Hexosamine biosynthetic pathway mutations cause neuromuscular transmission defect

Abstract

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.

Figure 1

Phenotype of CMS Patients in this Study and Linkage to Chromosome 2p12-p15 (A) Relative frequency of signs and symptoms in our patient cohort (n = 23). The following abbreviations are used: RNS, repetitive nerve stimulation; EMG, electromyography; CK, creatine kinase. (B) Patient LGM10.4 showing proximal muscle weakness and hyperlordotic posture at prolonged arm elevation. (C) Patient LGM12.3 showing scapular winging. (D) Genome-wide linkage analysis of family LGM3. Analysis was performed with MERLIN with the assumption of autosomal-recessive inheritance with complete penetrance and a disease allele frequency of 0.001.

Figure 2

Identification of GFPT1 Mutations (A) GFPT1 mutations detected in families used for candidate-gene sequencing. Chromatogram pairs of an unaffected individual and an affected patient are shown. The asterisks indicate the position of the mutations. Base and amino acid changes with the corresponding position in the nucleotide and protein sequence are given above chromatograms displaying GFPT1 mutations. The numbering of nucleotides and amino acids follows NM_002056.2 and NP_002047.2, which represent the short, ubiquitous isoform of GFPT1. Nucleotide numbering uses the A of the ATG translation initiation start site as nucleotide +1. (B) Domain structure of GFPT1 and position of mutations. GFPT1 contains a glutaminase and two sugar isomerase (SIS) domains. The site of insertion of 18 additional amino acids (hashed box) encoded by a muscle specific exon is indicated. Missense mutations are in black, truncating mutations are in red. The 3′-UTR mutation is not shown. The numbering of nucleotides and amino acids is as in (A) except for the c.719G>A (p.Trp240X) mutation in the muscle specific exon, for which we used numbering of the longer GFPT1-L sequence. aa is an abbreviation for amino acid. (C) Immunoblot of GFPT1 and O-GlcNAcylation in muscle biopsies of GFPT1 patients. Muscle lysates of patients LGM9.3, LGM5.5, LGM10.4, and LGM11.3 and two healthy control individuals were immunoblotted with an anti-GFPT1 antibody (top), the RL2 antibody (detects single N-acetylglucosamine at serine or threonine residues, middle) and an anti-actin antibody (as loading control, bottom). GFPT1 band intensities were normalized to actin bands in the same lane. Total GFPT1 levels (GFPT1-L and the shorter ubiquitous isoform) in patients' muscles were reduced to 29%, 8%, 9%, and 26% of the level in control muscle 1. The levels of O-linked N-acetylglucosamine on proteins were also markedly decreased in patients' muscles. The membrane was first decorated with the anti-GFPT1 antibody and later reprobed with the RL2 and anti-actin antibodies.

Figure 3

Gpft1 Knockdown in Zebrafish Embryos (A–D) Live embryos at 24 and 48 hpf injected with the translation-blocking gfpt1 antisense morpholino oligo MO1 (A and B) and noninjected controls (C and D). Note increased tail curvature in gfpt1 morphants. (E–P) Whole-mount immunostainings of zebrafish embryos. Slow-twitch muscle fiber structure in 48 hpf gfpt1-MO1 injected morphants (E–G) and noninjected controls (H–J). Embryos were stained for AChR (α−bungarotoxin) and slow-twitch muscle fibers (anti-slow muscle myosin heavy chain [slow MyHC], F59 antibody). Gfpt1 morphants show severe muscle fiber defects with detachment of fibers from the vertical myoseptum. Somites have lost their chevron shape. (K–P) Neuromuscular junctions in 48 hpf gfpt1 morphants (K–M) and noninjected controls (N–P). Embryos were stained for AChR (α−bungarotoxin) and presynaptic nerve endings (anti-SV2 antibody). In controls, motor axons have branched out into all parts of the somite where they form contacts with AChR clusters. In gfpt1-MO1 injected embryos AChR clusters are predominantly located along the medial surface of the somite, and motor axons form fewer branches than in controls. The scale bars represent 50 μm.