PARsylation-mediated ubiquitylation: lessons from rare hereditary disease Cherubism.

Modification of proteins by ADP-ribose (PARsylation) is catalyzed by the poly(ADP-ribose) polymerase (PARP) family of enzymes exemplified by PARP1, which controls chromatin organization and DNA repair. Additionally, PARsylation induces ubiquitylation and proteasomal degradation of its substrates because PARsylation creates a recognition site for E3-ubiquitin ligase. The steady-state levels of the adaptor protein SH3-domain binding protein 2 (3BP2) is negatively regulated by tankyrase (PARP5), which coordinates ubiquitylation of 3BP2 by the E3-ligase ring finger protein 146 (RNF146). 3BP2 missense mutations uncouple 3BP2 from tankyrase-mediated negative regulation and cause Cherubism, an autosomal dominant autoinflammatory disorder associated with craniofacial dysmorphia. In this review, we summarize the diverse biological processes, including bone dynamics, metabolism, and Toll-like receptor (TLR) signaling controlled by tankyrase-mediated PARsylation of 3BP2, and highlight the therapeutic potential of this pathway.

Molecular and cellular characterizations of human cherubism: disease aggressiveness depends on osteoclast differentiation.

BACKGROUND: Cherubism is a rare autosomal dominant disorder of the jaws caused by mutation of the SH3BP2 gene. The bone is replaced by a fibrous granuloma containing multinucleated giant cells. Cells of the cherubism granuloma have never been systematically analyzed. Hence, the aim of this study was to characterize the cells in human cherubism granulomas, to determine the osteoclastic characteristics of the multinucleated giant cells and to investigate the potential role of TNF-α in human cherubism. RESULTS: Seven granulomas were analyzed in pathology, molecular biology and immunohistochemistry. Granulomas were composed mainly of macrophages or osteoclasts within a fibroblastic tissue, with few lymphoid cells. Myeloid differentiation and nuclear NFATc1 localization were both associated with disease aggressiveness. OPG and RANKL immunohistochemical expression was unexpected in our specimens. Five granuloma cells were cultured in standard and osteoclastogenic media. In culture, cherubism cells were able to differentiate into active osteoclasts, in both osteoclastogenic and standard media. IL-6 was the major cytokine present in the culture supernatants. CONCLUSION: Multinucleated giant cells from cherubism granulomas are CD68 positive cells, which differentiate into macrophages in non-aggressive cherubism and into osteoclasts in aggressive cherubism, stimulated by the NFATc1 pathway. This latter differentiation appears to involve a disturbed RANK-L/RANK/OPG pathway and be less TNF-α dependent than the cherubism mouse model.

Reciprocal stabilization of ABL and TAZ regulates osteoblastogenesis through transcription factor RUNX2.

Cellular identity in metazoan organisms is frequently established through lineage-specifying transcription factors, which control their own expression through transcriptional positive feedback, while antagonizing the developmental networks of competing lineages. Here, we have uncovered a distinct positive feedback loop that arises from the reciprocal stabilization of the tyrosine kinase ABL and the transcriptional coactivator TAZ. Moreover, we determined that this loop is required for osteoblast differentiation and embryonic skeletal formation. ABL potentiated the assembly and activation of the RUNX2-TAZ master transcription factor complex that is required for osteoblastogenesis, while antagonizing PPARγ-mediated adipogenesis. ABL also enhanced TAZ nuclear localization and the formation of the TAZ-TEAD complex that is required for osteoblast expansion. Last, we have provided genetic data showing that regulation of the ABL-TAZ amplification loop lies downstream of the adaptor protein 3BP2, which is mutated in the craniofacial dysmorphia syndrome cherubism. Our study demonstrates an interplay between ABL and TAZ that controls the mesenchymal maturation program toward the osteoblast lineage and is mechanistically distinct from the established model of lineage-specific maturation.

Defining a new aggressiveness classification and using NFATc1 localization as a prognostic factor in cherubism.

