PTCH1 Gene Mutations in Keratocystic Odontogenic

Tumors: A Study of 43 Chinese Patients and a Systematic

Review
Yan-Yan Guo1, Jian-Yun Zhang1, Xue-Fen Li2, Hai-Yan Luo1, Feng Chen2*, Tie-Jun Li1*
1 Department of Oral Pathology, Peking University School and Hospital of Stomatology, Beijing, China, 2 Central Laboratory, Peking University School and Hospital of
Stomatology, Beijing, China

Abstract
Background: The keratocystic odontogenic tumor (KCOT) is a locally aggressive cystic jaw lesion that occurs sporadically or
in association with nevoid basal cell carcinoma syndrome (NBCCS). PTCH1, the gene responsible for NBCCS, may play an
important role in sporadic KCOTs. In this study, we analyzed and compared the distribution pattern of PTCH1 mutations in
patients with sporadic and NBCCS-associated KCOTs.

Methods: We detected PTCH1 mutations in 14 patients with NBCCS-associated KCOTs and 29 patients with sporadic KCOTs
by direct sequencing. In addition, five electronic databases were searched for studies detecting PTCH1 mutations in
individuals with NBCCS-associated or sporadic KCOTs, published between January 1996 and June 2013 in English language.

Results: We identified 15 mutations in 11 cases with NBCCS-associated KCOTs and 19 mutations in 13 cases with sporadic
KCOTs. In addition, a total of 204 PTCH1 mutations (187 mutations from 210 cases with NBCCS-associated and 17 mutations
from 57 cases with sporadic KCOTs) were compiled from 78 published papers.

Conclusions: Our study indicates that mutations in transmembrane 2 (TM2) are closely related to the development of
sporadic KCOTs. Moreover, for the early diagnosis of NBCCS, a genetic analysis of the PTCH1 gene should be included in the
new diagnostic criteria.

Citation: Guo Y-Y, Zhang J-Y, Li X-F, Luo H-Y, Chen F, et al. (2013) PTCH1 Gene Mutations in Keratocystic Odontogenic Tumors: A Study of 43 Chinese Patients
and a Systematic Review. PLoS ONE 8(10): e77305. doi:10.1371/journal.pone.0077305
Editor: Jingwu Xie, Indiana University School of Medicine, United States of America
Received August 15, 2013; Accepted September 4, 2013; Published October 21, 2013
Copyright: ß 2013 Guo et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted
use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported the National Natural Science Foundation of China (grant numbers 81030018, 30872900, 30901680) and the Doctoral Fund of
Ministry of Education of China (grant number 20120001110043). The funders had no role in study design, data collection and analysis, decision to publish, or
preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: moleculecf@gmail.com (FC); litiejun22@vip.sina.com (TJL)

Introduction 9q22.3-31 and consists of 23 coding exons spanning approximately

74 kb and encoding a 1447-amino-acid transmembrane (TM)
Originally described as cysts [1], odontogenic keratocysts glycoprotein. The Patched protein is involved in the Hedgehog
(OKCs) were recategorized as neoplastic lesions under the name (Hh) signaling pathway, a key regulator of body patterning and
‘‘karatocystic odontogenic tumors’’ (KCOTs) in the 2005 edition organ development during embryogenesis [13]. Patched and
of the World Health Organization Classification of Head and Smoothened (Smo) are thought to act as subunits of a putative Hh
Neck Tumors [2]. KCOTs are locally aggressive jaw cystic lesions receptor complex: Smo functions as the transducing subunit, and
that have putative growth potential and a propensity for its activity is blocked by a direct interaction with Patched, the
recurrence [3,4]. They may occur in isolation or in association ligand-binding subunit [14,15]. Misregulation of the signaling
with nevoid basal cell carcinoma syndrome (NBCCS, also known caused by inactivation of PTCH1 is involved in the development of
as Gorlin syndrome; OMIM No. 109400). NBCCS is a rare NBCCS and some related sporadic tumors, supporting the
autosomal dominant disorder with an estimated prevalence of 1/ hypothesis that PTCH1 functions as a tumor suppressor gene
55600 [5] to 1/256000 [6]. Affected patients present with a [10]. Several studies have demonstrated the presence of PTCH1
spectrum of developmental abnormalities, including palmar or mutations in patients with NBCCS [12] as well as in some
plantar pits, calcification of the falx cerebri, bifid ribs, and an sporadic tumors, including BCCs [16], medulloblastoma [17], and
increased susceptibility to different neoplasms, such as multiple KCOTs [18].
basal cell carcinomas (BCCs), KCOTs, medulloblastoma, and The Patched protein has several important functional domains.
ovarian fibroma [7–9]. Two large extracellular loops (ECLs) are required for the binding
The human homolog of the Drosophila segment polarity gene of N-Shh to the Patched protein, and glycosylation sites in the two
PTCH1 (OMIM No. 601309) has been identified as the gene large ECLs are required for N-Shh binding [19]. Martin et al.
responsible for NBCCS [10–12]. PTCH1 has been mapped to (2001) and Strutt et al. (2001) suggested that the sterol-sensing

