Polymorphisms of VDR gene and risk of gastric cardiac adenocarcinoma in Chinese population

Abstract

Vitamin D receptor (VDR) gene polymorphisms have been reported to increase susceptibility to some malignant tumors, yet the effect on gastric cardiac adenocarcinoma susceptibility remains unknown. Here, we conducted a hospital-based case-control study to examine the correlation of single nucleotide polymorphisms of VDR rs2107301T>C, rs2228570C>T, rs1989969C>T and rs11568820 G>A and gastric cardiac adenocarcinoma susceptibility. A total 330 cases and 608 controls were enrolled in the study. Using ligation detection reaction, we found that the variant alleles of the four polymorphisms were not associated with risk of gastric cardiac adenocarcinoma. Further stratified analyses showed that there was an increased risk associated with VDR rs1989969 polymorphism among patients who were drinking or aged <60. The haplotypes VDR T_rs2107301T_rs2228570C_rs1989969G_rs11568820 reduced the susceptibility. This study demonstrated that VDR rs1989969 polymorphism was involved in the carcinogenesis of gastric cardiac adenocarcinoma, especially increased the risk in the younger and alcohol drinking Chinese population.

INTRODUCTION

Gastric cardia adenocarcinoma (GCA) remains one of the most common malignant tumor worldwide [1]. Although the incidence of noncardia gastric cancer has declined steadily, the incidence and mortality of GCA are continuously increasing [2]. The etiology of GCA comprises interactions of multiple environmental and genetic factors. Environmental factors including cigarette smoking and alcohol consumption [3, 4], 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 D₃ [1,25(OH)₂D₃] is the hormonally active form of vitamin D, which has been shown to inhibit prostate, breast and colon cancer cell progression [5]. Consistently, amounting evidence has indicated the correlation of the Vitamin D Receptor (VDR) and cancer. The antineoplastic effect of 1,25(OH)₂D₃ requires the expression of VDR in tumor cells [6]. Increased tumor VDR expression is associated with a better prognosis in various types of cancer [7, 8]. 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 [6, 9–12].

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 [13]. 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

Characteristics of the study subjects

The characteristics of the study subjects, including demographics and environmental risk factors, are presented in Table 1. The cases and controls were well matched in gender and age (χ² 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).

Table 1: Distribution of selected demographic variables and risk factors in GCA cases and controls

Variable	Cases (n = 330)		Controls (n = 608)		pa
	n	%	n	%
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 χ² test and student t test; Bold values are statistically significant (p <0.05).

As shown in Table 2, 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). 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).

Table 2: 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 Scorea	5	5	No data	No data
TFBSb	—	—	—	Y
Splicing (ESE or ESS)	—	Y	—	—
nsSNP	—	Y	—	—
MAFc 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 HWEd test in our controls	0.690	0.347	0.718	0.574
Genotyping methode	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

As demonstrated in Table 3, 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).

Table 3: 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
	n	%	n	%
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

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 4–7, 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.

Table 4: 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 ORb (95% CI); p; phc
	CC	CT	TT	CT+TT	CC	CT	TT	CT+TT	TT vs. (CT+CC)
Sex
Male	92/189	98/170	28/46	126/216	1.00	1.19 (0.83-1.69); p: 0.345; ph:0.523	1.26 (0.74-2.14); p: 0.405; ph:0.575	1.20 (0.86-1.68); p: 0.283; ph:0.734	1.15 (0.70-1.91); p: 0.579; ph:0.412
Female	43/89	42/90	15/19	57/109	1.00	1.00 (0.59-1.70); p: 0.999; ph:0.523	1.67 (0.76-3.66); p: 0.199; ph:0.575	1.12 (0.68-1.84); p: 0.660; ph:0.734	1.67 (0.80-3.50); p: 0.172; ph:0.412
Age
<60	31/90	42/65	15/14	57/79	1.00	1.85 (1.03-3.33); p: 0.041; ph:0.031	2.97 (1.24-7.13); p: 0.015; ph:0.023	2.05 (1.18-3.57); p: 0.011; ph:0.010	2.19 (0.97-4.97); p: 0.060; ph:0.096
≥60	104/188	98/195	28/51	126/246	1.00	0.90 (0.64-1.27); p: 0.560; ph:0.031	0.99 (0.59-1.67); p: 0.977; ph:0.023	0.92 (0.67-1.27); p: 0.621; ph:0.010	1.04 (0.64-1.71); p: 0.865; ph:0.096
Smoking status
Never	88/197	87/189	25/48	112/237	1.00	1.01 (0.70-1.46); p: 0.962; ph:0.482	1.12 (0.64-1.97); p: 0.696; ph:0.345	1.03 (0.73-1.46); p: 0.863; ph:0.359	1.11 (0.65-1.90); p: 0.692; ph:0.451
Ever	47/81	53/71	18/17	71/88	1.00	1.24 (0.74-2.10); p: 0.415; ph:0.482	1.90 (0.87-4.15); p: 0.107; ph:0.345	1.37 (0.84-2.24); p: 0.213; ph:0.359	1.71 (0.82-3.56); p: 0.154; ph:0.451
Alcohol consumption
Never	99/205	93/206	30/47	123/253	1.00	0.90 (0.63-1.27); p: 0.538; ph:0.057	1.31 (0.77-2.23); p: 0.315; ph:0.835	0.97 (0.70-1.35); p: 0.865; ph:0.100	1.39 (0.84-2.28); p: 0.202; ph:0.647
Ever	36/73	47/54	13/18	60/72	1.00	1.90 (1.04-3.46); p: 0.036; ph:0.057	1.45 (0.60-3.50); p: 0.409; ph:0.835	1.78 (1.01-3.14); p: 0.045; ph:0.100	1.05 (0.46-2.40); p: 0.903; ph: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).

