BRAF Mutations in Iranian Patients with Papillary Thyroid Carcinoma

Papillary thyroid cancer or papillary thyroid carcinoma (PTC) is the most common thyroid cancer that is account for 80-90% of all thyroid cancer cases (Kimura et al., 2003; Xing et al., 2005; Yoon et al., 2013). This thyroid cancer is occasionally occurs in women aged 30-40 years old and it is also prevalent in children and patients whose exposed to radiation in head and neck area. These types of tumors are frequently un-encapsulated and show more propensities to metastasis into lymph nodes that may lead to production of cystic structures near thyroid which are diagnosed sorely. Genetic alterations are involved in thyroid carcinogenesis. Recently, occurrence of activation mutations with high frequencies is discovered in BRAF gene (Kimura et al., 2003; Soares et al., 2003; Xu et al., 2003; Jeong et al., 2012; Li, 2012). About 43 somatic mutations in BRAF gene were discovered which are associated with human cancers and located in exon 11 and 15 (Davies et al., 2002; Pakneshan et al., 2013). Serine/Threonineprotein kinase B-RAF or protooncogene B-RAF or in brief B-RAF is a human protein which is coded by BRAF gene (Sithanandam et al., 1990; 1992). Raf kinase is a member of Ras-Raf-MEK-ERKMAP pathway which plays role in cellular proliferation, differentiation and programmed cell death (Peyssonnaux


Introduction
Papillary thyroid cancer or papillary thyroid carcinoma (PTC) is the most common thyroid cancer that is account for 80-90% of all thyroid cancer cases (Kimura et al., 2003;Xing et al., 2005;Yoon et al., 2013).This thyroid cancer is occasionally occurs in women aged 30-40 years old and it is also prevalent in children and patients whose exposed to radiation in head and neck area.These types of tumors are frequently un-encapsulated and show more propensities to metastasis into lymph nodes that may lead to production of cystic structures near thyroid which are diagnosed sorely.Genetic alterations are involved in thyroid carcinogenesis.Recently, occurrence of activation mutations with high frequencies is discovered in BRAF gene (Kimura et al., 2003;Soares et al., 2003;Xu et al., 2003;Jeong et al., 2012;Li, 2012).About 43 somatic mutations in BRAF gene were discovered which are associated with human cancers and located in exon 11 and 15 (Davies et al., 2002;Pakneshan et al., 2013).
Serine/Threonine-protein kinase B-RAF or protooncogene B-RAF or in brief B-RAF is a human protein which is coded by BRAF gene (Sithanandam et al., 1990;1992).Raf kinase is a member of Ras-Raf-MEK-ERK-MAP pathway which plays role in cellular proliferation, differentiation and programmed cell death (Peyssonnaux

BRAF Mutations in Iranian Patients with Papillary Thyroid Carcinoma
Nastran Ranjbari 1 , Sara Almasi 1 , Javad Mohammadi-asl 2 , Fakher Rahim 3 * and Eychène, 2001).Three types of Raf genes are present in mammalian, among them BRAF is the strongest one in respect to activation of signal pathway which is located on chromosome 7 (Sithanandam et al., 1992;Avruch et al., 2001;Mercer and Pritchard, 2003).Some inherited mutations in BRAF may cause birth defect; alternatively other acquired mutations may lead to cancers such as: non-hodgkin lymphoma, colorectal cancer, malignant melanoma, papillary thyroid cancer, non-small cell lung carcinoma and adenocarcinoma of lung (Davies et al., 2002).BRAF mutations probably are involved in tumor initiation.Most of the PTCs are removed by surgery in early stages regardless to genotype.Many studies indicate that PTC carrying BRAF mutations are predominant among cases representing during advanced stage.Two mutation hot spots were discovered in BRAF gene exon 11 and 15, the most common point mutation is T1796A transversion which occurs in exon 15 while lead to glutamine for valine substitution (V600E).V600E somatic mutation codes constitutively active B-RAF kinase.Among differentiated neoplasm, this mutation is most discovered in PTC and approximately involved in 44% of all cases (Xing et al., 2005).Consequently, we examine BRAF V600E mutation frequencies in PTC patients to determine presence of relationship between occurrences of above mutation and PTC.

Subjects
Totally, 63 formalin fixed paraffin embedded (FFPE) Papillary Thyroid Carcinoma tumor tissues filed at Imam Khomeini hospital of Ahvaz, southwest Iran, were collected.These FFPE samples were obtained from PTC patients whom were diagnosed between 2000-2010 during biopsy or surgery.Thirty six out of 63 patients were female while other 44 patients were male.Most cases regardless their sexes were in 30-40 age range.Some of these patients whose were diagnosed in Ahvaz city were not living in Ahvaz but they came from other neighbor cities.This study was approved by the Review Board of the School of Medicine, Ahvaz Jundishapur University of Medical Sciences.

Genotyping
FFPE blocks were sectioned to 10 µm thickness by microtome and collected in 1.5 ml tubes.These micro-sectioned paraffin embedded tissues were treated by xylene to remove the paraffin.Genomic DNA was extracted by QIAamp DNA FFPE Tissue kit (Cat No: 56404).

