title: Targeted resequencing identifies PTCH1 as a major contributor to ocular developmental anomalies and extends the SOX2 regulatory network.
abstract: Ocular developmental anomalies (ODA) such as anophthalmia/microphthalmia (AM) or anterior segment dysgenesis (ASD) have an estimated combined prevalence of 3.7 in 10,000 births. Mutations in SOX2 are the most frequent contributors to severe ODA, yet account for a minority of the genetic drivers. To identify novel ODA loci, we conducted targeted high-throughput sequencing of 407 candidate genes in an initial cohort of 22 sporadic ODA patients. Patched 1 (PTCH1), an inhibitor of sonic hedgehog (SHH) signaling, harbored an enrichment of rare heterozygous variants in comparison to either controls, or to the other candidate genes (four missense and one frameshift); targeted resequencing of PTCH1 in a second cohort of 48 ODA patients identified two additional rare nonsynonymous changes. Using multiple transient models and a CRISPR/Cas9-generated mutant, we show physiologically relevant phenotypes altering SHH signaling and eye development upon abrogation of ptch1 in zebrafish for which in vivo complementation assays using these models showed that all six patient missense mutations affect SHH signaling. Finally, through transcriptomic and ChIP analyses, we show that SOX2 binds to an intronic domain of the PTCH1 locus to regulate PTCH1 expression, findings that were validated both in vitro and in vivo. Together, these results demonstrate that PTCH1 mutations contribute to as much as 10% of ODA, identify the SHH signaling pathway as a novel effector of SOX2 activity during human ocular development, and indicate that ODA is likely the result of overactive SHH signaling in humans harboring mutations in either PTCH1 or SOX2.
authors.affiliation: CHU Toulouse, Service de Génétique Médicale, Hôpital Purpan, 31059 Toulouse, France; Université Paul-Sabatier Toulouse III, EA-4555, 31000 Toulouse, France; Inserm U1056, 31000 Toulouse, France;
authors.forename: Nicolas
authors.initials: N
authors.lastname: Chassaing
authors.affiliation: Center for Human Disease Modeling, Duke University Medical Center, Durham, North Carolina 27701, USA; Department of Pediatrics and Department of Cell Biology, Duke University Medical Center, Durham, North Carolina 27701, USA;
authors.forename: Erica E
authors.initials: EE
authors.lastname: Davis
authors.affiliation: Center for Human Disease Modeling, Duke University Medical Center, Durham, North Carolina 27701, USA;
authors.forename: Kelly L
authors.initials: KL
authors.lastname: McKnight
authors.affiliation: Center for Human Disease Modeling, Duke University Medical Center, Durham, North Carolina 27701, USA;
authors.forename: Adrienne R
authors.initials: AR
authors.lastname: Niederriter
authors.affiliation: Université Paul-Sabatier Toulouse III, EA-4555, 31000 Toulouse, France; CHU Toulouse, Service d'Ophtalmologie, Hôpital Purpan, 31059 Toulouse, France;
authors.forename: Alexandre
authors.initials: A
authors.lastname: Causse
authors.affiliation: Institut de Génétique et Développement, CNRS UMR6290, Université de Rennes 1, IFR140 GFAS, Faculté de Médecine, 35043 Rennes, France; Laboratoire de Génétique Moléculaire, CHU Pontchaillou, 35043 Rennes Cedex, France;
authors.forename: Véronique
authors.initials: V
authors.lastname: David
authors.affiliation: Université Paul-Sabatier Toulouse III, EA-4555, 31000 Toulouse, France;
authors.forename: Annaïck
authors.initials: A
authors.lastname: Desmaison
