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Cancer Biomarkers 15 (2015) 27–32 DOI 10.3233/CBM-140442 IOS Press
Genetic variants at 6p21.1 are associated with head and neck cancer in Chinese Han population Ruixia Wanga,1, Longbiao Zhua,c,1, Yu Zhanga, Limin Miaoa , Hongxia Mab,c , Hua Yuana,c,∗ and Ning Chena,∗ a
Jiangsu Key Laboratory of Oral Diseases, Nanjing Medical University, Nanjing, Jiangsu, China Department of Epidemiology and Biostatistics, MOE Key Laboratory of Modern Toxicology, School of Public Health, Nanjing Medical University, Nanjing, Jiangsu, China c Section of Clinical Epidemiology, Jiangsu Key Lab of Cancer Biomarkers, Prevention and Treatment, Cancer Centre, Nanjing Medical University, Nanjing, Jiangsu, China b
Abstract. BACKGROUND: A recent study synthesized several published genome-wide association studies (GWAS) on three types of cancers and identified variants at 6p21.1 and 7p15.3 as candidate susceptibility loci for multiple types of human cancers. However, the role of these loci in the development of head and neck cancer (HNC) is still unclear. METHODS: To evaluate the relationships between genetic variants in these regions and HNC risk, we genotyped two common SNPs rs2494938 at 6p21.1 and rs2285947 at 7p15.3 in a case-control study with a total of 503 HNC cases and 900 controls in Han Chinese. RESULTS: We found that rs2494938 at 6p21.1 was associated with a significantly increased risk of HNC in our population [AA vs. GG: adjusted odds ratio (OR) = 1.84, 95% confidence interval (CI) = 1.13–3.00, P = 0.014; AAvs.GA/GG: adjusted OR = 1.78, 95% CI = 1.10–2.87, P = 0.018]. However, no significant association was observed between rs2285947 at 7p15.3 and HNC risk. CONCLUSION: Our results suggest that genetic variants at 6p21.1 may play an important role in HNC development in Han Chinese, and rs2494938 may be a candidate marker for HNC susceptibility. Keywords: Variants, head and neck cancer, risk, susceptibility
1. Introduction Head and neck cancer (HNC), which includes cancers of oral cavity, oropharynx, hypopharynx and lar1 These
authors contributed equally to this work. authors: Ning Chen, Jiangsu Key Laboratory of Oral Diseases, Nanjing Medical University, Nanjing, Jiangsu, China. Tel.: +86 25 8503 1856; Fax: +86 25 8651 6414; E-mail: [email protected]; Hua Yuan, Jiangsu Key Laboratory of Oral Diseases, Nanjing Medical University, Nanjing, Jiangsu, China. Tel.: +86 25 8503 1875; Fax: +86 25 8503 1914; E-mail: rolener@gmail. com. ∗ Corresponding
ynx, is the sixth most common cancer in the world, with an incidence of approximately 500,000 cases per year [1]. Tobacco and alcohol use have been widely considered to be major risk factors of HNC [2,3]. However, only a small fraction of individuals with such risk factors ultimately develop HNC, suggesting that genetic susceptibility may play an important role in the development of HNC. Accumulative evidence has shown that genetic variants in important candidate pathways contribute to the risk of HNC, but the results from different studies were inconsistent and the exact mechanism is still unclear [4–7].
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R. Wang et al. / Genetic variants at 6p21.1 and head and neck cancer
In the past few years, genome-wide association studies (GWAS) has provided a powerful tool to investigate the genetic determinants of complex diseases and successfully identified hundreds of genetic markers related to the susceptibility to human cancer [8, 9]. Until now, most of the susceptibility loci are just associated with cancers, such as the regions of 8q24, 5p15.33 (TERT-CLPTM1L) and 9p21.3 (ANRIL) [10– 12]. Recently, Jin et al. utilized the existing GWAS data on lung cancer, noncardia gastric cancer (NCGC) and esophageal squamous-cell carcinoma (ESCC) to map pleiotropic loci contributing to these three types of cancer in Han Chinese populations, and identified 2 novel SNPs (rs2494938 at 6p21.1 and rs2285947 at 7p15.3) significantly associated with all three types of cancer [13]. Additionally, Li found that rs2285947 at 7p15.3 also contribute to the development of ovarian cancer in Han Chinese women [14]. These findings indicate rs2494938 at 6p21.1 located in the intron region of LRFN2 [MIM 612808] and rs2285947 at 7p15.3 located in the intron of DNAH11 [MIM 603339] are candidate susceptibility loci for multiple types of human cancer. However, it is unclear whether such evidence of pleiotropy is applicable for other human cancers, such as HNC. Therefore, to evaluate the role of these two genetic polymorphisms (rs2494938 at 6p21.1 and rs2285947 at 7p15.3) in HNC risk in Han Chinese and to investigate the pleiotropic of these two SNPs in head and neck cancer, we conducted a case-control study with 503 HNC cases and 900 healthy controls.
