Deleted in Colorectal Cancer (DCC) Gene Polymorphism is Associated with H. pylori Infection among Susceptible Malays from the North-Eastern Region of Peninsular Malaysia

Abstract

Background/aims:
Using genome-wide case-control association approach, the current study aimed to determine whether genetic polymorphism(s) is/are associated with H. pylori infection among ethnic Malays from the north-eastern region of Peninsular Malaysia, a region with an exceptionally low prevalence for H. pylori infection and gastric cancer.

Methodology:
Twenty-three Malay subjects positive for H. pylori confirmed with urease test and histology were enrolled as "cases" and 37 subjects negative for H. pylori were "controls". Both groups were matched for age and environmental risks. Extracted DNA samples (QIAGEN, Germany) from the venous blood of study subjects were genotyped using the Human Mapping 50k xbal array (Affymetrix, USA). High throughput downstream analyses were then used to determine the significant SNP(s) associated with H. pylori infection.

Results:
Out of 20,361 SNPs filtered using the genotype association test, the top 1% (203) significant SNPs were selected for functional enrichment analysis. Of the 15 "enriched" SNPs, the rs10502974 which was located within the intronic region of Deleted in Colorectal Cancer (DCC) gene was the SNP most significantly associated with H. pylori infection (p=0.00549).

Conclusions:
Ethnic Malays is genetically susceptible to H. pylori infection and is possibly mediated through a genetic variation in the DCC gene.

Full text

124 GASTROINTESTINAL
Hepato-Gastroenterology 2013; 60:124-128doi 10.5754/hge12471
© H.G.E. Update Medical Publishing S.A., Athens
ABSTRACT
Key Words:
H. pylori; DCC
gene; Single
nucleotide
polymorphism;
Genome wide
association
study,

Deleted in Colorectal Cancer (DCC)
Gene Polymorphism is Associated with
H. pylori Infection among Susceptible
Malays from the North-Eastern Region
of Peninsular Malaysia
Sathiya Maran1, Yeong Yeh Lee2, Shuhua Xu3,
Nur-Shafawati Rajab1, Norhazrini Hasan4, Nazri Mustaffa2, Noorizan Abdul Majid5 and
Zilfalil Bin Alwi5
1Human Genome Center, 2Department of Medicine,
4Department of Immunology and
5Department of Paediatrics, School of Medical Sciences,
Universiti Sains Malaysia, Kubang Kerian, Kelantan, Malaysia
3Chinese Academy of Sciences and Max Planck Society Partner Institute for
Computational Biology, Shanghai Institutes for Biological Sciences, Chinese
Academy of Sciences, Shanghai, China
Corresponding author: Yeong Yeh Lee, MD MRCP (UK) DTM&H MMed FRCPE FACP, School of Medical Sciences,
Universiti Sains Malaysia, 16150 Kubang Kerian, Kelantan, Malaysia;
Tel.: +6097663000, Fax: +6097648277; E-mail: justnleeyy@gmail.com
: Using genome-wide case-control
association approach, the current study aimed to de-
termine whether genetic polymorphism(s) is/are as-
sociated with H. pylori infection among ethnic Malays
from the north-eastern region of Peninsular Malaysia,
a region with an exceptionally low prevalence for H.
pylori infection and gastric cancer. 
Twenty-three Malay subjects positive for H. pylori
       -
rolled as “cases” and 37 subjects negative for H. py-
lori were “controls”. Both groups were matched for
age and environmental risks. Extracted DNA samples
(QIAGEN, Germany) from the venous blood of study
subjects were genotyped using the Human Mapping
50k xba1 array (Affymetrix, USA). High throughput
downstream analyses were then used to determine
   -
tion.        

