INTRODUCTION {#s1}
============

Gastric cardia adenocarcinoma (GCA) remains one of the most common malignant tumor worldwide \[[@R1]\]. Although the incidence of noncardia gastric cancer has declined steadily, the incidence and mortality of GCA are continuously increasing \[[@R2]\]. The etiology of GCA comprises interactions of multiple environmental and genetic factors. Environmental factors including cigarette smoking and alcohol consumption \[[@R3], [@R4]\], as well as genetic factors such as gene polymorphisms, have been implicated with GCA. Yet, the underlying etiological mechanisms of GCA are not fully understood.

The 1,25-dihydroxyvitamin D~3~ \[1,25(OH)~2~D~3~\] is the hormonally active form of vitamin D, which has been shown to inhibit prostate, breast and colon cancer cell progression \[[@R5]\]. Consistently, amounting evidence has indicated the correlation of the Vitamin D Receptor (VDR) and cancer. The antineoplastic effect of 1,25(OH)~2~D~3~ requires the expression of VDR in tumor cells \[[@R6]\]. Increased tumor VDR expression is associated with a better prognosis in various types of cancer \[[@R7], [@R8]\]. The association of single nucleotide polymorphisms (SNPs) in *VDR* (ApaI \[rs7975232\], BsmI \[rs1544410\], FokI \[rs10735810\], TaqI \[rs731236\] and cancer risk has been reported, yet the results were inconclusive \[[@R6], [@R9]--[@R12]\].

In our previous study, we have investigated the correlation of four SNPs *VDR* rs11568820 G\>A, *VDR* rs1989969 C\>T, *VDR* rs2107301 T\>C and *VDR* rs2228570 C\>T with esophageal squamous cell carcinoma development, and found that *VDR* rs2107301 T\>C polymorphism with alcohol drinking enhanced the risk of esophageal squamous cell carcinoma \[[@R13]\]. Considering GCA occurs in the immediate vicinity of esophagus, we hypothesized that these four SNPs are also related to GCA. Here, we performed a hospital-based case-control study to examine the genetic effects of these four SNPs on the development of GCA.

RESULTS {#s2}
=======

Characteristics of the study subjects {#s2_1}
-------------------------------------

The characteristics of the study subjects, including demographics and environmental risk factors, are presented in Table [1](#T1){ref-type="table"}. The cases and controls were well matched in gender and age (χ^2^ test, *p*=0.746 and 0.965, respectively). However, tobacco smoking rate was much higher in GCA patients as compared with the control subjects (36.67% vs. 27.96%, *p*=0.006). Alcohol drinking rate was higher, yet not significantly, in GCA patients than in control subjects (29.39% vs. 23.01%, *p*=0.072).

###### Distribution of selected demographic variables and risk factors in GCA cases and controls

  Variable                Cases (n = 330)   Controls (n = 608)   *p*^a^           
  ----------------------- ----------------- -------------------- -------- ------- -----------
  Age (years) mean ± SD   65.06 (±8.37)     64.19 (±6.66)        0.103            
  Age (years)                                                                     0.746
   \< 60                  89                26.97                170      27.96   
   ≥ 60                   241               73.03                438      72.04   
  Sex                                                                             0.965
   Male                   223               67.58                410      67.43   
   Female                 107               32.42                198      32.57   
  Tobacco use                                                                     **0.006**
   Never                  209               63.33                438      72.04   
   Ever                   121               36.67                170      27.96   
  Alcohol use                                                                     0.072
   Never                  233               70.61                462      75.99   
   Ever                   97                29.39                146      24.01   

^a^ Two-sided χ^2^ test and student t test; Bold values are statistically significant (*p* \<0.05).

As shown in Table [2](#T2){ref-type="table"}, the genotyping successful rates were ranging from 95.76% to 100.0% in GCA cases and from 95.39 to 99.18% in controls. Compared with the minor allele frequency (MAF) for Chinese in database for all four SNPs loci, the MAF in our controls was similar (Table [2](#T2){ref-type="table"}). In the control subjects, the genotype frequencies for these four polymorphisms reached Hardy-Weinberg equilibrium (*p*-value for HWE, all *p*\>0.05, Table [2](#T2){ref-type="table"}).

###### Primary information for *VDR* rs2107301 T\>C, rs2228570 C\>T, rs1989969 C\>T and rs11568820 G\>A polymorphisms

  Genotyped SNPs                              *VDR* rs2107301 T\>C   *VDR* rs2228570 C\>T   *VDR* rs1989969 C\>T   *VDR* rs11568820 G\>A
  ------------------------------------------- ---------------------- ---------------------- ---------------------- -----------------------
  Chromosome                                  12                     12                     12                     12
  Gene (ID)                                   VDR (7421)             VDR (7421)             VDR (7421)             VDR (7421)
  Function                                    Intron region          Missense               Intron region          Intergene region
  Chr Pos (Genome Build 36.3)                 46541837               46559162               46564277               46588812
  Regulome DB Score^a^                        5                      5                      No data                No data
  TFBS^b^                                     ---                    ---                    ---                    Y
  Splicing (ESE or ESS)                       ---                    Y                      ---                    ---
  nsSNP                                       ---                    Y                      ---                    ---
  MAF^c^ for Chinese in database              0.291                  0.482                  0.330                  0.453
  MAF in our controls (n = 608)               0.297                  0.456                  0.323                  0.433
  *p* value for HWE^d^ test in our controls   0.690                  0.347                  0.718                  0.574
  Genotyping method^e^                        LDR                    LDR                    LDR                    LDR
  \% Genotyping value                         95.52%                 95.52%                 98.19%                 98.08%

^a^ <http://www.regulomedb.org/>;

^b^ TFBS: Transcription Factor Binding Site (<http://snpinfo.niehs.nih.gov/snpinfo/snpfunc.htm>);

^c^ MAF: minor allele frequency;

^d^ HWE: Hardy--Weinberg equilibrium;

^e^ LDR: ligation detection reaction.

