Globally, skin cancer is one the most commonly diagnosed type of cancer in humans [1]. It accounts for around 40% of cancer cases [2,3]. The rate of incidence is low in India as compared to the western world. However, due to its large population, the absolute number of cases is estimated to be significant [4]. The most common types of skin cancers may be categorized into two major groups: melanoma and non-melanoma. Non-melanoma type of skin cancer (NMSC) arises from keratinocytes and is further divided into two sub-types: basal cell carcinomas (BCC) and squamous cell carcinomas (SCC). Globally, BCC is the most common type of skin cancer accounting for approximately 70% of all malignant diseases of the skin [5]. Various studies have consistently reported that SCC is the most prevalent type of skin cancer in India [4,6-8]. Reports 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 [11-13]. 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/b-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 [22-25]. 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.

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.

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.

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/b-catenin 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/b-catenin pathway, VitaminD/VDR binds to b-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.

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.