P25 Validation of novel secondary dystroglycanopathy genes using biochemical, cellular and zebrafish studies

Abstract Mutations in several known genes such as LARGE , POMT1 , POMT2 , POMGnT1 , FKRP , FKTN , DPM2 , DPM3 , and DOLK are capable of causing glycosylation defects in alpha-dystroglycan (ADG), an integral component of the dystrophin glycoprotein complex. This contributes to the pathogenesis of an inherited subset of muscular dystrophies known as the dystroglycanopathies, at least in part by reducing the laminin binding abilities of hypoglycosylated ADG. There are undoubtedly many more genes to be discovered which contribute to this defective glycosylation as only approximately 50% of dystroglycanopathy patients can be diagnosed as having a defect in any of these known genes. In a collaborative study with the UK10K Rare Disease Project from the Sanger Institute, using whole exome sequencing techniques, we identified two patients with compound heterozygous mutations in two novel candidate genes, GDP-Mannose pyrophosphorylase beta ( GMPPB ) and beta-1,3-N-acetylgalactosaminyltransferase 2 ( B3GalNT2 ). Studies of a larger cohort of patients identified additional sporadic affected cases. GMPPB is an enzyme which catalyses the production of the high energy nucleotide sugar donor GDP-mannose. B3GalNT2 is a glycosyltransferase which synthesises the carbohydrate structure GalNacbeta1–3GlcNac. Patients with B3GalNT2 and GMPPB mutations present phenotypically as congenital muscular dystrophy with brain involvement. In vitro studies indicate that mutations in both genes affect the glycosylation of ADG, leading to a reduction in the amount of functionally glycosylated alpha-dystroglycan. Knocking down the two candidate genes in zebrafish results in muscle degeneration, brain and eye abnormalities and impaired motility. This recapitulates the patients’ phenotypes to some extent, supporting the hypothesis that the mutations are causative. We will present the results of zebrafish morpholino knockdowns of these two genes as well as the results of the in vitro work.