Background Patients with Fabry disease (FD) and amenable mutations can be treated with the chaperone migalastat to restore endogenous α-galactosidase A (AGAL) activity. However, certain amenable mutations do not respond biochemically in vivo as expected. Here, we aimed to establish a patient-specific and mutation-specific cell model to evaluate the amenability to chaperone therapy in FD.
Methods Since current tests to determine amenability are limited to heterologous mutation expression in HEK293T cells with endogenous AGAL activity, we generated CRISPR/Cas9-mediated AGAL-deficient HEK293T cells as a basis for mutant overexpression. Furthermore, primary urinary cells from patients were isolated and immortalised as a patient-specific cell model system to evaluate the amenability to chaperone therapy.
Results Under treatment (>13 months), carriers of p.N215S (n=6) showed a significant reduction of plasma lyso-Gb3 (p<0.05). Lyso-Gb3 levels in carriers of p.L294S increased (p<0.05) and two patients developed severe albuminuria. Both missense mutations were amenable in wild-type HEK293T cells (p<0.05), but presented different responses in CRISPR/Cas9-mediated AGAL knockouts and immortalised urinary cells. Chaperone incubation resulted in increased AGAL activity (p<0.0001) and intracellular globotriaosylceramide (Gb3) reduction (p<0.05) in immortalised p.N215S cells but not in p.L294S and IVS2+1 G>A cells.
Conclusion We conclude that repeated AGAL activity measurements in patients’ white blood cells are mandatory to assess the in vivo amenability to migalastat. Plasma lyso-Gb3 might be an appropriate tool to measure the biochemical response to migalastat. Patients with low AGAL activities and increasing lyso-Gb3 levels despite in vitro amenability might not benefit sufficiently from chaperone treatment.
- primary cells
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ML and FS contributed equally.
Contributors ML and EB designed the experiments and wrote the paper. ML, FS, CN, PJB and MB conducted the experiments. ML, FS and EB acquired data. ML, FS, BS, MB, AZ, HS, S-MB, CA-B and EB analysed and interpreted the data. All authors revised the manuscript critically for important intellectual content.
Funding This work was supported by the fund 'Innovative Medical Research' of the University of Muenster Medical School (LE211702 and LE221704).
Competing interests ML received speaker honoraria, travel funding and research grants from Sanofi, Shire and Amicus Therapeutics. EB received research grants and speaker honoraria from Sanofi, Shire and Amicus Therapeutics. S-MB has received speaker honoraria and research grants from Shire Corporation. CA-B received research grants, speaker honoraria and travel funding from Shire, Sanofi and Biomarin Pharmaceuticals and honoraria and travel funding from Amicus Therapeutics.
Patient consent for publication Not required.
Provenance and peer review Not commissioned; externally peer reviewed.