DCC (deleted in colorectal carcinoma) gene variants confer increased susceptibility to
gallbladder cancer (Ref. No.: Gene-D-12-01446)
Rajani Raia, Kiran L. Sharmaa, Swati Tiwaria, Sanjeev Misrab, Ashok Kumarc, Balraj Mittala,⁎
aDepartment of Genetics, Sanjay Gandhi Post Graduate Institute of Medical Sciences (SGPGIMS), Lucknow, India
bDepartment of Surgical Oncology, CSMMU, Lucknow, India
cDepartment of Surgical Gastroenterology, Sanjay Gandhi Post Graduate Institute of Medical Sciences (SGPGIMS), Lucknow, India
a b s t r a c t a r t i c l ei n f o
Accepted 11 January 2013
Available online 23 January 2013
Tumor suppressor gene
Background and aim: GBC is a lethal and multifaceted disease. Deleted in colorectal carcinoma (DCC) is a well
known tumor suppressor gene. Recently a small genome-wide association study (GWAS) identified DCC to be
significantly associated with gallbladder cancer (GBC) susceptibility in a Japanese population sample.
However, the study sample size was small and lacked independent replication. Therefore, the present
study was carried out to replicate the association of two GWAS identified DCC SNPs (A>Grs4078288,
C>Trs7504990)and two other SNPs (C>Grs2229080and A>Grs714) previously associated with various cancers.
Methodology: The study was accomplished in 406 GBC cases and 260 healthy control samples from North
India. Genotyping was carried out by PCR-RFLP and Taqman genotyping assays. Statistical analysis was
performed by using SPSS ver16 and functional prediction of these variants was carried out using Bioinformatics
tools (FAST-SNP, F-SNP).
Result: We did not observe association with GWAS-identified SNPs of DCC but other SNPs showed significant
association. In addition, a DCC haplotype Grs2229080-Ars4078288-Crs7504990-Ars714conferred high risk of GBC in
India. The haplotype associated risk was independent of gallstone, sex or tobacco usages which are
well-known modifiers of GBC risk. Further analysis suggested DCC A>Grs714as a major risk conferring SNP in
the Indian population.
and the risk haplotype may be explored as a useful marker for GBC susceptibility.
© 2013 Elsevier B.V. All rights reserved.
Gallbladder cancer (GBC), though an uncommon but highly aggres-
sive and lethal malignancy, represents the most frequent malignancy
of the biliary tract (Eslick, 2010) and the seventh most common malig-
nant neoplasm of the digestive tract (Reid et al., 2007; Shi et al., 2011;
Stinton and Shaffer, 2012). GBC has pronounced geographical, ethnic
and gender-specific, predilection worldwide, with females having
threefold higher incidence than males (Rai et al., 2011a,b). It is multi-
factorial disease but the exact etiology of GBC is poorly understood.
Various factors like gallstones, chronic infections of the gallbladder,
obesity, reproductive hormones, dietary factor and hepatobiliary
anomalies have been associated with gallbladder carcinogenesis
(Lazcano-Ponce et al., 2001).
Carcinogenesis is a multifaceted process involving multiple genetic
changes such as the mutational activation of proto-oncogenes and
inactivation of tumor-suppressor genes (Fearon and Vogelstein, 1990;
Wang and Sun, 2010). However, there is very limited information
about the molecular changes involved in the pathogenesis of GBC.
