Assessing Splicing Variants in the PAX6 Gene: A Comprehensive Minigene Approach

Haploinsufficiency of the PAX6 gene causes aniridia, a congenital eye disorder characterised by the absence or malformation of the iris and foveal hypoplasia. Previous studies indicate that pathogenic splice variants account for up to 15% of all disease-causing PAX6 variants. However, this proportion may be significantly underestimated because the pathogenicity of splice variants can only be accurately established through experimental validation. In this study, we developed and validated a system of eight minigene constructions for the functional analysis of splicing variants in the PAX6 gene. This system covers all PAX6 coding exons and allows the analysis of any exon and most intronic variants of PAX6. Our comprehensive approach, employing fragment analysis and deep targeted sequencing, enabled us to accurately characterise 38 previously described PAX6 variants, including challenging cases with multiple splicing events. The application of our system revealed that the number of pathogenic splicing variants might be closer to 30% of all pathogenic PAX6 variants. This finding considerably reshapes our understanding of their significance in the genetic landscape of aniridia.