RESUMO
Antisense oligonucleotide (AON)-based splice modulation is the most widely used therapeutic approach to redirect precursor messenger RNA (pre-mRNA) splicing. To study the functional effect of human mutations affecting pre-mRNA splicing for which AON-based splice redirection would be a potential therapeutic option, humanized knock-in animal models are pivotal. A major limitation of using humanized animal models for this purpose is the reported poor recognition of human splice sites by the splicing machineries of other species. To overcome this problem, we provide a detailed guideline for the generation of functional humanized knock-in zebrafish models to assess the effect of mutation-induced aberrant splicing and subsequent AON-based splice modulation therapy .
Assuntos
Splicing de RNA , Peixe-Zebra , Animais , Humanos , Mutação , Oligonucleotídeos Antissenso/farmacologia , Precursores de RNA/genética , Peixe-Zebra/genética , Peixe-Zebra/metabolismoRESUMO
A substantial proportion of subjects with autosomal recessive retinitis pigmentosa (arRP) or Usher syndrome type II (USH2) lacks a genetic diagnosis due to incomplete USH2A screening in the early days of genetic testing. These cases lack eligibility for optimal genetic counseling and future therapy. USH2A defects are the most frequent cause of USH2 and are also causative in individuals with arRP. Therefore, USH2A is an important target for genetic screening. The aim of this study was to assess unscreened or incompletely screened and unexplained USH2 and arRP cases for (likely) pathogenic USH2A variants. Molecular inversion probe (MIP)-based sequencing was performed for the USH2A exons and their flanking regions, as well as published deep-intronic variants. This was done to identify single nucleotide variants (SNVs) and copy number variants (CNVs) in 29 unscreened or partially pre-screened USH2 and 11 partially pre-screened arRP subjects. In 29 out of these 40 cases, two (likely) pathogenic variants were successfully identified. Four of the identified SNVs and one CNV were novel. One previously identified synonymous variant was demonstrated to affect pre-mRNA splicing. In conclusion, genetic diagnoses were obtained for a majority of cases, which confirms that MIP-based sequencing is an effective screening tool for USH2A. Seven unexplained cases were selected for future analysis with whole genome sequencing.