Enzymatic diagnosis of Fabry disease using a fluorometric assay on dried blood spots: An alternative methodology

Fabry disease (FD, OMIM#301500) is an X-linked lysosomal storage disorder caused by the functional deficiency of α-galactosidase A, a lysosomal enzyme. A method to screen for FD in large populations has been developed using a fluorometric assay of α-galactosidase A activity in dried blood spots (DBS) on filter paper. However, results can be influenced by quenching of fluorescence by haemoglobin which, together with small sample size, may result in a low light emission signal.An alternative, simple and sensitive fluorometric assay was developed for the determination of α-galactosidase A activity in DBS. The assay uses 4-methylumbelliferyl-α-d-galactose as an artificial substrate. To minimize the risk of false-positives, zinc sulfate was used for protein precipitation to stop the enzymatic reaction and eliminate interfering species (hemoglobin). Samples from 209 individuals (60 hemizygotes, 68 heterozygotes, and 81 controls) were tested to establish reference values for the assay.The mean α-galactosidase A activity of the 81 controls was 9.1 ± 3.3 μmol h−1 L−1 (mean ± SD). All 60 hemizygotes affected with FD had AGAL activities below 1.7 μmol h−1 L−1 (0.2 ± 0.3 μmol h−1 L−1). For the 68 heterozygous females, AGAL activity ranged from 0 to 12.6 μmol h−1 L−1 (3.5 ± 2.7 μmol h−1 L−1). Two-thirds of the female patients could be identified using the enzymatic assay and a cut-off level of 40% of the median control value (<3.4 μmol h−1 L−1).Our fluorometric assay using zinc sulfate protein precipitation was shown to have similar sensitivity and robustness while reducing the risk of false positive results due to quenching of 4-MU fluorescence by haemoglobin.