Haplotype and genotypes of the VDR gene and cutaneous melanoma risk in non-Hispanic whites in Texas: A case–control study

Authors


Abstract

In a hospital-based case–control study of 805 non-Hispanic whites with cutaneous melanoma and 841 cancer-free age-, sex- and ethnicity-matched control subjects, 3 VDR polymorphisms (i.e., TaqI, BsmI and FokI) were genotyped using blood samples collected between 1994 and 2006. We tested the hypothesis that the haplotypes and combined genotypes of these polymorphisms were associated with melanoma risk by interacting with known risk factors. Haplotypes t-B-F (adjusted odds ratio [OR], 0.52; 95% confidence interval [CI], 0.34–0.80) and t-B-f (adjusted OR, 0.51; CI, 0.27–0.94) were associated with a reduced risk when compared to T-b-f. The combined genotypes Tt+tt/Bb+BB/Ff+ff (adjusted OR, 0.69; CI, 0.52, 0.90) and Tt+tt/Bb+BB/FF (adjusted OR, 0.58; CI, 0.43, 0.78) were also associated with reduced risk, whereas the combined genotype TT/Bb+BB/Ff+ff genotype (adjusted OR, 2.35; CI, 1.13, 4.98) was associated with increased risk when compared to TT/bb/Ff+ff genotypes. On multivariate analysis, only the TaqI polymorphism was an independent risk factor, while the FokI polymorphism interacted with skin color (p = 0.029), moles (p = 0.017) and first-degree relatives with any cancer (p = 0.013) in modifying risk. Together, these findings suggest that VDR polymorphisms may directly affect or modify the risk associated with known melanoma risk factors. Larger, population-based studies are needed to replicate our findings. © 2008 Wiley-Liss, Inc.

Interaction

Vitamin D is involved in a variety of biological processes including bone metabolism, immunomodulation and regulation of cell proliferation and differentiation.1–4 Vitamin D is also known to have a potential protective effect against cancers, including cutaneous melanoma,5, 6 a lethal skin cancer that has an increasing incidence in the United States over the last 30 years.7 Vitamin D exerts its tumor-suppressive effects by binding to the vitamin D receptor (VDR). A ubiquitously expressed intracellular polypeptide that belongs to the steroid/retinoid receptor superfamily of nuclear receptors, VDR specifically binds to 1,252D3 and interacts with target cell nuclei.8 The VDR protein is overexpressed in malignant melanocytes responsive to vitamin D's antiproliferative effects.2 Several studies have suggested that VDR polymorphisms may alter the functions of genes involved in cell division and adhesion,2, 9 thus implicating such polymorphisms in melanoma development.10

Located on chromosome 12q12-q14, VDR contains at least 5 promoter regions,11 8 protein-coding exons and 6 untranslated exons, all of these regions being alternatively spliced.12VDR at least has 196 single nucleotide polymorphisms (SNPs) (http://egp.gs.washington.edu/data/vdr/vdrxx.csnps.txt), of which 64 lie in the promoter region, 32 in the 3′ and 5′ untranslated regions and 2 synonymous and 2 nonsynonymous SNPs in the coding region. FokI (exon 2, rs10735810), BsmI (intron 8, rs1544410) and TaqI (exon 9, rs731236) are the 3 most frequently investigated SNPs for their associations with various cancers.13–15 Some studies also addressed gene-environment interactions since environmental factors (e.g., sunlight) can influence vitamin D production.16

Although ultraviolet light plays an important role in melanoma development,17, 18 only 3 studies to date have assessed the associations between this factor, VDR polymorphisms, and melanoma risk. Moreover, no studies have examined whether VDR polymorphisms modulate the risk associated with other established melanoma risk factors.19–21 Therefore, we conducted a relatively large case–control study of non-Hispanic whites (i.e., 805 patients with melanoma and 841 cancer-free controls) to determine whether the haplotypes and combined genotypes of VDR polymorphisms TaqI, BsmI and FokI are associated with melanoma risk, and whether these polymorphisms can modify the risk associated with known melanoma risk factors.

Material and methods

Study subjects

The study protocol was approved by The University of Texas M. D. Anderson Cancer Center institutional review board, and written informed consent was obtained from all subjects. The subject recruitment has been described previously.20, 22, 23 In brief, all patients with newly diagnosed, histologically confirmed24 and untreated cutaneous melanoma, who were referred to M. D. Anderson Cancer Center between May 1994 and February 2006, were recruited as case subjects. Because most of the patients (∼99%) were non-Hispanic whites, the few minority subjects who were recruited were excluded from analysis. Although there were no restrictions on patients' age or tumor stage, only those patients who were free of metastases or other cancers and agreed to donate a blood sample were included in our study. Approximately 85% of eligible patients recruited for our study agreed to participate. Cancer-free control subjects were recruited during the same period from among cancer-free visitors to M. D. Anderson Cancer Center who were accompanying patients to our outpatient clinics, were not seeking medical care, and were not related by blood to the patients. Approximately 90% of eligible control subjects recruited for our study agreed to participate. The control subjects were matched by frequency to case subjects by age (±5 years), sex and ethnicity.