a pool of individuals participating in a communitybased screening program for non-infectious diseases conducted in Jiangsu Province. All participants were genetically unrelated, ethnic Han Chinese population. Each individual was interviewed by the trained interviewers to gather demographic information and environment exposure history, such as age, sex, smoking, and drinking consumption. Individuals who smoked one cigarette per day for over 1 year were considered as smokers and those who had three or more alcohol drinks a week for over 6 months were defined as alcohol drinkers. After the interview, approximately 5 ml of venous blood sample was collected from each study participant. Finally, 503 incident HNC cases and 900 frequency-matched controls were included in this study. 2.2. Genotyping
2. Materials and methods
Genomic DNA was extracted from a leukocyte pellet by proteinase K digestion and followed by phenol– chloroform extraction and ethanol precipitation. SNPs genotyping was performed using the TaqMan allelic discrimination assay on the platform of 7900HT Realtime PCR System (Applied Biosystems, Foster City, CA). The allelic discrimination results were shown in Supplemental Fig. 1. Two negative controls (no DNA) were included in each 384-well reaction plate and the genotyping results were determined by using SDS 2.3 Allelic Discrimination Software (Applied Biosystems). Moreover, 10% of samples (50 cases and 90 controls) were randomly selected to repeat and the accordance rate reached 100%.
2.1. Study populations
2.3. Statistical analysis
This case-control study was approved by the institutional review board of Nanjing Medical University. Participant recruitment was described previously. In brief, all patients with histopathologically confirmed HNC were consecutively recruited from Jiangsu Stomatological Hospital and the First Affiliated Hospital of Nanjing Medical University, Nanjing, China, since January 2009 to April 2012. There was no restriction of age, sex, histology or stage in patients [15]. The criteria for the recruitment of HNC patients included: (1) Han Chinese; (2) without previous malignant tumor in any other organs; (3) histopathologically confirmed diagnosis. Cancer free controls, frequency matched to cases on age (± 5 years) and sex, were recruited from
Hardy-Weinberg equilibrium of SNPs was evaluated using the goodness – of- fit χ2 test among the control subjects. Two-sided χ2 tests were used to evaluate differences in the distributions of demographic characteristics, selected variables and genotypes between the cases and controls. The associations of SNPs with HNC risks were estimated by computing the crude and adjusted odds ratios (ORs) and their 95% confidence intervals (CIs) using logistic regression analyses. All the statistical analyses were performed with the Statistical Analysis System software (v.9.1.3; SAS Institute, Cary, NC). Two-sided tests were generally used for statistical analysis and P < 0.05 was considered as the level of statistical significance.
R. Wang et al. / Genetic variants at 6p21.1 and head and neck cancer
29
Table 1 Primary information and minor allele frequencies (MAFs) of selected SNPs SNP rs2285947 rs2494938
Location 7p15.3 6p21.1
Base change G/A G/A
MAFa 0.267 0.233
MAFb 0.249 0.210
HWE 0.073 0.902
Genotyping rate (%) 96.67 97.44
MAF = minor allele frequency; HWE = Hardy-Weinberg equilibrium; CHB = Han Chinese in Beijing. a MAF in dbSNP database (CHB); b MAF in our controls. Table 2 Analysis of association between the SNPs and risk of HNC SNP rs2285947 GG GA AA Dominant model Recessive model Additive model rs2494938 GG GA AA Dominant model Recessive model Additive model
Cases
Adjusted OR(95%CI)a
Pa
57.47 35.17 7.36 42.53 7.36 −
1.00 1.20 (0.94–1.52) 1.01 (0.64–1.58) 1.16 (0.93–1.46) 0.94 (0.60–1.45) 1.09 (0.91–1.30)
0.141 0.980 0.191 0.764 0.361
62.34 33.30 4.34 37.64 4.34
1.00 1.10 (0.87–1.40) 1.84 (1.13–3.00) 1.18 (0.94–1.49) 1.78 (1.10–2.87) 1.22 (1.01–1.47)
0.431 0.014 0.147 0.018 0.036
Controls
N
%
N
%
262 199 34 233 34 −
52.93 40.20 6.87 47.07 6.87 −
500 306 64 370 64 −
291 173 36 209 36 −
58.20 34.60 7.20 41.80 7.20 −
547 292 38 330 38
CI = confidence interval; OR = odds ratio. a Adjusted by age, sex, smoking status and alcohol status. Significant values (P < 0.05) are in bold.
