Vitamin D receptor gene polymorphism and risk of skin cancer patients of Kashmiri population ( India ) : A case-control study

© Our Dermatol Online 3.2018 233 How to cite this article: Qadir J, Majid S, Rashid F, Hassan I, Sajad P, Bhat YJ, Din I, Bhat AA, Wani HA, Farooq R. Vitamin D receptor gene polymorphism and risk of skin cancer patients of Kashmiri population (India): A case-control study. Our Dermatol Online. 2018;9(3):233-240. Submission: 28.10.2017; Acceptance: 04.01.2018 DOI: 10.7241/ourd.20183.2 Vitamin D receptor gene polymorphism and risk of skin cancer patients of Kashmiri population (India): A case-control study

also indicate that skin cancer especially non-melanoma skin cancer (NMSC) is on rise in India [9].However, clinical spectrum of skin cancer in Kashmir valley bears a different tale from the rest of the country due to its geography, climate, dietary habits and socio culture.The incidence of NMSC in Kashmir valley among males and females has been reported to be 2.7% and 2.8% respectively [10].Genetic as well as environmental factors play an important role in the development of cancers [11][12][13].Strong associations has been observed between the deficient circulating levels of vitamin D and increased risk of various types of cancers like breast, colon, [14,15] and ovarian [16,17].Kashmiri people are prone to vitamin D deficiency due different topographical, geographical and climatic conditions [18].Association between vitamin D levels and risk of skin cancer have been examined in several studies and provide an insight into the positive role of vitamin D deficiency in the development of skin cancer especially NMSC.
Vitamin D modulates various cancer related signaling pathways and acts via binding to its intranuclear receptor vitamin D receptor (VDR) there by altering the gene expression of various proteins involved in the process of proliferation, differentiation and regulation of cell cycle [19].VDR contributes to the signaling of hedgehog (Hh) and Wnt/β-catenin pathways that plays an important role in proliferation and differentiation of keratinocytes [20].Several studies have reported that VDR gene is significantly associated with the frequency of occurrence of various types of cancers [19].VDR is encoded by a large gene (>100kb) located on chromosome 12q12-q14 [21].Whereas Fok1 polymorphism, (rs2228570) is present at the first potential site start in exon 2 of the VDR gene [22][23][24][25].This polymorphism alters an ACG codon that is located ten base pairs upstream from the translation start codon and results in the generation of an additional start codon.A change in the sequence from C to T allele in the translation site leads to generation of a polymorphic variant (TT).If the initiation of translation starts from this alternative site (thymine variant), the resultant product is three amino acid longer VDR protein of 247 amino acids that exerts less transcriptional activity as compared to the wild type (CC) [22].Several studies report that there is a significant association between the Fok1 polymorphism and the risk of various types of cancer [26].The Fok1 polymorphism is considered to be an independent risk marker as it has no Linkage Disequilibrium with any of the other VDR polymorphisms [27,28].Keeping in view the role played by vitamin D and VDR gene in various cancers, we aim to assess the contribution of vitamin D and Fok1 polymorphism and its association with skin cancer in Kashmiri population.

MATERIALS AND METHODS
A total of 68 histopathologically confirmed newly diagnosed skin cancer patients attending the Department of Dermatology, Government Medical College (GMC), Srinagar, were included in this study.A pool of 65 normal healthy controls were also recruited from the same hospital that belonged to the same geographical area, ethnic background and were of matching sex and age group.The controls did not have a previous diagnosis of any type of cancer and had maintained a healthy life style.The subjects included farmers, labours, employers, household, medical personals and students.A written informed consent was obtained from each recruited subject and the study was approved by the ethical committee of GMC, Srinagar.Among the cases, 55.88% were SCC, 39.70% included the BCC and 4.41% were melanoma.To avoid the experimental bias, melanoma type of skin cancer was excluded from the study.In cases, 58.82% were males and 41.17% females and the control consisted of 53.84% male and 46.15% female.Also, 60.3% cases were in the age group of greater than 50 and 39.7% of the cases were in age group of less than 50 years.In controls, 58.46% were in age group of greater than 50 and 41.53% were in the age group of less than 50.Out of 65 cases, 48.53% cases belonged to rural region and 51.47% cases belonged to urban region.In controls, 47.70% were from rural areas and 52.30% were from urban areas.Among the cases 25% had a family history of cancer.Blood sample (3ml) was collected in EDTA coated vials from both the study groups for plasma collection and DNA extraction.Sample collection was done from the month of March to October every year to avoid seasonal variation of Vitamin D levels.

