Increased T-Allele Frequency of 677 C > T Polymorphismin the Methylenetetrahydrofolate Reductase Gene

in Differentiated Thyroid Carcinoma

Semra Ozdemir,1 Fatma Silan,2 Zekiye Hasbek,3 Ahmet Uludag,2 Sinem Atik,2

Taner Erselcan,3 and Ozturk Ozdemir2,4

Background: Epigenetic alterations in the global DNA methylation status may be associated with an increasedrisk of some cancer types in humans. The methylenetetrahydrofolate reductase (MTHFR) gene is involved infolic acid metabolism and plays an essential role in inherited DNA methylation profiles. The common 677 C > Tand 1298 A > C polymorphisms in the MTHFR gene cause the production of a thermolabile enzyme with reducedfunction and, eventually, genomic DNA hypomethylation. The current preliminary study was designed todetermine the association between germ-line polymorphism in the MTHFR gene and differentiated thyroidcarcinoma (DTC). Methods: In the current case-control study of 60 thyroid carcinomas (TC); 45 papillary TC, 9follicular TC, and 6 DTC of an uncertain malignant potential were examined. Genomic DNA was extracted fromperipheral blood with EDTA, genotyped by a multiplex real-time polymerase chain reaction. Results: An ele-vated 2.33-fold risk was observed for DTC in individuals with the 677TT genotype when compared with thecontrol group (odds ratio [OR]: 1.92, 95% confidence interval [CI]: 1.03–3.58). Current DTC patients showedsimilar results as a control group for the 1298 A > C allele. No significant risk was detected for the homozygous1298CC genotype (CC vs. AA or AC) (OR: 1.30, 95% CI: 0.73–2.29). Conclusion: The current results are supportiveof the hypothesis that the homozygous MTHFR 677TT genotype increases the risk factor of developing thyroidcancer, and further large-scale studies are needed to validate this association.

Introduction

Thyroid cancer (TC) is the most common endocrinemalignancy, and it can be categorized as four main types:

papillary, follicular, medullary, and anaplastic. The papillaryand follicular types, also known as differentiated thyroidcancers (DTC), account for > 90% (Stephen et al., 2011). Theincidence of thyroid cancer has been increasing in recent de-cades (Hou et al., 2010). Currently, the application of molec-ular technologies has focused on genetics for the accuratediagnosis and therapy of cancer. Early and/or late stage typesof DTC may also be initiated by point mutations in differentproto-oncogenes, such as RET, B-RAF, NTRK1, and K-RAS(Namba et al., 2003; Weier et al., 2009; Kim et al., 2010; Moseset al., 2010; Bales and Chopra, 2011). Epigenetic silencingthrough aberrant DNA methylation of tumor suppressorgenes has been reported in TC (Russo et al., 2011). DNAmethylation is a semi-conservative mechanism that acts inepigenetic gene regulation and influences cellular develop-ment and function. It is well known that DNA methylation

plays a critical role in gene regulation and has been implicatedin the etiology of chronic diseases, including atherosclerosis,neural tissue degeneration, and cancer. The altered DNAmethylation status of different candidate genes may alsocause cancer development in a wide range of tissues in hu-mans (Schulz and Hoffmann 2009; Arslan et al., 2010; Ozdemiret al., 2010; Izmirli et al., 2011). Gene-specific DNA hypo-methylation has been reported under the condition of folatedeficiency (Curtin et al., 2011). James et al. have claimed thatthe levels of DNA methylation may depend on DNMTsactivity, which is regulated by the ratio of AdoMet toS-adenosylhomocysteine (SAM) by reversible biosynthesisfrom homocysteine ( James et al., 2002). Abnormalities in DNAmethylation have also been reported in cancer cells, includinghypermethylation of the promoter region of tumor suppressorgenes, as claimed by Luczak and Jagodzinski (Luczak andJagodzinski, 2006). The functional methylenetetrahydrofolatereductase (MTHFR) gene is located on chromosome 1p36.3and contains 11 exons, which are essential for the regulation ofDNA synthesis, methylation cycle, epigenetic regulation, and

Departments of 1Nuclear Medicine and 2Medical Genetics, Faculty of Medicine, Canakkale Onsekiz Mart University, Canakkale, Turkey.Departments of 3Nuclear Medicine and 4Medical Genetics, Faculty of Medicine, Cumhuriyet University, Sivas, Turkey.

GENETIC TESTING AND MOLECULAR BIOMARKERSVolume 16, Number 7, 2012ª Mary Ann Liebert, Inc.Pp. 780–784DOI: 10.1089/gtmb.2011.0347

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homocysteine concentration in solid tissues. SAM is a uni-versal precursor substrate that serves as a methyl donor, andmethionine is a key molecule in the biological methylationreactions. The MTHFR gene also plays a crucial role in regu-lating folate metabolism, which affects both DNA synthesisand methylation implicated in many types of cancer (Lu et al.,2011). Three common single-nucleotide polymorphisms(SNPs), such as C677T, A1298C, and G1793A, have been re-ported for the MTHFR gene that lead to thermolability and/orreduced enzyme activity (van der Put et al., 1998; Melo et al.,2006).

