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      Improving molecular diagnosis of aniridia and WAGR syndrome using customized targeted array-based CGH

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          Abstract

          Chromosomal deletions at 11p13 are a frequent cause of congenital Aniridia, a rare pan-ocular genetic disease, and of WAGR syndrome, accounting up to 30% of cases. First-tier genetic testing for newborn with aniridia, to detect 11p13 rearrangements, includes Multiplex Ligation-dependent Probe Amplification (MLPA) and karyotyping. However, neither of these approaches allow obtaining a complete picture of the high complexity of chromosomal deletions and breakpoints in aniridia. Here, we report the development and validation of a customized targeted array-based comparative genomic hybridization, so called WAGR-array, for comprehensive high-resolution analysis of CNV in the WAGR locus. Our approach increased the detection rate in a Spanish cohort of 38 patients with aniridia, WAGR syndrome and other related ocular malformations, allowing to characterize four undiagnosed aniridia cases, and to confirm MLPA findings in four additional patients. For all patients, breakpoints were accurately established and a contiguous deletion syndrome, involving a large number of genes, was identified in three patients. Moreover, we identified novel microdeletions affecting 3' PAX6 regulatory regions in three families with isolated aniridia. This tool represents a good strategy for the genetic diagnosis of aniridia and associated syndromes, allowing for a more accurate CNVs detection, as well as a better delineation of breakpoints. Our results underline the clinical importance of performing exhaustive and accurate analysis of chromosomal rearrangements for patients with aniridia, especially newborns and those without defects in PAX6 after diagnostic screening.

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          Most cited references 38

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          Aniridia.

          Aniridia is a rare congenital disorder in which there is a variable degree of hypoplasia or the absence of iris tissue associated with multiple other ocular changes, some present from birth and some arising progressively over time. Most cases are associated with dominantly inherited mutations or deletions of the PAX6 gene. This article will review the clinical manifestations, the molecular basis including genotype-phenotype correlations, diagnostic approaches and management of aniridia.
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            Long-range downstream enhancers are essential for Pax6 expression

            Pax6 is a developmental control gene with an essential role in development of the eye, brain and pancreas. Pax6, as many other developmental regulators, depends on a substantial number of cis-regulatory elements in addition to its promoters for correct spatiotemporal and quantitative expression. Here we report on our analysis of a set of mice transgenic for a modified yeast artificial chromosome carrying the human PAX6 locus. In this 420 kb YAC a tauGFP-IRES-Neomycin reporter cassette has been inserted into the PAX6 translational start site in exon 4. The YAC has been further engineered to insert LoxP sites flanking a 35 kb long, distant downstream regulatory region (DRR) containing previously described DNaseI hypersensitive sites, to allow direct comparison between the presence or absence of this region in the same genomic context. Five independent transgenic lines were obtained that vary in the extent of downstream PAX6 locus that has integrated. Analysis of transgenic embryos carrying full-length and truncated versions of the YAC indicates the location and putative function of several novel tissue-specific enhancers. Absence of these distal regulatory elements abolishes expression in specific tissues despite the presence of more proximal enhancers with overlapping specificity, strongly suggesting interaction between these control elements. Using plasmid-based reporter transgenic analysis we provide detailed characterization of one of these enhancers in isolation. Furthermore, we show that overexpression of a short PAX6 isoform derived from an internal promoter in a multicopy YAC transgenic line results in a microphthalmia phenotype. Finally, direct comparison of a single-copy line with the floxed DRR before and after Cre-mediated deletion demonstrates unequivocally the essential role of these long-range control elements for PAX6 expression.
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              3' deletions cause aniridia by preventing PAX6 gene expression.