SNPs were selected for functional enrichment analysis.
Of the 15 “enriched” SNPs, the rs10502974 which was
located within the intronic region of Deleted in Colo-
        -
cantly associated with H. pylori infection (p=0.00549).
 Ethnic Malays is genetically susceptible
to H. pylori infection and is possibly mediated through
a genetic variation in the DCC gene.
INTRODUCTION
In Asia, the prevalence of H. pylori remains high with
rates above 70% reported from China, Korea, Vietnam
and Turkey (1-3). Compelling evidence from epidemio-
logical studies exist for H. pylori as the carcinogen for
the development of non-cardia gastric cancer (4). The
inter-relationship between H. pylori infection, gastric
cancer and genetic susceptibilities between different
populations is unknown. It is however known that a high
prevalence of H. pylori infection and gastric cancer runs
in parallel with the exception of the Indian population.
Despite having a high prevalence of H. pylori infection,
the gastric cancer rate in the Indians is paradoxically
low (5). There are populations with a low prevalence
of H. pylori infection and also a low incidence of gas-
tric cancer, for example the Malay population from the
north-eastern region of Peninsular Malaysia (6-8); the
reasons for this remain largely elusive.
There is evidence that ethnic Malays are susceptible
to H. pylori infection but environmental factors, and in
particular diet, may confer some protection (9). That
local practices may limit H. pylori infection was sup-
ported by a lower prevalence rate of H. pylori infec-
tion observed in both Chinese and Indian immigrants
in Kelantan compared to other larger cities (15.8% in
Kelantan compared to 76.1% in Kuala Lumpur) (7,10).
It is of interest that the incidence rate for oesophageal
and gastric cancer among the Malays are considerably
lower than that of Western Europe and North America
(11,12). This suggests that there may be intrinsic genet-
ic factors within the ethnic Malays that confer protec-
tion against the bacterium.
Two recent studies comparing the genetics of H. py-
lori strains obtained from members of the three major
ethnic groups resident in Malaysia supported the notion
that H. pylori was introduced to the Malays from the
immigrant Han Chinese and South Indian populations
(13,14). Whether the ethnic Malays have inherent ge-
netic susceptibilities to H. pylori infection is not known.
The current study aimed to determine the gene(s) in
which variant(s) can increase the risk of H. pylori infec-
tion among ethnic Malays from the north-eastern region
of Peninsular Malaysia using a genome-wide case-con-
trol association study.
METHODOLOGY
Study subjects
Malay subjects (age range 20-80 years) requiring

125Hepato-Gastroenterology 60 (2013)
upper endoscopy for gastrointestinal symptoms were
screened for study eligibility. Only subjects presented
with similar symptoms (including dyspepsia and/or
abdominal discomfort) and requiring the upper endos-
copy examination to exclude gastro-duodenal ulcer dis-
ease were included to ensure no ascertainment bias.
All Malay subjects included into the study must have
been born in Kelantan, resided within the region for at
least 3 generations and from different families but with
similar socio-economic and sociocultural backgrounds.
Study subjects positive for H. pylori infection on con-

as “cases” and subjects negative for H. pylori infection
      
above inclusion criteria were recruited into the study.
Exclusion criteria included an intake of antibiotics 3
months prior to upper endoscopy test, positive family
history of H. pylori infection and gastric cancer, previ-
ous history of H. pylori infection, and chronic psychiat-
ric and medical conditions including cancer. Informed
consent was obtained from all subjects prior to their
enrolment in the study.
The exceptionally low rate H .pylori infection within
the region severely limits the number of H. pylori posi-
tive that could be collected. Only 23 Malay subjects were
  
the “controls” were sought to compensate for a low
         -
vironmental risks, only subjects matched for age, socio-
economic and sociocultural backgrounds were included.
Only 37 Malay subjects were eventually included as
“controls” as a result of strict inclusion criteria along
with drop-out subjects who did not consent to the study
and exclusion of poor blood samples. The study was ap-
proved by the Human Research and Ethics Committee of
Universiti Sains Malaysia.

All recruited subjects were called up by one of the
investigators (SM) to have their 1mL of venous blood
taken during the study day. Unlike conventional meth-
   
commercially available kits. The blood was collected in
EDTA bottle and was transported immediately to a fa-
cility (Human Genome Centre, Universiti Sains Malaysia,
Kelantan) to be stored at 4°C. Subsequently, DNA for all
recruited cases and controls was isolated using QIAamp
DNA Blood Mini Kit (QIAGEN, Hilden, Germany).

