Antisense Oligonucleotide Rescue of Deep-Intronic Variants Activating Pseudoexons in the 6-Pyruvoyl-Tetrahydropterin Synthase Gene

Abstract

We report two new 6-pyruvoyl-tetrahydropterin synthase splicing variants identified through genomic sequencing and transcript analysis in a patient with tetrahydrobiopterin deficiency, presenting with hyperphenylalaninemia and monoamine neurotransmitter deficiency. Variant c.243 + 3A>G causes exon 4 skipping. The deep-intronic c.164-672C>T variant creates a potential 5' splice site that leads to the inclusion of four overlapping pseudoexons, corresponding to exonizations of an antisense short interspersed nuclear element AluSq repeat sequence. Two of the identified pseudoexons have been reported previously, activated by different deep-intronic variants, and were also detected at residual levels in control cells. Interestingly, the predominant pseudoexon is nearly identical to a disease causing activated pseudoexon in the F8 gene, with the same 3' and 5' splice sites. Splice switching antisense oligonucleotides (SSOs) were designed to hybridize with splice sites and/or predicted binding sites for regulatory splice factors. Different SSOs corrected the aberrant pseudoexon inclusion, both in minigenes and in fibroblasts from patients carrying the new variant c.164-672C>T or the previously described c.164-716A>T. With SSO treatment PTPS protein was recovered, illustrating the therapeutic potential of the approach, for patients with different pseudoexon activating variants in the region. In addition, the natural presence of pseudoexons in the wild type context suggests the possibility of applying the antisense strategy in patients with hypomorphic PTS variants with the purpose of upregulating their expression to increase overall protein and activity.

Keywords: antisense oligonucleotides; pseudoexons; splicing; tetrahydrobiopterin.

FIG. 1.

PTS gene splicing profile in patient 1 fibroblasts. (A) RT-PCR analysis in C and patient 1 fibroblasts treated with (+) or without (−) puromycin. Shown on the right are the schematics of the identified bands confirmed by sequencing. (B) Sequence of the intron 2 region where the pseudoexons are located, showing the conserved GT and AG splice sites (in bold) used by the pseudoexons and the location of the novel variant. Shown below is the schematics of the identified pseudoexons along with the score of the splice sites according to HSF. C, control; HSF, Human Splice Finder; RT-PCR, reverse transcription polymerase chain reaction.

FIG. 2.

Minigene analysis of the novel splicing variants identified in patient 1. (A) Scheme of the pCR3.1 minigene used and RT-PCR analysis of WT and mutant (c.164–672C>T) minigenes in Hep3B and SH-SY5Y cells. (B) Scheme of the pSPL3 minigene used and RT-PCR analysis of WT and mutant (c.243 + 3A>G) minigenes in Hep3B and SH-SY5Y cells. The star indicates the location of the variants. WT, wild-type.

FIG. 3.

SSO treatment in minigenes. (A) Sequence of the intron 2 region showing the identified pseudoexons, the conserved GT and AG splice sites used, the location of the c.163–716A>T and c.164–672C>T variants, and the region targeted by the designed SSOs (black boxes). Binding sites for SRSF1 predicted by ESE finder are shown in light grey boxes along with their scores. (B, C) RT-PCR analysis in WT and mutant minigenes untransfected (−) or transfected with 50 nM of each SSO. SCR, scrambled SSO; SSO, splice switching antisense oligonucleotide.

FIG. 4.

SSO treatment in patients' fibroblasts. RT-PCR analysis, using primers to amplify a fragment from exon 1 to exon 3, in control and patient 1 (A) or patient 2 (B) fibroblasts, transfected with 50 nM of each SSO. Puromycin was used as NMD inhibitor. (C, D) show the graphical representation of the quantification of each band corresponding to the transcripts without pseudoexons (non-PE) and to the insertion of each pseudoexon, expressed as percentage of reads obtained after deep sequencing the region in puromycin treated samples. NMD, nonsense mediated mRNA decay.

FIG. 5.

PTPS protein analysis in patients' fibroblasts. Western blot analysis of the PTPS protein in untransfected (−) or transfected fibroblasts from patients 1 and 2, untreated (A) or treated (B) with puromycin. GAPDH was used as loading control. C, control fibroblasts; PTPS, 6-pyruvoyltetrahydropterin synthase.