              Aniridia is a panocular human eye malformation caused by heterozygous null mutations within PAX6, a paired-box transcription factor, or cytogenetic deletions of chromosome 11p13 that encompass PAX6. Chromosomal rearrangements also have been described that disrupt 11p13 but spare the PAX6 transcription unit in two families with aniridia. These presumably cause a loss of gene expression, by removing positive cis regulatory elements or juxtaposing negative DNA sequences. We report two submicroscopic de novo deletions of 11p13 that cause aniridia but are located >11 kb from the 3' end of PAX6. The clinical manifestations are indistinguishable from cases with chain-terminating mutations in the coding region. Using human x mouse retinoblastoma somatic cell hybrids, we show that PAX6 is transcribed only from the normal allele but not from the deleted chromosome 11 homolog. Our findings suggest that remote 3' regulatory elements are required for initiation of PAX6 expression.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, CA USA )
                1932-6203
                23 February 2017
                2017
                : 12
                : 2
                Affiliations
                [1 ]Department of Genetics & Genomics, Instituto de Investigación Sanitaria-Fundación Jiménez Díaz University Hospital- Universidad Autónoma de Madrid (IIS-FJD, UAM), Madrid, Spain
                [2 ]Centre for Biomedical Network Research on Rare Diseases (CIBERER), ISCIII, Madrid, Spain
                [3 ]Institute of Medical & Molecular Genetics (INGEMM), Hospital Universitario La Paz, Universidad Autónoma de Madrid, IdiPAZ, Madrid, Spain
                Queen Mary Hospital, HONG KONG
                Author notes

                Competing Interests: The authors have declared that no competing interests exist.

                • Conceptualization: EV JN PL CA.

                • Formal analysis: MP EV.

                • Funding acquisition: PL CA MC.

                • Investigation: FBK MP MC RMA.

                • Methodology: EV.

                • Resources: FBK ILS JN PL CA.

                • Supervision: JN PL CA MC.

                • Validation: CV CVM MJT ILS.

                • Visualization: MC.

                • Writing – original draft: FBK MC.

                • Writing – review & editing: FBK MP EV ILS JN MJT PL CA MC.

                ‡ These authors are joint senior authors on this work.

                Article
                PONE-D-16-39611
                10.1371/journal.pone.0172363
                5322952
                28231309
                © 2017 Blanco-Kelly et al

                This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                Page count
                Figures: 3, Tables: 1, Pages: 13
                Product
                Funding
                Funded by: Cátedra de Patrocinio HU-FJD-UAM "Medicina Genómica"
                Award Recipient :
                Funded by: Instituto de Salud Carlos III/FEDER
                Award ID: CP12/03256
                Award Recipient :
                Funded by: MINECO
                Award ID: SAF2013-46943-R
                Award Recipient :
                Funded by: Fundacion Mutua Madrileña
                Award Recipient :
                Funded by: Instituto de salud Carlos III - RETICS
                Award ID: RD12/0034/0010
                Funded by: Instituto de Salud Carlos III - CIBERER
                Award ID: 06/07/0036 and INTRA/07/704.1
                Award Recipient :
                This work was supported by several grants from the Spanish Centre for Biomedical Network Research on Rare Diseases (CIBERER) (06/07/0036 and INTRA/07/704.1), Instituto de Salud Carlos III (Spanish Ministry of Health)/FEDER (CP12/03256), RETICS (RD09/0076/00101 and RD12/0034/0010), Ministry of Economy and Competitiveness/FEDER (MINECO, SAF2013-46943-R), and the Cátedra de Patrocinio HU-FJD-UAM "Medicina Genómica" and Fundación Mutua Madrileña. MC is sponsored by the Miguel Servet Program (CP12/03256) from Instituto de Salud Carlos III (Spanish Ministry of Health). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Research Article
                Biology and Life Sciences
                Computational Biology
                Comparative Genomics
                Biology and Life Sciences
                Genetics
                Genomics
                Comparative Genomics
                Biology and Life Sciences
                Cell Biology
                Chromosome Biology
                Chromosomes
                Medicine and Health Sciences
                Diagnostic Medicine
                Biology and Life Sciences
                Cell Biology
                Chromosome Biology
                Chromosomal Aberrations
                Deletions
                Biology and Life Sciences
                Genetics
                Genetic Loci
                Biology and Life Sciences
                Genetics
                Human Genetics
                Biology and Life Sciences
                Genetics
                Genomics
                Human Genomics
                Medicine and Health Sciences
                Oncology
                Cancers and Neoplasms
                Blastomas
                Nephroblastoma
                Custom metadata
                Array CGH and structural variant data have been deposited in the ClinVar (accession ID SUB2327789), Database of Genomic Variants archive (accession ID estd232), the Human PAX6 Allelic Variant Database - LOVD ( http://lsdb.hgu.mrc.ac.uk/home.php?select_db=PAX6) and Figshare (DOI. 10.6084/m9.figshare.4629760).

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