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 PTCH1 Mutations in Keratocystic Odontogenic Tumors

domain (SSD) of Patched is required to negatively regulate were confirmed by cloning purified PCR products into the plasmid
Smoothened (Smo) activity, indicating a role of the SSD in vector pCR2.1 (Invitrogen, Carlsbad, CA, USA). After transform-
mediating the vesicular trafficking of PTCH to regulate Smo ing competent Escherichia coli strain TOP10 with the recombinant
activity [20,21]. Additionally, its large intracellular loop (ICL) vectors, white colonies were selected and grown overnight in 3 mL
participates in a G2/M checkpoint by regulating the localization of of LB medium with ampicillin. Sequencing was performed on an
M-phase promoting factor [22]. The C-terminal region of Patched ABI PRISM 3100 Genetic Analyzer (Applied Biosystems, Foster
has been shown to induce apoptotic cell death during spinal cord City, CA, USA) with M13 universal primers. Identified mutations
development [23]. were confirmed by reverse sequencing and at least two additional
The distribution pattern of PTCH1 mutations has been independent PCR products.
analyzed in patients with NBCCS as well as in several tumors
including BCCs, BCCs with xeroderma pigmentosum syndrome Restriction Enzyme Analysis
(XP-BCCs), and sporadic medulloblastomas [24]. They found that To define unreported PTCH1 mutations (missense mutations, in
PTCH1 mutations were identified throughout nearly the entire particular) as novel mutations rather than rare polymorphisms, we
sequence, with a high frequency of mutations clustered in the two tested 100 unrelated control DNAs by restriction enzyme analysis.
large extracellular loops and the large intracellular loop [24]. Digestion of the PCR products with MspI, BanII or BaeGI (New
NBCCS cases and each class of tumor analyzed revealed a England Biolabs, Beverly, MA, USA) was performed as recom-
different distribution of mutations in the various Patched protein mended by the supplier. The digested DNA was electrophoresed
domains [24]. PTCH1 gene harbors mutational hot spot regions, on 2.5% agarose gels and stained with nucleic acid stain.
including a slippage-sensitive sequence in the N-terminus [24].
However, few reports have described the distribution pattern in Literature Review
KCOTs, especially sporadic KCOTs. Thus, the aim of the present We performed a systematic search from January 1996 to June
study was to investigate PTCH1 gene mutations in 29 sporadic and 2013 using five electronic databases (PubMed, ScienceDirect,
14 NBCCS-associated KCOTs. Additionally, to systematically OviSP, Web of Science, and the Human Gene Mutation
analyze and compare the distribution pattern of PTCH1 mutations Database). Additional studies were identified by searching the
in NBCCS-associated and sporadic KCOTs, we conducted a reference lists of the included publications. The keywords used
systematic review of published studies evaluating PTCH1 muta- were ‘‘PTCH1’’ (or its synonyms) and ‘‘NBCCS or KCOT’’ (or
tions in patients with KCOTs. their synonyms). This review was conducted and reported
according to the PRISMA (Preferred Reporting Items for
Materials and Methods Systematic Reviews and Meta-Analyses) Statement issued in
2009 (Checklist S1) [25].
Subjects and Samples Studies were included if they concerned PTCH1 gene mutations
KCOT samples from 43 unrelated Chinese patients (14 patients in patients with sporadic or NBCCS-associated KCOTs. A list of
with NBCCS-related and 29 with sporadic KCOTs) were exclusionary indications is shown in Fig. 1. Only one entry was
obtained from Peking University, School and Hospital of made when the same mutation in a single patient was reported in
Stomatology (Beijing, China). Fresh KCOT tissue specimens different papers (for example: c.2186A.T was first reported by
and corresponding peripheral blood samples were collected and Ponti et al. [26] and subsequently by Pastorino et al. [27]).
immediately stored at 280uC for subsequent polymerase chain Separate entries were made for each patient as recurrent
reaction (PCR) and sequencing analysis. Peripheral blood control mutations when the same mutation was reported in apparently
samples were obtained from 100 normal volunteers from the unrelated patients (for example: c.403C.T identified in different
Blood Transfusion Center at Peking University First Hospital. The patients [18,28]). Nucleotide and protein names were given for
protocol for this study was approved by the Ethics Committee of each mutation according to current nomenclature guidelines
Peking University’s Health Science Center. Written informed (http://www.hgvs.org/mutnomen/). Nucleotide numbering was
consent was obtained from all study participants. based on GenBank entry NM_000264.3, where the A of the ATG
initiation codon represents nucleotide +1. The amino acid
DNA Extraction and PCR numbering was based on GenBank entry NP_000255.2.
Genomic DNA was extracted from the frozen tissue specimens
and peripheral blood samples was extracted with a QIAamp DNA Statistical Analysis
Mini Kit (Qiagen, Hilden, Germany), and 22 coding exons (exons Data were analyzed using SPSS software (ver. 13.0), and
2–23) and the exon-intron boundaries of PTCH1 were amplified significant differences between categorical groups were determined
by PCR with specific primers. Two primer sets were used using the chi-squared test or Fisher’s exact test, while numerical
separately to amplify exons 14 and 23 because of the relatively groups were assessed using the independent t-test. P values,0.05
large size of the exons. PCR was performed in a final reaction were considered to indicate statistical significance. All statistical
volume of 50 mL, containing approximately 100 ng of template tests were two-sided.
DNA, 200 mM dNTPs, 10 mM each primer and 1.25 U of Ex Taq
DNA polymerase (Takara, Kyoto, Japan) in PCR buffer. Results
Amplification was performed in a thermal cycler (Eppendorf,
Hamburg, Germany) using the following cycling parameters: Clinical Manifestations of Patients with NBCCS-associated
initial incubation at 94C for 5 min; 35 cycles of denaturation at KCOTs
94uC for 30 s, annealing at 56–65uC for 30 s, and elongation at The male to female ratio among the 14 patients with NBCCS-
72uC for 30 s; and final elongation at 72uC for 7 min. associated KCOTs was 3.67:1. To improve the comparability of
age factors, we used age of onset (age at diagnosis minus duration)
Mutation Analysis by Direct and Clonal Sequencing in the following statistical analysis. The individuals ranged in onset
The amplified products were sequenced directly with the age from 8 to 44 years (mean 20.57610.71; median 18.50 years;
primers used for the original PCR. Insertion or deletion mutations Fig. 2A).

