Gorlin-Like Phenotype in a Young Girl With a De Novo PTCH2 Variant Mutation of Uncertain Significance.

ABSTRACT: Gorlin syndrome, also known as basal cell nevus syndrome, is an autosomal dominant genetic disorder that predisposes humans to tumors. In most cases, this syndrome results from inactivating mutations in the patched homologue 1 gene. Basal cell carcinomas are one of the main characteristics of this syndrome and serve as a major diagnostic criterion. Gorlin syndrome shows a variable phenotype, and recently, other less common mutations in the suppressor of fused homologue or patched homologue 2 genes have been documented in individuals with this syndrome. We present the case of a patient with early-onset basal cell carcinomas and a mild Gorlin syndrome phenotype, attributed to a de novo patched homologue 2 variant of uncertain significance, which has not been previously reported in the literature.

Reduced PTCH2 expression is associated with glioma development through its regulation of the PTEN/AKT signaling pathway.

Mutations in the human protein patched homolog (PTCH) gene have been demonstrated to be associated with cancer development in several types of malignancy. However, the underlying mechanism of PTCH-associated cancer development remains poorly understood, to the best of our knowledge. In the present study, the expression of PTCH2 in glioma tumor tissues from The Cancer Genome Atlas (TCGA) database and clinical patients with glioma were measured. Reduced expression levels of PTCH2 were observed in patients with glioma with poor prognose. In vitro, overexpression of PTCH2 significantly suppressed the proliferation and invasion of the glioma cell lines, LN229 and U87-MG. Mechanistically, PTCH2 upregulated the expression of tumor suppressor PTEN, thereby leading to the suppression of pro-survival AKT signals in glioma. Reduced expression of PTEN and enhanced expression of AKT promoted glioma development in vitro and in vivo. Blockade of PTCH2/AKT signals efficiently strengthened the anticancer effects of chemotherapy and prolonged the survival time in tumor-bearing mice, which provided a novel insight into potential treatment strategies for glioma in the clinic.

Inherited rare and common variants in PTCH1 and PTCH2 contributing to the predisposition to reproductive cancers.

PTCH1 and PTCH2 are associated with nevoid basal cell carcinoma syndrome and basal cell carcinoma. We determined the prevalence of their common and rare variants in 877 patients with various reproductive cancers and 296 healthy subjects. Using targeted next-generation sequencing, we found significantly statistical associations of the minor alleles at seven common variants of PTCH1 and PTCH2 with a decreased risk of reproductive cancers (P = 9.69 × 10-12). Among these variants, two haplotype blocks in high linkage disequilibrium were consisted of rs2277184, rs2066829 and rs2236405 sites at PTCH1 and rs3795720, rs11573590 and rs11211040 sites at PTCH2. Single marker and haplotype-based analysis consistently revealed a decreased risk of reproductive cancers especially breast and prostate cancers in the subjects carrying the minor alleles, and on the contrary, an increased risk for major alleles. Healthy control subjects showed a higher rate of rare variants than that of cancer patients (P = 0.017). Notably, two frameshift variants (p.Ser391* and p.Cys101Alafs*48) of PTCH2 with deleterious effects were found in only four cancer patients. Higher frequencies of variants of PTCH genes might have a protective role against the development of reproductive cancers, whereas rare deleterious variants of PTCH2 might predispose a carrier to reproductive cancers.

PTCH2 is not a strong candidate gene for gorlin syndrome predisposition.

A number of case/family reports have proposed PTCH2 as a putative Gorlin Syndrome (GS) gene, but evidence to support this is lacking. We assessed our cohort of 21 PTCH1/SUFU negative GS families for PTCH2 variants and assessed current evidence from reported cases/families and population data. In our PTCH1/SUFU variant negative GS cohort (25% of total), no pathogenic or likely pathogenic PTCH2 variants were identified. In addition, none of the previously published PTCH2 variants in GS families/cases could be considered pathogenic or likely pathogenic using current guidelines. The absence of clear pathogenic variants in GS families and the high frequency of Loss-of-function (LoF) variants in the general population, including the presence of homozygous LoF variants without a clinical phenotype, mean that it is untenable that PTCH2 is a GS gene. PTCH2 should not be included in panels for genetic diagnosis of GS.

