SeyyedMohammadRazavi MohammadRezaPourreza AbbasMoridnia LalehShariati MaryamSoltani MohammadAminTabatabaiefar ZhalehMohsenifar Geneticstudyofthe BRAF generevealsnewvariantsandhighfrequencyoftheV600EmutationamongIranianameloblastomapatients

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Genetic study of the BRAF gene reveals new variants and high frequency of the V600E mutation among Iranian ameloblastoma patients

Maryam Soltani


| Mohammad Amin Tabatabaiefar


| Zhaleh Mohsenifar


| Mohammad Reza Pourreza


| Abbas Moridnia


| Laleh Shariati



Seyyed Mohammad Razavi


1Department of Oral and Maxillofacial Pathology, School of Dentistry, Isfahan University of Medical Sciences, Isfahan, Iran

2Department of Genetics and Molecular Biology, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran

3Pediatric Inherited Diseases Research Center, Research Institute for Primordial Prevention of Noncommunicable Disease, Isfahan University of Medical Sciences, Isfahan, Iran

4Department of General Pathology, School of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran

5Department of Immunology, Dezful University of Medical Sciences, Dezful, Iran

6Isfahan Cardiovascular Research Center, Cardiovascular Research Institute, Isfahan University of Medical Sciences, Isfahan, Iran

7Dental Implant Research Center, Isfahan University of Medical Sciences, Isfahan, Iran


Seyyed Mohammad Razavi, Department of Oral and Maxillofacial Pathology, School of Dentistry, Isfahan University of Medical Sciences, Isfahan, Iran.


Funding information

This study was funded by research deputy of Isfahan University of Medical Sciences (grant number: 384502).

Background: Ameloblastoma is a benign, slow-growing and locally invasive tumor. It is one of the most prevalent odontogenic tumors, with an incidence rate of 1% of all oral tumors and approximately 18% of odontogenic tumors. A group of genes have been investigated in patients with ameloblastoma. The BRAF V600E mutation has been implicated as the most common mutation in ameloblastoma. The presence or absence of this mutation has been associated with several clinicopathological properties, including location, age at diagnosis, histology, and prognosis. Although some populations have been investigated so far, little data are available on the Ira- nian population. The current research was launched to study the BRAF V600E muta- tion among a cohort of Iranian patients with ameloblastoma.

Methods: In this clinicopathological and molecular biology study, a total of 19 for- malin-fixed, paraffin-embedded tissues were studied. DNA extraction was per- formed, followed by PCR-sequencing of exons 10 and 15 of the BRAF gene to identify mutations. In silico analysis was performed for the identified variants.

Results were analyzed by T test, Chi-square, and Fisher ’s exact test.

Results: Totally, 12 of 19 samples (63%) harbored the p. V600E hotspot mutation.

In addition, we identified several variants, two of which were novel. The c.1769T >G (p. V590G) and c.1751C >T (p.L584F) as the novel variants showed a possible dam- aging effect by in silico analysis. No variant was found within exon 10.

Conclusions: Our study confirms the role of BRAF mutations in ameloblastoma in the Iranian patients studied.


ameloblastoma, BRAF, gene, Iran

1 | I N T R O D U C T I O N

Ameloblastoma is a benign, slow-growing and locally invasive tumor.

It is one of the most prevalent odontogenic tumors with an inci- dence rate of 1% of all oral tumors and approximately 18% of odon- togenic tumors. It is able to perforate the cortical bone of the jaw to

cause facial asymmetry.1-3 Ameloblastoma is known to originate from the remaining tooth lamina and to reduce enamel epithelium, epithelial cell rests of Malassez, or the basal layer cells. It often affects the mandible (80%-85% of cases).2,3Ameloblastomas are cat- egorized into unicystic, multicystic (solid), peripheral, and malignant subtypes. The conventional solid or multicystic subtype, with an



© 2017 John Wiley & Sons A/S.