After giving informed consent, all subjects completed a self-administered questionnaire that elicited information on demographic factors (e.g., age, sex, education and income), ethnicity, medical history, family history and sunlight exposure history (i.e., tanning ability, lifetime number of severe sunburns and freckling in the sun as a child).25 Then, each subject was interviewed in-person to assess his or her host characteristics (e.g., natural hair, eye and skin color) as well as self-reported skin conditions (e.g., color, moles and pigmented nevi). After each interview, a sample of blood (30 mL) was drawn from the subject and collected in a heparinized tube.

Genotyping

Genotyping was performed as follows. First, 1 mL of each whole blood sample was centrifuged to isolate a leukocyte cell pellet from the buffy coat fraction. Genomic DNA was extracted from the pellet, purified using a DNA blood mini kit (Qiagen, Valencia, CA) and assayed for purity and concentration by spectrophotometry (i.e., absorbance at 260 and 280 nm). Next, DNA fragments of VDR containing the TaqI,26BsmI27 and FokI10, 19 polymorphisms were amplified by polymerase chain reaction, subjected to restriction fragment length polymorphism analysis and sequenced (Fig. 1). Approximately 10% of samples were genotyped a second time; the repeat genotyping results agreed completely with the initial results.

Figure 1.

VDR gene structure and locations and genotypes of selected polymorphisms.

Statistical methods

The χ2 test was used to evaluate case–control differences in the frequency distributions of selected demographic variables, known risk factors and each allele and genotype of the VDR polymorphisms. Because skin color was self-assessed on the screening questionnaire on a scale of 1 (light) to 10 (dark), skin colors were categorized as fair (1 or 2), brown (3 or 4) or dark (5–10); the aim was to obtain similar numbers of observations in each stratum to facilitate further stratification analysis. Some subjects did not provide information about some variables (e.g., hair color, eye color, skin color, tanning ability, number of sunburns, freckling, pigmented nevi and family history of skin cancer); the missing variables for those subjects were treated as missing data on multivariate analysis. The linkage disequilibrium for each SNP of interest (i.e., TaqI, BsmI and FokI) was calculated, and the polymorphism haplotypes for each subject were reconstructed on the basis of the known TaqI, BsmI and FokI genotypes. Because of the potential effect of locus–locus interactions of the polymorphisms on melanoma risk, associations between risk and the haplotypes and combined genotypes of the 3 polymorphisms were also evaluated.

Crude and adjusted odds ratios (ORs) and associated 95% confidence intervals (CIs) were determined by univariate and multivariate unconditional logistic regression analyses. Multivariate adjustments were made, where appropriate, for age, sex and other known risk factors. ORs, CIs and p-values for interactions and trend tests were obtained from multivariate logistic regression models.

The null hypotheses of multiplicative gene–gene interactions were tested, and departures from multiplicative interaction models were assessed empirically. A more-than-multiplicative interaction was suggested when OR11 > OR10 * OR01.28 To assess potential departures from a multiplicative model, interaction terms between variables were modeled according to standard unconditional logistic regression techniques. Finally, to determine whether the main effect of the VDR polymorphisms was independent of other known risk factors, selected variables were included in the multivariate logistic regression analyses of data from only those subjects who completely answered their questionnaires.29

Two models were fitted. The first model included age, sex and the 3 polymorphisms of interest, the aim being to control for any potential effects due to associations among the polymorphisms. The second model was to exclude the polymorphism that showed no statistically significant association with risk in the first model and then include all other known risk factors, the aim being to assess further the independent effects of the polymorphisms. A p-value of ≤0.05 was considered statistically significant. All tests were two-sided and were performed using SAS software (version 9.13; SAS Institute, Cary, NC).

Results

Population characteristics and risk factors

The initial analysis included all cases (n = 805) and controls (n = 841). The 2 groups had similar age (p = 0.37), sex (p = 0.16), education (p = 0.99) and household income (p = 0.35) (Table I). The similar age and sex distributions implied adequate frequency matching. Because some subjects did not completely answer their questionnaires, the numbers of subjects in some risk factor strata were less than the total number of subjects in the study. Nevertheless, our results were consistent with previous findings by others.30–32 Except for skin color (p = 0.16), the frequencies of known melanoma risk factors were significantly higher among cases than among controls and were associated with 1.55- to 7.78-fold increased melanoma risk (Table II). Subjects with these risk factors were placed into dichotomized groupings for further stratification and assessment of interactions in multivariate logistic regression analyses.

