Association of CYP2D6 *4 gene polymorphism with early papillary thyroid carcinoma [ CYP2D6 *4 gen polimor ﬁ zminin erken papiller

Objectives: CYP2D6 is highly polymorphic and a common variant CYP2D6 *4 results in the generation of poor meta-bolizer enzyme. The CYP2D6 *4 variant has been associated with altered susceptibility to several cancers. The aim of the present case-control study aims to investigate the association between CYP2D6 *4 polymorphism and the risk of papillary thyroid carcinoma (PTC). Materials and methods: Astudypopulationof97caseswith PTC and 120 controls were included in the study. Genotyping was done by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method to detect the presence of CYP2D6 *4. Results: The CYP2D6 *4 was associated with signi ﬁ cantly


Introduction
Although thyroid carcinomas constitute only 1% of all malignancies in humans; they are responsible for more than 90% of all endocrine cancers [1].The majority of thyroid cancers are epithelial tumors that arise from thyroid follicular cells.They are mainly classified into three subtypes: papillary thyroid carcinoma (PTC), follicular thyroid carcinoma (FTC) and anaplastic thyroid carcinoma (ATC).Differently, medullary thyroid carcinoma (MTC) derives from thyroid parafollicular (C) cells [2].
PTC, the commonest type of endocrine malignancy, is a differentiated cancer and its incidence has risen a lot in recent decades [3].PTC is classified into three major variants.Those are classic (CVPTC), follicular (FVPTC) and tall cell variants (TCVPTC).The genetic alterations of the signaling proteins in the mitogen-activated protein kinase (MAPK) pathway, including RET, ALK, RAS and BRAF are closely related with PTC [4].Many additional tests used together with thyroid specimens have focused on biomarkers in this signaling pathway [5].Although more advanced molecular tests are convenient, BRAF mutation is the most crucial biomarker for the diagnosis of PTC [6].The molecular mechanisms that participate in PTC tumorigenesis and progression are not fully understood.Thus, elucidation of the underlying molecular mechanisms will be useful for the advancement of new diagnosis and treatment strategies for PTC.
Cytochrome P450s (CYPs) are mostly located in the liver and metabolize xenobiotics to non-toxic or carcinogenic metabolites [7].CYP2D6 (debrisoquine hydroxylase) is a member of CYP family and the CYP2D6 gene takes place on chromosome 22q13.1 [8].CYP2D6, takes part in the metabolism of drugs and bioactivation of several procarcinogens and neurotoxins [9].The gene encoding the CYP2D6 enzyme is very polymorphic with more than 100 variants, and the allele frequencies vary between ethnic populations [10].People may be sorted as poor, intermediate, extensive, or ultrarapid metabolizers depending on the CYP2D6 genotype combinations [11].Among the variant alleles with nonfunctional activity, CYP2D6*4 (G1934A) is the most common one [10,12].A single nucleotide polymorphism at the splice site of intron 3 and exon 4 leads to the development of a deficient enzyme variant (rs3892097; c.1846G>A) (also called as CYP2D6*4 isoform) and consequently result in a poor metabolizer status [13].The poor metabolizer genotype has been shown to have effect on susceptibility to various types of cancers including head and neck [14], acute lymphoblastic leukemia (ALL) [15], prostate [16] and breast cancer [17]; but only a few studies have been studied for thyroid cancer [18].In the present study, we aimed to evaluate the contribution of CYP2D6*4 polymorphism to the susceptibility of PTC in a Turkish population considering their demographic and histopathological characteristics.

Patient selection
Paraffin-embedded thyroid tissue samples of 97 cases were provided from the department of pathology, Cerrahpaşa Faculty of Medicine.The cases operated with PTC between 2000 and 2010 were involved in the study.The selected cases had tumors greater than 10 mm.Sixtyseven patients had CVPTC, and 30 had FVPTC.Tumors were evaluated and classified according to the WHO recommendations, 2004.Since BRAF V600E is a somatic mutation, the analysis in our previous study was performed from tumor tissue samples [19].Therefore, the genotyping of CYP2D6 was performed with DNA extracted from these tissue samples.In addition, CYP2D6 is inherited as an autosomal recessive trait [20], genotypes are the same in tissue and blood, so the genotyping of controls were conducted on DNA extracted from blood samples taken from healthy controls that matched the age of the patients.The ethics approval was acquired from the local ethical committee.Informed consent was taken from whole cases and controls.

