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PTCH1 and SMO Gene Alterations in Keratocystic Odontogenic Tumors Department of Oral Pathology, Hospital and School of Stomatology, Peking University, 22 South Zhongguancun Avenue, Haidian District, Beijing 100081, P.R. China Correspondence: * corresponding author, litiejun22{at}vip.sina.com
Keratocystic odontogenic tumors (KCOTs, previously known as odontogenic keratocysts) are aggressive jaw lesions that may occur in isolation or in association with nevoid basal cell carcinoma syndrome (NBCCS). Mutations in the PTCH1 (PTCH) gene are responsible for NBCCS and are related in tumors associated with this syndrome. Mutations in the SMO gene have been identified in basal cell carcinoma and in medulloblastoma, both of which are features of NBCCS. To clarify the role of PTCH1 and SMO in KCOTs, we undertook mutational analysis of PTCH1 and SMO in 20 sporadic and 10 NBCCS-associated KCOTs, and for SMO, 20 additional cases of KCOTs with known PTCH1 status were also included. Eleven novel (1 of which occurred twice) and 5 known PTCH1 mutations were identified. However, no pathogenic mutation was detected in SMO. Our findings suggest that mutations are rare in SMO, but frequent in PTCH1 in sporadic and NBCCS-associated KCOTs. Abbreviations: NBCCS, nevoid basal cell carcinoma syndrome; KCOTs, keratocystic odontogenic tumors; BCCs, basal cell carcinomas.
Key Words: PTCH1 • SMO • nevoid basal cell carcinoma syndrome • keratocystic odontogenic tumors • PTCH
Keratocystic odontogenic tumors (KCOTs, previously known as odontogenic keratocysts) are aggressive, non-inflammatory jaw cysts with a putative high growth potential and a propensity for recurrence (Browne, 1971; Li et al., 1994). Considerable insight into their genesis has come from the discovery that mutations of the PTCH1 gene (PTCH; MIM# 601309) are associated with nevoid basal cell carcinoma syndrome (NBCCS; MIM# 109400), an autosomal-dominant disorder presenting a spectrum of developmental abnormalities such as palmar/plantar pits, calcified falx cerebri, bridged sella, bifid ribs, and an increased susceptibility to different neoplasms, including multiple basal cell carcinomas (BCCs), KCOTs, medulloblastoma, and ovarian fibroma (Hahn et al., 1996; Johnson et al., 1996). Mutations in PTCH1 are associated with the majority of NBCCS and are found in tumors associated with this syndrome, as has been most convincingly demonstrated for BCCs and medulloblastomas (Raffel et al., 1997; Wolter et al., 1997). In addition to PTCH1, somatic mutations in the SMO gene (MIM# 601500) have also been identified in BCCs and in medulloblastomas (Reifenberger et al., 1998; Xie et al., 1998). These findings provide additional insight into the role of the sonic hedgehog pathway in NBCCS and associated tumors. Hedgehog signaling is a key regulator of embryonic development controlling cellular proliferation and fate. Binding of sonic hedgehog (SHH) to its receptor, patched (PTCH1), is thought to relieve normal inhibition by PTCH1 of smoothened (SMO), a seven-span transmembrane protein with homology to a G-protein-coupled receptor (Stone et al., 1996). Thus, loss of PTCH1 function by the inactivation of PTCH1 mutations, as well as aberrant activation of SMO by the activation of SMO mutations, could cause constitutive, ligand-independent signal transduction that may lead to neoplastic growth (Toftgard, 2000). KCOTs are among the most prominent features of NBCCS, which are found in 65–100% of affected individuals (Gorlin, 1987). This fact leads to the obvious hypothesis that KCOTs are caused by genetic alterations, both in syndromic and sporadic cases (Lench et al., 1997; Barreto et al., 2000; Pavelic et al., 2001; Ohki et al., 2004). To assess further the role of PTCH1 and SMO in the pathogenesis of KCOTs, we describe here the screen of PTCH1 and SMO gene mutations in a large series of Chinese persons with sporadic and NBCCS-associated KCOTs.
Participants and Tumors In total, 50 KCOT samples from 50 unrelated Chinese persons were obtained from Peking University Hospital and School of Stomatology, 20 of which have been previously described for PTCH1 mutations (11 PTCH1 mutations were identified in 5 of 14 sporadic and 6 of 6 NBCCS-associated KCOTs) (Gu et al., 2006; Yuan et al., 2006). The remaining samples included 20 sporadic and 10 NBCCS-associated KCOTs. Additionally, a total of ten affected relatives and 12 unaffected family members belonging to the kindreds of five NBCCS probands (NB9, NB10, NB12, NB13, NB16) were also investigated for the familial segregation of the mutations identified. Diagnosis of NBCCS was established according to previously described clinical criteria (Kimonis et al., 1997). Fresh tissue specimens were collected and frozen at –80°C. Peripheral blood was collected from all participants and 100 unaffected donors after they provided informed consent. The study protocol was approved by the Ethical Committee of Peking University Health Science Center.
