Fabry disease is an X-linked lysosomal storage disorder caused by mutations in the gene encoding alpha-galactosidase A (alpha-Gal A), with consequent accumulation of its major glycosphingolipid substrate, globotriaosylceramide (GL-3). Over 500 Fabry mutations have been reported; approximately 60% are missense. The iminosugar 1-deoxygalactonojirimycin (DGJ, migalastat hydrochloride, AT1001) is a pharmacological chaperone that selectively binds alpha-Gal A, increasing physical stability, lysosomal trafficking, and cellular activity. To identify DGJ-responsive mutant forms of alpha-Gal A, the effect of DGJ incubation on alpha-Gal A levels was assessed in cultured lymphoblasts from males with Fabry disease representing 75 different missense mutations, one insertion, and one splice-site mutation. Baseline alpha-Gal A levels ranged from 0 to 52% of normal. Increases in alpha-Gal A levels (1.5- to 28-fold) after continuous DGJ incubation for 5 days were seen for 49 different missense mutant forms with varying EC(50) values (820 nmol/L to >1 mmol/L). Amino acid substitutions in responsive forms were located throughout both structural domains of the enzyme. Half of the missense mutant forms associated with classic (early-onset) Fabry disease and a majority (90%) associated with later-onset Fabry disease were responsive. In cultured fibroblasts from males with Fabry disease, the responses to DGJ were comparable to those of lymphoblasts with the same mutation. Importantly, elevated GL-3 levels in responsive Fabry fibroblasts were reduced after DGJ incubation, indicating that increased mutant alpha-Gal A levels can reduce accumulated substrate. These data indicate that DGJ merits further evaluation as a treatment for patients with Fabry disease with various missense mutations.

Purpose:Fabry disease is an X-linked lysosomal storage disorder caused by mutations in the α-galactosidase A gene. Migalastat, a pharmacological chaperone, binds to specific mutant forms of α-galactosidase A to restore lysosomal activity.Methods:A pharmacogenetic assay was used to identify the α-galactosidase A mutant forms amenable to migalastat. Six hundred Fabry disease–causing mutations were expressed in HEK-293 (HEK) cells; increases in α-galactosidase A activity were measured by a good laboratory practice (GLP)-validated assay (GLP HEK/Migalastat Amenability Assay). The predictive value of the assay was assessed based on pharmacodynamic responses to migalastat in phase II and III clinical studies.Results:Comparison of the GLP HEK assay results in in vivo white blood cell α-galactosidase A responses to migalastat in male patients showed high sensitivity, specificity, and positive and negative predictive values (≥0.875). GLP HEK assay results were also predictive of decreases in kidney globotriaosylceramide in males and plasma globotriaosylsphingosine in males and females. The clinical study subset of amenable mutations (n = 51) was representative of all 268 amenable mutations identified by the GLP HEK assay.Conclusion:The GLP HEK assay is a clinically validated method of identifying male and female Fabry patients for treatment with migalastat.Genet Med 19 4, 430–438.

Fabry disease is caused by mutations in the gene (GLA) that encodes α-galactosidase A (α-Gal A). The iminosugar AT1001 (GR181413A, migalastat hydrochloride, 1-deoxygalactonojirimycin) is a pharmacological chaperone that selectively binds and stabilizes α-Gal A, increasing total cellular levels and activity for some mutant forms (defined as “responsive”). In this study, we developed a cell-based assay in cultured HEK-293 cells to identify mutant forms of α-Gal A that are responsive to AT1001. Concentration-dependent increases in α-Gal A activity in response to AT1001 were shown for 49 (60%) of 81 mutant forms. The responses of α-Gal A mutant forms were generally consistent with the responses observed in male Fabry patient-derived lymphoblasts. Importantly, the HEK-293 cell responses of 19 α-Gal A mutant forms to a clinically achievable concentration of AT1001 (10 µM) were generally consistent with observed increases in α-Gal A activity in peripheral blood mononuclear cells from male Fabry patients orally administered AT1001 during Phase 2 clinical studies. This indicates that the cell-based responses can identify mutant forms of α-Gal A that are likely to respond to AT1001 in vivo. Thus, the HEK-293 cell-based assay may be a useful aid in the identification of Fabry patients with AT1001-responsive mutant forms. Hum Mutat 32:1–13, 2011. © 2011 Wiley-Liss, Inc.

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