Thesis

Production of sialic acid affected by GNE gene mutations

Hereditary Inclusion Body Myopathy (HIBM) is an autosomal recessive disorder characterized by adult onset muscle-wasting, affecting both proximal and distal muscles. HIBM is caused by different mutations in the GNE gene including the common Middle Eastern founder allele, p.M712T. The GNE gene is located on chromosome 9p13.3 of humans. The GNE gene encodes a bifunctional rate limiting enzyme UDP N-acetyl glucosamine 2-epimerase/N-acetyl mannosamine kinase (UDP-GNE/MNK). This enzyme catalyzes the first two steps in the sialic acid biosynthetic pathway. Mutations in the GNE gene may lead to, either decreased sialic acid biosynthesis and reduced sialylation of a variety of proteins like alpha-dystroglycan, Neprilysin and NCAM, (Huizing et al., Broccolini et al., Ricci et al.,) or to increased sialic acid production. Glycosylation defects have recently become recognized as an important cause of muscular dystrophy (Muntoni et al.). The functional capacity of sialic acid production can be restored by introduction and expression of the wild-type GNE gene. In order to demonstrate this, I used lectin resistant Chinese Hamster Ovary (CHO) cells (Lec3), which lack GNE/MNK activity. The CHO cells were transfected with pUMVC3-GNE recombinant constructs expressing either a wild-type GNE, D176V, R263L, M265T, V572L, M712T or R266Q mutant insert. CHO cells transfected with the R263L and R266Q GNE expression plasmid had an increase in sialic acid production. Those transfected with the D176V, M265T, V572L, and M712T GNE expression plasmid showed significantly lower amounts of sialic acid production. We intend to use these data to construct a model for gene therapy in mice and investigate its safety and effectiveness in the alleviation of muscle degeneration manifested in HIBM.

Relationships

Items