DNA Isolation and Mutation Analysis
Total RNA Isolation and RT-PCR
We detected 16 PTCH1 mutations (1 of which was identified twice) in 5 of 20 sporadic and 8 of 10 NBCCS-associated KCOTs, 11 of which were novel (Table  ). The 16 mutations consisted of 6 frameshift, 2 nonsense, 3 aberrant splicing, 4 missense, and 1 in-frame duplication mutations. In addition, 13 previously described polymorphism sites of the PTCH1 gene were identified (APPENDIX). In contrast, no pathogenic mutation was found in the splicing and coding regions of the SMO gene in the total cohort of 50 KCOT samples tested. But 8 SMO polymorphisms were identified in 18 out of 50 participants, among which 3 (c.536C>T, c.IVS537+18C>T, c.582A>G) were newly identified (APPENDIX).
Somatic PTCH1 Mutations in Sporadic KCOTs We identified 6 novel (c.983delA, c.1325dupT, c.1558_1574del, c.2635delG, c.1247C>G, c.3162dupG) and 1 known (c.403C>T) PTCH1 mutations in 5 out of 20 sporadic KCOTs investigated. All mutations were somatic because of their absence in the matching blood samples. Two cases (KC19, KC21) were found to be compound heterozygous for 2 different frameshift mutations (c.[983delA(+)1325dupT] and c.[1558_1574del (+)2635delG]). Another novel frameshift mutation (c.3162dupG) was detected in a sporadic case (KC33). All these frameshift mutations were predicted to cause premature termination of PTCH1. One known nonsense mutation (c.403C>T) demonstrated a homozygous pattern in a tumor (KC30) and was absent in peripheral blood. The 2 concomitant polymorphisms (c.2913T>C, c.3141T>G) detected in this case were homozygous in a tumor but heterozygous in blood (Fig. 1 ). Analysis of these data suggests loss of 1 allele of PTCH1 and mutation of the other, since it is unlikely that both alleles harbor the same point mutations. One novel missense mutation (c.1247C>G) identified in a sporadic individual (KC22) also occurred in one of the 3 KCOT lesions of a NBCCS individual (NB12, Table ). This mutation was absent in the matching constitutional DNA of NB12. It affected a moderately conserved amino acid and was not detected in a total of 200 control chromosomes; therefore, it is unlikely to be a rare polymorphism.
Germ-line PTCH1 Mutations in Persons with NBCCS In total, 9 germ-line PTCH1 mutations (5 novel and 4 known) were identified in seven out of ten persons with NBCCS. One known nonsense mutation (c.2619C>A) was detected in a person with NBCCS (NB9) and was found to segregate with the disease in his family. Another known frameshift mutation (c.2196_2197delCT) was identified in NB11. This person also had a known missense mutation (c.863G>A), which hits a highly conserved residue (APPENDIX). One known missense mutation (c.3499G>A) was detected in NB16 and was found to segregate with the disease in the family. Two novel mutations, one missense (c.3440T>G) and one in-frame duplication (c.3244_3246dup), were detected in two persons (NB13, NB14), respectively. All these missense/in-frame duplication mutations were located in highly conserved regions across species (APPENDIX) and were not detected in a total of 200 control chromosomes. We have therefore classified these variants as pathogenic.
Four variants identified in four persons with NBCCS (c.1347+6G>A, NB7; c.1504-1G>A, NB9; c.2251-3C>G, NB8; c.2560+1G>T, NB10) occurred at the exon-intron junction. Splicing events in NB7 and NB9 were analyzed by RT-PCR with primers designed to amplify exons 8–13. An RNA sample from KC15 (no PTCH1 mutation detected) was used as control. The expected wild-type RT-PCR product of 744 bp and a shorter fragment (588 bp) that derives from the skipping of exon 10 were detected in all cases investigated (KC15, NB7, NB9) (Fig. 2A
We previously reported 11 PTCH1 mutations in 5 of 14 sporadic and 6 of 6 NBCCS-associated KCOTs (Gu et al., 2006; Yuan et al., 2006). Here we present 16 additional PTCH1 mutations in 5 of 20 sporadic and 8 of 10 NBCCS-associated KCOTs. In total, we have detected 26 PTCH1 mutations (2 mutations, c.2619C>A and c.1247C>G, occurred twice) in 10 out of 34 (29.4%) sporadic and 14 out of 16 (87.5%) NBCCS-associated KCOTs. The 26 mutations consisted of 10 frameshift, 2 nonsense, 3 aberrant splicing, 4 in-frame insertion/deletion/ duplication, and 7 missense mutations (APPENDIX). In line with a recent review on PTCH1 mutation (Lindstrom et al., 2006), 13 of the detected mutations were predicted to result in premature termination of PTCH1 protein, and a significantly higher frequency of mutations (9/26) was clustered into the two large extracellular loops where hedgehog ligand binding occurs. Another ’hot’ region was the highly conserved sterol-sensing domain (5/26), which harbors the transmembrane domains 2–6. Through careful analysis, however, no apparent genotype–phenotype correlations could be established. Although no PTCH1 mutation hot-spots have been reported in the literature, we identified here 2 recurrent PTCH1 