Table 5: Stratified analyses between VDR rs2228570 polymorphism and GCA risk by sex, age, smoking status and alcohol consumption

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

Table 6: Stratified analyses between VDR rs2107301 polymorphism and GCA risk by sex, age, smoking status and alcohol consumption

Variable	VDR rs2107301 (case/control)a				Adjusted ORb (95% CI); p; phc
	CC	CT	TT	CT+TT	CC	CT	TT	CT+TT	TT vs. (CT+CC)
Sex
Male	21/30	86/174	105/187	191/361	1.00	0.706 (0.38-1.31); p:0.266; ph:0.947	0.802 (0.437-1.47); p:0.476; ph:0.510	0.756 (0.421-1.36); p:0.756; ph:0.983	0.934 (0.669-1.31); p:0.669; ph:0.995
Female	11/19	43/72	50/98	93/170	1.00	1.032 (0.45-2.37); p:0.942; ph: 0.947	0.881 (0.39-1.99); p:0.762; ph: 0.510	0.945 (0.43-2.07); p:0.887; ph: 0.983	1.163 (0.72-1.88); p:0.536; ph:0.995
Age
<60	9/15	36/71	40/73	76/144	1.00	0.845 (0.34-2.12); P:0.719; ph: 0.607	0.913 (0.37-2.27); p:0.845; ph:0.784	0.880 (0.37-2.1); p:0.773; ph:0.735	0.955 (0.56-1.62); p:0.864; ph:0.746
≥60	23/34	93/175	115/212	208/387	1.00	0.786 (0.44-1.41); p:0.419; ph0.607	0.802 (0.45-1.43); p:0.452; ph: 0.784	0.795 (0.456-1.39); p:0.416; ph: 0.735	1.023 (0.74-1.41); p:0.889; ph:0.746
Smoking status
Never	24/36	94/176	104/204	198/380	1.00	0.801 (0.45-1.42); p:0.448; ph:0.753	0.765 (0.433-1.35); p:0.354; ph:0.248	1.279 (0.74-2.21); p:0.374; ph:0.216	1.092 (0.78-1.51); p:0.598; ph:0.205
Ever	8/13	35/70	51/81	86/151	1.00	0.813 (0.31-2.14); p:0.674; ph: 0.753	1.023 (0.397-2.64); p:0.962; ph: 0.248	0.925 (0.37-2.32); p:0.869; ph:0.216	0.823 (0.49-1.37); p:0.452; ph:0.205
Alcohol consumption
Never	25/42	103/189	116/208	219/397	1.00	0.916 (0.53-1.58); p:0.753; ph:0.959	0.937 (0.54-1.62); p:0.815; ph:0.204	0.927 (0.55-1.56); p:0.775; ph:0.159	0.994 (0.73-1.36); p:0.968; ph:0.147
Ever	7/7	26/57	39/77	65/134	1.00	0.456 (0.15-1.43); p:0.172; ph: 0.959	0.506 (0.166-1.55); p:0.226; ph: 0.204	0.485 (0.16-1.44); p:0.18; ph:0.159	1.018 (0.57-1.80); p:0.951; ph: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).

Table 7: Stratified analyses between VDR rs11568820 polymorphism and GCA risk by sex, age, smoking status and alcohol consumption

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

Linkage disequilibrium analyses in both controls and cases were conducted as shown in Table 8. D’ and r² were analyzed and showed that there were weak correlations between the four loci.

