Abstract. Background: Recent evidence indicates that
single nucleotide polymorphisms (SNPs) in the Cox-2 gene
may modulate the risk of colorectal adenoma development.
Patients and Methods: We explored possible associations
between Cox-2 polymorphisms and risk of adenoma
development in an African American case–control study
comprising 72 cases of advanced adenomas and 146 polyp-
free controls. An exhaustive approach of genotyping 13
haplotype-tagging SNPs (ht SNPs) distributed over the entire
COX-2 gene was used. Results: Statistically significant
inverse associations were observed between the heterozygous
genotypes at the 5229 G>T polymorphism in intron 5 [odds
ratio (OR)=0.42; confidence interval (CI)=0.19-0.92;
p=0.03] and at the 10935 A>G polymorphism in the 3’
flanking region downstream from the poly A signals
(OR=0.39;CI=0.18-0.83;p=0.01) and the risk for colorectal
adenoma development. Conclusion: The data from our pilot
study suggest that allelic variants of the COX-2 gene
significantly influence the risk of adenoma development in
the African American population.
Colon cancer accounts for approximately 10% of all cancer-
related deaths and remains the third deadliest killer among
cancer types in the United States (1). Epidemiological data
show that African Americans have higher age-specific
incidence and mortality rates and lower 5-year survival rates
compared to Caucasians (2). Although reasons for this
disparity are not clear, evidence implicates genetic,
environmental and lifestyle factors as contributors to this
multi-factorial disease (3).
There is mounting evidence that chronic inflammation is
involved in the etiology of cancer. Previous studies have
reported an association between genetic variants of pro-
inflammatory genes and the risk of developing colorectal
adenoma and carcinoma (4-6). One such gene, encoding the
enzyme cyclooxygenase-2 (Cox-2) plays a significant role
in inflammation and carcinogenesis (7). Epidemiological
observations as well as randomized prevention clinical trials
have provided evidence for a significant role of Cox-2 in
colon carcinogenesis (8-11). More recently, several studies
have explored association between genetic variants of Cox-2,
alone or in interaction with environmental factors, and risk
of developing colorectal adenoma/carcinoma mostly in
Relatively few studies have addressed the influence of
genetic variants of Cox-2 on cancer risk in the African
Americans replacing the amino acid valine with alanine at
position 511 in exon 10 of Cox-2 has been described to
reduce the risk of colorectal cancer (16, 17). Another study
reported different patterns of association between the
genetic variants in the regulatory regions of Cox-2 and
prostate cancer risk in three different ethnic populations
including African Americans (18). This observation is not
surprising as patterns of genetic polymorphisms may vary
within and between populations. The haplotype block
structures of human genome containing regions of high
linkage disequilibrium are of shorter size and reduced
diversity in African Americans compared to Caucasians
(19). To better understand the significance of genetic
variations in the Cox-2 gene in influencing colorectal cancer
risk in African Americans, we used a case–control study of
advanced adenomas to exhaustively analyze a possible
*Both authors contributed equally to this work.
Correspondence to: Iqbal U. Ali, Molecular Oncology Program,
Panjwani Center for Molecular Medicine and Drug Research,
International Center for Chemical and Biological Sciences,
University of Karachi, Karachi, Pakistan. e-mail: email@example.com
Key Words: Cox-2 polymorphisms, colorectal adenoma, African
ANTICANCER RESEARCH 28: 3119-3124 (2008)
Protective Effect of Cox-2 Allelic Variants on Risk of Colorectal
Adenoma Development in African Americans
HASSAN ASHKTORAB1*, SHIRLEY TSANG2*, BRIAN LUKE3, ZHONGHE SUN2,
LUCILE ADAM-CAMPBELL1, JOHN KWAGYAN1, RICHARD POIRIER4, SHAHINA AKTER1,
AHMAD AKHGAR1, DUANE SMOOT1, DAVID J. MUNROE2and IQBAL UNNISA ALI4
1Department of Medicine and Cancer Center, Howard University College of Medicine, Washington, D.C.;
2Laboratory of Molecular Technology, and
3Advanced Biomedical Computing Center, SAIC-Frederick, Frederick, MD;
4Division of Cancer Prevention, National Cancer Institute, Bethesda, MD, U.S.A.
association between Cox-2 polymorphisms and colorectal
nucleotide polymorphisms (htSNPs) in the Cox-2 gene.
Materials and Methods
Patient selection. The study was approved by the Howard University
Institutional Review Board. Study participants were recruited from
patients referred for colonoscopy to the gastroenterology division at
Howard University Hospital between September 2000 and October
2003. Indications for colonoscopy included rectal bleeding, irregular
bowel habit, weight loss, family history of colon polyp/cancer,
personal history of colon polyp and routine screening. Cases were
eligible if colonoscopy resulted in a first diagnosis of colorectal
adenomatous or hyperplastic polyp as confirmed by histology.
malabsorption, any cancer, current or past chemotherapy or
interferon treatment were excluded. Patients with distal or proximal
polyps and with adenomatous or hyperplastic pathology, as
determined by independent pathologists were selected as cases.