Cherubism is a rare genetic disease characterized by bilateral giant cell reparative granuloma of the jaws consisting of a fibrotic stroma with giant multinucleated cells (GMCs) and osteoclastic features. Cherubism severity is highly variable, and recurrence after surgery is the most important risk. Currently, there are no prognostic indicators. The aims of this study were to evaluate the osteoclastogenesis phenotype by histologic examination of nuclear factor of activated T cells 1 (NFATc1) localization and tartrate-resistant acid phosphatase (TRAP) activity and to correlate the results to disease aggressiveness to define prognostic indicators. Based on cherubism evolution 1 year after surgery, 3 classes of cherubism aggressiveness were identified: mild (group A), moderate (group B), and severe (group C). Histologically, in grade A and B cherubism lesions, GMCs were negative for both TRAP activity and NFATc1 nuclear localization. In contrast, in grade C cherubism lesions, GMCs were all positive for TRAP activity and NFATc1 nuclear localization and displayed osteoclast-like features. Other histopathologic findings were not different among the 3 groups. Our results establish that TRAP activity and NFTAc1 nuclear localization are associated with aggressive cherubism and therefore could be added to routine pathologic examination to aid in prognosis and management of the disease. The finding of NFATc1 nuclear localization in aggressive tumors supports the addition of anticalcineurin treatment to the therapeutic arsenal for cherubism.

[Molecular and Cellular Pathogenesis of Cherubism].

Study of rare genetic disorder often provides fundamental insights into the pathology of common diseases. Cherubism is a rare craniofacial disorder in children characterized by the destruction of maxillary and mandibular bones due to expansile fibrous inflammatory lesions. Genetic study of cherubism families discovered that gain-of-function mutations in the signaling adaptor protein SH3BP2 are responsible for cherubism. Analysis of the mouse model revealed that cherubism is an autoinflammatory disorder that is caused by dysregulated signaling pathway mediated by toll-like receptors and spleen tyrosine kinase. Recent study of the SH3BP2-deficient mice showed that SH3BP2 plays important roles in bone resorption in mouse models of inflammatory arthritis. These results establish SH3BP2 as a key player in the osteoimmune system beyond its role in a rare inherited disorder and suggest that the signaling pathway mediated by SH3BP2 is involved in the pathogenesis of common inflammatory bone diseases such as rheumatoid arthritis.

Etanercept administration to neonatal SH3BP2 knock-in cherubism mice prevents TNF-α-induced inflammation and bone loss.

Cherubism is a genetic disorder of the craniofacial skeleton caused by gain-of-function mutations in the signaling adaptor protein, SH3-domain binding protein 2 (SH3BP2). In a knock-in mouse model for cherubism, we previously demonstrated that homozygous mutant mice develop T/B cell-independent systemic macrophage inflammation leading to bone erosion and joint destruction. Homozygous mice develop multiostotic bone lesions whereas cherubism lesions in humans are limited to jawbones. We identified a critical role of tumor necrosis factor α (TNF-α) in the development of autoinflammation by creating homozygous TNF-α-deficient cherubism mutants, in which systemic inflammation and bone destruction were rescued. In this study, we examined whether postnatal administration of an anti-TNF-α antagonist can prevent or ameliorate the disease progression in cherubism mice. Neonatal homozygous mutants, in which active inflammation has not yet developed, were treated with a high dose of etanercept (25 mg/kg, twice/week) for 7 weeks. Etanercept-treated neonatal mice showed strong rescue of facial swelling and bone loss in jaws and calvariae. Destruction of joints was fully rescued in the high-dose group. Moreover, the high-dose treatment group showed a significant decrease in lung and liver inflammatory lesions. However, inflammation and bone loss, which were successfully treated by etanercept administration, recurred after etanercept discontinuation. No significant effect was observed in low-dose-treated (0.5 mg/kg, twice/week) and vehicle-treated groups. In contrast, when 10-week-old cherubism mice with fully active inflammation were treated with etanercept for 7 weeks, even the high-dose administration did not decrease bone loss or lung or liver inflammation. Taken together, the results suggest that anti-TNF-α therapy may be effective in young cherubism patients, if treated before the inflammatory phase or bone resorption occurs. Therefore, early genetic diagnosis and early treatment with anti-TNF-α antagonists may be able to prevent or ameliorate cherubism, especially in patients with a mutation in SH3BP2.

SH3BP2 cherubism mutation potentiates TNF-α-induced osteoclastogenesis via NFATc1 and TNF-α-mediated inflammatory bone loss.

