Hypoglycosylation of α-dystroglycan in patients with hereditary IBM due to GNE mutations

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Abstract

Hereditary inclusion body myopathy (HIBM) is an adult onset neuromuscular disorder associated with mutations in the gene UDP-N-acetylglucosamine-2-epimerase/N-acetylmannosamine kinase (GNE), whose product is the rate limiting bi-functional enzyme catalyzing the first two steps of sialic acid biosynthesis. Loss of GNE activity in HIBM is thought to impair sialic acid production and interfere with proper sialylation of glycoconjugates, but it remains unclear how such a defect would lead to muscle destruction and muscle weakness. Hypoglycosylation of α-dystroglycan, a central protein of the skeletal muscle dystrophin–glycoprotein complex, results in disturbed interactions with extracellular matrix proteins. This has recently been identified as the pathomechanism involved in several congenital muscular dystrophies. We examined the glycosylation status of α-dystroglycan in muscle biopsies of four HIBM patients of non-Iranian Jewish origin (one American, two Indians, and one Greek). Two of these patients carry novel compound heterozygous GNE mutations on exon 2 and exon 9. All four muscle biopsies showed absent or markedly reduced immunolabeling with two different antibodies (VIA4 and IIH6) to glycosylated epitopes of α-dystroglycan. Normal labeling was found using antibodies to the core α-dystroglycan protein, β-dystroglycan, and laminin α-2. These findings resemble those found for other congenital muscular dystrophies, suggesting that HIBM may be a “dystroglycanopathy,” and providing an explanation for the muscle weakness of patients with GNE mutations.

Introduction

Hereditary inclusion body myopathy (HIBM; OMIM 600737) is an autosomal recessive neuromuscular disorder characterized by slowly progressive myopathic weakness and atrophy. The disease usually manifests in the second or third decade with foot drop and progressive muscle weakness that involves all limbs but nearly always spares the quadriceps muscles. Histologically, the muscle fibers degenerate and form rimmed vacuoles, especially in atrophic areas [1], [2], [3]. HIBM results from mutations in the UDP-N-acetylglucosamine-2-epimerase/N-acetylmannosamine kinase gene (GNE) on chromosome 9p12–13 [4]. A Japanese variant of HIBM, distal myopathy with rimmed vacuoles (DMRV), is allelic to HIBM and displays mutations in GNE [5]. GNE is the rate limiting, bi-functional enzyme that catalyzes the first two steps committed toward sialic acid biosynthesis [6]. Sialic acid serves as the terminal, charged sugar on glycoproteins and glycolipids, and loss of GNE activity interferes with proper sialylation of cell surface glycoconjugates [7]. However, evidence of impaired sialylation of muscle glycoproteins has not been demonstrated in the muscles of patients with HIBM.

A reasonable candidate for such undersialylation is α-dystroglycan, an essential component of the dystrophin–glycoprotein complex (DGC). Within the DGC, α-dystroglycan binds components of the extracellular matrix, thereby linking it to the actin-associated cytoskeleton via β-dystroglycan and dystrophin [8]. α-Dystroglycan is heavily glycosylated with predominantly O-linked glycans, including rare O-mannosyl linked glycans consisting of ser/thr–mannose–N-acetylglucosamine–galactose–sialic acid. These glycans appear to be critical for interactions with laminin and other extracellular matrix ligands [8], [9]. Abnormal dystroglycan–ligand interactions, some of which occur because of improper glycosylation of α-dystroglycan, provide the explanation for the pathogenesis of several muscular dystrophies, including Fukuyama’s congenital muscular dystrophy (FCMD), Muscle–Eye–Brain disease (MEB), and Walker–Warburg syndrome (WWS) [8], [10]. Perturbation of α-dystroglycan–ligand interactions in muscle fibers, perhaps due to inadequate sialylation of α-dystroglycan as a result of GNE mutations, could explain the clinical manifestations of muscle weakness in HIBM. Here we report markedly reduced α-dystroglycan staining, using glycan-recognizing antibodies, in the skeletal muscle specimens of four HIBM individuals, including two previously unreported patients with novel GNE mutations.

Section snippets

Subjects

Patient #1 is a 39-year-old woman diagnosed at the National Institutes of Health Clinical Center in 1993 as the first non-Iranian Jewish, white American patient with quadriceps-sparing inclusion body myopathy [2]. No consanguinity was present in the family. The initial manifestation was bilateral foot drop at the age of 22 years, followed by progressive, diffuse, limb muscle weakness, and atrophy, except for the quadriceps muscles and abductors of the hips. The same disease began in her older

Results

Mutation analysis on genomic DNA from four HIBM patients revealed compound heterozygous mutations in GNE for all four patients. Each patient has one mutation in the epimerase and one in the kinase domain of GNE. Patient #1 carried V216A and A631V mutations, as reported previously [11]. Patient #2 carried R11W and V696M mutations. Patient #3 carried C303X and V696M; similar mutations have been reported by Eisenberg et al. [12]. Patient #4 had two novel mutations, R202L on exon 2 and G559R on

Discussion

In skeletal muscle membranes, both α- and β-dystroglycan participate in the multimeric dystrophin–glycoprotein complex (DGC). This complex maintains the structural stability of the sarcolemma during cycles of contraction and relaxation by linking cytoskeletal actin (via dystrophin) to components of the extracellular matrix (via α-dystroglycan) [8], [9], [14]. Recent studies have shown that aberrant glycosylation of α-dystroglycan is the underlying biochemical defect in several congenital

Note added in proof

Since submission of the paper, we have repeated the experiments using different (non-commercial) lots of IIH6 and VIA-4 antibodies against a- dystroglycan kindly provided to us by Dr. Campbell. These antibodies do not distinguish a difference between the HIBM patients versus controls. This indicates that there exist differences in epitope recognition between lots of the VIA4 and IIH6 monoclonal antibodies. This observation generates additional interest in the disease and the need to identify

Acknowledgements

The authors greatly appreciate the gift of α-dystroglycan core antibody from Dr. Stephan Kroger.

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