3. Results Characteristics of the 503 HNC cases and 900 controls are shown previously [15]. No significant difference was found in the distributions of age (P = 0.906), sex (P = 0.207) and smoking status (P = 0.191). Among all HNC cases, 382 (75.9%) had primary tumors of the oral cavity, 9 (1.8%) of the oropharynx, 97 (19.3%) of the larynx and 15 (3.0%) of the others. In addition, 445(88.5%) cases were classified as squamous cell carcinoma while 58 (11.5%) were considered as other histological types, including undifferentiated carcinoma, adenocarcinoma and undetermined cancer. No significant differences were detected on age, sex and smoking status between the cases and controls (P = 0.906, 0.207 and 0.191, respectively). However, cases were more likely to be drinkers than controls (46.7 vs. 32.3%, P < 0.001). The position, minor allele frequency (MAF), and the genotyping rate of the two selected SNPs are presented in Table 1. The observed genotype frequencies for these two SNPs were all in Hardy–Weinberg equilibrium in the controls (P = 0.073 and 0.902 for rs2285947 and rs2494938, respectively), and calling rates were all more than 95.0%. The genotype frequencies of the two SNPs in the cases and the controls are summarized in Table 2. With adjustment for
age, sex, smoking, and alcohol status, multivariate logistic regression analysis revealed that subjects carrying rs2494938 AA genotype had a significantly increased risk of HNC (adjusted OR = 1.84, 95% CI = 1.13–3.00, P = 0.014), compared with those with the rs2494938 GG genotype. Moreover, in the recessive and additive genetic models, we also found similar associations (recessive model: adjusted OR = 1.78, 95% CI = 1.10–2.87, P = 0.018; additive model: adjusted OR = 1.22, 95% CI = 1.01–1.47, P = 0.036). However, no significant association was observed between rs2285947 genotypes and risk of HNC (Table 2). Further, stratification analyses by age, sex, smoking status, drinking status, tumor sites, and histology were conducted to evaluate the potential association of genetic variants of rs2494938 with risk HNC (Table 3). Although the protective effect of rs2494938 AA genotypes were statistically significant in some groups, heterogeneity test showed that there was no significant heterogeneity (P > 0.05) between every two strata, suggesting no risk effect modification by the variables under investigation. 4. Discussion In this study, we evaluated the associations of genetic variants at 6p21.1 (rs2494938 G>A) and 7p15.3
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R. Wang et al. / Genetic variants at 6p21.1 and head and neck cancer Table 3 Stratified analysis for associations between variant genotypes of rs2494938 and HNC risk Variables Age, yr 60 > 60 Sex Females Males Smoking status Never Ever Drinking status Never Ever Tumor site Oral cavity Histology Squamous cell carcinoma
Cases (n = 500) GG/AG AA
Control (n = 877) GG/AG AA
Adjusted OR (95%CI)a
P for heterogeneity
237 227
17 19
438 401
19 19
1.64 (0.82–3.27) 1.84 (0.95–3.60)
0.814
155 309
16 20
266 573
11 27
2.62 (1.18–5.80) 1.43 (0.78–2.64)
0.237
231 231
25 11
467 372
22 16
2.30 (1.26–4.18) 1.19 (0.52–2.76)
0.209
240 222
24 12
566 273
24 14
2.32 (1.28–4.22) 1.20 (0.54–2.70)
0.197
349
30
839
38
1.98 (1.18–3.30)
0.223
413
30
839
38
1.67 (1.01–2.76)
0.668
CI = confidence interval; OR = odds ratio. a Adjusted by age, sex, smoking status and alcohol status. Significant values (P < 0.05) are in bold.