In the current case-control study, we aimed at finding apossible role of the germ-line functional gene MTHFR poly-morphism in susceptibility to DTC.

Materials and Methods

Patients and clinical diagnosis

Blood samples with EDTA from 60 thyroid cancer patientswho had undergone total thyroidectomy and 50 healthycontrol individuals were used in the current study. In a total of60 thyroid cancer patients, there were 45 papillary thyroidcarcinoma (PTC) (75%), 9 folicullar thyroid carcinoma (FTC)(15%), 6 DTC (10% (11 men (18.3%))), and 49 women (81.7%)with a mean age-min–max; 55.25 – 3.22 (28–75); 50 healthyadult individuals used as the control group; [21 men (42%), 29women (58%), and mean age-min–max; 86.60 – 2.82 (65–77)]were included in the current results. Both groups were geno-typed for MTHFR SNPs (C677T and A1298C) and compared.Individuals with any familial cancer history were excludedfrom the control group.

Mutation analysis

Peripheral blood samples containing EDTA from the pa-tients and the control group were used for genomic DNAisolation. Total genomic DNA was extracted from each pe-ripheral blood sample by the Magna Pure Compact (Roche)and Invitek kit extraction techniques (Invitek�; Invisorb spinblood). Two polymorphic regions of the MTHFR gene (C677Tand A1298C) were amplified by a real-time polymerase chainreaction (PCR), LightCycler 2.0 methods (Roche). Briefly,LightCycler FastStart DNA Master HybProbes, master mix(water, PCR-grade, MgCl2, stock solution, Primer mix, andHybProbe mix), and DNA template were used for real-timeamplification. The multiple PCR consisted of a denaturationstep of 10 min at 95�C; followed by 40 cycles of 5 s at 95�C, 10 sat 55�C, and 15 s at 72�C; and a melting step of 20 s at 95�C,20 s at 40�C, a continuous mode at 85�C, a cooling step of 30 sat 40�C for MTHFR C677T (rs1801133), and a denaturationstep of 10 min at 95�C; followed by 40 cycles of 5 s at 95�C, 10 sat 62�C, and 6 s at 72�C; and a melting step of 30 s at 72�C, 20 sat 95�C, 1 s at 40�C, 30 s at 40�C, and 1 s at 85�C; and coolingstep of 30 s at 40�C for MTHFR A1298C (rs1801131). A soft-ware program (LightCycler 2.0, Roche) was used for the de-tection of the mutated and normal genotype profiles of targetgenes in the current DTC patient and the control groups.

Statistical analysis

The estimate risk was examined by multivariate logisticregression analysis. Mutational variables were analyzed byusing Fisher’s exact test. Results were given as the mean

(standard deviation [SD]). The software SPSS for Windowsversion 12.0 was used to perform statistical analysis. TheMann–Whitney U and v2 and chi-square tests were used toanalyze the differences between the patients and controls.Odds ratio (OR) and p-values for T- and C-allele frequencieswere used to estimate the risk for the MTHFR gene SNPs incurrent patients with DTC.

Results

The current preliminary case-control study was designed todetermine the association between germ-line point mutationsin the MTHFR gene and DTC. By the multiplex real-time PCRtechnique, we evaluated two common SNPs of the MTHFRgene in DTC patients, and the results were compared with thehealthy control group (Fig. 1). The estimate risk was examinedby multivariate logistic regression analysis. Statistically, theTT homozygous genotype in the 677 C > T SNP was associ-ated with a 2.33-fold significance in the increase of the risk forDTC patients.

Clinicopathologic data and follow-up knowledge

A total of 60 DTC patients [49F (81.7%) and 11M (18.3%)]; 45PTC (75%), 9 FTC (15%) and 6 uncertain malignant potential(10%), mean age 55.25 – 13.22 (28–75), were clinically diag-nosed and treated. Peripheral blood-EDTA samples fromhealthy controls and DTC patients were examined for geno-typing in the current study. The subtypes and some clinicalcharacteristics, such as mean age, sex distribution, smoke sta-tus, and familial cancer history of patients and control groups,are provided in Table 1. Both smoking status and familialcancer history showed statistical significance in the studiedDTC group when compared with the control group ( p < 0.01).Sixteen of the (26.6%) current DTC patients were heavysmokers (package per a day). This situation was significant(OR: 1.289, 95% confidence interval [CI]: 0.530–3.187) whencompared with the control group (Table 1). Twenty-two of the(36.6%) DTC patients reported a familial cancer history. Thissituation was also statistically significant (OR: 1.648, 95% CI:0.724–3.747) when compared with the control group (Table 1).The genotype analysis and statistical results for both MTHFR677 C > T and 1298 A > C SNPs were demonstrated in Tables 2and 3. The studied DTC patients showed 28 (46.6) CC, 25(41.7%) CT and 7 (11.7%) TT genotypes for 677 C > T SNP. TheC-allele frequency was 0.675, and the T-allele frequency was0.325 for 677 C > T SNP in the DTC patients (Table 2). An ele-vated 2.33-fold risk was observed for DTC in individuals withthe 677TT genotype (OR: 1.92, 95% CI: 1.03–3.58), ( p = 0.01).