Using candidate geneassociation studies, we have previously identified
several genetic variants in cholesterol metabolism, inflammation,
hormonal and DNA-repair pathway as playing a significant role in vul-
nerability to GBC (Sharma et al., 2012; Srivastava et al., 2010, 2011a,b,
2012). Recently, a genome-wide association study (GWAS) showed a
significantassociationbetween DCC gene polymorphisms and GBC sus-
ceptibility in a Japanese population sample (Cha et al., 2012). However,
Previously, several studies have highlighted the potential role of
DCC as a tumor suppressor gene (Castets et al., 2011; Krimpenfort
et al., 2012; Mazelin et al., 2004; Minami et al., 1997). Moreover,
genetic alterations in the DCC gene, including frequent homozygous
Gene 518 (2013) 303–309
Abbreviations: DCC, deleted in colorectal carcinoma; GWAS, genome-wide association
PCR-RFLP, polymerase chain reaction-restriction fragment length polymorphism; SPSS,
statistical package for social sciences; FDR, False discovery rate; LD, linkage disequilibrium;
LOH, loss of heterozygosity; OR, odds ratio; CI,confidence interval; HWE, Hardy–Weinberg
⁎ Corresponding author at: Department of Genetics, Sanjay Gandhi Post Graduate
Institute of Medical Sciences, Raebareilly Road, Lucknow-226014, India. Tel.: +91 522
2494322; fax: +91 522 2668017, +91 522 2668973.
E-mail addresses: email@example.com, firstname.lastname@example.org (B. Mittal).
0378-1119/$ – see front matter © 2013 Elsevier B.V. All rights reserved.
Contents lists available at SciVerse ScienceDirect
journal homepage: www.elsevier.com/locate/gene
deletions of the 5′ end, several insertions, point mutations and aber-
rant gene and protein expression have been a priori reported to be
associated with metastasis in a variety of human cancers. The dele-
tions at 18q21 loci (including DCC) were also suggested to have an
important role in the development of gallbladder carcinoma; howev-
er, patho-physiological role of DCC in gallbladder carcinogenesis has
not been clarified (Wistuba and Albores-Saavedra, 1999). Additionally,
SNPs are known to exhibit population/ethnic specific pattern of associ-
ation. Therefore, in this present study, we have included the two SNPs
as reported in GWAS (C>Trs7504990and A>Grs4078288) and two other
SNPs (A>Grs714and C>Grs2229080) studied in other cancers to find out
their association with GBC in a large cohort from North India, a region
with very high incidence of GBC.
2.1. Ethics statement
The study protocol was approved by the institutional ethical com-
mittee of Sanjay Gandhi Post Graduate Institute of Medical Sciences
(SGPGIMS). The enrolment of subjects was carried out according to
the norms of the World's Association Declaration of Helsinki. A writ-
ten informed consent was taken from all participants recruited in the
study. A personal interview was conducted with all the subjects and
information on demographic characteristics, such as gender, age,
occupation, demographic features and tobacco usage was collected
by a custom-designed questionnaire.
2.2. Study population
As in our previous hospital based case control study, a total of 666
subjects, consisting of 406 GBC patients and 260 control subjects
were enrolled in this study from the Department of Gastroenterology
of Sanjay Gandhi Postgraduate Institute of Medical Sciences, a tertiary
care hospital in Lucknow, Uttar Pradesh, India and Department of Sur-
gical Oncology, CSSMU Lucknow, Uttar Pradesh, India (Sharma et al.,
2012). Cancer diagnosis for all cases was confirmed by histopathology
and fine needle aspirated cell cytology. Healthy controls were also
recruited from the general population who came for their routine
health checkups or minor illness such as fever, common headache,
or minor surgery. All the controls were free from any prior history
of cancer, precancerous lesions and gallstones proven by ultrasonog-
raphy and were frequency-matched to GBC patients on age, gender
and ethnicity. Individuals with silent gallstones detected by ultraso-
nography were excluded from the controls. All unrelated subjects
were of North Indian ethnicity. To test the possibility for population
stratification, genomic control method was used as described by
(Devlin and Roeder (1999). Staging of cancer was authenticated
according to the AJCC/UICC staging.
