Abstract
Vitamin D has potent anticancer properties, especially against gastrointestinal cancers. Group-specific component (GC), a key member of vitamin D pathway proteins, could bind to and transport vitamin D to target organs. As a polymorphic protein, two common coding single nucleotide polymorphisms (SNP) [Glu416Asp (rs7041) and Thr420Lys (rs4588)] were identified in its gene. These SNPs have been associated to circulating vitamin D levels and several cancer risks in different populations. However, there is no report on their role in gastrointestinal cancer development among Chinese to date. Therefore, we examined the association between these variants and risk of gastrointestinal cancers in a case-control cohort including 964 patients with four gastrointestinal cancers (hepatocellular carcinoma, esophageal cancer, gastric cancer and colorectal cancer) and 1187 controls. Odds ratios and 95% confidence intervals were estimated by logistic regression. We found that GC Thr420Lys polymorphism has significant impact on the risk of developing gastrointestinal cancers, especially colorectal cancer. Additionally, subjects who carrying GC Asp416-Lys420 haplotype, which contains the at-risk 420Lys allele, also showed significantly increased risk to develop gastrointestinal cancers. In conclusion, our study demonstrated that common genetic variants and haplo-types in GC may influence individual susceptibility to gastrointestinal cancers in Chinese population.
Keywords: Group-specific component, single nucleotide polymorphism, gastrointestinal cancer, susceptibility
Introduction
Vitamin D has potent anticancer properties, especially against gastrointestinal cancers. Reduced risk for colorectal cancer (CRC) was firstly correlated to vitamin D levels in vivo in 1980 [1]. This inverse relationship between vitamin D intake or circulating 25-hydroxyvitamin D [25 (OH)D], a biomarker of vitamin D status and CRC was supported in both cohort studies [2-5] and case-control studies [6-8], although the association did not reach statistical significance in all reports. Similar inverse correlation was observed between vitamin D and esophageal cancer (EC) risk [5, 9]. Although inconsistent results on relationship between vitamin D and gastric cancer (GCa) were reported [5, 10], Ikezaki S et al did show that 24R.25-dihydroxyvitamin D3 (a vitamin D3 derivative) exerts chemopreventive effects on glandular stomach carcinogenesis in rats [11]. For hepatocellular carcinoma (HCC), there are few epidemiology studies on role of vitamin D in oncogenesis of liver. However, 1,25-dihydroxyvitamin D3 (another vitamin D3 derivative) exhibited significantly anti-HCC activities ex vivo and in vivo [12-14], indicating the potential protective role of vitamin D in HCC development.
As a key protein of vitamin D pathway, group-specific component (GC, also known as vitamin D-binding protein) could bind to vitamin D and its plasma metabolites and transport them to target tissues. In addition, GC can be converted into a macrophage activating factor (GcMAF) by the stepwise action of β-galactosidase of B cells and sialidase of T cells [15]. During inflammation, GcMAF could stimulate phagocytotic activity of macrophages [16]. Interestingly, GcMAF also shown antitumor activities in mice [17] and may be a potential immunotherapeutic reagent for metastatic breast cancer [18]. All these evidence suggest that GC may play an important part during carcinogenesis, alone or in a combination manner with vitamin D.
The GC gene, which coding human GC, is polymorphic. There are two common non-synonymous coding single nucleotide polymorphisms (SNP) [Glu416Asp (rs7041) and Thr420Lys (rs4588)] in this gene. Glu416Asp and Thr420Lys have been shown to alter plasma concentrations of 25(OH)D in candidate gene studies [19, 20]. Recently, genome wide association studies (GWAS) also shown that GC rs2282679, which is in strong linkage disequilibrium (LD) with Glu416Asp, was associated with serum 25(OH)D levels [21, 22]. In the previous reports, selected genetic variants in the GC gene, including Glu416Asp and Thr420Lys, have been investigated in breast cancer [23-25], prostate cancer [26], CRC [27] and basal cell carcinomas [28]. However, to date, there is no systematic evaluation on how common Glu416Asp and Thr420Lys SNPs are involved in development of gastrointestinal cancers in Chinese. Therefore, we specifically examined whether these two non-synonymous SNPs are associated with development of four gastrointestinal cancers (EC, HCC, GCa and CRC) in the current study. In addition, the role of GC haplotypes in development of gastrointestinal cancers was also evaluated.