The isolated DNA from all recruited cases (n=23)
and controls (n=37) were processed and genotyped
using the Affymetrix 50k xba1 array (Affymetrix, USA)
according to the instructions provided in the Affym-
etrix GeneChip Human Mapping 100K Assay Manual
(15). DNA samples with <90% genotype call rate were
dropped from the analysis. Single nucleotide polymor-
phisms (SNPs) that had a minor allele frequency of <5%,
that failed to genotype in >5% of samples, and with a
Hardy-Weinberg Equilibrium p value of <0.5 were also
excluded from subsequent analysis.

       
the SNPs was performed with the Bayesian Robust Line-

algorithm from the Affymetrix® Genotyping Console™
software version 4.0 (Affymetrix, USA). Quality control
-
tering tool of SVS Golden Helix Bioinformatics Tools ver-
sion 7.4 (Golden Helix Inc, Montana). The genotype as-
sociation test between the genes and H. pylori infection
was evaluated for every single SNP in each gene with
SVS Golden Helix Bioinformatics Tools. False Discovery
Rate (FDR) and Bonferroni adjustments were then used
for multiple hypothesis testing.
      -
cant SNP(s) associated with H. pylori infection were
carried out using The Database for Annotation, Visuali-
 -
sion 6.7 (SAIC-Frederick Inc, Frederick, MD, USA). Data
analysis using SVS Golden Helix Bioinformatics Tools
and enrichment analysis using the DAVID software are
-
-
ic, phenotypic and clinical data more validly and much
easier (16). The “enriched” SNPs were then tested using
the FAMHAP program (IMBIE, Bonn, Germany) which

RESULTS
A total of 56,000 SNPs were generated using the

with the SVS Golden Helix Software resulted in 20,361
SNPs which were different between study groups. Out of
20,361 SNPs from the genotype association test, the top
 
were selected for functional gene enrichment analysis.
      -
notation Clustering Analysis” tool within the DAVID
software allowed a division of 80 annotation clusters
of genes based on enrichment scores. The annotation
cluster with the highest enrichment score of 3.89 was
       
which this cluster has 14 GOTERMS (Table 1). Out of
the 14 GOTERMS obtained, the GOTERM of “Cell Projec-


The group with “Cell Projection Morphogenesis” con-
sisted of 15 SNPs; the x2 p value of these SNPs was deter-
mined using the FAMHAP program (17) (Table 2). Mean-
while, the functions of these SNPs were determined
using the DAVID’s “Functional Annotation Chart” tool
(18) (Table 2). Of these 15 SNPs, the SNP rs10502974
-
ation with H. pylori infection. The SNP rs10502974 was
mapped to the chromosome 18q21within the intronic
region of the Deleted in Colorectal Carcinoma (DCC)
gene (19).
DISCUSSION
Genome wide association study using the Affymetrix
50k xba1 array can generate a large number of SNPs,
but this is not useful to detect any association with dis-
eases or infection. These SNPs can be “enriched” using
computational techniques, in our case the DAVID bioin-
-
cant SNP(s) associated with H. pylori infection among
the susceptible Malays. There were 15 candidate SNPs
after enrichment analysis and the rs10502974 was the

In the current study, the location for SNP rs10502974
was found to be in the intronic region of 18q21.3 of DCC
gene (19). Intronic polymorphisms harbor functional
   
genes that host them (20,21).
The DCC gene encodes a 170 to 190kDa protein of the
Immunoglobulin superfamily. The role of DCC gene as a
tumor suppressor in particular colorectal cancer has re-
mained controversial over many years. It is known that
DCC and netrin-1 are essential for axonal guidance but
more recently DCC has been found also to function as a
dependence receptor and to induce apoptosis via this
mechanism (22,23). Adhesion of the H. pylori bacteri-

essential steps for the H. pylori infection in human be-
ings (24). In 2006, Martin et al. showed that the protein
products from the DCC gene could stimulate cell-cell
DCC gene and H. pylori in Ethnic Malays

126 Hepato-Gastroenterology 59 (2012)
adhesion (25). Studies had also shown that expression

pylori infected gastric tissues (26).
Alteration of tumor suppressor function of the DCC
gene in susceptible Malay individuals to H. pylori may
potentially increase the risk for developing gastric can-
cer. Studies had shown that there is aberrant methyla-
tion of the DCC gene observed in the course of gastric
carcinogenesis (27,28). The current study provides a
hint that there is a genetic link between H. pylori in-
fection and gastric cancer mediated by the DCC gene
variant among susceptible Malays and possibly other
susceptible populations. This will be validated and con-

are currently performing.
There are limitations to be addressed in the current
         
H. pylori infection rate among the local Malays and the
strict inclusion and exclusion criteria severely limited
subject recruitment. However, the well-matched group
in terms of environmental risks compensates for the
      
gene is involved in gastric carcinogenesis and is also
shown to be involved with H. pylori susceptibility in our
study suggests a possible genetic linkage between the
two. Secondly, further validation studies in a larger co-
     -
    -
tional protein analysis or gene expression analysis may

gene. These studies are currently ongoing in our center.
In conclusion, the current study suggests that ethnic
Malays from the north-eastern region of Peninsular
Malaysia, a region known for extremely low prevalence
of H. pylori infection, are genetically susceptible to the
bacterium, possibly mediated through a genetic varia-
tion in the DCC gene.







