Original article 273
Melanoma risk is associated with vitamin D receptor gene polymorphisms Katarina Zeljica,f,*, Lidija Kandolf-Sekulovicb,e,*, Gordana Supica,e, Janko Pejovicc, Marijan Novakovicd,e, Zeljko Mijuskovicb,e and Zvonko Magica,e Previous studies have reported that vitamin D receptor (VDR) gene polymorphisms are associated with the occurrence of various cancers, including melanoma. The aim of the current study was to investigate the association of VDR gene polymorphisms with melanoma risk, clinicopathological characteristics, and vitamin D levels. The study group included 117 patients (84 patients with superficial spreading melanoma and 33 patients with nodular melanoma). The control group included 122 sex-matched and age-matched healthy-blood donors of the same ethnicity. VDR gene polymorphisms FokI, EcoRV, TaqI, and ApaI were genotyped by real-time PCR. In 60 patients, the total 25-hydroxyvitamin D levels were evaluated in serum samples by direct chemiluminescence. Associations among parameters were considered to be significant if the P value was less than 0.05. Significant differences in the frequencies of VDR genotypes were observed between cases and the control group for FokI and TaqI polymorphisms (P < 0.0001; P = 0.005, respectively). Heterozygous Ff as well as mutant FF genotypes of the FokI polymorphism were associated with increased melanoma risk compared with the wild-type form [odds ratio (OR) = 3.035, P = 0.003; OR = 9.276, P < 0.0001, respectively]. A significantly increased melanoma risk was observed for the heterozygous Tt (OR = 2.302, P = 0.011) and the mutated variant tt
(OR = 3.697, P = 0.003) of the TaqI polymorphism in comparison with the wild-type genotype. None of the polymorphisms studied was associated with clinicopathological characteristics and vitamin D serum level. Our results suggest that FokI and TaqI polymorphisms in the VDR gene may be considered as potential biomarkers for melanoma susceptibility. Low vitamin D levels in melanoma patients indicate the need for vitamin D supplementation. Melanoma Res 24:273–279 c 2014 Wolters Kluwer Health | Lippincott Williams & Wilkins.
Introduction
Numerous studies have shown that vitamin D exerts antiproliferative, prodifferentiation, proapoptotic, and angiogenesis inhibitor effects [8,9]. There are also some data, which require further confirmation, on the use of vitamin D as a preventive and therapeutic cancer agent, supported by preclinical and clinical studies in different cancer types, including melanoma [10].
Cutaneous melanoma, in its advanced stage, is one of the most aggressive and, despite recent advances in treatment, still incurable diseases, with a median survival of less than 1 year [1–3]. The incidence of melanoma is increasing worldwide [4] and the main risk factors for its development are ultraviolet radiation, number of nevi, and skin phototype [5]. Genetic predisposition plays a significant role in the development of this cancer [5–7]. Apart from high-penetrance genes such as CDK4 and CDK6 and low-penetrance genes linked to the phenotype such as MC1R, well known for their role in melanoma development, considerable efforts have been made to discover other potential biomarkers that can be used reliably to help identify individuals at risk or predict the disease course.
All supplementary data is available directly from the corresponding author. c 2014 Wolters Kluwer Health | Lippincott Williams & Wilkins 0960-8931
Melanoma Research 2014, 24:273–279 Keywords: melanoma risk, single nucleotide polymorphisms, VDR gene, vitamin D level a
Laboratory for Molecular Genetics, Institute for Medical Research, bClinic for Dermatovenereology, cCenter for Biochemistry, dClinic for Plastic Surgery and Burns, Military Medical Academy, eFaculty of Medicine, Military Medical Academy, University of Defense and fDepartment of Genetics and Evolution, Faculty of Biology, University of Belgrade, Belgrade, Serbia Correspondence to Katarina Zeljic, PhD, Laboratory for Molecular Genetics, Institute for Medical Research, Military Medical Academy, Crnotravska 17, 11 000 Belgrade, Serbia Tel: + 381 63 84 52 350; fax: + 381 11 266 27 22; e-mails: [email protected], [email protected] *Katarina Zeljic and Lidija Kandolf-Sekulovic contributed equally to the writing of this article. Received 30 December 2013 Accepted 11 February 2014
Previous studies have reported the proapoptotic effects of vitamin D and cell migration inhibition in human melanoma cell lines in vitro [11]. In melanoma patients living in areas with low insolation, suboptimal concentrations of vitamin D were linked to greater Breslow thickness and melanoma progression [12]. Similar results were also found in areas with high insolation, such as Spain, where in the majority of melanoma patients at diagnosis, suboptimal vitamin D concentrations were also detected [13]. In contrast, in a Danish study, higher concentrations of vitamin D were found to be a biomarker for increased sun exposure and risk factor for melanoma DOI: 10.1097/CMR.0000000000000065
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274 Melanoma Research 2014, Vol 24 No 3
development [14]. No data have been reported on the vitamin D levels in melanoma patients from southeast Europe, an area with continental climate, low insolation during the winter, and high insolation in the summer. In this study, we report for the first time in southeast Europe vitamin D levels from our group of melanoma patients. The vitamin D functions are mediated through the nuclear vitamin D receptor (VDR), which acts as a ligand-inducible transcription factor [15]. The VDR protein is coded by the VDR gene (chromosome location 12q12–14) [15], which is expressed in both normal and malignant melanocytes [3]. Numerous single nucleotide polymorphisms (SNPs) have been identified in the VDR gene, which might compromise receptor functionality and consequently contribute toward cancer. The EcoRV (rs4516035) polymorphism is located in the VDR gene promoter region without known functional effects. FokI (rs2228570) is the most studied VDR gene polymorphism with functional effects. Namely, the mutated form results in the synthesis of a VDR protein shorter by three amino acids, which has increased transactivation capacity in comparison with the wild type [15,16]. ApaI (rs7975232) and TaqI (rs731236) polymorphisms, located in intron 8, are silent SNPs found in linkage disequilibrium (LD) [15]. A number of studies have reported an association between VDR gene polymorphisms and the risk of occurrence of various cancers, such as breast [17], colon [18], and oral cancer [19], but also melanoma [3,13,20]. Despite the important role of vitamin D in carcinogenesis, genetic variants in the VDR gene in melanoma have been investigated in a limited number of studies. As the data from previous studies are inconsistent, the aim of the current study was to investigate the association between VDR gene polymorphisms and the risk of occurrence of melanoma as well as the association with clinicopathological characteristics, prognosis, and vitamin D serum levels in melanoma patients from Serbia.
Patients and methods Study group and samples
The study group comprised 117 patients (56 men, 61 women, median age 54 years) with newly diagnosed melanoma in 2011 at the Melanoma Center, Military Medical Academy, Belgrade, Serbia. Eighty-four patients had superficial spreading melanoma and 33 patients had nodular melanoma. Patients with other melanoma subtypes were excluded from the study. Melanoma risk factors were recorded on the basis of a structured questionnaire containing data on sun exposure, physician recorded skin phototype, eye color, number of nevi, presence and number of solar lentigo, family history of melanoma and other cancers, and personal history of skin cancer. Clinicopathological characteristics were retrieved from the hospital database, and the following parameters were recorded: age, sex, melanoma subtype, tumor location, Breslow thickness, ulceration, pT stage, and clinical stage at diagnosis. Disease staging was performed
according to the current AJCC classification from 2009. The patients were followed up for a median of 26 months (3–43 months). The control group included 122 healthy-blood donors, White individuals of the same ethnicity with no personal cancer history, matched in sex and age. Peripheral blood samples were collected from both the patient and the control groups and stored at – 201C until DNA isolation. This study was approved by the Ethics Committee of the Military Medical Academy, according to the Helsinki Declaration (2008). All individuals provided informed consent for the use of biological samples for research purposes. DNA isolation
DNA was isolated from the peripheral blood samples using a Blood Prep kit at the AbiPrism 6100 NucPrep Station (Applied Biosystems, Foster City, California, USA) following the manufacturer’s instructions. DNA samples were stored at – 201C until further analysis. Genotyping
Polymorphisms EcoRV (rs4516035), FokI (rs2228570), ApaI (rs7975232), and TaqI (rs731236) in the VDR gene were genotyped by the allelic discrimination method using the TaqMan SNP Genotyping Assays (Applied Biosystems). Data analyses were carried out using SDS Software on a real-time PCR 7500 system (Applied Biosystems). Measurement of serum 25-hydroxyvitamin D
In 60 melanoma patients, the total 25-hydroxyvitamin D level was measured by direct chemiluminescence (Advia Centaur XP; Siemens, Erlangen, Germany) in serum samples collected at diagnosis and up to 6 months after melanoma diagnosis during the winter months. Insufficient levels of 25-hydroxyvitamin D were defined as less than 20 ng/ml (< 50 nmol/l) [13]. Statistical analysis
Collected data were analyzed using SPSS software (version 20.00; IBM Inc., Chicago, Illinois, USA). Binary logistic regression was used for odds ratio (OR) calculation with 95% confidence interval (CI). All reported ORs and 95% CIs were adjusted for sex and age. Dominant, recessive, and log-additive genetic inheritance models were tested. Hazard ratio was calculated by the Cox proportional hazard regression analysis with 95% CI. Kaplan–Meier survival curves were compared using the log-rank test. Using Haploview software (version 4.2) [21], pairwise LD (D0 and r2 values), haplotype frequencies, and departure from Hardy–Weinberg equilibrium were determined. Haplotype block was identified using the option ‘solid spine block definition’. Permutation testing to calculate corrected P values for multiple testing of 1000
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VDR gene polymorphisms in melanoma Zeljic et al.