Materials and methods
Study cohorts
This study consisted of 964 patients with gastrointestinal cancers (237 HCC cases, 289 EC cases, 192 GCa cases and 246 CRC cases) and 1187 healthy controls. HCC, EC, and GCa patients were consecutively recruited between January 2009 and July 2011, at Huaian No. 2 Hospital, Huaian, Jiangsu Province, China. CRC patients were recruited between January 2009 and October 2011, at Huaian No. 2 Hospital. Controls were cancer-free individuals selected from a community cancer-screening program for early detection of cancer conducted during the same time period as the patients were collected. Controls were matched to patients on age (± 5 years) and sex. All subjects were ethnic Han Chinese. At recruitment, informed consent was obtained from each subject and each participant was then interviewed to collect the information on demographic characteristics and lifetime history of tobacco use. This study was approved by the institutional Review Board of Huaian No. 2 Hospital.
Polymorphism genotyping
GC Asp416Glu and Thr420Lys genotypes were examined using PCR-based restriction fragment length polymorphism (RFLP). In PCR-RFLP genotyping, the primers used for amplifying DNA segments containing either GC Asp416Glu or Thr420Lys were 5'-AGA TCT GAA ATG GCT ATTAT TTT GCA-3'/5'-TTG CCA GTT CCG TGG GTG AG-3’ or 5'-TCT GAA ATG GCT ATT ATT TTG CAT T-3'/5'-TGT TAA CCA GCT TTG CCA GTT AC-3', respectively. The underline nucleotides are mismatch nucleotides introduced to create an Eco147I site or an HpyCH4IV site. Restriction enzyme Eco147I (Fermentas) or HpyCH4IV (New England Biolabs) was used to distinguish GC Asp416Glu or Thr420Lys genotypes, respectively. A 15% random sample was reciprocally tested by direct sequencing, and the reproducibility was 99.9%.
Statistics
Pearson's X2 test was used to examine the differences in demographic variables, smoking status and genotype distributions of GC Asp416Glu and Thr420Lys polymorphisms between patients and controls. Associations between genotypes and risk of the development of gastrointestinal cancers were estimated by odds ratios (ORs) and their 95% confidence intervals (95% CIs) computed using unconditional logistic regression model. Smokers were considered current smokers if they smoked up to 1 year before the date of cancer diagnosis or if they smoked up to 1 year before the date of the interview for control subjects. Subjects who never smoked or smoked less than 1 year before the date of cancer diagnosis for case patients or the date of interview for control subjects were defined as nonsmokers. All ORs were adjusted for age, sex, or smoking status, where it was appropriate. A P value of less than 0.05 was used as the criterion of statistical significance, and all statistical tests were two-sided. All abovementioned analyses were performed with SPSS software (version 16.0). Haplo.stats [29] was also used to assess the relative effects of GC haplotypes on gastrointestinal cancers and adjusted for sex, age, and smoking status.
Results
Subject characteristics
As shown in Table 1, there were no statistically significant differences in the distributions of sex, age and smoking status between patients and controls, suggesting that the frequency matching was adequate. However, smokers were over-represented in gastrointestinal cancer patients compared with controls (all P < 0.01).
Table 1.