Published by John Wiley & Sons Ltd J Oral Pathol Med. 2018;47:86–90.


incidence of 86% of cases, is more infiltrative with a higher rate of recurrence.1 Their histological variations consist of follicular, plexi- form, acanthomatous, granular cell, basal cell, and desmoplastic types, among which the follicular and plexiform types are the most common types.4

A group of genes have been investigated in patients with ameloblastoma. However, further investigations are required to detect the mechanisms of oncogenesis and molecular pathogenesis.

Mutations influencing several genes within the MAPK pathway are currently known to occur in a large number of cases. The biological importance of these mutations is pronounced by their high fre- quency and pattern of mutual exclusivity. The hedgehog and FGFR- MAPK pathway components have been reported to be expressed during tooth development and in ameloblastomas.5-7Moreover, anal- ysis using gene expression microarrays, immunohistochemistry, and quantitative RT-PCR have, in particular, indicated the differential expression of Hedgehog pathway genes in ameloblastomas.8,9

Both in vitro and anecdotal clinical data suggest MAPK pathway inhibition as a promising treatment option for ameloblastoma.10Vari- able sensitivity of primary ameloblastoma cells to EGFR-targeted drugs in vitro has been reported.9The BRAF V600E mutation (15 of 24 samples, 63%) was found in the cell line overexpressing EGFR but resistant to EGFR inhibition. These data provide a novel insight into the poorly recognized molecular pathogenesis of ameloblastoma and offer a rationale for testing the drugs targeting EGFR or mutant BRAF as novel therapies for ameloblastoma.9

More than 40 different mutations have been reported in the BRAF gene related to human cancer.11 Interestingly, the BRAF V600E mutation has been implicated as the most common mutation (90% of all BRAF mutations) in ameloblastoma resulting in constitu- tive activation of the gene.12The presence or absence of this muta- tion has been associated with several clinicopathological properties, including location, age at diagnosis, histology, and prognosis.10

Although there are several studies to address the prevalence of the BRAF V600E mutation in ameloblastoma patients,5,9,13,14 they do show that the mutation frequency differs in different popula- tions; Iran is a big country with a population about 80 million peo- ple. However, there is no report on the role of BRAF in ameloblastoma. Therefore, the current research was launched to study the BRAF V600E mutation among a cohort of Iranian patients with ameloblastoma.

2 | M A T E R I A L S A N D M E T H O D S

2.1 | Subjects

This study was approved by the Institutional Review Board of Isfa- han University of Medical Sciences. Altogether, 23 families were recruited from the Department of Oral Pathology and Al Zahra Pathology Laboratory affiliated to Isfahan University of Medical Sciences and Department of Pathology, Shahid Beheshti University of Medical Sciences. All the centers had protocols for taking informed written consent from the patients before banking their

archive samples. All families were of Persian ethnicity. A detailed clinical history was taken.

2.2 | DNA extraction

Totally, 23 formalin-fixed, paraffin-embedded tissues were recruited in this study. Tissue sections were deparaffinized with xylene followed by genomic DNA extraction using QIAamp DNA FFPE Tissue Kit (Qiagen, Germany) following the manufacturer’s protocol. The concentration and purity of DNA were measured by Nanodrop 1000 Spectrophot- ometer (Thermo Fisher Scientific Inc., Waltham, MA, USA) and 1.2%

agarose gel, and 50 ng of the genomic DNA was used for PCR.

2.3 | Mutation screening of BRAF

Sequencing of the selected BRAF gene exons 10 and 15 was per- formed to identify mutations. The primers for the exons were designed to include exon-intron boundaries. The exons were ampli- fied using 29 Master Mix Red (Ampliqon, Copenhagen, Denmark) by standard PCR programs (Table 1). Primers were designed using Oligo (version National Biosciences Inc.). Primer sequences and PCR conditions are available upon request.

