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Abstract
Previous studies have investigated the relationship between blood type and pancreatic
cancer, but little research has been done to determine how blood type correlates with the
development of colon cancer. The fucosyltransferase2 (FUT2) gene is involved in the
production and secretion of ABH blood group antigens along with mucin into the lumen of the
colorectal tract. These antigens serve as carbon sources and anchoring sites for bacteria that are
normally present in our gut and aid with digestion. However, a mutation in the FUT2 gene
inhibits secretion of the ABH blood group antigens. The absence of these antigens could lead to
a decrease in the number of normal bacteria in the gut, in turn allowing pathogenic, potentially
cancer-causing, bacteria to colonize the colorectal tract. Therefore, we examined the relationship
between the rs516246 SNP in the FUT2 gene, the microbial composition of the gut and the
presence of colorectal adenomas (CRAs), which are precursors to colorectal cancer. We
hypothesized that individuals with the variant allele (T) of the rs516246 SNP would be more
likely to have CRAs and would have different bacterial profiles than those with the wildtype
allele (C). We genotyped the rs516246 SNP and sequenced bacterial DNA in over 600 cases and
controls using genomic DNA extracted from blood and biopsies respectively. We found no
significant overall correlation between the variant genotype and the risk of developing CRAs, but
that the variant genotype does affect the bacterial profile. Genotype was also linked to the level
of expression of inflammation and mucin genes. It was expected that a change in the mucin
secreted would increase inflammation in epithelial cells of the colorectal tract, because the
mucus layer covering epithelial cells may be less capable of preventing pathogenic bacteria from
penetrating the cells. While the presence of a T allele was significantly associated with increased
expression of TLR4, which activates the immune system, it was also significantly associated
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with decreased expression of the inflammation marker IL-10. Our findings suggested that the
rs516246 FUT2 variant alters bacterial composition and inflammation of the colorectal tract.
Further studies are needed in order to analyze additional FUT2 SNPs and confirm their role in
colorectal adenomas and cancer.
Introduction
Colorectal cancer is one of the leading causes of cancer death worldwide (Torre et al.,
2015) and the third leading cause of cancer death in the United States, in both females and males
(American Cancer Society, 2014). This disease usually progresses slowly, and the transition
from normal epithelium to adenocarcinoma (Figure 1) can sometimes take 20 years (American
Cancer Society, 2014). One of the stages of colorectal cancer is the adenoma (Lao et al., 2011),
which was the focus of this study.
Figure 1. The progression of colorectal cancer from normal gut epithelium to polyps/adenomas,
to cancer. Source: (Lao et al., 2011)
Colorectal cancer can be hereditary, but it can also affect individuals with no family
history of the disease, as in sporadic colorectal cancer (Haghighi et al., 2009). Risk factors for
sporadic colorectal cancer include lifestyle choices such as physical inactivity, smoking (Torre et
al., 2015) and high meat intake (Roberts-Thomson et al., 1999). The impact that these
environmental factors have on an individual can vary depending on the genetic polymorphisms
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that are present (Roberts-Thomson et al., 1999). One genetic polymorphism that could affect the
impact of environmental factors is the rs516246 SNP in the fucosyltransferase 2 (FUT2) gene,
which is involved in the production of ABH blood group antigens (Jaff, 2010).
