Identification And Functional Study Of A Pathogenic Mutation In A Family With Ossifying Fibroma Of Jaw

Backgroud: Ossifying Fibroma(OF)is a rare benign fibrous bone tumor usually occurs in adolescents,mostly in the jaw,and can easily lead to tooth displacement and maxillofacial deformity.Hereditary OF is mostly reported as a concomitant phenotype of hyperparathyroidism-jaw tumor syndrome(HPT-JT).The mutation of the tumor suppressor gene Cell Division Cycle 73(CDC73)was considered to be the main pathogenic factor of this syndrome,but its specific pathogenic mechanism has not been clarified.Aim: To screen and identify the pathogenic locus of a family with simple nonsyndromic OF in the jaw,and to study its mechanism in the occurrence of the disease.Methods and Results: 1.Whole exome sequencing was used to screen candidate pathogenic genes in a family with nonsyndromic OF in the jaw,a total of 15 candidate genes were screened,and one non-coding gene was removed.Sanger sequencing was used to analyze the common genes in the family and segregation validation confirmed that heterozygous CDC73:c.2T>C(p.0)was the pathogenic variant in this family.2.The effect of CDC73:c.2T>C(p.0)mutation on protein function in the family was studied by western blotting and immunofluorescence.The results revealed that the mutation caused the transcribed m RNA to be completely unable to synthesize protein followed by down-regulated expression of CDC73 in protein level.Integrating the CDC73 mutation sites that have been reported in the Clin Var database and clearly related to OF in the jaw,it was found that all of them would cause premature termination of CDC73 protein translation.Combined with lower expression of CDC73 detected by immunohistochemistry in pathological tissues,it could be suggested that the pathogenesis of OF is closely related to the insufficient expression of complete CDC73.3.RNA interference was used to inhibit the expression of CDC73 in HEK293 cells.CCK8 and flow cytometry revealed that down-regulation of CDC73 significantly promoted cell proliferation,and inhibited cell apoptosis.4.Via CRISPR-Cas9 technology,we obtained a CDC73 heterozygous knockout cell line constructed in human osteosarcoma cell MG63.Alkaline phosphatase staining,detection and alizarin red staining experiments proved that the down-regulation of CDC73 caused by the heterozygous knockout of CDC73 could significantly inhibit the osteogenic differentiation ability of cells.Combined with bioinformatics analysis,it is confirmed that the activation of TGF-β signaling pathway plays an intermediate regulatory role in this progression,and inhibition of TGF-βsignaling pathway can restore the mineralization ability of CDC73 heterozygous knockout cells,but not the alkaline phosphatase level.5.Cdc73 knockout and c.2T>C point mutant mice were successfully constructed by CRISPR-Cas9,and it was found that both CDC73 homozygous knockout and homozygous mutation were lethal.The analysis of growth and development revealed that the weight gain of CDC73 knockout heterozygotes was significantly lower than that of wild-type mice,but no significant difference in CDC73:c.2T>C heterozygotes;while no differentin parathyroid hormone levels revealed in each type of mice,and no tumor was found in the organs including jaw,kidney and uterus.Primary bone marrow mesenchymal stem cells were extracted and detected,and it was found that CDC73 knockout heterozygotes and CDC73:c.2T>C heterozygotes showed higher Smad2/3,p-Smad2/3,and Type I collagen precursor-Collagen IA1 expression than wild type,suggested the activation of TGF-β signaling to a certain degree.In addition,up-regulated expression of P21 was detected in heterozygotes of both CDC73 knockout and mutation mice,suggesting the role of cellular senescence in OF progression.Conclusion: CDC73:c.2T>C(p.0)heterozygous variant is the causative locus in this family with nonsyndromic OF in the jaw,which mainly causes lower expression of CDC73 in protein level;down-regualtion of CDC73 inhibits the maturation and mineralization of osteoblast via activating TGF-β signaling,and eventually leads to the occurrence of OF.