Table 8: Linkage disequilibrium analyses of VDR rs2228570, rs1989969, rs11568820 and rs2107301 in control and case groups

	Control			Case
	rs2228570	rs1989969	rs11568820	rs2228570	rs1989969	rs11568820
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
r2
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

As shown in Table 9, haplotype analysis was also conducted and haplotypes were from the genotypes of VDR polymorphisms. The haplotype analysis indicated that the VDR T_rs2107301C_rs2228570C_rs1989969G_rs11568820 was the most common haplotype in both groups (17.27% in case group, 14.88% in control group). Compared with the haplotype T_rs2107301C_rs2228570C_rs1989969G_rs11568820, the haplotypes VDR T_rs2107301T_rs2228570C_rs1989969G_rs11568820 were more common in the controls (0.143) than in the case group (0.113) with significant difference (p=0.038). T_rs2107301T_rs2228570C_rs1989969G_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.

Table 9: VDR haplotype frequencies (%) in cases and controls and risk of GCA

Haplotypes	Cases (n = 660)		Controls (n = 1216)		Crude OR (95% CI)	p
	n	%	n	%
VDR Trs2107301Crs2228570Crs1989969Grs11568820	114	17.27	181	14.88	1.00
VDR Trs2107301Trs2228570Crs1989969Grs11568820	75	11.36	174	14.31	0.68 (0.48-0.98)	0.038
VDR Trs2107301Crs2228570Crs1989969Ars11568820	54	8.18	123	10.12	0.70 (0.47-1.04)	0.075
VDR Trs2107301Trs2228570Trs1989969Ars11568820	74	11.21	114	9.38	1.03 (0.71-1.50)	0.875
VDR Crs2107301Crs2228570Crs1989969Grs11568820	59	8.94	115	9.46	0.82 (0.55-1.21)	0.305
VDR Trs2107301Trs2228570Crs1989969Ars11568820	44	6.67	86	7.07	0.81 (0.53-1.25)	0.346
VDR Crs2107301Crs2228570Crs1989969Ars11568820	31	4.70	65	5.35	0.76 (0.47-1.23)	0.264
VDR Trs2107301Crs2228570Trs1989969Grs11568820	29	4.39	63	5.18	0.73 (0.44-1.20)	0.218
VDR Trs2107301Crs2228570Trs1989969Ars11568820	36	5.45	62	5.10	0.92 (0.58-1.48)	0.736
VDR Trs2107301Trs2228570Trs1989969Grs11568820	34	5.15	58	4.77	0.93 (0.57-1.51)	0.771
VDR Crs2107301Trs2228570Crs1989969Grs11568820	27	4.09	50	4.11	0.86 (0.51-1.45)	0.565
VDR Crs2107301Crs2228570Trs1989969Grs11568820	15	2.27	31	2.55	0.77 (0.40-1.49)	0.433
VDR Crs2107301Trs2228570Crs1989969Ars11568820	20	3.03	29	2.38	1.10 (0.59-2.03)	0.773
VDR Crs2107301Trs2228570Trs1989969Ars11568820	17	2.58	25	2.06	1.08 (0.56-2.09)	0.820
VDR Crs2107301Crs2228570Trs1989969Ars11568820	24	3.64	23	1.89	1.66 (0.89-3.07)	0.109
VDR Crs2107301Trs2228570Trs1989969Grs11568820	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

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_rs2107301T_rs2228570C_rs1989969G_rs11568820 haplotype was associated with a significantly reduced risk of GCA.

Recently, accumulating evidence showed 1,25(OH)₂D₃, the hormonally active form of vitamin D, participates in apoptosis, cell proliferation and inflammation in cancer [5]. 1,25(OH)₂D₃ could restrain cancer cell growth by inducing their differentiation, by arresting cells in the G₀/G₁ phase of cell cycle or by induction of apoptotic cell death. Additionally, 1,25(OH)₂D₃ also has an impact on angiogenesis, thereby limiting the invasiveness of cancer cells [6]. 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 [10], ovarian [14, 15], melanoma [16] and other malignancies [10, 17, 18]. 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) [10, 19, 20], yet the results are inconsistent. In contrast to the findings that VDR gene polymorphisms seem not related to the esophageal adenocarcinoma (EAC) risk development [21], we previously showed a significantly increased risk of esophageal squamous cell carcinoma associated with VDR rs2107301 T>C polymorphism among patients who were drinking [13]. 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 [22]). 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 [23]. Interestingly, vitamin D [(1,25(OH)₂D₃] – VDR signaling pathway also stimulates the IP3 and DAG generation via phospholipase C [24], 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) [22], 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 [25]. 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_rs2107301T_rs2228570C_rs1989969G_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_rs2107301T_rs2228570C_rs1989969G_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

Ethical approval of the study protocol

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

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

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

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 χ² 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 (χ²) 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.

Abbreviations

VDR: vitamin D receptor; GCA: gastric cardiac adenocarcinoma; LD: linkage disequilibrium; OR: odds ratio; CI: confidential interval; SNPs: single-nucleotide polymorphisms.

ACKNOWLEDGMENTS

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).