Cherubism (OMIM# 118400) is a genetic disorder with excessive jawbone resorption caused by mutations in SH3 domain binding protein 2 (SH3BP2), a signaling adaptor protein. Studies on the mouse model for cherubism carrying a P416R knock-in (KI) mutation have revealed that mutant SH3BP2 enhances tumor necrosis factor (TNF)-α production and receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclast differentiation in myeloid cells. TNF-α is expressed in human cherubism lesions, which contain a large number of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells, and TNF-α plays a critical role in inflammatory bone destruction in homozygous cherubism mice (Sh3bp2(KI/KI) ). The data suggest a pathophysiological relationship between mutant SH3BP2 and TNF-α-mediated bone loss by osteoclasts. Therefore, we investigated whether P416R mutant SH3BP2 is involved in TNF-α-mediated osteoclast formation and bone loss. Here, we show that bone marrow-derived M-CSF-dependent macrophages (BMMs) from the heterozygous cherubism mutant (Sh3bp2(KI/+) ) mice are highly responsive to TNF-α and can differentiate into osteoclasts independently of RANKL in vitro by a mechanism that involves spleen tyrosine kinase (SYK) and phospholipase Cγ2 (PLCγ2) phosphorylation, leading to increased nuclear translocation of NFATc1. The heterozygous cherubism mutation exacerbates bone loss with increased osteoclast formation in a mouse calvarial TNF-α injection model as well as in a human TNF-α transgenic mouse model (hTNFtg). SH3BP2 knockdown in RAW264.7 cells results in decreased TRAP-positive multinucleated cell formation. These findings suggest that the SH3BP2 cherubism mutation can cause jawbone destruction by promoting osteoclast formation in response to TNF-α expressed in cherubism lesions and that SH3BP2 is a key regulator for TNF-α-induced osteoclastogenesis. Inhibition of SH3BP2 expression in osteoclast progenitors could be a potential strategy for the treatment of bone loss in cherubism as well as in other inflammatory bone disorders.

New insights into the molecular and cellular functions of poly(ADP-ribose) and PARPs.

Poly(ADP-ribose) polymerases (PARPs) are enzymes that transfer ADP-ribose groups to target proteins and thereby affect various nuclear and cytoplasmic processes. The activity of PARP family members, such as PARP1 and PARP2, is tied to cellular signalling pathways, and through poly(ADP-ribosyl)ation (PARylation) they ultimately promote changes in gene expression, RNA and protein abundance, and the location and activity of proteins that mediate signalling responses. PARPs act in a complex response network that is driven by the cellular, molecular and chemical biology of poly(ADP-ribose) (PAR). This PAR-dependent response network is crucial for a broad array of physiological and pathological responses and thus is a good target for chemical therapeutics for several diseases.

Cherubism: best clinical practice.

Cherubism is a skeletal dysplasia characterized by bilateral and symmetric fibro-osseous lesions limited to the mandible and maxilla. In most patients, cherubism is due to dominant mutations in the SH3BP2 gene on chromosome 4p16.3. Affected children appear normal at birth. Swelling of the jaws usually appears between 2 and 7 years of age, after which, lesions proliferate and increase in size until puberty. The lesions subsequently begin to regress, fill with bone and remodel until age 30, when they are frequently not detectable.Fibro-osseous lesions, including those in cherubism have been classified as quiescent, non-aggressive and aggressive on the basis of clinical behavior and radiographic findings. Quiescent cherubic lesions are usually seen in older patients and do not demonstrate progressive growth. Non-aggressive lesions are most frequently present in teenagers. Lesions in the aggressive form of cherubism occur in young children and are large, rapidly growing and may cause tooth displacement, root resorption, thinning and perforation of cortical bone.Because cherubism is usually self-limiting, operative treatment may not be necessary. Longitudinal observation and follow-up is the initial management in most cases. Surgical intervention with curettage, contouring or resection may be indicated for functional or aesthetic reasons. Surgical procedures are usually performed when the disease becomes quiescent. Aggressive lesions that cause severe functional problems such as airway obstruction justify early surgical intervention.

Loss of Tankyrase-mediated destruction of 3BP2 is the underlying pathogenic mechanism of cherubism.

Cherubism is an autosomal-dominant syndrome characterized by inflammatory destructive bony lesions resulting in symmetrical deformities of the facial bones. Cherubism is caused by mutations in Sh3bp2, the gene that encodes the adaptor protein 3BP2. Most identified mutations in 3BP2 lie within the peptide sequence RSPPDG. A mouse model of cherubism develops hyperactive bone-remodeling osteoclasts and systemic inflammation characterized by expansion of the myelomonocytic lineage. The mechanism by which cherubism mutations alter 3BP2 function has remained obscure. Here we show that Tankyrase, a member of the poly(ADP-ribose)polymerase (PARP) family, regulates 3BP2 stability through ADP-ribosylation and subsequent ubiquitylation by the E3-ubiquitin ligase RNF146 in osteoclasts. Cherubism mutations uncouple 3BP2 from Tankyrase-mediated protein destruction, which results in its stabilization and subsequent hyperactivation of the SRC, SYK, and VAV signaling pathways.

