Identification of the polymorphism rs4072037 of MUC1 gene in patients with gastric cancer

JOURNAL OF MEDICAL RESEARCH

IDENTIFICATION OF THE POLYMORPHISM RS4072037 OF
MUC1 GENE IN PATIENTS WITH GASTRIC CANCER
Nguyen Thi Ngoc Lan, Nguyen Van Tan, Ta Thanh Van, Dang Thi Ngoc Dung
Hanoi Medical University
Gastric cancer, a malignant disease, is the fourth most common cancer and second most common
cause of cancer death. The pathogenesis of gastric cancer is a complex process, including Helicobacter
pylori infection, genetic factors as well as many other factors. Among them, Single Nucleotide Polymorphism
(SNP) is one of the most interesting topics nowadays. Few studies on rs4072037 polymorphisms of
the MUC1 gene have been done in different populations, particularly in Asian populations, which have
indicated its role and significance as a susceptibility factor to gastric cancerL. Our objective was to see if
this varitation can be detected in Vietname patients with gastric cancer. Methods: 130 patients diagnosed
of gastric cancer and 130 healthy controls were recruited from the 4 largest hospitals in the North
Vietnam. Genetic polymorphism at rs4072037 was determined by PCR-RFLP. Results: Three types of
SNPs were found as the allele type (G vs A: OR = 0.42, 95% CI: 0.25-0.72), the heterologeneous gene
type (AG vs. AA, OR = 0.47, 95% CI: 0.28-0.77) and dominant gene type (AG + AA vs. GG: OR = 0.67,
95% CI: 0.47-0.97). Conclusions: Allele G of rs4072037 associated with the reduction of gastric cancer risk.
Key words: gastric cancer, polymorphism, rs4072037, MUC1 gene

I. INTRODUCTION
In 2012, there were about 1 million new

to other South- East Asian countries [1]. The

cases of gastric cancer (6.8% of all incident

mortality rates of gastric cancer for both males

cancer cases). As the third leading cause of

and females were also among the highest of

cancer death and the fifth most common cancer

cancers in Vietnam, surpassed only by liver

worldwide, gastric cancer can be considered

cancer and lung cancer [13]. This has been

one of the world’s pressing medical challenges

argued to be attributable to the high prevalence

[2]. More than 70% of gastric cancer cases

of Helicobacter pylori (seen in as much as 75%

occur in developing countries, especially

of Vietnamese adults), in combination with

in East Asia. Vietnam had the highest rate

other risk factors such as smoking, obesity,

compared to other South-East Asian countries

socioeconomic status and genetic factors [8],

In Vietnam, the ASR has been reported to

[16]. A single nucleotide polymorphism, or

be about 16.3 per 100,000 people of both

SNP, is a variation at a single position in a DNA

sexes, which was the highest rate compared

sequence among individuals. One variant gene
is called SNP if more than 1% of a population

Corresponding author: Nguyen Thi Ngoc Lan, Ha-

does not carry the same nucleotide at a specific

noi Medical University

position in the DNA sequence. SNP may occur

Email:
Received: 27/11/2018
Accepted: 12/03/2019

8

within coding or noncoding regions. They
can relate to cancer-causing mechanisms in

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sporadic cancer and SNPs have a important
significance for community health.

One-hundred

and

thirty

patients

with

gastric cancer were selected by the following

The rs4072037 A > G SNP located in the

criteria: 1) being diagnosed with gastric cancer

5′ UTR of the 2nd exon of MUC1 gene is

(histopathology confirmation); 2) agreeing

predicted to have an effect on the splicing

to participate in the study; 3) being able to

of the primary transcripts, which in turn

communicate with the interviewers. One-

determines the type of variants. The results

hundred and thirty subjects without tumor

of researches demonstrated that the G allele

(controls) were recruited from endoscopy

belongs to variant 2, while the A allele belongs

departments (endoscopy confirmation with

to variant 3. The structural differences between

normal or only benign, acute gastritis). Medical

these two variants lead to insertion/deletion

records of patients and controls were collected

of nine amino acids encoded by the second

under the agreement of the administrative

exon, which are involved in the N-terminal

departments of the 4 hospitals.

signal peptide. This differential signal peptide

2. SNP genotype

may lead to a different function of the encoded
variant protein. Also, the A allele reduces
the transcriptional activity, which may result
in decreased MUC1 expression [7]. The
rs4072037 also influences the transcriptional
activity of the MUC1 gene promoter; the A
allele associated with gastric cancer reduced
the transcriptional activity, which may result in
decreased MUC1 expression [10]. Therefore,
low expression of MUC1 may cause a
reduction in its barrier function in the stomach
and subsequently increases gastric cancer
susceptibility.
The purpose of this study is to find whether
rs4072037 is a risk factor in Vietnamese
patients with gastric cancer.