DNA extraction from tissue and peripheral blood samples
After microscopic evaluation, the regions of the tissues that contained the tumoral tissues on the stained slides and paraffin blocks were determined.Thereafter, the tumoral areas were dissected and taken into sterile tubes.DNA extraction from the tumoral tissues and peripheral blood samples were done by using spin column kits (Purelink Genomic DNA Mini Kit, Invitrogen), as suggested by the manufacturer.

Statistical analysis
We performed sample size calculation using the "PS Power and Sample Size Calculation" package program (version 3.0) [22], with inputs of p0 (probability of exposure in controls) and p1 (probability of exposure in cases) from the Ensemble genome browser.The Type I error probability (α) was used as 0.05.According to the results of this analysis, the study sample of 217 subjects (97 patients and 120 controls) provided adequate statistical power (0.640) to show association of CYP2D6*4 gene polymorphism and risk of papillary thyroid carcinoma.The statistical calculations were done using SPSS version 20.0 Software (SPSS Inc., Chicago, IL, USA).The association between CYP2D6*4 and the gender and histopathologic parameters of the thyroid specimens were analyzed by Chi-square test.The relationship between CYP2D6*4 and age was assessed by Student's t-test.Odds ratios (OR) with 95% confidence interval (CI) were determined for the relationship between CYP2D6*4 and risk of PTC.A p value of <0.05 was regarded as statistically significant.

Results
All of the patients with PTC had occult tumors.The patients had no secondary tumors.The mean age was 42.37 ± 10.52 years in patients with PTC and 40.33 ± 8.32 years in controls (p>0.05).The female to male ratio was greater in the PTC group when compared with the controls but there was no significance (in PTC group: 71/26, in control group: 79/41; p>0.05).Our patient group consisted of two variant subgroups: CVPTC and FVPTC.The distributions of tumor variants for gender and age were alike.The frequency distributions of PTC patients according to tumor stages were 31 (32%) PT1, 15 (15.4%)PT2, 39 (40.2%)PT3 and 12 (12.4%)PT4.The 'A' allele (CYP2D6*4) frequency was higher in patients who had encapsulated tumors, but it was not statistically significant (56.2 vs. 33.3%,p=0.111).The frequency distribution of histopathologic parameters among papillary thyroid carcinoma cases is given in Table 1.The differences were not significant between the PTC patient and control groups, when age and sex distributions were analyzed (p>0.05).The distributions of genotypes and alleles of CYP2D6 are shown in Table 2 and Figure 1(a).The frequency of 'A' allele was greater in cases with PTC than in control group (OR: 1.995, 95% CI: 1.060-3.752,p=0.031).Moreover the 'A' allele frequency was higher in patients with age ≤50 years when compared to those with age >50 (OR: 2.380, 95% CI: 1.191-4.755,p=0.013).The age-dependent distributions of genotypes and alleles of CYP2D6 are shown in Figure 1(b).
The distribution of CYP2D6 polymorphism was not consistent with HWE (p<0.05) in both patients and control groups.The 'A' allele frequency was higher among the patients who had lymphocyte infiltration in their tumorsurrounding tissues, but there was no statistical significance (p=0.211).Genotype and allele frequencies were not significantly different between patients with the classical and the follicular variant of PTC.CYP2D6 genotype and allele

