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| Journal of the New Zealand Medical Association,
08-June-2012, Vol 125 No 1356
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Robert W Bentley, Dayle A Keown, Richard B Gearry, Vicky A
Cameron, Jacqui Keenan, Rebecca L Roberts, Andrew S Day
Colorectal cancer (CRC) is the second most commonly
diagnosed cancer in New Zealand (NZ), with over 2500 new cases of CRC registered
in 2007.1 The role of vitamin D and its
biological effects mediated through the vitamin D receptor (VDR) in the
development of CRC is not entirely clear. Some studies have indicated that
individuals with CRC have insufficient levels of vitamin
D.2
Whilst vitamin D may be synthesised as vitamin D3 in the
skin following exposure to ultraviolet light, it can also be obtained from
dietary sources3 and it has been shown that
vitamin D supplementation or an increase in the intake of foods with high
vitamin D levels may play a role in the prevention of
CRC.4,5
The active form of vitamin D (1, 25-dihydroxyvitamin D3) is
bound by the intracellular VDR. This complex binds and interacts with
target-cell nuclei (at VDR elements) to produce a variety of biological
effects.6
Recent research has indicated that vitamin D may play a role
as a key regulator of innate immunity in
humans.7–9 Vitamin D is also shown to
suppress CRC development and growth by affecting cell proliferation,
differentiation, apoptosis, and
angiogenesis.4
The VDR gene maps to a region on chromosome
1210. Association studies of single nucleotide
polymorphisms (SNPs) in the VDR gene suggest that these variants may
influence CRC risk.11–14
Despite the high rates of CRC in the NZ population and
associations of VDR gene polymorphisms with CRC risk reported elsewhere, very
little research has been carried out in order to define the frequency of these
variants in the general NZ population or in NZ CRC disease cohorts.
The aim of this study was therefore to screen for genetic
variation of the three SNPs rs2228570 (also known as rs10735810; Fok1), rs731236
(Taq1), and rs11568820 (Cdx2) of the VDR gene in a well-defined
population of individuals with CRC and compare their incidence to a healthy
control population, in order to determine the contribution of
VDR
polymorphisms to CRC in NZ.
MethodStudy participants—DNA from
patients who had been diagnosed with CRC (N=200) was obtained from the
Christchurch Tissue Bank (New Zealand). DNA was extracted from Whatman FTA Elute
Cards (GE Healthcare, UK) using the manufacturers’ recommended protocols.
Briefly, a 3.0 mm disc from the FTA Elute Card was washed with 500 mcl of
sterile H2O by pulse vortexing and then incubated
at 95°C for 20 minutes in 30 mcl sterile
H2O. The eluted DNA was separated from the FTA
matrix by centrifugation and stored at -20ºC until analysed.
Control DNA (N=200) was obtained from the Canterbury
Healthy Volunteers for the Study of Heart Disease
project.15 Samples were selected by age- and
gender-matching to the CRC patients. At the time of recruitment they had no
personal history of cancer of any type or self-reported family history of
cancer. Median follow-up was 5.9 years (range 0.1–8.7yrs).
Ethical considerations—Each
participant provided written, informed consent. Ethical approval for use of
these samples was covered by the Upper South A Ethics Committee (Reference
CTY/01/05/062, and URA/10/09/068).
Genotyping—Genotyping of SNPs
rs11568820 (Cdx2), rs2228570 (aka rs10735810, Fok1) and rs731236 (Taq1) was
performed using pre-designed Taqman® SNP
genotyping assays (Applied Biosystems, Foster City, CA) in a
Lightcycler® 480 II (Hoffmann La Roche,
Basel, Switzerland). 384-well plates with 4.8μl reaction volumes (2 μl
genomic DNA, 2.8 μl Taqman® master mix) were used.
Cycling conditions for all SNP assays were 10 minutes
at 95°C, 40 cycles of 15 sec at 92°C and 1 min at 60ºC, and 30
seconds of cooling at 40°C. Results were analysed using Lightcycler®
480 (version 1.5.0) software. The accuracy of the genotyping assay was confirmed
by repeat analysis of 10% of samples. Concordance between original and repeat
genotype calls was 99%.
