Cancer Epidemiology Biomarkers & Prevention 17, 1007, April 1, 2008. doi: 10.1158/1055-9965.EPI-08-0023
© 2008 American Association for Cancer Research
Single Nucleotide Polymorphisms in Obesity-Related Genes and the Risk of Esophageal Cancers
James D. Doecke,
Zhen Zhen Zhao,
Mitchell S. Stark,
Adèle C. Green,
Nicholas K. Hayward,
Grant W. Montgomery,
Penelope M. Webb,
David C. Whiteman and for the Australian Cancer Study
Queensland Institute of Medical Research, Brisbane, Queensland, Australia
Requests for reprints: David C. Whiteman, Division of Population Studies and Human Genetics, Queensland Institute of Medical Research, PO Royal Brisbane Hospital, Brisbane, Queensland 4029, Australia. Phone: 61-7-3362-0279; Fax: 61-7-3845-3503. E-mail: david.whiteman{at}qimr.edu.au
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Abstract
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Rates of adenocarcinoma of the esophagus (EAC) and esophagogastric junction (EGJAC) have been rising rapidly in recent decades, in contrast to the declining rates of esophageal squamous cell carcinomas (ESCC). Obesity is a major risk factor for both EAC and EGJAC, but not ESCC, and there is speculation that obesity promotes adenocarcinoma development through endocrine and related pathways. We therefore compared the prevalence of 12 single nucleotide polymorphisms (SNPs) in nine candidate genes previously implicated in obesity pathways (LEP, LEPR, ADIPOQ, POMC, PPAR
, PPAR
, RXR, GHRL, and INSIG2) in a large Australian case-control study comprising DNA samples from 260 EAC cases, 301 EGJAC cases, 213 ESCC cases, and 1,352 population controls. No SNPs were associated with EGJAC or ESCC. Although several SNPs seemed to be associated with EAC on crude analysis [ADIPOQ (rs1501299), LEP (5'-untranslated region), PPAR (H447H), and GHRL (M72L)], effect sizes were modest and none of the associations was significant after correcting for multiple comparisons. Further, we found no consistent evidence that any of the genotypes were associated with risk of EAC or EGJAC within strata of body mass index (<25.0 kg/m2, 25.0-29.9 kg/m2, >30 kg/m2). In conclusion, our data suggest that these SNPs do not play a major role in esophageal carcinogenesis. (Cancer Epidemiol Biomarkers Prev 2008;17(4):1007–12)
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Introduction
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The incidence of adenocarcinomas of the esophagus (EAC) and esophagogastric junction (EGJAC) has been rising in many Western populations, whereas the incidence of esophageal squamous cell carcinomas (ESCC) has been declining (1-3). Obesity has been identified as a major risk factor for EAC and EGJAC (4-8), and it is likely that the increasing prevalence of obesity (9) underlies the rising incidence of these cancers. Because obesity has been associated with increased risks of several cancers (10), there is speculation that obesity may promote tumor development through endocrine and related pathways. Leading candidate genes include leptin (LEP; ref. 11), leptin receptor (LEPR; ref. 12), adiponectin (ADIPOQ; ref. 13), proopiomelanocortin (POMC; ref. 14), peroxisome proliferative-activated receptor (PPAR; ref. 15), peroxisome proliferative-activated receptor (PPAR; ref. 16), retinoid X receptor (RXR; ref. 17), ghrelin (GHRL; ref. 18), and insulin-induced gene 2 (INSIG2; ref. 19). To address the hypothesis that single nucleotide polymorphisms (SNPs) in these genes are specifically associated with EAC, we investigated possible associations between SNPs in these genes and risk of developing EAC, EGJAC, and ESCC in a population-based case-control study conducted in Australia.
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Materials and Methods
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Study Population
Full details of the study have been published previously (8). Briefly, we recruited patients diagnosed between July 1, 2002 (July 1, 2001 in the state of Queensland) and June 30, 2005 with histologically confirmed EAC, EGJAC, or ESCC. Simultaneously, we recruited 1,580 matched controls selected at random from a population register. Detailed information was collected about height, weight 1 y before diagnosis (before recruitment for controls), cigarette smoking, alcohol consumption, and frequency of symptoms of gastroesophageal acid reflux, as reported previously (8, 20). Complete epidemiologic data and DNA were available for 260 EAC cases, 301 EGJAC cases, 213 SCC cases, and 1,352 controls. Approval was obtained from the human research ethics committees of the Queensland Institute of Medical Research and participating hospitals throughout mainland Australia and written informed consent was obtained from all participants.
SNP Selection and Genotyping
Twelve SNPs were selected from nine obesity-related genes: LEP, LEPR, ADIPOQ, POMC, PPAR, PPAR, RXR, GHRL, and INSIG2. SNP selection was based on previously published results showing allelic associations between these polymorphisms and obesity (11-22). Polymorphisms included six coding region nonsynonymous mutations, two synonymous mutations, three 5' and 3' SNPs, and one intronic SNP (Table 1
). Genotyping was conducted using the Sequenom iPLEX protocol (Sequenom) as previously published (20).