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 PTCH1 Mutations in Keratocystic Odontogenic Tumors

Figure 1. Flow chat of the selection process for the extensive review.
doi:10.1371/journal.pone.0077305.g001

The total number of lesions was 45, from 1 to 5, and the average found to carry two concomitant mutations while NB24 was found
number of lesions was 3.21 (Table 1). Of the lesions, 18/45 to carry two somatic truncation-causing mutations (c.3156_
(40.0%) were located in the maxilla and 27/45 (60.0%) in the 3163delins19 and c.2709_2713delTAAAC), identified in different
mandible. Among the 14 cases, only one patient had a single lesion KCOTs. One mutation in patient NB21 was a germline
of KCOT; however, the scope of the lesion in this patient was truncation mutation in exon 2 (c.233G.A) and the other was a
wide, approximately from the right mandibular angle to the left somatic truncation mutation in exon 19 (c.3253dupA), resulting in
mandibular angle. premature stops at codon 78 and 1144 respectively. Two germline
mutations were identified in patient NB22, one was a truncation
Clinical Manifestations of Patients with Sporadic KCOTs mutation in exon 6 (c.873C.G) and the other was a non-
The male to female ratio among the 29 patients with sporadic truncation mutation in exon 15 (c.2479A.G). Also, one mutation
KCOTs was 1.07:1. The age range of the 29 affected persons was in patient NB23 was a germline non-truncation mutation in exon
13 to 71 years (mean 32.03616.75; median 25.0 years; Fig. 2A). 11 (c.1526G.A) and the other was a somatic splice-site mutation
The mean age of patients with sporadic KCOTs was significantly in exon 19 (c.394+3_+20del18). Additionally, 6 of 14 cases
higher than that of patients with systematic KCOTs (t = (42.86%; NB21, NB24, NB25, NB27, NB30, and NB31) suffered
-2.333,P = 0.025). All the 29 cases had a single lesion (Table 2). from cleft lip or plate, an incidence significantly higher than that in
Of 29 lesions, only 2 (6.90%) were located in the maxilla and 27 previous studies [9,29,30]. All six cases were found to carry
(93.10%) in the mandible. PTCH1 mutations (Table 1). Patient NB25 carried a non-
truncation mutation (c.3180_3185del6). One truncation mutation
PTCH1 Mutations in Patients with NBCCS-associated and (c.1342_1345delCTCA) was identified in patient NB27. One
Sporadic KCOTs truncation mutation (c.387G.A) was detected in patient NB30.
We identified 11 germline and 4 somatic mutations in 11 of 14 Patient NB31 carried a truncation mutation (c.2464dupC) in the
(78.57%) patients with NBCCS-associated KCOTs (Table 1). second large ECL (ECL2). In addition, A G.C substitution at
Additionally, 12 mutations (80.0%) had not been previously nucleotide 1531 in exon 11, which resulted in a glycine to arginine
reported in the literature and consisted of 7 truncation-causing, 3 substitution at codon 511, was detected in a 32-year-old male
non-truncation-causing, and 2 splice-site mutations. Of the 12 new patient with NBCCS (NB29). This mutation was not detected in
mutations, 5 were distributed in the two large ECLs (ECL1 and 100 control normal volunteers by direct sequencing.
ECL4), 4 in the TMs, 2 in the N-terminus, and 1 in the fourth ICL The 14 affected patients could be divided into two groups:
(ICL4). Furthermore, three cases (NB21, NB22, and NB23) were mutation and non-mutation. The mean age (17.55 years) of the

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 PTCH1 Mutations in Keratocystic Odontogenic Tumors

Figure 2. The onset age distribution of patients with NBCCS-associated and sporadic KCOTs. (A) The age distribution of 14 patients with
syndromic KCOTs was significantly lower than in 29 patients with sporadic KCOTs (P = 0.025). (B) In syndromic KCOTs, the age distribution in the