Plasma vitamin D estimation
Vitamin D status was measured by estimating concentrations of 25-hydroxyvitaminD (25(OH)D) in the plasma.The circulating concentration of 25(OH) D in the range of 30-50ng/ml is considered necessary for optimal health [29].The healthy controls whose 25(OH)D levels were in the range of 27-53ng/ml were included in the study.Plasma 25(OH)D levels were estimated by automated chemilumnescent microparticle immunoassay (CMIA) method by ARCHITECT25-OH vitamin D assay (Abbott laboratories illino is, USA Ref 3L52-25).

Genotype Analysis
Genomic DNA was extracted from the blood samples by using Quick-gDNA TM MicroPrepkit (Zymo Research, The Epigenetics Company, USA) according to given protocol.The concentration of extracted DNA was measured in a spectrophotometer at 260nm wave length by using the formula: DNAμg/ml=A 260 x50xdilution factor.The purity of DNA was checked by using A 260 /A 280 .VDR Fok1 genotype was analysed by PCR-RFLP using specific primers Forward 5'-AGCTGGCCCTGGCACTGACTCTGCTCT-3'and Reverse 5'-ATGGAAACACCTTGCTTCTT CTCCCTC-3' for amplification of 265bp of DNA segment [30].PCR amplification was carried out in a 50-μl volume containing 50-150ng genomic DNA; 1X PCR buffer containing 2mM MgCl 2 (Biotools, B&M Labs, S.A. Madrid, Spain); 0.2mM dNTPs (Biotools, B&M Labs, S.A. Madrid, Spain); 1.5Units of Taq polymerase; 2pmol/μl of forward and reverse primers (Eurofins Genomics India Pvt Ltd).The PCR cycle conditions were as follows: Initial denaturation at 94°C for 10 minutes followed by 35 cycles of denaturation at 94°C for 45 seconds, annealing at 60°C for 45 seconds, extention for 72°C for 45 seconds and final extention at 72°C for 5 minutes.PCR products were verified on 2% agarose gel and analysed under a UV illuminator.The amplicons were digested with FastDigest Fok1 restriction enzyme (Thermo Scientific, (EU) Lithuania) (1U at 37°C for 15-20 minutes).DNA fragments were subjected to electrophoresis on a 3.5% agarose gel for resolution.Genotyping of the samples were confirmed via sequencing by Sanger method (SciGenom Labs Pvt Ltd, Cochin, Kerala).

Statistical Analysis
The vitamin D levels were analysed using independent t-test.The χ 2 -test was used to compare the allelic and genotypic frequencies.The association of the VDR genotype with the risk of skin cancer were estimated by computing the odds ratios (OR) and 95%confidence intervals (95%CI).A p-value of <0.05 was considered as statistically significant.Statistical analysis was done using SPSS version16.0(SPSS, Inc., Chicago IL, USA).

RESULT
Vitamin D levels and Fok1 polymorphism in VDR gene were evaluated in skin cancer cases.The calculated mean age of the skin cancer cases was 52.5±8.7 years whereas it was 49.3±9.25 years among the controls.The general characteristics of the studied subjects are given in Table 1.Interestingly higher number of SCC cases (55.88%) followed by BCC (39.70%) were observed when the skin cancer cases were classified into groups.Further, age distribution of cases showed that there was high incidence of skin cancer in the age group of ≥50 years as compared to <50 years.The incidences of skin cancer were also found to be higher in males as compared to females.However, the rates of incidences were comparable among of urban and rural cases.

Plasma vitamin D levels
The mean plasma 25(OH)D levels were significantly lower among skin cancer cases when compared to normal healthy controls (21.05±9.67ng/mlvs 38.88±7.29ng/ml,P<0.05, Fig. 1).However, no significant difference was observed in 25(OH)D levels between SCC and BCC type of skin cancer (Table 2).In cases, the plasma 25(OH) D levels were found significantly lower among the age group of ≥50 years as compared to <50 years (P<0.05).While, no significant difference in 25(OH)D levels was observed between the male and female cases (Table 2).