Statistical Analyses
We calculated Hardy-Weinberg equilibrium and allele frequencies and assessed linkage disequilibrium using Haploview (21). Minor allele frequencies between case and control groups were tested using 
2 analyses, with P values presented in Table 1. To estimate the relative risk of cancer associated with each SNP, we calculated the odds ratio (OR) and 95% confidence interval (95% CI) using multivariable logistic regression in Statistical Analysis System version 9.1 (SAS Institute, Inc.). Initial models were adjusted only for age and sex; final models included terms for age, sex, body mass index (BMI), smoking history, and frequency of gastroesophageal reflux symptoms 10 y before diagnosis. Additional adjustment for alcohol consumption, frequency of nonsteroidal anti-inflammatory drug use, and education made essentially no difference to the risk estimates. In further analyses, we assessed genotypic associations within strata of BMI using standard WHO cut points (22). We assessed statistical significance at = 0.05, which became 0.004 after Bonferroni correction (0.05/12).
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Results
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All SNPs were in Hardy-Weinberg equilibrium. Table 1 shows genotype and allele frequencies for each SNP in controls compared with the three case groups. P values are presented for the minor allele comparisons. Although several SNPs seemed to be associated with EAC {ADIPOQ (rs1501299), LEP [5'-untranslated region (UTR)], PPAR (H447H), and GHRL (M72L)}, these were not statistically significant following Bonferroni correction.
After stratification for BMI, the effect sizes for PPAR, PPAR, and GHRL markedly increased for persons with BMI <25 kg/m2 (Table 2
, EAC only), whereas the OR for POMC was significantly increased in the BMI 
30 kg/m2 group. There were no other consistent patterns across BMI strata for EAC, and none for EGJAC or ESCC.
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Table 2. Associations between polymorphisms in multiple obesity-related genes and the risk of EAC, EGJAC, and ESCC and BMI
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Discussion
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The Australian Cancer Study constitutes one of the largest available collections of tissue and risk factor data from esophageal cancer patients and has an estimated 80% power to detect ORs between 1.4 and 2.0 for allele frequencies between 0.05 and 0.45. Thus, our case-control sample had adequate power to find moderate risk alleles in the selected genes if they existed. Of the 12 SNPs tested, we identified modest positive associations for two mutations [PPAR (H447H) and GHRL (M72L)] and a sizable inverse association for one SNP [ADIPOQ (rs1501299)]; however, none of the associations was statistically significant at the Bonferroni corrected threshold. To the best of our knowledge, this is the first study to comprehensively evaluate obesity-related SNPs in relation to risk of esophageal cancer. A recent review (23) examined all studies before February 2007 regarding polymorphisms and risk of EAC and ESCC. From 100 studies reviewed, no obesity-related genes were examined.
We further investigated the potential role of selected SNPs within strata of BMI. We observed some evidence of decreasing risks of EAC associated with LEP (5'-UTR), RXR (A140A), and GHRL (M72L) with increasing adiposity; however, these trends were not statistically significant. The other SNPs tested displayed inconsistent trends across BMI strata, indicating no relationship with esophageal cancer risk regardless of BMI.
In summary, we tested 12 SNPs across three groups of cases compared with controls. We found no convincing associations between any of the SNPs tested and risk of EGJAC or SCC. However, the finding of weak associations between LEP, ADIPOQ, and GHRL and risk of EAC suggests that further investigation of the SNPs tested here, and other SNPs within the same genes, may be warranted.
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Appendix A The Australian Cancer Study: Esophageal Cancer
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Investigators: David C. Whiteman, M.B.B.S., Ph.D.; Penelope M. Webb, M.A., D.Phil.; Adele C. Green, M.B.B.S., Ph.D.; Nicholas K. Hayward, Ph.D.; Peter G. Parsons, Ph.D.; and David M. Purdie, Ph.D.
Clinical collaborators: B. Mark Smithers, F.R.A.C.S.; David Gotley, F.R.A.C.S., Ph.D.; and Andrew Clouston, F.R.A.C.P., Ph.D.
Project Manager: Suzanne Moore, R.N., M.P.H.
Research Nurses: Suzanne O'Brien, R.N., M.P.H.; Ellen Minehan, R.N.; Deborah Roffe, R.N.; Sue O'Keefe, R.N.; Suzanne Lipshut, R.N.; Gabby Connor, R.N.; Hayley Berry, R.N.; Frances Walker, R.N.; Teresa Barnes, R.N.; Janine Thomas, R.N.; Linda Terry, R.N., M.P.H.; Michael Connard, B.Sc.; Leanne Bowes, B.Sc.; Mary Rose Malt, R.N.; and Jo White, R.N.
Laboratory staff: Peter Schultz, Lauren Aoude, Loralie Parsonson, and Stephen Walsh.
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Acknowledgments
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We thank Shahram Sadeghi, M.D., and Harish Babu, M.D., for assisting with pathology abstractions; Nirmala Pandeya, M.Med.Sci., for data cleaning and programming; and all the study participants.
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Footnotes
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Grant support: Queensland Cancer Fund project grant and National Health and Medical Research Council of Australia Program grant 199600. N.K. Hayward, P.M. Webb, and D.C. Whiteman are supported by Research Fellowships from the National Health and Medical Research Council of Australia.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 1/14/08;
accepted 2/12/08.
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Abstract
Introduction
