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PAX6 haploinsufficiency causes aniridia, a congenital eye disorder that involves the iris, and foveal hypoplasia. Comprehensive screening of the PAX6 locus, including the non-coding regions, by next-generation sequencing revealed four deep-intronic variants with potential effects on pre-RNA splicing. Nevertheless, without a functional analysis, their pathogenicity could not be established. We aimed to decipher their impact on the canonical PAX6 splicing using in vitro minigene splicing assays and nanopore-based long-read sequencing. Two multi-exonic PAX6 constructs were generated, and minigene assays were carried out. An aberrant splicing pattern was observed for two variants in intron 6, c.357+136G>A and c.357+334G>A. In both cases, several exonization events, such as pseudoexon inclusions and partial intronic retention, were observed due to the creation or activation of new/cryptic non-canonical splicing sites, including a shared intronic donor site. In contrast, two variants identified in intron 11, c.1032+170A>T and c.1033-275A>C, seemed not to affect splicing processes. We confirmed the high complexity of alternative splicing of PAX6 exon 6, which also involves unreported cryptic intronic sites. Our study highlights the importance of integrating functional studies into diagnostic algorithms to decipher the potential implication of non-coding variants, usually classified as variants of unknown significance, thus allowing variant reclassification to achieve a conclusive genetic diagnosis. [ABSTRACT FROM AUTHOR]

Photodynamic therapy (PDT) constitutes a cancer treatment modality based on the administration of a photosensitizer, which accumulates in tumor cells. The subsequent irradiation of the tumoral area triggers the formation of reactive oxygen species responsible for cancer cell death. One of the compounds approved in clinical practice is methyl-aminolevulinate (MAL), a protoporphyrin IX (PpIX) precursor intermediate of heme synthesis. We have identified the mitotic catastrophe (MC) process after MAL-PDT in HeLa human carcinoma cells. The fluorescence microscopy revealed that PpIX was located mainly at plasma membrane and lysosomes of HeLa cells, although some fluorescence was also detected at endoplasmic reticulum and Golgi apparatus. Cell blockage at metaphase-anaphase transition was observed 24 h after PDT by phase contrast microscopy and flow cytometry. Mitotic apparatus components evaluation by immunofluorescence and Western blot indicated: multipolar spindles and disorganized chromosomes in the equatorial plate accompanied with dispersion of centromeres and alterations in aurora kinase proteins. The mitotic blockage induced by MAL-PDT resembled that induced by two compounds used in chemotherapy, taxol and nocodazole, both targeting microtubules. The alterations in tumoral cells provided evidence of MC induced by MAL-PDT, resolving mainly by apoptosis, directly or through the formation of multinucleate cells.