Subjects
A family consisting of 21 biologically related members, 3 of whom were female patients with cherubism, was identified in Northern China. Seventeen members were recruited for the present study as four male members II-8, II-1, III-1 and III-4 were missing. As shown in Figure 1, affected member II-7 was the proband of cherubism, aged 41 years, with a slowly progressive upward displacement of both eyes and an intraorbital mass bilaterally (Figure 2A). An irregular mandible was noticed at the age of 10 years. Her facial appearance was typically cherubic. Radiological examinations demonstrated bilateral multi-cystic lesions both in mandible and in maxilla compatible with cherubism. She was promptly referred to a maxillofacial service. An incisional biopsy was performed. Histological examination revealed many giant cells in a vascularized fibrous stroma. The diagnosis of cherubism was confirmed, and surgical operations, such as curettage and osteoplasty, were then performed. Although cherubism is generally self-limiting and subsides with age, the patient described here was unusual in that the orbital involvement arose in adulthood after the jaw lesions had subsided. A physical examination revealed symmetrical enlargement of the jaws, exophthalmos with diplopia and a slight upward turning of the eyes (Figure 2A). The mandibular angles were also observed (Figure 2B). Her visual activity and field were impaired. Computed tomography (CT) scans showed bilateral multi-cystic lesions in the maxilla and mandible (Figure 2C). The CT scans also revealed that the lesions consisted mainly of soft tissue masses with a ballooned cortex. Furthermore, the lesion of the both maxilla extended into the orbit and then affected the eyeball position (Figure 2C). However, there was no sign of compression of the optic canal by the lesion. Three-dimensional CT scans revealed that the symmetrically expanded anterior walls of the maxilla and the symmetrically expanded mandible had a soap bubble appearance as described previously by James and co-workers [2] (Figure 2D).
The affected member II-9 is a sister of the member II-7, aged 37 years. She was diagnosed as having cherubism at the age of 7 years and she had fibrous dysplastic tissue curetted from her bilateral mandibles. A pathological examination confirmed clusters of multinucleated giant cells within a fibrous stroma, consistent with the diagnosis of cherubism. Because she had undergone a surgery operation, her facial appearances looked well when her sample was taken for this study (Figure 3A and 3B). However, the jaw panoramic radiophotography showed partial calcifications of anterior mandible and mandibular angles (Figure 3C).
The affected member III-7 is the daughter of the member II-9, aged 14 years. She developed bilateral cystic lesions of the mandible when she was 8 or 9 years old. At the age of 9 years, there were visible lesions over the bilateral mandibular angles and radiographs showed symmetric multi-cystic lesions. Osteoplasty of the affected jaws was performed. Her facial appearances did not show severe abnormalities (Figure 4A and 4B) although the jaw panoramic radiophotography revealed symmetric multi-cystic alterations and impacted right mandibular canine teeth. Defect of left mandibular canine, second molar teeth and tooth root was also observed (Figure 4C).
To identify a disease-causing mutation, we also recruited 100 unrelated healthy subjects as control for DNA analysis.
All the subjects gave written informed consent for the present study, including clinical examinations, blood collection and genetic analysis. Written consent was also obtained from the patients and their relatives for publication of study. This study was approved by the local institutional review boards.
Sequencing of the SH3BP2 gene
Genomic DNA for the genetic analysis was extracted from peripheral venous blood samples by conventional methods. The primers used for PCR amplification of exon 9 of the gene were designed based on its DNA sequence (accession numbers NT003023 and NM006081), including 5'-TgA gCT TTT TAg ggT CAC Agg-3'and 5'-ggC TTT ACA Tgg TgC TgT gT-3'. The PCR amplification was performed in a 25-μL reaction volume containing 100 ng of genomic DNA, 2.5 μL of 10X PCRx amplification buffer, 2.5 μL of 2.5 mM of dNTP mixture, 10 μM of each primer, and 1.0 U of Taq DNA polymerase. The conditions used for the PCR amplification included denaturation at 94°C for 30 seconds, followed by 40 cycles at 94°C for 30 seconds, 62°C for 30 seconds and 72°C for 30 seconds, and final elongation at 72°C for 8 minutes. The purified PCR products were used as templates for direct sequencing by a fluorescent dye-terminator cycle sequencing method with an ABI PRISM Big Dye terminator cycle sequencing ready reaction kit (Applied Biosystems, USA). Nucleotide sequences were determined by an ABI 3730 XL automated DNA sequencer (Applied Biosystems, USA).
Identification of mutations
Genomic DNA sequencing was performed in all 17 subjects recruited in this study. A missense mutation was identified in exon 9 of the SH3BP2 gene in patients with cherubism. It was an A1517G base change, which leads to the D419G amino acid substitution. Of 17 family members, the A1517G base change was shown only in 3 affected individuals (affected members II-7, II-9 and III-7). We did not observe such a mutation in 100 unrelated controls. It is very likely that the A1517G base change is a disease-causing mutation.