(rs2285947 G>A) with HNC susceptibility in a case– control study with 503 HNC cases and 900 controls in Han Chinese populations. We found that rs2494938 at 6p21.1 was significantly associated with HNC risk in our population, suggesting the pleiotropy of this SNP in the susceptibility to HNC. In stratification analyses, the associations between rs2285947 and HNC risk was significant in the subgroup with female (dominant model: OR = 2.62, 95%CI = 1.18–5.80), with nonsmokers (dominant model: OR = 2.30, 95%CI = 1.26– 4.18), and non-drinkers (dominant model: OR = 2.32, 95%CI = 1.28–4.22). However, no significant heterogeneity was detected between the stratified subgroups. The positive SNP rs2494938 at 6p21.1 is in the intron region of LRFN2 (encoding leucine-rich repeat and fibronectin type III domain-containing protein 2), a member of synaptic adhesion-like molecules (SALMs) family that interacts with the N-methyl D-aspartate (NMDA) receptor NR1 subunit through its extracellular or TM1 domains [16]. And SNPs in strong LD with rs2494938 at 6p21.1 are also located in the initial region (including promoter, exon 1, and intron 1) of LRFN2. Recent studies have found that LRFN2 is able to subvert hematopoietic differentiation to increase erythropoiesis, and may also be involved in colony forming units-fibroblast regulation [17]. NMDA receptors are glutamate receptors, consisting of NR1 and NR2 (2A to 2D) subunits [18,19]. Interestingly, it has been reported that the NR1 expression level was significantly associated with the lymph node metastasis, tumor size and cancer stage of oral squamous cell carcinoma [20]. Our study revealed the association between rs2494938 and HNC risk, which was con-
sistent with the findings in other three cancer types by Jin et al. [13]. So far, the evidence indicated that LRFN2 might work as a susceptibility gene by means of LRFN2-NMDA receptor pathway for multiple cancers, but the exact mechanism of such loci in the development of HNC still needs further investigation. The other SNP, rs2285947 at 7p15.3, was also reported to be associated with multiple cancers risk in the study by Jin et al. [13]. The SNP rs2285947, localized to 7p15.3, is a G→A change located in the intron of DNAH11 (Dynein, axonemal, heavy chain 11). Dyneins are composed of several heavy, light, and intermediate chains [21]. Studies have shown that dyneins are specifically required for the activation of MKK3/6 and p38 MAPKs [22]. The p38 MARK pathway plays an additional role in the induction of the immune and inflammatory responses [23,24] as well as in the regulation of cell differentiation, migration and survival [25,26]. Li found that rs2285947 was associated with increasing ovarian cancer risk in Han Chinese women and this result was consistent with Jin [14]. However, in this case-control study, we failed to observe any significant association of this SNP with risk of HNC or subgroup of HNC, suggesting rs2285947 may not contribute to the risk of HNC, but we could not completely exclude the possibility that the association between 7p15.3 variants and HNC risk and our analysis did not have enough statistical power to detect it. Large studies are warranted to clarify the associations of 7p15.3 with HNC risk. In the present study, several limitations need to be addressed. First of all, the statistical power was relatively low due to the small sample size, especially
R. Wang et al. / Genetic variants at 6p21.1 and head and neck cancer
in stratification analysis. Secondly, we recruited HNC cases from hospitals and selected controls from communities, which might result in potential selection bias. However, we applied a rigorous epidemiological design in selecting study subjects and used further statistical adjustment for known risk factors to minimize potential biases. Third, the biological mechanism of genetic variants at 6p21.1 was not further investigated in this study. In summary, this study indicated that rs2494938 at 6p21.1 had significant associations with HNC risk in Han Chinese, consistent with the findings in lung cancer, non-cardia gastric cancer, and esophageal squamous-cell carcinoma by Jin et al., which further supported the pleiotropy of this SNP in multiple cancers. Further larger epidemiological studies and functional characterizations are needed to confirm these findings and identify the specific mechanism of such loci in the development of human cancer.
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Acknowledgements National Natural Science Foundation of China (813 02361), A Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD, 2014-37), Jiangsu Natural Science Foundation (BK2011764), Specialized Research Fund for the Doctoral Program of Higher Education (Grant No. 20133234120013)
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Supplemental material
Supplemental Fig. 1. Allelic Discrimination plots for rs2494938 and rs2285947 assays. Allele X(1) is shown in red and allele Y(2) is shown in blue. Line green represents a mixture of the two alleles.