Aberrant activation of the Hedgehog signaling pathway in granulosa cells from patients with polycystic ovary syndrome.

The molecular mechanism that triggers polycystic ovary syndrome (PCOS) is mysterious. Abnormal development of ovarian granulosa cells (GCs) is one of the causes of PCOS. Herein, our study was carried out using RNA-seq to detect the different gene expression levels in ovarian GCs between three patients with PCOS and four normal controls. To verify the RNA-seq data, GCs from 22 patients with PCOS and 21 controls with normal ovulation were collected to perform the RT-PCR analysis. Hedgehog signaling pathway (Hh) members, Ihh and Ptch2 were abnormally highly expressed in the PCOS tissue (PT). The qPCR also indicated that the expression levels of Hh signaling pathway downstream members, Ptch1, Gli1, and Gli2 in the PT were significantly higher than those in the normal tissue (NT). Besides, the expression of TNF-α mRNA in PCOS patients was higher than that in the control group. Through the chromatin immunoprecipitation assay (ChIP), we found that the Gli1-IP-DNA enriched from the granular cells of PCOS patients was higher than that of the control group. Finally, the Hh signaling pathway inhibitor, cyclopamine, can decrease the apoptosis of PCOS ovarian granulosa cells. These results suggest that abnormal activation of Hh signaling pathway, especially Ihh signal, may have a profound influence on PCOS.

Basal cell nevus syndrome: From DNA to therapeutics.

Basal cell nevus syndrome, also known as Gorlin syndrome, is a hereditary cancer syndrome associated with multiple basal cell carcinomas, congenital defects, and nondermatologic tumors. This disease is autosomal dominant with variable expressivity and is caused by abnormalities in the sonic hedgehog signaling pathway. Management requires a multidisciplinary approach and should include the biopsychosocial needs of patients and their families. Genetic testing is necessary to confirm an unclear diagnosis, evaluate at-risk relatives, and assist with family planning.

Sustained hedgehog signaling in medulloblastoma tumoroids is attributed to stromal astrocytes and astrocyte-derived extracellular matrix.

Aberrant activation of the hedgehog (Hh) signaling pathway is associated with the formation of medulloblastoma (MB), the most common malignant pediatric brain tumor. However, tumor cells from human and mouse MB can not be passaged or preserved after being adherently cultured. Moreover, Hh signaling in MB cells is inactivated in such culture. Here we demonstrate that MB cells are capable of forming tumoroids (tumor spheroids) in vitro under optimized conditions, which can be further passaged and cryopreserved. More importantly, MB cells maintain Hh pathway activation and cell proliferation in tumoroids. Our studies further reveal that tumoroids-forming capacity of MB cells relies on astrocytes, a major component of the MB microenvironment. Astrocytes facilitate the formation of MB tumoroids by secreting sonic hedgehog (Shh) and generating astrocyte-derived extracellular matrix. These findings demonstrate the critical role of stromal astrocytes in supporting the survival and proliferation of MB cells in vitro. This study establishes a valid model for long-term culture of primary MB cells, which could be greatly beneficial for future investigation of MB tumorigenicity and the development of improved approaches to treat MB.

Gorlin-like phenotype in a patient with a PTCH2 variant of uncertain significance.

Gorlin syndrome, also known as Nevoid Basal-Cell Carcinoma Syndrome (NBCCS), is an autosomal dominant tumor predisposition syndrome that presents early in life with characteristic congenital malformations and tumors. This syndrome most commonly results from germline mutations of the PTCH1 tumor suppressor gene, which shows high penetrance and great intra and interfamilial phenotypic variability, as well as the SUFU tumor suppressor gene. Recently, the PTCH2 gene has also been implicated as a cause of Gorlin syndrome. Notably, these patients displayed milder phenotypes of Gorlin syndrome when considered against PTCH1 and SUFU-related disease. We report a patient with a novel PTCH2 mutation inherited from his father. The proband displays several minor diagnostic features of Gorlin syndrome, supporting the pathogenic role of this gene. Features in the proband include macrocephaly, a wide face, prominent forehead, hypertelorism/telecanthus, large eyes, cleft lip and palate, thin vertical palmar creases, penoscrotal inversion, and a hyperpigmented spot on his penis. His father displays macrocephaly, several nevi on his back and shoulders, and a single palmar pit on his left hand, raising suspicion for Gorlin syndrome. Whole exome sequence (trio) found that the proband and father are heterozygous for NM_003738.4:c.3347C>T;p.(Pro1116Leu) in exon 21 of PTCH2, found also in his mildly affected brother. This semi-conservative amino acid substitution has been reported in the literature, but its significance is unclear. Notably, the proband, brother, and father do not meet clinical criteria for Gorlin syndrome. However, the clinical findings described in this family support the association between PTCH2 mutations and Gorlin-like phenotypes.