On the basis of recent GWAS we selected two SNPs (C>Trs7504990
and A>Grs4078288) that showed strong association with GBC (Cha
et al., 2012). Two more DCC SNPs were selected from the studies
reported in other cancers (Kong et al., 2001; Minami et al., 1997;
Toma et al., 2009). Genomic DNA was isolated from 5 ml peripheral
blood leukocytes according to a standard salting out method. The
genotypes for A>Grs714and C>Grs2229080polymorphism were deter-
mined by the polymerase chain reaction (PCR)-restriction fragment
length polymorphism (RFLP) method. The PCR reaction conditions,
primers used, restriction pattern, and restriction enzymes used for
studied polymorphisms were as described (Hall et al., 2003; Toma
et al., 2009). For C>Trs7504990 and A>Grs4078288 polymorphism
genotyping, predesigned TaqMan® assays were performed with ABI
7500HT Fast Sequence Detection System (Applied Biosystems, Foster
As a negative control, PCR mix without DNA sample was used to
ensure contamination free PCR product. Samples that failed to geno-
type were scored as missing. Genotyping was performed without
knowledge of the case or control status. Ten percent of samples
from patients and controls including samples for each genotype
were tested twice by different laboratory personnel and by sequenc-
ing to evaluate the quality of genotyping which showed 100%
2.4. Statistical analysis
Effective sample sizes for case–control study, and for obtaining
80% power was calculated by Quanto 1.1 ver. software (Gauderman
and Morrison, 2006) using minor allele frequency data from
HapMap (http://hapmap.ncbi.nlm.nih.gov/). All statistical analyses
were performed using SPSS software version 16.0 (SPSS, Chicago, IL,
USA). Statistical analysis of the haplotype estimation and linkage dis-
equilibrium was conducted using the SNPstat software (Sole et al.,
Demography of patients and controls is presented as means and
standard deviations for continuous measures and frequencies or
absolute value and percentages for categorical measures. For observed
genotype frequencies for all the polymorphisms in controls, the
chi-square goodness of fit test was used for any deviation from
exact test was used to compare the differences in demographic vari-
ables and genotype distributions of the polymorphisms between cases
and controls. Riskestimates were calculated for codominant, dominant,
and recessive genetic models using the most common homozygous
genotype as reference. Binary logistic regression analysis was used for
allanalysis variables to estimateoddsratio(OR)and 95% confidence in-
terval (CI) adjusted for age and gender to estimate the risk of gallblad-
der cancer with the polymorphisms. A two-tailed p-value of less than
0.05 was considered a statistical significant result. We applied false dis-
suggested by Carlson et al. (2009). FDR based p value correction strikes
a low rate of false-positive findings.
2.5. In silico analysis
The putative functional effects were determined in both coding
and non-coding regions of the DCC gene by online web servers
FASTSNP (http://fastsnp.ibms.sinica.edu.tw) and F-SNP http://compbio.
cs.queensu.ca/F-SNP (Lee and Shatkay, 2008; Yuan et al., 2006).
3.1. Population characteristics
The demographic characteristics of GBC patients with respect to
their age and gender matched controls are presented in Table 1. The
mean age of 406 GBC and 260 controls was (52.67±10.43 and
47.96±10.79, respectively) comparable and demonstrated no statis-
tically significant differences. Most of the GBC patients were in
advanced stages of cancer (stage III and stage IV). Of the 406 GBC
cases, 5.4% had stage II adenocarcinoma, 49.3% had stage III and
45.3% had stage IV. About 31% of the GBC patients were tobacco
users (either in the form of smoking or chewing, or both). Gallstones
were found to be present in 50.5% of GBC patients. All cancer and
gallstone patients were incident cases and none of the controls had
family history of cancer. The majority of the female patients in our
study were housewives and male patients were not engaged in any
R. Rai et al. / Gene 518 (2013) 303–309
hazardous occupations. Among GBC, 37% of the cases had early age of
onset, i.e. b50 years.
3.2. Allelic distribution of studied polymorphisms in controls
The distribution of DCC polymorphisms (C>Grs2229080,A>Grs4078288,
C>Trs7504990and A>Grs714) are shown in Table 2. The observed geno-
type frequencies of all the studied polymorphisms in controls were in
accordance with Hardy–Weinberg equilibrium (p>0.05).