mutation group was significantly lower than in non-mutation group (P = 0.037), whereas in sporadic KCOTs, no significant difference was detected
between the groups (P = 0.537). ‘‘N-KCOT’’ indicates ‘‘NBCCS-associated KCOT’’; ‘‘S-KCOT’’ indicates ‘‘sporadic KCOT’’; ‘‘M’’ indicates ‘‘Mutation group’’;
and ‘‘Non-M’’ indicates ‘‘Non-mutation group’’ (Error bars6SD, * P,0.05).
doi:10.1371/journal.pone.0077305.g002

mutation group was significantly younger than that (31.67 years) of normal volunteers by restriction enzyme analysis. The abnormal
the non-mutation group (Fig. 2B; t = 22.350, P = 0.037). All the restriction sites present in the PCR products from KC44 and
patients under the age of 20 years carried mutations. Four patients KC46 were absent in the control DNAs. Thus, the two missense
over 20 years of age were identified to carry mutations whereas mutations were unlikely to be polymorphisms.
three patients over 20 years of age carried no mutations (Fig. 2B). Similarly, the 28 affected cases could be further categorized into
Particularly, patient NB32, a 28-year-old patient with typical two groups: mutation and non-mutation. The mean age (34.23
NBCCS phenotypes (such as multiple KCOTs and bilamellar years) of the mutation group was slightly higher than that (30.25
calcification of the falx cerebri), was found to carry no PTCH1 years) in non-mutation group (Fig. 2B), but the difference was not
mutations and had only a single KCOT. Additionally, in the statistically significant (t = 0.630, P = 0.537). Moreover, only two
mutation group, 14 lesions were found in the maxilla while 23 in cases (KC51 and KC54) occurred in the maxilla; both carried
the mandible. In the non-mutation group, four lesions were found truncation mutation (c.2430delA and c.262_265delTTTA, respec-
in the maxilla while five in the mandible (Table 1). No significant tively).
difference was detected between the two groups in terms of the
lesion distribution (Pearson x2 = 0.057,P = 0.811). Literature Search Results
We identified 19 somatic PTCH1 mutations in 13 of 29 Our search strategy retrieved 858 reports, 751 of which were
(44.83%) patients with sporadic KCOTs (Table 2). No germline
excluded on the basis of the title and abstract. After carefully
mutation was detected in any patient. Additionally, six cases (6/28,
examining the full text of the remaining 107 articles, 78 articles
21.43%) were found to carry two concomitant mutations. Of those
(187 mutations from 210 cases with NBCCS-associated and 17
mutations, 17 (89.47%) had not been previously reported,
mutations from 57 cases with sporadic KCOTs) were included.
consisting of 13 truncation, 2 non-truncation, and 2 splice-site
Fig. 1 presents the flowchart of the search process. Tables S1-2
mutations. Of the 17 new mutations, seven were distributed in
summarizes the key characteristics of each PTCH1 mutation.
ECL1 and ECL4, five in TMs, two in N-terminus, one in the first
intracellular loop (ICL1), one in ECL2 and one in ICL5.
Additionally, novel missense mutations (c.536T.C and Distribution Pattern of PTCH1 Gene Mutations in Patients
c.3346G.C) were respectively identified in patients KC44 and with Sporadic and NBCCS-associated KCOTs
KC46. The two mutations introduced a novel restriction enzyme Including the mutations found in our study, we compiled a list
site for MspI and BanII, creating two fragments, thus confirming of 238 mutations (202 from patients with NBCCS-associated
the presence of the mutation. To characterize the two novel KCOTs and 36 from patients with sporadic KCOTs). Large
missense mutations, we tested 100 control DNA samples from deletions and duplications, although important in the pathogenesis

