Abstract
Keratocystic odontogenic tumours (KOCTs) are common benign cystic tumours that arise sporadically or associated with nevoid basal cell carcinoma syndrome (NBCCS). PTCH mutation can be found in sporadically or NBCCS associated KOCTs. Few PTCH mutations in families with non-syndromic KOCTs have been reported. Through PCR and gene sequence analysis, the authors discovered one missense mutation c.3277G>C in exon 19 of PTCH gene in a Chinese family with non-syndromic KOCTs. This mutation causes one highly conserved glycine residue transit to arginine on the 10th transmembrane region of PTCH protein. This work revealed that the missense mutation of PTCH is the causative and dominant gene of KOCTs in this family.
Keratocystic odontogenic tumours (KOCTs) are common benign cystic tumours that have the potential to behave aggressively and destroy portions of the jaws. In 2005, WHO renamed odontokeratocysts to KOCTs . The majority of cysts arises sporadically and in single form on the jaws of middle-aged people . When associated with nevoid basal cell carcinoma syndrome (NBCCS), these lesions appear earlier, often during the first or second decade of life, and may be (often) multiple and easily recrudescent . NBCCS is a rare autosomal dominant disease characterized by multiple basal cell carcinomas, KOCTs of the jaws, palmar and/or plantar pits, calcifications of the falx cerebri, skeletal anomalies, and a variety of other benign or malignant tumours. It is now thought that PTCH , the human homolog of the Drosophila patched gene, is responsible for this syndrome. PTCH (GenBank accession number: NC_000009.10 ) is a tumour suppressor gene located at chromosome 9q22.3, the genomic sequence is about 64 kb and at least 24 exons (including 23 coding exons), and it encodes a 1447 amino acid transmembrane protein that is thought to be a receptor for Hedgehog (Hh) .
Since the discovery in 1996 that PTCH is a gene responsible for NBCCS , about 280 mutations, to the authors’ knowledge, have been reported worldwide . Mutations of PTCH have not only been found in NBCCS, but have also been found in other sporadic malignancies, including sporadic keratocystic odontogenic tumours .
In their clinical work, the authors have found that patients in some families only have multiple KOCTs without the other symptoms of NBCCS. These types of disease were nominated as familial KOCTs. According to the literature review, the mutations of PTCH in these families have seldom been investigated; only one germline mutation of PTCH in one Chinese family has been reported . In this article, the authors report a germline mutation of PTCH gene in another Chinese family with non-syndromic KOCTs.
Materials and methods
With the informed consent of all nine family members, peripheral blood samples were collected. Genomic DNA was isolated from blood samples with QIAmp Blood Mid Kit (Qiagen, German). Twenty-one pairs of primers were designed and used for the amplification of the 23 exons of the PTCH gene ( Table 1 ).
