CDC73 gene mutations in sporadic ossifying fibroma of the jaws

Background The tumor suppressor gene CDC73 was found to be associated with hyperparathyroidism-jaw tumor syndrome (HPT-JT), which is characterized by parathyroid adenoma or carcinoma, ossifying fibroma (OF) of the jaws, and renal and uterine lesions. Mutations in CDC73 have also been frequently detected in sporadic parathyroid carcinomas and renal tumors. However, the prevalence and range of CDC73 mutations in sporadic OFs have not been established. Methods We directly sequenced coding and flanking splice junctional regions of CDC73 in 40 cases of sporadic OF of the jaws. We also used immunohistochemistry to detect parafibromin, the protein product of CDC73, in those cases. Results Two novel CDC73 mutations were identified in 2 of the 40 cases (5 %). Both were somatic mutations located in exon 1 of the coding region. Strong parafibromin expression was detected in all 40 cases, irrespective of the presence of CDC73 mutations. Conclusions Mutations inCDC73 were rare in sporadic OF of the jaws, but may affect the pathogenesis of a small subset of tumors of this type.


Background
According to the World Health Organization, it is proposed that benign fibro-osseous lesions be divided into 3 categories, including fibrous dysplasia, ossifying fibroma, and osseous dysplasia [1,2]. The most common forms of BFOL are ossifying fibroma (OF) and fibrous dysplasia (FD) and they manifest different clinical courses and should be treated differently [3].
Ossifying fibroma is a benign tumor thought to arise from the periodontal ligament, which potential of continuous growth if not treated [4,5]. It is a slowgrowing, generally lack of symptoms, but can cause serious cosmetic and functional problems. [6] OF can occur in almost any bone in the craniofacial region, predominantly in the premolar-molar region of the mandible, with adult women more frequently affected [7]. Radio-graphically, OF usually presents a wellcircumscribed radiolucency with varying degrees of calcification that rarely erodes or displaces teeth [4]. Ossifying fibromas are typically encountered as solitary lesions, multiple and/or familial lesions rarely present in clinic [8]. In some instances, OF can be destructive and risk for recurrence, as a result of which completely surgical enucleating is needed [9].
FDs are genetic and non-inheritable, caused by missense mutations that occur post-zygotically in the gene, GNAS, which is located on chromosome 20q13 and codes for the a-subunit of the stimulatory G-protein, Gs [10]. The mutations generate a somatic mosaic and the resulting proteins display reduced GTPase activity and, consequently, increased activation of adenylyl cyclase. Therefore, mutated cells constitutively generate high levels of cAMP and have a high rate of proliferation [11]. The differential diagnostic value of GNAS in FD is well recognized. In previous studies, our research group demonstrated that, in a total of 307 cases of FD and 23 cases of OF, the mutation rate of GNAS in patients with FD was approximately 86 % (264/307), while GNAS mutations were not found in patients with OF. [12] Little is known about the etiology of OF.
Hyperparathyroidism-jaw tumor (HPT-JT) syndrome is an autosomal dominant, multiple neoplasia syndrome which is characterized by parathyroid adenoma or carcinoma, ossifying fibroma of the jaws, renal and uterine lesions [13,14]. Between 30 and 40 % of individuals with HPT-JT also develop OFs, which are distinct from the "brown" tumors associated with severe hyperparathyroidism.. The HPT-JT locus was mapped to chromosome 1q24-q32; the putative gene, designated first as hyperparathyroidism type 2 (HRPT2) and then as CDC73, encodes a 531 amino acid protein called parafibromin. [15] Parafibromin is ubiquitously expressed and is evolutionarily conserved. It is the human homologue of yeast Cdc73, which is a component of the yeast RNA polymerase II/Paf1 complex that is important for histone modification and post-transcriptional events [16,17]. Mutations in CDC73 can be detected in approximately 58 % of probands with clinical features of HPT-JT syndrome [15]. In one series, which included four cases with OFs, mutations in CDC73 were found in two of the cases [18].
Collectively, these data suggested that CDC73 may be a key factor in the etiology of HPT-JT syndrome and its related tumors, including OFs. The prevalence and range of CDC73 mutations in sporadic OFs remain to be determined, in view of the limited number of cases examined to date. The goal of the present study was to identify CDC73 mutations in a group of Chinese patients presenting with sporadic OFs and to examine the possible role of CDC73 in the pathogenesis and diagnosis of OFs.

Subjects and samples
The fresh tumor specimens and peripheral blood samples of 40 sporadic OFs were obtained from the Department of Oral Pathology, Peking University Hospital and School of Stomatology, during 2003-2015. The diagnosis was made according to the WHO classification of odontogenic tumors [2]. Samples were only collected if the patient's family history was negative and PTH serum level was normal, to exclude cases of HPT-JT related OF. Detailed information regarding these cases is provided in Table 1.

DNA Extraction and Polymerase Chain Reaction (PCR)
Genomic DNA was extracted from frozen samples (25 mg) of neoplastic tissue and peripheral blood by using a DNeasy Tissue Kit (Qiagen Sciences, Maryland, USA). The 17 coding exons of CDC73 were amplified as 15 different fragments with primers derived from the flanking intronic or 3′/5′UTR regions, to allow the detection of mutations that occurred in coding regions or that affected splicing, as previously described [19]. The condition of PCR was used as follows: initial denaturation at 95°C for 5 min; 35 cycles of denaturation at 95°C for 30 s, annealing at 58-62°C for 30 s, elongation at 72°C for 30 s; and a final extension at 72°C for 7 min.

Direct sequencing
The amplified PCR products were gel-purified and directly sequenced. When nucleotides insertion or deletion was detected, clone sequencing by the plasmid vector was used for confirmation. The method of clone sequencing were carried out by previously described [20]. All detected mutations were confirmed by reverse sequencing and at least two independent experiments.