Associations between risk of GCA and four polymorphisms {#s2_2}
-------------------------------------------------------

As demonstrated in Table [3](#T3){ref-type="table"}, the single locus analyses showed no statistically significant difference in genotype frequencies of four SNPs between the cases and the controls (*p*\>0.05). There are no correlation between these four polymorphic sites with the risk of GCA as evaluated by the logistic regression analyses (Table [3](#T3){ref-type="table"}).

###### Logistic regression analyses of associations between *VDR* rs2107301 T\>C, rs2228570 C\>T, rs1989969 C\>T and rs11568820 G\>A polymorphisms and risk of GCA

  Genotype                Cases (n = 330)   Controls (n = 608)   Crude OR (95%CI)   *p*     Adjusted OR ^a^ (95%CI)   *p*                        
  ----------------------- ----------------- -------------------- ------------------ ------- ------------------------- ------- ------------------ -------
  *VDR* rs2107301 T\>C                                                                                                                           
   TT                     155               49.05                285                49.14   1.00                              1.00               
   TC                     129               40.82                246                42.41   0.96 (0.72-1.29)          0.805   0.97 (0.72-1.29)   0.819
   CC                     32                10.13                49                 8.45    1.20 (0.74-1.95)          0.461   1.19 (0.73-1.95)   0.492
  CC vs. TC vs. TT                                                                                                                               0.683
   TC+CC                  161               50.95                295                50.86   1.00 (0.76-1.32)          0.980   1.00 (0.76-1.32)   0.982
   TT+TC                  284               89.87                531                91.55   1.00                              1.00               
   CC                     32                10.13                49                 8.45    1.22 (0.77-1.95)          0.403   1.21 (0.75-1.94)   0.436
   C allele               193               30.54                344                29.66                                                        
  *VDR* rs2228570 C\>T                                                                                                                           
   CC                     97                30.70                166                28.62   1.00                              1.00               
   CT                     153               48.42                299                51.55   0.88 (0.64-1.20)          0.412   0.89 (0.65-1.23)   0.488
   TT                     66                20.89                115                19.83   0.98 (0.66-1.46)          0.928   0.97 (0.65-1.45)   0.895
  TT vs. CT vs. CC                                                                                                                               0.666
   CT+TT                  219               69.30                414                71.38   0.91 (0.67-1.22)          0.515   0.92 (0.68-1.24)   0.567
   CC+CT                  250               79.11                465                80.17   1.00                              1.00               
   TT                     66                20.89                115                19.83   1.07 (0.76-1.50)          0.706   1.05 (0.74-1.47)   0.799
   T allele               285               45.09                529                45.60                                                        
  *VDR* rs1989969 C\>T                                                                                                                           
   CC                     135               42.45                278                46.10   1.00                              1.00               
   CT                     140               44.03                260                43.12   1.11 (0.83-1.48)          0.486   1.10 (0.82-1.48)   0.521
   TT                     43                13.52                65                 10.78   1.36 (0.88-2.11)          0.165   1.36 (0.88-2.11)   0.172
  TT vs. CT vs. CC                                                                                                                               0.369
   CT+TT                  183               57.55                325                53.90   1.16 (0.88-1.53)          0.290   1.15 (0.87-1.52)   0.314
   CC+CT                  275               86.48                538                89.22   1.00                              1.00               
   TT                     43                13.52                65                 10.78   1.29 (0.86-1.95)          0.220   1.30 (0.86-1.96)   0.221
   T allele               226               35.53                390                32.34                                                        
  *VDR* rs11568820 G\>A                                                                                                                          
   GG                     99                30.00                193                32.71   1.00                              1.00               
   GA                     162               49.09                283                47.97   1.12 (0.82-1.52)          0.488   1.11 (0.81-1.53)   0.500
   AA                     69                20.91                114                19.32   1.18 (0.80-1.73)          0.399   1.20 (0.81-1.77)   0.361
  AA vs. GA vs. GG                                                                                                                               0.666
   GA+AA                  231               70.00                397                67.29   1.13 (0.85-1.52)          0.397   1.14 (0.85-1.53)   0.390
   GG+GA                  261               79.09                476                80.68   1.00                              1.00               
   AA                     69                20.91                114                19.32   1.10 (0.79-1.54)          0.563   1.12 (0.80-1.58)   0.503
   A allele               300               45.45                511                43.31                                                        

^a^ Adjusted for age, sex, smoking status and alcohol consumption.

Stratification analyses of four polymorphisms and risk of GCA {#s2_3}
-------------------------------------------------------------

To further assess the effects of *VDR* rs11568820 G\>A, rs1989969 C\>T, rs2107301 T\>C and rs2228570 C\>T on GCA risk according to different sex, smoking, age and alcohol drinking status, stratification analyses were conducted as shown in Tables [4](#T4){ref-type="table"}, [5](#T5){ref-type="table"}, [6](#T6){ref-type="table"}, [7](#T7){ref-type="table"}, respectively. In association with the *VDR* rs1989969 C\>T polymorphism, we further identified two significantly increased risk factors of GCA, which are age\<60 or alcohol drinking.

