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Human Genetics

, Volume 137, Issue 10, pp 831–846 | Cite as

Implication of non-coding PAX6 mutations in aniridia

  • Julie Plaisancié
  • M. Tarilonte
  • P. Ramos
  • C. Jeanton-Scaramouche
  • V. Gaston
  • H. Dollfus
  • D. Aguilera
  • J. Kaplan
  • L. Fares-Taie
  • F. Blanco-Kelly
  • C. Villaverde
  • C. Francannet
  • A. Goldenberg
  • I. Arroyo
  • J. M. Rozet
  • C. Ayuso
  • N. Chassaing
  • P. Calvas
  • M. Corton
Original Investigation

Abstract

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.

Keywords

Aniridia Eye development PAX6 Non-coding mutation Cis-regulatory region 5′UTR Minigene assay 

Notes

Acknowledgements

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).

Ethical approval

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.

Supplementary material

439_2018_1940_MOESM1_ESM.docx (18 kb)
Supplementary material 1 (DOCX 17 KB)
439_2018_1940_MOESM2_ESM.pdf (425 kb)
Supplementary material 2 (PDF 424 KB)
439_2018_1940_MOESM3_ESM.tif (2.4 mb)
Online resource 3. Structure of the hybrid minigene construct PAX6_ex1-4 and splicing outcomes obtained for the different 5’UTR variants studied. A. Structure of the minigene PAX6_ex1-4, derived from the exon trapping pSLP3 vector, indicating the included full exons and intronic regions (dashed line indicate shortened sequences of intron 2), the donor (5’) and acceptor (3’) splicing sites (SS) and the genomic context of the 5 studied variants on 5’ UTR. Vector exon specific primers SD6-pSPL3_F and SA2-pSPL3_R are also showed. B-D. Schematic representation of splicing events and transcripts generated by wild type and mutant minigenes PAX6_ex1-4. Anomalous splicing events are shown in red. B. Canonical splicing events on the wild-type construct. C. Aberrant splicing events observed on the mutant construct for the c.-129+1G>A variant. D. Aberrant splicing event obtained for the PAX6_ex1-4 constructs for 4 different variants in canonical acceptor or donor splicing sites. (TIF 2418 KB)
439_2018_1940_MOESM4_ESM.docx (15 kb)
Supplementary material 4 (DOCX 15 KB)
439_2018_1940_MOESM5_ESM.tif (967 kb)
Online resource 5. Identification of 3’ regulatory deletions downstream of PAX6 in patients with isolated aniridia. Targeted array-based comparative genomic hybridization (aCGH) or NGS-derived depth-read analysis identified deletions involving telomeric deletions to PAX6 in 9 families. All the 3’ PAX6 deletions reported to date are represented. The red colored bars represent the genomic positions of the deletions in our families. The blue colored bars showed the published genomic coordinates for 16 additional cases from the literature. The green bars indicate a cluster of cis-regulatory regions located in intronic positions of ELP4 and PAX6. The SIMO and E180 elements are represented in red. A minimum shared region of 18 Kb (chr11:31648248-31666340) for all the 3’ PAX6 deletions, except one, was also delimited using vertical lines. (TIFF 967 KB)
439_2018_1940_MOESM6_ESM.tif (94 kb)
Online resource 6. Semi-quantitative analysis of transcripts from minigene assays for the variant c.-129+1G>A in exon 2. RT-PCR were performed using vector specific primers (FAM-labelled SD6_pSPL3_F and SA2__pSPL3_R) and analyzed by capillary electrophoresis. Fragment sizes (bp) and relative fluorescent units are indicated on the x- and y-axes, respectively. Capillary electropherograms of the splicing events are showed for the different minigene constructs: A) the empty vector, B) wild type, obtaining the expected canonical transcript (CT), C) the c.-129+1G>A in HEK-293 cell line and D) the c.-129+1G>A in ARP-19 cell line. At least 5 different splicing events were obtained and confirmed by sequencing: i) two aberrant larger transcripts due to different partial retentions of intron 2 (Ins_I2) by the disruption of the 5’SS and the use of different deep intronic cryptic donors; ii) three aberrant shorter isoforms for the exon skipping of exon 2 (∆E2), exon2-3 (∆E2-E3) or exon 2-4 (∆E2-E4). Some low abundant artefactual isoforms for each splicing events were also observed (represented by *) corresponding to an alternative use of a cryptic acceptor site on the pSPL3, located downstream of SA2, leading to the inclusion of additional 114 nucleotides of vector sequence (TIF 93 KB)
439_2018_1940_MOESM7_ESM.tif (96 kb)
Online resource 7. Semi-quantitative analysis of transcripts from minigene assays for four variants in exon 3. RT-PCR was performed using vector specific primers (FAM-labelled SD6_pSPL3_F and SA2__pSPL3_R) and analyzed by capillary electrophoresis. Fragment sizes (bp) and relative fluorescent units are indicated on the x- and y-axes, respectively. Capillary electropherograms of the splicing events are showed for the different minigene constructs transfected in the photoreceptor derived ARPE-19 cell line: A) the empty vector, B) wild type, obtaining the expected canonical transcript (CT), C) the variant c.-128-2del, D) the variant c.-52+1G>A, E) the variant c.-52+5del and F) the variant c.-52+3_-52+6delinsTG, for which the skipping of exon was observed (∆3). Similar pattern was obtained for HEK-293 cell line (data not shown). Some low abundant artefactual isoforms for each canonical or aberrant splicing events were also observed (represented by *) corresponding to an alternative use of a cryptic acceptor site on the pSPL3, located downstream of SA2, leading to the inclusion of additional 114 nucleotides of vector sequence (TIF 96 KB)

