Abstract Body: Introduction: Fabry disease is an inborn error of metabolism caused by mutations in the alpha-galactosidase gene (GLA) located on the X chromosome, resulting in intracellular accumulation of globotriaosylceramide and systemic tissue injury. In addition to enzyme replacement therapy, chaperone therapy is effective for many missense mutations. One female patient had c.547G>A mutation, which has been reported as p.G183S, and we checked the indication for migalastat, a pharmacological chaperone. However, migalastat reactivity testing using HEK293 cells transfected with mutant cDNA had not been performed for this mutation.
Aims: We analyzed the c.547G>A mutation and examined the efficacy of migalastat for this mutation.
Methods: Peripheral blood mononuclear cells from patients were transfected with reprogramming factors using episomal vectors to generate induced pluripotent stem (iPS) cells. Cardiomyocytes were induced from the iPS cells. The mutation site was located at the 3' end of exon 3, and splicing mutation was suspected. Thus, PCR amplification was performed between exon 2 and exon 5 of the cDNA, and the PCR product was sequenced to confirm the mRNA sequence generated in the patient's cells. Real-time PCR was performed using Taqman probe to amplify sites unaffected by the mutation, and expression levels of normal and mutant GLA were compared. iPS cells were treated with migalastat and the effect on α-galactosidase activity was examined.
Results: Since the patient was female, five iPS cell lines expressing only normal GLA and four iPS cell lines expressing only mutant GLA were established due to X-chromosome inactivation. Mutant cDNA was found to have a deletion of 36 bases, part of exon 3, resulting in a deletion of 12 amino acids. Real-time PCR result showed that the GLA expression level of mutant GLA-expressing cells was about half that of normal GLA-expressing cells, which was comparable to the GLA expression level of the cells from a healthy subject. Mutant GLA-expressing iPS cells had no α-galactosidase activity, which was not increased by treatment with migalastat.
Conclusions: In patient-derived iPS cells and iPS cell-derived cardiomyocytes, c.547G>A produced p.C172_G183del, not p.G183S, and migalastat was ineffective. Further therapeutic development is needed for patients with this mutation.