PCR conditions were as follows: Each reaction contained 1lL MgCl2 (50 mM), 2.5lL Taq PCR buffer (109), 0.5 lL of each of the primers (10 PM), 0.1lL Taq DNA polymerase (5 U/lL), 0.5 lL dNTP

T A B L E 1 Demographic and pathological features of the studied sample

Sample # Age Sex Type Variant

3 87 M FOLI V600E

5 58 M FOLI V600E

6 46 M PLXI V600E

9 51 M PLXI V600E

10 52 F PLXI

12 30 M PLXI V600E

15 16 F PLXI

16 30 M PLXI V600E

17 58 F FOLI G606E

20 78 M FOLI V600E

22 35 M FOLI

23 50 F PLXI V600E

24 22 M FOLI V600E G606E

25 31 M FOLI

26 37 F FOLI V600E

28 33 M FOLI

29 41 M FOLI V600E G606E

30 45 F FOLI L584Pa V590Ga

33 26 F PLXI V600E

MAN, mandible; MAX, maxilla; F, female; M, male; FOLI, follicular; PLXI, plexiform; ACAN, acanthomatous.

aNew variant.


mix (10 mM), and 1lL DNA (about 50 ng). The total volume was adjusted to 25lL by ddH2O. A touchdown thermal program was performed as follows: one cycle of denaturation at 95°C for 3 min- utes; five touchdown cycles of denaturation at 94°C for 40″, anneal- ing at 64°C for 40″ in the first cycle with 1°C reduction per cycle, and extension at 72°C for 45″; 27 cycles of denaturation at 94°C for 45″, annealing at 60°C for 40″, and extension at 72°C for 45″; and a final extension step of 72°C for 7 minutes. PCR products were investigated using agarose gel electrophoresis and sequenced bidi- rectionally using ABI 3130XL automated sequencer (Applied Biosys- tems, Foster City, California, USA). Sequences were compared with the reference genomic sequence NG_007873.3 for variant detection using the SeqMan software version 5.00© (DNASTAR, Madison, WI, USA).

2.4 | Pathogenicity analyses

Pathogenicity of the identified variants was studied based on the nature of variants, evolutionary conservation of substituted amino acid. After identification of each variant, Ensemble, dbSNP, and 1000 genome database, NHLBI GO exome sequencing project (ESP), exome aggregation consortium (ExAC), and the Human Gene Muta- tion Database (HGMD) as well as the literature were investigated for previously known variants. Sequence variant numbering was based on the reference transcript sequence NM_004333 for the BRAF gene. All novel variants were named according to the guidelines of the human variation society ( Possible patho- genic effects of the novel variants were checked by MutationTaster ( In addition, FATHMM (http://fath, PolyPhen-2 (http://genetics.bwh.harvard.ed u/pph2/), PROVEAN (, PANTHER ( SIFT (, ConSurf (, and KinMut2 (http://kinmut2.bioinf were used for in silico prediction of the variant effects.

3 | R E S U L T S

We used 23 patients with multicystic ameloblastoma lesion, and DNA was extracted. However, upon PCR, only 19 samples showed satisfactory results after amplification on agarose gel after different conditions were examined. Demographic and pathological features of the studied samples are shown in Table 1. None of the samples had positive family history.

3.1 | Sequencing results for the BRAF gene

Only 19 of the 23 samples showed acceptable results in molecular studies and were included. From the 19 samples that were sequenced, 12 had an exon 15 heterozygous mutation named c.1799T>A (p.V600E); therefore, the prevalence of the mutation among ameloblastoma patients was 63%. Figure 1A shows this

variant. The pathogenicity prediction of all identified variants was checked via online software tools (Table 2).

In terms of tissue distribution of the mutation, six cases were of plexiform and six were of follicular type. Thus, there was no signifi- cant difference in the distribution of the mutation. The average age of the patients harboring the mutation was 43.6 years vs those with- out the mutation (38.6 years of age). In addition, we identified three other variants including G606E, L584P, and V590G (Figure 1B-D), two of which, L584P and V590G were novel. The variants V590G and G606E showed possible damaging effects by in silico analysis.

The novel variants, L584P and V590G, also were suggested to be of pathogenic consequence by the software tools except for KinMut2 tool for L584P.