Category Term p value Fold
Enrichment
GOTERM_BP_FAT Cell projection
morpogenesis 1.67x10-06 5.019
GOTERM_BP_FAT Cell part
morphogenesis 2.80x10-06 4.803
GOTERM_BP_FAT Cell morphogenesis 2.83x10-05 3.684
GOTERM_BP_FAT Cell projection
 4.14x10-05 3.564
GOTERM_BP_FAT Axon guidance 4.63x10-05 6.896
GOTERM_BP_FAT Neuron projection
morphogenesis 5.70x10-05 4.619
GOTERM_BP_FAT Neuron projection
development 6.70x10-05 4.163
GOTERM_BP_FAT Cellular component
morphogenesis 9.75x10-05 3.304
GOTERM_BP_FAT Axonogenesis 1.21x10-04 4.672
GOTERM_BP_FAT
Cell morphogenesis
involved in
differentiation
1.91x10-04 4.032
GOTERM_BP_FAT 1(5.6) 0 1(5.6)
GOTERM_BP_FAT
Cell morphogenesis
involved in neuron
differentiation
2.32x10-04 4.315
GOTERM_BP_FAT Neuron development 8.76x10-04 3.144
GOTERM_BP_FAT Neuron differentiation 0.007 2.433
GOTERM_BP_FAT Cell motion 0.123 1.726
ACKNOWLEDGEMENTS
This work was supported by Fundamental Research
Grant Scheme (FRGS) 203/PPSP/6171121, 1001/
PPSP/812016 and 1001/PPSP/8122022 Universiti
Sains Malaysia. Shuhua Xu was supported by the
National Science Foundation of China (30971577,
31171218) and Shanghai Rising-Star Program
(11QA1407600). Shuhua Xu also gratefully acknowl-
edges the support of K.C. Wong Education Foundation,
Hong Kong. We also would like to thank Wenfei Jin
and Dongsheng Lu from Chinese Academy of Sciences
and Max Planck Society Partner Institute for Computa-
tional Biology for their remarkable help in GWAS data
analysis.
Sathiya Maran, Yeong Yeh Lee et al.

127Hepato-Gastroenterology 60 (2013)
H. pylori

 rsID  
χ2

SNP_A-1753707 rs10496563  2q14 0.067 regulation of epidermal proliferation
and skin tumorigenesis
SNP_A-1647724 rs1268328 RAB3A interacting protein
(rabin3) 12q14.3 0.864 
membrane transport
SNP_A-1661578 rs9319171 SLIT and NTRK-like family 13q31.1 0.494 suppresses neurite outgrowth
SNP_A-1671688 rs4474385 ankyrin G 10q21 0.418
cell motility, activation, proliferation,
contact and the maintenance of

SNP_A-1681788 rs10503813 clusterin 8p21-p12 0.389
lipid transport, epithelial cell
differentiation, tumorigenesis, and
apoptosis
SNP_A-1699297 rs10502974 DCC 18q21.3 0.00549 tumor suppressor
SNP_A-1698719 rs1533949  2q21 0.761 axonal outgrowth and fasciculation
SNP_A-1758143 rs221493 neurexin 3 14q31 0.556 involved in cell recognition molecules in
the nerve terminal
SNP_A-1677344 rs10487849 Neuronal cell adhesion
molecule 7q31.1-q31.2 0.13 axon growth
SNP_A-1749641 rs2120441 one cut homebox 1 15q21.1-q21.2 0.153 regulator of hepatocyte specific genes
SNP_A-1721159 rs1941158 protein tyrosine phosphatase 18p11.2 0.552 signal transduction and growth control
SNP_A-1751019 rs10435337 reelin 7q22 0.891
modulating the structure and function
of retinal synaptic circuitry and of the
central nervous system and synaptic
plasticity
SNP_A-1667186 rs200032 SEMA5A 5p15.2 0.206 involved in axonal guidance during
neural development
SNP_A-1657528 rs2896092 tetratricopeptide repeat
domain 8 14q31.3 0.469 planar cell polarity