Table I. Demographic Distributions of Non-Hispanic Whites in a Hospital-Based Case–Control Study of Cutaneous Melanoma, Texas, 1994–2006
Variables1Cases (n = 805)Controls (n = 841)p2
No.%No.%
  • 1

    The numbers of subjects in some of the strata were less than the total number of subjects included in our study, because some subjects did not provide complete information in their screening questionnaires.

  • 2

    Two-sided χ2 test.

Age (years)    0.36
 <3912315.312114.4 
 39–5019524.218922.5 
 51–6025832.025830.7 
 >6022928.527332.4 
Sex    0.16
 Male51463.856567.2 
 Female29136.227632.8 
Education (years)    0.99
 ≤1423932.723532.7 
 >1449367.448467.3 
Household income (yearly)    0.35
 <$35,00014420.312718.4 
 ≥$35,00056479.756481.6 
Table II. Age- and Sex-Adjusted Odds Ratios for Selected Cutaneous Melanoma Risk Factors, Texas, 1994–2006
VariablesCases (n = 805)1Controls (n = 841)1p2Odds ratio and 95% confidence interval
No.%No.%Crude odds ratio95% Confidence intervalAdjusted odds ratio395% Confidence interval
  • 1

    The numbers of subjects in some of the strata were less than the total number of subjects included in our study, because some subjects did not provide complete information in their screening questionnaires.

  • 2

    Two-sided χ2 test.

  • 3

    Adjusted by age and sex.

Hair color    <0.01  
 Black or brown48366.057980.9 1.0Referent1.0Referent
 Blond(e) or red24934.013719.1 2.181.71, 2.772.151.69, 2.74
Eye color    <0.01   
 Other42458.053474.4 1.0Referent1.0Referent
 Blue30742.018425.6 2.101.68, 2.632.091.68, 2.62
Skin color    0.16    
 Black or brown32844.834748.1 1.0Referent1.0Referent
 Fair40455.237451.9 1.140.93, 1.411.130.92, 1.40
Tanning ability    <0.01    
 Good17323.626737.1 1.0Referent1.0Referent
 Moderate or poor55976.445362.9 1.911.52, 2.401.901.51, 2.39
Lifetime sunburn with blistering    <0.01  
 None19126.429645.5 1.0Referent1.0Referent
 ≥1 Time53373.642154.5 1.961.57, 2.451.951.56, 2.45
Freckling in the sun as a child    <0.01  
 No34347.142158.6 1.0Referent1.0Referent
 Yes38552.929741.4 1.591.29, 1.961.551.25, 1.91
Moles    <0.01  
 No17521.728934.4 1.0Referent1.0Referent
 Yes63078.355265.6 1.891.51, 2.351.881.51, 2.34
Atypical nevi    <0.01  
 No74091.983298.9 1.0Referent1.0Referent
 Yes658.191.1 8.124.02, 16.47.783.84, 15.8
First-degree relatives with any cancer    <0.01  
 No34342.643151.3 1.0Referent1.0Referent
 Yes46157.441048.7 1.421.17, 1.721.451.19, 1.76

VDR allele and genotype distributions and association with melanoma risk

Allele and genotype frequencies of the polymorphisms of interest are presented in Table III. Genotype distributions among controls were consistent with the Hardy-Weinberg equilibrium (p = 0.49 for TaqI, p = 0.31 for BsmI and p = 0.64 for FokI). TaqI alleles t and BsmI alleles B were significantly less frequent among cases than among controls (0.370 vs. 0.429 [p < 0.01] and 0.394 vs. 0.431 [p = 0.03], respectively), whereas the FokI allele f was more frequent, though not so significant (0.378 vs. 0.356 [p = 0.20]). This suggested that t, B and F might protect carriers against melanoma or T, b and f might put them at risk. Moreover, the t and B genotypes (i.e., Tt+tt and Bb+BB) were consistently less frequent among cases than among controls (p < 0.05 for both) and were associated with a significantly lower melanoma risk (i.e., a protective effect) for Tt+ttvs.TT genotypes (adjusted OR [CI], 0.68 [0.56, 0.83] and Bb+BBvs.bb genotypes (adjusted OR [CI], 0.72 [0.58, 0.90]), (Table III). In contrast, the f genotypes (i.e., ff+Ff) were significantly more frequent among cases than among controls and were associated with a significantly greater melanoma risk than was the FF genotype (adjusted OR [CI], 1.25 [1.03, 1.53]) (Table III).

Table III. Genotype and Allele Frequencies of the VDR Polymorphisms among Non-Hispanic Whites in a Case–Control Study of Cutaneous Melanoma, Texas, 1994–2006
VDR genotypeCase (n = 805)Control (n = 841)1p2Odds ratio and 95% confidence interval
No.%No.%Crude odds ratio95% Confidence intervalAdjusted odds ratio395% Confidence interval
  • 1

    The observed distribution of genotype frequency among the control subjects appeared to be in Hardy Weinberg equilibrium (χ2 = 0.49, p = 0.49 for TaqI; χ2 = 1.04, p = 0.31 for BsmI; and χ2 = 0.64, p = 0.43 for FokI).