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 PTCH1 Mutations in Keratocystic Odontogenic Tumors

Table 1. Clinical manifestations and PTCH1 mutations in 14 cases with NBCCS-associated KCOTs.

Sex/ Number of Exon/

Patient Onset age Lesions Nomenclature c. Nomenclature p. Mutation type Intron no. Characterization Structure
Maxilla Mandible

NB21d M/11 2 3 c.233G.A p.Trp78X Missense mutations Exon2 Germline N-terminus

c.3253dupA p.Ile1085AsnfsX60 Small duplications Exon19 Somatic TM10
NB22 M/11 1 2 c.873C.G p.Tyr291X Nonsense mutations Exon6 Germline ECL1
c.2479A.Ga p.Ser827Gly Missense mutations Exon15 Germline ECL4
NB23 M/14 1 3 c.394+3_+20del18 Splice-site mutations Intron2 Somatic N-terminus
c.1526G.Ab p.Gly509Asp Missense mutations Exon11 Germline TM4
NB24d M/14 1 2 c.3156_3163delins19 p.Ala1053LeufsX2 Indel mutations Exon18 Somatic ICL4
c.2709_2713delTAAAC p.Lys904AlafsX10 Small out-of-frame Exon17 Somatic ECL4
deletions
NB25d M/25 0 2 c.3180_3185del6 p.Ala1061_Leu1062del Small in-frame Exon19 Germline TM9
deletions
NB26 F/30 1 2 c.534_545del12 p.His178_Ala182delinsGln Small in-frame Exon3 Germline ECL1
deletions
NB27d M/8 1 2 c.1342_1345delCTCA p.Leu448CysfsX7 Small out-of-frame Exon9 Germline TM2
deletions
NB28 M/12 2 2 c.584+1G.A Splice-site mutations Intron3 Germline ECL1
NB29 M/32 1 1 c.1531G.C p.Gly511Arg Missense mutations Exon11 Germline TM4
NB30d F/10 1 2 c.387G.Ac p.Trp129X Nonsense mutations Exon2 Germline ECL1
d
NB31 M/26 3 2 c.2464dupC p.Leu822ProfsX7 Small duplications Exon15 Germline ECL4
NB32 M/28 0 1 Not identified
NB33 M/23 1 2 Not identified
NB34 F/44 3 1 Not identified

a
This mutation has been reported in a holoprosencephaly patient [40].
b
This mutation has been reported in a NBCCS patient [41].
c
This mutation has been reported in a NBCCS patient [42].
d
Those patients suffered from cleft lip or plate.
Abbreviations: NB, NBCCS.
doi:10.1371/journal.pone.0077305.t001