Expression profile of sonic hedgehog signaling-related molecules in basal cell carcinoma.

Basal cell carcinoma (BCC) is the most common human cancer, characterized by aberrant activation of the hedgehog (HH) signaling pathway resulting from mutations in the patched 1 (PTCH1) or smoothened (SMO) genes. In the present study, to uncover the expression profile of HH signaling-related molecules, we thoroughly examined the mRNA and protein expression levels of six molecules including GLI1, GLI2, PTCH1, PTCH2, SHH, and SMO in BCC and various other cutaneous tumors. Real-time PCR analysis demonstrated that BCC showed remarkably enhanced mRNA expression of all HH molecules, except SMO compared to other skin tumors. However, immunohistochemical analysis revealed that only GLI1 protein was specifically upregulated in BCC, while the other HH-related proteins did not show any significant differences between the tumors. Notably, other skin malignancies such as squamous cell carcinoma, sebaceous carcinoma, and malignant melanoma showed no GLI1 expression and there was no difference in GLI1 expression between the BCC subtypes. In addition, GLI1 and GLI2 expression were strongly associated with the hair follicle stem cell markers, LGR4 and LGR5, which are known target genes of the Wnt pathway. Our results suggest that GLI1 has the potential to be a diagnostically useful marker for differentiating BCC from other skin malignancies and an interaction between the HH and Wnt signaling pathways may be involved in the development of BCCs.

PDS5B regulates cell proliferation and motility via upregulation of Ptch2 in pancreatic cancer cells.

Pds5b (precocious dissociation of sisters 5B) is involved in both tumorigenesis and cancer progression; however, the functions and molecular mechanisms of Pds5b in pancreatic cancer (PC) are unknown. Several approaches were conducted to investigate the molecular basis of Pds5b-related PC progression, including transfection, MTT, FACS, western blotting, wound healing assay, transwell chamber invasion assay, and immunohistochemical methods. Pds5b overexpression inhibited cell growth and induced apoptosis, whereas the inhibition of Pds5b promoted growth of PC cells. Moreover, Pds5b overexpression inhibited cell migration and invasion, while the downregulation of Pds5b enhanced cell motility. Furthermore, reduced Pds5b expression was associated with survival in PC patients. Mechanistically, Pds5b positively regulated the expression of Ptch2 to influence the Sonic hedgehog signaling pathway. Consistently, Ptch2 downregulation enhanced cell growth, migration, and invasion, while inhibiting cell apoptosis. Notably, the downregulation of Ptch2 abolished Pds5b-mediated anti-tumor activity in PC cells. Strikingly, Pds5b expression was positively associated with levels of Ptch2 in PC patient samples, suggesting that the Pds5b/Ptch2 axis regulates cell proliferation and invasion in PC cells. Our findings indicate that targeting Pds5b and Ptch2 may represent a novel therapeutic approach for PC.

Functionally Distinctive Ptch Receptors Establish Multimodal Hedgehog Signaling in the Tooth Epithelial Stem Cell Niche.

