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Orofacial clefts (OFCs) represent the most prevalent congenital craniofacial anomalies, significantly impacting patients' appearance, oral function, and psychological well-being. Among these, non-syndromic OFCs (NSOFCs) are the most predominant type, with the etiology attributed to a combination of genetic and environmental factors. Rare variants of key genes involved in craniofacial development-related signaling pathway are crucial in the occurrence of NSOFCs, and our recent studies have identified PTCH1, a receptor-coding gene in the Hedgehog signaling pathway, as a causative gene for NSOFCs. However, the role of PTCH2, the paralog of PTCH1, in pathogenesis of NSOFCs remains unclear. Here, we perform whole-exome sequencing to explore the genetic basis of 144 sporadic NSOFC patients. We identify five heterozygous variants of PTCH2 in four patients: p.L104P, p.A131G, p.R557H, p.I927S, and p.V978D, with the latter two co-occurring in a single patient. These variants, all proven to be rare through multiple genomic databases, with p.I927S and p.V978D being novel variants and previously unreported. Sequence alignment suggests that these affected amino acids are evolutionarily conserved across vertebrates. Utilizing predictive structural modeling tools such as AlphaFold and SWISS-MODEL, we propose that these variants may disrupt the protein's structure and function. In summary, our findings suggest that PTCH2 may be a novel candidate gene predicted to be associated with NSOFCs, thereby broadening the spectrum of causative genes implicated in the craniofacial anomalies., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: [Yue Zhang reports financial support was provided by Natural Science Foundation of Xinjiang Uygur Autonomous Region. Huaxiang Zhao reports financial support was provided by National Natural Science Foundation of China. Yuxia Hou reports financial support was provided by National Natural Science Foundation of China. Wenbin Huang reports financial support was provided by Shenzhen Clinical Research Center for Oral Diseases. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.]., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

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., (© 2021. The Author(s).)

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., (Copyright © 2021. Published by Elsevier B.V.)

Embryogenesis is modulated by numerous complex signaling cascades, which are essential for normal development. The Hedgehog (Hh) signaling pathway is part of these central cascades. As a homolog of Patched (Ptch)-1, Ptch2 initially did not appear to be as important as Ptch1. Recent reports have revealed that Ptch2 plays a crucial role in ligand-dependent feedback inhibition of Hh signaling in vertebrates. The role of Ptch2 in facial development remains unclear. Here, we investigated the detailed expression pattern of Ptch2 during craniofacial development in murine embryos based on in situ hybridization (ISH) studies of whole-mounts and sections, immunohistochemistry (IHC), and quantitative real-time PCR. We found that both Ptch2 mRNA and protein expression increased in a dynamic pattern in the facial development at mouse embryonic days 11-14.5. Moreover, distinct expression of Ptch2 was observed in the structures of the facial region, such as the tooth germ, Meckel's cartilage, and the follicles of vibrissae. These data, combined with our work in the macrostomia family, suggest that Ptch2 may play a critical role in facial development., (Copyright © 2021 Elsevier GmbH. All rights reserved.)

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., (Copyright © 2020. Published by Elsevier Masson SAS.)

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., (Copyright © 2019 Elsevier B.V. All rights reserved.)

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., (©AlphaMed Press 2019.)

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.

Signaling through the Hedgehog (Hh) pathway is mediated by the Patched (Ptch) family of proteins. Although the vertebrate Ptch proteins Ptch1 and Ptch2 harbor two closely related transmembrane modules related to sterol-sensing domains (SSDs), the role of these closely related receptors in the Hh pathway are not equivalent. Ptch1 is essential for development and appears to be the principal receptor mediating responses to Hh ligands, whereas Ptch2 is nonessential, and its role in Hh-signaling remains ambiguous. We hypothesized that the SSDs of the Ptch proteins function as generic modules whose protein-specific activities are determined by the adjacent cytoplasmic and luminal domains. We first showed that individual N-terminal and C-terminal halves of Ptch1 associated noncovalently to mediate ligand-dependent regulation of Hh signaling. The analogous regions of Ptch2 also interacted noncovalently but did not repress the Hh pathway. However, the SSD of Ptch2 were capable of repressing Hh signaling, as determined using chimeric proteins where the SSDs of Ptch1 were replaced by those from Ptch2. Replacement of the SSDs of Ptch1 with the analogous regions from the cholesterol transporter NPC1 failed to produce a chimeric protein capable of Hh repression. Further refinement of the specific regions in Ptch1 and Ptch2 revealed that specific cytoplasmic domains of Ptch1 were necessary but not sufficient for repression of Hh signaling and that the two principal luminal domains of Ptch1 and Ptch2 were interchangeable. These data support a model where the SSDs of the Ptch family proteins exhibit generic activities and that the adjacent cytoplasmic and luminal domains determine their protein-specific activities., (© 2018 Fleet and Hamel.)

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.

Although many marker genes for germ cell differentiation have been identified, genes that specifically regulate primordial germ cell (PGC) generation are more difficult to determine. In the current study, we confirmed that C2EIP is a PGC marker gene that regulates differentiation by influencing the expression of pluripotency-associated genes such as Oct4 and Sox2. Knockout of C2EIP during embryonic development reduced PGC generation efficiency 1.5-fold, whereas C2EIP overexpression nearly doubled the generation efficiency both in vitro and in vivo. C2EIP encodes a cytoplasmic protein that interacted with PTCH2 at the intracellular membrane, promoted PTCH2 ubiquitination, activated the Hedgehog (HH) signaling pathway via competitive inhibition of the GPCR-like protein SMO, and positively regulated PGC generation. Activation and expression of C2EIP are regulated by the transcription factor STAT1, histone acetylation, and promoter methylation. Our data suggest that C2EIP is a novel, specific indicator of PGC generation whose gene product regulates embryonic stem cell differentiation by activating the HH signaling pathway via PTCH2 modification.

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., (Copyright © 2018 by the American Society of Nephrology.)

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.

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.

Smoothened (Smo) inhibition by Patched (Ptch) is central to Hedgehog (Hh) signaling. Ptch, a proton driven antiporter, is required for Smo inhibition via an unknown mechanism. Hh ligand binding to Ptch reverses this inhibition and activated Smo initiates the Hh response. To determine whether Ptch inhibits Smo strictly in the same cell or also mediates non-cell-autonomous Smo inhibition, we generated genetically mosaic neuralized embryoid bodies (nEBs) from mouse embryonic stem cells (mESCs). These experiments utilized novel mESC lines in which Ptch1, Ptch2, Smo, Shh and 7dhcr were inactivated via gene editing in multiple combinations, allowing us to measure non-cell autonomous interactions between cells with differing Ptch1/2 status. In several independent assays, the Hh response was repressed by Ptch1/2 in nearby cells. When 7dhcr was targeted, cells displayed elevated non-cell autonomous inhibition. These findings support a model in which Ptch1/2 mediate secretion of a Smo-inhibitory cholesterol precursor., Competing Interests: The authors declare that no competing interests exist.