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Polymorphisms in PTCH1 Affect the Risk of Ameloblastoma

¹ Kyoto University Graduate School of Medicine, Department of Oral and Maxillofacial Surgery,
² Department of Clinical Genetics Unit,
³ Department of Biomedical Ethics, and
⁴ Department of Biostatistics, Graduate School of Medicine, Kyoto University, Syogoin-Kawahara-cho, Sakyou-ku, Kyoto 606-8501, Japan;

Correspondence: ^* corresponding author, takahask{at}kuhp.kyoto-u.ac.jp

	ABSTRACT

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Ameloblastoma is the most common odontogenic tumor, but the genetic nature of the changes in the tumor cells has been unclear. Mutations of CTNNB1 or PTCH1 are observed in many human tumors. Both CTNNB1 and PTCH1 are important in tooth development and are expressed in ameloblastoma. The aim of this study was to investigate whether genetic alterations of CTNNB1 and PTCH1 are present in ameloblastoma. We investigated 14 cases of ameloblastoma. The polymorphisms found in the ameloblastoma patients were further examined in a subsequent case-control study. We found a CTNNB1 mutation in one case of plexiform-type ameloblastoma. CGG triplet repeat-number polymorphism (CGG7/CGG8) in the 5'-untranslated region of PTCH1 was observed. The proportion of CGG8 alleles was significantly higher in the ameloblastoma group. The results of this study indicate a possible relationship between the CGG8 allele in PTCH1 and the risk for ameloblastoma.

Key Words: ameloblastoma • PTCH1 • CTNNB1 • polymorphism • Mantel trend test

	INTRODUCTION

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Ameloblastoma is the most common odontogenic tumor, and has the potential to behave aggressively locally, with a high risk of recurrence (Kramer et al., 1992; Melrose, 1999; Sciubba et al., 2001). It shows a variety of histopathological appearances and unpredictable biological behavior. A series of genetic alterations appears to promote the development of tumors by means of multiple steps (Vogelstein et al., 1988). Recently, several studies have reported genetic and cytogenetic alterations in ameloblastoma (Jaaskelainen et al., 2002; Shibata et al., 2002; Sekine et al., 2003; Kumamoto et al., 2004; Nodit et al., 2004). However, genetic changes in ameloblastoma are still poorly understood.

CTNNB1 functions as a transcriptional activator of the WNT signaling pathway. CTNNB1-mediated signaling can be constitutively activated by amino acid substitution caused by a mutation in exon 3 of the CTNNB1 (Polakis, 2000). Mutations in this region are observed in many human tumors. A recent report demonstrated that adamantinomatous craniopharyngioma (Sekine et al., 2002), which shows histological similarity to ameloblastoma (Bernstein and Buchino, 1983), harbored a CTNNB1 mutation in a hot spot of exon 3 (Sekine et al., 2002). The WNT signaling pathway is also involved in the signaling network mediating the epithelial-mesenchymal interactions in tooth morphogenesis (Miletich and Sharpe, 2003).

PTCH1 is a human homologue of the Drosophila segment polarity gene patched, and consists of 23 exons encoding a protein with 1447 amino acid residues (Ingham et al., 2000). This protein is believed to be a receptor for a secreted molecule, Sonic Hedgehog (Stone et al., 1996). Mutation of this gene was found to be responsible for the nevoid basal cell carcinoma syndrome (NBCCS) (Hahn et al., 1996), which is often associated with multiple odontogenic keratocysts. Subsequently, mutations of this gene were also found in sporadic odontogenic keratocysts (Barreto et al., 2000), in addition to tumors such as sporadic basal cell carcinoma, medulloblastoma, primitive neuro-ectodermal tumor, breast cancer, colon cancer, and meningioma (Toftgard, 2000). Furthermore, recently, several reports have demonstrated that PTCH1 is expressed at various levels in ameloblastoma (Barreto et al., 2002; Kumamoto et al., 2004).

Taken together, these findings have sparked intense interest in the roles of CTNNB1 and PTCH1 in ameloblastoma pathogenesis. Therefore, the aim of this study was to investigate whether alterations of the CTNNB1 and PTCH1 are present in ameloblastoma.