Distribution of selected characteristics among gastrointestinal cancer patients and controls
| Hepatocellular carcinoma | Esophageal cancer | Gastric cancer | Colorectal cancer | |||||
|---|---|---|---|---|---|---|---|---|
| Variable | Cases/Control | Pa | Cases/Control | P* | Cases/Control | P* | Cases/Control | P* |
| 237/315 | 289/337 | 192/204 | 246/331 | |||||
| Age (year) | 0.702 | 0.897 | 0.209 | 0.377 | ||||
| <59 | 121/166 | 155/179 | 99/118 | 134/168 | ||||
| >59 | 116/149 | 134/158 | 93/86 | 112/163 | ||||
| Sex | 0.587 | 0.616 | 0.481 | 0.420 | ||||
| Male | 189/257 | 204/244 | 138/153 | 185/239 | ||||
| Female | 48/58 | 85/93 | 54/51 | 61/92 | ||||
| Smoking status | <0.001 | <0.001 | <0.001 | <0.001 | ||||
| No | 83/177 | 67/169 | 51/114 | 81/183 | ||||
| Yes | 154/138 | 222/168 | 141/90 | 165/148 | ||||
Two-sided x2 test
Genotype distributions of GC Asp416Glu and Thr420Lys polymorphisms
Genotyping results are shown in Table 2. The allele frequencies for GC 416Glu and 420Lys, respectively, were 0.281 and 0.301 in healthy controls as well as 0.283 and 0.336 in gastrointestinal cancer patients. All observed GC Asp416Glu and Thr420Lys genotype frequencies in both controls and patients conform to Hardy-Weinberg equilibrium (HWE) (PHWE > 0.05), except GC Asp416Glu in the GCa case-control cohort (PHWE = 0.04). Linkage disequilibrium analysis showed that these two SNPs are in strong linkage, with D’ = 1.00 and r2 = 0.18. Distributions of these GC Asp416Glu and Thr420Lys genotypes were then compared among patients and controls. Frequencies of 416Asp/Asp, Asp/Glu and Glu/Glu genotypes among all gastrointestinal cancer patients were not significantly different from those among controls (x2 = 0.500, P = 0.779; degrees of freedom = 2). Significant difference was observed for 420Thr/Thr, Thr/Lys and Lys/Lys genotypes between gastrointestinal cancer patients and controls (x2 = 10.38, P = 0.006; degrees of freedom = 2). In the stratified analyses, frequencies of GC 420Thr/Thr, Thr/Lys and Lys/Lys genotypes were significantly different among CRC patients and controls (x2 = 15.65, P = 0.0004; degrees of freedom = 2), with the Lys/Lys variant being more prevalent in patients than in controls (13.4% vs. 4.5%).
Table 2.
GC genotype frequencies among patients and controls and their association with risk of gastrointestinal cancers
| Cancer type | Asp416Glu(rs7041T/G) | Thr420Lys (rs4588 C/A) | ||||||
|---|---|---|---|---|---|---|---|---|
| Genotype | Cases/Controlsb | 0Ra (95% Cl) | P | Genotype | Cases/Controlsb | 0Ra (95% Cl) | P | |
| Hepatocellular carcinoma | 237/315 | 237/315 | ||||||
| Asp/Asp | 117/152 | Reference | Thr/Thr | 101/142 | Reference | |||
| Asp/Glu | 98/139 | 1.07 (0.75-1.53) | 0.697 | Thr/Lys | 111/148 | 1.05 (0.73-1.49) | 0.808 | |
| Glu/Glu | 22/24 | 0.88 (0.64-1.21) | 0.432 | Lys/Lys | 25/25 | 1.17 (0.86-1.60) | 0.310 | |
| Ptrendc | 0.920 | Ptrendc | 0.356 | |||||
| Esophageal cancer | 289/337 | 289/337 | ||||||
| 148 | 148/188 | Reference | Thr/Thr | 148/159 | Reference | |||
| 119 | 119/128 | 0.82 (0.57-1.16) | 0.260 | Thr/Lys | 108/144 | 0.78(0.55-1.12) | 0.181 | |
| 22 | 22/21 | 1.00 (0.69-1.44) | 0.997 | Lys/Lys | 33/34 | 0.96(0.72-1.28) | 0.769 | |
| Ptrendc | 0.232 | Ptrendc | 0.616 | |||||
| Gastric cancer | 192/204 | 192/204 | ||||||
| Asp/Asp | 98/99 | Reference | Thr/Thr | 74/88 | Reference | |||
| Asp/Glu | 86/89 | 1.11(0.71-1.73) | 0.663 | Thr/Lys | 89/92 | 1.24(0.78-1.99) | 0.367 | |
| Glu/Glu | 8/16 | 1.45 (0.89-2.36) | 0.133 | Lys/Lys | 29/24 | 1.25(0.89-1.75) | 0.200 | |
| Ptrendc | 0.310 | Ptrendc | 0.249 | |||||
| Colorectal cancer | 246/331 | 246/331 | ||||||
| Asp/Asp | 123/171 | Reference | Thr/Thr | 113/182 | Reference | |||
| Asp/Glu | 107/132 | 0.86 (0.65-1.24) | 0.472 | Thr/Lys | 100/134 | 1.35 (0.92-1.71) | 0.269 | |
| Glu/Glu | 16/28 | 1.28 (0.67-2.48) | 0.484 | Lys/Lys | 33/15 | 3.41 (1.85-6.57) | 1.01×10-4 | |
| Ptrendc | 0.956 | Ptrendc | 0.0009 | |||||
| Total | 964/1187 | 964/1187 | ||||||
| Asp/Asp | 486/610 | Reference | Thr/Thr | 436/571 | Reference | |||
| Asp/Glu | 410/488 | 0.97 (0.83-1.13) | 0.682 | Thr/Lys | 408/518 | 1.07 (0.92-1.25) | 0.383 | |
| Glu/Glu | 68/89 | 1.02 (0.88-1.17) | 0.841 | Lys/Lys | 120/98 | 1.15 (1.02-1.30) | 0.020 | |
| Ptrendc | 0.845 | Ptrendc | 0.013 | |||||
Data were calculated by unconditional logistic regression, adjusted for age, sex, and smoking status where it was appropriate. For all cancer together, the analyses were also adjusted for age, sex, and smoking status;
Number of subjects;
Tests for trend of odds were 2-sided and based on likelihood ratio tests assuming a multiplicative model.