simulations was performed using Haploview software. Haplotype association analysis was carried out using Thesias software (version 3.1) [22]. Calculated P values were two-tailed and considered significant when P values were less than 0.05.
Results Association between vitamin D receptor single nucleotide polymorphisms, melanoma risk, and clinicopathological characteristics
Genotype and allele frequencies of polymorphisms analyzed in the VDR gene are presented in Table 1. Significant differences in the VDR allele and genotype frequencies between the case and the control groups were observed for the FokI and TaqI polymorphisms (P < 0.0001; P < 0.0001, respectively) (Table 1). Adjusted OR analysis showed that the heterozygous Ff as well as the mutated FF genotype of the FokI polymorphism were associated with an increased risk of melanoma compared with the wild-type genotype (OR = 2.860, P = 0.004; OR = 9.033, P < 0.0001, respectively) (Table 2). Significantly increased risk of melanoma was observed for the heterozygous Tt (OR = 2.323, P = 0.011) and the mutated variant tt (OR = 3.558, P = 0.003) of the TaqI polymorphism compared with the wild-type genotype (Table 2). Mutated allele carriers (combined heterozygous and mutated genotypes) were associated with a significantly increased risk of melanoma compared with the wild-type genotype for the FokI and TaqI polymorphisms (OR = 4.015, P < 0.0001; OR = 2.616, P = 0.002, respectively) (Table 2). None of these polymorphisms was associated with clinicopathological characteristics: melanoma type, Breslow thickness, ulceration, tumor size, presence of regional or distant metastases, and stage at diagnosis (Supplementary Table 1). Cox regression analysis showed that Breslow thickness, tumor size, nodal status, and stage were significant indicators for overall survival (Table 3). However, the VDR gene SNPs studied here were not associated with overall survival (Kaplan–Meier survival curves not shown). Table 1
275
Haplotype analysis and linkage disequilibrium
Pairwise LD coefficients (D0 and r2 values) for VDR gene SNPs, on the basis of genotypes of 239 individuals in the case–control study and in each of the case and control groups separately, were calculated and plotted (Fig. 1). VDR ApaI and TaqI polymorphisms were in LD in the cases (D0 = 0.59, r2 = 0.17) and the control group (D0 = 1, r2 = 0.32), as well as the case and control groups combined (D0 = 0.78, r2 = 0.24) (Fig. 1).
Association between haplotypes and melanoma risk and clinicopathological characteristics
In further analyses, we considered haplotypes of VDR ApaI and TaqI polymorphisms found in LD. Thus, the EcoRV and FokI polymorphisms were excluded from the study, according to the LD findings. Permutation and raw P values are presented in Table 4. A significant difference in haplotype frequencies between melanoma cases and controls was observed for At, aT, and at haplotypes. The at haplotype was significantly more frequent in melanoma patients than in the control group, where haplotype at was not detected (6 vs. 0%, raw P = 0.003, permutation P = 0.003). The At haplotype was more prevalent in melanoma patients (39 vs. 32%, raw P = 0.040, permutation P = 0.210), whereas the aT haplotype was more prevalent in the control group (40 vs. 29%, raw P = 0.030, permutation P = 0.095). The association between identified haplotypes and risk of melanoma and clinicopathological characteristics was estimated using the Thesias software. The aT haplotype was associated with an increased risk of melanoma (haplotype OR = 1.781, P = 0.006) in comparison with the most frequent At haplotype (Table 4). A marginally significant trend toward an association between the AT haplotype and an increased risk of melanoma was found (haplotype OR = 1.594, P = 0.052) (Table 4). No significant associations between any of the identified haplotypes and clinicopathological characteristics were observed (data not shown).
Genotype and allele distribution of vitamin D receptor gene polymorphisms in melanoma patients and the control group
Genes/SNPs EcoRV/rs4516035
FokI/rs2228570
ApaI/rs7975232
TaqI/rs731236
Genotype
Cases (N)
Controls (N)
P
Allele
Cases (N)
Controls (N)
P
ee Ee EE ff Ff FF aa Aa AA TT Tt tt
27 66 24 17 60 40 21 41 55 33 62 22
34 51 37 46 62 14 29 41 52 59 48 15
0.067
e E
120 114
119 125
0.583
< 0.0001
f F
94 140
154 90
< 0.0001
0.533
a A
83 151
99 145
0.251
0.006
T t
128 106
166 78
0.003
N, total number of cases/controls; SNP, single nucleotide polymorphism. P < 0.05 is shown in bold.
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276 Melanoma Research 2014, Vol 24 No 3
Table 2
Vitamin D receptor gene polymorphisms association with the risk of melanoma
Genes/SNPs EcoRV/rs4516035
FokI/rs2228570
ApaI/rs7975232
TaqI/rs731236
Genotype
Cases [N (%)]
Controls [N (%)]
ee Ee EE Dominantb Recessivec Log-additive ff Ff FF Dominantb Recessivec Log-additive aa Aa AA Dominantb Recessivec Log-additive TT Tt tt Dominantb Recessivec Log-additive
27 (23) 66 (56) 24 (21) 90 (77) 93 (79) – 17 (15) 60 (51) 40 (34) 100 (85) 77 (66) – 21 (18) 41 (35) 55 (47) 96 (82) 62 (53) – 33 (28) 62 (53) 22 (19) 84 (72) 95 (81) –
34 (28) 51 (42) 37 (30) 88 (72) 85 (70) – 46 (38) 62 (51) 14 (11) 76 (62) 108 (89) – 29 (24) 41 (34) 52 (43) 93 (76) 70 (57) – 59 (48) 48 (39) 15 (13) 63 (52) 107 (88) –
Adjusted ORa (95% CI) 1.000 1.745 1.058 1.463 0.654 1.020 1.000 2.860 9.033 4.015 3.986 2.920 1.000 1.573 2.118 1.868 1.537 0.700 1.000 2.323 3.558 2.616 2.293 1.970
(0.894–3.409) (0.433–2.524) (0.767–2.793) (0.344–1.245) (0.690–1.500) (1.397–5.855) (3.496–23.339) (1.983–8.128) (1.929–8.237) (1.850–4.590) (0.714–3.464) (0.965–4.650) (0.916–3.813) (0.871–2.714) (0.490–1.020) (1.219–4.427) (1.502–8.428) (1.433–4.775) (1.064–4.493) (1.300–2.970)
P Reference 0.103 0.899 0.248 0.196 0.930 Reference 0.004 < 0.0001 < 0.0001 < 0.0001 < 0.0001 Reference 0.261 0.061 0.086 0.138 0.061 Reference 0.010 0.004 0.002 0.034 0.001
CI, confidence interval; N, total number of cases/controls; SNP, single nucleotide polymorphism; OR, odds ratio. Adjusted for sex and age. Heterozygous and mutated genotypes combined versus the wild-type genotype. c Mutated genotype versus heterozygous and the wild-type genotype combined. P < 0.05 is shown in bold. a
b
and serum levels of 25-hydroxyvitamin D (Supplementary Table 3).