  • 2

    Two-sided χ2 test for distributions of either genotype or allele frequency.

  • 3

    Odds ratios were adjusted for age and sex in a logistic regression model.

  • 4

    Two-sided χ2 test for distribution of three genotypes.

  • 5

    Two-sided χ2 test for distribution of combined genotypes.

  • 6

    Two-sided χ2 test for allele distribution.

TaqI    <0.014    
 TT33041.026932.0 1Referent1Referent
 Tt35544.142250.2 0.690.55, 0.860.690.56, 0.86
 tt12014.915017.8 0.650.49, 0.870.660.49, 0.87
 Tt + tt47559.057268.0<0.0150.680.55, 0.830.680.56, 0.83
 t Allele frequency0.3700.429<0.016    
BsmI    0.024    
 bb30537.926531.5 1Referent1Referent
 Bb36645.542750.8 0.750.60, 0.920.750.60, 0.93
 BB13416.614917.7 0.780.59, 1.040.780.59, 1.05
 Bb + BB50062.157668.5<0.0150.750.58, 0.920.720.58, 0.90
 B Allele frequency0.3940.4310.036    
FokI    0.054    
 FF28735.734440.9 1Referent1Referent
 Ff42753.039647.1 1.291.05, 1.591.301.05, 1.60
 ff9111.310112.0 1.080.78, 1.491.080.78, 1.49
 Ff + ff51864.449759.10.0351.251.02, 1.531.251.03, 1.53
 f Allele frequency0.3780.3560.206  

Association between VDR haplotypes or combined genotypes and melanoma risk

TaqI, BsmI and FokI polymorphisms were in linkage disequilibrium (t and B alleles: D′ = 0.918, R2 = 0.855, p < 0.001; t and F alleles: D′ = 0.039, R2 = 0.001, p < 0.001; B and F alleles: D′ = 0.027, R2 = 0.001, p < 0.001), suggesting a potentially joint effect of the haplotypes of the 3 VDR polymorphisms on melanoma risk. Eight hypothetical haplotypes were estimated based on the observed genotypes (Table IV). However, the overall distributions of these haplotypes did not significantly differ between cases and controls (p = 0.381). When the Tbf haplotype was used as the referent (the T, b and f alleles being putatively associated with increased melanoma risk), the haplotypes tBF and tBf were both associated with a significantly reduced melanoma risk (adjusted OR [CI], 0.52 [0.34, 0.80] and 0.51 [0.27, 0.94], respectively) (Table IV). This suggested that the tB haplotype was protective, regardless of the f allele's presence or absence.

Table IV. Age- and Sex-Adjusted Odds Ratios for Association Between Cutaneous Melanoma Risk and Presence of Haplotypes and Combined Genotypes of VDR TAQI, BSMI and FOKI in Non-Hispanic Whites, Texas, 1994–2006
 CasesControlsOdds ratio and 95% confidence interval
TaqIBsmIFokINo.%No.%Crude odds ratio95% Confidence intervalAdjusted odds ratio395% Confidence interval
  • 1

    Halotype cases n = 1610 alleles; controls n = 1682 alleles.

  • 2

    Combined genotype cases n = 805; controls n = 841.

  • 3

    Odds ratios were adjusted for age and sex in a logistic regression model.

Haplotype1          
 Tbf32920.431418.71.0Referent1.0Referent
 TbF58636.460936.20.690.42, 1.120.690.42, 1.11
 TBf161.080.53.810.51, 28.54.020.53, 30.4
 TBF362.2291.71.470.50, 4.311.480.50, 4.36
 tbF191.2221.30.390.09, 1.730.390.09, 1.72
 tbf130.8120.71.060.14, 7.881.090.14, 8.07
 tBF37823.542425.20.520.33, 0.790.520.34, 0.80
 tBf23314.526415.70.510.27, 0.940.510.27, 0.94
Combined genotype2          
 TTbbFf + ff18122.514216.91.0Referent1.0Referent
 TTbbFF9912.310512.50.740.52, 1.050.740.52, 1.05
 TTBb + BBFf + ff303.7101.22.351.11, 4.982.351.13, 4.98
 TTBb + BBFF202.5121.41.310.62, 2.761.310.62, 2.77
 Tt + ttBbFf + ff182.2121.41.180.55, 2.521.180.55, 2.54
 Tt + ttBbFF70.960.70.920.30, 2.780.940.31, 2.85
 Tt + ttBb + BBFf + ff28935.933339.60.680.52, 0.890.690.52, 0.90
 Tt + ttBb + BBFF16120.022126.30.570.42, 0.770.580.43, 0.78

When the putative risk genotypes (i.e., TT, bb and ff+Ff) were combined and used as the referent (TT/bb/ff+Ff), only the Tt+tt/Bb+BB/Ff+ff and Tt+tt/Bb+BB/FF genotypes were associated with a significantly reduced melanoma risk (adjusted OR [CI], 0.69 [0.52, 0.90] and 0.58 [0.43, 0.78], respectively), whereas the TT/Bb+BB/Ff+ff genotype was associated with a significantly increased risk (adjusted OR [CI], 2.35 [1.13, 4.98]). Together, these findings suggested that the Tt+tt/Bb+BB genotypes were protective, consistent with the effect of the tB haplotype, and that the Bb+BB genotypes were not protective in the presence of the TT genotype (Table IV).