3.3. Association of DCC polymorphisms with GBC
Table 2 shows the risk of GBC in relation to each of the SNPs of
DCC. On comparing the genotype frequency distribution of A>Grs714
polymorphism in GBC patients with that of controls, the heterozygous
variants (GA), homozygous variant genotypes (AA) and AA+AG
showed statistically significant increased risk for developing GBC
[FDR Pcorr=0.005; OR=1.84; FDR Pcorr=0.042; OR=1.65 and
FDR Pcorr=0.005; OR=1.79, respectively]. At allele level also the
A allele showed significantly increased risk of GBC as compared
with controls [FDR Pcorr=0.015; OR=1.37]. However, for DCC
C>Grs2229080polymorphism, the variant containing genotype GC, CC
and CC+GC, showed low risk for GBC as compared with controls
and it was found to be statistically significant [FDR Pcorr=0.017;
OR=0.64, FDR Pcorr 0.019; OR=0.38 and FDR Pcorr 0.008; OR=
0.61, respectively]. In the recessive model also, the CC genotype
showed borderline significantly low risk of GBC vs. controls [FDR
Pcorr=0.046; OR=0.47]. By contrast, no significant difference was
observed in the DCC C>Trs7504990and DCC A>Grs4078288polymor-
phisms in any group both at genotype and allele levels whereas, the
GG genotype of DCC A>Grs4078288polymorphism showed statistically
significant association only in the recessive model [FDR Pcorr 0.043;
3.4. Linkage disequilibrium and haplotype analysis of DCC polymorphism
in case and control groups
On LD analysis, DCC C>Trs7540990was found to be in strong LD
with DCC A>Grs4078288(D′=0.8412). DCC A>Grs714was in moderate
linkage with DCC A>Grs4078288(D′=0.6675) and in close linkage dis-
equilibrium with DCC C>Trs7504990(D′=0.8459) [Table 3]. However,
DCC A>Grs4078288and DCC C>Trs7540990SNPs were found to be in
Demographic profile of the study subjects.
Variables GBC patientsHealthy controls (HC)
N (%)N (%)
Overall frequency distribution of DCC C>Grs2229080, A>Grs4078288C>Trs7504990and A>Grs714polymorphism in GBC and healthy controls.
Genotype/allele Healthy controls (HC)
p-Value FDR Pcorr
DCC C>Grs2229080genotypes/alleles (age and sex adjusted)
GG+GC vs. CC
DCCA>Grs4078288genotypes/alleles (age and sex adjusted)
AA+AG vs. GG
DCC C>Trs7504990genotypes/alleles (age and sex adjusted)
DCC A>Grs714genotypes/alleles (age and sex adjusted)
GBC—gallbladder cancer, OR—odds ratio, CI—confidence interval. Significant values are given in bold.
R. Rai et al. / Gene 518 (2013) 303–309
weak or marginal LD with DCC C>Grs2229080(D′=0.5677 and 0.5166,
respectively). These suggest that four SNPs examined might be divided
into two haplotype blocks.
Ten inferred haplotype combinations were constructed for the four
polymorphisms in the DCC gene including A>Grs714, A>Grs4078288,
C>Trs7504990and C>Grs2229080. The haplotypes comprising the major
alleles were taken as references and the differences in the frequencies
of haplotypes between patients and controls were tested using
chi-square test (Table 3).
The haplotypeanalysis ofthefourstudied SNPs of DCC revealed that
the frequency of Grs2229080-Ars4078288-Crs7504990-Ars714and Grs2229080-
Grs4078288-Crs7504990-Ars714haplotype was significantly higher in GBC
as compared with that in controls (10% v/s 4% and 3% vs. 1%) and con-
ferred high risk for GBC (FDR Pcorr p=0.008; OR=2.55 and FDR Pcorr
p=0.042; OR=2.74, respectively]. Global haplotype analysis also
pointed to as a statistically significant difference between GBC cases
and controls based on the distribution pattern of the 4 haplotypes
(FDR Pcorr=0.004, Table 4).
