Associations between Genes in the One-Carbon Metabolism
Pathway and Advanced Colorectal Adenoma Risk in
Individuals with Low Folate Intake
Summer S. Han1, Laura Y. Sue1, Sonja I. Berndt1, Jacob Selhub2, Laurie A. Burdette3,
Philip S. Rosenberg1, and Regina G. Ziegler1
Background: Folate is essential for one-carbon metabolism, a pathway required by DNA synthesis,
methylation, and repair. Low dietary and circulating folate and polymorphic variation in this pathway are
associated with increased risk of colorectal adenoma and cancer.
1,331 cases of advanced colorectal adenoma, identified by sigmoidoscopy at baseline, and 1,501 controls from
one-carbon genes and adenoma risk in all subjects and stratified by folate intake. We applied the Adaptive
Rank Truncated Product (ARTP) method to assess statistical significance at the gene and pathway levels.
Results: Folate intake was inversely associated with advanced colorectal adenoma risk [odds ratio (OR)
by quartile ¼ 0.85, P ¼ 1.9 ? 10?5]. We found no statistically significant associations between one-carbon
genes and adenoma risk in all subjects. As hypothesized, we observed a statistically significant pathway-
level association (P ¼ 0.038) in the lowest quartile of folate; no significant associations were found in higher
quartiles. Several genes including adenosine deaminase (ADA) and cysteine dioxygenase (CDO1) con-
tributed to this signal (gene-level P ¼ 0.001 and 0.0073, respectively). The most statistically significant SNP
was rs244072 in ADA (P ¼ 2.37 ? 10?5).
Conclusions and Impact: Stratification by dietary folate and application of the ARTP method revealed
interact with associations between common variants in one-carbon metabolism genes and colorectal adenoma
risk. Cancer Epidemiol Biomarkers Prev; 21(3); 417–27. ?2012 AACR.
Rates of colorectal cancer incidence and mortality have
decreased in recent years; however, colorectal cancer
remains the third most common cancer and third leading
cause of cancer-related death in both men and women in
the United States (1). Although most colorectal adenoma-
tous polyps will not develop into cancer, these are com-
monly accepted as precursor lesions of colorectal cancer
(2, 3), and screening by colonoscopy or sigmoidoscopy
and polyp excision is standard of care for adults ages 50
and older (4, 5).
Folate is essential for one-carbon metabolism, a path-
way required by DNA synthesis, methylation, and repair
(6). Folate is a water-soluble B-vitamin found in leafy
green vegetables, beans and peas, many fruits, and forti-
fied grains and cereals. In epidemiologic studies, low
folate intake and low circulating folate levels have con-
sistently been associated with increased risk of colorectal
adenoma and colorectal cancer (7–11).
Genes in the folate-mediated one-carbon metabolism
pathway have been of interest as candidate genes for
influencing colorectal adenoma risk (10, 12–15). Studies
of these folate-related genes and folate intake may reveal
patterns of risk. There is expanding discussion of the
possible importance of gene–environment interaction in
Cancer Epidemiology and Genetics, National Cancer Institute, NIH,
on Aging, Tufts University,Boston, Massachusetts; and3Core Genotyping
Facility at the Advanced Technology Center, Division of Cancer Epidemi-
Note: Supplementary data for this article are available at Cancer Epide-
miology, Biomarkers & Prevention Online (http://cebp.aacrjournals.org/).
S.S. Han and L.Y. Sue contributed equally to this work.
Corresponding Author: Summer S. Han, Biostatistics Branch, Division of
Cancer Epidemiology and Genetics, National Cancer Institute, 6120 Exec-
utive Blvd., EPS/8089, Bethesda, MD 20892. Phone: 301-435-3994; Fax
301-402-0081; E-mail: email@example.com
?2012 American Association for Cancer Research.