The studied DTC patients also showed 22 (36.6) AA, 33(55.0%) AC, and 5 (8.4%) CC genotypes for a second SNP,1298 A > C in the MTHFR gene (Table 3). The A-allele fre-quency was 0.700, and the T-allele frequency was 0.300 forSNP in the patients group. The difference in genotype fre-quency distribution between the DTC cases and controls wasnot significant for this SNP ( p = 0.18), (Tables 1 and 3). Mul-tivariate analysis demonstrated an increased risk of DTC forthe 677 C > T homozygous genotype TT for the results pre-sented. Despite some limitations, the results indicated that theindividuals with the 677TT genotype had an 11.7% higher riskof having DTC. No evidence of such an association was ob-served in the DTC patients with the A > C polymorphism atcodon 1298 in the current results.

MTHFR GENE, SNP, INCREASED T ALLELE, DTC 781

Discussion

Thyroid cancers of a follicular cell origin account for themajority (95%) of all thyroid cancers and represent the mostcommon type of endocrine neoplasia. DTC is the most com-mon malignancy of the thyroid gland and involves somemolecular ethiological parameters, such as point mutations inthe proto-oncogenes of BRAF V600E, KRAS, RET, functionalgenes of MTHFR, MDR1, and epigenetic alterations of severaltumor suppressor gene abnormalities (Schulz and Hoffmann2009; Fard-Esfahani et al., 2011; Moura et al., 2011; Vineis et al.,2011). The genetic parameters may contribute to the individualdifferences in the epigenetic regulation of cancer susceptibility,

drug efficacy, and cytotoxicity. The functional MTHFR genecatalyzes the conversion of 5,10-methylenetetrahydrofolate(5,10-methylene-THF) to 5-methyl-tetrahydrofolate (5-methylTHF) and plays a crucial role in folate metabolism and sys-temic methyl sources. Therefore, a decrease in 5-methyl THF inthe MTHFR dysfunction and/or hypofunction leads to a de-crease of the available SAM, which results in DNA hypo-methylation and may play a critical role in carcinogenesis(Goyette et al., 1998; Weisberg et al., 1998; Liu et al., 2011; Tsaiet al., 2011; Zacho et al., 2011). Reduced enzyme activity (*50–60%) was reported by Dong et al. for the individuals withheterozygous profiles for both the 677 C > T and 1298 A > Calleles (Dong et al., 2010). Curtin et al. suggest that folate

FIG. 1. This figure shows real-time—polymerase chain reaction profiles of wild and mutated genotypes for MTHFR 677C > T and 1298 A > C single-nucleotide polymorphisms in the current differentiated thyroid carcinoma patients.

Table 1. Clinical Findings and Smoking Status of Current Controls

and Patients with Differentiated Thyroid Carcinomas

Patients Statistically comparison

Characteristics PTC FTC DTC (UMP) Control p-value OR (CI 95%)

Tumor type (n/%) 45 (75%) 9 (15%) 6 (10%) –Gender (n/%)

F 49 (81.7%) 29 (58%)M 11 (18.3%) 21 (42%)

The mean age(Min - Max)

55.25 – 3.22 (28–75) 68.60 – 2.82 (65–77)

Treatment dose(mCi) (Min - Max)

112.16 – 13.22 (100–154) –

Smoke(Package per a day)

Yes: 16 (26.6) Yes: 11 (22%) 0.286a 1.289 (0.530–3.187)No: 44 (73.4) No: 39 (18%)

Familial cancer history(first degree) (n/%)

Yes: 22 (36.6%) Yes: 13 (26%) 0.116a 1.648 (0.724–3.747)No: 38 (63.4%) No: 37 (74%)

aChi-square test is significant, p > 0.01.PTC, papillary thyroid carcinoma; FTC, folicullar thyroid carcinoma; DTC (UMP), Well-differentiated thyroid carcinoma of uncertain

malignant potential; OR, odds ratio; CI, confidence interval.

