In Vitro and In Vivo Amenability to Migalastat in Fabry Disease

Abstract

Migalastat (1-deoxygalactonojirimycin) is approved for the treatment of Fabry disease (FD) in patients with an amenable mutation. Currently, there are at least 367 amenable and 711 non-amenable mutations known, based on an in vitro good laboratory practice (GLP) assay. Recent studies demonstrated that in vitro amenability of mutations did not necessarily correspond to in vivo amenability of migalastat-treated patients. This discrepancy might be due to (methodological) limitations of the current GLP-HEK assay. Currently, there are several published comparable cell-based amenability assays, with partially different outcomes for the same tested mutation, leading to concerns in FD-treating physicians. The aim of this review is to elucidate the idea of amenability assays from their beginning, starting with patient-specific primary cells to high-throughput assays based on overexpression. Consequently, we compare methods of current assays, highlighting their similarities, as well as their pros and cons. Finally, we provide a literature-based list of α-galactosidase A mutations, tested by different assays to provide a comprehensive overview of amenable mutations as a good basis for the decision-making by treating physicians. Since in vitro amenability does not always correspond with in vivo amenability, the treating clinician has the responsibility to monitor clinical and laboratory features to verify clinical response.

Graphical abstract

Figure 1

Schematic Overview of the Basic Principles of HEK293 Amenability Assays HEK assays can be separated into five steps. Step 1: cloning of GLA mutations using site-directed mutagenesis. Step 2: transfection of seeded HEK293 cells with a mock control (negative control; necessary for AGAL background substraction), wild-type AGAL (positive control; reference activity is set to 100%), and the GLA mutation of interest. Step 3: incubation of cells with or without DGJ. Step 4: cell lysis to release overexpressed AGAL. Step 5: AGAL activities are measured and compared to each other. Depending on the assay protocols, various sub-steps can be included in between, such as wash-outs, determination of transfection efficiencies, among others. AGAL/GLA, α-galactosidase A; DGJ, 1-deoxygalactonojirimycin; WT, wild-type.

Figure 2

Comparison of the GLP-HEK Assay with In-House Assays (A–C) Pearson correlation of α-galactosidase A activities presented from (A) baseline activities without DGJ, (B) total activities after incubation with DGJ, and (C) total activity increase over baseline in % of wild-type (WT) activity after incubation with DGJ. (D) Bland-Altman analysis of the in-house assays compared to the GLP-HEK assay for total activity increase over baseline. The dotted lines represent the 95% confidence intervals. Only amenable mutations according to the GLP-HEK assay were included and compared to data available from in-house assays (Lukas et al., n = 77 mutations; Oommen et al., n = 54 mutations; Lenders et al., n = 12 mutations). Data were extracted from Benjamin et al., Lukas et al., Oommen et al., and Lenders et al. GraphPad Prism v8.0 software (GraphPad, La Jolla, CA, USA) was used for statistical analysis and visualization.

Figure 3

Work-Up to Assess Clinical Amenability in Patients Treated with Migalastat Biochemical response after migalastat initiation should already be measurable after 3 months of treatment by increasing AGAL activities and decreasing plasma lyso-Gb₃ levels. Regular follow-up examinations should include echocardiography of the heart to assess left ventricular hypertrophy (LVH) and left ventricular masses (LVMs), determination of eGFR and proteinuria/albuminuria measures for kidney function, brain MRI to assess cerebrovascular events (CVEs) and white matter lesions (WMLs), a pain questionnaire for FD-specific pain and gastrointestinal symptoms, and α-galactosidase A (AGAL) activity and globotriaosylsphingosine (lyso-Gb₃) measurements in blood.