Use of a cell-free system to determine UDP-N-acetylglucosamine 2-epimerase and N-acetylmannosamine kinase activities in human hereditary inclusion body myopathy

Hereditary inclusion body myopathy (HIBM) is an autosomal recessive neuromuscular disorder associated with mutations in uridine diphosphate (UDP)-N-acetylglucosamine (GlcNAc) 2-epimerase (GNE)/N-acetylmannosamine (ManNAc) kinase (MNK), the bifunctional and rate-limiting enzyme of sialic acid biosynthesis. We developed individual GNE and MNK enzymatic assays and determined reduced activities in cultured fibroblasts of patients, with HIBM harboring missense muta-tions in either or both the GNE and MNK enzymatic domains. To assess the effects of individual mutations on enzyme activ-ity, normal and mutated GNE/MNK enzymatic domains were synthesized in a cell-free in vitro transcription–translation system and subjected to the GNE and MNK enzymatic assays. This cell-free system was validated for both GNE and MNK activities, and it revealed that mutations in one enzymatic