Activation of cryptic donor splice sites by non-coding and coding <i>PAX6</i> variants contributes to congenital aniridia.

Abstract

The paired-domain transcription factor paired box gene 6 (PAX6) causes a wide spectrum of ocular developmental anomalies, including congenital aniridia, Peters anomaly and microphthalmia. Here, we aimed to functionally assess the involvement of seven potentially non-canonical splicing variants on missplicing of exon 6, which represents the main hotspot region for loss-of-function <i>PAX6</i> variants. By locus-specific analysis of <i>PAX6</i> using Sanger and/or targeted next-generation sequencing, we screened a Spanish cohort of 106 patients with <i>PAX6</i>-related diseases. Functional splicing assays were performed by in vitro minigene approaches or directly in RNA from patient-derived lymphocytes cell line, when available. Five out seven variants, including three synonymous changes, one small exonic deletion and one non-canonical splice variant, showed anomalous splicing patterns yielding partial exon skipping and/or elongation. We describe new spliceogenic mechanisms for <i>PAX6</i> variants mediated by creating or strengthening five different cryptic donor sites at exon 6. Our work revealed that the activation of cryptic <i>PAX6</i> splicing sites seems to be a recurrent and underestimated cause of aniridia. Our findings pointed out the importance of functional assessment of apparently silent <i>PAX6</i> variants to uncover hidden genetic alterations and to improve variant interpretation for genetic counselling in aniridia.