Based on these criteria, 72 patients qualified as cases. Controls had
to be free of all polyps and self-described with no previous history
of colorectal adenomas/cancer. All patients were as African
Americans. Clinical and demographic data collected on each patient
included race, gender, past medical history, family history of
witha historyofinflammatorybowel disease,
colorectal polyp/cancer and information on smoking, alcohol
consumption and medication use. DNA was extracted from samples
from 72 patients and 146 controls.
Genotyping. The htSNPs of the Cox-2 gene for the population of
African American descent, together with the respective primers and
probes used in this study are displayed in Table I. The positions of the
polymorphisms refer to the Genbank entry AY382629 and as detailed
at http://pga.gs.washington.edu/data/ptgs2/. All assays were designed
and developed using Assay-by-Design (Applied Biosystems Inc, CA,
USA). All oligo primers and probes were synthesized by Applied
Biosystems, Inc. Assays were validated and optimized using in-house
collected human DNA samples. Positive control DNAs of known
genotypes as well as a no-template control were run on each assay
plate for quality control. All SNPs were tested by the Taqman assay
using the MGB chemistry (Applied Biosystems, Inc.) and the ABI
7900HT Sequence Detector. SDS 2.1 (Applied Biosystems, Inc.) was
used to determine the genotype calls. Specific experimental details
about genotyping will be provided upon request from the authors.
Data analysis. Odds ratios (ORs) were estimated using logistic
regression models with the PROC LOGISTIC function of the SAS
software package (version 9.1; SAS Institute, Cary, NC, USA)
adjusting for gender and smoking. Departure from Hardy–Weinberg
equilibrium was assessed by comparing the expected to observed
genotype frequencies using the asymptomatic Pearson’s χ2test.
ANTICANCER RESEARCH 28: 3119-3124 (2008)
Table I. Polymorphisms, primers and probes used in this study.
PolymorphismForward primers Reverse primersReporter _VICReporter _FAM
Numbers for polymorphism refer to positions in the Genbank entry AY382629 and as detailed at http://pga.gs.washington.edu/data/
Characteristics of the study population and the association
with colorectal cancer in this group of cases and controls are
displayed in Table II. The only highly significant positive
association was observed between current smokers and the
risk of adenoma development [odds ratio (OR)=2.73, 95%
confidence interval (CI) =1.20-6.19, p=0.03). A non-
significant association was also present between former
smokers and adenoma development (OR=1.52, CI=0.81-
The data of the association analysis for the main effect of
the 13 polymorphisms distributed over the entire Cox-2 gene
are displayed in Table III. Of the 13 htSNPs, an intronic
polymorphism and another in the 3’ flanking region (FR),
when adjusted for gender and smoking, were associated with
a lower risk of adenomas. Adjusting for age and smoking
also resulted in very similar associations (data not shown).
Individuals with the heterozygous genotype at the intron 5-
5229 had a statistically significant decrease in the risk of
developing adenomas (OR=0.42, CI=0.19-0.92, p=0.03).
Similarly, a highly significant protective effect for adenoma
risk was observed in individuals with the heterozygous
genotype at position 3’FR-10935 (OR=0.39, CI=0.18-0.83,
p=0.01). The risk of adenoma in individuals with the variant
homozygous genotypes at intron 5-5229 and 3’ FR-10935,
however, was no different from that of the control group.
Ashktorab et al: Cox-2 Variants and Adenoma Risk in African Americans
Table III. COX-2 genotypes and the risk of advanced colorectal
Ancestral alleles are treated as wild-type. OR, odds ratio; CI, confidence
interval. Values are adjusted for gender and smoking.
Table II. Characteristics of cases and controls.
Characteristic Cases Controls OR95% CI
Body mass index
OR, odds ratio; CI, confidence interval.
Besides the polymorphisms at intron 5- 5229 and 3’FR-
10935, two other polymorphisms in the promoter region
showed a trend for a protective effect for adenoma
development. There was a marginally significant lower risk
of adenoma development in individuals with the rare
homozygous genotype at the –861 position (OR=0.29,
CI=0.08-1.04, p=0.06) and a statistically non-significant
protective trend for the risk of adenomas (OR=0.29,
CI=0.06-1.38, p=0.12) in individuals with another rare
homozygous variant at the –663 polymorphism (Table III).
To our knowledge, our pilot study represents the first
exhaustive approach to determine the influence of genetic
variants of Cox-2 on the risk of colorectal adenoma
development in African Americans. We evaluated 13 htSNPs
with a minor allele frequency ranging between 0.13-0.43 that
were distributed over the entire Cox-2 gene and captured
most common variations in the African American population.