F I G U R E 1 Chromatograms of variants identified in exon 15 in the ameloblastoma patients. (A) represents substitution of T to A at position 1799 of coding DNA (p.V600E), (B) shows the substitution of G to A at position 1817 of coding DNA (G606E), (C) indicates a novel variant in which C is replaced by T at coding DNA position 1751 (L584P), and (D) illustrates replacement of T to G at position 1769 at coding DNA level (V590G)


4 | D I S C U S S I O N

Ameloblastoma is a benign, locally invasive tumor with complex eti- ology.1-3 In this descriptive study, Iranian patients with ameloblas- toma were analyzed for the presence of the BRAF V600E mutation.

Totally, 12 of 19 patients were found to carry this mutation. The BRAF gene has been reported to be mutated in some human cancers including colorectal cancer,15-17 malignant melanoma,18-24 patients with Langerhans cell histiocytosis,25 papillary thyroid carcinoma,26 non-small-cell lung carcinoma, non-Hodgkin lymphoma, and lung adenocarcinoma.11 The BRAF mutations have been implicated in ameloblastoma. In 2014, three separate reports demonstrated recur- rent MAPK mutations in ameloblastoma, with the most common mutation being BRAF V600E. The findings reported in these studies suggest a new paradigm for the diagnostic classification and treat- ment of ameloblastomas. Two of these reports indicated BRAF muta- tions at a similar frequency (64% and 63%; 54/84 and 15/24), while a third showed a lower frequency (46%; 13/28).5,9,14

Diniz et al. evaluated BRAF V600E in multicystic, unicystic, and desmoplastic ameloblastomas. In their study, 17 ameloblastoma sam- ples were included. Fourteen of 17 (82%) ameloblastomas showed BRAF V600E mutation, 5/6 (83%) in unicystic, 7/9 (78%) in multicys- tic, and 2/2 in desmoplastic ameloblastomas.13In our study on multi- cystic ameloblastoma, 12 of 19 samples harbored the p. V600E hotspot mutation. Therefore, the prevalence of this mutation among the studied Iranian ameloblastoma cases is reported to be 63%.

Sweeney et al. argued that BRAF-mutated tumors may have indica- tions for location, histologic pattern (plexiform vs follicular), and pos- sible prognosis. In this regard, 80% of ameloblastomas with the plexiform histologic pattern were BRAF wild-type.13However, Brown et al. reported no relationship between follicular/plexiform pattern and genotype. Plexiform histology was significantly more prevalent among BRAF wild-type tumors (62%) than among BRAF-mutated tumors (35%; P=.02).5 In our study, in term of tissue distribution of the mutation, six of eight cases were of plexiform and six of 11 were of the follicular type (P=.63). Thus, there was no significant differ- ence in the distribution of the mutation. This could be due to small sample size and population-related differences. Sweeney et al. found that BRAF mutations occurred in younger patients with a mean age at diagnosis of 34.5 years compared to 53.6 in BRAF wild-type cases

(P=.0001).13In our study, the average age of the patients harboring the mutation was 43.6 years vs those without the mutation (38.6 years of age) (P=.385). Thus, no significant relationship was found between the average age and carrying the mutation.

Sweeney et al.14 found that BRAF mutations are predominant in tumors of the mandible (75%). In a study, BRAF mutations were shown to occur much more frequently in the mandible and only rarely in the maxilla (5.6%), while 43% of BRAF wild-type tumors arose in the maxilla. 64% of BRAF wild-type tumors arose in the maxilla as well.5 In our study, no significant difference was found between tumor types and the position of the jaw (mandibular and maxillary) (P=.99).

Cancer is a multistep process. Simultaneous occurrence of two variants (V600E with G606E and L584F with V590G) in the same gene may simply be a reflection of the instability of the genome.