chronic disease etiology, and how to assess efficiently
studies have found associations between colorectal
adenoma risk and common polymorphisms in methyle-
netetrahydrofolate reductase (MTHFR; 12, 15) and thy-
midylate synthase (TYMS; 13, 14), along with evidence of
gene–environment interaction; specifically some of these
genes among individuals with low folate intake and
(19, 20). Recently, there have been several genome-wide
association studies (GWAS) for colorectal cancer (21–23),
but so far no GWAS have been reported for colorectal
In this study, we investigated associations between
advanced colorectal adenomas and 82 one-carbon metab-
olism genes, selected on the basis of the known biology of
this pathway and an extensive literature search. We stud-
Colorectal and Ovarian Cancer Screening Trial (PLCO), a
by the National Cancer Institute (NCI). We systematically
looked for associations with each of 882 tag single nucle-
otide polymorphisms (SNP) in these genes in the entire
population, and then examined associations by level of
folate intake. To further investigate gene- and pathway-
level associations both in the entire population and by the
folate levels, we applied the Adaptive Rank Truncated
and women, ages 55 to 74 years, were enrolled from 10
different centers in the United States between 1993 and
2001 (24). All subjects included in this study had been
assigned to the screening arm of the trial, completed a
baseline risk factor questionnaire, provided a blood spec-
imen at baseline, and consented to participate in etiologic
studies of cancer and related diseases. In addition, sub-
jects had completed at baseline a food frequency ques-
tionnaire, called the PLCO Dietary Questionnaire (DQx).
Cases were all those participants with a histologically
at baseline sigmoidoscopy. Advanced colorectal adeno-
mas were defined as adenomas more than 1 cm in size,
with villous characteristics, and/or with high-grade
dysplasia, and carcinomas in situ. Controls were random-
ly selected from those participants without distal colorec-
tal polyps at baseline sigmoidoscopy, and frequency
matched to cases on age at baseline, sex, and race. A total
(950 men and 551 women) were included in this analysis.
A subset (n ¼ 500 cases and 500 controls) was chosen
for folate measurements in baseline plasma. During the
PLCO randomization period (1993–2001), the United
States had required that enriched breads, cereals, flours,
corn meals, pastas, rice, and other grain products be
fortified with folic acid, specifically by January 1, 1998
from those advanced adenoma cases with baseline blood
draws either before December, 1997 or after January, 1998
so that plasma folate values would represent circulating
levels either prior or subsequent to the mandatory folate
the cases on date of baseline blood draw.
Folate intake and alcohol consumption were derived
intake during the past year of 137 food items, including
beer, wine, and hard liquor, and typical portion sizes for
77 of the food items (24).
We calculated 7 measures of folate intake, in micro-
grams per day, all of which were based on the single DQx
administered to each participant at the time of study
entry. Two of the dietary folate estimates were based on
databases: the first for the U.S. food supply prior to folate
fortification in 1998 and the second for the U.S. food
supply after folate fortification. The USDA databases
relied on microbiologic folate assays to estimate the bio-
availability of the folate naturally occurring in foods and
the synthetic folic acid added in fortification. A third
measure, of total folate, combined vitamin supplement
information from the DQx with dietary folate estimates
based on the postfortification USDA database.
Four University of Minnesota food composition data-
bases from the Nutrition Data Systems for Research soft-
ware (NDS-R) were also used; all were for the U.S. food
natural folate in foods and the second estimated synthetic
folate added to foods. The third and fourth combined
natural and synthetic folate occurring in foods by bio-
availability basedonmicrobiologic assaysandbyweight-
ing synthetic folate as 1.67 times more bioavailable than
natural folate, respectively.
Unless otherwise specified, for all statistical analyses,
we used dietary folate estimated with the USDA prefor-
tification food composition database. Quartile cut points
were based on the frequency distribution in the study
controls. The quartiles of dietary folate intake were 0 to
and more than 466 mg/d (Q4).
For the subset of 500 cases and 500 controls, heparin
plasma was assayed for folate, using a Lactobacillus casei
microbial assay, at the USDA Human Nutrition Research
Center on Aging at Tufts University (26). Plasma collec-
tions from 4 subjects were used for quality control. Four
anonymized quality control samples were randomly
inserted into each of the 28 assay batches, with 2 of the
Han et al.
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One-Carbon Metabolism Genes and Colorectal Adenoma
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