Structural basis and sequence rules for substrate recognition by Tankyrase explain the basis for cherubism disease.

The poly(ADP-ribose)polymerases Tankyrase 1/2 (TNKS/TNKS2) catalyze the covalent linkage of ADP-ribose polymer chains onto target proteins, regulating their ubiquitylation, stability, and function. Dysregulation of substrate recognition by Tankyrases underlies the human disease cherubism. Tankyrases recruit specific motifs (often called RxxPDG "hexapeptides") in their substrates via an N-terminal region of ankyrin repeats. These ankyrin repeats form five domains termed ankyrin repeat clusters (ARCs), each predicted to bind substrate. Here we report crystal structures of a representative ARC of TNKS2 bound to targeting peptides from six substrates. Using a solution-based peptide library screen, we derive a rule-based consensus for Tankyrase substrates common to four functionally conserved ARCs. This 8-residue consensus allows us to rationalize all known Tankyrase substrates and explains the basis for cherubism-causing mutations in the Tankyrase substrate 3BP2. Structural and sequence information allows us to also predict and validate other Tankyrase targets, including Disc1, Striatin, Fat4, RAD54, BCR, and MERIT40.

Enhancement of B-cell receptor signaling by a point mutation of adaptor protein 3BP2 identified in human inherited disease cherubism.

Tyrosine phosphorylation of adaptor protein c-Abl-Src homology 3 (SH3) domain-binding protein-2 (3BP2, also referred to SH3BP2) positively regulates the B-cell antigen receptor (BCR)-mediated signal transduction, leading to the activation of nuclear factor of activated T cells (NFAT). Here we showed the effect of the proline to arginine substitution of 3BP2 in which is the most common mutation in patients with cherubism (P418R) on B-cell receptor signaling. Comparing to the wild type, overexpression of the mutant form of 3BP2 (3BP2-P416R, corresponding to P418R in human protein) enhanced BCR-mediated activation of NFAT. 3BP2-P416R increased the signaling complex formation with Syk, phospholipase C-γ2 (PLC-γ2), and Vav1. In contrast, 3BP2-P416R could not change the association with the negative regulator 14-3-3. Loss of the association mutant that was incapable to associate with 14-3-3 could not mimic BCR-mediated NFAT activation in Syk-deficient cells. Moreover, BCR-mediated phosphorylation of extracellular signal regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) was not affected by P416R mutation. These results showed that P416R mutation of 3BP2 causes the gain of function in B cells by increasing the interaction with specific signaling molecules.

Cherubism gene Sh3bp2 is important for optimal bone formation, osteoblast differentiation, and function.

INTRODUCTION: Cherubism is a human genetic disorder that causes bilateral symmetrical enlargement of the maxilla and the mandible in children. It is caused by mutations in SH3BP2. The exact pathogenesis of the disorder is an area of active research. Sh3bp2 knock-in mice were developed by introducing a Pro416Arg mutation (Pro418Arg in humans) in the mouse genome. The osteoclast phenotype of this mouse model was recently described. METHODS: We examined the bone phenotype of the cherubism mouse model, the role of Sh3bp2 during bone formation, osteoblast differentiation, and osteoblast function. RESULTS: We observed delays in early postnatal development of homozygous Sh3bp2(KI/KI) mice, which exhibited increased growth plate thickness and significantly decreased trabecular bone thickness and bone mineral density. Histomorphometric and microcomputed tomography analyses showed bone loss in the cranial and appendicular skeletons. Sh3bp2(KI/KI) mice also exhibited a significant decrease in osteoid formation that indicated a defect in osteoblast function. Calvarial osteoblast cell cultures had decreased alkaline phosphatase expression and mineralization, suggesting reduced differentiation potential. Gene expression of osteoblast differentiation markers such as collagen type I, alkaline phosphatase, and osteocalcin were decreased in osteoblast cultures from Sh3bp2(KI/KI) mice. CONCLUSIONS: These data suggest that Sh3bp2 regulates bone homeostasis through not only osteoclast-specific effects, but also through effects on osteoblast differentiation and function.