CDC73 mutations
Results from the mutational analysis of CDC73 in 40 cases of OF are summarized in Table 1. Two novel CDC73 mutations were identified in two cases. Both mutations were somatic and located in exon 1. The mutation in case 1 was a frameshift mutation (c.13_16delCTTA), which leads to a premature stop codon at position 15. The frameshift mutation also causes the substitution of the fifth amino acid, leucine, which was the first affected amino acid, with an alanine (p.Leu5Alafs*15). The novel mutation found in case 2, c.8_10delAC-GinsCT, was a frameshift mutation that leads to a premature stop codon at position 18. This mutation causes the substitution of the third amino acid, aspartic acid, with an alanine (p.Asp3Alafs*18) (Fig. 1).

Immunohistochemical detection of parafibromin expression
Using immunohistochemical analysis, we found that all cases of sporadic OF showed strong parafibromin expression in the nucleus, and that some showed parafibromin expression in the cytoplasm of the spindleshaped lesion cells, including those cells that were carriers of a CDC73 mutation (Fig. 2).

Discussion
Parafibromin, the product of CDC73, is believed to be important for embryonic development and tumorigenesis  The other novel mutation, c.8_10delACGinsCT (highlighted in small box), was found in case 2. The frame shifted from the third amino acid residue aspartic acid that is changed into alanine [15]. Homozygous CDC73 knockout mutations in mice led to in utero death by stage E6.5. Conditional knockout of both CDC73 alleles in E8.5 or older mice resulted in the retardation of embryonic growth and increased apoptosis, whereas in adult mice it resulted in cachexia and death within 20 days [22]. Parafibromin is a ubiquitously expressed nuclear protein [21]. In humans, parafibromin interacts with RNA polymerase II via the human PAF1 complex, which also includes human Paf1, CTR9, and Leo1. This complex regulates a number of key transcriptional events, including transcription initiation, transcript elongation, and post-transcriptional events, including mRNA maturation and maintenance of poly (A) tail length [16,17]. Mutations in CDC73 have been frequently detected in patients with HPT-JT and also occur in 20-29 % of individuals with apparently sporadic parathyroid carcinoma [23]. However, the frequency of CDC73 mutations in sporadic parathyroid adenomas is low, at 0-4 %, indicating that CDC73 mutations likely confer an aggressive growth potential and may result in malignant transformation of parathyroid cells [24]. CDC73 mutations have been detected in between 0 and 33 % of individuals with familial isolated primary hyperparathyroidism [23]. Somatic CDC73 mutations have also been found in sporadic renal tumors [25].
The frequency of CDC73 mutations in individuals with sporadic OF of the jaws has not been extensively studied. In the present study, two somatic mutations were identified in 40 cases of OF (5 %). This result suggested a possible pathogenetic role for CDC73 in some cases of sporadic OFs. Previously, a few cases of sporadic OF was found to harbor a mutation in CDC73 [18,26]. Further studies are needed to clarified the exact frequency of CDC73 mutation in sporadic OF due to limitation of sample size.
The CDC73 mutations identified in this study included two novel somatic mutations (c.13_16delCTTA and c.8_10delACGinsCT), which caused frameshifts and premature truncations of the protein. This finding is consistent with previous studies showing that over 75 % of the reported CDC73 mutations are frameshift or nonsense mutations that are predicted to result in either the truncation of the parafibromin protein or in loss of the translated protein through nonsense-mediated mRNA decay.
Somatic mutations of CDC73 were predominantly located in exons 1 and 2 (55 and 21 %, respectively), in contrast to germline mutations (22 and 16 %, respectively) mainly detected in HPT-JT, which were predominantly located in exon 7 (30 %). Only one somatic mutation has been detected in exon 7 to date [24]. The two mutations that we identified in OF both occurred in exon 1, and a previous study on OF [18] also identified a somatic mutation in exon 1. The basis of the differences in the distribution of the germline and somatic CDC73 mutations remains to be elucidated.
In vitro studies have shown that parafibromin acts as a tumor suppressor. Overexpression of parafibromin inhibits the proliferation of NIH3T3 and HEK293 cells, increases G1 arrest and apoptosis in HeLa cells, and downregulates expression of the cell cycle regulator, cyclin D1 [27][28][29]. Likewise, RNAi-mediated inhibition of parafibromin expression in HeLa cells resulted in increased S-phase entry with reduction in basal apoptosis and increase in expression of the proto-oncogene, c-myc [30,31].
According to Knudson's two-hit model for tumor suppressor genes [32] two mutations, one occurring in each of the two alleles of a gene, or one mutation in one allele of a tumor suppressor gene accompanied by the allelic Fig. 2 Parafibromin staining by immunohistochemistry. All tumors, without (a) or with (b) CDC73 mutation (*200), showed strong nuclear and cytoplasmic staining in both fibroblasts and osteoblasts within the lesion loss of the remaining wild-type allele, are required to trigger neoplasm formation. Biallelic inactivation in CDC73 has been detected in tumors in HPT-JT kindred and in sporadic parathyroid carcinomas and renal carcinomas [15]. Since parafibromin is ubiquitously expressed, biallelic inactivation of CDC73 can be detected immunohistochemically by the diffuse loss of nuclear expression. This is a common feature in parathyroid carcinoma [33,34].
However, the two-hit model may not apply to sporadic OF. First, only one mutation was detected in each case, even in the case with two somatic mutations reported by Pimenta [18]. Second, normal parafibromin expression was detected by immunohistochemistry. The effect of mutated CDC73 on sporadic OFs needs to be studied further.

Conclusions
Although CDC73 mutations are rare in sporadic OFs of the jaws, CDC73 may play a role in the pathogenesis of a small subset of tumors.