###### Stratified analyses between *VDR* rs1989969 C\>T polymorphism and GCA risk by sex, age, smoking status and alcohol consumption

  Variable              *VDR* rs1989969 C \> T (case/control)^a^   Adjusted OR^b^ (95% CI); *p*; *p*~h~^c^                                                                                                                                                                               
  --------------------- ------------------------------------------ ----------------------------------------- ------- --------- ------ ------------------------------------------------ ------------------------------------------------ ------------------------------------------------ --------------------------------------------
  Sex                                                                                                                                                                                                                                                                                    
   Male                 92/189                                     98/170                                    28/46   126/216   1.00   1.19 (0.83-1.69); *p*: 0.345; *p*~h~:0.523       1.26 (0.74-2.14); *p*: 0.405; *p*~h~:0.575       1.20 (0.86-1.68); *p*: 0.283; *p*~h~:0.734       1.15 (0.70-1.91); *p*: 0.579; *p*~h~:0.412
   Female               43/89                                      42/90                                     15/19   57/109    1.00   1.00 (0.59-1.70); *p*: 0.999; *p*~h~:0.523       1.67 (0.76-3.66); *p*: 0.199; *p*~h~:0.575       1.12 (0.68-1.84); *p*: 0.660; *p*~h~:0.734       1.67 (0.80-3.50); *p*: 0.172; *p*~h~:0.412
  Age                                                                                                                                                                                                                                                                                    
   \<60                 31/90                                      42/65                                     15/14   57/79     1.00   1.85 (1.03-3.33); ***p*: 0.041; *p*~h~:0.031**   2.97 (1.24-7.13); ***p*: 0.015; *p*~h~:0.023**   2.05 (1.18-3.57); ***p*: 0.011; *p*~h~:0.010**   2.19 (0.97-4.97); *p*: 0.060; *p*~h~:0.096
   ≥60                  104/188                                    98/195                                    28/51   126/246   1.00   0.90 (0.64-1.27); *p*: 0.560; *p*~h~:0.031       0.99 (0.59-1.67); *p*: 0.977; *p*~h~:0.023       0.92 (0.67-1.27); *p*: 0.621; *p*~h~:0.010       1.04 (0.64-1.71); *p*: 0.865; *p*~h~:0.096
  Smoking status                                                                                                                                                                                                                                                                         
   Never                88/197                                     87/189                                    25/48   112/237   1.00   1.01 (0.70-1.46); *p*: 0.962; *p*~h~:0.482       1.12 (0.64-1.97); *p*: 0.696; *p*~h~:0.345       1.03 (0.73-1.46); *p*: 0.863; *p*~h~:0.359       1.11 (0.65-1.90); *p*: 0.692; *p*~h~:0.451
   Ever                 47/81                                      53/71                                     18/17   71/88     1.00   1.24 (0.74-2.10); *p*: 0.415; *p*~h~:0.482       1.90 (0.87-4.15); *p*: 0.107; *p*~h~:0.345       1.37 (0.84-2.24); *p*: 0.213; *p*~h~:0.359       1.71 (0.82-3.56); *p*: 0.154; *p*~h~:0.451
  Alcohol consumption                                                                                                                                                                                                                                                                    
   Never                99/205                                     93/206                                    30/47   123/253   1.00   0.90 (0.63-1.27); *p*: 0.538; *p*~h~:0.057       1.31 (0.77-2.23); *p*: 0.315; *p*~h~:0.835       0.97 (0.70-1.35); *p*: 0.865; *p*~h~:0.100       1.39 (0.84-2.28); *p*: 0.202; *p*~h~:0.647
   Ever                 36/73                                      47/54                                     13/18   60/72     1.00   1.90 (1.04-3.46); ***p*: 0.036; *p*~h~:0.057**   1.45 (0.60-3.50); *p*: 0.409; *p*~h~:0.835       1.78 (1.01-3.14); ***p*: 0.045; *p*~h~:0.100**   1.05 (0.46-2.40); *p*: 0.903; *p*~h~:0.647

^a^ The genotyping was successful in 318 (96.36%) GCA cases and 603 (99.18%) controls for *VDR* rs1989969 C\>T;

^b^ Adjusted for age, sex, smoking status and alcohol consumption (besides stratified factors accordingly) in a logistic regression model;

^c^ *p*~h~ for heterogeneity; Bold values are statistically significant (*p* \<0.05).

###### Stratified analyses between *VDR* rs2228570 polymorphism and GCA risk by sex, age, smoking status and alcohol consumption