Association between GC Asp416Glu and Thr420Lys polymorphisms and gastrointestinal cancer risks
No significant association between GC Asp416Glu or Thr420Lys SNPs and EC, HCC or GCa risk was observed (Ptreand of EC = 0.232 for Asp416Glu or 0.616 for Thr420Lys; Ptreand of HCC = 0.920 for Asp416Glu or 0.356 for Thr420Lys; Ptreand of GCa = 0.310 for Asp416Glu or 0.249 for Thr420Lys) (Table 2). However, there was a significant correlation between the GC Thr420Lys genetic variant and CRC risk (Ptreand = 0.0009) (Table 2). In detail, carriers of GC 420Lys/Lys genotype shown significantly elevated risk to develop CRC compared with 420Thr/Thr carriers (OR = 3.41, 95% CI = 1.85-6.57, P = 1.01×10-4). Although increased risk in carriers of 420Thr/Lys genotype was also found compared with 420Thr/Thr carriers, the association did not reach statistical significance (OR = 1.35, 95% CI = 0.92-1.71, P = 0.269). No such association existed between CRC and GC Asp416Glu SNP (Ptre and = 0.956) (Table 2). In the combined analysis of all gastrointestinal cancer patients and controls, we found that a 1.15-fold increased risk to develop gastrointestinal cancers in subjects carrying GC 420Lys/Lys genotype compared with 420Thr/ Thr carriers (95% CI = 1.02-1.30, P = 0.020) after adjustment of age, sex, and smoking status (Table 2). However, there was no significant association between GC Asp416Glu polymorphism and gastrointestinal cancer risk (Table 2). We also examined whether there are gene-smoking interactions, but the results were negative (data not shown).
GC Asp416Glu and Thr420Lys haplotypes and gastrointestinal cancer risks
Haplotype analyses shown that, compared with GC Asp416-Thr420 haplotype, a significantly increased gastrointestinal cancer risk was observed in carriers of Asp416-Lys420 haplotype (OR = 1.22, 95% CI = 1.04-1.39, P = 0.015). However, there was no significant association between GC GIu416-Thr420 haplotype and gastrointestinal cancer risk (OR = 1.11, 95% CI = 0.95-1.27, P = 0.273) (Table 3). Considering the haplotype frequency of Glu416-Lys420 was only 0.1% in cases or controls, we did not calculate its OR and 95% CI (Table 3). For CRC, compared with GC Asp416-Thr420 haplotype, a significantly increased gastrointestinal cancer risk was observed in carriers of Asp416-Lys420 haplotype (OR = 1.57, 95% CI = 1.19-2.17, P = 0.002) or Glu416-Thr420 haplotype and gastrointestinal cancer risk (OR = 1.43, 95% CI = 1.08 -1.94, P = 0.023) (Table 4).
Table 3.
Distribution of GC hapiotypes frequencies among patients and controls and their association with gastrointestinal cancers
| Haplotypes | No. of chromosomes (%) | ORa (95% Cl) | Pb | |
|---|---|---|---|---|
| Patients | Controls | |||
| Huaian cohort | ||||
| Asp416-Thr420 | 790 (41.0) | 900 (37.9) | Reference | |
| Glu416-Thr420 | 542 (28.1) | 669 (28.2) | 1.11 (0.95-1.27) | 0.273 |
| Asp416-Lys420 | 594 (30.8) | 802 (33.8) | 1.22 (1.04-1.39) | 0.015 |
| GIU416-Lys420 | 2 (0.1) | 2 (0.1) | NC | NC |
NC, not calculated.
Adjusted for sex, age, and smoking;
After 1000 permutation test.
Table 4.
