Details

Autor(en) / Beteiligte

• Ebrahim, Hatim Y.
• Baker, Robert J.
• Mehta, Atul B.
• Hughes, Derralynn A.

Titel

Functional analysis of variant lysosomal acid glycosidases of Anderson-Fabry and Pompe disease in a human embryonic kidney epithelial cell line (HEK 293 T)

Ist Teil von

• Journal of inherited metabolic disease, 2012-03, Vol.35 (2), p.325-334

Ort / Verlag

Dordrecht: Springer Netherlands

Erscheinungsjahr

2012

Quelle

Wiley Blackwell Single Titles

Beschreibungen/Notizen

• The functional significance of missense mutations in genes encoding acid glycosidases of lysosomal storage disorders (LSDs) is not always clear. Here we describe a method of investigating functional properties of variant enzymes in vitro using a human embryonic kidney epithelial cell line. Site-directed mutagenesis was performed on the parental plasmids containing cDNA encoding for alpha-galactosidase A (α-Gal A) and acid maltase (α-Glu) to prepare plasmids encoding relevant point mutations. Mutant plasmids were transfected into HEK 293 T cells, and transient over-expression of variant enzymes was measured after 3 days. We have illustrated the method by examining enzymatic activities of four unknown α-Gal A and one α-Glu variants identified in our patients with Anderson-Fabry disease and Pompe diseases respectively. Comparison with control variants known to be either pathogenic or non-pathogenic together with over-expression of wild-type enzyme allowed determination of the pathogenicity of the mutation. One leader sequence novel variant of α-Gal A (p.A15T) was shown not to significantly reduce enzyme activity, whereas three other novel α-Gal A variants (p.D93Y, p.L372P and p.T410I) were shown to be pathogenic as they resulted in significant reduction of enzyme activity. A novel α-Glu variant (p.L72R) was shown to be pathogenic as this significantly reduced enzyme activity. Certain acid glycosidase variants that have been described in association with late-onset LSDs and which are known to have variable residual plasma and leukocyte enzyme activity in patients appear to show intermediate to low enzyme activity (p.N215S and p.Q279E α-Gal A respectively) in the over-expression system.