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SH3BP2-encoding exons involved in cherubism are not associated with central giant cell granuloma
Int. J. Oral Maxillofac. Surg. 2011; 40: 851–855 doi:10.1016/j.ijom.2011.04.003, available online at http://www.sciencedirect.com
Research Paper Genetics
SH3BP2-encoding exons involved in cherubism are not associated with central giant cell granuloma R. C. Teixeira, H. P. Horz, J. H. Damante, G. P. Garlet, C. F. Santos, R. L. M. Nogueira, R. B. Cavalcante, G. Conrads: SH3BP2-encoding exons involved in cherubism are not associated with central giant cell granuloma. Int. J. Oral Maxillofac. Surg. 2011; 40: 851–855. # 2011 International Association of Oral and Maxillofacial Surgeons. Published by Elsevier Ltd. All rights reserved. Abstract. Central giant cell granuloma (CGCG) is a benign lesion with unpredictable biological behaviour ranging from a slow-growing asymptomatic swelling to an aggressive lesion associated with pain, bone and root resorption and also tooth displacement. The aetiology of the disease is unclear with controversies in the literature on whether it is mainly of reactional, inflammatory, infectious, neoplasic or genetic origin. To test the hypothesis that mutations in the SH3BP2 gene, as the principal cause of cherubism, are also responsible for, or at least associated with, giant cell lesions, 30 patients with CGCG were recruited for this study and subjected to analysis of germ line and/or somatic alterations. In the blood samples of nine patients, one codon alteration in exon 4 was found, but this alteration did not lead to changes at the amino acid level. In conclusion, if a primary genetic defect is the cause for CGCG it is either located in SH3BP2 gene exons not yet related to cherubism or in a different gene.
The central giant cell granuloma (CGCG) was first described by JAFFE in 195314, as a giant-cell reparative granuloma of the jaw bones. It is an intra-osseous lesion resulting from a local reparative reaction of unknown aetiology. It occurs mainly in the mandible in patients aged 10–25 years and in most cases the appearance is a problem and causes the patient to seek treatment3,7. The World Health Organization (WHO) has defined CGCG as an intra-osseous lesion consisting of cellular fibrous tissue 0901-5027/080851 + 05 $36.00/0
that contains multiple foci of haemorrhage, aggregations of multinucleated giant cells and occasional trabeculae of woven bone with an incidence rate of 1.1/ million population/year6. The histological appearance of CGCG is similar to that of brown tumours seen in hyperparathyroidism, which should be excluded by performing differential diagnostic laboratory analysis. The true nature of CGCG remains speculative and there is considerable controversy in the literature4,5.
R. C. J. H. C. F. R. B.
Teixeira1,2, H. P. Horz1, Damante2, G. P. Garlet3, Santos3, R. L. M. Nogueira4, Cavalcante5, G. Conrads1
1 Division of Oral Microbiology and Immunology, Department of Operative and Preventive Dentistry & Periodontology, and Department of Medical Microbiology, Medical Faculty, RWTH Aachen University, Germany; 2 Department of Stomatology, Bauru School of Dentistry, University of Sa˜o Paulo, Bauru, Sa˜o Paulo, Brazil; 3Department of Oral Biology, Bauru School of Dentistry, University of Sa˜o Paulo, Bauru, Sa˜o Paulo, Brazil; 4Oral and Maxillofacial Surgery, Federal University of Ceara´, Fortaleza, Ceara´, Brazil; 5Department of Oral Pathology, University of Fortaleza, Fortaleza, Ceara´, Brazil
Key words: central giant cell granuloma; SH3BP2-encoding exons; cherubism. Accepted for publication 8 April 2011 Available online 16 June 2011
Conversely, cherubism (Mendelian Inheritance in Man (MIM) code 118400) was thought to be a familial disease affecting 100% of males and up to 70% of females8. In a study of a Turkish family, 8 DE LANGE et al. showed that in one male and one female the mutation was present without any present or past signs and symptoms of cherubism. This could indicate that, contrary to earlier literature, the mutation described does not have 100% penetrance in males.
# 2011 International Association of Oral and Maxillofacial Surgeons. Published by Elsevier Ltd. All rights reserved.
852
Teixeira et al.