Table 3 Univariate analysis of clinicopathological variables of melanoma patients according to the Cox regression model 95% CI Variables Sex Age Melanoma type Ulceration Breslow thickness pT stage Nodal status Clinical stage at diagnosis EcoRV rs4516035 FokI rs2228570 ApaI rs7975232 TaqI rs731236
HR
Lower
Upper
P
0.699 0.973 0.241 1.175 1.206 2.655 2.673 2.607 0.781 0.827 1.970 0.871
0.156 0.927 0.054 0.283 1.029 1.024 1.335 1.058 0.257 0.277 0.596 0.288
3.125 1.022 1.077 4.879 1.414 6.884 5.353 6.427 2.373 2.475 6.513 2.630
0.639 0.281 0.062 0.825 0.021 0.045 0.006 0.037 0.662 0.735 0.266 0.806
CI, confidence interval; HR, hazard ratio. P < 0.05 is shown in bold.
25-Hydroxyvitamin D serum level
The 25-hydroxyvitamin D serum level was measured in 60 melanoma patients 3–6 months from diagnosis during the winter months. Of melanoma patients, 31% had 25-hydroxyvitamin D levels less than 25 nmol/l, 60% in a range of 26–50 nmol/l, whereas in only 9% of patients concentrations were higher than 50 nmol/l. Thus, a serum level of 25-hydroxyvitamin D was evaluated as a dichotomous variable and classified into two categories: less than 25 nmol/l and at least 25 nmol/l. There was no association between 25-hydroxyvitamin D serum level and VDR gene polymorphisms (Supplementary Table 2). The absence of an association was observed for the clinicopathological characteristics of melanoma patients
Discussion Ultraviolet radiation plays an important role in melanomagenesis and, at the same time, it is necessary for the skin vitamin D synthesis, the main source of vitamin D in humans. The UVR action spectrum is almost the same for direct DNA damage and vitamin D synthesis – it belongs to the UVB spectrum. Hence, there is a complex relationship between UVR exposure, vitamin D levels, VDR polymorphisms, and development of melanoma. This could explain the variable association of these factors in different populations from different latitudes, and so far, these have been reported for the UK, Spain, USA, and Denmark. In this study, we have explored these associations, particularly VDR polymorphisms and vitamin D levels, in samples in Serbia, representing the population of southeast Europe. In our study, significant differences in both genotype distribution and allele frequency of FokI and TaqI VDR gene polymorphisms between the cases and control groups indicate their potential role in melanoma susceptibility. The results of the current study indicate that heterozygous and mutated genotypes, as well as the variant allele carriers of FokI polymorphisms, could be associated with an increased risk of melanoma in comparison with the wild-type genotype. Thus, it could be assumed that wild-type FokI could play a protective role in melanoma susceptibility. Our findings are in
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VDR gene polymorphisms in melanoma Zeljic et al. 277
Fig. 1
18
8 9
2
78
Block 1 (0 kb) 3 4 2
0 0
18 1
rs731236
1
rs7975232
rs2228570
Block 1 (0 kb) 3 4
rs4516035
2
rs731236
rs2228570
1
rs7975232
rs4516035
(a)
24 2
0
16
16 1
2
59
1
Block 1 (0 kb) 3 4 2
0
12 5
rs731236
1
rs7975232
rs2228570
Block 1 (0 kb) 3 4
rs4516035
2
rs731236
rs2228570
1
rs7975232
rs4516035
(b)
17 0
0
19
11 15
rs731236
2
rs7975232
1 0
12 8
rs2228570
Block 1 (0 kb) 3 4
rs4516035
2
rs731236
rs2228570
1
rs7975232
rs4516035
(c)
Block 1 (0 kb) 3 4 32
1 1
1 0
Pairwise linkage disequilibrium plots of analyzed polymorphisms in the vitamin D receptor gene for (a) cases and the control group taken together, (b) cases group, (c) control group. D0 and r2 values are presented in percentages. The shaded boxes on the left side correspond to the paired D0 values between the single nucleotide polymorphisms (SNPs). The shaded boxes on the right side correspond to the paired r2 between the SNPs.
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278 Melanoma Research 2014, Vol 24 No 3
Table 4 Haplotype frequencies and haplotype odds ratio analysis for vitamin D receptor gene polymorphisms, compared with the most common haplotype N (%) Haplotypes At aT AT at
Total 85 84 63 7
(36) (35) (26) (3)
Association P value
Cases
Controls
46 35 29 7
39 50 34 0
(39) (29) (25) (6)
(32) (40) (28) (0)
Raw
Permutation
HOR (95% CI)
P
0.040 0.030 0.336 0.003
0.210 0.095 0.769 0.003
Reference 1.781 (1.176–2.698) 1.594 (0.996–2.551) 0.362 (0.003–44.178)
0.006 0.052 0.678
N, total number of cases/controls; HOR, haplotype odds ratio; CI, confidence interval. P < 0.05 is shown in bold.
agreement with the results from previous studies on melanoma [23–26] and basal cell carcinoma [27], although contrasting the results for oral cancer patients from the Serbian population [19]. It is known that the mutated form of FokI results in the synthesis of a shorter receptor form with higher transactivation capacity as a transcription factor [15]. Different findings of other studies examining various cancer types could be explained by the gene and cell type-specific transactivation capacity of the FokI mutated form [15].
previous findings in melanoma [2,25,29] and oral carcinoma [19]. However, a previous study has reported that the EcoRV mutated genotype is associated with a decreased risk of cutaneous melanoma compared with the wild-type form [30]. In another study, the wild-type allele of EcoRV SNP was associated with an increased susceptibility to melanoma, risk for metastasis, and poor patient outcome [31]. Conflicting data from the different studies warrant further analysis to elucidate the role of the EcoRV polymorphism in melanoma susceptibility.
Variant allele carriers of TaqI polymorphism were associated with an increased risk of melanoma, which is consistent with findings from other study [3]. In contrast, a recent study has reported no association of the TaqI polymorphism with the risk of melanoma [28]. The inconsistency between studies could be explained by population differences as well as study group size. Our data suggest that the wild-type genotype might protect carriers against melanoma. It is known that the TaqI polymorphism does not alter the amino acid sequences of the VDR protein [3]. Thus, the exact mechanism of how the TaqI polymorphism could influence the risk of melanoma remains unclear. It could be assumed that the mutated variant is itself functional or is in LD with other functionally relevant polymorphisms in the VDR gene. Further investigations on the functional effects of VDR polymorphisms on the risk of melanoma are warranted.
Studies on the association between VDR gene polymorphisms and the risk of melanoma are not consistent in the literature. These discrepancies may be because of differences in serum vitamin D levels, sun exposure, population differences, and sample size [3]. A study carried out in a Danish population showed that elevated plasma calcidiol level was associated with an increased risk of nonmelanoma and melanoma cancer [14]. In contrast, higher vitamin D serum levels were associated with lower Breslow thickness and better survival of melanoma patients in a UK study [12]. In the first study from a country with high insolation (Spain), suboptimal vitamin D levels were also found in melanoma patients at diagnosis, but the levels were higher than those reported in the UK study. In our study, the vitamin D levels in winter months were from 20 to 75 nmol/l in 68.6% of patients and less than 20 nmol/l in 31.4% of patients (median age 48.5 years). Low vitamin D levels were also found in postmenopausal women in this region in 88.4% of patients [32]. Apart from the possible insufficient sun exposure, these data also indicate a probable insufficient vitamin D intake in our population, and this was reported previously for the region of central and eastern Europe [33], possibly linked to socioeconomic factors, compared with western European countries [33]. This further emphasizes the necessity of identifying melanoma patients with suboptimal vitamin D levels, and advising them on vitamin D supplementation as after the diagnosis of melanoma, rigorous sun protection is advised, and therefore, vitamin D status could deteriorate further.
VDR ApaI and TaqI polymorphisms were found in LD in our study group, which is in agreement with the literature data [15]. Among the haplotypes identified, the aT haplotype was significantly associated with an increased risk of melanoma. The ApaI polymorphism was not associated with the risk of melanoma itself, whereas in the haplotype block with the T allele of TaqI SNP, association with increased susceptibility to melanoma was observed. These findings indicate that genetic association studies should include haplotype-based analysis. In addition, it has been shown that ApaI and TaqI polymorphisms could influence VDR mRNA stability [15], which could be a possible explanation for the haplotype association observed in the current study. The lack of association of the EcoRV polymorphism with the risk of melanoma observed in our research is consistent with
There was no association between vitamin D serum level and VDR gene polymorphisms analyzed nor with clinicopathological characteristics in melanoma patients, which is consistent with findings from other studies. Because of the small number of patients, the previously
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VDR gene polymorphisms in melanoma Zeljic et al. 279
described interaction of VDR polymorphism and vitamin D level could not be explored [12].
