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Background 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 PAX6 variants. Methods By locus-specific analysis of PAX6 using Sanger and/or targeted next-generation sequencing, we screened a Spanish cohort of 106 patients with PAX6-related diseases. Functional splicing assays were performed by in vitro minigene approaches or directly in RNA from patient-derived lymphocytes cell line, when available. Results 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. Conclusion We describe new spliceogenic mechanisms for PAX6 variants mediated by creating or strengthening five different cryptic donor sites at exon 6. Our work revealed that the activation of cryptic PAX6 splicing sites seems to be a recurrent and underestimated cause of aniridia. Our findings pointed out the importance of functional assessment of apparently silent PAX6 variants to uncover hidden genetic alterations and to improve variant interpretation for genetic counselling in aniridia. [ABSTRACT FROM AUTHOR]

Background: Pulmonary hypoplasia, Diaphragmatic anomalies, Anophthalmia/microphthalmia and Cardiac defects delineate the PDAC syndrome. We aim to identify the cause of PDAC syndrome in patients who do not carry pathogenic variants in RARB and STRA6 , which have been previously associated with this disorder., Methods: We sequenced the exome of patients with unexplained PDAC syndrome and performed functional validation of candidate variants., Results: We identified bi-allelic variants in WNT7B in fetuses with PDAC syndrome from two unrelated families. In one family, the fetus was homozygous for the c.292C>T (p.(Arg98*)) variant whereas the fetuses from the other family were compound heterozygous for the variants c.225C>G (p.(Tyr75*)) and c.562G>A (p.(Gly188Ser)). Finally, a molecular autopsy by proxy in a consanguineous couple that lost two babies due to lung hypoplasia revealed that both parents carry the p.(Arg98*) variant. Using a WNT signalling canonical luciferase assay, we demonstrated that the identified variants are deleterious. In addition, we found that wnt7bb mutant zebrafish display a defect of the swimbladder, an air-filled organ that is a structural homolog of the mammalian lung, suggesting that the function of WNT7B has been conserved during evolution for the development of these structures., Conclusion: Our findings indicate that defective WNT7B function underlies a form of lung hypoplasia that is associated with the PDAC syndrome, and provide evidence for involvement of the WNT-β-catenin pathway in human lung, tracheal, ocular, cardiac, and renal development., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2023. No commercial re-use. See rights and permissions. Published by BMJ.)