Continuous growth of the mouse incisor teeth is due to the life-long maintenance of epithelial stem cells (SCs) in their niche called cervical loop (CL). Several signaling factors regulate SC maintenance and/or their differentiation to achieve organ homeostasis. Previous studies indicated that Hedgehog signaling is crucial for both the maintenance of the SCs in the niche, as well as for their differentiation. How Hedgehog signaling regulates these two opposing cellular behaviors within the confinement of the CL remains elusive. In this study, we used in vitro organ and cell cultures to pharmacologically attenuate Hedgehog signaling. We analyzed expression of various genes expressed in the SC niche to determine the effect of altered Hedgehog signaling on the cellular hierarchy within the niche. These genes include markers of SCs (Sox2 and Lgr5) and transit-amplifying cells (P-cadherin, Sonic Hedgehog, and Yap). Our results show that Hedgehog signaling is a critical survival factor for SCs in the niche, and that the architecture and the diversity of the SC niche are regulated by multiple Hedgehog ligands. We demonstrated the presence of an additional Hedgehog ligand, nerve-derived Desert Hedgehog, secreted in the proximity of the CL. In addition, we provide evidence that Hedgehog receptors Ptch1 and Ptch2 elicit independent responses, which enable multimodal Hedgehog signaling to simultaneously regulate SC maintenance and differentiation. Our study indicates that the cellular hierarchy in the continuously growing incisor is a result of complex interplay of two Hedgehog ligands with functionally distinct Ptch receptors. Stem Cells 2019;37:1238-1248.

Hotspot DAXX, PTCH2 and CYFIP2 mutations in pancreatic neuroendocrine neoplasms.

Mutations in DAXX/ATRX, MEN1 and genes involved in the phosphoinositide-3-kinase/Akt/mammalian target of rapamycin (PI3K/Akt/mTOR) pathway have been implicated in pancreatic neuroendocrine neoplasms (pNENs). However, mainly mutations present in the majority of tumor cells have been identified, while proliferation-driving mutations could be present only in small fractions of the tumor. This study aims to identify high- and low-abundance mutations in pNENs using ultra-deep targeted resequencing. Formalin-fixed paraffin-embedded matched tumor-normal tissue of 38 well-differentiated pNENs was sequenced using a HaloPlex targeted resequencing panel. Novel amplicon-based algorithms were used to identify both single nucleotide variants (SNVs) and insertion-deletions (indels) present in >10% of reads (high abundance) and in <10% of reads (low abundance). Found variants were validated by Sanger sequencing. Sequencing resulted in 416,711,794 reads with an average target base coverage of 2663 ± 1476. Across all samples, 32 high-abundance somatic, 3 germline and 30 low-abundance mutations were withheld after filtering and validation. Overall, 92% of high-abundance and 84% of low-abundance mutations were predicted to be protein damaging. Frequently, mutated genes were MEN1, DAXX, ATRX, TSC2, PI3K/Akt/mTOR and MAPK-ERK pathway-related genes. Additionally, recurrent alterations on the same genomic position, so-called hotspot mutations, were found in DAXX, PTCH2 and CYFIP2. This first ultra-deep sequencing study highlighted genetic intra-tumor heterogeneity in pNEN, by the presence of low-abundance mutations. The importance of the ATRX/DAXX pathway was confirmed by the first-ever pNEN-specific protein-damaging hotspot mutation in DAXX. In this study, both novel genes, including the pro-apoptotic CYFIP2 gene and hedgehog signaling PTCH2, and novel pathways, such as the MAPK-ERK pathway, were implicated in pNEN.

Traitement médical des carcinomes basocellulaires avancés: Medical treatment of advanced basal cell carcinoma.

Until recently, advanced BCC were only accessible to a highly morbid surgery not necessarily proving to be carcinologic, and leaving terrible dysmorphic sequelae hard to accept by the patient. Another possibility, the only one in case of metastatic BCC, was chemotherapy which efficacy has never been proven in a clinical trial. Radiotherapy is most often not accessible because of previous radiotherapy or because of the localization or the extension of the lesion. The discovery of the importance of the sonic hedgehog pathway in the physiopathology of BCC has opened a new strategy with the development of targeted anti SMO drugs inactivating the pathway. Two molecules have become available following Phase I and II studies: vismodegib (Erivedge®) the first in class indicated for locally advanced and metastatic BCC and sonidegib (Odomzo®) indicated only for locally advanced BCC. The pharmacokinetic profiles of sonidegib and vismodegib showed several differences. No head to head comparative studies are available between these two drugs. Their pivotal phase II studies had similar study designs and endpoints. The objective response rate (ORR) by central review for vismodegib was 47.6% (95% CI 35.5-60.6) at 21 months follow-up. The ORR for sonidegib according to central review at 18 months follow-up is 56.1% (95% CI 43.3-68.3). Although both treatments share a similar adverse event profile with possible numerically differences in incidence, most patients will discontinue hedgehog inhibitors treatment in the long term because of side effects. Some resistant cases to these drugs have been described but are rather rare. In case of resistance or bad tolerability to the drug future hopes rely on immunotherapy currently under investigation. © 2018. Published by Elsevier Masson SAS. All rights reserved. Cet article fait partie du numéro supplément Prise en charge des carcinomes basocellulaires difficiles à traiter réalisé avec le soutien institutionnel de Sun Pharma.

