Assessment of Gene Variant Amenability for Pharmacological Chaperone Therapy with 1-Deoxygalactonojirimycin in Fabry Disease

Abstract

Fabry disease is one of the most common lysosomal storage disorders caused by mutations in the gene encoding lysosomal α-galactosidase A (α-Gal A) and resultant accumulation of glycosphingolipids. The sugar mimetic 1-deoxygalactonojirimycin (DGJ), an orally available pharmacological chaperone, was clinically approved as an alternative to intravenous enzyme replacement therapy. The decision as to whether a patient should be treated with DGJ depends on the genetic variant within the α-galactosidase A encoding gene (GLA). A good laboratory practice (GLP)-validated cell culture-based assay to investigate the biochemical responsiveness of the variants is currently the only source available to obtain pivotal information about susceptibility to treatment. Herein, variants were defined amenable when an absolute increase in enzyme activity of ≥3% of wild type enzyme activity and a relative increase in enzyme activity of ≥1.2-fold was achieved following DGJ treatment. Efficacy testing was carried out for over 1000 identified GLA variants in cell culture. Recent data suggest that about one-third of the variants comply with the amenability criteria. A recent study highlighted the impact of inter-assay variability on DGJ amenability, thereby reducing the power of the assay to predict eligible patients. This prompted us to compare our own α-galactosidase A enzyme activity data in a very similar in-house developed assay with those from the GLP assay. In an essentially retrospective approach, we reviewed 148 GLA gene variants from our former studies for which enzyme data from the GLP study were available and added novel data for 30 variants. We also present data for 18 GLA gene variants for which no data from the GLP assay are currently available. We found that both differences in experimental biochemical data and the criteria for the classification of amenability cause inter-assay discrepancy. We conclude that low baseline activity, borderline biochemical responsiveness, and inter-assay discrepancy are alarm signals for misclassifying a variant that must not be ignored. Furthermore, there is no solid basis for setting a minimum response threshold on which a clinical indication with DGJ can be justified.

Misfolding of Lysosomal α-Galactosidase a in a Fly Model and Its Alleviation by the Pharmacological Chaperone Migalastat

“…The accumulation leads to tissue damage and eventual organ failure. There are more than 1000 known mutations in the GLA gene [ 36 , 50 ]. Most of the mutations disrupt the hydrophobic core of the protein, presumably leading to protein misfolding and degradation in the ER [ 31 , 51 , 52 , 53 ].…”

“…Migalastat is an analogue of α-Gal A substrate that binds to the active site of the enzyme [ 30 , 31 ]. Treatment with migalastat was shown to increase enzyme activity in cell culture and in mice expressing amenable mutations [ 32 , 33 , 34 , 35 , 36 , 37 ].…”

In Vitro and In Vivo Amenability to Migalastat in Fabry Disease

“…Interestingly, when comparing outcomes for amenable mutations (according to the GLP-HEK assay) with data from the in-house assays by Lukas et al. 39 and Oommen et al., 36 the in-house assay by Lukas et al. fits better to the GLP-HEK assay ( Figure 2 ).…”

“… 34 In their recent meta study, the authors adopted the current GLP-HEK assay criteria (absolute increase by ≥3% of wild-type AGAL activity and a ≥1.2-fold increase of baseline enzymatic activity) and identified 89 out of 178 mutations as amenable. 34 , 35 , 39 Although used as a basis for future assays, the setup has some limitations that need to be considered. Since migalastat is an inhibitor, a wash-out step after incubation is very important.…”

“…Only amenable mutations according to the GLP-HEK assay were included and compared to data available from in-house assays (Lukas et al., 39 n = 77 mutations; Oommen et al., 36 n = 54 mutations; Lenders et al., 26 n = 12 mutations). Data were extracted from Benjamin et al., 10 Lukas et al., 39 Oommen et al., 36 and Lenders et al. 26 GraphPad Prism v8.0 software (GraphPad, La Jolla, CA, USA) was used for statistical analysis and visualization.…”