Two polymorphisms located in intron 5 and in the 3’ FR
showed a protective effect for adenoma development.
A reduced risk of adenoma development in African
Americans in carriers of the heterozygous genotype at intron
5-5229 in the Cox-2 gene is consistent with the previous
finding of the protective effect of this polymorphism on
development of colorectal adenoma in Caucasians (12).
Interestingly, a Swedish study reported a protective effect of
the same polymorphism (rs20432) (referred to as position
+3100) for prostate cancer (20). It is also noteworthy that,
similar to our study in African Americans, the heterozygous
but not the variant homozygous genotype at intron 5-5229 had
an inverse association with prostate cancer risk in a Swedish
population (20). Intronic sequences are believed to harbor
transcriptional regulatory elements. The intronic variants may
therefore modulate disease risk by regulating gene expression,
gene splicing, or transcript stability (21). A protective effect
of the variant G allele at intron 5-5229 of the Cox-2 gene in
colorectal and prostate cancer may indicate a transcription
regulatory role of intronic sequences. Alternatively, the intron
5-5229 polymorphism may be in linkage disequilibrium with
a nearby functional polymorphism.
Another polymorphism with a protective effect for
adenoma development was detected at position 10935 in the
3’ flanking region of the Cox-2 gene. This is located
downstream of the polyadenylation signal and AU-rich
elements. Previously, disease-associated variants have been
described in the 3’ flanking region of genes that affect
transcription factor-binding sites (22). Polymorphisms in
the Cox-2 gene located upstream of the 3’FR-10935
position have been reported to have cancer-modulating
effect. Especially, both positive and negative association of
the 3’ UTR-8494 polymorphism with various types of
cancer has been widely reported (12). In particular, the
heterozygous, but not the variant homozygous genotype at
3’ UTR-10494 was found to be protective for prostate
cancer in a Swedish population (20). Although, there was
no evidence of a risk-modulating effect of the previously
reported 8494 or 10494 variants of the Cox-2 gene in our
small study, the protective effect of the nearby 3’ FR-10935
polymorphism underscores the significance of allelic
variants in the 3’ regulatory region of the Cox-2 gene in
affecting the risk of cancer development.
In summary, our study underscores the relevance of genetic
variants in the regulatory regions of Cox-2 in modulating
cancer risk. In the absence of any information on the
functional significance of the intron 5-5229 and 3’FR-10935
polymorphisms in development of colorectal adenoma in
African Americans, future studies with larger numbers of
cases and controls will be necessary to rule out the possibility
that the protective effect of Cox-2 variants in the regulatory
regions on adenoma development is a chance finding.
This work was supported by Grant #CA102681, funded by
the National Cancer Institute and GCRC.
1 Jemal A, Siegel R, Ward E, Murray T, Xu J and Thun MJ:
Cancer statistics, 2007. CA Cancer J Clin 57: 43-66, 2007.
Kauh J, Brawley OW and Berger M: Racial disparities in
colorectal cancer. Curr Probl Cancer 31: 123-133, 2007.
Satia JA, Galanko JA and Rimer BK: Methods and strategies to
recruit African Americans into cancer prevention surveillance
studies. Cancer Epidemiol Biomarkers Prev 14: 718-721, 2005.
Macarthur M, Sharp L, Hold GL, Little J and El-Omar EM: The
role of cytokine gene polymorphisms in colorectal cancer and
their interaction with aspirin use in the northeast of Scotland.
Cancer Epidemiol Biomarkers Prev 14: 1613-1618, 2005.
Sansbury LB, Bergen AW, Wanke KL, Yu B, Caporaso NE,
Chatterjee N, Ratnasinghe L, Schatzkin A, Lehman TA,
Kalidindi A, Modali R and Lanza E: Inflammatory cytokine gene
polymorphisms, nonsteroidal anti-inflammatory drug use, and
risk of adenoma polyp recurrence in the polyp prevention trial.
Cancer Epidemiol Biomarkers Prev 15: 494-501, 2006.
Gunter MJ, Canzian F, Landi S, Chanock SJ, Sinha R and
Rothman N: Inflammation-related gene polymorphisms and
colorectal adenoma. Cancer Epidemiol Biomarkers Prev 15:
Cao Y and Prescott SM: Many actions of cyclooxygenase-2 in
cellular dynamics and in cancer. J Cell Physiol 190: 279-286, 2002.
Baron JA: Epidemiology of non-steroidal anti-inflammatory
drugs and cancer. Prog Exp Tumor Res 37: 1-24, 2003.