Patched-2 functions to limit Patched-1 deficient skin cancer growth.

PURPOSE: Basal cell carcinoma (BCC) is one of the most common skin cancers, and is typically driven by an aberrantly activated Hedgehog (Hh) pathway. The Hh pathway is regulated by interactions between the Patched-1 (Ptch1) and Smoothened (Smo) receptors. Smo is an activating receptor and is subject to inhibition by Ptch1. Following ligand binding to Ptch1, its inhibitory action is relieved and pathway activation occurs. This receptor interaction is pivotal to restraining uncontrolled cellular growth. Both receptors have been found to be frequently mutated in BCCs. Ptch2 is a Ptch1 paralog that exhibits overlapping functions in both normal development and tissue homeostasis. As yet, its contribution to cancer growth is poorly defined. Here we set out to assess how Ptch2 inhibits BCC growth. METHODS: We used several in vitro readouts for transcriptional and chemotactic Hh signaling in BCC-derived ASZ001 cells, and a novel xenograft model to assess in vivo BCC tumor growth. Gene editing by TALEN was used to untangle the different Ptch2-dependent responses to its ligand sonic hedgehog (Shh). RESULTS: We first defined the signaling competence of Ptch2 in Ptch1-deficient ASZ001 cells in vitro, and found that Ptch2 ligand binding drives their migration rather than eliciting a transcriptional response. We found that subsequent targeting of Ptch2 abrogated the chemotaxic effect. Next, we tested the contribution of Ptch2 to in vivo tumor growth using a xenograft model and found that reduced Ptch function results in increased tumor growth, but that selective pressure appatently acts against complete Ptch2 ablation. CONCLUSIONS: We conclude that like Ptch1, Ptch2 exerts a tumor-suppressive function in BCC cells, and that after targeting of both paralogs, ligand-independent activation of the Hh pathway contributes to tumor growth.

Congenital embryonal rhabdomyosarcoma caused by heterozygous concomitant PTCH1 and PTCH2 germline mutations.

The sonic hedgehog (SHH) signaling pathway has been shown to play important roles in embryogenesis, cell proliferation as well as in cell differentiation. It is aberrantly activated in various common cancers in adults, but also in pediatric neoplasms, such as rhabdomyosarcoma (RMS) and atypical teratoid/rhabdoid tumors (AT/RTs). Dysregulation and germline mutation in PATCHED1 (PTCH1), a receptor for SHH, is responsible for the Gorlin Syndrome, a familial cancer predisposing syndrome including RMS. Here, we report a newborn diagnosed with congenital embryonal RMS. Whole-exome sequencing (WES) identified the presence of two heterozygous germline mutations in two target genes of the SHH signaling pathway. The PTCH1 mutation p.(Gly38Glu) is inherited from the mother, whereas the PTCH2 p.(His622Tyr) mutation is transmitted from the father. Quantitative RT-PCR expression analysis of GLI and SMO, key players of the SHH pathway, showed significantly increase in the tumor tissue of the patient and also enrichment in the germline sample in comparison to the parents indicating activation of the SHH pathway in the patient. These findings demonstrate that SHH pathway activity seems to play a role in eRMS as evidenced by high expression levels of GLI1 RNA transcripts. We speculate that PTCH2 modulates tumorigenesis linked to the PTCH1 mutation and is likely associated with the congenital onset of the RMS observed in our patient.

Activated Hedgehog-GLI Signaling Causes Congenital Ureteropelvic Junction Obstruction.