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Cancer Biomarkers 15 (2015) 27–32 DOI 10.3233/CBM-140442 IOS Press
Genetic variants at 6p21.1 are associated with head and neck cancer in Chinese Han population Ruixia Wanga,1, Longbiao Zhua,c,1, Yu Zhanga, Limin Miaoa , Hongxia Mab,c , Hua Yuana,c,∗ and Ning Chena,∗ a
Jiangsu Key Laboratory of Oral Diseases, Nanjing Medical University, Nanjing, Jiangsu, China Department of Epidemiology and Biostatistics, MOE Key Laboratory of Modern Toxicology, School of Public Health, Nanjing Medical University, Nanjing, Jiangsu, China c Section of Clinical Epidemiology, Jiangsu Key Lab of Cancer Biomarkers, Prevention and Treatment, Cancer Centre, Nanjing Medical University, Nanjing, Jiangsu, China b
Abstract. BACKGROUND: A recent study synthesized several published genome-wide association studies (GWAS) on three types of cancers and identified variants at 6p21.1 and 7p15.3 as candidate susceptibility loci for multiple types of human cancers. However, the role of these loci in the development of head and neck cancer (HNC) is still unclear. METHODS: To evaluate the relationships between genetic variants in these regions and HNC risk, we genotyped two common SNPs rs2494938 at 6p21.1 and rs2285947 at 7p15.3 in a case-control study with a total of 503 HNC cases and 900 controls in Han Chinese. RESULTS: We found that rs2494938 at 6p21.1 was associated with a significantly increased risk of HNC in our population [AA vs. GG: adjusted odds ratio (OR) = 1.84, 95% confidence interval (CI) = 1.13–3.00, P = 0.014; AAvs.GA/GG: adjusted OR = 1.78, 95% CI = 1.10–2.87, P = 0.018]. However, no significant association was observed between rs2285947 at 7p15.3 and HNC risk. CONCLUSION: Our results suggest that genetic variants at 6p21.1 may play an important role in HNC development in Han Chinese, and rs2494938 may be a candidate marker for HNC susceptibility. Keywords: Variants, head and neck cancer, risk, susceptibility
1. Introduction Head and neck cancer (HNC), which includes cancers of oral cavity, oropharynx, hypopharynx and lar1 These
authors contributed equally to this work. authors: Ning Chen, Jiangsu Key Laboratory of Oral Diseases, Nanjing Medical University, Nanjing, Jiangsu, China. Tel.: +86 25 8503 1856; Fax: +86 25 8651 6414; E-mail: [email protected]; Hua Yuan, Jiangsu Key Laboratory of Oral Diseases, Nanjing Medical University, Nanjing, Jiangsu, China. Tel.: +86 25 8503 1875; Fax: +86 25 8503 1914; E-mail: rolener@gmail. com. ∗ Corresponding
ynx, is the sixth most common cancer in the world, with an incidence of approximately 500,000 cases per year [1]. Tobacco and alcohol use have been widely considered to be major risk factors of HNC [2,3]. However, only a small fraction of individuals with such risk factors ultimately develop HNC, suggesting that genetic susceptibility may play an important role in the development of HNC. Accumulative evidence has shown that genetic variants in important candidate pathways contribute to the risk of HNC, but the results from different studies were inconsistent and the exact mechanism is still unclear [4–7].
c 2015 – IOS Press and the authors. All rights reserved ISSN 1574-0153/15/$27.50
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R. Wang et al. / Genetic variants at 6p21.1 and head and neck cancer
In the past few years, genome-wide association studies (GWAS) has provided a powerful tool to investigate the genetic determinants of complex diseases and successfully identified hundreds of genetic markers related to the susceptibility to human cancer [8, 9]. Until now, most of the susceptibility loci are just associated with cancers, such as the regions of 8q24, 5p15.33 (TERT-CLPTM1L) and 9p21.3 (ANRIL) [10– 12]. Recently, Jin et al. utilized the existing GWAS data on lung cancer, noncardia gastric cancer (NCGC) and esophageal squamous-cell carcinoma (ESCC) to map pleiotropic loci contributing to these three types of cancer in Han Chinese populations, and identified 2 novel SNPs (rs2494938 at 6p21.1 and rs2285947 at 7p15.3) significantly associated with all three types of cancer [13]. Additionally, Li found that rs2285947 at 7p15.3 also contribute to the development of ovarian cancer in Han Chinese women [14]. These findings indicate rs2494938 at 6p21.1 located in the intron region of LRFN2 [MIM 612808] and rs2285947 at 7p15.3 located in the intron of DNAH11 [MIM 603339] are candidate susceptibility loci for multiple types of human cancer. However, it is unclear whether such evidence of pleiotropy is applicable for other human cancers, such as HNC. Therefore, to evaluate the role of these two genetic polymorphisms (rs2494938 at 6p21.1 and rs2285947 at 7p15.3) in HNC risk in Han Chinese and to investigate the pleiotropic of these two SNPs in head and neck cancer, we conducted a case-control study with 503 HNC cases and 900 healthy controls.