15
The rapid increase in the incidence of melanoma and its high mortality requires comprehensive research on the risk factors associated with melanoma. Our results suggest an association between FokI and TaqI polymorphisms in the VDR gene and the risk of melanoma, implicating their potential application as molecular markers for the management of patients at high risk for the development of this cancer and their increased surveillance. Further analysis on vitamin D intake and serum calcidiol levels of melanoma patients, as well as gene–gene and gene–environment interactions, would provide additional information on the importance and role of the VDR gene in melanoma susceptibility.
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Acknowledgements Conflicts of interest
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There are no conflicts of interest. 23
References 1
2
3
4 5
6 7 8 9 10 11 12
13
14
De Souza CF, Xander P, Monteiro AC, Silva AG, da Silva DC, Mai S, et al. Mining gene expression signature for the detection of pre-malignant melanocytes and early melanomas with risk for metastasis. PLoS One 2012; 7:e44800. Gapska P, Scott RJ, Serrano-Fernandez P, Mirecka A, Rassoud I, Gorski B, et al. Vitamin D receptor variants and the malignant melanoma risk: a population-based study. Cancer Epidemiol 2009; 33:103–107. Li C, Liu Z, Zhang Z, Strom SS, Gershenwald JE, Prieto VG, et al. Genetic variants of the vitamin D receptor gene alter risk of cutaneous melanoma. J Invest Dermatol 2007; 127:276–280. Tsao H, Chin L, Garraway LA, Fisher DE. Melanoma: from mutations to medicine. Genes Dev 2012; 26:1131–1155. Rastrelli M, Alaibac M, Stramare R, Chiarion Sileni V, Montesco MC, Vecchiato A, et al. Melanoma m (zero): diagnosis and therapy. ISRN Dermatol 2013; 2013:616170. Regad T. Molecular and cellular pathogenesis of melanoma initiation and progression. Cell Mol Life Sci 2013; 70:4055–4065. Bis S, Tsao H. Melanoma genetics: the other side. Clin Dermatol 2013; 31:148–155. Deeb KK, Trump DL, Johnson CS. Vitamin D signalling pathways in cancer: potential for anticancer therapeutics. Nat Rev Cancer 2007; 7:684–700. Field S, Newton-Bishop JA. Melanoma and vitamin D. Mol Oncol 2011; 5:197–214. Osborne JE, Hutchinson PE. Vitamin D and systemic cancer: is this relevant to malignant melanoma? Br J Dermatol 2002; 147:197–213. Gandini S, Francesco F, Johanson H, Bonanni B, Testori A. Why vitamin D for cancer patients? Ecancermedicalscience 2009; 3:160. Newton-Bishop JA, Beswick S, Randerson-Moor J, Chang YM, Affleck P, Elliott F, et al. Serum 25-hydroxyvitamin D3 levels are associated with Breslow thickness at presentation and survival from melanoma. J Clin Oncol 2009; 27:5439–5444. Ogbah Z, Visa L, Badenas C, Rios J, Puig-Butille JA, Bonifaci N, et al. Serum 25-hydroxyvitamin D3 levels and vitamin D receptor variants in melanoma patients from the Mediterranean area of Barcelona: 25-hydroxyvitamin D3 levels and VDR variants in melanoma patients from Barcelona. BMC Med Genet 2013; 14:26. Afzal S, Nordestgaard BG, Bojesen SE. Plasma 25-hydroxyvitamin D and risk of non-melanoma and melanoma skin cancer: a prospective cohort study. J Invest Dermatol 2013; 133:629–636.
24
25
26
27
28
29
30
31
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Uitterlinden AG, Fang Y, Van Meurs JB, Pols HA, Van Leeuwen JP. Genetics and biology of vitamin D receptor polymorphisms. Gene 2004; 338:143–156. Whitfield GK, Remus LS, Jurutka PW, Zitzer H, Oza AK, Dang HT, et al. Functionally relevant polymorphisms in the human nuclear vitamin D receptor gene. Mol Cell Endocrinol 2001; 177:145–159. Mishra DK, Wu Y, Sarkissyan M, Sarkissyan S, Chen Z, Shang X, et al. Vitamin D receptor gene polymorphisms and prognosis of breast cancer among African-American and Hispanic women. PLoS One 2013; 8:e57967. Kupfer SS, Anderson JR, Ludvik AE, Hooker S, Skol A, Kittles RA, et al. Genetic associations in the vitamin D receptor and colorectal cancer in African Americans and Caucasians. PLoS One 2011; 6:e26123. Zeljic K, Supic G, Stamenkovic Radak M, Jovic N, Kozomara R, Magic Z. Vitamin D receptor, CYP27B1 and CYP24A1 genes polymorphisms association with oral cancer risk and survival. J Oral Pathol Med 2012; 41:779–787. Santonocito C, Capizzi R, Concolino P, Lavieri MM, Paradisi A, Gentileschi S, et al. Association between cutaneous melanoma, Breslow thickness and vitamin D receptor BsmI polymorphism. Br J Dermatol 2007; 156:277–282. Barrett JC, Fry B, Maller J, Daly MJ. Haploview: analysis and visualization of LD and haplotype maps. Bioinformatics 2005; 21:263–265. Tregouet DA, Garelle V. A new JAVA interface implementation of THESIAS: testing haplotype effects in association studies. Bioinformatics 2007; 23:1038–1039. Hutchinson PE, Osborne JE, Lear JT, Smith AG, Bowers PW, Morris PN, et al. Vitamin D receptor polymorphisms are associated with altered prognosis in patients with malignant melanoma. Clin Cancer Res 2000; 6:498–504. Mocellin S, Nitti D. Vitamin D receptor polymorphisms and the risk of cutaneous melanoma: a systematic review and meta-analysis. Cancer 2008; 113:2398–2407. Randerson-Moor JA, Taylor JC, Elliott F, Chang YM, Beswick S, Kukalizch K, et al. Vitamin D receptor gene polymorphisms, serum 25-hydroxyvitamin D levels, and melanoma: UK case–control comparisons and a meta-analysis of published VDR data. Eur J Cancer 2009; 45:3271–3281. Li C, Liu Z, Wang LE, Gershenwald JE, Lee JE, Prieto VG, et al. Haplotype and genotypes of the VDR gene and cutaneous melanoma risk in nonHispanic whites in Texas: a case–control study. Int J Cancer 2008; 122:2077–2084. Lesiak A, Norval M, Wodz-Naskiewicz K, Pawliczak R, Rogowski-Tylman M, Sysa-Jedrzejowska A, et al. An enhanced risk of basal cell carcinoma is associated with particular polymorphisms in the VDR and MTHFR genes. Exp Dermatol 2011; 20:800–804. Schafer A, Emmert S, Kruppa J, Schubert S, Tzvetkov M, Mossner R, et al. No association of vitamin D metabolism-related polymorphisms and melanoma risk as well as melanoma prognosis: a case–control study. Arch Dermatol Res 2012; 304:353–361. Barroso E, Fernandez LP, Milne RL, Pita G, Sendagorta E, Floristan U, et al. Genetic analysis of the vitamin D receptor gene in two epithelial cancers: melanoma and breast cancer case–control studies. BMC Cancer 2008; 8:385. Povey JE, Darakhshan F, Robertson K, Bisset Y, Mekky M, Rees J, et al. DNA repair gene polymorphisms and genetic predisposition to cutaneous melanoma. Carcinogenesis 2007; 28:1087–1093. Halsall JA, Osborne JE, Epstein MP, Pringle JH, Hutchinson PE. The unfavorable effect of the A allele of the vitamin D receptor promoter polymorphism A-1012G has different mechanisms related to susceptibility and outcome of malignant melanoma. Dermatoendocrinology 2009; 1:54–57. Vuceljic M, Ilic-Stojanovic O, Lazovic M, Grajic M. Vitamin D and parathyroid hormone in relation to bone mineral density in postmenopausal women. Vojnosanit Pregl 2012; 69:243–248. Novakovic R, Cavelaars AE, Bekkering GE, Roman-Vinas B, Ngo J, Gurinovic M, et al. Micronutrient intake and status in Central and Eastern Europe compared with other European countries, results from the EURRECA network. Public Health Nutr 2013; 16:824–840.
