Mutations in the O-mannosyltransferase gene POMT1 give rise to the severe neuronal migration disorder Walker-Warburg syndrome

Abstract

Walker-Warburg syndrome (WWS) is an autosomal recessive developmental disorder characterized by congenital muscular dystrophy and complex brain and eye abnormalities. A similar combination of symptoms is presented by two other human diseases, muscle-eye-brain disease (MEB) and Fukuyama congenital muscular dystrophy (FCMD). Although the genes underlying FCMD (Fukutin) and MEB (POMGnT1) have been cloned, loci for WWS have remained elusive. The protein products of POMGnT1 and Fukutin have both been implicated in protein glycosylation. To unravel the genetic basis of WWS, we first performed a genomewide linkage analysis in 10 consanguineous families with WWS. The results indicated the existence of at least three WWS loci. Subsequently, we adopted a candidate-gene approach in combination with homozygosity mapping in 15 consanguineous families with WWS. Candidate genes were selected on the basis of the role of the FCMD and MEB genes. Since POMGnT1 encodes an O-mannoside N-acetylglucosaminyltransferase, we analyzed the possible implication of O-mannosyl glycan synthesis in WWS. Analysis of the locus for O-mannosyltransferase 1 (POMT1) revealed homozygosity in 5 of 15 families. Sequencing of the POMT1 gene revealed mutations in 6 of the 30 unrelated patients with WWS. Of the five mutations identified, two are nonsense mutations, two are frameshift mutations, and one is a missense mutation. Immunohistochemical analysis of muscle from patients with POMT1 mutations corroborated the O-mannosylation defect, as judged by the absence of glycosylation of alpha-dystroglycan. The implication of O-mannosylation in MEB and WWS suggests new lines of study in understanding the molecular basis of neuronal migration.

Figure 1

Brain abnormalities in patients with WWS who have POMT1 mutations. A, Normal brain of fetus at 21 wk gestation. B, Brain from an affected fetus from family 4 at 19 wk gestation. Note the rough cobblestone brain surface, the cerebellar hypoplasia (arrow), and the abnormal vasculature. C, Midsagital T1–weighted magnetic-resonance image from an affected child from family 2, showing enlarged ventricles, lack of corpus callosum, absence of pons, and rudimentary cerebellar vermis. D–F, Haematoxylin and eosin staining of paraffin-embedded brain sections. D, Fetal brain at 21 wk gestation, showing normal lamination of the cortex: meninges (M), lamina molecularis (I), lamina granularis externa (II), lamina pyramidalis (III), lamina granularis interna (IV), lamina ganglionaris (V), lamina multiformis (VI), and intermediate zone (IZ). E, Brain section from affected fetus from family 4, showing abnormal cortical lamination with disruption of the glia limitans (**), and protrusion of the migrating neuroblasts into the subarachnoid space (*). SZ = subventricular zone. F, Higher magnification of panel E, showing the subarachnoid-space invasion by glio-vascular neuronal tissue. Glia limitans is shown with an arrow.

Figure 2

POMT1 gene mutations in families with WWS. For each of the families with POMT1 mutations, the pedigrees are shown, as well as two chromatograms, corresponding to a patient and a control DNA from the normal population. Patients for whom sequencing results are shown are marked by an arrow. A, Family 1. The patient is homozygous for a 226G→A mutation (Gly76Arg). A multiple-sequence alignment of the POMT1 peptide sequence from different species around glycine 76 is shown. * = not known. B, Families 2 and 3, carrying the same homozygous mutation, 907C→T (Gln303Stop). This mutation creates a new BfaI site, which was used to analyze the segregation of this mutation in family 3. The 180-bp amplicon is digested into 23- and 157-bp products for the wild-type allele, and the mutant allele is digested into products of 110, 47, and 23 bp. Undigested (−) and digested (+) products were loaded for the control. C, Sequence analysis of family 4 reveals homozygosity of 2110InsG (Val703fs). The bottom portion shows the comparison of the C-terminal peptide sequence from different species, as well as the predicted protein encoded by the 2110InsG and 2167InsG (Gly722fs) alleles. The mutation is marked by an arrow. * = predicted stop codon. D, Patient with WWS from family 6, who is a compound heterozygote for the POMT1 mutations 1283T→A (Val428Asp) and 2167InsG (Gly722fs). * = not known. E, MZ twins from family 7, who are homozygous for the 1153C→T (Gln385Stop) mutation. This mutation disrupts a PvuII site, which was used to study the segregation in the family. The 178-bp amplicon is digested into 78- and 100-bp products for the wild-type allele, and into 49-, 51-, and 78-bp products for the mutant allele.

Figure 3

Immunofluorescence staining of skeletal muscle from a control individual and from patients with WWS from families 2 and 4. Note the normal expression of laminin-α2 and β-dystroglycan in WWS. Variable staining intensities were observed with anti-α-dystroglycan antibody specific for the C-terminal peptide sequence, with most muscle fibers showing a marked reduction in α-dystroglycan staining. No α-dystroglycan staining was observed in patient muscle by using an antibody thought to recognize the O-glycan motif.

Figure 4

Representation of the only O-mannose–linked glycan motif found in brain, muscle, and peripheral nerve. The big circle represents the protein, to which several sugar residues are added in a sequential manner by the catalytic activity of specific glycosyltransferases. POMT1 catalyzes the addition of the first residue (mannose), whereas POMGnT1 catalyzes the second step, the linkage of N-acetylglucosamine in β-1,2. B4GALTs (Amado et al. 1999) and ST3GALs (Tsuji 1996) are the families of enzymes that are able to catalyze the third and fourth steps, respectively, of this O-glycan synthesis. The specific enzymes that catalyze these steps have not been assigned yet.