Statistical analysis—A web-based
calculator (http://ihg2.helmholtz-muenchen.de/cgi-bin/hw/hwa1.pl)
was used to test for deviations from Hardy-Weinberg Equilibrium (HWE) and to
perform Chi-squared and odds ratio analyses. Associations were considered
significant if p<0.05.
ResultsControls and CRC patients were age, gender and ethnicity
matched. In the case and control groups, 94 samples (47%) were female. The
median age by gender was the same in control and case groups (72 yrs). The
average age by gender for case and control groups was 69.5±0.4 yrs. Samples
were from New Zealand Caucasians of European origin.
DNA samples from 199 CRC patients, and 191(rs2228570) or 182
(rs731236 and rs11568820) DNA samples from healthy controls were successfully
genotyped. Minor allele frequencies are shown in Table 1. Hardy-Weinberg
equilibrium was seen for the three SNPs in case and control groups
(p=0.14–0.73), indicating that allele and genotype frequencies do not
deviate from expectation.
The allelic frequencies (p=0.43–0.73) and genotypic
distribution (p=0.15–0.90) of the three VDR SNPs were not significantly
associated with disease (Table 1).
Table 1. Genotype and allele frequencies of
VDR SNPs in CRC patients and healthy controls
aMAF = Minor Allele
Frequency.
bThe alleles
constituting the genotype are denoted as 1 or 2.
Furthermore, no significant differences for allelic
frequencies of the three SNPs were revealed in subgroup analysis by age
(above/below median age of 72 yrs; p=0.38–0.91), gender
(p=0.22–0.88), or age/gender (p=0.33–0.93).
DiscussionAssociation studies of VDR SNPs with different forms of
cancer, including CRC, have indicated that they may influence disease risk
risk,11–14 and that the frequency of
these SNPs varies with ethnicity .
Little research has been performed in the New Zealand
population to determine the distribution and association of VDR SNPs with CRC.
The minor allele frequencies (MAFs) of Taq1 (CRC 42.5% and HC 37.7%) and Cdx2
(CRC 21.9% and HC 20.%) in our study were in agreement with the MAFs reported
for these SNPs in other studies on populations of European
origin.13, 16
In contrast, the MAF of Fok1 was higher in our CRC patients
(40.5%) and healthy controls (37.7%) than the MAF reported in French
(33%)16 and UK
(31%)17 populations. Reasons for this
discordance are unknown, but may be due to subtle population stratification.
The lack of any significant overall or subgroup association
of these SNPs of the VDR gene with CRC does not indicate a role for
these variants in CRC in NZ Caucasians of European origin. However, previous
studies have indicated that risk conferred by SNPs of the VDR gene may
be modified by calcium intake, vitamin D uptake, dietary
fat18–20 and body mass index
(BMI).21 Our CRC samples were sourced from
tissue bank samples lacking this supporting information.
Further research in a larger cohort taking these factors
into account may clarify the nature of this gene/environment interaction in the
NZ population.
Competing interests: None
declared.
Author information: Robert W Bentley,
Research Fellow1; Dayle A Keown, Research
Student2; Richard B Gearry, Associate Professor
Gastroenterology1; Vicky A Cameron Associate
Research Professor1; Jacqui Keenan, Senior
Research Fellow2; Rebecca L Roberts, Senior
Research Fellow1,3; Andrew S Day, Professor
Paediatric Gastroenterology4,5
Acknowledgements:
This research was funded by the Cancer Society of New Zealand
(Canterbury/West Coast branch). Control samples were from the Canterbury Healthy
Volunteers for the Study of Heart Disease—funded by the Health Research
Council of New Zealand. RLR is the recipient of a Sir Charles Hercus Health
Research Fellowship. The authors also thank Helen Morrin (Curator of the
Christchurch Tissue Bank) for her support and technical input.
Correspondence: Dr Robert Bentley,
Department of Paediatrics, University of Otago, PO Box 4345, Christchurch 8140,
New Zealand. Fax: +64 3 364 0009; email: Robert.Bentley@otago.ac.nz
References:
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