Intrinsic ureteropelvic junction obstruction is the most common cause of congenital hydronephrosis, yet the underlying pathogenesis is undefined. Hedgehog proteins control morphogenesis by promoting GLI-dependent transcriptional activation and inhibiting the formation of the GLI3 transcriptional repressor. Hedgehog regulates differentiation and proliferation of ureteric smooth muscle progenitor cells during murine kidney-ureter development. Histopathologic findings of smooth muscle cell hypertrophy and stroma-like cells, consistently observed in obstructing tissue at the time of surgical correction, suggest that Hedgehog signaling is abnormally regulated during the genesis of congenital intrinsic ureteropelvic junction obstruction. Here, we demonstrate that constitutively active Hedgehog signaling in murine intermediate mesoderm-derived renal progenitors results in hydronephrosis and failure to develop a patent pelvic-ureteric junction. Tissue obstructing the ureteropelvic junction was marked as early as E13.5 by an ectopic population of cells expressing Ptch2, a Hedgehog signaling target. Constitutive expression of GLI3 repressor in Ptch1-deficient mice rescued ectopic Ptch2 expression and obstructive hydronephrosis. Whole transcriptome analysis of isolated Ptch2+ cells revealed coexpression of genes characteristic of stromal progenitor cells. Genetic lineage tracing indicated that stromal cells blocking the ureteropelvic junction were derived from intermediate mesoderm-derived renal progenitors and were distinct from the smooth muscle or epithelial lineages. Analysis of obstructive ureteric tissue resected from children with congenital intrinsic ureteropelvic junction obstruction revealed a molecular signature similar to that observed in Ptch1-deficient mice. Together, these results demonstrate a Hedgehog-dependent mechanism underlying mammalian intrinsic ureteropelvic junction obstruction.

Lentivirus-mediated silencing of the PTC1 and PTC2 genes promotes recovery from spinal cord injury by activating the Hedgehog signaling pathway in a rat model.

This study aimed to investigate the effect of Patched-1 (PTC1) and PTC2 silencing in a rat model, on Hedgehog (Hh) pathway-mediated recovery from spinal cord injury (SCI). An analytical emphasis on the relationship between the sonic hedgehog (Shh) pathway and nerve regeneration was explored. A total of 126 rats were divided into normal, sham, SCI, negative control (NC), PTC1-RNAi, PTC2-RNAi and PTC1/PTC2-RNAi groups. The Basso, Beattie and Bresnahan (BBB) scale was employed to assess hind limb motor function. Quantitative real-time polymerase chain reaction and western blotting were performed to examine the mRNA and protein levels of PTC1, PTC2, Shh, glioma-associated oncogene homolog 1 (Gli-1), Smo and Nestin. Tissue morphology was analyzed using immunohistochemistry, and immunofluorescent staining was conducted to detect neurofilament protein 200 (NF-200) and glial fibrillary acidic protein (GFAP). The PTC1/PTC2-RNAi group displayed higher BBB scores than the SCI and NC groups. Shh, Gli-1, Smo and Nestin expression levels were elevated in the PTC1/PTC2-RNAi group. PTC1 and PTC2 mRNA and protein expression was lower in the PTC1/PTC2-RNAi group than in the normal, sham and SCI groups. Among the seven groups, the PTC1/PTC2-RNAi group had the largest positive area of NF-200 staining, whereas the SCI group exhibited a larger GFAP-positive area than both the normal and the sham groups. The Shh pathway may provide new insights into therapeutic indications and regenerative recovery tools for the treatment of SCI. Activation of the Hh signaling pathway by silencing PTC1 and PTC2 may reduce inflammation and may ultimately promote SCI recovery.

Multi-layered mutation in hedgehog-related genes in Gorlin syndrome may affect the phenotype.