II. MATERIALS AND METHODS
1. Study subjects
A cross-sectional study was conducted
from May 2016 to May 2018 in four hospitals
in Hanoi, Vietnam, include: National Cancer
Hospital, Hanoi Medical University Hospital,

108 Military Central Hospital, and Viet Duc
Hospital.

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A 20 – minute interview via telephone or
face-to-face was conducted with each patient,
in which a research questionnaire was used
to

collect

information

socioeconomic

regarding

patient’s

characteristics,

disease

characteristics. The purpose, the benefits,
the drawbacks, and the confidentiality aspect
of patients in the study were introduced to
the patients when they were asked to join the
study. Patients’s consent was ensured prior to
the interview.

Patients were asked to self-report their
information about gender, age, and medical
history.
Blood collection for SNP analysis
2ml peripheral blood was taken from each
patient and put into EDTA tube. All collected
samples were stored in appropriate condition
and transported to the Medical Laboratory
Quality Control – Hanoi Medical University for
gene analysis.
SNP genotypes:
Genomic DNA was
peripheral

blood

using

extracted

from

Exgene™

Blood

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SV (Gene All, Korea), according to the

perform the repeated assays, and the results

manufacturer’s

PCR-

were 100% concordant.

restriction fragment length polymorphism assay

3. Statistical methods

instructions.

The

was used to detect the SNP rsA4072037G
with the specific forward and reverse primers
5 ’ - A A G G C C TAT G G G C A G A G A G A - 3 ’
and

5’-ACGCTGCTGGTCATACTCAC-3’,

respectively. The 332-bp PCR products were
digested overnight with the restriction enzyme
AlwNI (New England BioLabs, Beverly, MA)
and then separated on 2% agarose gel. The
A allele resulted in 2 fragments of 223 and 109

bp, and the G allele produced 1 fragment of
332 bp.
About 10% samples which without knowing
the subject’s case or control status were
Genotyped by direct sequencing method
base on Sanger principle. Additionally, 10% of
the samples were also randomly selected to

Student’s t-test or chi-square test was used
to evaluate differences in the distributions of
demographic characteristics, selected variables
and genotypes of the rs4072037 between the
cases and controls. The associations between
the 3 genotypes of this SNP and risk of gastric
cancer were estimated by computing the odds
ratios (ORs) and their 95% confidence intervals
(CIs) with unconditional logistic regression
analyses. All the above statistical analyses
were performed with SPSS (v.16.0; IBM SPSS
Statistics).
4. Ethics approval
This study was approved by the Ethics
Council of Hanoi Medical University.

III. RESULTS
Table 1. The socioeconomic characteristics of patients
Case group

 

Control group

n

%

n

Male

79

60.8

69

53.1

Female

51

39.2

61

46.9

The average age

Mean

SD

Mean

SD

57.2

10.6

56.4

11.5

 

p

%  

Gender
0.21

0.51

Table 1 describes the socioeconomic characteristics of patients. There is no statistically
significant difference in gender between case and control groups (p = 0.21). The mean age of the
case group had no significant difference compared to the control group (p = 0.51)

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Figure 1. Electrophoresis of productions treatedby enzyme AlwNI
M: 100bp ladder; K1 đến K14: Samples of patients with gastric cancer; (+): Positive control and
(-): Negative control
Figure 1 describes electrophoresis results after using PCR-RFLP with K3, K5 samples have
GG genotype (332bp DNA fragment); K4, K7, K8, K11, K12: have AA genotype (223bp and 109bp
DNA fragments); K1, K2, K6, K9, K10, K13 and K14 have AG genotype (332bp, 223bp, 109bp DNA
fragments).

Figure 2. Sequencing results ofDNA fragment containing rs4072037
Figure 2 describes the results of sequencing analysis of K4, K5, K6 samples after PCR-RFLP.
Product bands are clear and easy to determine the genotypes of these patients. The results were
100% concordant.
Table 2. Genotype and allele characteristics of rs4072037
Case
n

 

p

Control

%

n

%

 

Genotype
AA

68

52.3

44

33.8

AG

48

36.9

73

56.2

GG

14

10.8

13

10

0.006

Allele

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Case
n

 

p

Control

%

n

%

 

A

184

70.8

161

61.9

G

76

29.2

99

38.1

0.033

Table 2 describes genotypes and alleles of rs4072037 between cases and controls. In the
case group, AA genotype has highest rate (52.3%) and was significantly differenent between the

distribution genotypes between cases and controls (p = 0.006). Allele A in case group was higher
than in control group with p = 0.033.
Table3. Gastric cancer risk of genotype and allele of rs4072037
Genotype and allele