of KCOTs, offered less detail in the distribution pattern of PTCH1 55.56%) were found in the TM domains, especially in the second
mutations because those mutations encompassed more extensive half of the protein in patients with sporadic KCOTs.
structural changes. Thus, these 15 large deletions and duplications
were excluded in the following statistical analysis. Different Mutation Types in Patients with Sporadic and
Compared with the predicted Patched protein sequence, the NBCCS-associated KCOTs
223 PTCH1 mutations (187 from NBCCS-associated and 36 from The predominant mutation type seen in both the NBCCS-
sporadic KCOTs) were distributed over the entire sequence. The associated and sporadic KCOT groups was the predicted
mutations (n = 187) from patients with NBCCS-associated truncation-causing mutation in the Patched protein, with no
KCOTs were mainly clustered in the two large extracellular loops statistically significant difference between the groups (113/187 vs.
(95/187, 50.80%; Fig. 3A and 4). Mutations were also found in the
25/36; Pearson x2 = 1.040; P = 0.308; Table 3). Similarly, no
SSD (16.58%), the large intracellular loop (8.56%) and the N-
significant difference in the distribution pattern of splice-site
terminal region (6.95%). Approximately half of the non-trunca-
mutations or mutations that did not cause truncation of the
tion-causing mutations (26/51, 50.98%) were located in the TM
Patched protein was observed between the groups.
domains, especially TM4. Additionally, most splice-site mutations
(11/23, 47.83%) were concentrated in the first large extracellular
loops. Discussion
Mutations (n = 36) from patients with sporadic KCOTs were In the present study, 29 new PTCH1 mutations were identified,
found predominantly in the two large extracellular loops (14/36, including 20 truncation, 5 non-truncation, and 4 splice-site
38.89%) and TM2 (7/36, 19.44%; Fig. 3B and 4). Compared with mutations. In general agreement with previous studies, most of
the mutations in patients with NBCCS-associated KCOTs, the the mutations identified were expected to lead to the synthesis of a
mutations from sporadic KCOTs were differently distributed, with truncated Patched protein. The PTCH1 mutations were widely
a significantly higher frequency of mutations in TM2 (7/36 vs. 8/ distributed along the entire sequence: 12 in the two large
187; Pearson x2 = 7.631,P = 0.006). Additionally, no mutation was extracellular loops, 8 in the SSD, 4 in the N-terminus region, 3
found in the large intracellular loop (0/36), compared to those in TM8-10, and 2 in ICL4 and ICL5. Those mutations enrich the
found in the NBCCS group (16/187; Pearson x2 = 2.158, spectrum of PTCH1 mutations in KCOTs, thus providing further
P = 0.142). Most non-truncation-causing mutations (5/9,

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 PTCH1 Mutations in Keratocystic Odontogenic Tumors

Table 2. Clinical manifestations and PTCH1 mutations in 29 cases with sporadic KCOTs.

Sex/
Onset Exon/
Patient age Number of Lesions Nomenclature c. Nomenclature p. Mutation type Intron no. Characterization Structure
Maxilla Mandible