The DCC
DCC gene and H. pylori in Ethnic Malays

128 Hepato-Gastroenterology 60 (2013)
REFERENCES
1. : Prevalence of Helicobacter

urban Beijing, China. Helicobacter 2009; 14:128-133.
2. : Epidemiology of
Helicobacter pylori infection and public health implications.
Helicobacter 2011; 16 Suppl 1:1-9.
3. : Current prevalence
of intestinal metaplasia and Helicobacter pylori infection in
dyspeptic adult patients from Turkey. Hepatogastroenterol-
ogy 2010; 57:1563-1566.
4. .: Helico-
bacter pylori infection and the risk of gastric carcinoma. N
Engl J Med 1991; 325:1127-1131.
5.  The enigma of Helico-
bacter pylori infection and gastric cancer. Indian J Gastroen-
terol 2010; 29:95-100.
6.  Helicobacter pylori
infection in north-eastern peninsular Malaysia. Evidence for
an unusually low prevalence. Scand J Gastroenterol 1994;
29:209-213.
7. : Further evidence
for an exceptionally low prevalence of Helicobacter pylori
infection among peptic ulcer patients in north-eastern penin-
sular Malaysia. Trans R Soc Trop Med Hyg 2001; 95(1):24-27.
8. : Chronic atrophic antral
gastritis and risk of metaplasia and dysplasia in an area with
low prevalence of Helicobacter pylori. Indian J Gastroenterol
2009; 28:49-52.
9. : Sociocultural and
dietary practices among Malay subjects in the north-eastern
region of Peninsular Malaysia: a region of low prevalence of
Helicobacter pylori infection. Helicobacter 2012; 17:54-61.
10.  The racial cohort phenomenon:
seroepidemiology of Helicobacter pylori infection in a multi-
racial South-East Asian country. Eur J Gastroenterol Hepatol
2001; 13:177-183.
11. 
DY: Incidence of esophageal carcinoma among Malays in
north-eastern region Peninsular Malaysia: an area with an
exceptionally low prevalence of Helicobacter pylori infection.
Dig Dis Sci 2011; 56:1438-1443.
12.  Differences in the pat-
tern of gastric carcinoma between north-eastern and north-

pylori prevalence. Med J Malaysia 2004; 59:560-561.
13. : Popu-
lation structure of Helicobacter pylori among ethnic groups
in Malaysia: recent acquisition of the bacterium by the Malay
population. BMC Microbiol 2009; 9:126.
14.  Distinct cagA EPIYA
motifs are associated with ethnic diversity in Malaysia and
Singapore. Helicobacter 2009; 14:256-263.
15. 
http://www.affymetrix.com/ [Accessed October 20, 2011].
16.  Systematic and
integrative analysis of large gene lists using DAVID bioinfor-
matics resources. Nat Protoc 2009; 4:44-57.
17.  Maximum-likelihood estimation of
haplotype frequencies in nuclear families. Genet Epidemiol
2004; 27:21-32.
18. : Bioinformatics
enrichment tools: paths toward the comprehensive functional
analysis of large gene lists. Nucleic Acids Res 2009; 37:1-13.
19. http://compbio.cs.queensu.ca/F-SNP/ [Ac-
cessed October 20, 2011].
20.Functional intronic polymorphisms: Buried
treasure awaiting discovery within our genes. Hum Genomics
2010; 4:284-288.
21.  The function of introns. Front Genet
2012; 3:55.
22.Midline axon guidance and human genetic
disorders. Clin Genet 2011; 80:226-234.
23.  DCC constrains tumour
progression via its dependence receptor activity. Nature
2011; 482:534-537.
24.  Molecular mechanisms
of gastric epithelial cell adhesion and injection of CagA by
Helicobacter pylori. Cell Commun Signal 2011; 9:28.
25. 
: DCC regulates cell adhesion in human colon

Cell Biol 2006; 85:769-783.
26. 
Helicobacter pylori infection is associated with downregula-
tion of E-cadherin, a molecule involved in epithelial cell adhe-
sion and proliferation control. J Clin Pathol 1998; 51:410-412.
27.: Methylation of the DCC
gene is lost in advanced gastric cancer. Anticancer Res 2010;
30:107-109.
28.  Alterations of tumor suppresor and tumor-related
genes in the development and progression of gastric cancer.
World J Gastroenterol 2006; 12:192-198.
Sathiya Maran, Yeong Yeh Lee et al.