Genotype distribution
The distribution of genotypic and allelic frequencies of Fok1 VDR polymorphism (C>T) were compared between the skin cancer cases (diagnosed ones) and controls.The alteration of C to T allele in the start codon of translation site created a restriction site in the amplified region which was digested by Fok1 restriction enzyme.The CC homozygote (wild) shows only one fragment of 265bp, while the TT homozygote (variant) with Fok1 restriction site generated two fragments of 196bp and 69bp.The heterozygous (CT) genotype displayed three fragments of 265bp, 196bp and 69bp (Fig. 2).cases and controls.When the age group of ≥50 years in cases of diagnosed subjects was evaluated, the frequency of mutant T allele was found to be 41.46% compared to 26.31% among controls with OR=1.983, 95%CI 1.01-3.89,P=0.045.While in age group of <50 years, no significant difference was found between the cases and controls.In females, the frequency of mutant T allele was 46.43% and 28.33% in cases and controls respectively and this observation showed a statistical significance of T allele among the cases when compared to controls (P=0.044).While, in males, no significant difference was observed between the cases and controls as shown in Table 4. Genotyping of the samples were confirmed via sequencing by Sanger method (SciGenom Labs Pvt Ltd, Cochin, Kerala) (Figs. 3 -5).

DISCUSSION
Vitamin D plays an important role in various cancer related signaling pathways.Vitamin D induces transcriptional activation or repression of target genes by binding to the VDR.In the epidermis, Hh and Wnt/βcatenin are the two important vitamin D signaling pathways that play an important role in proliferation and differentiation of keratinocytes.VitaminD/ VDR inhibits the Hh pathway in keratinocytes by suppressing the expression of Shhandgli1.In Wnt/βcatenin pathway, VitaminD/VDR binds to β-catenin and reduces its transcriptional activity.Therefore, VitaminD/VDR reduces the proliferation and induces the process of differentiation in keratinocytes thereby limiting their ability to induce tumors in the skin [20].
In keratinocytes, VitaminD/VDR regulates the proliferation in the basal layer of the epidermis and promotes sequential differentiation [31].A low circulatory level of 25(OH)D is the main marker of   vitamin D deficiency.The limited exposure to sun is considered the main cause of vitamin D deficiency.However, prolonged sun exposure will not increase the vitamin D levels further, as Holick et al documented that human skin has the intrinsic ability of vitamin D production [32].Harinarayan et al reported that the sunlight exposure between the hours 11a.m. to 2p.m. will promote adequate vitamin D formation in the skin [33].Usually, 20-30 minutes exposure in the sun two to four times a week is enough to maintain adequate levels of Vitamin D. The prolonged exposure to sunlight increases the risk of skin cancer as ultraviolet radiation in sufficient quantity can damage DNA, causing genetic mutations and results in abnormal cellular proliferation [34].People aged >50 years are prone to develop vitamin D deficiency due to various risk factors such as decreased dietary intake, diminished sunlight exposure, reduced skin thickness, impaired intestinal absorption and impaired hydroxylation in the liver and kidney [35].In the present study the plasma 25(OH)D levels were found to be significantly lower among skin cancer cases when compared to healthy controls (21.05±9.67 vs 38.8±7.29,P<0.05).Several studies reported that the basal line of 25(OH)D levels were lower in skin cancer patients as compared to the control group.Asgari et al., reported an increased risk for BCC with higher prediagnostic serum 25(OH)D levels, adjusted for sun exposure, in a nested case control study [36].Tang et al., reported that higher baseline 25(OH)D serum levels coincided with a decreased risk for NMSC [37].Likewise Van der Pols et al., found that there is a reduced risk for SCC type of skin cancer, in those with a history of skin cancer and whose vitamin D levels >75nmol/L [38].However, in the current study no significant difference was observed in plasma 25(OH)D levels between the SCC and BCC type of skin cancer.Plasma 25(OH)D levels were significantly lower among the cases of ≥50 years age group as compared to <50 years.Tang et al., reported that elderly men with 25(OH)D levels >75nmol/L were associated with the decreased risk for non-melanoma type of skin cancer (OR=0.53,P=0.026) [37].In this study no significant difference was observed in the plasma 25(OH)D levels between the male and female patients.The rate of skin cancer incidence was observed higher in males as compared to female as unprotected sun exposure was found usually higher in males as compared to females.Several studies have evaluated the role of Fok1 VDR polymorphism in skin cancer and have found that Fok-1 VDR gene polymorphism is as an important mediator in the development of skin cancer.A study reported that the Fok1 ff genotype was positively associated with an increased risk for each type of skin cancer [38].A meta-analysis conducted by Gandini et al., reported borderline significance with increased risk of f allele in NMSC while the ff genotype attributed to about 30% of the increased risk for the NMSC type of skin cancer [39].Consistent with this studies, our   findings suggest that Fok1 VDR polymorphism was significantly associated with the risk of skin cancer.The frequency of homozygous mutant Fok1 TT genotype and T allele was found to be statistically significant among the skin cancer cases as compared to controls but statistical association was limited to SCC type.In SCC type of skin cancer, the genotypic frequency of both heterozygous CT and homozygous TT mutant Fok1 polymorphism was found statistically significant in cases as compared to controls.This was consistent with a study conducted by Han et al., which showed the significantly positive association of Fok1 polymorphism with SCC risk among the woman [40].However, the association was found to be statistically in significant among BCC type of skin cancer.In ≥50 years of age group, the frequency of mutant T allele was observed to be statistically significant among the cases as compared to controls, while as, in <50 years of age group, no significant difference was found between the cases and controls.Similarly, among the males, no significant difference was found between the cases and controls, however, in females the frequency of mutant T allele was found statistically significant among the cases as compared to controls.The present study supports the notion that vitamin D deficiency and Fok1 VDR polymorphism may increase the risk of skin cancer and a strong association of Fok1 polymorphism was observed with SCC type of skin cancer.