Association between melanoma risk and polymorphism genotypes stratified by known risk factors

Because the FokI variants were associated with increased melanoma risk and the TaqI and BsmI variants with reduced risk, subjects bearing the protective TaqI and BsmI variant genotypes were further stratified by the Ff+ff and FF genotypes and all known melanoma risk factors (Table V). In the Ff+ff subgroup, the Tt+tt genotypes were associated with a significantly lower risk of melanoma than was the TT genotype, provided the carriers of the Tt+tt genotypes were old, male and blue-eyed; had not freckled in the sun as a child; or had no pigmented nevi. In contrast, the protective Bb+BB genotypes were associated with a significantly lower risk than was the bb genotype only if the carriers were old (Table V).

Table V. Association Between Cutaneous Melanoma Risk and VDR TAQI, BSMI and VDR FOKI Genotypes, Stratified by Risk Factors, in Non-Hispanic Whites, Texas, 1994–2006
VariablesFokI Ff + ff (no. cases/no. controls)1FokI FF (no. cases/no. controls)1
TaqIBsmITaqIBsmI
TTTt + ttOdds ratio2CI3p4bbBb + BBOdds ratio2CI3p4TTTt + ttOdds ratio2CI3p4bbBb + BBOdds ratio2CI3p4
  • 1

    The numbers of subjects in some of the strata were less than the total number of subjects included in our study, because some subjects did not provide the information.

  • 2

    Odds ratios were adjusted for age and sex in a logistic regression model.

  • 3

    CI, confidence interval.

  • 4

    p values for interaction were obtained from logistic regression models after adjustment for age, sex and the main effects of the interactive variables.

 Age (years)    0.03    0.01    0.80    0.71
  ≤5039/5575/781.350.80, 2.27 35/5179/821.400.82–2.38 87/56117/1210.620.41, 0.95 85/58119/1190.680.45, 1.04 
  >5080/6293/1490.500.33, 0.76 71/60102/1510.580.38–0.89 124/96190/2240.660.48, 1.92 114/96200/2240.760.54, 1.05 
 Sex    0.18    0.17    0.19    0.05
  Male78/73104/1570.620.41, 0.93 69/68113/1620.690.46–1.04 132/108200/2270.740.54, 1.02 121/111211/2240.890.64, 1.22 
  Female41/4464/701.000.58, 1.72 37/4368/711.110.64–1.93 79/44107/1180.500.32, 0.79 78/43108/1190.500.32, 0.78 
 Skin color         0.07    0.65    0.70
  Black or brown55/4372/860.660.39, 1.09 54/3868/710.550.32–0.92 82/61119/1570.550.36, 0.83 74/64127/1540.700.47, 1.06 
  Fair55/5381/1090.740.45, 1.19 44/5473–911.080.66–1.77 104/62164/1500.650.44, 0.96 104/61164/1510.650.44, 0.96 
 Hair color    0.45    0.81    0.05    0.03
  Black or brown70/77105/550.760.51, 1.15 64/73111/1590.810.53, 1.22 124/92184/2550.540.39, 0.75 119/93189/2540.580.42, 0.81 
  Blond(e) or red40/1848/390.570.28, 1.14 34/1854/390.730.36, 1.50 62/3199/491.040.60, 1.82 59/32102/481.200.69, 2.09 
 Eye color    0.12    0.26    0.38    0.44
  Other63/7793/1440.790.52, 1.21 57/7399/1480.860.56, 1.33 63/4693/600.550.39, 0.79 106/90162/2330.620.44, 0.87 
  Blue46/1760/510.420.21, 0.82 40/1766/510.530.27, 1.04 77/17144/510.730.45, 1.20 72/35129/810.790.48, 1.29 
 Tanning ability    0.44    0.05    0.74    0.83
  Good (high)30/3141/750.550.29, 1.05 31/2840/780.430.22, 0.83 40/4862/1130.670.40, 1.14 40/5062/1110.710.42, 1.20 
  Poor (low)80/65112/1190.770.51, 1.17 67/64125/1201.000.65, 1.53 146/75221/1930.600.42, 0.84 138/75229/1930.660.47, 0.92 
 Lifetime sunburn with blistering    0.83    0.92    0.42    0.93
  None34/3849/840.630.35, 1.14 31/3852/840.730.40, 1.32 38/5070/1240.750.45, 1.25 39/4869/1260.680.40, 1.14 
  ≥174/156105/1110.710.45, 1.10 65/52114/1150.790.50, 1.23 146/173208/1810.580.41, 0.82 137/77217/1770.700.49, 0.98 
 Freckling in the sun as a child    0.45    0.71    0.10    0.23
  No62/5671/1090.570.35, 0.91 54/5479/1110.690.42, 1.11 83/83127/1730.740.50, 1.08 80/82130/1740.770.53, 1.13 
  Yes48/3981/850.770.45, 1.30 44/3785/870.810.47, 1.38 102/40154/1330.460.30, 0.71 98/43158/1300.550.36, 0.84 
 Moles    0.94    0.67    0.35    0.65
  No25/3043/710.730.38, 1.41 21/3047/710.980.50, 1.92 40/4767/1410.570.34, 0.95 39/5368/1350.700.42, 1.16 
  Yes94/87125/1560.760.52, 1.11 85/81134/1620.810.55, 1.19 171/105240/2040.740.54, 1.01 160/101251/2080.790.57, 1.07 
 Dysplastic nevi    0.98    0.51    0.28    0.24
  No113/115155/2270.700.50, 0.97 100/110168/2320.800.57, 1.12 190/151282/3390.660.51, 0.86 178/153294/3370.750.57, 0.98 
  Yes6/213/0 NC 6/113/10.480.01, 40.2 21/125/60.070.01, 0.99 21/125/60.070.01, 0.98 
 First-degree relatives with any cancer    0.36    0.94    0.49    0.63
  No43/6767/1220.860.53, 1.40 42/6268/1270.790.48, 1.29 104/77129/1650.580.40, 0.85 96/77137/1650.680.46, 0.99 
  Yes76/50101/1050.640.41, 1.00 64/49113/1060.830.52, 1.31 107/75178/1800.700.49, 1.00 103/77182/1780.770.53, 1.10 