782 OZDEMIR ET AL.

supplementation may affect the initiation and progression ofmethylated rectal tumors in men in the presence of a variableMTHFR gene and produce endogenous/exogenous hormonaleffects (Curtin et al., 2011). Lu et al. claimed that the individualscarrying MTHFR 677CC genotypes have protective effectswith regard to esophageal squamous cell carcinoma. They alsoclaimed that patients with high folate consumption showed abetter survival rate when compared with the individuals witha low folate intake (Lu et al., 2011).

In the current preliminary study, we aimed at finding apossible linkage between a homozygous mutated (T allele)MTHFR gene and DTC. Genomic DNA was extracted fromperipheral blood and genotyped by real-time PCR methods. Thepresented results report the allele frequency of two polymorphiccodons in the systemic functional gene of MTHFR in TurkishDTC patients. Preliminary results of the current study showedthat a polymorphic T allele in the 677 C > T codon in the MTHFRgene may be associated with a high risk of thyroid cancer inhumans. These data suggest that MTHFR 677 C > T SNP mayplay a pivotal role in the development of DTC in humans, andthe results should be supported by larger studies in differentethnic populations. Subic et al. have reported that the associ-ation with etiological factors, such as the MTHFR 677 C > Tgenotype with high alcohol intake, solely or in interaction,has an impact on oral cancer risk (Supic et al., 2011). Hustadet al. have claimed that thyroid dysfunction affects the fo-late and homocysteine metabolisms in the presence ofMTHFR 677 C > T gene polymorphism by the modifying ri-boflavin cofactors synthesis (Hustad et al., 2004). Smoke and

genetic susceptibility of MTHFR are the two suspected factorsmost closely associated with DTC according to the presentedresults. The same results were speculated by Rouissi et al. fortobacco, polymorphic MTHFR, MTR, MTRR genotypes, andbladder tumor stage association (Rouissi et al., 2011). Folatesare common constituents in DNA synthesis, replication, re-pair, and DNA methylation reactions, as reported by Wanget al. (2008). Metabolic functional enzymes such as MTHFR,methionine synthase, and thymidylate synthase play a crucialrole in folate metabolism and DNA methylation processes.Altered methylation status, such as hypo DNA methylation,may induce genomic instability and promote cancers due tovariable MTHFR enzyme activity (Momparler and Bovenzi,2000; Sato and Meltzer, 2006).

The current results indicated that individuals with the 677TTgenotype had an 11.7% higher risk of having DTC than thosewith the 1928CC genotype. Furthermore, patients carrying bothcopies of the variant alleles (TT) showed a 2.33 times higher riskof developing DTC than their control counterparts. Althoughthe A1298C polymorphism was not associated with DTC risk, asignificant interaction with smoking and familial cancer historywas observed in the current DTC patients.

In conclusion, the C677T polymorphism was significantlyassociated with DTC risk. It is also possible to speculate thatthe person with homozygous T-alleles in the MTHFR gene hasa risk of developing epigenetic-mediated DTC due to the re-duced activity of the MTHFR enzyme and diminished methylsources. Our results should be supported by large-scalestudies of representative DTC patients.

Table 2. The Genotypes and Allele Frequency of Methylenetetrahydrofolate Reductase 677 C > TSingle-Nucleotide Polymorphism in Healthy Controls and Current

Differentiated Thyroid Carcinoma Patients

GROUP Allele frequency

Patients (n = 60) Control (n = 50) Patient Control

Gene SNP Codon Genotype n% n% C T C T

MTHFR C > T 677 CC 28 (46.6) 33 (66.0)CT 25 (41.7) 14 (28.0)TT 7 (11.7) 3 (6.0) 0.675 0.325 0.80 0.20

p–value 0.02a

OR (CI 95%) 1.92 (1.03–3.58)a

aChi-Square test is significant, p > 0.01.SNP, single-nucleotide polymorphism.

Table 3. The Allele Frequency of Methylenetetrahydrofolate Reductase 1298 A > C Single-Nucleotide

Polymorphism in Healthy Controls and Current Differentiated Thyroid Carcinoma Patients

Group Allele frequency

Patients (n = 60) Control (n = 50) Patient Control

Gene SNP Codon Genotype n% n% A C A C

MTHFR A > C A1298C AA 22 (36.6) 24 (48.0)AC 33 (55.0) 22 (44.0)CC 5 (8.4) 4 (8.0) 0.642 0.358 0.700 0.300

p-value 0.18OR (95% CI) 1.30 (0.73–2.29)

MTHFR GENE, SNP, INCREASED T ALLELE, DTC 783

Author Disclosure Statement

There is no conflict of interest statement.

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Address correspondence to:Semra Ozdemir, M.D., Ph.D.

Department of Nuclear MedicineFaculty of Medicine

Canakkale Onsekiz Mart University17100 Canakkale

Turkey

E-mail: semozdemir@yahoo.com

784 OZDEMIR ET AL.