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Melanoma risk is associated with vitamin D receptor gene polymorphisms Katarina Zeljica,f,*, Lidija Kandolf-Sekulovicb,e,*, Gordana Supica,e, Janko Pejovicc, Marijan Novakovicd,e, Zeljko Mijuskovicb,e and Zvonko Magica,e Previous studies have reported that vitamin D receptor (VDR) gene polymorphisms are associated with the occurrence of various cancers, including melanoma. The aim of the current study was to investigate the association of VDR gene polymorphisms with melanoma risk, clinicopathological characteristics, and vitamin D levels. The study group included 117 patients (84 patients with superficial spreading melanoma and 33 patients with nodular melanoma). The control group included 122 sex-matched and age-matched healthy-blood donors of the same ethnicity. VDR gene polymorphisms FokI, EcoRV, TaqI, and ApaI were genotyped by real-time PCR. In 60 patients, the total 25-hydroxyvitamin D levels were evaluated in serum samples by direct chemiluminescence. Associations among parameters were considered to be significant if the P value was less than 0.05. Significant differences in the frequencies of VDR genotypes were observed between cases and the control group for FokI and TaqI polymorphisms (P < 0.0001; P = 0.005, respectively). Heterozygous Ff as well as mutant FF genotypes of the FokI polymorphism were associated with increased melanoma risk compared with the wild-type form [odds ratio (OR) = 3.035, P = 0.003; OR = 9.276, P < 0.0001, respectively]. A significantly increased melanoma risk was observed for the heterozygous Tt (OR = 2.302, P = 0.011) and the mutated variant tt
(OR = 3.697, P = 0.003) of the TaqI polymorphism in comparison with the wild-type genotype. None of the polymorphisms studied was associated with clinicopathological characteristics and vitamin D serum level. Our results suggest that FokI and TaqI polymorphisms in the VDR gene may be considered as potential biomarkers for melanoma susceptibility. Low vitamin D levels in melanoma patients indicate the need for vitamin D supplementation. Melanoma Res 24:273–279 c 2014 Wolters Kluwer Health | Lippincott Williams & Wilkins.
Introduction
Numerous studies have shown that vitamin D exerts antiproliferative, prodifferentiation, proapoptotic, and angiogenesis inhibitor effects [8,9]. There are also some data, which require further confirmation, on the use of vitamin D as a preventive and therapeutic cancer agent, supported by preclinical and clinical studies in different cancer types, including melanoma [10].
Cutaneous melanoma, in its advanced stage, is one of the most aggressive and, despite recent advances in treatment, still incurable diseases, with a median survival of less than 1 year [1–3]. The incidence of melanoma is increasing worldwide [4] and the main risk factors for its development are ultraviolet radiation, number of nevi, and skin phototype [5]. Genetic predisposition plays a significant role in the development of this cancer [5–7]. Apart from high-penetrance genes such as CDK4 and CDK6 and low-penetrance genes linked to the phenotype such as MC1R, well known for their role in melanoma development, considerable efforts have been made to discover other potential biomarkers that can be used reliably to help identify individuals at risk or predict the disease course.
All supplementary data is available directly from the corresponding author. c 2014 Wolters Kluwer Health | Lippincott Williams & Wilkins 0960-8931
Melanoma Research 2014, 24:273–279 Keywords: melanoma risk, single nucleotide polymorphisms, VDR gene, vitamin D level a
Laboratory for Molecular Genetics, Institute for Medical Research, bClinic for Dermatovenereology, cCenter for Biochemistry, dClinic for Plastic Surgery and Burns, Military Medical Academy, eFaculty of Medicine, Military Medical Academy, University of Defense and fDepartment of Genetics and Evolution, Faculty of Biology, University of Belgrade, Belgrade, Serbia Correspondence to Katarina Zeljic, PhD, Laboratory for Molecular Genetics, Institute for Medical Research, Military Medical Academy, Crnotravska 17, 11 000 Belgrade, Serbia Tel: + 381 63 84 52 350; fax: + 381 11 266 27 22; e-mails: [email protected], [email protected] *Katarina Zeljic and Lidija Kandolf-Sekulovic contributed equally to the writing of this article. Received 30 December 2013 Accepted 11 February 2014
Previous studies have reported the proapoptotic effects of vitamin D and cell migration inhibition in human melanoma cell lines in vitro [11]. In melanoma patients living in areas with low insolation, suboptimal concentrations of vitamin D were linked to greater Breslow thickness and melanoma progression [12]. Similar results were also found in areas with high insolation, such as Spain, where in the majority of melanoma patients at diagnosis, suboptimal vitamin D concentrations were also detected [13]. In contrast, in a Danish study, higher concentrations of vitamin D were found to be a biomarker for increased sun exposure and risk factor for melanoma DOI: 10.1097/CMR.0000000000000065
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274 Melanoma Research 2014, Vol 24 No 3
development [14]. No data have been reported on the vitamin D levels in melanoma patients from southeast Europe, an area with continental climate, low insolation during the winter, and high insolation in the summer. In this study, we report for the first time in southeast Europe vitamin D levels from our group of melanoma patients. The vitamin D functions are mediated through the nuclear vitamin D receptor (VDR), which acts as a ligand-inducible transcription factor [15]. The VDR protein is coded by the VDR gene (chromosome location 12q12–14) [15], which is expressed in both normal and malignant melanocytes [3]. Numerous single nucleotide polymorphisms (SNPs) have been identified in the VDR gene, which might compromise receptor functionality and consequently contribute toward cancer. The EcoRV (rs4516035) polymorphism is located in the VDR gene promoter region without known functional effects. FokI (rs2228570) is the most studied VDR gene polymorphism with functional effects. Namely, the mutated form results in the synthesis of a VDR protein shorter by three amino acids, which has increased transactivation capacity in comparison with the wild type [15,16]. ApaI (rs7975232) and TaqI (rs731236) polymorphisms, located in intron 8, are silent SNPs found in linkage disequilibrium (LD) [15]. A number of studies have reported an association between VDR gene polymorphisms and the risk of occurrence of various cancers, such as breast [17], colon [18], and oral cancer [19], but also melanoma [3,13,20]. Despite the important role of vitamin D in carcinogenesis, genetic variants in the VDR gene in melanoma have been investigated in a limited number of studies. As the data from previous studies are inconsistent, the aim of the current study was to investigate the association between VDR gene polymorphisms and the risk of occurrence of melanoma as well as the association with clinicopathological characteristics, prognosis, and vitamin D serum levels in melanoma patients from Serbia.
Patients and methods Study group and samples
The study group comprised 117 patients (56 men, 61 women, median age 54 years) with newly diagnosed melanoma in 2011 at the Melanoma Center, Military Medical Academy, Belgrade, Serbia. Eighty-four patients had superficial spreading melanoma and 33 patients had nodular melanoma. Patients with other melanoma subtypes were excluded from the study. Melanoma risk factors were recorded on the basis of a structured questionnaire containing data on sun exposure, physician recorded skin phototype, eye color, number of nevi, presence and number of solar lentigo, family history of melanoma and other cancers, and personal history of skin cancer. Clinicopathological characteristics were retrieved from the hospital database, and the following parameters were recorded: age, sex, melanoma subtype, tumor location, Breslow thickness, ulceration, pT stage, and clinical stage at diagnosis. Disease staging was performed
according to the current AJCC classification from 2009. The patients were followed up for a median of 26 months (3–43 months). The control group included 122 healthy-blood donors, White individuals of the same ethnicity with no personal cancer history, matched in sex and age. Peripheral blood samples were collected from both the patient and the control groups and stored at – 201C until DNA isolation. This study was approved by the Ethics Committee of the Military Medical Academy, according to the Helsinki Declaration (2008). All individuals provided informed consent for the use of biological samples for research purposes. DNA isolation
DNA was isolated from the peripheral blood samples using a Blood Prep kit at the AbiPrism 6100 NucPrep Station (Applied Biosystems, Foster City, California, USA) following the manufacturer’s instructions. DNA samples were stored at – 201C until further analysis. Genotyping
Polymorphisms EcoRV (rs4516035), FokI (rs2228570), ApaI (rs7975232), and TaqI (rs731236) in the VDR gene were genotyped by the allelic discrimination method using the TaqMan SNP Genotyping Assays (Applied Biosystems). Data analyses were carried out using SDS Software on a real-time PCR 7500 system (Applied Biosystems). Measurement of serum 25-hydroxyvitamin D
In 60 melanoma patients, the total 25-hydroxyvitamin D level was measured by direct chemiluminescence (Advia Centaur XP; Siemens, Erlangen, Germany) in serum samples collected at diagnosis and up to 6 months after melanoma diagnosis during the winter months. Insufficient levels of 25-hydroxyvitamin D were defined as less than 20 ng/ml (< 50 nmol/l) [13]. Statistical analysis
Collected data were analyzed using SPSS software (version 20.00; IBM Inc., Chicago, Illinois, USA). Binary logistic regression was used for odds ratio (OR) calculation with 95% confidence interval (CI). All reported ORs and 95% CIs were adjusted for sex and age. Dominant, recessive, and log-additive genetic inheritance models were tested. Hazard ratio was calculated by the Cox proportional hazard regression analysis with 95% CI. Kaplan–Meier survival curves were compared using the log-rank test. Using Haploview software (version 4.2) [21], pairwise LD (D0 and r2 values), haplotype frequencies, and departure from Hardy–Weinberg equilibrium were determined. Haplotype block was identified using the option ‘solid spine block definition’. Permutation testing to calculate corrected P values for multiple testing of 1000
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VDR gene polymorphisms in melanoma Zeljic et al.
