Congenital myasthenic syndromes due to mutations in ALG2 and ALG14

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 linkage analysis, whole-exome and whole-genome sequencing to determine the underlying defect in patients with an inherited limb-girdle pattern of myasthenic weakness. We identify ALG14 and ALG2 as novel genes in which mutations cause a congenital myasthenic syndrome. Through analogy with yeast, ALG14 is thought to form a multiglycosyltransferase complex with ALG13 and DPAGT1 that catalyses the first two committed steps of asparagine-linked protein glycosylation. We show that ALG14 is concentrated at the muscle motor endplates and small interfering RNA silencing of ALG14 results in reduced cell-surface expression of muscle acetylcholine receptor expressed in human embryonic kidney 293 cells. ALG2 is an alpha-1,3-mannosyltransferase that also catalyses early steps in the asparagine-linked glycosylation pathway. Mutations were identified in two kinships, with mutation ALG2p.Val68Gly found to severely reduce ALG2 expression both in patient muscle, and in cell cultures. Identification of DPAGT1, ALG14 and ALG2 mutations as a cause of congenital myasthenic syndrome underscores the importance of asparagine-linked protein glycosylation for proper functioning of the neuromuscular junction. These syndromes form part of the wider spectrum of congenital disorders of glycosylation caused by impaired asparagine-linked glycosylation. It is likely that further genes encoding components of this pathway will be associated with congenital myasthenic syndromes or impaired neuromuscular transmission as part of a more severe multisystem disorder. Our findings suggest that treatment with cholinesterase inhibitors may improve muscle function in many of the congenital disorders of glycosylation.

Figure 1

Diagrammatic representation of the N-linked glycosylation pathway. ER = endoplasmic reticulum.

Figure 2

(A) Segregation of mutations ALG14 c.194C > T, p.Pro65Leu and c.310C > T, p.Arg104* within Family 1. (B) Conservation of ALG14 protein sequence within the vicinity of the p.Pro65Leu mutation. Alignment of protein sequence flanking Pro65 (red) from several species was carried using ClustalW. Colours indicate percentage identity and were introduced using JalView. Residue numbering is according to the human protein. (C) Representation of the ALG14 protein transmembrane topology and location of mutations. Protein structure was visualized using TEXtopo (Beitz, 2000).

Figure 3

Expression of ALG14 wild-type (WT) and p.Pro65Leu in HEK 293 cells. Complementary DNA expression constructs were transfected into HEK293 cells, and 48 h later, cells were lysed and subjected to western blot analysis using an antibody to ALG14. α-tubulin was used as a loading control.

Figure 4

Downregulation of ALG14 reduces surface expression of acetylcholine receptor in transfected HEK293 cells. (A) Silencing of ALG14 expressed in HEK 293 cells by small interfering RNAs (siRNA) targeted at ALG14 messenger RNA. Small interfering RNAs and the ALG14 expression construct were transfected into HEK 293 cells, and 48 h later, ALG14 expression analysed by western blot. (B) Two small interfering RNAs (1 and 10) targeted at ALG14 messenger RNA or ‘scrambled’ small interfering RNA were transfected into HEK 293 cells. Twenty-four hours later, the same cells were transfected with complementary DNAs encoding the human acetylcholine receptor α, β, δ and ε subunits, and 48 h later, cell-surface ¹²⁵I-α-bungarotoxin binding was determined (n = 3).

Figure 5

(A) Graphical representation of homozygous regions shared by Cases 3, 4 and 5 (autozygosity analysis performed before the birth of Case 6) generated using the AutoSNPa program. Shared blocks of homozygous single nucleotide polymorphisms are shown as red bars. The most significant homozygous region is on 9q31.1 (genomic location 100114051–105435311) is 27 Mb long and contains 283 genes (denoted by asterisk). (B) Sequence analysis in the c.214_226delGGGGACTGGCTGCinsAGTCCCCGGC p.72_75delGDWLinsSPR region for members of Family 2. Cases 3–6 are homozygous for this indel, whereas their parents (second cousins) and unaffected siblings are heterozygous. Asterisk denotes the individual in which exome sequencing was performed (Case 5). (C) Location of mutations for Families 2 and 3 in ALG2. ALG2 amino acid residues p.Val68 and p.72_75GDWL are conserved across species. Alignment of protein sequence flanking p.72_75GDWL from several species was carried out using ClustalW. Colours indicate per cent identity and were introduced using JalView. Residue numbering is according to the human protein.

Figure 6

Segregation of ALG2 c.203 T > G with disease within the pedigree of Family 3. Only the index case is homozygous for the variant. Sequence analysis in the c.203C > T region of five unaffected family members is shown. The index case (Case 7) and homozygous mutated nucleotide are indicated with arrows.

Figure 7

Reduced expression of the ALG2p.Val68Gly variant. (A) Proteins in lysates from two controls muscles biopsies with no N-glycosylation defect or from a muscle biopsy from Case 7 were subject to western blot analysis probed with an antibody against ALG2 (Aviva Systems Biology). (B) HEK 293 cells transfected with wild-type or mutant ALG2 were subject to similar western blot analysis. Transfection efficiency was normalized by co-transfection with EGFP. (C) Quantification of the data from transfected HEK 293 cells (P = 0.0197, t-test, n = 3).

Figure 8

Alg14 and Alg2 are enriched at endplate regions in mouse muscle sections. (A) Mouse muscle sections were stained with an anti-Alg14 antibody (Abgent), or (B) with an anti-Alg2 antibody (Santa Cruz Biotechnology). Secondary antibodies were Alexa Fluor®488 conjugated anti-rabbit from Invitrogen (green). AChRs at the neuromuscular junctions were visualized by staining with an Alexa Fluor®594 conjugated α-Bungarotoxin (red).