3.5. Modulation of risk in the presence or absence of gallstones in GBC
The haplotype analysis showed that the frequency of Grs2229080-
Ars4078288-Crs7504990-Ars714haplotypes was significantly higher and as-
sociated with increased GBC risk in both, GBC patients with gallstones
[10%; FDR Pcorr=0.009; OR=2.83] and GBC patients without stones
[10%; FDR Pcorr=0.038; OR=2.23] as compared with controls (4%).
ciated with GBC risk only in patients without stones [4%; FDR Pcorr=
0.008; OR=5.32], when compared with controls (1%). The global
haplotype association FDR Pcorr value for GBC patients with and with-
out stones is 0.021 and 0.003, respectively (Table 5).
3.6. Haplotype analysis of DCC polymorphism in case and control groups
after gender stratification
After gender stratification of cases and controls, Grs2229080-
Ars4078288-Crs7504990-Ars714haplotype was significantly more associated
withmarginalassociationinwomen[9%; FDR Pcorr=0.047; OR=2.12],
when compared with controls (4% each for males and females, Table 6).
Global haplotype analysis also indicated a statistically significant differ-
encebetweenGBC malesand controls basedonthedistributionpatterns
of the 4 haplotypes (FDR Pcorr 0.042) while in the case of females
the global haplotype association showed only borderline significance
3.7. In-silico analysis of genetic variants on gene activity
All the SNPs except the C>Grs2229080are located in the intronic
region. It is possible that these intronic SNPs may have influence on
splicing or transcription of the gene. However in-silico analysis
using FAST-SNP and F-SNP did not show any changes in transcription
factor binding site as well as gene expression, with C>Trs7504990and
A>Grs714polymorphism. Moreover, both SNPs were predicted to be
‘Benign’ and ‘Tolerated’ by PolyPhen and SIFT computational tools.
In case of A>Grs4078288polymorphism FAST SNP showed it as enhancer
damaging with medium to high risk. The Arg to Gly change by this SNP
results in change in splicing regulation (Table 7).
Our results showed that DCC A>Grs714variants genotype (AA and
AG) were significantly associated with an increased risk of GBC. On
the other hand DCC C>Grs2229080variants (CC and GC genotype)
were associated with protection from propensity to develop GBC,
but there was no association of DCC C>Trs7504990and A>Grs4078288
polymorphism with the GBC risk. At haplotype interface, GBC subjects
carrying DCC Grs2229080-Ars4078288-Crs7504990-Ars714 and Grs2229080-
Grs4078288-Crs7504990-Ars714 had substantially increased risk for GBC.
Therefore, SNPs A>Grs714and A>Grs4078288variants of DCC may be in-
volved in conferring elevated risk of GBC in North India. However, the
higher risk was not affected by gallstone status or gender of GBC pa-
tients. These facts corroborated the tumor suppression function of
DCC and gallstone independent pathway of gallbladder carcinogenesis.
Recently, GWAS by Cha et al. (2012) identified DCC SNP rs7504990
and multiple additional SNPs, near LOH harboring genomic region,
having strong associations with GBC susceptibility in Japanese patients.
Sincethey had limited sample size to detect other genetic variants with
modest or weak effects on susceptibility to GBC, they suggested inde-
pendent replication involving larger number of samples. To the best of
our knowledge, this is the first replication study of the GWAS that
has investigated the role of genetic variants in DCC genes using
multi-analytic approach to define individual risk profiles for GBC. In
our North-Indian cohort, we failed to find any significant association
Frequency distribution of haplotypes of DCC C>Grs2229080,A>Grs4078288C>Trs7504990and A>Grs714polymorphism in GBC and healthy controls.
Gallbladder cancer patients and healthy controls
Haplotypes Frequency p-ValueOR⁎(95%CI)
HC 260 (%)GBC 406 (%)
Global haplotype association p-value: 0.00041c
GBC—gallbladder cancer, OR—odds ratio, CI—confidence interval. Significant values are given in bold, FDR Pcorr=0.008a, 0.042b, 0.004c.
Linkage disequilibrium and D′ statistic.
D′ >0.5 shows LD, the higher the value of D′, the stronger the LD.
R. Rai et al. / Gene 518 (2013) 303–309
of GWAS identified DCC C>Trs7504990SNP with GBC but A>Grs4078288
SNP showed association only in the recessive model. The reason behind
the discrepancy could be due to the population variation. Also, the total
number of GBC cases recruited in the GWAS study was comparatively
smaller and severely unpowered as compared with the present study.