Table 1. Anamnestic details of CGCG patients subjected to this study. Clinical findings
Nonaggressive (16)
Aggressive (14)
Average age Sex M:F Location Mandible Maxilla Pain Rapid growth Root resorption Root displacement Cortical bone destruction
17.41 (6–29) 7:9
13.54 (5–23) 7:7
11 5 – – – 9 –
7 7 7 11 8 10 11
Cherubism is a painless, disfiguring disease primarily affecting the bones of the jaw. It was first described by JONES in 193315 but there are numerous subsequent reports11,16,23,25. Most of the reported cases of cherubism are hereditary with a Mendelian dominant mode of inheritance29. In the few reported cases of nonfamilial, sporadic, cherubism13 the differentiation from CGCG is even more difficult given that the clinical and histological features are highly similar9,17,26 and that the genetic cause is not obvious. The chromosomal mutation responsible for cherubism is located at position 4p16.321 encoding for the adapter protein SH3BP2 (Src Homology-3 Binding Protein-2)21,22,27, which was found capable of binding c-Abl via its SH3-domain1,10,24. UEKI et al.27 showed that this protein is expressed in the multinucleated stromal cells of soft fibrous tissue from cherubism lesions. The gene SH3BP2 consists of 13 exons and the mutations most frequently found in cherubism are located in exon 920,27. In a few case reports, other mutations were found in exons 3 or 42,3. It has been hypothesized that mutations in exon 9 may be responsible for CGCG as well, because of the strong clinical, radiological and histological correlation between cherubism and CGCG. In three different studies, no single mutation was found in exon 98,12,19. Instead, one somatic mutation in exon 11 was most recently reported for one patient in another study3. The above findings were either focused on a few patients and/or on a few exons, so the possibility of a manifest mutation (germ line or somatic) in the SH3BP2 gene as a possible cause for CGCG remains unclear. The aim of this study was to analyse all exons previously associated with cherubism and CGCG (i.e. exons 3, 4, 9 and 11) by testing 30 patients with different phenotypes of CGCG. Materials and methods
Thirty patients attending for treatment in the Batista Hospital, Fortaleza-CE-Brazil,
with a confirmed diagnosis of CGCG, were recruited. Patients gave their written informed consent and their inclusion was carried out in accordance with the Ethics Committee of the Bauru School of Dentistry, University of Sa˜o Paulo. The inclusion/exclusion criteria adopted were: discard cherubism and hyperparathyroidism brown tumour using analysis of familial history; clinical, radiographic and laboratory examinations (parathormone, alkaline phosphatase, calcium and potassium levels) and ascertain that sufficient data are available to classify the lesion as aggressive or nonaggressive (cortical destruction or expansion, uni- or multilocular images, presence of tooth dislocation and or dental resorption). The 30 CGCG patients (14 male, 16 female) recruited for this study were subjected to a detailed clinical and radiological examination and classified according to the clinical behaviour of the disease. The age of the patients ranged from 5 to 29 years (Table 1). The diagnosis was verified by clinical, laboratory, radiographic and histopathological examinations and all patients were submitted to the inclusion/exclusion criteria adopted. Of 30 patients, 12 presented lesions in the maxilla and 18 in the mandible. The posterior region of the mandible seemed to be more frequently affected with 13 cases. Fourteen patients presented aggressive lesions and 16 were classified as nonaggressive, according to the criteria established by CHUONG et al.4 (Table 1). Sample collection and DNA isolation
Peripheral venous blood samples were obtained from all 30 patients. From 14 patients, lesion tissue (fixed and embedded) could be acquired and it was subjected to genetic analysis. Tissue samples were not available from the other patients at this experimental phase of the study. For peripheral venous blood samples, genomic DNA was extracted using a QIAmp DNA Mini kit according to the manufacturer’s instructions. DNA
samples were frozen at 70 8C and transported on dry ice by an overnight delivery service to the Division of Oral Microbiology and Immunology, RWTH Aachen University Hospital, Aachen, Germany, where genetic analyses were performed. Formalin-fixed, paraffin-embedded (FFPE) tissue samples were also transported to the same institution, where DNA was extracted and purified with a QIAmp DNA Mini kit but using the ‘tissue protocol’ (QIAGEN, Hilden, Germany) according to the manufacturer’s instructions, with one modification: 0.8 g of zirconia–silica beads (0.1 mm in diameter; BioSpec, Bartlesville, OK, USA) was added prior to the addition of Proteinase K. Samples were then agitated in a FastPrep FP 120 instrument (Qbiogene, Carlsbad, CA, USA) at 6.5 m/s for 45 s. All further steps followed the original protocol. Polymerase chain reaction (PCR) amplification of initial DNA extracts from the FFPE tissues was not successful, so 10 ml of the DNA extracts were loaded on a 1% agarose-gel stained with ethidium bromide. After electrophoresis a DNA band of high molecular weight was excised out of the gel and the DNA subsequently purified using the QIAquick Gel Extraction Kit (QIAGEN, Hilden, Germany) according to the manufacturer’s instructions. The DNA concentration (A260) and purity (A260/A280) were calculated using a Gene Quant II photometer (Pharmacia Biotech, Cambridge, England). PCR conditions
Four pairs of primers deduced from the SH3BP2 gene sequence targeting exons 3, 4, 9 and 11 were used in this study (Table 2). For PCR, each reaction mixture contained 0.5 U of Taq DNA polymerase supplied with PCR buffer (Roche Applied Science, Penzberg, Germany), 0.2 mM dNTPs (Roche Applied Science, Penzberg, Germany), 0.5 mM of each primer, and 1 ml of template DNA (approximately 25 ng). Amplification was performed in a total volume of 50 ml in PCR reaction tubes using an Eppendorf Mastercycler (Eppendorf, Hamburg, Germany). The PCR cycling conditions were: 94 8C for 1 min, followed by 37 cycles at 94 8C for 30 s, 57 8C for 40 s, and 72 8C for 50 s, ending with a single 10-min extension step at 72 8C13. With these conditions, no product was obtained from blood sample DNA in about 32% of cases (38 out of 120 total runs). In these cases, the annealing temperature was decreased