In the FF subgroup, the Tt+tt genotypes were more likely to be associated with reduced risk in carriers who were young, female, black- or brown-skinned, black- or brown-haired or non-blue-eyed; had poor tanning ability, had ≥1 lifetime sunburn with blistering, had a childhood history of freckling in the sun, had no moles or pigmented nevi or had no family history of cancer. The same was generally true of the Bb+BB genotypes, except that being young was not a risk factor. Further tests for interaction were significant for age (p = 0.03 for TaqI and p = 0.01 for BsmI) among subjects carrying the Ff+ff genotype and for sex (p = 0.05 for BsmI) and hair color (p = 0.05 for TaqI and p = 0.03 for BsmI) among subjects carrying the FF genotype. However, these findings may have been due to chance since multiple tests were performed.

Multivariate analysis of association between polymorphisms and melanoma risk

All variables used in the initial analyses were fitted to 2 multivariate unconditional logistic models (Models 1 and 2) after simultaneous adjustment (Table VI). In the first model, which included the age, sex and polymorphism genotypes for all subjects, the t genotypes (Tt+ttvs.TT) and f genotypes (Ff+ffvs.FF), but not the B genotypes (Bb+BBvs.bb), were associated with a significantly reduced melanoma risk (OR [CI], 0.57 [0.39, 0.84] for TaqI and 1.27 [1.04, 1.55] for FokI). This suggested that the BsmI polymorphism was not an independent melanoma risk factor, consistent with the high-linkage disequilibrium between the t and B alleles. Consequently, BsmI was excluded from the second model, and all other selected risk factors were added to the multivariate logistic regression model. The second model included only data from subjects who provided complete questionnaire data (i.e., 712 case subjects and 707 control subjects). Most of the known risk factors were consequently found to be significant independent predictors of melanoma risk, the exceptions being age, sex, skin color and childhood freckling. Because skin color may be represented by hair or eye color and freckling by the number of sunburns in the same model, and because there was a high correlation between these variables in our study (data not shown), variance in the model was reduced by excluding skin color and freckling from the final model (Table VI). As a result, the VDR TaqI t variant genotypes assessed in the final model were associated with a significantly reduced melanoma risk (OR [CI], 0.68 [0.54, 0.86]), whereas the FokI f variant genotypes were not (1.16 [0.92–1.46]) (Table VI). Further tests for interaction revealed significant associations between the FokI f genotypes and skin color (p = 0.029), moles (p = 0.017) and a family history of cancer (p = 0.013) but not between the TaqI t genotypes and the same variables (data not shown). Since multiple tests were performed, these interactions are only suggestive and require validation in larger future studies.

Table VI. Multivariate Logistic Regression Analysis of Associations Between VDR TAQI, BSMI and FOKI Genotype Frequencies and Cutaneous Melanoma Risk in Non-Hispanic Whites, Texas, 1994–2006
VariablesβWald χ2pOdds ratio95% Confidence interval
  • 1

    The numbers of subjects included in this model were less than the total number of subjects included in our study, because this model only included subjects who provided complete information in their screening questionnaires.

