Functional Analysis of Complex Structural and Splice-Altering Variants in the Gene Towards the Personalized Antisense-Based Therapy for Mucopolysaccharidosis Type VI Patients.

Mucopolysaccharidosis Type VI (MPS VI) is a lysosomal storage disorder associated with biallelic pathogenic variants in the gene. Herein, we present three patients with biochemical and clinical pictures of MPS VI, for whom routine molecular genetic analysis using Sanger sequencing of failed to identify one or both causative variants. RNA analysis of patients' samples revealed alterations of the wild-type mRNA isoform in all cases, and one case required further analysis using whole genome sequencing. As a result, we identified one complex structural variant, which is a 52-kb insertion of the gene fragment in the Intron 4, derived from nonallelic homologous recombination and leading to premature transcription termination, a recurrent deep intronic variant leading to pseudoexon activation and an intragenic deletion altering the integrity and splicing of the Exon 2. Using a minigene-based cellular model, we demonstrated that the identified pseudoexon can be efficiently blocked by antisense molecules incorporated into modified U7 small nuclear RNAs and circular RNAs. The same approach was used to block the overlapping polymorphic pseudoexon in the gene and increase the amount of wild-type mRNA isoform approximately twofold.