  Variable              *VDR* rs2228570 (case/control)^a^   Adjusted OR^b^ (95% CI); *p*; *p*~h~^c^                                                                                                                                                                     
  --------------------- ----------------------------------- ----------------------------------------- ------- --------- ------ --------------------------------------------- --------------------------------------------- -------------------------------------------- ---------------------------------------------
  Sex                                                                                                                                                                                                                                                                   
   Male                 101/205                             71/113                                    40/73   141/278   1.00   1.112 (0.71-1.71); *p*: 0.646; *p*~h~:0.572   0.872 (0.54-1.42); *p*:0.581; *p*~h~:0.559    1.013 (0.66-1.55); *p*:0.953; *p*~h~:0.965   0.807 (0.56-1.16); *p*: 0.242; *p*~h~:0.984
   Female               52/94                               26/53                                     26/42   78/136    1.00   0.894 (0.49-1.62); *p*:0.711; *p*~h~: 0.572   0.792 (0.40-1.56); *p*:0.501; *p*~h~:0.559    0.857 (0.49-1.50); *p*:0.590; *p*~h~:0.965   0.855 (0.49-1.48); *p*: 0.574; *p*~h~:0.984
  Age                                                                                                                                                                                                                                                                   
   \<60                 45/78                               25/48                                     15/33   60/111    1.00   1.269 (0.62-2.58); *p*:0.511; *p*~h~:0.945    1.146 (0.53-2.49); *p*: 0.732; *p*~h~:0.245   1.222 (0.62-2.40); *p*:0.561; *p*~h~:0.746   1.038 (0.58-1.85); *p*:0.900; *p*~h~:0.741
   ≥60                  108/221                             72/118                                    51/82   159/303   1.00   0.786 (0.52-1.19); *p*:0.258; *p*~h~:0.945    0.981 (0.62-1.55); *p*: 0.935; *p*~h~:0.245   0.854 (0.58-1.27); *p*: 0.431; *p*~h~:0746   1.163 (0.82-1.65); *p*: 0.399; *p*~h~:0.741
  Smoking status                                                                                                                                                                                                                                                        
   Never                108/221                             69/117                                    45/78   153/299   1.00   0.847 (0.55-1.31); *p*:0.452; *p*~h~:0.062    1.022 (0.64-1.64); *p*:0.927; *p*~h~:0.722    0.908 (0.60-1.37); *p*:0.643; *p*~h~:0.788   0.868 (0.61-1.24); *p*:0.43; *p*~h~:0.200
   Ever                 45/78                               28/49                                     21/37   66/115    1.00   1.016 (0.53-1.95); *p*:0.961; *p*~h~:0.062    1.007 (0.49-2.04); *p*:0.985; *p*~h~:0.722    1.013 (0.55-1.86); *p*:0.967; *p*~h~:0.788   1.004 (0.58-1.75); *p*:0.988; *p*~h~:0.200
  Alcohol consumption                                                                                                                                                                                                                                                   
   Never                122/228                             72/125                                    50/86   172/314   1.00   0.920 (0.61-1.39); *p*:0.693; *p*~h~:0.051    0.991(0.63-1.56); *p*:0.968; *p*~h~:0.893     0.945(0.64-1.39); *p*:0.777; *p*~h~:0.513    0.951 (0.67-1.34); *p*:0.775; *p*~h~:0.143
   Ever                 31/71                               25/41                                     16/29   47/100    1.00   0.791 ( 0.37-1.66); *p*:0.536;*p*~h~:0.051    1.105 (0.50-2.43); *p*:0.803; *p*~h~:0.893    0.906 (0.45-1.80); *p*:0.78; *p*~h~:0.513    0.771 (0. 42-1.41); *p*:0.399; *p*~h~:0.143

^a^ The genotyping was successful in 316 (95.75%) GCA cases and 603 (95.70%) controls *VDR* rs2228570 C\>T;

^b^ Adjusted for age, sex, smoking status and alcohol consumption (besides stratified factors accordingly) in a logistic regression model;

^c^ *p*~h~ for heterogeneity; bold values are statistically significant (*p* \<0.05).

###### Stratified analyses between *VDR* rs2107301 polymorphism and GCA risk by sex, age, smoking status and alcohol consumption

  Variable              *VDR* rs2107301 (case/control)^a^   Adjusted OR^b^ (95% CI); *p*; *p*~h~^c^                                                                                                                                                                          
  --------------------- ----------------------------------- ----------------------------------------- --------- --------- ------ --------------------------------------------- ---------------------------------------------- ---------------------------------------------- ---------------------------------------------
  Sex                                                                                                                                                                                                                                                                        
   Male                 21/30                               86/174                                    105/187   191/361   1.00   0.706 (0.38-1.31); *p*:0.266; *p*~h~:0.947    0.802 (0.437-1.47); *p*:0.476; *p*~h~:0.510    0.756 (0.421-1.36); *p*:0.756; *p*~h~:0.983    0.934 (0.669-1.31); *p*:0.669; *p*~h~:0.995
   Female               11/19                               43/72                                     50/98     93/170    1.00   1.032 (0.45-2.37); *p*:0.942; *p*~h~: 0.947   0.881 (0.39-1.99); *p*:0.762; *p*~h~: 0.510    0.945 (0.43-2.07); *p*:0.887; *p*~h~: 0.983    1.163 (0.72-1.88); *p*:0.536; *p*~h~:0.995
  Age                                                                                                                                                                                                                                                                        
   \<60                 9/15                                36/71                                     40/73     76/144    1.00   0.845 (0.34-2.12); *P*:0.719; *p*~h~: 0.607   0.913 (0.37-2.27); *p*:0.845; *p*~h~:0.784     0.880 (0.37-2.1); *p*:0.773; *p*~h~:0.735      0.955 (0.56-1.62); *p*:0.864; *p*~h~:0.746
   ≥60                  23/34                               93/175                                    115/212   208/387   1.00   0.786 (0.44-1.41); *p*:0.419; *p*~h~0.607     0.802 (0.45-1.43); *p*:0.452; *p*~h~: 0.784    0.795 (0.456-1.39); *p*:0.416; *p*~h~: 0.735   1.023 (0.74-1.41); *p*:0.889; *p*~h~:0.746
  Smoking status                                                                                                                                                                                                                                                             
   Never                24/36                               94/176                                    104/204   198/380   1.00   0.801 (0.45-1.42); *p*:0.448; *p*~h~:0.753    0.765 (0.433-1.35); *p*:0.354; *p*~h~:0.248    1.279 (0.74-2.21); *p*:0.374; *p*~h~:0.216     1.092 (0.78-1.51); *p*:0.598; *p*~h~:0.205
   Ever                 8/13                                35/70                                     51/81     86/151    1.00   0.813 (0.31-2.14); *p*:0.674; *p*~h~: 0.753   1.023 (0.397-2.64); *p*:0.962; *p*~h~: 0.248   0.925 (0.37-2.32); *p*:0.869; *p*~h~:0.216     0.823 (0.49-1.37); *p*:0.452; *p*~h~:0.205
  Alcohol consumption                                                                                                                                                                                                                                                        
   Never                25/42                               103/189                                   116/208   219/397   1.00   0.916 (0.53-1.58); *p*:0.753; *p*~h~:0.959    0.937 (0.54-1.62); *p*:0.815; *p*~h~:0.204     0.927 (0.55-1.56); *p*:0.775; *p*~h~:0.159     0.994 (0.73-1.36); *p*:0.968; *p*~h~:0.147
   Ever                 7/7                                 26/57                                     39/77     65/134    1.00   0.456 (0.15-1.43); *p*:0.172; *p*~h~: 0.959   0.506 (0.166-1.55); *p*:0.226; *p*~h~: 0.204   0.485 (0.16-1.44); *p*:0.18; *p*~h~:0.159      1.018 (0.57-1.80); *p*:0.951; *p*~h~:0.147