(LD) with other SNPs resulting in functional change or in regulatory
regions affecting the expression/transcription of genes or may generate
alternative splicing, we have selected two other SNPs of DCC gene
which have been widely studied in colorectal and other cancers.
The AA genotype and A allele of DCC A>Grs714polymorphism have
been a priori found to be associated with increased risk of colorectal
cancer (Toma et al., 2009). As this locus has been studied as LOH
marker associated with decreased expression of DCC and linked to
progression of many cancers (Enomoto et al., 1995; Khan et al.,
2011; Mattar et al., 2004), our result suggests that the individuals car-
rying the GA genotype may be more susceptible for LOH. Since LOH
provides an independent genetic pathway in gallbladder carcinogen-
esis (Wistuba et al., 2001; Yoshida et al., 2000) and a frequent mech-
anism for loss of DCC function, it may increase the risk for GBC.
Previous studies have also attributed the role of DCC in gallbladder
carcinogenesis and laid down a conjecture of plausible relationship
between DCC polymorphisms and risk of GBC. In addition to normal
and metaplastic epithelia, LOH of DCC has been also observed in 31–
45% of GBC, suggesting it as a frequent and early event in GBC
(Wistuba et al., 1995; Yoshida et al., 2000). Hidaka et al. (1999)
suggested that LOH at 18q (DCC) has a role in its tumor progression.
Inactivation of the tumor-suppressor gene has been widely shown to
play an important role in the development of a variety of human can-
cers including GBC.
DCC gene has long been a familiar bona fide tumor-suppressor
gene and late gatekeeper which limits tumor progression (Castets et
al., 2011). It encodes netrin-1 receptors (DCC protein) which belongs
to the functional dependence receptor family and induces apoptosis
in the absence of their ligand binding, but blocks apoptosis (or in-
duces survival) when engaged by netrin-1 (Mehlen et al., 1998).
Thus, tumor suppressor activity of DCC is attributed to its ability to
trigger tumor cell apoptosis when ligands are limited (during metas-
tasis or tumor growth beyond local blood supply) (Castets et al.,
2011; Mehlen et al., 1998). It is inactivated or down regulated in
many kinds of advanced tumors (prostate, breast, endometrial, ovar-
ian, esophageal, testicular, glial, neuroblastoma and hematological
malignancies) and is associated with loss of heterozygosity or epige-
netic silencing (Goldschneider and Mehlen, 2010; Krimpenfort et al.,
2012; VanZomeren-Dohm et al., 2011). Aberrant expression of DCC
occurred more frequently in the cases of later clinical stage and
higher pathological grade (Meimei et al., 2011), and is associated
with marked increase in aggressiveness, worse prognosis and decreased
response to adjuvant chemotherapy. Moreover, re-establishment of DCC
expression suppresses tumorigenicity (Krimpenfort et al., 2012). DCC
Haplotype analysis DCC C>Grs2229080, A>Grs4078288,C>Trs7504990, A>Grs714in gallbladder cancer patients after gender stratification and healthy controls.
Haplotypes Haplotype analysis of DCC in gallbladder cancer and healthy
Haplotype analysis DCC in gallbladder cancer and healthy
HC (89) GBC (121) p-ValueOR⁎(95%CI)HC (171)GBC (285) p-ValueOR⁎(95%CI)
Global haplotype association p-value:0.034c
Global haplotype association p-value:0.051
GBC—gallbladder cancer, HC—healthy controls, OR—odds ratio, CI—confidence interval.
Significant values are given in bold, FDR Pcorr=0.035a, 0.047=0.05b, 0.0420c.
Haplotype analysis DCC C>Grs2229080, A>Grs4078288,C>Trs7504990, A>Grs714in gallbladder cancer (with stone and without stone) patients and healthy controls.
