[Fabry disease: enzymatic screening using dried blood spots on filter paper]

Fabry disease (FD) is an X-linked lysosomal storage disorder caused by mutations in the GLA gene, which leads to a deficient activity of α-galactosidase A. α-galactosidase A activity can be assayed on dried blood spots on filter paper but the original method has been associated with a number of false positive due in great part to quenching of fluorescence. Here, we describe an adaptation of the original fluorimetric method reducing quenching of the fluorescence.

A simple and sensitive fluorimetric method has been described for the determination of the α-galactosidase A activity in dried blood spots on filter paper, convenient for screening of FD in at-risk populations. The procedure uses 4-methylumbelliferyl-α-D-galactose, as a synthetic substrate for the enzyme. In this study, protein precipitation was added after incubation both to stop the enzymatic reaction and eliminate interfering proteins. With the novel method the risk of false-positive due to fluorescence quenching was minimized. A cut-off level of 2.1 μmol.h(-1).L(-1) (control values: 5.6 ± 2.0 μmol.h(-1).L(-1), mean ± SD) was chosen corresponding to 40 % of median control value. In all 60 hemizygotes males, α-gal A activities were below 1.1 μmol.h(-1).L(-1) (0.11 ± 0.2 μmol.h(-1).L(-1)). In the 68 heterozygous females, α-gal A activity ranged from 0 to 7.8 μmol.h(-1).L(-1) (2.2 ± 1.7 μmol.h(-1).L(-1)). Using the improved methodology, one third of the females were not identified using the enzymatic assay, due to significant residual enzyme activity.

This improved method for the assay of α-gal A was robust and reduced the number of false-positive results. It can be applied for the screening of at-risk populations. α-galactosidase A enzymatic assay should not be used for screening for FD in women or, if used, should be interpreted cautiously together with the results of genotyping.