^a^ The genotyping was successful in 316 (95.75%) GCA cases and 603 (95.39%) controls for *VDR* rs1989969 C\>T;

^b^ Adjusted for age, sex, smoking status and alcohol consumption (besides stratified factors accordingly) in a logistic regression model;

^c^ *p*~h~ for heterogeneity; bold values are statistically significant (*p* \<0.05).

###### Stratified analyses between *VDR* rs11568820 polymorphism and GCA risk by sex, age, smoking status and alcohol consumption

  Variable              *VDR* rs11568820 (case/control)^a^   Adjusted OR^b^ (95% CI); *p*; *p*~h~^c^                                                                                                                                                                     
  --------------------- ------------------------------------ ----------------------------------------- -------- --------- ------ --------------------------------------------- -------------------------------------------- -------------------------------------------- ----------------------------------------------
  Sex                                                                                                                                                                                                                                                                    
   Male                 49/85                                100/184                                   74/126   174/310   1.00   0.943 (0.65-1.45); *p*: 0.787; *p*~h~:0.399   1.019 (0.65-1.60); *p*:0.936; *p*~h~:0.352   0.974(0.65-1.45); *p*:0.895; *p*~h~:0.927    0.943 (0.66-1.34); *p*:0.743; *p*~h~:0.895
   Female               20/29                                62/99                                     25/67    87/166    1.00   0.908 (0.47-1.74); *p*:0.772; *p*~h~: 0.399   0.541 (0.26-1.13); *p*:0.098; *p*~h~:0.352   0.760(0.41-1.42); *p*:0.389; *p*~h~:0.927    1.72 (1.00-2.94); *p*:0.057^e^; *p*~h~:0.895
  Age                                                                                                                                                                                                                                                                    
   \<60                 14/34                                48/69                                     27/60    75/129    1.00   1.689 (0.82-3.48); *p*:0.153; *p*~h~:0.799    1.093 (0.51-2.36); *p*:0.821; *p*~h~:0.151   1.412 (0.71-2.79); *p*:0.322; *p*~h~:0.824   1.338 (0.77-2.33); *p*:0.302; *p*~h~:0.844
   ≥60                  55/80                                114/214                                   72/133   186/347   1.00   0.775 (0.51-1.17); *p*:0.224; *p*~h~: 0.799   0.787 (0.50-1.23); *p*:0.295; *p*~h~:0.151   0.780 (0.53-1.15); *p*:0.207; *p*~h~:0.824   1.062 (0.75-1.49); *p*:0.732; *p*~h~:0.844
  Smoking status                                                                                                                                                                                                                                                         
   Never                50/83                                120/208                                   65/135   185/343   1.00   0.958 (0.63-1.45); *p*:0.839; *p*~h~:0.214    0.799 (0.51-1.26); *p*:0.338; *p*~h~:0.097   0.895 (0.60-1.33); *p*:0.582; *p*~h~:0.200   1.213 (0.85-1.72); *p*:0.280; *p*~h~:0.191
   Ever                 19/31                                42/75                                     34/58    76/133    1.00   0.914 (0.46-1.81); *p*:0.796; *p*~h~:0.214    0.956 (0.47-1.95); *p*:0.902; *p*~h~:0.097   0.932 (0.49-1.76); *p*:0.829; *p*~h~:0.200   0.982 (0.58-1.66); *p*:0.945; *p*~h~:0.191
  Alcohol consumption                                                                                                                                                                                                                                                    
   Never                58/84                                126/220                                   73/145   199/365   1.00   0.829 (0.56-1.24); *p*:0.359; *p*~h~:0.176    0.729 (0.47-1.13); *p*:0.156; *p*~h~:0.060   0.790 (0.54-1.15); *p*:0.218; *p*~h~:0.150   1.202 (0.86-1.68); *p*:0.282; *p*~h~:0.145
   Ever                 11/30                                36/63                                     26/48    62/111    1.00   1.558 (0.69-3.48); *p*:0.277; *p*~h~:0.176    1.477 (0.64-3.42); *p*:0.361; *p*~h~:0.060   1.523 (0.71-3.25); *p*:0.274; *p*~h~:0.150   0.933 (0.52-1.69); *p*:0.818; *p*~h~:0.145

^a^ The genotyping was successful in 330 (100%) GCA cases and 603 (97.04%) controls for *VDR* rs11568820 A\>G;

^b^ Adjusted for age, sex, smoking status and alcohol consumption (besides stratified factors accordingly) in a logistic regression model;

^c^ *p*~h~ for heterogeneity; bold values are statistically significant (*p* \<0.05).