a pool of individuals participating in a communitybased screening program for non-infectious diseases conducted in Jiangsu Province. All participants were genetically unrelated, ethnic Han Chinese population. Each individual was interviewed by the trained interviewers to gather demographic information and environment exposure history, such as age, sex, smoking, and drinking consumption. Individuals who smoked one cigarette per day for over 1 year were considered as smokers and those who had three or more alcohol drinks a week for over 6 months were defined as alcohol drinkers. After the interview, approximately 5 ml of venous blood sample was collected from each study participant. Finally, 503 incident HNC cases and 900 frequency-matched controls were included in this study. 2.2. Genotyping
2. Materials and methods
Genomic DNA was extracted from a leukocyte pellet by proteinase K digestion and followed by phenol– chloroform extraction and ethanol precipitation. SNPs genotyping was performed using the TaqMan allelic discrimination assay on the platform of 7900HT Realtime PCR System (Applied Biosystems, Foster City, CA). The allelic discrimination results were shown in Supplemental Fig. 1. Two negative controls (no DNA) were included in each 384-well reaction plate and the genotyping results were determined by using SDS 2.3 Allelic Discrimination Software (Applied Biosystems). Moreover, 10% of samples (50 cases and 90 controls) were randomly selected to repeat and the accordance rate reached 100%.
2.1. Study populations
2.3. Statistical analysis
This case-control study was approved by the institutional review board of Nanjing Medical University. Participant recruitment was described previously. In brief, all patients with histopathologically confirmed HNC were consecutively recruited from Jiangsu Stomatological Hospital and the First Affiliated Hospital of Nanjing Medical University, Nanjing, China, since January 2009 to April 2012. There was no restriction of age, sex, histology or stage in patients [15]. The criteria for the recruitment of HNC patients included: (1) Han Chinese; (2) without previous malignant tumor in any other organs; (3) histopathologically confirmed diagnosis. Cancer free controls, frequency matched to cases on age (± 5 years) and sex, were recruited from
Hardy-Weinberg equilibrium of SNPs was evaluated using the goodness – of- fit χ2 test among the control subjects. Two-sided χ2 tests were used to evaluate differences in the distributions of demographic characteristics, selected variables and genotypes between the cases and controls. The associations of SNPs with HNC risks were estimated by computing the crude and adjusted odds ratios (ORs) and their 95% confidence intervals (CIs) using logistic regression analyses. All the statistical analyses were performed with the Statistical Analysis System software (v.9.1.3; SAS Institute, Cary, NC). Two-sided tests were generally used for statistical analysis and P < 0.05 was considered as the level of statistical significance.
R. Wang et al. / Genetic variants at 6p21.1 and head and neck cancer
29
Table 1 Primary information and minor allele frequencies (MAFs) of selected SNPs SNP rs2285947 rs2494938
Location 7p15.3 6p21.1
Base change G/A G/A
MAFa 0.267 0.233
MAFb 0.249 0.210
HWE 0.073 0.902
Genotyping rate (%) 96.67 97.44
MAF = minor allele frequency; HWE = Hardy-Weinberg equilibrium; CHB = Han Chinese in Beijing. a MAF in dbSNP database (CHB); b MAF in our controls. Table 2 Analysis of association between the SNPs and risk of HNC SNP rs2285947 GG GA AA Dominant model Recessive model Additive model rs2494938 GG GA AA Dominant model Recessive model Additive model
Cases
Adjusted OR(95%CI)a
Pa
57.47 35.17 7.36 42.53 7.36 −
1.00 1.20 (0.94–1.52) 1.01 (0.64–1.58) 1.16 (0.93–1.46) 0.94 (0.60–1.45) 1.09 (0.91–1.30)
0.141 0.980 0.191 0.764 0.361
62.34 33.30 4.34 37.64 4.34
1.00 1.10 (0.87–1.40) 1.84 (1.13–3.00) 1.18 (0.94–1.49) 1.78 (1.10–2.87) 1.22 (1.01–1.47)
0.431 0.014 0.147 0.018 0.036
Controls
N
%
N
%
262 199 34 233 34 −
52.93 40.20 6.87 47.07 6.87 −
500 306 64 370 64 −
291 173 36 209 36 −
58.20 34.60 7.20 41.80 7.20 −
547 292 38 330 38
CI = confidence interval; OR = odds ratio. a Adjusted by age, sex, smoking status and alcohol status. Significant values (P < 0.05) are in bold.