It has been suggested that blood type in humans is associated with the risk of developing
various types of cancer. Wolpin et al. (2009) reported that non-O blood group is related to an
increased risk of pancreatic cancer. A later study found that the overall risk of developing cancer
is lower in those with blood group O, while the greatest risk of developing cancer is associated
with blood group A (Zhang et al., 2014). Blood type data from individuals with various types of
cancer, including pancreatic, breast, colorectal, ovarian and nasopharyngeal, was used in this
study (Zhang et al., 2014). Two genes that are involved in the production of ABH blood group
antigens are fucosyltransferase 1 (FUT1) and FUT2 (Jaff, 2010). FUT1 is an enzyme that is
responsible for producing ABH antigens that are presented on the surfaces of red blood cells,
while FUT2 is an enzyme that is responsible for the production and secretion of ABH antigens
into bodily fluids, such as the mucus of the gut (Wacklin et al., 2011). The H antigens produced
by the FUT1 and FUT2 enzymes are precursors for the A and B antigens (Jaff, 2010). These
blood group antigens that are secreted, along with mucin, into the lumen of the colon are
oligosaccharides that serve as carbon sources and anchoring sites for gut bacteria (Mäkivuokko
et al., 2012). Non-secretors, or those with a genetic mutation in the FUT2 gene, do not secrete
ABH blood group antigens, so these antigens would not be present in the colorectal tract (Jaff,
2010). It has been found that beneficial bacteria, such as Bifidobacteria, are more abundant in
the colorectal tracts of secretors than non-secretors (Wacklin et al., 2011), which suggests that
other, pathogenic bacteria may colonize the gut of non-secretors. These pathogenic bacteria may
also be better able to infect epithelial cells of the colorectal tract in non-secretors because a lack
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of secretion of ABH antigens would change the composition of the protective mucus layer that
lines the lumen of the gut (Tong et al., 2014). Therefore, non-secretors may be at a higher risk
for colorectal cancer, as research has indicated that the gut microbiome may play a highly
significant role in the development of this disease (Louis et al., 2014). The rs516246 SNP in the
FUT2 gene is a good indicator of the secretor status of an individual. It can be inferred that those
with the wildtype cytosine (C) at the site of this SNP in both alleles have the secretor status,
while those with the variant thymine (T) at this same site in both alleles have the non-secretor
status (Okunola et al., 2015). Those who are heterozygous and have a C at the rs516246 SNP in
one allele and a T at this SNP in the other allele most likely experience some secretion of blood
group antigens.
We hypothesized that the rs516246 SNP is associated with the risk of developing
colorectal adenomas and that this association may be modified by bacteria, which utilize the
antigens produced by FUT2. In addition, the differences in the levels of secretion of these
antigens into the mucin of the gut may affect levels of inflammation caused by pathogenic
bacteria in the gut.
In this study we examined the relationship between the FUT2 C>T polymorphism
(rs516246), microbial gut composition, levels of gene expression of inflammatory markers
(Interleukin 10, Toll-Like Receptor 4, Mucin 2) and the presence of colorectal adenomas.
Methods
Study Population
The study population consisted of 628 consenting subjects between the ages of 50 and 80
who were undergoing screening colonoscopy at UNC Hospitals as part of the Diet and Health
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Study V (DHSV). The study was approved by the UNC School of Medicine IRB. Rectal
biopsies and blood samples used in this study were obtained during the procedure. After
collection, blood was centrifuged in order to separate the plasma, buffy coat and red blood cells.
The buffy coat containing the white blood cells was collected and stored at -80°C until use in the
current study.
DNA Extraction from Buffy Coat
DNA Extraction was performed using the Gentra Puregene Blood Kit (Qiagen) according
to the kit protocol. Buffy coat samples were thawed at 37 °C, after which residual red blood cells
were lysed with Red Blood Cell Lysis solution. The white blood cells were then pelleted by
centrifugation and lysed with Cell Lysis solution in order to isolate genomic DNA. The solution
was treated with RNase A Solution to achieve RNA-free DNA. Proteins were separated from the
DNA with Protein Precipitation Solution, and isopropanol was added to each sample to coalesce
the DNA strands. The DNA was then pelleted, dried and later resuspended in DNA Hydration
Solution. The concentration of DNA obtained from each sample was measured using a
NanoDrop Spectrophotometer, and the DNA was stored at -4 °C.
Allelic Discrimination Assay
Individual samples were genotyped using a TaqMan SNP Genotyping Assay
(ThermoFisher Scientific). A total of 10 ng of DNA from each sample were added to an
individual well of a 384-well plate and combined with TaqMan Genotyping Master Mix
(ThermoFisher Scientific) and FUT2-specific primers and probes to target the rs516246 SNP.