Model 1 (805 cases and 841 controls)
  Age (years)−0.0072.590.110.990.99, 1.00
  Sex (male vs. female)0.131.510.221.140.93, 1.40
  VDR BsmI (Bb + BB vs. bb)0.211.110.291.230.84, 1.82
  VDR FokI (Ff + ff vs. FF)0.245.390.021.271.04, 1.55
  VDR TaqI (Tt + tt vs. TT)−0.578.200.0040.570.39, 0.84
Model 2 (712 cases and 707 controls)1
  Age (years)−0.010.110.741.000.99, 1.01
  Sex (male vs. female)0.080.420.521.080.88, 1.37
  Eye color (other vs. blue)0.5821.7<0.0011.791.40, 2.29
  Hair color (blond[e] or red vs. black or brown)0.4611.3<0.0011.581.21, 2.07
  Lifetime sunburns with blistering (≥1 vs. 0)0.4512.9<0.0011.561.23, 1.99
  Tanning ability after prolonged sun exposure (moderate or poor vs. good)0.378.550.0031.451.13, 1.86
  Moles (yes vs. no)0.6527.9<0.0011.921.51, 2.44
  Pigmented nevi (yes vs. no)1.7020.9<0.0015.462.64, 11.3
  Family history of skin cancer (yes vs. no)0.275.250.021.311.04, 1.65
  VDR FokI (Ff + ff vs. FF)0.151.550.211.160.92, 1.46
  VDR TaqI (Tt + tt vs. TT)−0.3810.30.0010.680.54, 0.86

Discussion

In this hospital-based case–control study of cutaneous melanoma, we found that TaqI t and BsmI B variant genotypes of the VDR gene (Tt+tt and Bb+BB, respectively) were associated with a reduced risk of melanoma and FokI f variant genotypes (Ff and Ff+ff) with an increased risk when compared to the TT, bb and FF genotypes, respectively. The tBF and tBf haplotypes were associated with a significantly lower melanoma risk than was the Tbf haplotype. The VDR FokI polymorphisms appeared to interact with other known risk factors to modulate the melanoma risk associated with those factors, while the VDR TaqI polymorphism appeared to exert its protective (i.e., risk-reducing) effect independently of other risk factors.

The VDR protein is expressed in both melanocytes and melanoma cells, and 1,25-[OH]2D3 can apparently inhibit the growth of both normal and malignant melanocytes in vitro.2, 5, 33 However, malignant transformation may inhibit the anticancer actions of 1,252D3 for reasons that include genetic polymorphisms of the VDR gene.34 The VDR gene comprises 9 exons harboring several polymorphisms, including a poly-A microsatellite in the 3′ flanking region,35 changes in intron 8 that generate BsmI36 and ApaI restriction enzyme sites,37 a synonymous change at codon 352 in exon 9 that generates a TaqI restriction enzyme site38 and a 5′ FokI site in exon 2.8 No apparent association has been found between the TaqI or Bsm1 polymorphisms and altered functional activities. Nevertheless, both of TaqI and BsmI polymorphisms located near to the 3′ end of the gene, thus are thought to affect mRNA stability and VDR gene transcription regulation.39 Among the VDR polymorphisms, the FokI single-nucleotide polymorphism of the translation start site is the only one that results in a VDR protein with a different structure.40 This polymorphism is characterized by the presence of either 2 ATG start codons separated by 6 nucleotides in the long f-VDR allele or only 1 start codon due to a T-to-C substitution in the most 50 ATG codon, resulting in a 3-aa shorter F-VDR protein (424 aa instead of 427 aa).41

To date, 2 other groups have reported their studies on TaqI, BsmI or FokI polymorphisms in evaluating their association with melanoma risk19–21 but generated mixed results. In the earliest study of the TaqI polymorphism in 316 melanoma patients and 108 control subjects, neither the Tt nor the combined Tt+tt genotype was associated with altered melanoma risk when compared to the TT genotype.19 However, in our large study, we found that the t genotypes were in fact associated with a lower melanoma risk. In the Nurses' Health Study, investigators examined the association between melanoma risk and BsmI polymorphism in 219 melanoma patients and 873 controls and found no association between the two.21 However, in our study, we found an association between Bb+BB genotypes and reduced melanoma risk only in women who carried the FF genotype and not in men (Table V). We found the Ff and Ff+ff genotypes to be associated with increased melanoma risk. Interestingly, even though this finding was consistent with published data from the other group,19 it was not consistent with the finding in the Nurses' Health Study of an association (though not significant) between only the ff genotype and higher melanoma risk.21

