SH3BP2-encoding exons involved in cherubism are not associated with central giant cell granuloma


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 J affe in 1953 , 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 treatment .

The World Health Organization (WHO) has defined CGCG as an intra-osseous lesion consisting of cellular fibrous tissue 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/year . 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 literature .

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 females . In a study of a Turkish family, de L ange 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.

Cherubism is a painless, disfiguring disease primarily affecting the bones of the jaw. It was first described by J ones in 1933 but there are numerous subsequent reports . Most of the reported cases of cherubism are hereditary with a Mendelian dominant mode of inheritance . In the few reported cases of nonfamilial, sporadic, cherubism the differentiation from CGCG is even more difficult given that the clinical and histological features are highly similar and that the genetic cause is not obvious.

The chromosomal mutation responsible for cherubism is located at position 4p16.3 encoding for the adapter protein SH3BP2 (Src Homology-3 Binding Protein-2) , which was found capable of binding c-Abl via its SH3-domain . U eki et al. 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 9 . In a few case reports, other mutations were found in exons 3 or 4 .

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 9 . Instead, one somatic mutation in exon 11 was most recently reported for one patient in another study . 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 São 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 C huong et al. ( Table 1 ).

Table 1
Anamnestic details of CGCG patients subjected to this study.
Clinical findings Nonaggressive (16) Aggressive (14)
Average age 17.41 (6–29) 13.54 (5–23)
Sex M:F 7:9 7:7
Mandible 11 7
Maxilla 5 7
Pain 7
Rapid growth 11
Root resorption 8
Root displacement 9 10
Cortical bone destruction 11

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 °C 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 μl 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 ( A 260) and purity ( A 260/ A 280) 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 μM of each primer, and 1 μl of template DNA (approximately 25 ng). Amplification was performed in a total volume of 50 μl in PCR reaction tubes using an Eppendorf Mastercycler (Eppendorf, Hamburg, Germany).

Table 2
Primers for amplifying SH3BP2 exons (I mai et al. ) used in this study.
Primer Primer sequence Size of PCR product (bp)
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Feb 5, 2018 | Posted by in Oral and Maxillofacial Surgery | Comments Off on SH3BP2-encoding exons involved in cherubism are not associated with central giant cell granuloma
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