authors.affiliation: Université de Toulouse; INSA, UPS, INP, LISBP, F-31077 Toulouse, France; INRA, UMR792, Ingénierie des Systèmes Biologiques et des Procédés, F-31400 Toulouse, France; CNRS, UMR5504, F-31400 Toulouse, France; Plateforme Biopuces de la Génopole de Toulouse Midi Pyrénées, INSA/DGBA 135, 31077 Toulouse, France;
authors.forename: Sophie
authors.initials: S
authors.lastname: Lamarre
authors.affiliation: Service de Génétique Médicale, Hôpital Jeanne de Flandre, 59037 Lille, France;
authors.forename: Catherine
authors.initials: C
authors.lastname: Vincent-Delorme
authors.affiliation: Service de Génétique Clinique, Hôpital Sud, 35200 Rennes, France;
authors.forename: Laurent
authors.initials: L
authors.lastname: Pasquier
authors.affiliation: Service de Génétique Médicale, Hôpital Arnaud de Villeneuve, 34295 Montpellier, France;
authors.forename: Christine
authors.initials: C
authors.lastname: Coubes
authors.affiliation: Service de Génétique Médicale, Hôpital Pellegrin, 33076 Bordeaux Cedex, France; Université Bordeaux Segalen, Laboratoire MRGM, 33076 Bordeaux, France;
authors.forename: Didier
authors.initials: D
authors.lastname: Lacombe
authors.affiliation: Service de Génétique, Hospices Civils de Lyon, Groupement Hospitalier Est, 69677 Bron, France; INSERM U1028 UMR CNRS 5292, UCBL, CRNL TIGER Team, 69677 Bron Cedex, France;
authors.forename: Massimiliano
authors.initials: M
authors.lastname: Rossi
authors.affiliation: Service d'Ophtalmologie, Hôpital Necker Enfants Malades, 75015 Paris, France;
authors.forename: Jean-Louis
authors.initials: JL
authors.lastname: Dufier
authors.affiliation: Service de Génétique Médicale, Hôpitaux Universitaires de Strasbourg, 67091 Strasbourg, France;
authors.forename: Helene
authors.initials: H
authors.lastname: Dollfus
authors.affiliation: INSERM U781 & Department of Genetics, Paris Descartes University, 75015 Paris, France;
authors.forename: Josseline
authors.initials: J
authors.lastname: Kaplan
authors.affiliation: Center for Human Disease Modeling, Duke University Medical Center, Durham, North Carolina 27701, USA; Department of Pediatrics and Department of Cell Biology, Duke University Medical Center, Durham, North Carolina 27701, USA;
authors.forename: Nicholas
authors.initials: N
authors.lastname: Katsanis
authors.affiliation: Université Paul-Sabatier Toulouse III, EA-4555, 31000 Toulouse, France; INSERM, UMR_S910, Aix-Marseille University, Faculté de Médecine, 13385 Marseille, France;
authors.forename: Heather C
authors.initials: HC
authors.lastname: Etchevers
authors.affiliation: INSERM unit 1048, I2MC, Team 12, 31432 Toulouse, France.
authors.forename: Stanislas
authors.initials: S
authors.lastname: Faguer
authors.affiliation: CHU Toulouse, Service de Génétique Médicale, Hôpital Purpan, 31059 Toulouse, France; Université Paul-Sabatier Toulouse III, EA-4555, 31000 Toulouse, France; Inserm U1056, 31000 Toulouse, France;
authors.forename: Patrick
authors.initials: P
authors.lastname: Calvas
mesh_terms: D000483:Alleles; D000818:Animals; D016022:Case-Control Studies; D004195:Disease Models, Animal; D005124:Eye Abnormalities / Q000235:genetics* / Q000378:metabolism*; D005786:Gene Expression Regulation; D053263:Gene Regulatory Networks; D056426:Genetic Loci; D006579:Heterozygote; D006801:Humans; D009154:Mutation; D000072081:Patched-1 Receptor / Q000235:genetics* / Q000378:metabolism; D010641:Phenotype; D055748:SOXB1 Transcription Factors / Q000378:metabolism*; D017422:Sequence Analysis, DNA; D015027:Zebrafish
chemical_list: D000072081:Patched-1 Receptor; D055748:SOXB1 Transcription Factors