Clinical Endocrinology (2016) 84, 431–437

doi: 10.1111/cen.12804

ORIGINAL ARTICLE

Correlation between polymorphisms of BRAF gene and papillary thyroid carcinoma Rui Jiang*, Chunming Zhao†, Hao Xu‡, Miaoqing Zhao§, Xiaogang Sun*, Xinyu Wang* and Wei Song* *Department of Oncology, Provincial Hospital Affiliated to Shandong University †Department of Ophthalmology, Provincial Hospital Affiliated to Shandong University ‡Department of Thyroid and Breast Surgery, Provincial Hospital Affiliated to Shandong University, and §Department of Pathology, Provincial Hospital Affiliated to Shandong University, Jinan, China

Introduction Summary Background Papillary thyroid carcinoma (PTC), which accounts for 80% of all thyroid cancers, has an increasing incidence over these years. Single nucleotide polymorphisms (SNPs) of BRAF were considered to be one of well-established risk factors leading to development of PTC. The aim of this study was to investigate whether the common mutations of BRAF could elevate significantly the risk of PTC in a Chinese population. Methods Four SNPs (rs11762469, rs17623204, rs1267636 and rs3748093) of BRAF were selected through our filter by Haploview 4.2 software with HapMap databases. We used the polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP) to genotype the four SNPs in blood samples of 618 subjects (206 patients with PTC and 412 healthy controls). The correlation between BRAF polymorphisms and PTC risk was assessed using student t-test and chisquare test. Results The results showed that mutation in rs3748093 was significantly associated with an increased risk of PTC in allele model (A allele vs. T allele, OR = 168, 95% CI = 116–243, P = 0006), dominant model (TA + AA vs TT, OR = 164, 95% CI = 108–248, P = 0019) and homozygote model (AA vs. TT, OR = 294, 95% CI = 100–861, P = 0040). However, the other three SNPs (rs11762469, rs17623204 and rs1267636) were shown to have no association with the risk of PTC. Conclusions Our results indicated that polymorphism of rs3748093*A was significantly correlated with an increased risk of PTC in a Chinese population. Further investigation on the aetiological mechanism of PTC is needed to validate our results. (Received 28 November 2014; returned for revision 9 January 2015; finally revised 14 April 2015; accepted 19 April 2015)

Correspondence: Rui Jiang, Department of Oncology, Provincial Hospital Affiliated to Shandong University, No. 324 Jing Wu Road, Jinan 250021, China. Tel.: +86-0531-68777153; E-mail: [email protected] R. Jiang and C. Zhao contributed to this work equally. © 2015 John Wiley & Sons Ltd

Papillary thyroid carcinoma (PTC) is one of the most common malignant tumours in the endocrine system, accounting for 80% of all the thyroid cancers. PTC was reported as the most curable cancer among all the clinical carcinomas with approximately 90% 10-year survival rates nowadays. Due to lack of effective diagnosis for PTC, studies found that the incidence rate of PTC increased substantially in the past 30 decades,1–3 and the 10-year mortality rate of PTC is approximately 10% according to the recent epidemiological studies.4,5 However, the pathogenesis of PTC still remain unclear. In previous studies, it is reported that people with the habits of smoking and alcohol drinking have higher risk of developing PTC than those without such habits.6 Moreover, studies found that radiation of ultrasound was also the risk factor for PTC.1,6,7 In past decades, gene mutations (such as GSTs, VDR and XRCC3) and gene rearrangement (e.g. PTC and RET) were shown to be associated with increased risk of PTC.8–10 BRAF polymorphisms were recently found to be correlated with thyroid cancer risk.11–13 The activation of mitogen-activated protein kinase (MAPK) signalling pathway was observed in the PTC patients with typical genetic alterations14,15. The mutation or rearrangement of BRAF may activate the MAPK as well as the other downstream effectors in the MAPK signalling pathway.16,17 And BRAF further influences the cellular growth by disturbing the ordinance of cell differentiation or proliferation. Besides, BRAF is a member of the Raf gene family and presents as the potent effector in response to the signals.18,19 Polymorphisms of BRAF were reported to be the most studied aetiological polymorphisms for PTC susceptibility, which can be found in almost 50% of PTC cases.20–22 Hence, some SNPs that were significantly associated with PTC risk could be considered as effective prognostic or diagnostic marker for PTC.23 Additionally, Meyer et al. found that the polymorphisms of BRAF could contribute to the susceptibility of several cancers, such as mucinous ovarian cancer, melanoma, colon cancer and PTC.24 Furthermore, many researchers performed pre-operative assessment of surgical treatment according to BRAF mutation, and BRAF V600E had already been used in some clinical prognosis despite of 431

432 R. Jiang et al. controversies.25–28 However, the study about other polymorphisms of BRAF still needs to be investigated. Therefore, in this study, we hypothesized that genetic variants of BRAF involved in the pathogenesis of PTC. We aimed at assessing the association between four BRAF genetic variants (rs11762469, rs17623204, rs1267636 and rs3748093) and PTC susceptibility in a Chinese population.