KC43 F 0 1 c.1359_1362delCTGT p.Cys454X Small out-of-frame Exon10 Somatic TM2

deletions
c.1493_1494insTG p.Thr499GlufsX44 Small in-frame Exon10 Somatic ECL2
insertions
KC44 M 0 1 c.536T.C p.Leu179Pro Missense mutations Exon3 Somatic ECL1
c.1164dupC p.Glu389ArgfsX48 Small duplications Exon8 Somatic ECL1
KC45 M 0 1 c.2638_2668del31 p.Gly880ProfsX13 Small out-of-frame Exon16 Somatic ECL4
deletions
c.260_271delinsAA p.Leu87X Indel mutations Exon2 Somatic N-terminus
KC46 F 0 1 c.3346G.C p.Val1116Leu Missense mutations Exon20 Somatic ICL5
c.1127_1143del17 p.Phe376CysfsX55 Small out-of-frame Exon8 Somatic ECL1
deletions
KC47 M 0 1 c.202-3C.T Splice-site mutations Intron1 Somatic N-terminus
c.1362_1375del14 p.Cys454TrpfsX38 Small out-of-frame Exon10 Somatic TM2
deletions
KC48 M 0 1 c.1327delG p.Ala443ProfsX13 Small out-of-frame Exon9 Somatic TM2
deletions
c.1344_1347delCATG p.Met449SerfsX6 Small out-of-frame Exon9 Somatic TM2
deletions
KC49 M 1 0 c.407dupT p.Ser137LysfsX3 Small duplications Exon3 Somatic ECL1
KC50 M 0 1 c.3081dupG p.Leu1028AlafsX117 Small duplications Exon18 Somatic TM8
KC51 F 0 1 c.2430delA p.Asp811ThrfsX19 Small out-of-frame Exon15 Somatic ECL4
deletions
KC52 M 0 1 c.1063_1067+11del16 Splice-site mutations Exon7/Intron7 Somatic ECL1
KC53 F 0 1 c.2908G.Ta p.Glu970X Nonsense mutations Exon18 Somatic ECL4
KC54 F 1 1 c.262_265delTTTAb p.Phe88AsnfsX28 Small out-of-frame Exon2 Somatic N-terminus
deletions
KC55 M 0 1 c.1394_1396delinsA p.Ser465X Indel mutations Exon10 Somatic ICL1
KC56 F 0 1 Not identified
KC57 F 0 1 Not identified
KC58 M 0 1 Not identified
KC59 F 0 1 Not identified
KC60 F 0 1 Not identified
KC61 M 0 1 Not identified
KC62 M 0 1 Not identified
KC63 F 0 1 Not identified
KC64 M 0 1 Not identified
KC65 F 0 1 Not identified
KC66 F 0 1 Not identified
KC67 M 0 1 Not identified
KC68 F 0 1 Not identified
KC69 F 0 1 Not identified
KC70 M 0 1 Not identified
KC71 F 0 1 Not identified

a
This mutation has been reported in a NBCCS patient [34].
b
This mutation has been reported in a NBCCS patient [43].
Abbreviations: KC, KCOT.
doi:10.1371/journal.pone.0077305.t002

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 PTCH1 Mutations in Keratocystic Odontogenic Tumors

Figure 3. Distribution pattern of PTCH1 mutations in relation to the different domains of the Patched protein. (A) NBCCS-associated
KCOTs (n = 174). (B) Sporadic KCOTs (n = 36). The thick line indicates the SSD.
doi:10.1371/journal.pone.0077305.g003

information about the pathogenesis of syndromic and sporadic Thus, it is not surprising that syndromic KCOTs presented at a
KCOTs. younger age, and were multifocal with widespread sites of
NBCCS is a hereditary condition caused by mutations in involvement located in the maxilla and mandible, whereas
PTCH1 gene and transmitted in an autosomal dominant manner sporadic KCOTs presented at an older age, and were unifocal
with high penetrance and variable expressivity. In the present with local lesions mainly located in the mandible.
study, 10 of 14 (71.43%) individuals with syndromic KCOTs The mutation rate of PTCH1 gene in NBCCS patients varied
carried at least a germline PTCH1 mutation. Because the initial from 29.0 to 100% [32–34]. However, the mutation rate of
tumor-causing mutation is inherited through the germline, it is PTCH1 gene in sporadic KCOTs was approximately 28.6% [18].
already present in every cell of the body [31]. However, all 19 Here, we identified 34 mutations in 11 of 14 (78.57%) syndromic
PTCH1 mutations identified in sporadic KCOTs were somatic. and 13 of 29 (44.83%) sporadic KCOTs patients. Additionally,

Figure 4. Proportion of PTCH1 mutations in important domains of the Patched protein. ECL1 indicates the first large extracellular loop
(amino acids 122–436); TM2 denotes the second transmembrane region (amino acids 437–457); SSD stands for the sterol-sensing domain (amino
acids 438–598); ECL4 denotes the second large extracellular loop (amino acids 770–1027); * P,0.05.
doi:10.1371/journal.pone.0077305.g004

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 PTCH1 Mutations in Keratocystic Odontogenic Tumors