simulations was performed using Haploview software. Haplotype association analysis was carried out using Thesias software (version 3.1) [22]. Calculated P values were two-tailed and considered significant when P values were less than 0.05.
Results Association between vitamin D receptor single nucleotide polymorphisms, melanoma risk, and clinicopathological characteristics
Genotype and allele frequencies of polymorphisms analyzed in the VDR gene are presented in Table 1. Significant differences in the VDR allele and genotype frequencies between the case and the control groups were observed for the FokI and TaqI polymorphisms (P < 0.0001; P < 0.0001, respectively) (Table 1). Adjusted OR analysis showed that the heterozygous Ff as well as the mutated FF genotype of the FokI polymorphism were associated with an increased risk of melanoma compared with the wild-type genotype (OR = 2.860, P = 0.004; OR = 9.033, P < 0.0001, respectively) (Table 2). Significantly increased risk of melanoma was observed for the heterozygous Tt (OR = 2.323, P = 0.011) and the mutated variant tt (OR = 3.558, P = 0.003) of the TaqI polymorphism compared with the wild-type genotype (Table 2). Mutated allele carriers (combined heterozygous and mutated genotypes) were associated with a significantly increased risk of melanoma compared with the wild-type genotype for the FokI and TaqI polymorphisms (OR = 4.015, P < 0.0001; OR = 2.616, P = 0.002, respectively) (Table 2). None of these polymorphisms was associated with clinicopathological characteristics: melanoma type, Breslow thickness, ulceration, tumor size, presence of regional or distant metastases, and stage at diagnosis (Supplementary Table 1). Cox regression analysis showed that Breslow thickness, tumor size, nodal status, and stage were significant indicators for overall survival (Table 3). However, the VDR gene SNPs studied here were not associated with overall survival (Kaplan–Meier survival curves not shown). Table 1
275
Haplotype analysis and linkage disequilibrium
Pairwise LD coefficients (D0 and r2 values) for VDR gene SNPs, on the basis of genotypes of 239 individuals in the case–control study and in each of the case and control groups separately, were calculated and plotted (Fig. 1). VDR ApaI and TaqI polymorphisms were in LD in the cases (D0 = 0.59, r2 = 0.17) and the control group (D0 = 1, r2 = 0.32), as well as the case and control groups combined (D0 = 0.78, r2 = 0.24) (Fig. 1).
Association between haplotypes and melanoma risk and clinicopathological characteristics
In further analyses, we considered haplotypes of VDR ApaI and TaqI polymorphisms found in LD. Thus, the EcoRV and FokI polymorphisms were excluded from the study, according to the LD findings. Permutation and raw P values are presented in Table 4. A significant difference in haplotype frequencies between melanoma cases and controls was observed for At, aT, and at haplotypes. The at haplotype was significantly more frequent in melanoma patients than in the control group, where haplotype at was not detected (6 vs. 0%, raw P = 0.003, permutation P = 0.003). The At haplotype was more prevalent in melanoma patients (39 vs. 32%, raw P = 0.040, permutation P = 0.210), whereas the aT haplotype was more prevalent in the control group (40 vs. 29%, raw P = 0.030, permutation P = 0.095). The association between identified haplotypes and risk of melanoma and clinicopathological characteristics was estimated using the Thesias software. The aT haplotype was associated with an increased risk of melanoma (haplotype OR = 1.781, P = 0.006) in comparison with the most frequent At haplotype (Table 4). A marginally significant trend toward an association between the AT haplotype and an increased risk of melanoma was found (haplotype OR = 1.594, P = 0.052) (Table 4). No significant associations between any of the identified haplotypes and clinicopathological characteristics were observed (data not shown).
Genotype and allele distribution of vitamin D receptor gene polymorphisms in melanoma patients and the control group
Genes/SNPs EcoRV/rs4516035
FokI/rs2228570
ApaI/rs7975232
TaqI/rs731236
Genotype
Cases (N)
Controls (N)
P
Allele
Cases (N)
Controls (N)
P
ee Ee EE ff Ff FF aa Aa AA TT Tt tt
27 66 24 17 60 40 21 41 55 33 62 22
34 51 37 46 62 14 29 41 52 59 48 15
0.067
e E
120 114
119 125
0.583
< 0.0001
f F
94 140
154 90
< 0.0001
0.533
a A
83 151
99 145
0.251
0.006
T t
128 106
166 78
0.003
N, total number of cases/controls; SNP, single nucleotide polymorphism. P < 0.05 is shown in bold.
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276 Melanoma Research 2014, Vol 24 No 3
Table 2
Vitamin D receptor gene polymorphisms association with the risk of melanoma
Genes/SNPs EcoRV/rs4516035
FokI/rs2228570
ApaI/rs7975232
TaqI/rs731236
Genotype
Cases [N (%)]
Controls [N (%)]
ee Ee EE Dominantb Recessivec Log-additive ff Ff FF Dominantb Recessivec Log-additive aa Aa AA Dominantb Recessivec Log-additive TT Tt tt Dominantb Recessivec Log-additive
27 (23) 66 (56) 24 (21) 90 (77) 93 (79) – 17 (15) 60 (51) 40 (34) 100 (85) 77 (66) – 21 (18) 41 (35) 55 (47) 96 (82) 62 (53) – 33 (28) 62 (53) 22 (19) 84 (72) 95 (81) –
34 (28) 51 (42) 37 (30) 88 (72) 85 (70) – 46 (38) 62 (51) 14 (11) 76 (62) 108 (89) – 29 (24) 41 (34) 52 (43) 93 (76) 70 (57) – 59 (48) 48 (39) 15 (13) 63 (52) 107 (88) –
Adjusted ORa (95% CI) 1.000 1.745 1.058 1.463 0.654 1.020 1.000 2.860 9.033 4.015 3.986 2.920 1.000 1.573 2.118 1.868 1.537 0.700 1.000 2.323 3.558 2.616 2.293 1.970
(0.894–3.409) (0.433–2.524) (0.767–2.793) (0.344–1.245) (0.690–1.500) (1.397–5.855) (3.496–23.339) (1.983–8.128) (1.929–8.237) (1.850–4.590) (0.714–3.464) (0.965–4.650) (0.916–3.813) (0.871–2.714) (0.490–1.020) (1.219–4.427) (1.502–8.428) (1.433–4.775) (1.064–4.493) (1.300–2.970)
P Reference 0.103 0.899 0.248 0.196 0.930 Reference 0.004 < 0.0001 < 0.0001 < 0.0001 < 0.0001 Reference 0.261 0.061 0.086 0.138 0.061 Reference 0.010 0.004 0.002 0.034 0.001
CI, confidence interval; N, total number of cases/controls; SNP, single nucleotide polymorphism; OR, odds ratio. Adjusted for sex and age. Heterozygous and mutated genotypes combined versus the wild-type genotype. c Mutated genotype versus heterozygous and the wild-type genotype combined. P < 0.05 is shown in bold. a
b
and serum levels of 25-hydroxyvitamin D (Supplementary Table 3).
