The prevalent deep intronic c. 639Â +Â 919 GÂ >Â A GLA mutation causes pseudoexon activation and Fabry disease by abolishing the binding of hnRNPA1 and hnRNP A2/B1 to a splicing silencer

- We investigate the mechanism of GLA pseudoexon activation in Fabry disease.
- The prevalent c.639 + 919 G > A mutation disrupts the binding of hnRNP A1/A2 to an ESS.
- The GLA ESS is a general inhibitory motif able to inhibit also other pseudoexons.
- HnRNP F/H are also involved in the regulation as enhancers of the pseudoexon.
- SSOs might be a relevant therapy option as they restore normal GLA splicing.

Fabry disease is an X-linked recessive inborn disorder of the glycosphingolipid metabolism, caused by total or partial deficiency of the lysosomal α-galactosidase A enzyme due to mutations in the GLA gene. The prevalent c.639 + 919 G > A mutation in GLA leads to pathogenic insertion of a 57 bp pseudoexon sequence from intron 4, which is responsible for the cardiac variant phenotype. In this study we investigate the splicing regulatory mechanism leading to GLA pseudoexon activation. Splicing analysis of GLA minigenes revealed that pseudoexon activation is influenced by cell-type. We demonstrate that the wild-type sequence harbors an hnRNP A1 and hnRNP A2/B1-binding exonic splicing silencer (ESS) overlapping the 5′splice site (5′ss) that prevents pseudoexon inclusion. The c.639 + 919 G > A mutation disrupts this ESS allowing U1 snRNP recognition of the 5′ss. We show that the wild-type GLA 5′ss motif with the ESS is also able to inhibit inclusion of an unrelated pseudoexon in the FGB gene, and that also in the FGB context inactivation of the ESS by the c.639 + 919 G > A mutation causes pseudoexon activation, underscoring the universal nature of the ESS.Finally, we demonstrate that splice switching oligonucleotide (SSO) mediated blocking of the pseudoexon 3′ss and 5′ss effectively restores normal GLA splicing. This indicates that SSO based splicing correction may be a therapeutic alternative in the treatment of Fabry disease.