	MATERIALS & METHODS

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Subjects
All of the tumor samples and blood samples were obtained at the Department of Oral and Maxillofacial Surgery, Kyoto University Hospital, in accordance with a protocol approved by our Institutional Review Board (the medical ethics committee of Kyoto University). We enrolled 14 ameloblastoma patients and 35 unrelated volunteers who were free from odontogenic disease. This sample size was adequate to carry out the screening of the entire coding region of exon 23 of PTCH by direct sequencing. The ameloblastoma samples consisted of eight cases of the plexiform type (including two malignant cases), four cases of the follicular type, one case of the desmoplastic type, and one case of the basal cell type. In this study, we treated two malignant cases as being in the subclass of plexiform-type ameloblastoma, due to their histological appearance (Table 1). The mean (± SD) age of the 14 patients, nine of whom were men, was 41.4 ± 18.2 yrs.

DNA Isolation
Total genomic DNA from the frozen tumor samples was isolated by the use of a DNA Extraction WB kit (Wako, Osaka, Japan). Total genomic DNA from peripheral leukocytes was extracted with the use of a QIAamp^®DNA Blood Midi Kit (QIAGEN, Hilden, Germany). All procedures were performed according to the manufacturer’s instructions.

Polymerase Chain-reaction and Sequencing
Exons 1a, 3, 5–11, and 15–20 were amplified by the use of the previously described primer pairs (Hahn et al., 1996). We used newly designed primers to analyze exons 1b, 2, 4, 12, 13, 14, 21, and 22 of PTCH1 as well as exon 3 of CTNNB1. For exon 23, we used a novel reverse primer, while we used the reported forward primer. The newly designed primers are as follows: 5'-AGCAGCGGCTGGTCTGTCAAC-3' and 5'-GGAGAGGTGT GAGTGACTGTG-3' for exon 1b, 5'-AGCCCCCCATGACGC TCAGATC-3' and 5'-AGCCAGGCTCTAGGTGTGCGCTG-3' for exon 2, 5'-AGCTCTGCTCGTTTTGACAGATGC-3' and 5'-TTCCCAGAAGCAGTCCAAAGGTG-3' for exon 4, 5'-TAATGCCAGCATGATAAGCTG-3' and 5'-AGCTCTAAACA CAGGCATTTC-3' for exon 12, 5'-CGGTTTCAAATGCTT CAAGAG-3' and 5'-TTCTCCACACCAGCACAAACC-3' for exon 13, 5'-CTCTCCTAAGTCAGAGCTGTG-3' and 5'-GGAGGACTGAAATGTATCATAC-3' for exon 14, 5'-AACTGCGGTTGGATAACAGCC-3' and 5'-GTTTACTGAA GAACCACCAGC-3' for exon 21, 5'-TAGAGAAGGGG AGGTTAATAC-3' and 5'-CTGTACCTTATCTCTGCATCC-3' for exon 22, 5'-AGGAGAACCTTGTCCTCCTCTTTG-3' for exon 23 of PTCH1, and 5'-CCAATCTACTAATGCTAATACTG-3' and 5'-CTCCATTCTGACTTTCAGTAAGGC-3' for exon 3 of CTNNB1. The polymerase chain-reaction (PCR) for exon 1b was carried out by the use of LA Taq^TM with GC Buffer (TaKaRa, Ohtsu, Japan) according to the manufacturer’s instructions. The PCR reaction conditions for this exon consisted of 35 cycles of denaturation at 94°C for 30 sec, annealing at 60°C for 30 sec, and extension at 72°C for 2 min on a GeneAmp^® PCR System9700 (Applied Biosystems, Foster City, CA, USA). For the other exons, PCR was carried out with the use of LA Taq^®, and the reaction conditions consisted of 35 cycles of denaturation at 94°C for 30 sec, annealing at 54°C for 30 sec, and extension at 72°C for 30 sec. The sequence of the isolated PCR products was determined by means of the Big Dye Terminator system (Applied Biosystems). The sequencing reaction products were purified by means of CENTRI-SEP Spin Columns (PRINCETON SEPARATIONS, INC., Foster City, CA, USA). The sequences of both the sense and antisense strands of the sequencing reaction products were determined by means of an Applied Biosystems 3100 Genetic Analyzer.

