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Brain malformations and retinal dysplasia in a subset of congenital muscular dystrophies (CMDs), dystroglycanopathies, are caused by disruptions of the basement membranes.Reduced extracellular matrix binding by the hypoglycosylated α-dystroglycan because of mutations in several glycosyltransferases is the underlying molecular defects.It is not known how the basement membrane becomes disrupted.We hypothesized that assembly of the basement membrane is reduced in rate thus causing a physically compromised basement membrane.To test this hypothesis, we analyzed assembly of extracellular matrix on cultured neural stem cells and physical properties of the mutant basement membrane by atomic force microscopy.Laminin assembly on the protein O-mannose β1,2-N-acetylglucosaminyltransferase 1 (POMGnT1) knockout neural spheres was reduced when compared to the wildtype.When incubated with Matrigel, extracellular matrix (ECM) molecules including all 4 major components of the basement membrane, laminin, collagen Ⅳ, perlecan, and nidogen co-aggregated.Rate of ECM aggregation was reduced on POMGnT1 knockout neural sphere as revealed by slower growth in aggregate size when compared to the wildtype.Immunofluorescence staining and proteomic comparison revealed that the mutant basement membrane exhibited compositional changes from the wildtype.Atomic force microscopic analysis revealed that the mutant basement membrane had reduced elastic modulus with surface topography showing bigger valleys than the controls.Thus, disruptions of the basement membrane in dystroglycanopathies may be caused by its weakened strength resulting from biochemical changes in composition and physical changes in structure and reduced assembly rate.