Discussion
PTC incidence rate has increased worldwide, in recent decades.The development of new diagnostic techniques provided detection of even small tumors [23].Humans are exposed to many different chemicals as a result of modern life.The biotransformation of those xenobiotics are mainly done through two enzymatic pathways: phase 1 and phase 2. A plenty of polycyclic aromatic hydrocarbons, pesticides, herbicides, and drugs are metabolized by CYPs [24].CYPs are highly polymorphic and there is an inter-individual variability in the capacity to detoxify carcinogens or activate procarcinogens.
CYP2D6*4 which is the most prevalent polymorphism among Caucasians and creates a deficient protein [25].Poor metabolizers are not capable of producing functional CYP2D6 enzymes.Therefore the substrates of the enzyme accumulate to a large extent without being metabolized [26].Insufficient capacity to detoxify toxic compounds causes DNA and cellular damage due to the genomic instability resulting from the construction of chemical elements linked to DNA and protein molecules.Consequently, the possibility of developing cancer increases [27].
First, in 1984, Ayesh et al. reported that CYP2D6 enzyme activity had an effect on cancer, in lung cancer patients [28].Thereafter, many studies were conducted on investigating the influence of CYP2D6 on susceptibility to various kinds of cancer.However, the influence of CYP2D6 allelic variants in different types of cancer has remained controversial.While some studies suggested a possible role for the poor metabolizer genotype in the development of cancer, other studies could not find evidence for this.The CYP2D6*4 allele was not reported to effect the susceptibility to bladder [29] or gastric cancer [30] risk in Tunisian   The data was shown as n, %.
and Chinese patients, respectively.No association was reported with CYP2D6*4 and the risk of acute leukemia AML and ALL in Turkish cases [31], but a risk was found in Brazilian ALL cases [15].The influence of CYP2D6*4 on breast cancer susceptibility is also controversial [17,32].On the other hand, CYP2D6*4 allele frequency was significantly reduced in patients who had tumors in their pituitary glands [25].The CYP2D6*4 allele was found to have a protective effect on susceptibility to PTC in Portuguese [18].In contrast, we found that presence of CYP2D6*4 polymorphism might be a risk determinant for PTC in patients with especially age ≤50 years in Turkish population.The diversity in ethnic and geographical conditions as well as the genetic backgrounds and environmental exposures may have an impact on the risk of PTC associated with the CYP2D6 genotype.Lemos et al. reported that, although they have different morphological structures, the two major variants CVPTC and FVPTC had similar CYP2D6 genotype and allele frequencies Likewise, no difference was found between the early and late tumor stage, or multifocality [18].The findings of the current study were similar to their findings.
In this study, the distribution of CYP2D6*4 allele frequency was not concordant with Hardy-Weinberg equilibrium (HWE) in both patients and control groups.This may be due to the relatively small size of our study groups [33].Moreover, according to the Hardy-Weinberg principle, factors such as mutations, migration, lack of natural selection and non-random mating may cause genotypic and allele frequencies in the population to remain constant from generation to generation [34].
Silveira et al. reported that, since the CYP2D6 gene takes a role in the detoxification of carcinogenic compounds; and thus due to the lack of enzymatic activity genotoxic metabolites gets accumulated in phase I metabolism process thereupon the risk of ALL increases [15].Here, we agree with Silveira et al. that presumably the accumulated genotoxic metabolites forming as a consequence of poor metabolizing enzyme activity may take a role in the development of PTC.

Conclusion
To our knowledge, there is no study other than ours investigating the relationship between CYP2D6*4 allele and histopathological characteristics and risk of PTC, in Turkish patients.Our results may provide new insight to understand the molecular causes of thyroid cancer susceptibility.The major limitation of this study was the relatively low number of participants in the patient and control groups.Despite the fact that the results may provide clinical impact, our data needs confirmation by increasing the number of cases in the study.
[19]ar Aday et al.: Association of CYP2D6*4 gene polymorphism frequencies were not significantly different between patients with the CVPTC and FVPTC or between at early (I/II) and late (III/IV) stage tumors.BRAF V600E mutation analysis of the same patient group was analyzed in our previous study[19].Fifty-four (55.67%) of the PTC patients had BRAF V600E mutation.According to the histological type of the tumor, the BRAF V600E mutation was present in 65.67% of cases of CVPTC (44 of 67) and in 33.33% (10 of 30) of FVPTC.The frequency of BRAF V600E mutation was statistically higher

Table  :
CYPD genotype and allele frequencies in patients with papillary thyroid carcinoma (PTC) and in controls.Intermediate Metabolizing Enzyme; PME, Poor Metabolizing Enzyme.Chi-square method was used to compare the groups.The data was shown as n, %. *p=.,X  =.; OR=. (% CI: .-.).

Table  :
Relationship between BRAF VE mutation and CYPD*.