Missense mutations in β-1,3-N-acetylglucosaminyltransferase 1 (B3GNT1) cause Walker-Warburg syndrome

Abstract

Several known or putative glycosyltransferases are required for the synthesis of laminin-binding glycans on alpha-dystroglycan (αDG), including POMT1, POMT2, POMGnT1, LARGE, Fukutin, FKRP, ISPD and GTDC2. Mutations in these glycosyltransferase genes result in defective αDG glycosylation and reduced ligand binding by αDG causing a clinically heterogeneous group of congenital muscular dystrophies, commonly referred to as dystroglycanopathies. The most severe clinical form, Walker-Warburg syndrome (WWS), is characterized by congenital muscular dystrophy and severe neurological and ophthalmological defects. Here, we report two homozygous missense mutations in the β-1,3-N-acetylglucosaminyltransferase 1 (B3GNT1) gene in a family affected with WWS. Functional studies confirmed the pathogenicity of the mutations. First, expression of wild-type but not mutant B3GNT1 in human prostate cancer (PC3) cells led to increased levels of αDG glycosylation. Second, morpholino knockdown of the zebrafish b3gnt1 orthologue caused characteristic muscular defects and reduced αDG glycosylation. These functional studies identify an important role of B3GNT1 in the synthesis of the uncharacterized laminin-binding glycan of αDG and implicate B3GNT1 as a novel causative gene for WWS.

Figure 1.

Schematic representation of B3GNT1 chromosomal position, protein structure and localization of mutations. (A) Ideogram of chromosome 11 showing the localization of the SNPs flanking the shared homozygous region in the patients. (B) Zoom-in of the 5.2 Mb homozygous region. (C) Gene structure of B3GNT1 showing the 5′ UTR, two coding exons separated by the intron and the 3′ UTR. (D) Protein structure of B3GNT1 showing the topological domains, the conserved glycosyltransferase domain and the position of the missense mutations M1 (p.N390D) and M2 (p.A406V). (E) Pedigree of family WWS-31. Individuals that were available for study are identified by their lab number. The mutation status is indicated below each individual (+, present, −, absent, NA, not available).

Figure 2.

(A–D) MRI at 4 months of age. Sagittal T2W image (A) reveals hydrocephalus, a hypoplastic ‘Z’-shaped brainstem (arrow) and a hypoplastic, dysplastic vermis. Coronal T1W image (B) demonstrates absence of the septal leaflets, vertical hippocampi and fusion of the forniceal columns in the midline (arrow). Axial T2W image (C) shows focal cobblestone lissencephaly of the occipital cortex (arrow). The subjacent white matter is abnormally increased in signal intensity. A shunt is present in the posterior horn of the right lateral ventricle. In addition to ventriculomegaly and focal cobblestone lissencephaly, coronal T2W image (D) reveals cysts (arrow) within the dysplastic cerebellum. (E) Patient (P) and control (C) muscle homogenates were used for a laminin overlay assay (LO). β-Dystroglycan (β-DG) staining was used as loading control.

Figure 3.

Cellular localization of wild-type and mutant B3GNT1. Wild-type and mutant variants of EGFP-tagged B3GNT1 (green) colocalize with the Golgi marker Giantin (red). Scale bar represents 10 µm.

Figure 4.

Flow cytometry analysis of transfected PC3 cells. PC3 cells transfected with an empty vector are used as control (A). In B3GNT1 WT transfected PC3 cells (B) the percentage of IIH6-positive cells is significantly higher than in PC3 cells transfected with an empty vector (A, F). The percentages of IIH6-positive cells in B3GNT1 M1 (C), B3GNT1 M2 (D) and B3GNT1 M1M2 (E) transfected PC3 cells are comparable with the percentage of the PC3 cells transfected with the empty vector (A, summary in F), indicating that glycosylation is affected. (F) Bar chart showing the relative amount of IIH6-positive cells, taking the empty vector control as standard (n = 3, *P < 0.05, one sample T-test). Error bars show the standard deviation.

Figure 5.

Knockdown of zebrafish b3gnt1 causes muscle defects and reduced glycosylation of αDG. (A and B) RT-PCR results showing that b3gnt1 is expressed throughout zebrafish embryonic development (A) but greatly reduced in 48 hpf embryos treated with 6 ng or 9 ng of b3gnt1 morpholino (2c, two-cell stage; Shd, shield stage; d, days post fertilization) (B), compared with β-actin loading control; arrow indicates aberrantly spliced b3gnt1 transcripts. (C) Western blot using IIH6 antibody to detect the αDG glycosylation state (Glyco. αDag1) in 48 hpf wild-type (wt), b3gnt1 morphant (bMO) or dag1 morphant (dMO) embryos. Knockdown of b3gnt1 causes hypoglycosylation of αDG compared with wild-type. Ponceau staining (PonS) loading control shown below. (D) Fluorescent confocal microscopy images of 48 hpf wild-type (top) and b3gnt1 morphant (bottom) embryos stained with phalloidin (green), and the corresponding DIC images. Loss of function of b3gnt1 results in disrupted MTJs as indicated by βDG immunoreactivity (red) and muscle fibres spanning multiple segments. (E) Compromised sarcolemmal integrity precedes fibre detachment in b3gnt1 morpholino-treated embryos. Fluorescent confocal microscopy image of a 48 hpf embryo, previously injected with b3gnt1 splice-blocking morpholino, treated with EBD (top panel) to highlight muscle fibres with disrupted sarcolemma (arrows). The corresponding DIC image is shown in the middle panel. Representative images of identified muscle lesions from three independent experiments; scale bar represents 50 µm.