Linkage disequilibrium analyses {#s2_4}
-------------------------------

Linkage disequilibrium analyses in both controls and cases were conducted as shown in Table [8](#T8){ref-type="table"}. *D*' and *r*^2^ were analyzed and showed that there were weak correlations between the four loci.

###### Linkage disequilibrium analyses of *VDR* rs2228570, rs1989969, rs11568820 and rs2107301 in control and case groups

               Control   Case                            
  ------------ --------- ------- ------- ------- ------- -------
  *D*'                                                   
   rs2107301   0.289     0.224   0.136   0.202   0.105   0.015
   rs2228570   \-        0.213   0.069   \-      0.206   0.138
   rs1989969   \-        \-      0.249   \-      \-      0.366
  *r*^2^                                                 
   rs2107301   0.029     0.010   0.006   0.015   0.003   0.000
   rs2228570   \-        0.026   0.004   \-      0.029   0.019
   rs1989969   \-        \-      0.039   \-      \-      0.092

Haplotype analysis of VDR polymorphisms and susceptibility of GCA {#s2_5}
-----------------------------------------------------------------

As shown in Table [9](#T9){ref-type="table"}, haplotype analysis was also conducted and haplotypes were from the genotypes of *VDR* polymorphisms. The haplotype analysis indicated that the *VDR* T~rs2107301~C~rs2228570~C~rs1989969~G~rs11568820~ was the most common haplotype in both groups (17.27% in case group, 14.88% in control group). Compared with the haplotype T~rs2107301~C~rs2228570~C~rs1989969~G~rs11568820~, the haplotypes *VDR* T~rs2107301~T~rs2228570~C~rs1989969~G~rs11568820~ were more common in the controls (0.143) than in the case group (0.113) with significant difference (*p*=0.038). T~rs2107301~T~rs2228570~C~rs1989969~G~rs11568820~ haplotype was associated with a significantly reduced risk of GCA (OR=0.68, 95%CI=0.48-0.98, *p*=0.038). We also further conducted other haplotypes and risk of GCA, but no association was observed between patients and controls.

###### *VDR* haplotype frequencies (%) in cases and controls and risk of GCA

  Haplotypes                                                Cases (n = 660)   Controls (n = 1216)   Crude OR (95% CI)   *p*                        
  --------------------------------------------------------- ----------------- --------------------- ------------------- ------- ------------------ -------
  *VDR* T~rs2107301~C~rs2228570~C~rs1989969~G~rs11568820~   114               17.27                 181                 14.88   1.00               
  *VDR* T~rs2107301~T~rs2228570~C~rs1989969~G~rs11568820~   75                11.36                 174                 14.31   0.68 (0.48-0.98)   0.038
  *VDR* T~rs2107301~C~rs2228570~C~rs1989969~A~rs11568820~   54                8.18                  123                 10.12   0.70 (0.47-1.04)   0.075
  *VDR* T~rs2107301~T~rs2228570~T~rs1989969~A~rs11568820~   74                11.21                 114                 9.38    1.03 (0.71-1.50)   0.875
  *VDR* C~rs2107301~C~rs2228570~C~rs1989969~G~rs11568820~   59                8.94                  115                 9.46    0.82 (0.55-1.21)   0.305
  *VDR* T~rs2107301~T~rs2228570~C~rs1989969~A~rs11568820~   44                6.67                  86                  7.07    0.81 (0.53-1.25)   0.346
  *VDR* C~rs2107301~C~rs2228570~C~rs1989969~A~rs11568820~   31                4.70                  65                  5.35    0.76 (0.47-1.23)   0.264
  *VDR* T~rs2107301~C~rs2228570~T~rs1989969~G~rs11568820~   29                4.39                  63                  5.18    0.73 (0.44-1.20)   0.218
  *VDR* T~rs2107301~C~rs2228570~T~rs1989969~A~rs11568820~   36                5.45                  62                  5.10    0.92 (0.58-1.48)   0.736
  *VDR* T~rs2107301~T~rs2228570~T~rs1989969~G~rs11568820~   34                5.15                  58                  4.77    0.93 (0.57-1.51)   0.771
  *VDR* C~rs2107301~T~rs2228570~C~rs1989969~G~rs11568820~   27                4.09                  50                  4.11    0.86 (0.51-1.45)   0.565
  *VDR* C~rs2107301~C~rs2228570~T~rs1989969~G~rs11568820~   15                2.27                  31                  2.55    0.77 (0.40-1.49)   0.433
  *VDR* C~rs2107301~T~rs2228570~C~rs1989969~A~rs11568820~   20                3.03                  29                  2.38    1.10 (0.59-2.03)   0.773
  *VDR* C~rs2107301~T~rs2228570~T~rs1989969~A~rs11568820~   17                2.58                  25                  2.06    1.08 (0.56-2.09)   0.820
  *VDR* C~rs2107301~C~rs2228570~T~rs1989969~A~rs11568820~   24                3.64                  23                  1.89    1.66 (0.89-3.07)   0.109
  *VDR* C~rs2107301~T~rs2228570~T~rs1989969~G~rs11568820~   7                 1.06                  17                  1.40    0.65 (0.26-1.63)   0.361

With the order of *VDR* rs2107301 T\>C, rs2228570 C\>T, rs1989969 C\>T and rs11568820 G\>A in gene position.