3. Results Characteristics of the 503 HNC cases and 900 controls are shown previously [15]. No significant difference was found in the distributions of age (P = 0.906), sex (P = 0.207) and smoking status (P = 0.191). Among all HNC cases, 382 (75.9%) had primary tumors of the oral cavity, 9 (1.8%) of the oropharynx, 97 (19.3%) of the larynx and 15 (3.0%) of the others. In addition, 445(88.5%) cases were classified as squamous cell carcinoma while 58 (11.5%) were considered as other histological types, including undifferentiated carcinoma, adenocarcinoma and undetermined cancer. No significant differences were detected on age, sex and smoking status between the cases and controls (P = 0.906, 0.207 and 0.191, respectively). However, cases were more likely to be drinkers than controls (46.7 vs. 32.3%, P < 0.001). The position, minor allele frequency (MAF), and the genotyping rate of the two selected SNPs are presented in Table 1. The observed genotype frequencies for these two SNPs were all in Hardy–Weinberg equilibrium in the controls (P = 0.073 and 0.902 for rs2285947 and rs2494938, respectively), and calling rates were all more than 95.0%. The genotype frequencies of the two SNPs in the cases and the controls are summarized in Table 2. With adjustment for
age, sex, smoking, and alcohol status, multivariate logistic regression analysis revealed that subjects carrying rs2494938 AA genotype had a significantly increased risk of HNC (adjusted OR = 1.84, 95% CI = 1.13–3.00, P = 0.014), compared with those with the rs2494938 GG genotype. Moreover, in the recessive and additive genetic models, we also found similar associations (recessive model: adjusted OR = 1.78, 95% CI = 1.10–2.87, P = 0.018; additive model: adjusted OR = 1.22, 95% CI = 1.01–1.47, P = 0.036). However, no significant association was observed between rs2285947 genotypes and risk of HNC (Table 2). Further, stratification analyses by age, sex, smoking status, drinking status, tumor sites, and histology were conducted to evaluate the potential association of genetic variants of rs2494938 with risk HNC (Table 3). Although the protective effect of rs2494938 AA genotypes were statistically significant in some groups, heterogeneity test showed that there was no significant heterogeneity (P > 0.05) between every two strata, suggesting no risk effect modification by the variables under investigation. 4. Discussion In this study, we evaluated the associations of genetic variants at 6p21.1 (rs2494938 G>A) and 7p15.3
30
R. Wang et al. / Genetic variants at 6p21.1 and head and neck cancer Table 3 Stratified analysis for associations between variant genotypes of rs2494938 and HNC risk Variables Age, yr 60 > 60 Sex Females Males Smoking status Never Ever Drinking status Never Ever Tumor site Oral cavity Histology Squamous cell carcinoma
Cases (n = 500) GG/AG AA
Control (n = 877) GG/AG AA
Adjusted OR (95%CI)a
P for heterogeneity
237 227
17 19
438 401
19 19
1.64 (0.82–3.27) 1.84 (0.95–3.60)
0.814
155 309
16 20
266 573
11 27
2.62 (1.18–5.80) 1.43 (0.78–2.64)
0.237
231 231
25 11
467 372
22 16
2.30 (1.26–4.18) 1.19 (0.52–2.76)
0.209
240 222
24 12
566 273
24 14
2.32 (1.28–4.22) 1.20 (0.54–2.70)
0.197
349
30
839
38
1.98 (1.18–3.30)
0.223
413
30
839
38
1.67 (1.01–2.76)
0.668
CI = confidence interval; OR = odds ratio. a Adjusted by age, sex, smoking status and alcohol status. Significant values (P < 0.05) are in bold.
