Implication of non-coding PAX6 mutations in aniridia
There is an increasing implication of non-coding regions in pathological processes of genetic origin. This is partly due to the emergence of sophisticated techniques that have transformed research into gene expression by allowing a more global understanding of the genome, both at the genomic, epigenomic and chromatin levels. Here, we implemented the analysis of PAX6, whose coding loss-of-function variants are mainly implied in aniridia, by studying its non-coding regions (untranslated regions, introns and cis-regulatory sequences). In particular, we have taken advantage of the development of high-throughput approaches to screen the upstream and downstream regulatory regions of PAX6 in 47 aniridia patients without identified mutation in the coding sequence. This was made possible through the use of custom targeted resequencing and/or CGH array to analyze the entire PAX6 locus on 11p13. We found candidate variants in 30 of the 47 patients. 9/30 correspond to the well-known described 3′ deletions encompassing SIMO and other enhancer elements. In addition, we identified numerous different variants in various non-coding regions, in particular untranslated regions. Among these latter, most of them demonstrated an in vitro functional effect using a minigene strategy, and 12/21 are thus considered as causative mutations or very likely to explain the phenotypes. This new analysis strategy brings molecular diagnosis to more than 90% of our aniridia patients. This study revealed an outstanding mutation pattern in non-coding PAX6 regions confirming that PAX6 remains the major gene for aniridia.
KeywordsAniridia Eye development PAX6 Non-coding mutation Cis-regulatory region 5′UTR Minigene assay
We acknowledge generous support from the families published in this article. We thank Salvador Marti and Virginia Corrochano from CIBERER Biobank (Valencia, Spain) for their help in the generation of LCLs. This work was supported by Gêniris funding awarded to Dr Julie Plaisancié, Retina France funding awarded to Jean-Michel Rozet and Patrick Calvas, Spanish Institute of Health Carlos III (ISCIII)/European Regional Development Fund (ERDF) (PI17_01164 and CP12/03256), Spanish Ministry of Economy and Competitiveness/ERDF (MINECO, SAF2013-46943-R), Spanish Federation of Rare Diseases (FEDER) and Mutua Madrileña Foundation, funding awarded to Marta Cortón, CIBERER (06/07/0036), the University Chair UAM-IIS-FJD of Genomic Medicine, the Ramon Areces Foundation and Regional Government of Madrid (CAM, B2017/BMD3721), funding awarded to Carmen Ayuso. Marta Cortón is sponsored by the Miguel Servet Program (CP12/03256 and CPII17_00006) from ISCIII and Maria Tarilonte received a Conchita Rabago PhD fellowship.
Compliance with ethical standards
Conflict of interest
The authors declare that they have no competing interests (Note: Nucleotide sequence data reported are available in the ClinVar database under the accession numbers: SCV000803674 to SCV000803681).
All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.
*Human PAX6 Mutation Database: http://lsdb.hgu.mrc.ac.uk/home.php?select_db=PAX6.
*HGMD Professional: http://www.biobase-international.com/product/hgmd.
*Human Slicing Finder: http://www.umd.be/HSF.
*SPARN software: http://tools.genes.toronto.edu.
*The Human PAX6 mutation Database: http://lsdb.hgu.mrc.ac.uk/home.php?select_db=PAX6.
*CIBERER Spanish Variant Server: http://csvs.babelomics.org.
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