There are several possible reasons for the apparent discrepancy between our results and those reported by others. One is the relatively larger size of our control population, and another is the potential for selection bias in our control population. Our study, with its 805 melanoma cases and 841 control subjects, is the largest study so far to have addressed the possible association between VDR polymorphisms and melanoma risk; in addition, the VDR t, B and f allele frequencies we report here are similar to those reported previously in a large meta-analysis of studies in whites.14 Therefore, we believe it unlikely that the association between the VDR polymorphisms and melanoma risk demonstrated in our study was biased by our selection of controls. A third possible reason for the discrepancy between our findings and those previously reported is variations in the serum vitamin D levels of study subjects between studies. Indeed, the serum vitamin D level may have affected our results. Unfortunately, none of the studies published so far gathered data on serum vitamin D levels in their subjects. A fourth possible reason is recall biases in exposure data, to which a retrospective study might be prone. Thus, larger, population-based studies are needed to verify our findings.

The functional significance of the VDR TaqI polymorphisms is unknown. As a synonymous change in exon 9, the TaqI polymorphism does not cause the amino acid substitution (http://egp.gs.washington.edu/data/vdr/vdrxx.csnps.txt; http://snp500cancer.nci.nih.gov/snplist.cfm). In addition, no apparent association has been found between the BsmI polymorphism and altered functional activities.42 Nevertheless, these polymorphisms might be functional themselves or in linkage disequilibrium with other functional SNPs and associated with melanoma risk. Indeed, previous in vitro functional studies have revealed the baT haplotype (haplotype of Bsm1/ApaI/TaqI) inserted in transfection constructs resulted in lower reporter gene activity compared to BAt43 and associated with low VDR mRNA expression,44 which is in agreement with our findings that both of the BsmI B allele and TaqI t allele are protective against melanoma in our study. VDRFokI is the only polymorphism that is not linked to any of the other VDR polymorphisms.41 A study recently provided evidence that the VDRFokI polymorphism affects immune cell behavior, with a more active immune system for the short F-VDR protein.45 Consistent with this functional study, we found Ff+ff genotype associated with significantly increased melanoma risk, which might be due to the f allele-related reduced anti-tumor immune activity. However, since some in vitro data may not accurately reflect the biologic environment, in which a marker may be acting in humans, our consideration regarding the putative function of VDR polymorphisms should be adequately validated in further functional studies.

As some epidemiologic studies have suggested that, adequate vitamin D levels (including sunlight induced) may provide very important protection against colon, breast and prostate cancers.46–48 However, its protection against skin cancer is a more complex issue. One potential complication is that ultraviolet light exposure not only promotes vitamin D-3 (cholecalciferol) synthesis in the skin but also increases the risk of skin cancer by inducing DNA damage. Therefore, it is very important to consider gene-environment interactions as well as locus–locus interactions when studying associations between VDR polymorphisms and melanoma risk. For example, 1 recent case-only analysis study revealed an association between the TaqI tt genotype and reduced prostate cancer risk, but only in association with high sun exposure.16 However, in our study, we found that the TaqI t variant genotypes exerted their protective effects independently of other genotypes and known risk factors. Meanwhile, the effect of FokI genotypes on melanoma risk appeared to be independent of the TaqI polymorphism but dependent on other known risk factors, suggesting that some environmental modification of the VDR gene may have occurred. Indeed, we found that the FokI polymorphism interacted with the known melanoma risk factors of skin color, moles and family history of cancer. However, because of our study's limited size and current uncertainty about the biological mechanisms underlying such interactions, these findings are only suggestive. Again, larger, population-based and functional studies are necessary to validate these interactions.

Our study has several limitations. First, it was a hospital-based case–control study in which selection of the unrepresentive population and retrospective collection of exposure data may have led to uncontrolled biases. Second, despite being the largest study of its kind ever published, our study was still too underpowered to detect gene–gene or gene-environment interactions. Third, the self-reporting of skin conditions by both case and control subjects created an additional source of potential bias. Finally, like most previous studies on the subject, ours could not account for serum vitamin D and thus did not allow for genotype–phenotype correlation analysis. These limitations can only be overcome in large, well-designed prospective studies that gather data on both genotypes and phenotypes of vitamin D metabolism.

In summary, the VDRTaqI, BsmI and FokI polymorphisms and their combined variant genotypes do affect melanoma risk. The VDR TaqI polymorphism alters risk independently of BsmI, FokI and other known melanoma risk factors, while the VDR FokI polymorphism may modify it through interaction with sun exposure-related melanoma risk factors. Larger, population-based studies are needed to confirm these findings.

Acknowledgements

We thank Ms. Margaret Lung, Mr. Cesar A. Maldonado and Ms. Amanda Franco for assistance in recruiting subjects; Mr. Zhaozheng Guo, Ms. Yawei Qiao, Mr. Jianzhong He and Ms. Kejing Xu for laboratory assistance; Ms. Monica Domingue for assistance in preparing the article; and Ms. Jude Richard, Ms. ELS, for editing the article.

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