Mutations in CGCG Table 2. Primers for amplifying SH3BP2 exons (IMAI et al.13) used in this study. Size of PCR product (bp)
Primer
Primer sequence
Exon3F Exon3R
50 -AGGGTCCTCACAGTGGGTCTT-30 50 -GCTTCGGGAAAGCACTTTCC-30
289
Exon4F Exon4R
50 -GGCCATCCCTATAGCTGTGA-30 50 -ACACCCCCAGTCACCTTGCA-30
214
Exon9F Exon9R
50 -CCGTGTCTGACAGTGAAATGG-30 50 -ATGGCTTTCCCACCACCTGT-30
222
Exon11F Exon11R
50 -AAGGTCCGTGTGAAAGCTGCC-30 50 -ACAGCCTGGGTGTGTGGAGA-30
201
to 54 8C, which allowed successful PCR amplification. DNA from a few tissue samples that could not be amplified using the above conditions was subjected to a real time PCR using a LightCycler 2.0 (Roche, Penzberg, Germany) with the following conditions: 95 8C for 10 min, followed by
[(Fig._1)TD$IG]
50 cycles at 95 8C for 10 s, 56 8C for 10 s, and 72 8C for 20 s.
Sequence analysis
Aliquots of the amplicons (10 ml) were examined by electrophoresis on a 1% agarose-gel and purified using the QIAquick PCR Purification Kit (QIAGEN, Hilden, Germany). Bi-directional sequencing of the purified amplicons was performed using the Big Dye terminator cycle sequencing kit (Applied Biosystems, Foster City, CA, USA) and an automatic capillary DNA sequencer (API Prism 310; Applied Biosystems). The quality of all sequences was visually inspected using Vector NTI Advance 9.0 (Invitrogen Life Science Software, MD, USA). The identities of the gene sequences were confirmed by searching the international sequence databases using the Blast program (URL: http://www.ncbi.nlm.nih.gov/blast/). Results Identification of mutations
PCR amplicons of the expected size were generated from all DNA samples extracted from blood (n = 30) and from paraffinembedded tissue (n = 14). No samples had mutations in exons 3, 9, and 11. Interestingly and for the first time reported here, one alteration was found in exon 4 in nine blood samples and corresponding tissue samples. This alteration (position 300 T>C) implied the shift from triplet CAT to CAC, both encoding for the same amino acid, histidine. With one exception, all patients were homozygotic for both the wildtype (GenBank accession number: NT_006051.18) and the alteration (Fig. 1). Discussion Fig. 1. Representative partial chromatograms of exon 4 of the SH3BP2 gene showing at position 300 a T>C alteration. n = number of patients.
Although the aetiology of CGCG remains unknown, the similar histological features of CGCG and cherubism suggest that both
853
diseases may have a similar genetic background. Since cherubism is caused by germ line mutations in the SH3BP2 gene18,21,22,27 the authors searched for possible SH3BP2 mutations in CGCG patients. LIETMAN et al.19 analysed 10 giant cell bone tumours and nine giant cell reparative granulomas. The authors did not find any mutation in exon 9 of SH3BP2 and showed that the SH3BP2 transcript and protein were produced abundantly. In one case, they found a smaller (but not further explained) SH3BP2 transcript in granuloma cells, leading to a truncated protein, and they concluded that a deletion mutation in SH3BP2 outside exon 9 must exist. UEKI et al.28 suggested that the SH3BP2 gene is involved in the regulation of osteoblast and osteoclast activities. As a consequence, mutations in this gene may lead to a dysfunction interfering with the regulatory pathway of osteoclastogenesis (e.g. by a pathological activation of osteoclasts)11,22. IDOWU et al.12 and DE LANGE et al.7 analysed 15 and four patients, respectively, and also found no mutation in exon 9. In 2003, an exon 9 associated mutation in the SH3BP2 gene was described in a case of nonfamilial cherubism13 and more recently an exon 3 associated mutation in one aggressive case of sporadic cherubism2 as well as an exon 4 associated mutation3. Studying four patients with CGCG lesions in the lower jaw, the same group found a somatic mutation in exon 11 in one patient3. As this patient had a nonaggressive small unilocular radiolucent lesion, this mutation could be related to a specific phenotype, but the data were too scarce to draw definite conclusions. The authors performed a more comprehensive study by searching for mutations in all four exons previously found to be involved and by including a higher number of patients and phenotypes (i.e. 30 patients with aggressive or nonaggressive lesions and with different localisation in the jaws and different radiological findings). In particular, this study included, amongst others, 10 patients with a similar phenotype to that linked with a somatic mutation in exon 113. The absence of the same mutation in the present patients (tissue and blood) indicates that a strict relationship between mutation and phenotype seems not to exist. It remains unclear whether spontaneous mutations (i.e. in exon 11) in tissue could be responsible for the development of CGCG. The authors found one codon alteration in exon 4 in the blood samples of nine patients but, as a third position mutation, with no consequence at the amino acid
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level as it still translated to histidine. This alteration (as well as the wildtype version) was identical for both alleles (homozygotic) in all but one patient (Fig. 1). The high prevalence of this alteration suggests that it is relatively preserved but whether characteristic for different ethnic groups could not be ascertained. Even though tissue samples from only 14 of 30 patients were investigated in this study, the lack of mutations, strongly indicates no role for the exons analysed in the development of CGCG. The authors thus consider these 14 tissue samples as sufficiently representative. The authors have shown that all exons previously implicated with cherubism were not mutated in the present 30 patients with CGCG. Given the Mendelian nature of cherubism and its phenotypical similarity to CGCG it is important to expand the search for possible mutations to exons in SH3BP2 not investigated so far but also to consider other proteins that may interact with SH3BP2.