Mutation Screen
We analyzed genomic DNA from tumor samples, and if some alterations were detected, we used genomic DNA from peripheral blood to confirm whether the genetic alteration was a germ line alteration or a somatic one.

Statistical Analysis
We compared observed with histological-type polymorphisms, and then performed a case-control study of polymorphisms to investigate a risk factor for ameloblastoma by comparing ameloblastoma patients with healthy controls.

We used the observed genotypic distributions to calculate the allelic frequencies. The frequencies of single-nucleotide polymorphisms (SNPs) and genotypic distributions of SNPs were compared between cases and controls by means of the chi-square test (without the Yates correction). We used the Mantel trend test to test whether the risk of ameloblastoma correlated with the CGG8 carrying status. Odds ratios (ORs) and 95% confidence intervals (95%CI) were calculated. A p-value of < 0.05 was considered to indicate statistical significance. All tests were two-tailed. We analyzed the data using Stat View for Macintosh, version 5.0J (SAS Institute Inc., Cary, NC, USA). The Mantel trend test and calculations of odds ratios were performed by PCSAS8.02.

	RESULTS

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Mutation Screen
Coding Region of PTCH1
To determine whether PTCH1 (Genbank U59464) was inactivated by mutations in ameloblastoma, we amplified and sequenced the entire coding region of PTCH1 in 14 cases of ameloblastoma. We could not detect any somatic mutations in the protein coding region or exon-intron boundaries of PTCH1.

Exon 3 of CTNNB1
We next screened mutations in exon 3 of CTNNB1 (NM001904). Only one case of ameloblastoma (case 3) was confirmed to harbor CTNNB1 alteration TCT (Ser) > TGT (Cys) of Ser³³ in exon 3 (Fig., A). In the other samples, we could not find any alteration in that region. We found no alteration in the genomic DNAs from the peripheral blood of case 3, so we concluded that the alteration was a somatic mutation. The histological features of case 3 were typical of plexiform-type ameloblastoma, i.e., interdigitating cords of epithelial cells and scant stellate reticulum (Fig., B).

View larger version (105K): [in this window] [in a new window]   Figure. CTNNB1 mutation in plexiform-type ameloblastoma. (A) Sequence of exon 3 of CTNNB1 in case 3. Codon 33 was affected in 1 allele. Alteration from TCT to TGT caused an amino acid substitution from serine to cysteine at the glycogen synthase kinase-3β (GSK3-β)-phosphorylation site of CTNNB1. (B) Histological features of case 3. This case shows typical plexiform-type ameloblastoma characterized by interdigitating cords of epithelial cells and scant stellate reticulum. Original magnification: x100. Scale bar: 100 µm.

Case-Control Study
Single-nucleotide Polymorphisms (SNPs)
To evaluate the association of each bi-allelic polymorphism (C2222T, G2678A, A3583T, and T3944C) of PTCH1 with ameloblastoma, we performed a case-control study. We found no significant difference in allele frequency or genotypic distribution between the group of 14 ameloblastoma cases and the group of 35 controls (data not shown).

5'-UTR Triplet Repeat-number Polymorphism (CGG7/CGG8)
The frequency of the CGG8 repeat allele in the ameloblastoma patients was significantly different from that in the controls (p = 0.03, chi-square test) (Table 2).

	DISCUSSION

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We identified 4 SNPs of PTCH1 as causing amino acid substitutions (Ala741Val, Arg893His, Thr1195Ser, Pro1315Leu) in 14 patients with ameloblastoma, while no somatic mutations in the coding region or exon-intron boundary of PTCH1 could be found by direct sequencing. As for Pro1315Leu, it was claimed that this PTCH1 polymorphism might modulate the association between the use of oral contraceptives and breast cancer risk (Chang-Claude et al., 2003). However, in this investigation we could not observe a significant difference between ameloblastoma patients and controls regarding these 4 SNPs. These SNPs may not be responsible for the genetic mechanism of ameloblastoma, or the lack of a significant difference may have resulted from the small study size. For this issue to be resolved, an extensive epidemiological study must be performed.

