Families Investigation, Genetic Analysis And Molecular Mechanism On Cherubism

Cherubism (CBM; MIM 18400) is a rare, familial disease in which the trait is transmitted in an autosomal dominant fashion , although several sporadic cases have been described. The penetrance is high, but the precise estimate will depend on whether clinical or radiological diagnostic criteria are used. Males are more commonly and more severely affected than females. Affected individuals are normal at birth. Usually, the disease manifests in early childhood (at age 2—5 years) and becomes more marked until puberty, at which time the bony lesions begin to regress. However, the distortion of the jaw during childhood leads to permanent dental abnormalities. Other complications, such as visual disturbance (due to lesions within the orbital bones) and deafness, have also been reported but are relatively rare.The characteristic radiographic findings in CBM are well-defined multilocular areas of diminished density, often very extensive, with a few irregular bony septa. In the adult, the multilocular rarefactions become replaced by sclerosis, with progressive calcification. The lesions are usually restricted to the mandibular and maxillary regions. Histopathological evaluation of the lesions shows proliferating fibrous connective tissue containing numerous multinucleated giant cells. The clinical and histological features of cherubism may sometimes present problems in diagnostic distinction from giant-cell tumor, giant-cell granuloma, ossitying fibroma, fibrous dysplasia of the jaw, and Paget's disease of bone.The pathogenesis of cherubism remains controversial. No cause and effect relationship with trauma, infection, or hemorrhage has ever been verified. In keeping with the hereditary nature of the condition, Anderson et al (1962) suggested that a genetically induced biochemical abnormality stimulates the giant cell lesion characteristic ofcherubism. Zachariades et al (1985) speculated cherubism was a manifestation of fibrous dysplasia and that it represented a perivascular fibrosis that resulted in reduced oxygenation of bone and an alteration of the mesenchyme occurring during the development of bone. Other theories presented over the years include latent hyperparathyroidism and aberration of ossification in membranous bone-producing fibrous dysplastic lesions.Recently, Tiziani et al. (1999) and Mangion et al. (1999) mapped a cherubism locus to 4pl6. Tiziani et al. (1999) used a genomewide search in a 3-generation family. Three other families affected with cherubism were also genotyped and were mapped to the same locus. The combined lod score was 4.21 at a recombination fraction of zero, and the locus spanned an interval of approximately 22 cM. In their 3-generation family, the mandible in males was more severely affected than the maxilla, whereas in females the maxilla was more severely affected. On average, the clinical onset of the disease was earlier in females (5.5 years of age) than in males (10.6 years of age). The description of the 14-year-old female proband with maxillary enlargement that started at the age of 5 included enlarged submandibular lymph nodes. Using 2 families with clinically, radiologically, and/or histologically proved cherubism, Mangion et al. (1999) also performed a genomewide linkage search and localized the gene to 4pl6.3, with a maximum multipoint lod score at 5.64. Critical meiotic recombinants placed the gene in a 3-cM interval between D4S127 and the telomere of 4p.By linkage and haplotype analysis of 12 families, Ueki et al. (2001) refined the cherubism locus to a 1.5-megabase interval between markers D4S127 and D4S115. By sequencing cDNA and genomic DNA from affected and unaffected members, they detected point mutations that caused amino acid substitutions in the SH3BP2 gene in 12 families. All mutations were in exon 9 and affected 3 amino acids within a 6-amino acid sequence (RSPPDG) located 31 to 36 amino acids upstream of the SH2 domain and 205 to 210 amino acids downstream of the SH3-binding domain. Mutations in pro418 (to leu, arg, or his) were the most common and occurred in 8 families. Other mutations resulted ingly420 being replaced by glu or arg and in arg415 being replaced by pro or gin. SH3BP2 lies within a region that is frequently deleted in individuals with Wolf-Hirschhorn syndrome. Haploinsufficiency of SH3BP2 in individuals with that syndrome does not result in cherubism or cherubism-like characteristics. This finding and the clustering of amino acid missense mutations in SH3BP2 support the hypothesis that the mutations in SH3BP2 lead to a gain of function or act in a dominant-negative manner. The onset of the abnormalities of cherubism and their organ-restricted