The plate was loaded onto a Bio-Rad CFX Real-Time PCR machine for one cycle of 95 °C for
10 minutes, followed by 40 cycles of 95 °C for 10 seconds and 60 °C for 60 seconds. A plate
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read was performed upon completion of the run. Each of the two alleles in the FUT2 gene of
each DNA sample were determined by Bio-Rad CFX Manager software. One possible allele
contained a C at the rs516246 SNP, and the other possible allele contained a T at the position of
this SNP. The two different alleles were distinguished through the use of probes, which were
small strands of nucleotides attached to a fluorescent reporter dye molecule and a quencher dye
molecule (Livak 1999). While attached to the probe, the level of fluorescence of the fluorescent
reporter dye molecule was kept low by the quencher dye molecule (Livak 1999). Only when the
fluorescent reporter dye molecule was detached from the probe and separated from the quencher
dye molecule was it able to emit a high level of fluorescence (Figure 2) (Livak 1999). VIC
reporter dye was attached to probe one, which was complementary to the C allele. FAM reporter
dye was attached to probe two, which was complementary to the T allele. During PCR, these
probes attached to the site of the rs512646 SNP in their complementary allele. Forward and
reverse primers then attached to either end of the region of DNA containing the SNP of interest,
and Taq polymerase added on nucleotides to the primers (Figure 2). When the Taq polymerase
reached the probe, the fluorescent reporter dye molecule was cleaved, causing the fluorescence
emitted by this molecule to increase in intensity. In cases where the complementary sequence to
one of the probes was not present, such as in homozygotes for the rs516246 SNP, that particular
probe did not bind to the DNA. Its reporter dye molecule remained attached to the probe, and it
did not fluoresce at a high level. The amount of fluorescence reported by each of the two dye
molecules in each individual well was measured during PCR. The level of fluorescence of each
dye molecule corresponded to the frequency of each allele. In this way the genotype of each
individual sample was determined to be either homozygous for the wildtype nucleotide,
homozygous for the variant nucleotide or heterozygous at the location of the rs516246 SNP.
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Figure 2. Cleavage of the fluorescent reporter dye during an
allelic discrimination assay. Source: (Livak, 1999)
Bacterial DNA Extraction and Illumina Sequencing
Both human and bacterial DNA were extracted from colorectal biopsy samples using a
Qiagen DNeasy Blood and Tissue Kit with a modified kit protocol that included lysozyme and
bead-beating (McCoy et al., 2013).
The Illumina library was then created using two different PCR reactions. First-step PCR
(PCR1) was performed using primers designed to amplify the V2 region of the 16S bacterial
rRNA gene. For PCR1, one reaction was performed for each sample using Phusion HighFidelity Master Mix (Life Technologies, Carlsbad, CA. PCR1 product was diluted 20-fold for
use as a template for second-step PCR (PCR2).
PCR2 primers contained an Illumina index barcode sequence, Illumina adapter sequence
and a tag sequence. There were two sets of PCR2 primers, and each PCR2 reaction received one
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of each, resulting in a dual-indexed product. One reaction was performed for each sample using
Phusion High-Fidelity Master Mix.
PCR product was visualized on an E-Gel 96 (Life Technologies, Carlsbad, CA) to check
all samples for amplification. All samples with positive amplification were included in the
library. All samples were normalized to 25 ng/µl using the SequalPrep Normalization Kit (Life
Technologies, Carlsbad, CA). The library was cleaned using AxyPrep Mag Beads (Fadrosh,
2014). Bacterial sequences were filtered for quality control and processed in Qiime (Caporaso,
2010).
Expression of Inflammatory Markers
Human RNA was extracted from colorectal biopsy samples using a Qiagen RNeasy Mini
Kit. The RNA was loaded into a chip and run on the Agilent 2100 bioanalyzer using the Agilent
6000 Nano Kit, in order to determine the concentration and fragment size of the RNA in each
sample. The RNA for each sample was then diluted to a concentration of 500 ng per 10 μl. A
Promega RQ1 RNase-Free DNase Kit was used to digest any DNA contaminants in the RNA
samples. Reverse transcription PCR was performed using an Invitrogen Cloned AMV FirstStrand cDNA Synthesis Kit in order to create cDNA from the RNA samples. The cDNA was
used as a template for real time PCR, along with a Qiagen RT² qPCR Primer Assay, which
included primers specific to the genes of interest. The Primer Assay was mixed with template
cDNA and 𝑖𝑇𝑎𝑞 𝑇𝑀 Universal SYBR® Green and run on a Bio-Rad CFX384 Real Time PCR
Detection System (Kang et al., 2013).
Statistical Analysis
Comparisons of general characteristics of the study population, genotype distribution,
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bacterial abundance and gene expression between adenoma cases and controls with no adenomas
were assessed by t-tests for continuous variables and chi squared test for categorical variables.