mutations, one of which (c.2619C>A), a germ-line nonsense mutation seen in two unrelated Chinese families, has also been previously reported in a French person with NBCCS (Boutet et al., 2003). Thus, analysis of our data, together with reports from other groups (Lench et al., 1997; Barreto et al., 2000; Ohki et al., 2004), indicates that defects of PTCH1 are involved in the pathogenesis of syndromic as well as sporadic KCOTs. The PTCH1 gene is thought to function as a tumor suppressor gene, at least in some of the malignancies associated with NBCCS, as has been most convincingly demonstrated for BCCs (Gailani et al., 1996). The molecular analyses of KCOTs and BCCs in NBCCS showed that a two-hit hypothesis is applicable to their pathogenesis (Levanat et al., 1996; Barreto et al., 2000). In the present study, we demonstrated that two persons with the syndrome carried 2 different mutations (NB9, nonsense plus aberrant splicing; NB11, frameshift and missense mutations), respectively. In one sporadic case (KC30), we found that the tumor may lose the normal copy of PTCH1 while retaining a mutant copy (c.403C>T). Two other sporadic cases (KC19, KC21) showed 2 coincident frameshift mutations, respectively. These results indicate the possibility that inactivation of PTCH1 via a two-hit mechanism may occur in a subset of KCOTs. Alternative splicing in PTCH1 is thought to be a complex event, since multiple isoforms of PTCH1 mRNA by alternative first exons have been identified (Kogerman et al., 2002; Nagao et al., 2005a). Additional tissue-specific or disease-related splicing variants have also been described involving exons 1–5, exon 10, and exon 12b (Nagao et al., 2005b). We describe here an alternate splicing product of exon-10-skipping in sporadic and NBCCS-associated KCOTs with/without PTCH1 mutations. A previous study observed identical consistent alternate splicing of exon 10 in all cultured normal and NBCCS patient lymphocyte and normal keratinocyte cell lines (Smyth et al., 1998). Its effect on PTCH1 function, however, is currently unknown. Whether it is a common phenomenon or is functional in another context remains to be elucidated. This study also identified 3 instances of disease-associated aberrant splicing in PTCH1. The mutation in NB7 at the donor splice site of intron 9 (c.1347+6G>A) resulted in the skipping of exons 9 and 10, which leads to destruction of extracellular loop 1 and the removal of transmembrane domains 2–3. Another instance of aberrant splicing identified in NB9 (c.1504-1G>A) was the skipping of exons 10 and 11, which ablated transmembrane domains 2–4. Although the functional consequences of these 2 aberrant splicing changes are yet to be investigated, the importance of extracellular loop 1 in binding of the SHH ligand and the role of the sterol-sensing domain, which is made up of transmembrane domains 2–6, in mediating the potent modulating effect of cholesterol on SHH/PTCH signaling suggest that the receptor form lacking exons 9–10 or exons 10–11 may show altered signaling properties. The mutation in NB10 at the 5' end of intron 15 (c.2560+1G>T) resulted in the entire skipping of exon 15 and premature truncation of PTCH1. The non-detection of PTCH1 mutations in many sporadic KCOTs and even in some typical familial cases also underlines the non-exploration of other genetic events. The idea can be examined in more detail in the context of SHH signaling, whereby PTCH1 acts to restrain the activity of the G-protein-coupled receptor, SMO. Inactivation of PTCH1 allows for hedgehog ligand-independent activation of SMO, with the subsequent activation of transcription factors of the GLI family. Given that activation of SMO, like inactivation of PTCH1, up-regulates transcription of hedgehog target genes, it is not surprising that activating mutations in the SMO gene have been identified in BCCs and in medulloblastomas. However, the failure to detect SMO mutation in a total of 50 KCOTs (including 16 NBCCS-associated cases), as demonstrated here in this study, suggests that it is an extremely rare event in this tumor. The role of other components of SHH signaling remains to be elucidated.
The first two authors contributed equally to this work. We gratefully acknowledge the participants and their families for their cooperation. This work was supported by Research Grants from the National Nature Science Foundation of China (30625044 and 30572048) and the Specialized Research Fund for the Doctoral Program of Higher Education (20050001110).
 authors contributing equally to this work  A supplemental appendix to this article is published electronically only at http://jdr.iadrjournals.org/cgi/content/full/87/6/575/DC1. Received for publication September 21, 2007. Revision received January 19, 2008. Accepted for publication February 22, 2008.
Journal of Dental Research, Vol. 87, No. 6,
575-579 (2008)
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ABSTRACT
INTRODUCTION