Haplotypes Haplotype analysis of DCC in gallbladder cancer patients
without stone and healthy controls
Haplotype analysis of DCC in gallbladder cancer patients with
stone and healthy controls
HC (260)GBC (201)p-Value OR⁎(95%CI) HC (260)GBC (205)p-Value OR⁎(95%CI)
Global haplotype association p-value: 0.00012d
Global haplotype association p-value: 0.013e
GBC—gallbladder cancer, HC—healthy controls, OR—odds ratio, CI—confidence interval.
Significant values are given in bold, FDR P corr=0.038a, 0.009b, 0.008c, 0.003d, 0.021e.
R. Rai et al. / Gene 518 (2013) 303–309
polymorphism has been previously reported to be associated with colo-
rectal carcinoma. These facts organize the cornerstone for the possible
involvement of DCC gene variants in gallbladder tumorigenesis.
Previously, codon 201 polymorphism involving substitution of Arg
with Gly of DCC has been seen more frequently in colorectal cancer
patients in advanced stages (Akkiprik et al., 2007; Zhang et al.,
2003). However, the SNP proved to be low risk in GBC and there
was no contribution of the SNP towards risk haplotype.
Since three of the studied SNPs are intronic, they do not affect
amino acid coding and therefore do not directly affect protein func-
tion. Our in silico and eQTL analysis also failed to find any functional
consequence or association of above intronic SNPs with expression
of the gene, except very low-low risk of DCC A>Grs4078288polymor-
phism affecting individuals' susceptibility for gallbladder carcinoma.
By contrast, in the present study, A>Grs714emerged as major SNP
conferring GBC risk. One potential explanation for observed associa-
tion between these SNP and GBC may be that DCCrs714is in close link-
age disequilibrium with DCCrs7504990(D′=0.8459) which in turn was
in close LD with DCCrs4078288(D′=0.8412). It is likely that these SNPs
of DCC may not be conferring direct effects on GBC susceptibility and
there may be complex interactions between these SNPs and the effect
may be mediated by A>G DCCrs714acting in a stronger manner in
combination rather than individually. As discussed earlier, this SNP
is closely associated with LOH. Moreover, tumor suppressor genes
are reported to be frequently inactivated by a mutation at one allele
and a deletion of a second allele (Knudson, 1989). Therefore, loss of
heterozygosity of DCC associated with A>Grs714 polymorphism
resulting in aberrant expression of DCC may also be a major mecha-
nism involving GBC pathogenesis and progression. However, exact
functional effect of these SNPs on the expression of DCC still requires
In the present context, A>Grs714SNP may be useful as marker for
GBC risk. Since loss of 18q is a marker for poor prognosis in many can-
cers, DCC status has the potential to define a group of patients who
may benefit from specific treatment regimes (Popat and Houlston,
2005). However, the present study may require validation in other
populations like native Indians of South America, China and other
high risk ethnic groups with high incidence of GBC.
The present study could not replicate the GWAS identified DCC
A>Grs4078288,C>Trs7504990as independent risk SNPs with GBC in a
large cohort from North India. Instead, it revealed a positive associa-
tion between the DCC haplotypes and GBC risk. Although, sample
size in the present study is sufficient to yield 80% power, it is limited
particularly in subgroup analysis, but we have applied FDR to mini-
mize false positive associations. The study may, however, be replicated
in other populations for establishing a clear role of the tumor-
suppressor gene, DCC in the pathobiology of GBC. The global estimate
of the extent of genetic changes leading to GBC will help in ascertaining
individuals at higher risk for GBC and should be useful for discovery of
new markers for translational research.
The authors thank the DBT and the ICMR, Government of India for
financial support to carry out the present study.
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Results of F-SNP and FAST: SNP for all the studied polymorphisms.
Result of F-SNP Result of FAST SNP
Genetic variationFunctional category Prediction tool Prediction resultFS score Possible functional effectsRisk
Protein codingPolyphen Probably damaging0.616 Missense (non-conservative) Medium–high
Splicing regulationSplicing regulation
No functional informationIntronic enhancerVery low–low
No functional information
No functional information
Intronic with no known function
Intronic with no known function
R. Rai et al. / Gene 518 (2013) 303–309
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