Steinbach G, Lynch PM, Phillips RK, Wallace MH, Hawk E,
Gordon GB, Wakabayashi N, Saunders B, ShenY, Fujimura T, Su
LK and Levin B: The effect of celecoxib, a cyclooxygenase-2
inhibitor, in familial adenomatous polyposis. N Engl J Med 342:
ANTICANCER RESEARCH 28: 3119-3124 (2008)
10 Bertagnolli MM, Eagle CJ, Zauber AG, Redston M, Solomon Download full-text
SD, Kim K, Tang J, Rosenstein RB, Wittes J, Corle D, Hess TM,
Woloj GM, Boisserie F, Anderson WF, Viner JL, Bagheri D,
Burn J, Chung DC, Dewar T, Foley TR, Hoffman N, Macrae F,
Pruitt RE, Saltzman JR, Salzberg B, Sylwestrowicz T, Gordon
GB and Hawk ET: Celecoxib for the prevention of sporadic
colorectal adenomas. N Engl J Med 355: 873-884, 2006.
11 Arber N, Eagle CJ, Spicak J, Racz I, Dite P, Hajer J, Zavoral M,
Lechuga MJ, Gerletti P, Tang J, Rosenstein RB, Macdonald K,
Bhadra P, Fowler R, Wittes J, Zauber AG, Solomon SD and Levin
B: Celecoxib for the prevention of colorectal adenomatous
polyps. N Engl J Med 355: 885-895, 2006.
12 Ali IU, Luke BT, Dean M and Greenwald P: Allellic variants in
regulatory regions of cyclooxygenase-2: association with
advanced colorectal adenoma. Br J Cancer 93: 953-959, 2005.
13 Koh WP, Yuan JM, van den Berg D, Lee HP and Yu MC:
Interaction between cyclooxygenase-2 gene polymorphism and
dietary n-6 polyunsaturated fatty acids on colon cancer risk: the
Singapore Chinese Health Study. Br J Cancer 90: 1760-1764,
14 Ulrich CM, Whitton J, Yu JH, Sibert J, Sparks R, Potter JD and
Bigler J: PTGS2 (COX-2) -765G > C promoter variant reduces
risk of colorectal adenoma among nonusers of nonsteroidal anti-
inflammatory drugs. Cancer Epidemiol Biomarkers Prev 14:
15 Tan W, Wu J, Zhang X, Guo Y, Liu J, Sun T, Zhang B, Zhao D,
Yang M, Yu D and Lin D: Associations of functional
lipoxygenase with risk of occurrence and advanced disease status
of colorectal cancer. Carcinogenesis 28: 1197-1201, 2007.
16 Lin HJ, Lakkides KM, Keku TO, Reddy ST, Louie AD, Kau IH,
Zhou H, Gim JS, Ma HL, Matthies CF, Dai A, Huang HF, Materi
AM, Lin JH, Frankl HD, Lee ER, Hardy SI, Herschman HR,
Henderson BE, Kolonel LN, Le Marchand L, Garavito RM,
Sandler RS, Haile RW and Smith WL: Prostaglandin H synthase
2 variant (Val511Ala) in African Americans may reduce the risk
for colorectal neoplasia. Cancer Epidemiol Biomarkers Prev 11:
17 Sansbury LB, Millikan RC, Schroeder JC, North KE, Moorman
PG, Keku TO, de Cotret AR, Player J and Sandler RS: COX-2
polymorphism, use of nonsteroidal anti-inflammatory drugs, and
risk of colon cancer in African Americans (United States).
Cancer Causes Control 17: 257-266, 2006.
18 Panguluri RC, Long LO, Chen W, Wang S, Coulibaly A, Ukoli F,
Jackson A, Weinrich S, Ahaghotu C, Isaacs W and Kittles RA:
COX-2 gene promoter haplotypes and prostate cancer risk.
Carcinogenesis 25: 961-966, 2004.
19 Costas J, Salas A, Phillips C and Carracedo A: Human genome-
wide screen of haplotype-like blocks of reduced diversity. Gene
349: 219-225, 2005.
20 Shahedi K, Lindstrom S, Zheng SL, Wiklund F, Adolfsson J, Sun
J, Augustsson-Balter K, Chang BL, Adami HO, Liu W, Gronberg
H and Xu J: Genetic variation in the COX-2 gene and the
association with prostate cancer risk. Int J Cancer 119: 668-672,
21 Fedorova L and Fedorov A: Introns in gene evolution. Genetica
118: 123-131, 2003.
22 Chen JM, Ferec C and Cooper DN: A systematic analysis of
disease-associated variants in the 3’ regulatory regions of human
protein-coding genes II: the importance of mRNA secondary
structure in assessing the functionality of 3’ UTR variants. Hum
Genet 120: 301-333, 2006.
Received May 14, 2008
Revised July 25, 2008
Accepted August 4, 2008
Ashktorab et al: Cox-2 Variants and Adenoma Risk in African Americans