Table 3 Univariate analysis of clinicopathological variables of melanoma patients according to the Cox regression model 95% CI Variables Sex Age Melanoma type Ulceration Breslow thickness pT stage Nodal status Clinical stage at diagnosis EcoRV rs4516035 FokI rs2228570 ApaI rs7975232 TaqI rs731236
HR
Lower
Upper
P
0.699 0.973 0.241 1.175 1.206 2.655 2.673 2.607 0.781 0.827 1.970 0.871
0.156 0.927 0.054 0.283 1.029 1.024 1.335 1.058 0.257 0.277 0.596 0.288
3.125 1.022 1.077 4.879 1.414 6.884 5.353 6.427 2.373 2.475 6.513 2.630
0.639 0.281 0.062 0.825 0.021 0.045 0.006 0.037 0.662 0.735 0.266 0.806
CI, confidence interval; HR, hazard ratio. P < 0.05 is shown in bold.
25-Hydroxyvitamin D serum level
The 25-hydroxyvitamin D serum level was measured in 60 melanoma patients 3–6 months from diagnosis during the winter months. Of melanoma patients, 31% had 25-hydroxyvitamin D levels less than 25 nmol/l, 60% in a range of 26–50 nmol/l, whereas in only 9% of patients concentrations were higher than 50 nmol/l. Thus, a serum level of 25-hydroxyvitamin D was evaluated as a dichotomous variable and classified into two categories: less than 25 nmol/l and at least 25 nmol/l. There was no association between 25-hydroxyvitamin D serum level and VDR gene polymorphisms (Supplementary Table 2). The absence of an association was observed for the clinicopathological characteristics of melanoma patients
Discussion Ultraviolet radiation plays an important role in melanomagenesis and, at the same time, it is necessary for the skin vitamin D synthesis, the main source of vitamin D in humans. The UVR action spectrum is almost the same for direct DNA damage and vitamin D synthesis – it belongs to the UVB spectrum. Hence, there is a complex relationship between UVR exposure, vitamin D levels, VDR polymorphisms, and development of melanoma. This could explain the variable association of these factors in different populations from different latitudes, and so far, these have been reported for the UK, Spain, USA, and Denmark. In this study, we have explored these associations, particularly VDR polymorphisms and vitamin D levels, in samples in Serbia, representing the population of southeast Europe. In our study, significant differences in both genotype distribution and allele frequency of FokI and TaqI VDR gene polymorphisms between the cases and control groups indicate their potential role in melanoma susceptibility. The results of the current study indicate that heterozygous and mutated genotypes, as well as the variant allele carriers of FokI polymorphisms, could be associated with an increased risk of melanoma in comparison with the wild-type genotype. Thus, it could be assumed that wild-type FokI could play a protective role in melanoma susceptibility. Our findings are in
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VDR gene polymorphisms in melanoma Zeljic et al. 277
Fig. 1
18
8 9
2
78
Block 1 (0 kb) 3 4 2
0 0
18 1
rs731236
1
rs7975232
rs2228570
Block 1 (0 kb) 3 4
rs4516035
2
rs731236
rs2228570
1
rs7975232
rs4516035
(a)
24 2
0
16
16 1
2
59
1
Block 1 (0 kb) 3 4 2
0
12 5
rs731236
1
rs7975232
rs2228570
Block 1 (0 kb) 3 4
rs4516035
2
rs731236
rs2228570
1
rs7975232
rs4516035
(b)
17 0
0
19
11 15
rs731236
2
rs7975232
1 0
12 8
rs2228570
Block 1 (0 kb) 3 4
rs4516035
2
rs731236
rs2228570
1
rs7975232
rs4516035
(c)
Block 1 (0 kb) 3 4 32
1 1
1 0
Pairwise linkage disequilibrium plots of analyzed polymorphisms in the vitamin D receptor gene for (a) cases and the control group taken together, (b) cases group, (c) control group. D0 and r2 values are presented in percentages. The shaded boxes on the left side correspond to the paired D0 values between the single nucleotide polymorphisms (SNPs). The shaded boxes on the right side correspond to the paired r2 between the SNPs.
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278 Melanoma Research 2014, Vol 24 No 3
Table 4 Haplotype frequencies and haplotype odds ratio analysis for vitamin D receptor gene polymorphisms, compared with the most common haplotype N (%) Haplotypes At aT AT at
Total 85 84 63 7
(36) (35) (26) (3)
Association P value
Cases
Controls
46 35 29 7
39 50 34 0
(39) (29) (25) (6)
(32) (40) (28) (0)
Raw
Permutation
HOR (95% CI)
P
0.040 0.030 0.336 0.003
0.210 0.095 0.769 0.003
Reference 1.781 (1.176–2.698) 1.594 (0.996–2.551) 0.362 (0.003–44.178)
0.006 0.052 0.678
N, total number of cases/controls; HOR, haplotype odds ratio; CI, confidence interval. P < 0.05 is shown in bold.
agreement with the results from previous studies on melanoma [23–26] and basal cell carcinoma [27], although contrasting the results for oral cancer patients from the Serbian population [19]. It is known that the mutated form of FokI results in the synthesis of a shorter receptor form with higher transactivation capacity as a transcription factor [15]. Different findings of other studies examining various cancer types could be explained by the gene and cell type-specific transactivation capacity of the FokI mutated form [15].
previous findings in melanoma [2,25,29] and oral carcinoma [19]. However, a previous study has reported that the EcoRV mutated genotype is associated with a decreased risk of cutaneous melanoma compared with the wild-type form [30]. In another study, the wild-type allele of EcoRV SNP was associated with an increased susceptibility to melanoma, risk for metastasis, and poor patient outcome [31]. Conflicting data from the different studies warrant further analysis to elucidate the role of the EcoRV polymorphism in melanoma susceptibility.
Variant allele carriers of TaqI polymorphism were associated with an increased risk of melanoma, which is consistent with findings from other study [3]. In contrast, a recent study has reported no association of the TaqI polymorphism with the risk of melanoma [28]. The inconsistency between studies could be explained by population differences as well as study group size. Our data suggest that the wild-type genotype might protect carriers against melanoma. It is known that the TaqI polymorphism does not alter the amino acid sequences of the VDR protein [3]. Thus, the exact mechanism of how the TaqI polymorphism could influence the risk of melanoma remains unclear. It could be assumed that the mutated variant is itself functional or is in LD with other functionally relevant polymorphisms in the VDR gene. Further investigations on the functional effects of VDR polymorphisms on the risk of melanoma are warranted.
Studies on the association between VDR gene polymorphisms and the risk of melanoma are not consistent in the literature. These discrepancies may be because of differences in serum vitamin D levels, sun exposure, population differences, and sample size [3]. A study carried out in a Danish population showed that elevated plasma calcidiol level was associated with an increased risk of nonmelanoma and melanoma cancer [14]. In contrast, higher vitamin D serum levels were associated with lower Breslow thickness and better survival of melanoma patients in a UK study [12]. In the first study from a country with high insolation (Spain), suboptimal vitamin D levels were also found in melanoma patients at diagnosis, but the levels were higher than those reported in the UK study. In our study, the vitamin D levels in winter months were from 20 to 75 nmol/l in 68.6% of patients and less than 20 nmol/l in 31.4% of patients (median age 48.5 years). Low vitamin D levels were also found in postmenopausal women in this region in 88.4% of patients [32]. Apart from the possible insufficient sun exposure, these data also indicate a probable insufficient vitamin D intake in our population, and this was reported previously for the region of central and eastern Europe [33], possibly linked to socioeconomic factors, compared with western European countries [33]. This further emphasizes the necessity of identifying melanoma patients with suboptimal vitamin D levels, and advising them on vitamin D supplementation as after the diagnosis of melanoma, rigorous sun protection is advised, and therefore, vitamin D status could deteriorate further.
VDR ApaI and TaqI polymorphisms were found in LD in our study group, which is in agreement with the literature data [15]. Among the haplotypes identified, the aT haplotype was significantly associated with an increased risk of melanoma. The ApaI polymorphism was not associated with the risk of melanoma itself, whereas in the haplotype block with the T allele of TaqI SNP, association with increased susceptibility to melanoma was observed. These findings indicate that genetic association studies should include haplotype-based analysis. In addition, it has been shown that ApaI and TaqI polymorphisms could influence VDR mRNA stability [15], which could be a possible explanation for the haplotype association observed in the current study. The lack of association of the EcoRV polymorphism with the risk of melanoma observed in our research is consistent with
There was no association between vitamin D serum level and VDR gene polymorphisms analyzed nor with clinicopathological characteristics in melanoma patients, which is consistent with findings from other studies. Because of the small number of patients, the previously
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VDR gene polymorphisms in melanoma Zeljic et al. 279
described interaction of VDR polymorphism and vitamin D level could not be explored [12].