CONCLUSION
Vitamin D deficiency is found to be associated with different types of cancers including skin cancer.This study suggests a possible association of vitamin D deficiency and Fok1 VDR polymorphism in skin cancer development, especially for SCC.Our study gives a strong impression that vitamin D has a protective effect against the development of skin cancer.However, there is need of independently large population-based prospective studies to validate our findings and to facilitate rigorous analyses of subgroups.

Figure 1 :
Figure 1: Represents the mean plasma 25(OH)D levels in skin cancer patients and controls.sData are represented as mean±95% CI, independent t-test.P<0.05 is considered as statistically signifi cant.

Figure 3 :
Figure 3: Representative electropherogram sequencing result of the Fok1 VDR gene polymorphism in exon 2, arrow indicates the presence of homozygous wild genotype (CC) at the polymorphic site.

Figure 4 :
Figure 4: Representative electropherogram sequencing result of the Fok1 VDR gene polymorphism in exon 2, arrow indicates the presence of heterozygous genotype (CT) at the polymorphic site.

Figure 5 :
Figure 5: Representative electropherogram sequencing result of the Fok1 VDR gene polymorphism in exon 2, arrow indicates the presence of mutated homozygous genotype (TT) at the polymorphic site.

Table 1 :
General characteristics of study population (cases and controls) P<0.05 is considered as statistically signifi cant, by chi square test.
The frequency of CC, CT and TT genotypes among the cases were 33.82%, 47.06% and 19.11% while in controls it was found to be 53.84%,38.46%and7.7% respectively Table3.The genotypic frequency of CC vs TT and CT+TT was found statistically significant among the cases when compared with normal healthy controls with a p-value of 0.016 and P=0.02 respectively.The mutant T allele was found to be a risk factor for skin cancer with OR=2.018, 95%CI1.205-3.379,P=0.007.When the subjects were classified further into groups, it was observed that the frequency of CC, CT and TT genotypes in SCC type were 26.3%, 55.2% and 21% respectively and this pattern of distribution showed statistical significance among the SCC cases as compared to controls (P<0.05).While in BCC type the frequency of CC, CT and TT genotypes were 44.4%, 40.7% and 15% respectively.However no statistical significance was observed between the BCC

Table 2 :
Represents plasma 25(OH)D ng/ml levels in variables of skin cancer cases and controls

Table 3 :
Represents genotypic and allelic frequencies of Fok1 VDR gene among the skin cancer cases and controls and their association P<0.05 is considered as statistically signifi cant, by chi square test.

Table 4 :
Represents genotypic and allelic frequencies of Fok1 VDR gene in age and gender variables of skin cancer cases and controls and their association with risk of skin cancer *P<0.05 is considered as statistically signifi cant, by chisquare test