DISCUSSION {#s3}
==========

In the current study, by multivariable logistic analysis, we demonstrated that there was no significant association between the polymorphisms of *VDR* rs11568820 G\>A, rs1989969 C\>T, rs2107301 T\>C andrs2228570 C\>T and the risk of GCA in Chinese population. Yet, notably, we detected an increased risk of GCA among alcohol drinking or younger patients (\<60 of age) who carried *VDR* rs1989969 C\>T genotype. Interestingly, the *VDR* T~rs2107301~T~rs2228570~C~rs1989969~G~rs11568820~ haplotype was associated with a significantly reduced risk of GCA.

Recently, accumulating evidence showed 1,25(OH)~2~D~3~, the hormonally active form of vitamin D, participates in apoptosis, cell proliferation and inflammation in cancer \[[@R5]\]. 1,25(OH)~2~D~3~ could restrain cancer cell growth by inducing their differentiation, by arresting cells in the G~0~/G~1~ phase of cell cycle or by induction of apoptotic cell death. Additionally, 1,25(OH)~2~D~3~ also has an impact on angiogenesis, thereby limiting the invasiveness of cancer cells \[[@R6]\]. As the key component of the vitamin D metabolism process, VDR similarly participates in the regulation of cancer development. SNPs of *VDR* gene have been shown correlated with cancers of the breast, prostate, colon \[[@R10]\], ovarian \[[@R14], [@R15]\], melanoma \[[@R16]\] and other malignancies \[[@R10], [@R17], [@R18]\]. In more than 200 specific *VDR*--SNPs, the most frequently associated with carcinogenesis are FokI (rs2228570), BsmI (rs1544410), TaqI (rs731236), ApaI (rs7975232) and Cdx2 (rs11568820) \[[@R10], [@R19], [@R20]\], yet the results are inconsistent. In contrast to the findings that *VDR* gene polymorphisms seem not related to the esophageal adenocarcinoma (EAC) risk development \[[@R21]\], we previously showed a significantly increased risk of esophageal squamous cell carcinoma associated with *VDR* rs2107301 T\>C polymorphism among patients who were drinking \[[@R13]\]. Therefore, in this study, we sought to verify our hypothesis that SNPs in *VDR* gene is related to GCA since it occurs in the anatomical vicinity of esophagus. Similarly, none of the four polymorphic sites was associated with the change of susceptibility to GCA, but a remarkable increased risk of GCA was found among alcohol drinking but younger patients (\<60 years of age) who carried *VDR* rs1989969 C\>T genotype.

Previous studies have shown the correlation of several SNPs with GCA (summarized in \[[@R22]\]). PLCE1 (rs2274223) A\>G SNP causes a missense variation in the protein phospholipase-Cε-1, which generates two critical messengers \[inositol 1,4,5-triphosphate (IP3) and diacylglycerol (DAG)\], thereby affecting cell growth, differentiation and gene expression \[[@R23]\]. Interestingly, vitamin D \[(1,25(OH)~2~D~3~\] -- VDR signaling pathway also stimulates the IP3 and DAG generation via phospholipase C \[[@R24]\], which may probably underlie its correlation with GCA. Other genetic variants significantly associated with the risk of GCA included PRKAA1 (rs13361707), IL1B (rs16944), TNF (rs1800629) and MDM2 (rs2279744) \[[@R22]\], involving the signal transduction, inflammation, apoptosis aspects. However, the connections between these SNPs with our findings remain obscure.

As compared with the homozygote for the common allele, men who were homozygote for the rare allele for *VDR* rs2107301 have higher risk of prostate cancer \[[@R25]\]. In contrast, *VDR* rs2107301 was not associated with GCA in either single locus analyses or the stratified analyses in the current study. Instead, we demonstrated that *VDR* rs1989969 C\>T polymorphism increased the risk of GCA among younger patients or alcohol drinkers, exemplifying the significance of the environment and genetic risk factors interact and both contribute to the carcinogenesis.

Our study showed the *VDR* T~rs2107301~T~rs2228570~C~rs1989969~G~rs11568820~ haplotype was associated with a significantly reduced risk of GCA, which indicated that polymorphism in single locus might not significantly modify the risk of cancer. The chain effect lying in different loci leads to a more profound impact which could regulate the risk of cancer.

Between ethnic groups, the frequencies of genetic polymorphisms do vary. In our study, the allele frequency of *VDR* rs1989969 was 0.323 in 608 control subjects, which is consistent with that in the Chinese Han (0.330) in the SNP Database, but lower than that of African (0.510) and Caucasian (0.410) population (<http://www.ncbi.nlm.nih.gov/SNP>).

Considering *VDR* rs1989969 C\>T mutant alleles in the control group, ORs, GCA samples and control samples, the power of our analysis (α= 0.05) was 0.999 in 330 GCA cases and 608 controls with an OR of 2.05 in age\<60 subgroup, and 0.983 with an OR of 1.78 in the drinking subgroup (PS, version 3.0, 2009, available at <http://biostat.mc.vanderbilt.edu/twiki/bin/view/Main/PowerSampleSize>).