characteristics may be related to dental developmental processes in children, when signals unique to the mandible and maxilla are transmitted through the extracellular matrix, triggered by the eruption of secondary teeth.The study includes the following three parts:Part I The aggressive form of cherubism in two Chinese families: case reports and clinicopathological featuresCherubism is a benign lesion that causes painless symmetrical enlargement of the jaws, usually with self-limiting and a familial tendency. We describe two cases of the aggressive form of cherubism in two Chinese families with unusual extremely extensive swelling in the bilateral mandible, typical pathological nature and apparent familial history. The disorder affected three and four individuals in the two families respectively. Clinical and radiographic examination manifested an outstanding over-enlarged mandible and characteristic multilocular radiolucencies. Histopathological findings showed that, besides varying numbers of multinucleated giant cells in a stroma of fibroblasts and the eosinophilic cuffing surrounding some vessels, actively proliferating areas with clear mitoschisis and rich fibroblasts as well as relative dormant areas with loose fibrous tissue and bone were also presented in microscopic fields of the lesion. Aesthetic and functional aspects were greatly improved by surgical operation on the case.Part II Expression of c-Src and comparison of cytologic features in cherubism, central giant cell granuloma and giant cell tumorCherubism is a rare hereditary multinuclear giant cell (MGC)-rich lesion of the jaws.Similar areas of these cells accumulation can also be seen in central giant cell granuloma (CGCG) and giant cell tumor (GCT). The nature and the pathogenesis of the cytologic components in these lesions are uncertain. The aim of this study is to clarify the osteoclastic features of the MGCs in CBM and CGCG of the jaw bone, as well as in GCT of the long bone. The patients of 12 CBM, 24 CGCGs in the jaw bones and 37 GCTs in the long bones were diagnosed by the clinical and histologic features. Histromorphometric differences in MGCs were compared among these three lesions. Tissue specimens of these cases were examined by enzyme histochemical staining for tartrate-resistant acid phosphatase (TRAP), immunohistochemistry and in situ hybridization for the expression of c-Src in protein and mRNA level. The ages of CBM were significant younger than those of CGCGs and GCTs. There are no quantitative differences among the lesions at the histological level, and all of cases were considerable variation in the size of the giant cell, even within the same patient. Expression of c-Src mRNA and protein was detected in these samples that both the MGCs and a fraction of mononuclear cells were strongly positive. Enzyme histochemical study showed that the c-src positive cells were strong staining by TRAP, no matter whether in CBM and CGCG of the jaw or in GCT of the long bone. These results suggest that MGCs in the two types of the jaws and one type of the long bone expressed the phenotypic characteristics of osteoclasts. Expression of c-Src in these MGCs-rich lesions indicates that this factor might locally regulate bone metabolism in CBM, CGCG and GCT.Part HI Expression of c-Abl and mutation detection of c-Abl-binding SH3BP2 gene in cherubismThe latest genetic research has mapped cherubism (CBM) to chromosome 4pl6.3. Furthermore, point mutations in the gene encoding c-Abl-binding protein SH3BP2 were demonstrated. The aim of this study is to elucidate whether a mutation in SH3BP2 gene in Chinese familial cherubism . Expression of c-Abl is analyzed to clarify the nature of lesions from this part. Mutation of SH3BP2 gene was detected by PCR and DNA sequencing in two familial cherubism, 12 cases of CBM were examined byimmunohistochemistry for the expression of c-Abl and enzyme histochemical study for the tartrate-resistant acid phosphatase ( TRAP).Direct DNA sequencing for SH3BP2 gene was carried out in 5 affected and 20 unaffected with CBM within two families. Mutational alteration was not detected at exon 1-13 or exon-intron boundaries of SH3BP2 gene in any of these 25 individuals. Immunochistochemical and enzyme histochemical studies showed that both the multinuclear giant cells and a fraction of mononuclear cells in the lesion were strongly positive for c-Abl and TRAP.Exon 9 in SH3BP2 gene is not the mutational hotspots of Chinese familial patients with CBM. Expression of cytoplasmic c-Abl production in the multinuclear giant cells and a fraction of mononuclear cells suggests that this factor might be involved in osteoclastic differentiation, activiation and locally regulate bone metabolism in cherubism.