Understanding the Consequences of Deficient Glutamine-Fructose-6-Phosphate Transaminase 1 (GFPT1) Expression in Congenital Myasthenic Syndromes (CMS) and Elucidating New Therapeutic Strategies

Abstract

Congenital myasthenic syndromes (CMS) are early onset, inheritable neuromuscular disorders caused by mutations in proteins required for neuromuscular junction (NMJ) development, maintenance, function, and motor endplate organization. Clinically, CMS are heterogeneous but typically manifest as fatigable weakness affecting facial, bulbar, ocular, respiratory, limb, and/or girdle muscles, with severity ranging from transient to permanent impairment depending on the underlying genetic defect. More than 40 genes have been implicated in CMS pathogenesis. Among these, glutamine–fructose 6 phosphate transaminase 1 (GFPT1) encodes the rate limiting enzyme of the hexosamine biosynthetic pathway (HBP). Biallelic GFPT1 mutations cause a limb girdle predominant CMS, yet the molecular basis of disease and effective treatments remain poorly understood. We hypothesized that GFPT1 deficiency generates a hypoglycosylated cellular environment that impairs neuromuscular function.

Using integrated biochemical, proteomic, and functional analyses, we define the molecular consequences of GFPT1 deficiency. We demonstrate that GFPT1 CMS is a bona fide glycosylation disorder marked by impaired N linked glycosylation and altered protein O GlcNAcylation. Notably, we identify the first mis glycosylated peptide in GFPT1 CMS, located within the δ subunit of the acetylcholine receptor (AChR), providing direct evidence that reduced HBP flux disrupts glycoprotein maturation essential for NMJ transmission. Building on this mechanistic insight, we establish two metabolic strategies that restore glycosylation: galactose supplementation, which partially rescues muscle protein glycosylation, normalizes NMJ and skeletal muscle morphology, and improves behavioral outcomes; and AMDH2 knockdown, which increases O GlcNAcylation in GFPT1 deficient cellular models.

Collectively, this work defines the first molecular signature of mis glycosylation in GFPT1 CMS, establishes two therapeutic strategies to restore glycan homeostasis, and identifies serglycin as a potential biomarker. These discoveries advance the mechanistic understanding of glycosylation dependent neuromuscular disease and lay the groundwork for targeted therapeutic development in GFPT1 CMS and related disorders.