Case

Control

OR

95% CI

AG vs AA

48/68

73/44

0.42

0.25 - 0.72*

GG + AG vs AA

62/68

86/44

0.47

0.28 - 0.77*

G vs A

76/184

99/161

0.67

0.47 - 0.97*

*p < 0.05
Unconditional logistic regression analysis revealed that the rs4072037 AG heterozygote was
associated with a significant decrease in the risk of gastric cancer (OR: 0.42, 95% CI: 0.25 – 0.72),
and the rs4072037 GG + AG was associated with a significantly decrease risk (OR: 0.47, 95%CI:
0.28 - 0.77) and the rs4072037 A allele was ascociated with a significantly decreased risk (OR =
0.67, 95%CI: 0.47 - 0.97) (Table 3).

IV. DISCUSSION
In this study, the association of genetic
variants of rs4072037 in MUC1 gene with
gastric cancer susceptibility was found in
130 gastric cancer cases and 130 controls in
Vietnamese population. The results show that
MUC1 rs4072037 G allele was significantly
associated with decreased risk of gastric
cancer. This suggests the rs4072037 SNP can
be considered as a marker for diagnosis and

screening for Vietnamese patients with gastric
cancer risk..
The mean age of the case group was similar
to other research population in Vietnam and
East Asia region. Le Viet Nho et al’s research
(2014) had average age 58.9 ± 13.8, and 59.2
12

± 11.9 in Jeong O et al’s research (2011) [5],
[3]. The gender ratio in our research was 1.55/1
which was lower than the studies conducted
by Trinh Hong Son and Wanebo (1.75/1) [14],
[15].
In the case group, the rate of AA, AG and
GG genotypes was as 52.31%, 36.92% and
10.77%, respectively. In control group, the
highest rate was the AG genotype with 56.15%,
followed by AA and GG with 33.85% and
10%, respectively. There was a stastistically
significant difference between genotype
distributions between these groups. Genotype
distribution in our research was similar to other
researches such as Hye-Rim Song (2014),
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Hanze Zhang et al (2011), AA genotype was
the most common genotype in research
population 79.2% and 74.2%, respectively,

less common were AG genotype 19.1% and
23%, respectively [12], [17]. The genotype
rate in control group was similar to research
of Yanbin Jia et al (2010). Both groups have
AG genotype with highest common rate, up to
51.6% [4]. Our results show that rs4072037
G allele leads to reduce gastric cancer in the
allele type (G vs A: OR = 0.42, 95% CI: 0.25
- 0.72), in the heterologous gene type (AG
vs. AA, OR = 0.47, 95% CI: 0.28 - 0.77), in
dominant gene type (AG + AA vs. GG: OR =

in the stomach, and subsequent increases in
gastric cancer susceptibility.
This strongly demonstrated rs4072037 G
allele was a protective factor in gastric cancer.
These findings suggested that this SNP may
be used as biomarker to screen Vietnamese
patients with gastric cancer risk.

V. CONCLUSION
The SNP rs4072037 G allele has a
significant association with reduced risk of
gastric cancer and this SNP may be used as
biomarker to screen Vietnamese patients with
gastric cancer risk.

0.67, 95% CI: 0.47 - 0.97). These results were

Acknowledgments

similar to the results of research groups from

This research is funded by Vietnam National
Foundation for Science and Technology
Development (NAFOSTED) under grant
number 106-YS.02-2015.37.
We wish to thank clinicians from National
Cancer Hospital, Hanoi Medical University
Hospital, 108 Military Central Hospital, and Viet
Duc Hospital for their excellent assistance in
recruitment of patients.
We thank scientists and technichians of
Biochemistry Department and Quality Control
Center for Medical Laboratory at Hanoi Medical
University for their support during the research.

Japan, Korea and China with OR arrange from
0.26 - 0.69 [9], [17], [6].
As we know, MUC1 is a member of mucin
family and is a transmembrane mucin. It is a
multi-functional protein involved in mucosal
lubrication, protection from pathogens, signal
transduction and cell-cell interaction. MUC1
was considered as oncogene because it has
role in cell growth, anchorage independence,
cell migration, anti-apoptosis and drug
resistance of cancer cells [10], [11].
The rs4072037 A > G SNP effects on
the splicing of the primary transcripts and

determines the type of variants. The G allele
of this SNP determines variant 2, while the
A allele belong to variant 3. Two variants
differences about nine amino acids encoded
by the second exon and lead to a different
function of the encoded variant protein. Also,
the A allele reduces the transcriptional activity
and decreased MUC1 expression [7]. The
rs4072037 A allele associated with gastric
cancer reduced the transcriptional activity of the
gene product, which may result in decreased
MUC1 expression, reduction in barrier function
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