4.
5.
6.
7.
8.
Competing interests
None declared.
9.
Funding
This work was supported by the Brazilian grant agencies CAPES (BEX 2632/08-0) and the DAAD (Deutscher Akademischer Austausch Dienst).
10.
11.
Ethical approval
Approved by Ethical Committee from the University of Sa˜o Paulo, Brazil. 12.
Acknowledgement. The authors thank Ilse Seyfarth for various forms of assistance.
References 1. Bell SM, Shaw M, Jou YS, Myers RM, Knowles MA. Identification and characterization of the human homologue of SH3BP2, an SH3 binding domain protein within a common region of deletion at 4p16.3 involved in bladder cancer. Genomics 1997: 44: 163–170. 2. Carvalho VM, Perdigao PF, Pimenta FJ, de Souza PE, Gomez RS, De Marco L. A novel mutation of the SH3BP2 gene in an aggressive case of cherubism. Oral Oncol 2008: 44: 153–155. 3. Carvalho VM, Perdigao PF, Amaral FR, de Souza PE, De Marco L, Gomez RS. Novel mutations in the SH3BP2 gene associated with sporadic central giant cell
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lesions and cherubism. Oral Dis 2009: 15: 106–110. Chuong R, Kaban LB, Kozakewich H, Perez-Atayde A. Central giant cell lesions of the jaws: a clinicopathologic study. J Oral Maxillofac Surg 1986: 44: 708–713. de Lange J, Van den Akker HP. Clinical and radiological features of central giant-cell lesions of the jaw. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2005: 99: 464–470. de Lange J, van den Akker HP, Klip H. Incidence and disease-free survival after surgical therapy of central giant-cell granulomas of the jaw in The Netherlands 1990–1995. Head Neck 2004: 26: 792– 795. de Lange J, van Maarle MC, van den Akker HP, Redeker EJ. DNA analysis of the SH3BP2 gene in patients with aggressive central giant cell granuloma. Br J Oral Maxillofac Surg 2007: 45: 499– 500. de Lange J, van Maarle MC, van den Akker HP, Redeker EJW. A new mutation in the SH3BP2 gene showing reduced penetrance in a family affected with cherubism. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2007: 103: 378–381. Flanagan AM, Nui B, Tinkler SM, Horton MA, Williams DM, Chambers TJ. The multinucleate cells in giant cell granulomas of the jaw are osteoclasts. Cancer 1988: 62: 1139–1145. Foucault I, Liu YC, Bernard A, Deckert M. The chaperone protein 143-3 interacts with 3BP2/SH3BP2 and regulates its adapter function. J Biol Chem 2003: 278: 7146–7153. Hyckel P, Berndt A, Schleier P, Clement JH, Beensen V, Peter H, Kosmehl H. Cherubism—new hypotheses on pathogenesis and therapeutic consequences. J Craniomaxillofac Surg 2005: 33: 61–68. Idowu BD, Thomas G, Frow R, Diss TC, Flanagan AM. Mutations in SH3BP2, the cherubism gene, were not detected in central or peripheral giant cell tumours of the jaw. Br J Oral Maxillofac Surg 2008: 46: 229–230. Imai Y, Kanno K, Moriya T, Kayano S, Seino H, Matsubara Y, Yamada A. A missense mutation in the SH3BP2 gene on chromosome 4p16.3 found in a case of nonfamilial cherubism. Cleft Palate Craniofac J 2003: 40: 632–638. Jaffe HL. Giant-cell reparative granuloma, traumatic bone cyst, and fibrous (fibro-oseous) dysplasia of the jawbones. Oral Surg Oral Med Oral Pathol 1953: 6: 159–175. Jones WA. Familial multilocular cystic disease of the jaws. Am J Cancer 1933: 17: 946–950. Jones AC, McGuff HS. Oral and maxillofacial pathology. Case of the month:
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cherubism. Tex Dent J 2009: 126: 268– 269. Kerley TR, Schow CE. Central giant cell granuloma or cherubism. Report of a case. Oral Surg Oral Med Oral Pathol 1981: 51: 128–130. Lietman SA, Kalinchinko N, Deng X, Kohanski R, Levine MA. Identification of a novel mutation of SH3BP2 in cherubism and demonstration that SH3BP2 mutations lead to increased NFAT activation. Hum Mutat 2006: 27: 717–718. Lietman SA, Prescott NL, Hicks DG, Westra WH, Levine MA. SH3BP2 is rarely mutated in exon 9 in giant cell lesions outside cherubism. Clin Orthop Relat Res 2007: 459: 22–27. Lo B, Faiyaz-Ul-Haque M, Kennedy S, Aviv R, Tsui LC, Teebi AS. Novel mutation in the gene encoding c-Ablbinding protein SH3BP2 causes cherubism. Am J Med Genet A 2003: 121A: 37– 40. Mangion J, Rahman N, Edkins S, Barfoot R, Nguyen T, Sigurdsson A, Townend JV, Fitzpatrick DR, Flanagan AM, Stratton MR. The gene for cherubism maps to chromosome 4p16.3. Am J Hum Genet 1999: 65: 151–157. Miah SM, Hatani T, Qu X, Yamamura H, Sada K. Point mutations of 3BP2 identified in human-inherited disease cherubism result in the loss of function. Genes Cells 2004: 9: 993–1004. Peters WJ. Cherubism: a study of twenty cases from one family. Oral Surg Oral Med Oral Pathol 1979: 47: 307–311. Ren R, Mayer BJ, Ciccheti P, Baltimore D. Identification of a ten-amino acid proline-rich SH3 binding site. Science 1993: 259: 1157–1161. Riefkohl R, Georgiade GS, Georgiade NG. Cherubism. Ann Plast Surg 1985: 14: 85–90. Singh-Wissmann K, Ferry JG. Transcriptional regulation of the phosphotransacetylase-encoding and acetate kinaseencoding genes (pta and ack) from Methanosarcina thermophila. J Bacteriol 1995: 177: 1699–1702. Ueki Y, Tiziani V, Santanna C, Fukai N, Maulik C, Garfinkle J, Ninomiya C, Doamaral C, Peters H, Habal M, Rhee-Morris L, Doss JB, Kreiborg S, Olsen BR, Reichenberger E. Mutations in the gene encoding c-Abl-binding protein SH3BP2 cause cherubism. Nat Genet 2001: 28: 125–126. Ueki Y, Lin CY, Senoo M, Ebihara T, Agata N, Onji M, Saheki Y, Kawai T, Mukherjee PM, Reichenberger E, Olsen BR. Increased myeloid cell responses to M-CSF and RANKL cause bone loss and inflammation in SH3BP2 ‘‘cherubism’’ mice. Cell 2007: 128: 71–83. Yamaguchi T, Dorfman HD, Eisig S. Cherubism: clinicopathologic features. Skeletal Radiol 1999: 28: 350–353.
Mutations in CGCG Address: Renata Cordeiro Teixeira Faculdade de Odontologia de Bauru – FOB/ USP
Departamento de Estomatologia Al. Dr. Octa´vio Pinheiro Brizolla 3 Cep: 17012 901 Bauru SP
Brazil Tel: +55 14 32358241 E-mail: [email protected]
855
Research Paper Genetics
SH3BP2-encoding exons involved in cherubism are not associated with central giant cell granuloma R. C. Teixeira, H. P. Horz, J. H. Damante, G. P. Garlet, C. F. Santos, R. L. M. Nogueira, R. B. Cavalcante, G. Conrads: SH3BP2-encoding exons involved in cherubism are not associated with central giant cell granuloma. Int. J. Oral Maxillofac. Surg. 2011; 40: 851–855. # 2011 International Association of Oral and Maxillofacial Surgeons. Published by Elsevier Ltd. All rights reserved. Abstract. Central giant cell granuloma (CGCG) is a benign lesion with unpredictable biological behaviour ranging from a slow-growing asymptomatic swelling to an aggressive lesion associated with pain, bone and root resorption and also tooth displacement. The aetiology of the disease is unclear with controversies in the literature on whether it is mainly of reactional, inflammatory, infectious, neoplasic or genetic origin. To test the hypothesis that mutations in the SH3BP2 gene, as the principal cause of cherubism, are also responsible for, or at least associated with, giant cell lesions, 30 patients with CGCG were recruited for this study and subjected to analysis of germ line and/or somatic alterations. In the blood samples of nine patients, one codon alteration in exon 4 was found, but this alteration did not lead to changes at the amino acid level. In conclusion, if a primary genetic defect is the cause for CGCG it is either located in SH3BP2 gene exons not yet related to cherubism or in a different gene.
