GNE Myopathy and Cell Apoptosis: A Comparative Mutation Analysis.

In a number of genetic disorders such as GNE myopathy, it is not clear how mutations in target genes result in disease phenotype. GNE myopathy is a progressive neuro-degenerative disorder associated with homozygous or compound heterozygous missense mutations in either epimerase or kinase domain of UDP-GlcNAc 2-epimerase/ManNAc kinase (GNE). This bifunctional enzyme catalyses the rate limiting step in sialic acid biosynthesis. Many mechanisms have been suggested as possible cause of muscle degeneration. These include hyposialylation of critical proteins, defects in cytoskeletal network, sarcomere organization and apoptosis. In order to elucidate the role of GNE in cell apoptosis, we have used HEK cell-based model system overexpressing pathologically relevant GNE mutations. These cells display a reduction in the levels of sialic acid-bound glycoconjugates. These mutants GNE overexpressing cells have defect in cell proliferation as compared to vector or wild-type GNE (wtGNE) controls. Moreover, effect of different GNE mutations on cell apoptosis was also observed using staining with annexin V-FITC and TUNEL assay. The downstream apoptosis signalling pathway involving activation of caspases and increased PARP cleavage were observed in all GNE mutant cell lines. In addition, morpho-structural changes in mitochondria in cells overexpressing different GNE mutants were noticed by transmission electron microscopy, and mitochondrial transmembrane potential was found to be altered in absence of functional GNE. Our results clearly indicate role of GNE in mitochondria-dependent cell apoptosis and provide insights into the pathomechanism of GNE myopathy.