Unexpected matrix diseases and novel therapeutic strategies.

Within the framework of a broad definition of the extracellular matrix (ECM), this review discusses three genetic disorders in which major pathogenetic features have been traced back to alterations in the levels/activities of matrix components. In each case, disease-associated alterations are found both intra- and extracellularly. The nature of the ECM involvement is surprising, offers an exciting therapeutic opportunity, and deepens our understanding of ECM-cell interactions. The first of these disorders, cherubism, is a case of inflammatory bone loss in the jaws of children for reasons that are surprisingly systemic in nature, considering the local nature of the disease. The primary defect involves an intracellular signaling molecule, but a major pathogenetic component and therapeutic target of the disease is the extracellular cytokine tumor necrosis factor alpha. The second disorder, Knobloch syndrome, is caused by recessive mutations in collagen XVIII. Although this protein has been classified as belonging to a group of structural macromolecules, the consequence of the mutations is impairment of cellular metabolism. The third disorder, infantile hemangioma, is a common tumor of capillary endothelial cells in infancy. The tumor appears within a few days/weeks after birth, grows rapidly over several months, and regresses over several years. The proliferative phase is the result of constitutively high levels of vascular endothelial cell growth factor (VEGF)-dependent signaling through VEGF receptor 2 (VEGFR2), but recent studies have led to the surprising conclusion that abnormalities in a cell-surface receptor complex controlling expression of the VEGF decoy receptor VEGFR1 is the underlying cause.

[Gene mutation and expression of SH-3BP-2 in cherubism].

OBJECTIVE: To detect the mutation and expression of SH-3BP-2 in Chinese patients of cherubism and to investigate the possible relationship of gene mutation and multinucleated giant cells in lesions. METHODS: Genomic DNA was extracted from paraffin-imbedded tissues and peripheral blood samples of 10 cases of cherubism (6 familial cherubism and 4 sporadic cherubism). SH-3BP-2 mutations were detected by PCR-direct sequencing. The nature of multinucleated giant cells in lesions was detected by enzyme histochemical staining and immunohistochemical staining using paraffin-imbedded tissues sections. The SH-3BP-2 protein was detected by immunohistochemical staining. RESULTS: Three missense mutations (G1520A, G1505A, G1505C) in exon 9 of SH-3BP-2 were identified which led to 3 transitions (Gly420Glu, Arg415Gln, Arg415Pro). There were no abnormalities in exon 3 of SH-3BP-2 except 1 case which had not PCR products. The protein SH-3BP-2, the calcitonin receptor and the tartrate-resistant acid phosphatase were detected in the cytoplasm of all multinucleated giant cells and parts of monokaryon matrix cells in 8 paraffin-imbedded samples. CONCLUSIONS: The SH-3BP-2 mutation may participate in the differentiation and maturation of osteoclast-like cells in the lesion of cherubism.

NFATc1 in mice represses osteoprotegerin during osteoclastogenesis and dissociates systemic osteopenia from inflammation in cherubism.

Osteoporosis results from an imbalance in skeletal remodeling that favors bone resorption over bone formation. Bone matrix is degraded by osteoclasts, which differentiate from myeloid precursors in response to the cytokine RANKL. To gain insight into the transcriptional regulation of bone resorption during growth and disease, we generated a conditional knockout of the transcription factor nuclear factor of activated T cells c1 (Nfatc1). Deletion of Nfatc1 in young mice resulted in osteopetrosis and inhibition of osteoclastogenesis in vivo and in vitro. Transcriptional profiling revealed NFATc1 as a master regulator of the osteoclast transcriptome, promoting the expression of numerous genes needed for bone resorption. In addition, NFATc1 directly repressed osteoclast progenitor expression of osteoprotegerin, a decoy receptor for RANKL previously thought to be an osteoblast-derived inhibitor of bone resorption. "Cherubism mice", which carry a gain-of-function mutation in SH3-domain binding protein 2 (Sh3bp2), develop osteoporosis and widespread inflammation dependent on the proinflammatory cytokine, TNF-alpha. Interestingly, deletion of Nfatc1 protected cherubism mice from systemic bone loss but did not inhibit inflammation. Taken together, our study demonstrates that NFATc1 is required for remodeling of the growing and adult skeleton and suggests that NFATc1 may be an effective therapeutic target for osteoporosis associated with inflammatory states.

Investigation of the SH3BP2 gene mutation in cherubism.