Multivariate analysis of bacteria data such as cluster analysis and non-multidimensional scaling
was performed using PRIMER-7 software as previously described (Shen et al., 2010).
Multivariate analysis permits the examination of relationships among multiple variables at the
same time. P-values <0.05 were deemed significant after adjustment for multiple comparisons.
Results
The purpose of the experiments described above was to determine if there is a correlation
between the rs516246 SNP, the types of bacteria present in the gut, the level of inflammation in
the gut and the presence of colorectal adenomas. The bacterial profile of the gut was determined
through the use of Illumina sequencing of extracted bacterial DNA, and the level of
inflammation of the gut was determined by measuring the level of expression of certain genes
involved in inflammation. The results of these experiments, along with the general
characteristics of the study population are given below.
The characteristics of subjects according to case/control status are given in Table 1.
Race, body mass index (BMI) and calories were evenly distributed among cases and controls,
however, age, sex and waist-hip ratio differed significantly between these two groups. There
was a higher proportion of males among subjects with colorectal adenomas, and these subjects
were also older and had a higher waist-hip ratio than those who did not present with colorectal
adenomas.
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Table 1. Descriptive Characteristics of Study Participants
General
Characteristics
Case (n=190)
Control (n=438)
P-value
Age (mean, SE)
56.5 (0.5)
54.9 (0.3)
0.005
Sex (% Male)
110 (58)
188 (43)
0.0006
Race (% White)
164 (86)
377 (86)
0.94
Waist-Hip Ratio
0.937 (0.006)
0.906 (0.004)
<0.0001
27.7 (0.4)
27.0 (0.3)
0.11
2,045 (66)
1,949 (39)
0.21
(mean, SE)
Body Mass Index
(mean, SE)
Calories (mean, SE)
The genotype distributions of FUT2 were in Hardy-Weinberg equilibrium, as determined
by the equation p² + 2pq + q² = 1, but there was no significant overall association between FUT2
genotype and presence of colorectal adenomas. The distribution of the CC, CT and TT
genotypes was 29%, 49% and 22% respectively for cases and 28%, 51% and 21% respectively
for controls. However, there were differences in bacterial profiles between those with the CC
genotype and those with the TT genotype that were borderline significant (P=0.07). As shown in
Figures 3a and 3b, the TT genotype is associated with reduced abundance of Ruminococcus
(P=0.02) and Dialister (P=0.04) and increased abundance of Eggerthella (P=0.06) compared to
the CC genotype. The genera of bacteria analyzed are divided into two graphs based on the scale
of the axis that best fits the data.
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Bacterial Abundance by Genotype
4.5
4
Percent Abundance
3.5
3
2.5
2
1.5
1
0.5
0
Bacteria
CC
CT
TT
Figure 3a. The distribution of six genera of bacteria by FUT2 genotype. There was a significant
reduction in Dialister (p=0.04) and a borderline significant increase in Eggerthella (p=0.06)
from CC to TT.
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Bacterial Abundance by Genotype
25
Percent Abundance
20
15
10
5
0
Ruminococcus
Blautia
Bacteria
CC
CT
TT
Figure 3b. The distribution of two genera of bacteria by FUT2 genotype. There was a
significant reduction in Ruminococcus (p=0.02) from CC to TT.
Table 2 displays the relationship between three inflammatory markers, FUT2 genotype at
the rs516246 SNP and the presence of colorectal adenomas. In cases, compared to controls, the
carriage of the T allele, compared to the CC genotype, was associated with reduced expression
levels of Interleukin-10 gene (IL-10), which produces an anti-inflammatory cytokine (Iyer et al.,
2012), and increased expression of Toll-Like Receptor 4 gene (TLR4), which produces a protein
that activates the innate immune system when bound to a pathogen (Chaudhuri et al., 2013).
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There was no statistically significant relationship between the Mucin 2 gene (MUC2) and FUT2
genotype.