We acknowledge that there are several limitations in the current study: First, restrained by the moderate sample size and lack of a validation cohort, the statistical power of our study was limited. Larger studies in multiple ethnical populations and various geographic locations are demanded to confirm the associations reported in our study. Second, the genetic effects of *VDR* polymorphisms on GCA susceptibility are probably caused by linkage disequilibrium (LD) with several functional variations within the *VDR* gene or with other closely linked genes. The SNPs we chose to study may not serve as a comprehensive representative of all the genetic variability of *VDR*, which entails further studies clarifying the genetic mechanism of GCA carcinogenesis by fine-mapping the susceptible region of the variants. Third, the study subjects recruited were from hospitals in the east part of China with same ethnicity, which may compromise its representativeness of the general population for potential inherited bias. Last but no least, the biological effects of VDR rs1989969 C\>T polymorphism on VDR function and the downstream signaling cascade remain unclear. Located on the second intron of VDR, rs1989969 may probably cause an alternative RNA splicing on VDR mRNA, thereby regulating the VDR protein function. Yet this speculation demands further investigations.

In conclusion, the GCA is associated with a variety of factors including gene, environment and life-style. Our findings that the increased risk of GCA was found among alcohol drinking and younger patients (\<60 years of age) who carried *VDR* rs1989969 C\>T genotype and the reduced risk of GCA for man with *VDR* T~rs2107301~T~rs2228570~C~rs1989969~G~rs11568820~ haplotype, should be interpreted with much caution. Further larger studies in multiple ethnical populations and various geographic locations are needed to verify our preliminary results.

MATERIALS AND METHODS {#s4}
=====================

Ethical approval of the study protocol {#s4_1}
--------------------------------------

This hospital-based case-control study was approved by the Review Board of Jiangsu University (Zhenjiang, China). We have complied with the World Medical Association Declaration of Helsinki regarding ethical conduct of research involving human subjects and/or animals. All subjects provided written informed consent to be included. Each participant agreed to donate 2ml of peripheral venous blood for the research project, which was performed by skilled nurses under strict aseptic condition to minimize potential risks on subjects' well being.

Study subjects {#s4_2}
--------------

The study included a total of 938 subjects. 330 patients with GCA were consecutively recruited from the Affiliated People\'s Hospital of Jiangsu University and Affiliated Hospital of Jiangsu University (Zhenjiang, China) between October 2010 and December 2012. The exclusion criteria were patients who previously had cancer, any metastasized cancer, radiotherapy or chemotherapy. The 608 controls were patients without cancer frequency-matched to the cases with regard to age (±5 years) and sex recruited from the two hospitals mentioned above during the same time period. Most of the controls were admitted to the hospitals for the treatment of trauma. Gender and age distribution had no significant difference between the case group and the control group, respectively.

Experienced and well-trained personnel interviewed each study subject with a pretested questionnaire. Demographic data and related risk factors were collected. 2mL samples of venous blood were collected from each subject with consent. Individuals who smoked one cigarette per day for ≥1 year were defined as "smokers". Subjects who consumed ≥3 alcoholic drinks a week for \>6 months were considered to be "alcohol drinkers".

Isolation of DNA and genotyping by ligation detection reaction {#s4_3}
--------------------------------------------------------------

Blood samples from patients and controls were collected using vacuum blood tube with Ethylene Diamine Tetraacetic Acid (EDTA). Genomic DNA was isolated from whole blood by using QIAamp DNA Blood Mini Kit (Qiagen, Berlin, Germany). Gene polymorphisms were analyzed by the ligation detection reaction (LDR) method with technical support from the Biowing Applied Biotechnology (Shanghai, China). 10% of the total samples were randomly selected to repeated analyses in order to maximize the probably error of the genotyping results and improve quality control.

Statistical analyses {#s4_4}
--------------------

Statistical analyses were performed using SPSS17.0 Statistical Package (2007, SPSS Inc., Chicago, IL). Hardy-Weinberg equilibrium for genotypes was tested by goodness-of-fit χ^2^ in control group. The distribution of *VDR* rs2107301 T\>C, rs2228570 C\>T, rs1989969 C\>T and rs11568820 G\>A genotypes was performed using the *chi*-square (χ^2^) test to examine statistical differences between patients and controls. The associations between these four SNPs and risk of GCA were estimated by computing the Odds ratios and confidence intervals (95%) using logistic regression analyses. Crude ORs and adjusted ORs when adjusting for age, sex, smoking and drinking status were also computed by using logistic regression analyses. Bilateral probability tests were taken, *p* value \<0.05 on behalf of the difference was statistically significant.

We thank Dr. Yiqun Chen (Biowing Applied Biotechnology, Shanghai, China) for technical support.

**CONFLICTS OF INTEREST**

The authors declare no conflicts of interest.

**GRANT SUPPORT**

This study was supported by the National Natural Science Foundation of China (81000028, 81370001, 81570031, 81300037, 81101889, 81472332, 81341006); the Key Research and Development Program of Jiangsu Province (BE2016714); the Natural Science Foundation of Jiangsu Province (BK2010333, BK2011481); the "333" Elitist Training Program, Jiangsu, China (BRA2013135); the "Six Talent Peaks" Training Program, Jiangsu, China (2014-WSN-078, 2015-WSN-117); the "Distinguished Medical Specialist" Program, Jiangsu, China; the "Innovative and Entrepreneurial Elite Team" Program (2016), Jiangsu, China; the research funding of Zhongshan Hospital (2016ZSLC15) and the Zhenjiang Social Development Program (SH2013039).

VDR

:   vitamin D receptor

GCA

:   gastric cardiac adenocarcinoma

LD

:   linkage disequilibrium

OR

:   odds ratio

CI

:   confidential interval

SNPs

:   single-nucleotide polymorphisms
