 11 patients, of which 12 are novel. Ten patients had slowly progressive limb-girdle weakness responsive to cholinergic agonists with onset between infancy and age 19 years. One patient (no. 6) harbored a nonsense mutation and a second mutation that disrupts the muscle-specific GFPT1 exon. This patient never moved in utero, was apneic and arthrogrypotic at birth, and was bedfast, tube-fed, and barely responded to therapy at age 6 years. Histochemical studies in 9 of 11 patients showed tubular aggregates in 6 and rimmed vacuoles in 3. Microelectrode studies of intercostal muscle endplates in 5 patients indicated reduced synaptic response to acetylcholine in 3 and severely reduced quantal release in patient 6. Endplate acetylcholine receptor content was moderately reduced in only one patient. The synaptic contacts were small and single or grape-like, and quantitative electron microscopy revealed hypoplastic endplate regions. Numerous muscle fibers of patient 6 contained myriad dilated and degenerate vesicular profiles, autophagic vacuoles, and bizarre apoptotic nuclei. Glycoprotein expression in muscle was absent in patient 6 and reduced in 5 others.

Conclusions: GFPT1-myasthenia is more heterogeneous than previously reported. Different parameters of neuromuscular transmission are variably affected. When disruption of muscle-specific isoform determines the phenotype, this has devastating clinical, pathologic, and biochemical consequences.

Figure 1. Patients with GFPT1-myasthenia and histochemical observations

(A) Patient 6 during infancy and (B) at age 4 years. Note facial diplegia, open mouth, protruding tongue, lack of head control, and strabismus. (C) Patient 2 at age 12 years showing Gowers sign. (D) Large tubular aggregates in patient 4 and (E) small and infrequent tubular aggregates in patient 2 shown by NADH dehydrogenase reaction. (F) Acid phosphatase–positive autophagic vacuoles in patient 6. (G) Type 1 fiber preponderance in patient 6, ATPase at pH 4.3. (H–K) Face-on views of EP regions. (H–J) The EPs are composed of small grape-like regions. (I and J) Paired localization of AChE (green) and AChR (red) in frozen section. (H and K) Localized AChE on fixed, teased muscle fibers. Bars = 50 μm in panels D–G and 20 μm in H–K. AChE = acetylcholinesterase; AChR = acetylcholine receptor; ATPase = adenosine triphosphatase; EP = endplate; NADH = nicotinamide adenine dinucleotide.

Figure 2. Electron microscopy findings

(A and B) Endplate regions in patients 1 and 2. Note nearly absent junctional folds and small nerve terminals. (C and D) Muscle fiber pathology in patient 6. (C) A large accumulation of dilated and degenerating vesicular profiles near a nucleus. (D) Multiple autophagic vacuoles harboring granular or globular debris and apoptotic nuclei, one of which is large and bizarre (asterisk). Bars = 1 μm in panels A, B, and D, and 2 μm in C.

Figure 3. GFPT1 mutations and immunoblots of muscle extracts

(A) Scaled linear model of GFPT1 with 3 domains and observed mutations. Predicted peptides of the mutant transcripts of patient 6 are shown separately. Novel mutations are in bold type. Red box represents muscle-specific exon. Numbering is based on the short isoform of GFPT1. (B) Immunoblots demonstrating expression of the ∼70-kD glycosylated protein with RL2 (left panel) and O-GlcNAc (right panel) antibodies in control and 7 patient muscles. Expression levels are normalized for myosin level in the transferred gel and by comparison to controls. Glycosylated protein expression is absent in patient 6 and close to normal in patient 5. SIS = sugar isomerase domain.