| Exons | Primers | Product size |
|---|---|---|
| Exon 1 | F:AGCGGCTGGTCTGTCAAC R: TGGACGGCTTTCCAGTG |
674 bp |
| Eoxn 2 | F: TGGCGAATATCTCTATCAA R: TGAATATTATCTGTGTCGAG |
600 bp |
| Exon 3 | F: TAATGCACTTGCTTTGTTAC R: CATTAATGGGCTTAGTGAGT |
600 bp |
| Exons 4 and 5 | F: ACTGAAATGGAACAAACAAT R: GAAAAGCAGATTAATGGTGT |
644 bp |
| Exons 6 and 7 | F: CGAGGATAACGGTTTAAGTA R: AGCCGTGTTACTTTACGAT |
804 bp |
| Eoxn 8 | F: CTAATGTTTTCCAGACACCT R: TGGGGTATATCTGTAAAAGG |
674 bp |
| Exon 9 | F: GACCACTTTTAAACCACTGT R: CCTGGAGTAAATACAGCACT |
562 bp |
| Exon 10 | F: CCTGGTCAATGACATACAT R: CCCTTGATGTAAAGTTTTGT |
548 bp |
| Exon 11 | F: GAGTCTTCCAGCTTATTTCA R: GAGAATGTGCACTTCAGG |
584 bp |
| Exon 12 | F: ACTCTATAAGGCAACAGACG R: GAGATGTCTGTCACTTAGCC |
689 bp |
| Exon 13 | F: GCAGAGATATGAAACATTCC R: TCCTATCAAATAGGCTCTTG |
594 bp |
| Exon 14 | F: GAACTCCAAAGGTTCTGTTA R: GCTGTGTAAAATGGGTATTC |
569 bp |
| Exon 15 | F: GCTGCTGCAGAAACAGTTCA R: TGGCAACACAAGACTGTCTCA |
698 bp |
| Exon 16 | F: ATTCTGACAATTGTTCTGGT R: AAGATCCAGTGCTTTCAGT |
570 bp |
| Exon 17 | F: CCAGAGTTTTAAACGAGAAG R: GCATTCAGATTTAATTGGTT |
596 bp |
| Exon 18 | F: CTTCAAGCTACAGGAGAGAG R: CTTTCATGCAAAGTTCTTCT |
583 bp |
| Exon 19 | F: GAAAGGAATCCAGAAATCTT R: TACTTTGCCTTCTACAGCTT |
525 bp |
| Exon 20 | F: TATATTTTGAGTGGAGATGA R: GTTCAAGATTTACGTCAACA |
538 bp |
| Exon 21 | F: TTGATTTAGAGGAACCAAAC R: TCCCTTCATGAACTAACACT |
538 bp |
| Exon 22 | F: GGAAGATGGCATTTGTATAG R: GTAGAGAAGGGGAGGTTAAT |
597 bp |
| Exon 23 | F: CTCTGGGGATAAAAGGTC R: GACTGAGTCTTTGGTGAAAC |
788 bp |
Polymerase chains reactions (PCRs) except exon 1 were performed in 15 μl volumes containing 20 ng of template DNA, 300 mM dNTP, 0.8 mM MgCl 2 , 1.5 mM Q-solution (Qiagen, German), 0.2 μM of each primer, 0.6 unit Taq polymerase (Takara, Japan) in 1.5 μl 10× PCR buffer (Qiagen, German). The cycling conditions (standard Touchdown System) are: 3 min at 94 °C, followed by 12 cycles of 40 s at 94 °C, 40 s at 63 °C (decreased 0.5 °C after each cycle), 1 min at 72 °C, followed by 30 cycles of 40 s at 94 °C, 40 s at 57 °C, 1 min at 72 °C.
PCR of exon 1 was performed in 15 μl volumes containing 20 ng of template DNA, 300 mM dNTP, 0.2 μM of each primer, 1.0 unit kod-plus polymerase in 1.5 μl 10× PCR buffer (special for kod-plus polymerase). The cycling conditions are: 2 min at 94 °C, followed by 14 cycles of 15 s at 94 °C, 30 s at 64 °C (decreased 0.5 °C after each cycle), 1 min at 68 °C, followed by 30 cycles of 15 s at 94 °C, 30 s at 57 °C, 1 min at 68 °C.
Sequencing reaction products were loaded onto an ABI 3730 Automatic Sequencer (Applied Biosystems, USA). This part of the technology was supported by the Chinese National Human Genome Center at Shanghai, China.
Results
The pedigree structure of this family is shown in Fig. 1 . The proband was a 29-year-old patient (III:1), a panoramic radiograph revealed a multiple cyst radiolucent lesion in the jaw ( Fig. 2 ). Odontogenic keratocysts (OKCs) and dentigerous cysts (DCs) were pathologically diagnosed in five affected members. No other symptoms or indications associated with NBCCS were found in this family, such as basal cell carcinomas, palmer or plantar pits, calcification of the falx cerebri, bifid or fused ribs, and tumour symptoms. All the patients in this family had had OKC extraction surgery at least once ( Table 2 , Fig. 3 ).