According to standards for reporting sequence variants in cancer, V600E is a variant with potential significance (second tier with evi- dence of level C) in ameloblastoma. The clinical significance of the novel variants is unknown.27 Notably, the V590G, L584F, and G606E exist in the same exon as V600E, thereby affecting the same domain (protein kinase domain). Available evidence supports that c.1751C>T (p.L584F) and c.1769T>G (p.V590G) are classified in vari- ants with uncertain significance category according to the ACMG guidelines.28Alternatively, none or one of these variants might have potential functional implications. Extending the sample size, recur- rence of the variants in other ameloblastoma tumor samples, qPCR, and kinase activity assessment could unravel the importance of these variants in ameloblastoma. Currently, it is not known whether the simultaneous occurrence of the variants within the BRAF gene would reflect additional selective advantage for the tumor or they may simply reflect tumor multistep nature and genomic instability.

5 | C O N C L U S I O N

In summary, for the first time in Iran, we studied BRAF mutations in a cohort of Iranian patients with ameloblastoma. We also report sev- eral variants some of which may have pathogenic effect. Our data confirm a significant role of the BRAF gene mutations among Iranian ameloblastoma patients.

T A B L E 2 List of variants found in BRAF among ameloblastoma patients and their pathogenicity investigation using software prediction tools Variant

c.1799T>A (V600E) c.1817G>A (p.G606E) c.1751C>T (p.L584F) c.1769T>G (p.V590G)

Score Prediction Score Prediction Score Prediction Score Prediction

SIFT 0 Damaging 0 Damaging 0.04 Damaging 0 Damaging

PROVEAN 4.781 Deleterious 7.376 Deleterious 6.470 Deleterious 6.215 Deleterious

MutationTaster2 121 Disease causing 98 Disease causing 22 Disease causing 109 Disease causing Polyphen2.0 0.971 Probably damaging 0.493 Possibly damaging 0.964 Probably damaging 1.000 Probably damaging

ConSurf 9 Conserved 8 Conserved 9 Conserved 9 Conserved

PANTHER 910 Probably damaging 910 Probably damaging 1036 Probably damaging 1036 Probably damaging

FATHMM 1.83 Cancer 1.80 Cancer 2.33 Cancer 2.45 Cancer

KinMut2 0.939 Disease 0.962 Disease 0.654 Neutral 0.769 Disease



We are deeply grateful to Isfahan University of Medical Sciences deputy of research due to their financial support (grant no 384502).

This study is a part of a thesis mainly done by Dr. Maryam Soltani.


The authors declare that they have no conflict of interest.


All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki Dec- laration and its later amendments or comparable ethical standards.

Informed consent: Informed consent was obtained from all indi- vidual participants included in the study.


1. Neville B, Damm DD, Allen C, Chi A Oral and Maxillofacial Pathology, 4th edn. St. Louis, MO: Elsevier; 2015.

2. Buchner A, Merrell PW, Carpenter WM. Relative frequency of central odontogenic tumors: a study of 1,088 cases from Northern California and comparison to studies from other parts of the world.

J Oral Maxillofac Surg. 2006;64:1343-1352.

3. Bataineh AB. Effect of preservation of the inferior and posterior bor- ders on recurrence of ameloblastomas of the mandible. Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 2000;90:155-163.

4. Kumamoto H. Molecular pathology of odontogenic tumors. J Oral Pathol Med. 2006;35:65-74.

5. Brown NA, Rolland D, McHugh JB, et al. Activating FGFR2-RAS- BRAF mutations in ameloblastoma. Clin Cancer Res. 2014;20:5517- 5526.

6. Heikinheimo K, Jee KJ, Niini T, et al. Gene expression profiling of ameloblastoma and human tooth germ by means of a cDNA microar- ray. J Dent Res. 2002;81:525-530.

7. Miyake T, Tanaka Y, Kato K, et al. Gene mutation analysis and immunohistochemical study of beta-catenin in odontogenic tumors.

Pathol Int. 2006;56:732-737.

8. Kumamoto H, Ohki K, Ooya K. Expression of Sonic hedgehog (SHH) signaling molecules in ameloblastomas. J Oral Pathol Med. 2004;


9. Kurppa KJ, Caton J, Morgan PR, et al. High frequency of BRAF V600E mutations in ameloblastoma. J Pathol. 2014;232:492-498.

10. Brown NA, Betz BL. Ameloblastoma: a review of recent molecular pathogenetic discoveries. Biomark Cancer. 2015;7(Suppl 2):19-24.

