Functional analysis in <i>Drosophila</i> indicates that the NBCCS/PTCH1 mutation G509V results in activation of smoothened through a dominant‐negative mechanism

Hime, Gary R.; Lada, Hania; Fietz, Michael; Gillies, Susan; Passmore, Abraham; Wicking, Carol; Wainwright, Brandon

doi:10.1002/dvdy.10499

Cited by 24 publications

(20 citation statements)

References 66 publications

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“…We also evaluated p.Leu360Arg, reported previously as a loss-of-function variant that is unable to complement PTCH1 function in murine Ptch1 −/− fibroblast cells (Bailey et al 2003). Considering the possibility that the PTCH1 variants in our ODA cohort could function as dominant negative alleles, we tested p.Gly509Val, a highly conserved change in the sterol-sensing domain that confers a dominant negative effect in Drosophila (Hime et al 2004). As negative controls for the assay, we evaluated a nonsynonymous change found commonly in population controls, p.Pro1315Leu, and a second C-terminal change, p. Pro1125Leu, shown previously to restore SHH signaling readout in vitro (Bailey et al 2003).…”

Section: Resultsmentioning

confidence: 99%

Targeted resequencing identifies PTCH1 as a major contributor to ocular developmental anomalies and extends the SOX2 regulatory network

et al. 2016

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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 highthroughput 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.

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Section: Resultsmentioning

confidence: 99%

Targeted resequencing identifies PTCH1 as a major contributor to ocular developmental anomalies and extends the SOX2 regulatory network

et al. 2016

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“…However, haploinsufficiency is not the only possible mechanism for a onehit tumorigenesis model. In vivo studies have shown that several mutant PTCH1 proteins (Ptc1130X, PtcG509V, and PtcD584N) could result in the activation of Hedgehog signaling through a dominant-negative mechanism despite the production of wild-type PTCH1 (23,32,33). PTCH1 may normally form a multimer in its active state, and the mutant PTCH1 might interact with wild-type PTCH1 to block its function or proper localization within the cells.…”

Section: Resultsmentioning

confidence: 99%

“…Analysis of tumors taken from the PTCH1 +/− mice revealed a retained wild-type allele of the gene and the absence of a second hit in these animals, indicating that haploinsufficiency of PTCH1 may be sufficient for tumor development (21,22). In addition, dominant-negative isoforms of PTCH1 mutations, which caused only one single null allele, have been identified to contribute to tumorigenesis, probably due to improper multimer protein or stabilization of SMO, another transmembrane protein interacting with PTCH1 in the Hedgehog signaling pathway (23). This is in stark contrast to Knudson's two-hit model, which states that both alleles of the tumor suppressor gene must be inactivated.…”

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confidence: 99%

Mechanisms of Inactivation of PTCH1 Gene in Nevoid Basal Cell Carcinoma Syndrome: Modification of the Two-Hit Hypothesis

Pan

Dong

Sun

et al. 2010

Clinical Cancer Research

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Purpose: PTCH1 has been identified as the gene responsible for nevoid basal cell carcinoma syndrome (NBCCS). Keratocystic odontogenic tumors (KCOT) are aggressive jaw lesions that may occur in isolation or in association with NBCCS. The aim of this study was to investigate the genetic and/or epigenetic mechanisms of inactivation of the PTCH1 gene in patients with NBCCS and related sporadic KCOTs.Experimental Design: Loss of heterozygosity was analyzed in 44 patients (15 NBCCS-related and 29 sporadic KCOTs), all of whom were previously analyzed for PTCH1 mutations. Allelic location was established in tumors carrying two coincident mutations. PTCH1 mRNA expression and promoter methylation status were analyzed in a panel of KCOTs to define the possible role of epigenetic effects on PTCH1 inactivation.Results: Although mutations and loss of heterozygosity of PTCH1 were frequently detected in both syndromic and nonsyndromic cases, hypermethylation of the PTCH1 promoter was not identified in the present series. Of all the 44 cases examined, 13 were identified to fit the two-hit model, 14 to conform to a one-hit model, and the remaining 17 cases showing no alteration in PTCH1.

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“…However, this effect was not observed with another mutation in the same codon G509R, indicating that removal of the glycine residue alone is insufficient to produce dominant-negative function. 22 The apparent differences between the effect of the G509R mutation in fly and vertebrate systems may truly reflect a fundamental difference in PTC signaling between Drosophila and vertebrates, or may be an artifact of the experimental methods used. In either event, both studies support the pathogenicity of the G509V mutation and indicate a key role for this codon in PTCH functioning.…”

Section: Discussionmentioning

confidence: 99%

Clinical testing for the nevoid basal cell carcinoma syndrome in a DNA diagnostic laboratory

Klein

Dykas

Bale

2005

Genetics in Medicine

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Purpose: This study determines which clinical features predict positive test results among samples submitted for DNA-based diagnostic nevoid basal cell carcinoma syndrome (NBCCS) testing, and further defines the mutational spectrum of the PTCH gene. Methods: DNA was extracted from peripheral blood leukocytes, and polymerase chain reaction products from exons 1 to 23 of the PTCH gene were directly sequenced. Pedigree phenotypic information was obtained by written questionnaire. Results: Among 106 presumably unrelated pedigrees, 44 independent mutations were found in 47 families. There were 11 nonsense mutations; 1 in-frame deletion; 17 deletions, 6insertions, and 1 deletion-insertion that generated frameshifts; 5 splice-site mutations; 1 in-frame duplication; and 2 presumptive missense mutations. Twenty-seven of 46 pedigrees (58.7%) with two or more typical radiographic or pathologic features of NBCCS tested positive for PTCH mutations. Of these, 26 had jaw cysts in combination with other characteristics or neoplasms including basal cell carcinomas, palmar pits, skeletal abnormalities, ocular abnormalities, medulloblastomas, cardiac or ovarian fibromas, calcification of the falx cerebri, polydactyly, cleft lip and/or palate, and agenesis of the corpus callosum or other central nervous system malformations. None of the

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Functional analysis in Drosophila indicates that the NBCCS/PTCH1 mutation G509V results in activation of smoothened through a dominant‐negative mechanism

Cited by 24 publications

References 66 publications

Targeted resequencing identifies PTCH1 as a major contributor to ocular developmental anomalies and extends the SOX2 regulatory network

Targeted resequencing identifies PTCH1 as a major contributor to ocular developmental anomalies and extends the SOX2 regulatory network

Mechanisms of Inactivation of PTCH1 Gene in Nevoid Basal Cell Carcinoma Syndrome: Modification of the Two-Hit Hypothesis

Clinical testing for the nevoid basal cell carcinoma syndrome in a DNA diagnostic laboratory

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