The central giant cell granuloma (CGCG) was first described by JAFFE in 195314, as a giant-cell reparative granuloma of the jaw bones. It is an intra-osseous lesion resulting from a local reparative reaction of unknown aetiology. It occurs mainly in the mandible in patients aged 10–25 years and in most cases the appearance is a problem and causes the patient to seek treatment3,7. The World Health Organization (WHO) has defined CGCG as an intra-osseous lesion consisting of cellular fibrous tissue 0901-5027/080851 + 05 $36.00/0
that contains multiple foci of haemorrhage, aggregations of multinucleated giant cells and occasional trabeculae of woven bone with an incidence rate of 1.1/ million population/year6. The histological appearance of CGCG is similar to that of brown tumours seen in hyperparathyroidism, which should be excluded by performing differential diagnostic laboratory analysis. The true nature of CGCG remains speculative and there is considerable controversy in the literature4,5.
R. C. J. H. C. F. R. B.
Teixeira1,2, H. P. Horz1, Damante2, G. P. Garlet3, Santos3, R. L. M. Nogueira4, Cavalcante5, G. Conrads1
1 Division of Oral Microbiology and Immunology, Department of Operative and Preventive Dentistry & Periodontology, and Department of Medical Microbiology, Medical Faculty, RWTH Aachen University, Germany; 2 Department of Stomatology, Bauru School of Dentistry, University of Sa˜o Paulo, Bauru, Sa˜o Paulo, Brazil; 3Department of Oral Biology, Bauru School of Dentistry, University of Sa˜o Paulo, Bauru, Sa˜o Paulo, Brazil; 4Oral and Maxillofacial Surgery, Federal University of Ceara´, Fortaleza, Ceara´, Brazil; 5Department of Oral Pathology, University of Fortaleza, Fortaleza, Ceara´, Brazil
Key words: central giant cell granuloma; SH3BP2-encoding exons; cherubism. Accepted for publication 8 April 2011 Available online 16 June 2011
Conversely, cherubism (Mendelian Inheritance in Man (MIM) code 118400) was thought to be a familial disease affecting 100% of males and up to 70% of females8. In a study of a Turkish family, 8 DE LANGE et al. showed that in one male and one female the mutation was present without any present or past signs and symptoms of cherubism. This could indicate that, contrary to earlier literature, the mutation described does not have 100% penetrance in males.
# 2011 International Association of Oral and Maxillofacial Surgeons. Published by Elsevier Ltd. All rights reserved.
852
Teixeira et al.
Table 1. Anamnestic details of CGCG patients subjected to this study. Clinical findings
Nonaggressive (16)
Aggressive (14)
Average age Sex M:F Location Mandible Maxilla Pain Rapid growth Root resorption Root displacement Cortical bone destruction
17.41 (6–29) 7:9
13.54 (5–23) 7:7
11 5 – – – 9 –
7 7 7 11 8 10 11
Cherubism is a painless, disfiguring disease primarily affecting the bones of the jaw. It was first described by JONES in 193315 but there are numerous subsequent reports11,16,23,25. Most of the reported cases of cherubism are hereditary with a Mendelian dominant mode of inheritance29. In the few reported cases of nonfamilial, sporadic, cherubism13 the differentiation from CGCG is even more difficult given that the clinical and histological features are highly similar9,17,26 and that the genetic cause is not obvious. The chromosomal mutation responsible for cherubism is located at position 4p16.321 encoding for the adapter protein SH3BP2 (Src Homology-3 Binding Protein-2)21,22,27, which was found capable of binding c-Abl via its SH3-domain1,10,24. UEKI et al.27 showed that this protein is expressed in the multinucleated stromal cells of soft fibrous tissue from cherubism lesions. The gene SH3BP2 consists of 13 exons and the mutations most frequently found in cherubism are located in exon 920,27. In a few case reports, other mutations were found in exons 3 or 42,3. It has been hypothesized that mutations in exon 9 may be responsible for CGCG as well, because of the strong clinical, radiological and histological correlation between cherubism and CGCG. In three different studies, no single mutation was found in exon 98,12,19. Instead, one somatic mutation in exon 11 was most recently reported for one patient in another study3. The above findings were either focused on a few patients and/or on a few exons, so the possibility of a manifest mutation (germ line or somatic) in the SH3BP2 gene as a possible cause for CGCG remains unclear. The aim of this study was to analyse all exons previously associated with cherubism and CGCG (i.e. exons 3, 4, 9 and 11) by testing 30 patients with different phenotypes of CGCG. Materials and methods
Thirty patients attending for treatment in the Batista Hospital, Fortaleza-CE-Brazil,