Distribution of GC hapiotypes frequencies among patients and controls and their association with colorectal cancer
| Haplotypes | No. of chromosomes (%) | ORa (95% Cl) | Pb | |
|---|---|---|---|---|
| Patients | Controls | |||
| Huaian cohort | ||||
| Asp416-Thr420 | 225 (45.8) | 242 (36.5) | Reference | |
| Glu416-Thr420 | 130 (26.5) | 194 (29.3) | 1.43 (1.08-1.94) | 0.023 |
| Asp416-Lys420 | 136(27.7) | 226 (34.2) | 1.57 (1.19-2.17) | 0.002 |
| GIU416-Lys420 | 0 (0.0) | 0 (0.0) | NC | NC |
NC, not calculated.
Adjusted for sex, age, and smoking;
After 1000 permutation test.
Discussion
In this study, we examined whether GC Asp416Glu or Thr420Lys genetic polymorphisms are associated with risk of developing gastrointestinal cancers. On the basis of analysis of a Chinese Han case-control cohort (964 incident patients with gastrointestinal cancers and 1187 control subjects), we demonstrated that GC Thr420Lys polymorphism has significant impact on the risk of developing gastrointestinal cancers, especially on CRC oncogenesis. Additionally, subjects who carrying GC Asp416-Lys420 haplotype, which contains the GC at-risk 420Lys allele, also shown significantly increased risk to develop gastrointestinal cancers, especially CRC.
GC is a serum glycoprotein that belongs to the albumin family. As a key member in vitamin D metabolism pathway, GC can bind to 25(OH)D and other blood vitamin D sterol metabolites and transports them in circulation to target organs, including gastrointestinal organs. It was found that Glu416Asp and Thr420Lys could alter plasma concentrations of 25(OH)D [19, 20]. Sinotte M et al found that the plasma 25 (OH)D concentration of GC 420Lys/Lys carriers was significantly lower than that of 420Thr/Thr carriers (Crude mean ± SE = 59.0 ± 2.4 vs. 67.2 ± 1.0, P = 0.0016; adjusted mean ± SE = 58.4 ± 2.0 vs. 67.2 ± 0.9, P < 0.0001) [19]. Similar results were observed in the Insulin Resistance Atherosclerosis Family Study [20]. GC Thr420Lys was associated with levels of 25(OH) D and 1,25(OH)2D in both Hispanics and African Americans participants at all three study centers [20]. These results suggest that the increased risk of GC 420Lys/Lys carriers to develop gastrointestinal cancers (especially CRC) may be due to their low circulating 25(OH)D levels compared to 420Thr/Thr carriers, since clinically deficient 25(OH)D levels have been associated to increased cancer risk. In addition, the activation of the vitamin D receptor pathway was correlated to increased endocytotic uptakes of the GC-25(OH)D-complex to cancer cells, which in turn leads to anticarcinogenic action of vitamin D [30]. Therefore, it is biologically plausible that subjects carrying 420Lys/ Lys genotype or Asp416-Lys420 haplotype have a lower uptake ability of GC-25(OH)D complex from circulation and, thus, a increased gastrointestinal cancer risk.
In the previous reports, three common phenotypic alleles (haplotypes) have been identified in the GC protein (Gc1s: Glu416-Thr420, Gc1f: Asp416-Thr420, and Gc2: Asp416-Lys420) [31, 32]. These GC haplotypes have been associated with differences in serum 25(OH)D concentration and affinity of GC to vitamin D metabolites. Compared with Asp416-Lys420 haplotype, GC Glu416-Thr420 and Asp416-Thr420 haplotypes were associated with higher levels of serum 25 (0H)D concentration as well as higher affinity of GC to vitamin D metabolites [32-34]. Consistent with these previous findings, we also identified the same three common haplotypes in Chinese population and found that Asp416-Lys420 haplotype was associated with significantly increased gastrointestinal cancer risk. This observation is biologically plausible since the Asp416-Lys420 haplotype carrier may be in vitamin D deficient status and their gastrointestinal cells more easily undergo malignant transformation compared to carriers of these other two common haploptypes.
In summary, our study demonstrates that GC Thr420Lys genetic polymorphism and Asp416-Lys420 haplotype are associated with increased risk of gastrointestinal cancers in Chinese Han population. However, as a hospital-based case-control study, our study may have selection bias and/or systematic error because the cases were recruited from a hospital and the controls were recruited from the community. Therefore, independent studies are needed to validate our findings.
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