15
The rapid increase in the incidence of melanoma and its high mortality requires comprehensive research on the risk factors associated with melanoma. Our results suggest an association between FokI and TaqI polymorphisms in the VDR gene and the risk of melanoma, implicating their potential application as molecular markers for the management of patients at high risk for the development of this cancer and their increased surveillance. Further analysis on vitamin D intake and serum calcidiol levels of melanoma patients, as well as gene–gene and gene–environment interactions, would provide additional information on the importance and role of the VDR gene in melanoma susceptibility.
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Acknowledgements Conflicts of interest
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There are no conflicts of interest. 23
References 1
2
3
4 5
6 7 8 9 10 11 12
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De Souza CF, Xander P, Monteiro AC, Silva AG, da Silva DC, Mai S, et al. Mining gene expression signature for the detection of pre-malignant melanocytes and early melanomas with risk for metastasis. PLoS One 2012; 7:e44800. Gapska P, Scott RJ, Serrano-Fernandez P, Mirecka A, Rassoud I, Gorski B, et al. Vitamin D receptor variants and the malignant melanoma risk: a population-based study. Cancer Epidemiol 2009; 33:103–107. Li C, Liu Z, Zhang Z, Strom SS, Gershenwald JE, Prieto VG, et al. Genetic variants of the vitamin D receptor gene alter risk of cutaneous melanoma. J Invest Dermatol 2007; 127:276–280. Tsao H, Chin L, Garraway LA, Fisher DE. Melanoma: from mutations to medicine. Genes Dev 2012; 26:1131–1155. Rastrelli M, Alaibac M, Stramare R, Chiarion Sileni V, Montesco MC, Vecchiato A, et al. Melanoma m (zero): diagnosis and therapy. ISRN Dermatol 2013; 2013:616170. Regad T. Molecular and cellular pathogenesis of melanoma initiation and progression. Cell Mol Life Sci 2013; 70:4055–4065. Bis S, Tsao H. Melanoma genetics: the other side. Clin Dermatol 2013; 31:148–155. Deeb KK, Trump DL, Johnson CS. Vitamin D signalling pathways in cancer: potential for anticancer therapeutics. Nat Rev Cancer 2007; 7:684–700. Field S, Newton-Bishop JA. Melanoma and vitamin D. Mol Oncol 2011; 5:197–214. Osborne JE, Hutchinson PE. Vitamin D and systemic cancer: is this relevant to malignant melanoma? Br J Dermatol 2002; 147:197–213. Gandini S, Francesco F, Johanson H, Bonanni B, Testori A. Why vitamin D for cancer patients? Ecancermedicalscience 2009; 3:160. Newton-Bishop JA, Beswick S, Randerson-Moor J, Chang YM, Affleck P, Elliott F, et al. Serum 25-hydroxyvitamin D3 levels are associated with Breslow thickness at presentation and survival from melanoma. J Clin Oncol 2009; 27:5439–5444. Ogbah Z, Visa L, Badenas C, Rios J, Puig-Butille JA, Bonifaci N, et al. Serum 25-hydroxyvitamin D3 levels and vitamin D receptor variants in melanoma patients from the Mediterranean area of Barcelona: 25-hydroxyvitamin D3 levels and VDR variants in melanoma patients from Barcelona. BMC Med Genet 2013; 14:26. Afzal S, Nordestgaard BG, Bojesen SE. Plasma 25-hydroxyvitamin D and risk of non-melanoma and melanoma skin cancer: a prospective cohort study. J Invest Dermatol 2013; 133:629–636.
24
25
26
27
28
29
30
31
32
33
Uitterlinden AG, Fang Y, Van Meurs JB, Pols HA, Van Leeuwen JP. Genetics and biology of vitamin D receptor polymorphisms. Gene 2004; 338:143–156. Whitfield GK, Remus LS, Jurutka PW, Zitzer H, Oza AK, Dang HT, et al. Functionally relevant polymorphisms in the human nuclear vitamin D receptor gene. Mol Cell Endocrinol 2001; 177:145–159. Mishra DK, Wu Y, Sarkissyan M, Sarkissyan S, Chen Z, Shang X, et al. Vitamin D receptor gene polymorphisms and prognosis of breast cancer among African-American and Hispanic women. PLoS One 2013; 8:e57967. Kupfer SS, Anderson JR, Ludvik AE, Hooker S, Skol A, Kittles RA, et al. Genetic associations in the vitamin D receptor and colorectal cancer in African Americans and Caucasians. PLoS One 2011; 6:e26123. Zeljic K, Supic G, Stamenkovic Radak M, Jovic N, Kozomara R, Magic Z. Vitamin D receptor, CYP27B1 and CYP24A1 genes polymorphisms association with oral cancer risk and survival. J Oral Pathol Med 2012; 41:779–787. Santonocito C, Capizzi R, Concolino P, Lavieri MM, Paradisi A, Gentileschi S, et al. Association between cutaneous melanoma, Breslow thickness and vitamin D receptor BsmI polymorphism. Br J Dermatol 2007; 156:277–282. Barrett JC, Fry B, Maller J, Daly MJ. Haploview: analysis and visualization of LD and haplotype maps. Bioinformatics 2005; 21:263–265. Tregouet DA, Garelle V. A new JAVA interface implementation of THESIAS: testing haplotype effects in association studies. Bioinformatics 2007; 23:1038–1039. Hutchinson PE, Osborne JE, Lear JT, Smith AG, Bowers PW, Morris PN, et al. Vitamin D receptor polymorphisms are associated with altered prognosis in patients with malignant melanoma. Clin Cancer Res 2000; 6:498–504. Mocellin S, Nitti D. Vitamin D receptor polymorphisms and the risk of cutaneous melanoma: a systematic review and meta-analysis. Cancer 2008; 113:2398–2407. Randerson-Moor JA, Taylor JC, Elliott F, Chang YM, Beswick S, Kukalizch K, et al. Vitamin D receptor gene polymorphisms, serum 25-hydroxyvitamin D levels, and melanoma: UK case–control comparisons and a meta-analysis of published VDR data. Eur J Cancer 2009; 45:3271–3281. Li C, Liu Z, Wang LE, Gershenwald JE, Lee JE, Prieto VG, et al. Haplotype and genotypes of the VDR gene and cutaneous melanoma risk in nonHispanic whites in Texas: a case–control study. Int J Cancer 2008; 122:2077–2084. Lesiak A, Norval M, Wodz-Naskiewicz K, Pawliczak R, Rogowski-Tylman M, Sysa-Jedrzejowska A, et al. An enhanced risk of basal cell carcinoma is associated with particular polymorphisms in the VDR and MTHFR genes. Exp Dermatol 2011; 20:800–804. Schafer A, Emmert S, Kruppa J, Schubert S, Tzvetkov M, Mossner R, et al. No association of vitamin D metabolism-related polymorphisms and melanoma risk as well as melanoma prognosis: a case–control study. Arch Dermatol Res 2012; 304:353–361. Barroso E, Fernandez LP, Milne RL, Pita G, Sendagorta E, Floristan U, et al. Genetic analysis of the vitamin D receptor gene in two epithelial cancers: melanoma and breast cancer case–control studies. BMC Cancer 2008; 8:385. Povey JE, Darakhshan F, Robertson K, Bisset Y, Mekky M, Rees J, et al. DNA repair gene polymorphisms and genetic predisposition to cutaneous melanoma. Carcinogenesis 2007; 28:1087–1093. Halsall JA, Osborne JE, Epstein MP, Pringle JH, Hutchinson PE. The unfavorable effect of the A allele of the vitamin D receptor promoter polymorphism A-1012G has different mechanisms related to susceptibility and outcome of malignant melanoma. Dermatoendocrinology 2009; 1:54–57. Vuceljic M, Ilic-Stojanovic O, Lazovic M, Grajic M. Vitamin D and parathyroid hormone in relation to bone mineral density in postmenopausal women. Vojnosanit Pregl 2012; 69:243–248. Novakovic R, Cavelaars AE, Bekkering GE, Roman-Vinas B, Ngo J, Gurinovic M, et al. Micronutrient intake and status in Central and Eastern Europe compared with other European countries, results from the EURRECA network. Public Health Nutr 2013; 16:824–840.
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