Table 2. Gene Expression Levels of Inflammatory Markers in Relation to FUT2 Genotype
Inflammatory
Marker
IL-10
Genotype
Case
Control
P-Value
CC (wildtype)
CT
TT
T allele
0.79
0.64
0.46
0.55
0.63
0.70
0.55
0.66
0.27
0.10
0.46
0.06
MUC2
CC (wildtype)
CT
TT
T allele
1.24
1.02
1.02
1.02
1.20
1.19
1.16
1.19
0.67
0.49
0.40
0.45
TLR4
CC (wildtype)
CT
TT
T allele
1.03
1.19
1.03
1.13
1.0
1.03
0.87
0.95
0.65
0.15
0.05
0.03
Discussion
In this study we determined the relationship between a FUT2 polymorphism (rs516246),
risk of colorectal adenomas, bacterial composition of the colorectal tract and inflammatory
markers. Although previous studies had found a correlation between blood type and pancreatic
cancer (Wolpin et al., 2009) and non-secretor status and oral cancer (Ensinck et al., 2013), we
did not find any overall association between the rs516246 FUT2 SNP and the risk of developing
colorectal adenomas. However, we found that the homozygous variant genotype (TT) is
associated with reduced abundance of Ruminococcus and Dialister and increased abundance of
Eggerthella compared to the homozygous wildtype genotype (CC). We also found a correlation
between the variant T allele, increased expression of TLR4 and decreased expression of IL-10
among cases, compared to controls.
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Previous studies reported significantly lower levels of Ruminococcus and Dialister in the
stool of patients with colorectal cancer compared to healthy individuals (Weir et al., 2013) and
high levels of Eggerthella in the gut of colorectal cancer patients (Wang et al., 2012). Therefore,
the association between the reduced abundance of the two former genera, the increased
abundance of Eggerthella and the variant FUT2 genotype could imply that the rs516246 FUT2
SNP affects the risk of developing colorectal cancer by way of altering the bacterial profile of the
gut. Although it is not completely understood how bacteria in the colorectal tract interact with
their human host, it is known that variations in the composition of the gut microbiome are
present in many diseases (Wu et al., 2015).
Not only does FUT2 affect the composition of the gut microbiome, but it also seems to
affect the function of these bacteria in the mucosal layer of the colorectal tract (Tong et al.,
2014). These functional changes have been shown to be accompanied by inflammation of the
intestinal walls (Tong et al., 2014). In this study we found a link between inflammation and the
presence of colorectal adenomas. Compared to controls, cases with the variant T allele displayed
lower expression levels of the anti-inflammatory gene IL-10 and higher expression levels of the
TLR4 gene, which produces a receptor that triggers the innate immunity response when it is
bound to a pathogen, than those with the homozygous wildtype genotype (CC). This may
suggest that a complex relationship exists between the gut microbiota, intestinal inflammation,
FUT2 and colorectal adenomas.
Although no overall association was found in this study between the rs516246 FUT2
SNP and the risk of developing colorectal adenomas, that does not discount FUT2 as being an
important factor in the progression of colorectal cancer, because this SNP is one of many
polymorphisms in the FUT2 gene. Sample size was not an issue in this study (n=628), but if we
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were not under time constraints, we would have liked to genotype more SNPs within the FUT2
gene.
In summary, our findings suggest that the rs516246 FUT2 variant alters bacterial
composition and inflammation of the colorectal tract. Further studies are needed in order to
analyze additional FUT2 SNPs and confirm their role in colorectal adenomas and cancer.
Acknowledgements
I would like to thank Dr. Temitope Keku for giving me the opportunity to complete this
study, for developing the hypothesis and for her continued support and input throughout the
process. I would like to thank Amber McCoy for teaching me all protocols and for completing
the bacterial DNA extractions and Illumina sequencing. I would like to thank Winifred Okunlola
for extracting and genotyping DNA from almost a third of the samples used in this study. I
would like to thank Dr. Joe Galanko for completing the data analysis for this study. I would like
to thank Félix Araújo-Pérez and the University of Maryland Institute for Genome Sciences for
determining cytokine gene expression in this study. I would like to thank Dr. Blaire Steinwand
for serving as my Biology Faculty Sponsor. I wish to acknowledge the Center of
Gastrointestinal Biology and Disease in the UNC School of Medicine for providing the research
laboratory for this study. This project was supported by the Tom and Elizabeth Long Excellence
Fund for Honors administered by Honors Carolina and by grants from the National Institutes of
Health NIH R01 CA04468, R01 CA136887 and P30 DK 034987.
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