Material & methods Study population This study was performed with strict protocol under approval of the Ethics Committee of Provincial Hospital Affiliated to Shandong University. Written informed consents were obtained from all participants enrolled in this study. All the recruited subjects were the Han Chinese. Finally, we enrolled a total of 618 subjects from Provincial Hospital Affiliated to Shandong University between September 2012 and June 2014. Among them, 206 subjects were diagnosed as patients with PTC, and the other 412 subjects were healthy controls recruited from visitors undergoing general check-up. All the healthy controls had been under the health screening, and their clinical characteristics (e.g. sex and age) were matched with the PTC cases, as shown in Table 1. Meanwhile, clinicopathological information was also collected from medical records, such as mean age at diagnosis, PTC variants, tumour node metastases (TNM) stage, tumour location, tumour size, lymph node involvement and metastasis. The detailed clinical characteristics are shown in Table 1. Then, we collected 5 ml venous blood sample from each eligible subject in EDTA tube. SNP selection The SNP selection was performed by retrieving the genotype data of unrelated individuals in Chinese population from the International HapMap Project SNP database (HapMap Data Rel 24/phaseII Nov08, on NCBI B36 Assembly, dbSNP b126). The linkage disequilibrium analysis was conducted using Haploview 4.2 software, and SNPs with minor allele frequency (MAF) A

rs3748093

129114T>A

F: 50 - GCAGGAAGAATATGAGTG-30 R: 30 -AGTCACAATCTCCGTTAG-50 F: 50 - AGTGGAGGATAGATGAAA-30 R: 30 -TTCATTATCGTAACCACGAC-50 F: 50 - CTTTCTTTTATCTCCTCA-30 R: 30 -TCGATTCTTCTTAATAATTC-50 F: 50 - AGGACGCCTCTTCCTGTG-30 R: 30 -ATTGTCGTCGTCGTGGTT-50

Global MAF

P-value of HWE

0221

0214

0141

0138

0061

0091

0046

0078

Table 2. Primers of BRAF genetic polymorphisms for PCR amplification

SNP, single nucleotide polymorphism; F, forward; and R, reverse.

Results Table 3. Associations between four common polymorphisms of BRAF gene and the risk of PTC

Genotype

Patients with PTC (n = 206)

rs11762469 (A>T) AA 118 AT 64 TT 24 AT+TT 88 AA+AT 182 TT 24 A allele 300 T allele 112 rs1267636 (A>G) AA 132 AG 68 GG 6 AG+GG 74 AA+AG 200 GG 12 A allele 332 G allele 80 rs17623204 (T>A) TT 167 TA 32 AA 7 TA+AA 39 TT+TA 199 AA 7 T allele 366 A allele 46 rs3748093 (T>A) TT 157 TA 41 AA 8 TA+AA 49 TT+TA 198 AA 8 T allele 355 A allele 57

Healthy controls (n = 412)

OR (95% CI)

228 140 33 173 368 33 596 206

Ref. 088 141 098 Ref. 147 Ref. 108

260 138 14 152 398 14 658 166

Ref. 097 084 096 Ref. 085 Ref. 096

313 90 9 99 403 9 716 108

Ref. 067 146 074 Ref. 158 Ref.

346 60 6 66 406 6 752 72

Ref. 151 294 164 Ref. 273 Ref. 168

OR, odds ratio; CI, confidence interval.

v2

P-value

(061–128) (079–249) (070–138)

0430 0370 0010

0511 0241 0920

(084–256)

1870

0171

(083–141)

0320

0574

(068–139) (031–225) (068–136)

0030 0120 0060

0870 0734 0813

(032–225)

0100

0748

(071–129)

0090

0762

(043–104) (053–398) (049–112)

3220 0550 2060

0073 0460 0151

(058–429)

0800

0370

0950

0330

(097–234) (100–861) (108–248)

3359 4215 5470

0067 0040 0019

(094–799)

3654

0056

(116–243)

7634

0006

Study characteristics We recruited a total of 618 subjects in this study. The subjects contained 206 patients with PTC (including 56 males and 150 females) and 412 normal controls (121 males and 291 females included). No significant difference was observed in gender among the participants (P = 0571). Furthermore, we separated the subjects into two groups by the age of 45. Overall, 101 patients with PTC and 182 healthy controls were under the age of 45, whereas 105 patients with PTC and 230 healthy controls were above the age of 45. No significance was observed in the difference of the age among the subjects (P = 0254). We also investigated the mean age of the patients with PTC at diagnosis (424  133). Besides, the family history was also considered to avoid the possible influence on the PTC risk. There is no obvious difference between the two groups (P = 0185). Furthermore, we collected detailed information about the clinical and histological information, which is shown in Table 1. Totally, 154 (75%) patients with PTC underwent to classic type of the histology, whereas the others were classified into types of sclerosant (288%), tall cells (288%), ossifils (77%) and follicular (1153%). The TNM stage was also considered due to its correlation with the PTC risk in previous studies: 74 (357%) in stage I, 15 (71%) in stage II, 95 (464%) in stage III and 22 (107%) in stage IV. According to the tumour location of PTC, it can be classified four types: 43% in the right lobe, 42% in the left lobe, 1% in the pyramidal lobe and 14% in isthmus. Tumour size mainly ranged from 10 to 40 mm (628%), 247% of small size (

Correlation between polymorphisms of BRAF gene and papillary thyroid carcinoma.

Papillary thyroid carcinoma (PTC), which accounts for 80% of all thyroid cancers, has an increasing incidence over these years. Single nucleotide poly...
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