11. Davies H, Bignell GR, Cox C, et al. Mutations of the BRAF gene in human cancer. Nature. 2002;417:949-954.

12. Van Dam SD, Unni KK, Keller EE. Metastasizing (malignant) ameloblastoma: review of a unique histopathologic entity and report of Mayo Clinic experience. J Oral Maxillofac Surg. 2010;68:


13. Diniz MG, Gomes CC, Guimaraes BV, et al. Assessment of BRAFV600E and SMOF412E mutations in epithelial odontogenic tumours. Tumour Biol. 2015;36:5649-5653.

14. Sweeney RT, McClary AC, Myers BR, et al. Identification of recur- rent SMO and BRAF mutations in ameloblastomas. Nat Genet.


15. Benlloch S, Paya A, Alenda C, et al. Detection of BRAF V600E muta- tion in colorectal cancer: comparison of automatic sequencing and real-time chemistry methodology. J Mol Diagn. 2006;8:540-543.

16. Deng G, Bell I, Crawley S, et al. BRAF mutation is frequently present in sporadic colorectal cancer with methylated hMLH1, but not in hereditary nonpolyposis colorectal cancer. Clin Cancer Res. 2004;10 (1 Pt 1):191-195.

17. Li WQ, Kawakami K, Ruszkiewicz A, Bennett G, Moore J, Iacopetta B. BRAF mutations are associated with distinctive clinical, pathologi- cal and molecular features of colorectal cancer independently of microsatellite instability status. Mol Cancer. 2006;5:2.

18. Bollag G, Hirth P, Tsai J, et al. Clinical efficacy of a RAF inhibitor needs broad target blockade in BRAF-mutant melanoma. Nature.


19. Chapman PB, Hauschild A, Robert C, et al. Improved survival with vemurafenib in melanoma with BRAF V600E mutation. N Engl J Med. 2011;364:2507-2516.

20. Das Thakur M, Salangsang F, Landman AS, et al. Modelling vemu- rafenib resistance in melanoma reveals a strategy to forestall drug resistance. Nature. 2013;494:251-255.

21. Gear H, Williams H, Kemp EG, Roberts F. BRAF mutations in con- junctival melanoma. Invest Ophthalmol Vis Sci. 2004;45:2484-2488.

22. Larsen AC, Dahmcke CM, Dahl C, et al. A retrospective review of conjunctival melanoma presentation, treatment, and outcome and an investigation of features associated with BRAF mutations. JAMA Ophthalmol. 2015;133:1295-1303.

23. Maldonado JL, Fridlyand J, Patel H, et al. Determinants of BRAF mutations in primary melanomas. J Natl Cancer Inst. 2003;95:


24. Nazarian R, Shi H, Wang Q, et al. Melanomas acquire resistance to B-RAF(V600E) inhibition by RTK or N-RAS upregulation. Nature.


25. Badalian-Very G, Vergilio JA, Degar BA, Rodriguez-Galindo C, Rollins BJ. Recent advances in the understanding of Langerhans cell histio- cytosis. Br J Haematol. 2012;156:163-172.

26. Namba H, Nakashima M, Hayashi T, et al. Clinical implication of hot spot BRAF mutation, V599E, in papillary thyroid cancers. J Clin Endo- crinol Metab. 2003;88:4393-4397.

27. Li MM, Datto M, Duncavage EJ, et al. Standards and guidelines for the interpretation and reporting of sequence variants in cancer: a joint consensus recommendation of the association for molecular pathology, American society of clinical oncology, and College of American Pathologists. J Mol Diagn. 2017;19:4-23.

28. Richards S, Aziz N, Bale S, et al. Standards and guidelines for the interpretation of sequence variants: a joint consensus recommenda- tion of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology. Genet Med. 2015;17:


How to cite this article: Soltani M, Tabatabaiefar MA, Mohsenifar Z, et al. Genetic study of the BRAF gene reveals a new variants and high frequency of the V600E mutation among Iranian ameloblastoma patients. J Oral Pathol Med.





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