with a confirmed diagnosis of CGCG, were recruited. Patients gave their written informed consent and their inclusion was carried out in accordance with the Ethics Committee of the Bauru School of Dentistry, University of Sa˜o Paulo. The inclusion/exclusion criteria adopted were: discard cherubism and hyperparathyroidism brown tumour using analysis of familial history; clinical, radiographic and laboratory examinations (parathormone, alkaline phosphatase, calcium and potassium levels) and ascertain that sufficient data are available to classify the lesion as aggressive or nonaggressive (cortical destruction or expansion, uni- or multilocular images, presence of tooth dislocation and or dental resorption). The 30 CGCG patients (14 male, 16 female) recruited for this study were subjected to a detailed clinical and radiological examination and classified according to the clinical behaviour of the disease. The age of the patients ranged from 5 to 29 years (Table 1). The diagnosis was verified by clinical, laboratory, radiographic and histopathological examinations and all patients were submitted to the inclusion/exclusion criteria adopted. Of 30 patients, 12 presented lesions in the maxilla and 18 in the mandible. The posterior region of the mandible seemed to be more frequently affected with 13 cases. Fourteen patients presented aggressive lesions and 16 were classified as nonaggressive, according to the criteria established by CHUONG et al.4 (Table 1). Sample collection and DNA isolation
Peripheral venous blood samples were obtained from all 30 patients. From 14 patients, lesion tissue (fixed and embedded) could be acquired and it was subjected to genetic analysis. Tissue samples were not available from the other patients at this experimental phase of the study. For peripheral venous blood samples, genomic DNA was extracted using a QIAmp DNA Mini kit according to the manufacturer’s instructions. DNA
samples were frozen at 70 8C and transported on dry ice by an overnight delivery service to the Division of Oral Microbiology and Immunology, RWTH Aachen University Hospital, Aachen, Germany, where genetic analyses were performed. Formalin-fixed, paraffin-embedded (FFPE) tissue samples were also transported to the same institution, where DNA was extracted and purified with a QIAmp DNA Mini kit but using the ‘tissue protocol’ (QIAGEN, Hilden, Germany) according to the manufacturer’s instructions, with one modification: 0.8 g of zirconia–silica beads (0.1 mm in diameter; BioSpec, Bartlesville, OK, USA) was added prior to the addition of Proteinase K. Samples were then agitated in a FastPrep FP 120 instrument (Qbiogene, Carlsbad, CA, USA) at 6.5 m/s for 45 s. All further steps followed the original protocol. Polymerase chain reaction (PCR) amplification of initial DNA extracts from the FFPE tissues was not successful, so 10 ml of the DNA extracts were loaded on a 1% agarose-gel stained with ethidium bromide. After electrophoresis a DNA band of high molecular weight was excised out of the gel and the DNA subsequently purified using the QIAquick Gel Extraction Kit (QIAGEN, Hilden, Germany) according to the manufacturer’s instructions. The DNA concentration (A260) and purity (A260/A280) were calculated using a Gene Quant II photometer (Pharmacia Biotech, Cambridge, England). PCR conditions
Four pairs of primers deduced from the SH3BP2 gene sequence targeting exons 3, 4, 9 and 11 were used in this study (Table 2). For PCR, each reaction mixture contained 0.5 U of Taq DNA polymerase supplied with PCR buffer (Roche Applied Science, Penzberg, Germany), 0.2 mM dNTPs (Roche Applied Science, Penzberg, Germany), 0.5 mM of each primer, and 1 ml of template DNA (approximately 25 ng). Amplification was performed in a total volume of 50 ml in PCR reaction tubes using an Eppendorf Mastercycler (Eppendorf, Hamburg, Germany). The PCR cycling conditions were: 94 8C for 1 min, followed by 37 cycles at 94 8C for 30 s, 57 8C for 40 s, and 72 8C for 50 s, ending with a single 10-min extension step at 72 8C13. With these conditions, no product was obtained from blood sample DNA in about 32% of cases (38 out of 120 total runs). In these cases, the annealing temperature was decreased
Mutations in CGCG Table 2. Primers for amplifying SH3BP2 exons (IMAI et al.13) used in this study. Size of PCR product (bp)
Primer
Primer sequence
Exon3F Exon3R
50 -AGGGTCCTCACAGTGGGTCTT-30 50 -GCTTCGGGAAAGCACTTTCC-30
289
Exon4F Exon4R
50 -GGCCATCCCTATAGCTGTGA-30 50 -ACACCCCCAGTCACCTTGCA-30
214
Exon9F Exon9R
50 -CCGTGTCTGACAGTGAAATGG-30 50 -ATGGCTTTCCCACCACCTGT-30
222
Exon11F Exon11R
50 -AAGGTCCGTGTGAAAGCTGCC-30 50 -ACAGCCTGGGTGTGTGGAGA-30
201
to 54 8C, which allowed successful PCR amplification. DNA from a few tissue samples that could not be amplified using the above conditions was subjected to a real time PCR using a LightCycler 2.0 (Roche, Penzberg, Germany) with the following conditions: 95 8C for 10 min, followed by
[(Fig._1)TD$IG]
50 cycles at 95 8C for 10 s, 56 8C for 10 s, and 72 8C for 20 s.