Cherubism is a rare developmental lesion of the jaw that is generally inherited as an autosomal dominant trait. Recent studies have revealed point mutations in the SH3BP2 gene in cherubism patients. In this study, we examined a 6-year-old Korean boy and his family. We found a Pro418Arg mutation in the SH3BP2 gene of the patient and his mother. A father and his 30-month-old younger brother had no mutations. Immunohistochemically, the multinucleated giant cells proved positive for CD68 and tartrate-resistant acid phosphatase (TRAP). Numerous spindle-shaped stromal cells expressed a ligand for receptor activator of nuclear factor kB (RANKL), but not in multinucleated giant cells. These results provide evidence that RANKL plays a critical role in the differentiation of osteoclast precursor cells to multinucleated giant cells in cherubism. Additionally, genetic analysis may be a useful method for differentiation of cherubism.

Adaptor protein 3BP2 and cherubism.

The adaptor protein 3BP2 (c-Abl Src homology 3 domain-binding protein-2, also referred to SH3BP2) is known to play a regulatory role in signaling from immunoreceptors. In mast cells, 3BP2 is rapidly tyrosine phosphorylated by the aggregation of the high affinity IgE receptor and the overexpression of its SH2 domain results in the dramatic suppression of IgE-mediated tyrosine phosphorylation of PLC-alpha, Ca2+ mobilization and degranulation. 3BP2 is a substrate of the protein-tyrosine kinase Syk, which phosphorylates it on Tyr174, Tyr183, and Tyr446 (in the mouse protein). Phosphorylation of Tyr183 promotes the activation of Rac1 through the interaction with the SH2 domain of Vav1. Phosphorylation of Tyr446 induces the binding to the SH2 domain of the upstream protein-tyrosine kinase Lyn and enhances its kinase activity. Thus, 3BP2 has a positive regulatory role in IgE-mediated mast cell activation. In lymphocytes, engagement of T cell or B cell receptors triggers tyrosine phosphorylation of 3BP2. Suppression of the 3BP2 expression by siRNA results in the inhibition of T cell or B cell receptor-mediated activation of NFAT. Genetic analyses reveal that 3BP2 is required for the proliferation of B cells and B cell receptor signaling. Point mutations of the 3BP2 gene cause the rare human inherited disorder cherubism, characterized by excessive bone resorption in the jaw bones. These mutations include substitution and deletion mutations of 3BP2. "Cherubism" mice exhibit increased myeloid cell responses to M-CSF and RANKL leading to the activation of osteoclasts. Further analysis could demonstrate that inhibition of 3BP2 might have therapeutic potential.

So-called hybrid central odontogenic fibroma/central giant cell lesion of the jaws. A report on seven additional cases, including an example in a patient with cherubism, and hypotheses on the pathogenesis.

BACKGROUND: Central odontogenic fibroma (COF) is characterized by poor to cellular fibroblastic proliferation and a variable odontogenic epithelial (OE) component. Central giant cell lesions (CGCL) are osteolytic fibroblastic proliferations characterized by osteoclast-like multinucleated giant cells (MGC). Rare examples of hybrid COF/CGCL have been described. Two pathogenetic theories prevail based on clinicopathologic characteristics. One regards the CGCL component as reactive to the COF, while the other regards the CGCL as inductive of a COF-like proliferation. The possibility of colliding tumors seems unlikely. METHODS AND MATERIALS: Seven patients with hCOF/CGCL, among them one with cherubism, were studied. Immunohistochemistry for cytokeratin 19 was applied to better appreciate the epithelial component. RESULTS: Six patients were males and one female and their age ranged from 15 to 73 years old. All lesions occurred in the premolars and molars of the mandible and presented as radiolucencies with primarily well-delineated borders. All patients underwent surgical excision and recurrences have not been reported to this date in 6 out of 7 patients (mean follow-up 60.6+/-36.25 months). The COF component predominated in 3 cases and the CGCL component in 3. Zones of collagen fibers featuring a whorling pattern and containing multiple nests of OE were present. In four cases there were hyalinized deposits in OE, while some foci of MGC contained few OE. CONCLUSIONS: Gender predilection in our series is in contrast with previously published reports. However, when all previously reported cases are reviewed there is still female predilection. The predominant site, as previously reported, is the tooth-bearing areas of the posterior mandible. This is the first report of hCOF/CGCL in cherubism. The pathogenesis of hCOF/CGCG remains obscure and molecular interactions would be of interest to be investigated.