Table 3. Different types of PTCH1 mutations in patients with The distribution pattern of PTCH1 mutations in NBCCS-
NBCCS-associated and sporadic KCOTs. associated and sporadic KCOTs had not been extensively
analyzed previously. To our knowledge, this is the first to
systematically analyze PTCH1 mutation distribution pattern in
Truncation Non-truncation Splice-site Total systematic and sporadic KCOTs. Careful analysis of the two
groups revealed several interesting findings. First of all, consistent
No. % of Total No. % of Total No. % of Total
with previous literature [24], PTCH1 mutation hot regions in
N-KCOT 113 61.49% 51 27.59% 23 10.92% 187 NBCCS-associated KCOTs involved the two large extracellular
S-KCOT 25 69.44% 9 25.00% 2 5.56% 36 loops, sterol-sensing domain (SSD), large intracellular loop, and
x2 test x2 = 1.040 x2 = 0.079 x2 = 0.785 the N terminal region (close to TM1), while PTCH1 mutation hot
regions in sporadic KCOTs involved the two large extracellular
P = 0.308 P = 0.778 P = 0.375
loops and TM2. Additionally, compared to mutations in NBCCS-
doi:10.1371/journal.pone.0077305.t003 associated KCOTs, a significantly higher frequency of mutations
in TM2 and no mutations in the large intracellular were found in
during the 14 cases with syndromic KCOTs, all patients under 20 sporadic KCOTs. Differences in the TM2 region were statistically
years of age were detected to carry PTCH1 mutations, whereas significant, while the mutations in the large intracellular loop were
some patients over 20 years of age did not carry mutations. It not. Seven mutations in patients with sporadic KCOTs were
indicated that PTCH1 gene might play a more important role in distributed in the TM2 region, six of which resulted in premature
the development of the younger age cases (#20 years) with truncation of Patched protein. Interestingly, a significantly lower
syndromic KCOTs, whereas the pathogenesis of the older age frequency of mutations in TM2 was also noted in sporadic basal
cases (.20 years) might be complicated and involved other as yet cell carcinomas and basal cell carcinomas with xeroderma
unknown genes. There were reports showing other genes, such as pigmentosum syndrome [24]. Taken together, these results
PTCH2 [35] and SUFU [36,37], might be involved in the indicate that mutations in TM2 might be closely related to the
pathogenesis of NBCCS. We suggested that KCOTs, as a development of sporadic KCOTs.
complex disease, might be caused by multiple genes and In conclusion, this is the first report to systematically analyze
environmental factors. To assess this, further study is needed to and compare the distribution patterns of PTCH1 mutations in
use other technologies, such as exome or whole-genome sequenc- patients with NBCCS-associated and sporadic KCOTs, especially
ing, to screen for additional genes. sporadic KCOTs. Our study indicates that mutations in TM2 are
Making a diagnosis of NBCCS in childhood patients could be closely related to the development of sporadic KCOTs. Moreover,
challenging because several signs of the syndrome develop over for the early diagnosis of NBCCS, genetic analysis of the PTCH1
time, including KCOTs, BCCs, calcification of the falx cerebri, gene might be included in new diagnostic criteria. Our findings
and ovarian fibromas. Patients affected with NBCCS at birth justify further investigation.
might present only some congenital developmental deformities
such as cleft lip or palate, and bifid, fused or accessory ribs. BCCs Supporting Information
most often appear between puberty and 35 years of age; the mean
age of onset is about 25 years of age [8]. Between 30% and 65% of Table S1 Literature review: 187 PTCH1 gene mutations in cases
patients with the syndrome have small asymmetric palmar or with NBCCS-associated KCOTs.
plantar pits by the age of 10 years; this percentage rises to 80% by (DOCX)
the age of 15 years [7,38]. The calcification of the falx cerebri can Table S2 Literature review: 17 PTCH1 gene mutations in cases
appear very early in life, and is often strikingly apparent from late with sporadic KCOTs.
childhood. Medulloblastoma characteristically presents during the (DOCX)
first two years of life, while in the general population, occurrence
peaks at 7 to 8 years of age. Ovarian fibromas generally develop in Checklist S1 PRISMA Checklist for this systematic
the teen years. In our study, two out of (14.3%) of patients develop review.
a cystic lesion by the age of 10 years and 7 out of 14 (50%) by the (DOC)
age of 20 years. Diagnostic criteria were presently based on the
most frequent and/or specific features of the syndrome [5,9]. Acknowledgments
Thus, the diagnosis time of most NBCCS cases is usually
postponed until the occurrence of symptoms. However, early We thank the patients and their families for specimen donation and their
support of our research.
diagnosis is often of great significance for the prevention and
prognosis of systematic tumors. The high detection rate of PTCH1
mutations among NBCCS patients, as demonstrated in the present Author Contributions
study as well as other previous ones [34,39], poses the possibility to Conceived and designed the experiments: TJL YYG. Performed the
include genetic analysis of PTCH1 in the new diagnostic criteria experiments: YYG. Analyzed the data: YYG FC HYL. Contributed
for early diagnosis. reagents/materials/analysis tools: JYZ XFL. Wrote the paper: YYG.

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