Double-target Antisense U1snRNAs Correct Mis-splicing Due to c.639+861C>T and c.639+919G>A GLA Deep Intronic Mutations

Abstract

Fabry disease is a rare X-linked lysosomal storage disorder caused by deficiency of the α-galactosidase A (α-Gal A) enzyme, which is encoded by the GLA gene. GLA transcription in humans produces a major mRNA encoding α-Gal A and a minor mRNA of unknown function, which retains a 57-nucleotide-long cryptic exon between exons 4 and 5, bearing a premature termination codon. NM_000169.2:c.639+861C>T and NM_000169.2:c.639+919G>A GLA deep intronic mutations have been described to cause Fabry disease by inducing overexpression of the alternatively spliced mRNA, along with a dramatic decrease in the major one. Here, we built a wild-type GLA minigene and two minigenes that carry mutations c.639+861C>T and c.639+919G>A. Once transfected into cells, the minigenes recapitulate the molecular patterns observed in patients, at the mRNA, protein, and enzymatic level. We constructed a set of specific double-target U1asRNAs to correct c.639+861C>T and c.639+919G>A GLA mutations. Efficacy of U1asRNAs in inducing the skipping of the cryptic exon was evaluated upon their transient co-transfection with the minigenes in COS-1 cells, by real-time polymerase chain reaction (PCR), western blot analysis, and α-Gal A enzyme assay. We identified a set of U1asRNAs that efficiently restored α-Gal A enzyme activity and the correct splicing pathways in reporter minigenes. We also identified a unique U1asRNA correcting both mutations as efficently as the mutation-specific U1asRNAs. Our study proves that an exon skipping-based approach recovering α-Gal A activity in the c.639+861C>T and c.639+919G>A GLA mutations is active.

Figure 1

Experimental strategy adopted in this work. (a) Schematic representation of GLA splicing reporter minigene. (b) Antisense sequences used in this study. (c) Schematic representation of the exon-skipping strategy for the GLA cryptic exon.

Figure 2

Screening of specific U1asRNA by RT-PCR analysis. Splicing assay and densitometric analysis of semiquantitative RT-PCRs of (a) wild-type, (b) c.639+861C>T, (c) c.639+919G>A minigenes transfected with or without the U1asRNAs. RT-PCR analysis of transcripts from U1-GLA5T, U1-GLA3A, U1-GLAWT (middle panel) and U1-GLAScramble (lower panel) were shown. (d) Schematic representation of both reporter GLA minigene cassette and chimeric U1 snRNAs. Arrows represent primer (not to scale). Data are expressed as mean (± standard deviation) of two independent experiments.

Figure 3

Western blot analysis of α-Gal A protein in transiently transfected COS-1 cells with (a) c.639+861C>T or (b) c.639+919G>A minigenes alone or in combination with specific U1asRNAs. Blots were carried out using 30 μg total protein with the anti-α-Gal A antibody (upper panels) and an anti-cyclophilin A antibody as a loading control (lower panels). The histograms represent the average amount (n = 2) of α-Gal A protein normalised over the amount of cyclophilin A protein, as assessed by densitometric quantification.

Figure 4

α-Gal A enzyme activities (expressed as nmol 4-MU released/mg protein/hour) in lysates that were prepared from transiently transfected COS-1 cells with minigenes: (a) c.639+861C>T, (b) c.936+919G>A and (c) wild-type with and without the supplementation of specific U1asRNAs. Data represent mean (± standard deviation) of three independent experiments (P < 0.05).