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Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  • Julie Plaisancié
    • 1
    • 2
  • M. Tarilonte
    • 3
    • 4
  • P. Ramos
    • 3
    • 4
  • C. Jeanton-Scaramouche
    • 1
  • V. Gaston
    • 1
  • H. Dollfus
    • 5
  • D. Aguilera
    • 3
    • 4
  • J. Kaplan
    • 6
  • L. Fares-Taie
    • 6
  • F. Blanco-Kelly
    • 3
    • 4
  • C. Villaverde
    • 3
    • 4
  • C. Francannet
    • 7
  • A. Goldenberg
    • 8
  • I. Arroyo
    • 4
    • 9
  • J. M. Rozet
    • 6
  • C. Ayuso
    • 3
    • 4
  • N. Chassaing
    • 1
    • 2
  • P. Calvas
    • 1
    • 2
  • M. Corton
    • 3
    • 4
  1. 1.Service de Génétique Médicale, Pavillon Lefebvre, Hôpital PurpanCHU ToulouseToulouse Cedex 9France
  2. 2.INSERM U1056Université Toulouse IIIToulouseFrance
  3. 3.Department of GeneticsInstituto de Investigacion Sanitaria-Fundacion Jimenez Diaz University Hospital-Universidad Autónoma de Madrid (IIS-FJD, UAM)MadridSpain
  4. 4.Center for Biomedical Network Research on Rare Diseases (CIBERER), ISCIIIMadridSpain
  5. 5.Centre de Référence pour les affections rares en génétique ophtalmologique, CARGOFilière SENSGENE, Hôpitaux Universitaires de StrasbourgStrasbourgFrance
  6. 6.Laboratoire de Génétique Ophtalmologique INSERM U1163Institut ImagineParisFrance
  7. 7.Service de Génétique MédicaleCHU EstaingClermont-FerrandFrance
  8. 8.Service de GénétiqueCHU de Rouen, Centre Normand de Génomique Médicale et Médecine PersonnaliséeRouenFrance
  9. 9.Department of GeneticsHospital of CáceresCáceresSpain

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