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.

Background: Walker-Warburg syndrome (WWS) is an autosomal recessive condition characterised by congenital muscular dystrophy, structural brain defects, and eye malformations. Typical brain abnormalities are hydrocephalus, lissencephaly, agenesis of the corpus callosum, fusion of the hemispheres, cerebellar hypoplasia, and neuronal overmigration, which causes a cobblestone cortex. Ocular abnormalities include cataract, microphthalmia, buphthalmos, and Peters anomaly. WWS patients show defective O-glycosylation of a-dystroglycan (a-DG), which plays a key role in bridging the cytoskeleton of muscle and CNS cells with extracellular matrix proteins, important for muscle integrity and neuronal migration. In 20% of the WWS patients, hypoglycosylation results from mutations in either the protein O-mannosyltransferase 1 (POMT1), fukutin, or fukutin related protein (FKRP) genes. The other genes for this highly heterogeneous disorder remain to be identified. Objective: To look for mutations in POMT2 as a cause of WWS, as both POMT1 and POMT2 are required to achieve protein O-mannosyltransferase activity. Methods: A candidate gene approach combined with homozygosity mapping. Results: Homozygosity was found for the POMT2 locus at 14q24.3 in four of 11 consanguineous WWS families. Homozygous POMT2 mutations were present in two of these families as well as in one patient from another cohort of six WWS families. Immunohistochemistry in muscle showed severely reduced levels of glycosylated a-DG, which is consistent with the postulated role for POMT2 in the O-mannosylation pathway. Conclusions: A fourth causative gene for WWS was uncovered. These genes account for approximately one third of the WWS cases. Several more genes are anticipated, which are likely to play a role in glycosylation of a-DG.

Walker-Warburg syndrome (WWS) is clinically defined as congenital muscular dystrophy accompanied by a variety of brain and eye malformations. It represents the most severe clinical phenotype in a spectrum of alpha-dystroglycan posttranslational processing abnormalities, which share a defect in laminin binding glycan synthesis1. Although six WWS causing genes have been described, only half of all patients can currently be diagnosed genetically2. A cell fusion complementation assay using fibroblasts from undiagnosed WWS individuals identified five novel complementation groups. Further evaluation of one group by linkage analysis and targeted sequencing identified recessive mutations in the isoprenoid synthase domain containing (ISPD) gene. Confirmation of the pathogenicity of the identified ISPD mutations was demonstrated by complementation of fibroblasts with wild-type ISPD. Finally, we show that recessive mutations in ISPD abolish the initial step in laminin binding glycan synthesis by disrupting dystroglycan O-mannosylation. This establishes a novel mechanism for WWS pathophysiology.

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Mutations in the dysferlin gene underlie a group of autosomal recessive muscle-wasting disorders denoted as dysferlinopathies. Dysferlin has been shown to play roles in muscle membrane repair and muscle regeneration, both of which require vesicle-membrane fusion. However, the mechanism by which muscle becomes dystrophic in these disorders remains poorly understood. Although muscle inflammation is widely recognized in dysferlinopathy and dysferlin is expressed in immune cells, the contribution of the immune system to the pathology of dysferlinopathy remains to be fully explored. Here, we show that the complement system plays an important role in muscle pathology in dysferlinopathy. Dysferlin deficiency led to increased expression of complement factors in muscle, while muscle-specific transgenic expression of dysferlin normalized the expression of complement factors and eliminated the dystrophic phenotype present in dysferlin-null mice. Furthermore, genetic disruption of the central component (C3) of the complement system ameliorated muscle pathology in dysferlin-deficient mice but had no significant beneficial effect in a genetically distinct model of muscular dystrophy, mdx mice. These results demonstrate that complement-mediated muscle injury is central to the pathogenesis of dysferlinopathy and suggest that targeting the complement system might serve as a therapeutic approach for this disease.

Dystroglycan is a cell membrane receptor that organizes the basement membrane by binding ligands in the extracellular matrix. Proper glycosylation of the α-dystroglycan (α-DG) subunit is essential for these activities, and lack thereof results in neuromuscular disease. Currently, neither the glycan synthesis pathway nor the roles of many known or putative glycosyltransferases that are essential for this process are well understood. Here we show that FKRP, FKTN, TMEM5 and B4GAT1 (formerly known as B3GNT1) localize to the Golgi and contribute to the O-mannosyl post-phosphorylation modification of α-DG. Moreover, we assigned B4GAT1 a function as a xylose β1,4-glucuronyltransferase. Nuclear magnetic resonance studies confirmed that a glucuronic acid β1,4-xylose disaccharide synthesized by B4GAT1 acts as an acceptor primer that can be elongated by LARGE with the ligand-binding heteropolysaccharide. Our findings greatly broaden the understanding of α-DG glycosylation and provide mechanistic insight into why mutations in B4GAT1 disrupt dystroglycan function and cause disease.DOI: http://dx.doi.org/10.7554/eLife.03941.001

Alkaptonuria (AKU) is an autosomal recessive disorder caused by the deficiency of homogentisate 1,2 dioxygenase (HGO) activity. AKU shows a very low prevalence (1:100,000-250,000) in most ethnic groups. One notable exception is in Slovakia, where the incidence of AKU rises to 1:19,000. This high incidence is difficult to explain by a classical founder effect, because as many as 10 different AKU mutations have been identified in this relatively small country. We have determined the allelic associations of 11 HGO intragenic polymorphisms for 44 AKU chromosomes from 20 Slovak pedigrees. These data were compared to the HGO haplotype data available in our laboratory for >80 AKU chromosomes from different European and non-European countries. The results show that common European AKU chromosomes have had only a marginal contribution to the Slovak AKU gene pool. Six of the ten Slovak AKU mutations, including the prevalent G152fs, G161R, G270R, and P370fs mutations, most likely originated in Slovakia. Data available for 17 Slovak AKU pedigrees indicate that most of the AKU chromosomes have their origins in a single very small region in the Carpathian mountains, in the northwestern part of the country. Since all six Slovak AKU mutations are associated with HGO mutational hot spots, we suggest that an increased mutation rate at the HGO gene is responsible for the clustering of AKU mutations in such a small geographical region.

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