During the course of sequencing the entire coding region and exon-intron boundaries of PTCH1, we found a triplet repeat-number polymorphism (CGG7/CGG8) located 4 bases upstream of the first methionine codon of exon 1b. Based on a comparison of 14 ameloblastoma patients with 35 controls, we made the interesting observation that the frequency of the CGG8 allele was significantly higher in the ameloblastoma group (Table 2). So we hypothesized that CGG8 allele-carrying status (CGG null, carrying 1 CGG, 2 CGGs) is associated with ameloblastoma risk. To confirm this hypothesis, we performed a case-control study. By using CGG7/7 carriers as the reference category, we could calculate the odds ratios (ORs) for the CGG7/8 genotype and CGG8/8 genotype. As a result, the OR for the CGG7/8 genotype was 2.8, while that for the CGG8/8 genotype was 7.7 (Table 3). This trend was tested by the Mantel trend test, and the p-value was found to be 0.04, which implies that the CGG8 allele influences the risk of ameloblastoma.

A polymorphism involving a CGG repeat in the PTCH1 gene was also reported recently (Nagao et al., 2004). They reported that the CGG8 allele frequency in the Japanese population was 13.7% (14/102). This result is similar to the frequency of 12.9% (9/70) in the present study. Therefore, we consider that this frequency represents that in the Japanese population. Furthermore, it was demonstrated that a longer repeat of the CGG element tends to induce higher expression in vitro, because of the efficiency of translation. This result suggests that individuals with the CGG8/8 genotype have higher levels of PTCH1 protein expression than those with CGG7/7. Indeed, it was reported that higher expression of PTCH1 protein in ameloblastoma was observed by immunohistochemistry (Barreto et al., 2002; Kumamoto et al., 2004). PTCH1 interacts with a member of the hedgehog signaling family as its receptor, and also is a target molecule of the pathway. PTCH1 expression is a marker of activation of the hedgehog pathway (Bale and Yu, 2001). Mutational inactivation of PTCH1, which is a negative regulator of the hedgehog pathway, leads to overexpression of the transcript, owing to failure of a negative feedback mechanism in basal cell carcinoma (Unden et al., 1997; Nagano et al., 1999). Recently, a model in which the ratio of bound to unbound Patched molecules determines the cellular response to hedgehog was proposed (Casali and Struhl, 2004). It seems that regulation of PTCH1 expression is one of the key molecular events in hedgehog signaling. In odontogenic cysts, including odontogenic keratocysts and follicular cysts, high frequencies of the CGG8 allele were also observed (our unpublished data). The number of CGG triple repeats might modulate the hedgehog signaling pathway via control of the level of expression of PTCH1 in odontogenic lesions.

	ACKNOWLEDGMENTS

 
This work was supported by Grant-in-Aid for Scientific Research (B) (14370668), Grant-in-Aid for Exploratory Research (14657521), Grant-in-Aid for Young Scientists (A) (14704048), and Grant-in-Aid for Scientific Research (C) (16591993).

	FOOTNOTES

 
A supplemental appendix to this article is published electronically only at http://www.dentalresearch.org.

Received for publication November 22, 2004. Revision received May 4, 2005. Accepted for publication May 31, 2005.

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Journal of Dental Research, Vol. 84, No. 9, 812-816 (2005)
DOI: 10.1177/154405910508400906


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This Article Abstract Figures Only Full Text (PDF) Data Supplement Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Add to Saved Citations Download to citation manager Request Permissions Request Reprints Add to My Marked Citations Citing Articles Citing Articles via Google Scholar Citing Articles via Scopus Google Scholar Articles by Kawabata, T. Articles by Iizuka, T. Search for Related Content PubMed PubMed Citation Articles by Kawabata, T. Articles by Iizuka, T. Pubmed/NCBI databases Gene GEO Profiles HomoloGene UniGene Substance via MeSH Genetics Home Reference Social Bookmarking                         What's this?