Sequence analysis
Aliquots of the amplicons (10 ml) were examined by electrophoresis on a 1% agarose-gel and purified using the QIAquick PCR Purification Kit (QIAGEN, Hilden, Germany). Bi-directional sequencing of the purified amplicons was performed using the Big Dye terminator cycle sequencing kit (Applied Biosystems, Foster City, CA, USA) and an automatic capillary DNA sequencer (API Prism 310; Applied Biosystems). The quality of all sequences was visually inspected using Vector NTI Advance 9.0 (Invitrogen Life Science Software, MD, USA). The identities of the gene sequences were confirmed by searching the international sequence databases using the Blast program (URL: http://www.ncbi.nlm.nih.gov/blast/). Results Identification of mutations
PCR amplicons of the expected size were generated from all DNA samples extracted from blood (n = 30) and from paraffinembedded tissue (n = 14). No samples had mutations in exons 3, 9, and 11. Interestingly and for the first time reported here, one alteration was found in exon 4 in nine blood samples and corresponding tissue samples. This alteration (position 300 T>C) implied the shift from triplet CAT to CAC, both encoding for the same amino acid, histidine. With one exception, all patients were homozygotic for both the wildtype (GenBank accession number: NT_006051.18) and the alteration (Fig. 1). Discussion Fig. 1. Representative partial chromatograms of exon 4 of the SH3BP2 gene showing at position 300 a T>C alteration. n = number of patients.
Although the aetiology of CGCG remains unknown, the similar histological features of CGCG and cherubism suggest that both
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diseases may have a similar genetic background. Since cherubism is caused by germ line mutations in the SH3BP2 gene18,21,22,27 the authors searched for possible SH3BP2 mutations in CGCG patients. LIETMAN et al.19 analysed 10 giant cell bone tumours and nine giant cell reparative granulomas. The authors did not find any mutation in exon 9 of SH3BP2 and showed that the SH3BP2 transcript and protein were produced abundantly. In one case, they found a smaller (but not further explained) SH3BP2 transcript in granuloma cells, leading to a truncated protein, and they concluded that a deletion mutation in SH3BP2 outside exon 9 must exist. UEKI et al.28 suggested that the SH3BP2 gene is involved in the regulation of osteoblast and osteoclast activities. As a consequence, mutations in this gene may lead to a dysfunction interfering with the regulatory pathway of osteoclastogenesis (e.g. by a pathological activation of osteoclasts)11,22. IDOWU et al.12 and DE LANGE et al.7 analysed 15 and four patients, respectively, and also found no mutation in exon 9. In 2003, an exon 9 associated mutation in the SH3BP2 gene was described in a case of nonfamilial cherubism13 and more recently an exon 3 associated mutation in one aggressive case of sporadic cherubism2 as well as an exon 4 associated mutation3. Studying four patients with CGCG lesions in the lower jaw, the same group found a somatic mutation in exon 11 in one patient3. As this patient had a nonaggressive small unilocular radiolucent lesion, this mutation could be related to a specific phenotype, but the data were too scarce to draw definite conclusions. The authors performed a more comprehensive study by searching for mutations in all four exons previously found to be involved and by including a higher number of patients and phenotypes (i.e. 30 patients with aggressive or nonaggressive lesions and with different localisation in the jaws and different radiological findings). In particular, this study included, amongst others, 10 patients with a similar phenotype to that linked with a somatic mutation in exon 113. The absence of the same mutation in the present patients (tissue and blood) indicates that a strict relationship between mutation and phenotype seems not to exist. It remains unclear whether spontaneous mutations (i.e. in exon 11) in tissue could be responsible for the development of CGCG. The authors found one codon alteration in exon 4 in the blood samples of nine patients but, as a third position mutation, with no consequence at the amino acid
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level as it still translated to histidine. This alteration (as well as the wildtype version) was identical for both alleles (homozygotic) in all but one patient (Fig. 1). The high prevalence of this alteration suggests that it is relatively preserved but whether characteristic for different ethnic groups could not be ascertained. Even though tissue samples from only 14 of 30 patients were investigated in this study, the lack of mutations, strongly indicates no role for the exons analysed in the development of CGCG. The authors thus consider these 14 tissue samples as sufficiently representative. The authors have shown that all exons previously implicated with cherubism were not mutated in the present 30 patients with CGCG. Given the Mendelian nature of cherubism and its phenotypical similarity to CGCG it is important to expand the search for possible mutations to exons in SH3BP2 not investigated so far but also to consider other proteins that may interact with SH3BP2.
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Competing interests
None declared.
9.
Funding
This work was supported by the Brazilian grant agencies CAPES (BEX 2632/08-0) and the DAAD (Deutscher Akademischer Austausch Dienst).
10.
11.
Ethical approval
Approved by Ethical Committee from the University of Sa˜o Paulo, Brazil. 12.
Acknowledgement. The authors thank Ilse Seyfarth for various forms of assistance.
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Mutations in CGCG Address: Renata Cordeiro Teixeira Faculdade de Odontologia de Bauru – FOB/ USP
Departamento de Estomatologia Al. Dr. Octa´vio Pinheiro Brizolla 3 Cep: 17012 901 Bauru SP
Brazil Tel: +55 14 32358241 E-mail: [email protected]
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