PCR-RFLP
Isolated DNA was amplified by PCR.The PCR was carried out using a specific pair of primers for exon 15 of the BRAF gene: 5'TCA TGA AGA CCT CAC AGT AAA AAT3' (Forward) and 5'TGG ATC CAG ACA ACT GT T CAA3' (Reverse) as designed previously by Takahashi et al.(2007) using the following PCR thermal program: initial denaturation at 95̊c for 5 minutes, followed by 40 cycles for denaturation at 95̊c for 30 seconds, annealing at 60̊c for 30 seconds and elongation at 72̊c for 30 seconds.After the last cycle, a final extension at 72̊c for 5 minutes was performed.PCR products were digested by TspRI (New England Biolabs, Ipswich, MA) restriction enzyme at 60̊c for 16 hours.TspRI is a restriction enzyme which recognizes and cuts the ACAGTGAAA restriction site in the wild type genotype.However, T1799A point mutation replaces T by A so enzyme cannot distinguish the new restriction site and intact 98 bp PCR products will remain.While in the presence of the normal genotype two distinct fragments with 46 and 52 bp lengths are produced.RFLP products were visualized on 8% poly acrylamide gel electrophoresis (PAGE) after ethidium bromide staining.DNA bands were distinguished by use of 100bp ladder (Gene on GmbH, Germany).

DNA Sequencing
DNA sequencing of the PCR-amplified product was carried out bi-directionally on an ABI 3130 automated sequencer (Applied Biosystems) (Bioneer, South Korea) using the same primers.The results were using Chromas lite version 2.0 software.

Statistical analysis
The results were represented as the frequency and percentage of each genotype.Results were shown as a genotype frequencies and percentages in Table 1.We also considered relationships between genotypes and age as well as sex.Data were analyzed using SPSS 16.0 computing program and relationships tasted by χ 2 test (Table 2).According to the tests results no significant relationships observed between sex or age and BRAF genotypes.

Results
We tested 63 isolated DNA from paraffin-embedded papillary thyroid cancers patient tissues to identify V600E mutation frequency in Ahvaz PTC patients.Using the PCR-RFLP and sequencing of PCR products, mutation were found in 49/63 (77.78%) of PTC cases (Table 1).All mutations were in heterozygote form while all of them contain TgA transversion at nucleotide 1796 during sequencing.Other samples showed normal homozygote genotype that was accounted for 14 out of 63 cases (22.22%) cases and we didn't observed any samples with mutant homozygote genotype as mentioned in following table.According to RFLP results heterozygote samples showed 3 bands: 46, 52 and 98bp when normal homozygote produced 2 fragments only include: 46 and 52 bp (Figure 1).Although there was a 1.5 fold increase in the risk of PTC related to the sex (OR: 1.507, 95%CI: 0.455-4.994,P=0.708), but the impact was not statistically    2).These findings suggest that the BRAF V600E mutation is involved in the carcinogenesis in most PTC patients especially whose diagnosed in Iran so it can be used as a selective marker for prediction of patient's prognosis and their response to chemotherapy.

Discussion
The BRAF mutations were reported in most of carcinomas while it's ranged 36-69% of papillary thyroid cancer (Kim et al., 2004).We have calculated the frequency of V600E mutation in Ahvaz PTC population during 2000-2010, which was equal to 77.78%.These findings confirmed results of many studies which had proved association between BRAF V600E mutation and PTC occurrence.Wide range for V600E frequency might be due to ethnical differences, environmental factor, genetic disposition of specific population, varies in disease stage, different mutation determination method and differences in samples size.However, estimation of BRAF mutation frequency in thyroid cancer patients may influence on thyroid cancer diagnosis and prognosis as a molecular biomarker.In many studies BRAF mutations which effect on response to chemotherapy were considered.These finding were revealed that presence of BRAF mutation was associated with absence of Cetuximab and Panitumab therapy.Cetuximab (Erbitux) and Panitumumab (Vectibix) are two monoclonal antibodies that inhibit EGFR which are used to treat CRC and PTC patients with chemotherapy (Meyerhardt and Mayer, 2005;Xing et al., 2005).PTC harbor BRAF mutations have a high recurrence rate and metastatic recurrence may defect radio-iodine avidity (Xing et al., 2005;Durante et al., 2007).Other studies also represented same results and mentioned that BRAF mutation-positive patients required more aggressive surgical and external radiation treatments for their recurrent disease (Dralle and Machens, 2008;Lalami and Awada, 2011).In conclusion, these results indicated that presence of BRAF mutation plays role in PTC mortality and morbidity, while it can effect on its treatment.

Figure 1 .
Figure 1.Position of RFLP Bands after Cutting by TspRI Restriction Enzyme to Identify V600E BRAF Mutation, from left F side 2 nd , 6 th and 8 th Samples are Heterozygous while 4 th Sample Shows Homozygous Genotype.M: marker, U: uncut, C: cut, M: mutant, N: normal, bp: base pair