(rs2285947 G>A) with HNC susceptibility in a case– control study with 503 HNC cases and 900 controls in Han Chinese populations. We found that rs2494938 at 6p21.1 was significantly associated with HNC risk in our population, suggesting the pleiotropy of this SNP in the susceptibility to HNC. In stratification analyses, the associations between rs2285947 and HNC risk was significant in the subgroup with female (dominant model: OR = 2.62, 95%CI = 1.18–5.80), with nonsmokers (dominant model: OR = 2.30, 95%CI = 1.26– 4.18), and non-drinkers (dominant model: OR = 2.32, 95%CI = 1.28–4.22). However, no significant heterogeneity was detected between the stratified subgroups. The positive SNP rs2494938 at 6p21.1 is in the intron region of LRFN2 (encoding leucine-rich repeat and fibronectin type III domain-containing protein 2), a member of synaptic adhesion-like molecules (SALMs) family that interacts with the N-methyl D-aspartate (NMDA) receptor NR1 subunit through its extracellular or TM1 domains [16]. And SNPs in strong LD with rs2494938 at 6p21.1 are also located in the initial region (including promoter, exon 1, and intron 1) of LRFN2. Recent studies have found that LRFN2 is able to subvert hematopoietic differentiation to increase erythropoiesis, and may also be involved in colony forming units-fibroblast regulation [17]. NMDA receptors are glutamate receptors, consisting of NR1 and NR2 (2A to 2D) subunits [18,19]. Interestingly, it has been reported that the NR1 expression level was significantly associated with the lymph node metastasis, tumor size and cancer stage of oral squamous cell carcinoma [20]. Our study revealed the association between rs2494938 and HNC risk, which was con-
sistent with the findings in other three cancer types by Jin et al. [13]. So far, the evidence indicated that LRFN2 might work as a susceptibility gene by means of LRFN2-NMDA receptor pathway for multiple cancers, but the exact mechanism of such loci in the development of HNC still needs further investigation. The other SNP, rs2285947 at 7p15.3, was also reported to be associated with multiple cancers risk in the study by Jin et al. [13]. The SNP rs2285947, localized to 7p15.3, is a G→A change located in the intron of DNAH11 (Dynein, axonemal, heavy chain 11). Dyneins are composed of several heavy, light, and intermediate chains [21]. Studies have shown that dyneins are specifically required for the activation of MKK3/6 and p38 MAPKs [22]. The p38 MARK pathway plays an additional role in the induction of the immune and inflammatory responses [23,24] as well as in the regulation of cell differentiation, migration and survival [25,26]. Li found that rs2285947 was associated with increasing ovarian cancer risk in Han Chinese women and this result was consistent with Jin [14]. However, in this case-control study, we failed to observe any significant association of this SNP with risk of HNC or subgroup of HNC, suggesting rs2285947 may not contribute to the risk of HNC, but we could not completely exclude the possibility that the association between 7p15.3 variants and HNC risk and our analysis did not have enough statistical power to detect it. Large studies are warranted to clarify the associations of 7p15.3 with HNC risk. In the present study, several limitations need to be addressed. First of all, the statistical power was relatively low due to the small sample size, especially
R. Wang et al. / Genetic variants at 6p21.1 and head and neck cancer
in stratification analysis. Secondly, we recruited HNC cases from hospitals and selected controls from communities, which might result in potential selection bias. However, we applied a rigorous epidemiological design in selecting study subjects and used further statistical adjustment for known risk factors to minimize potential biases. Third, the biological mechanism of genetic variants at 6p21.1 was not further investigated in this study. In summary, this study indicated that rs2494938 at 6p21.1 had significant associations with HNC risk in Han Chinese, consistent with the findings in lung cancer, non-cardia gastric cancer, and esophageal squamous-cell carcinoma by Jin et al., which further supported the pleiotropy of this SNP in multiple cancers. Further larger epidemiological studies and functional characterizations are needed to confirm these findings and identify the specific mechanism of such loci in the development of human cancer.
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Acknowledgements National Natural Science Foundation of China (813 02361), A Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD, 2014-37), Jiangsu Natural Science Foundation (BK2011764), Specialized Research Fund for the Doctoral Program of Higher Education (Grant No. 20133234120013)
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Supplemental material
Supplemental Fig. 1. Allelic Discrimination plots for rs2494938 and rs2285947 assays. Allele X(1) is shown in red and allele Y(2) is shown in blue. Line green represents a mixture of the two alleles.
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