Gorlin syndrome is a genetic disorder of autosomal dominant inheritance that predisposes the affected individual to a variety of disorders that are attributed largely to heterozygous germline patched1 (PTCH1) mutations. PTCH1 is a hedgehog (Hh) receptor as well as a repressor, mutation of which leads to constitutive activation of Hh pathway. Hh pathway encompasses a wide variety of cellular signaling cascades, which involve several molecules; however, no associated genotype-phenotype correlations have been reported. Recently, mutations in Suppressor of fused homolog (SUFU) or PTCH2 were reported in patients with Gorlin syndrome. These facts suggest that multi-layered mutations in Hh pathway may contribute to the development of Gorlin syndrome. We demonstrated multiple mutations of Hh-related genes in addition to PTCH1, which possibly act in an additive or multiplicative manner and lead to Gorlin syndrome. High-throughput sequencing was performed to analyze exome sequences in four unrelated Gorlin syndrome patient genomes. Mutations in PTCH1 gene were detected in all four patients. Specific nucleotide variations or frameshift variations of PTCH1 were identified along with the inferred amino acid changes in all patients. We further filtered 84 different genes which are closely related to Hh signaling. Fifty three of these had enough coverage of over ×30. The sequencing results were filtered and compared to reduce the number of sequence variants identified in each of the affected individuals. We discovered three genes, PTCH2, BOC, and WNT9b, with mutations with a predicted functional impact assessed by MutationTaster2 or PolyPhen-2 (Polymorphism Phenotyping v2) analysis. It is noticeable that PTCH2 and BOC are Hh receptor molecules. No significant mutations were observed in SUFU. Multi-layered mutations in Hh pathway may change the activation level of the Hh signals, which may explain the wide phenotypic variability of Gorlin syndrome.

UbcD1 regulates Hedgehog signaling by directly modulating Ci ubiquitination and processing.

The Hh pathway controls many morphogenetic processes in metazoans and plays important roles in numerous pathologies and in cancer. Hh signaling is mediated by the activity of the Gli/Ci family of transcription factors. Several studies in Drosophila have shown that ubiquitination by the ubiquitin E3 ligases Slimb and Rdx(Hib) plays a crucial role in controlling Ci stability dependent on the levels of Hh signals. If Hh levels are low, Slimb adds K11- and K48-linked poly-ubiquitin chains on Ci resulting in partial degradation. Ubiquitin E2 enzymes are pivotal in determining the topologies of ubiquitin chains. However, which E2 enzymes participate in the selective ubiquitination-degradation of Ci remains elusive. Here, we find that the E2 enzyme UbcD1 negatively regulates Hh signaling activity in Drosophila wing disks. Genetic and biochemical analyses in wing disks and in cultured cells reveal that UbcD1 directly controls Ci stability. Interestingly, UbcD1 is found to be selectively involved in Slimb-mediated Ci degradation. Finally, we show that the homologs of UbcD1 play a conserved role in modulating Hh signaling in vertebrates.

Fusion Oncogenes Are the Main Genetic Events Found in Sporadic Papillary Thyroid Carcinomas from Children.

BACKGROUND: Previous studies reported significant differences in the clinical presentation and outcomes of papillary thyroid carcinoma (PTC) in pediatric patients compared with adults. Previous studies have suggested that the clinicopathological differences observed between pediatric and adult PTCs may be due the existence of distinct genetic alterations. However, the knowledge of genetic events in pediatric PTCs is based primarily on studies in radiation-exposed PTCs or in the few studies that enrolled predominantly adolescent patients. The aim of this study was to characterize the known oncogenic alterations of the MAPK pathway found in adult and radiation-exposed PTCs in a cohort of predominantly sporadic pediatric PTC patients. METHODS: Thirty-five pediatric PTCs were screened for the most prevalent fusions (RET/PTC1, RET/PTC2, RET/PTC3, ETV6-NTRK3, and AGK-BRAF) and point mutations (BRAFV600E and NRASQ61) described in sporadic pediatric PTCs. The mutational status was correlated with clinicopathological data. RESULTS: Mutations were found in 20 out of 35 (57%) PTC cases. Fusion oncogenes were the main genetic alterations found. RET/PTC1-3 rearrangements were found in 13 (37%), ETV6-NTRK3 in 3 (9%), AGK-BRAF in 4 (11%), and BRAFV600E in 3 (9%). No mutation was found in NRASQ61. BRAFV600E was associated with older age and larger tumor size (p < 0.05), and RET/PTC3 was associated with a larger tumor size and multifocality (p < 0.05). CONCLUSIONS: The genetic signature in this cohort was remarkably different than that observed in adults. Although observed at a lower prevalence, the spectrum of mutations was quite similar to that described in radiation-exposed pediatric PTCs. As mutations were unidentifiable in over 40% of the PTC cases, more comprehensive studies conducted in these patients will help to decipher the genetic landscape of sporadic pediatric PTCs.