Download Association of (rs1801133) Polymorphism with Breast

International Journal of Pure and Applied Biomedical Sciences; Vol. 2016.1.2; pp. 24-35.
Association of (rs1801133) Polymorphism with Breast Cancer in
Egyptian Patients
Noha Mohamed Saida ,* , Afaf Mohammed El-saidb, Besheer El-Sayed
Bayomyc and Mona Mohammed Ibrahima
a
Biochemistry Division, Chemistry Department, Faculty of science, Zagazig University, Zagazig,
Egypt.
b
Children Hospital, faculty of Medicine-Mansoura University, Mansoura, Egypt.
c
Chemistry Department, Faculty of science, Zagazig University, Zagazig, Egypt.
Date of revised paper submission: 04/12/2016; Date of acceptance: 15/12/2016
Date of publication: 20/12/2016; *First Author / Corresponding Author; Paper ID: BS16203.
Reviewers: Baqri, S. R., India; Ping Chen, Yancheng, China
Abstract
This study provides a trial to find an association between breast cancer and MTHFR
gene. This gene is believed to be involved in the susceptibility to breast cancer.
This study is a case-control study for examining the role of MTHFR gene polymorphism (rs1801133)
in breast cancer patients. We used polymerase chain reaction allele refractory mutation system
polymorphism (PCR-ARMS) method for the genotyping.
Our results showed that the frequency of the minor allele of this site (T) is 53% in the breast cancer
cases and 40% in the controls. There is high significant difference between patients and controls in
genotypes distribution (p= 0.002). The results also, show significant association between breast
cancer development and minor genotype (TT) under homozygote and heterozygote models (OR =7.33;
CI 95% (1.46-36.66); P=0.011) and (OR =5.69; CI 95% (1.93-16.8); P= 0.001) respectively. In
addition, there is very high association between breast cancer risk and TT genotype under dominant
model (OR =5.85; CI 95% (2.00-17.12); P=0.00).
Our results suggested that MTHFR gene polymorphism (rs1801133) is important candidate
polymorphism for susceptibility to breast cancer in Egyptian population. We tried to go step by step in
ARMS-PCR method in genotyping study.
Keywords: Breast Cancer. (rs1801133), ARMS- PCR, Genotyping, Polymorphism C677T.
1. Introduction
The science revolution in discovering the genetic relation to the different diseases is
based on many studies comparing between patients and control. Revealing the variation between
patients and control requires genotyping to determine differences in the genetic make-up for each
individual [1]. Genotyping has a great importance in determining the association between gene
variants and diseases [2]. The various methods are used for genotyping as restriction fragment length
polymorphism identification (RFLP) of the genomic DNA, DNA sequencing, random amplified
polymorphic detection (RAPD) of the genomic DNA and allele refractory mutation system (ARMS).
These methods differ in cost, time and effort needed accuracy, lab and devices preparation [3-6].
Single nucleotide polymorphism (SNP) is the most common genotyping form used in
the studies. It measures the genetic variations of single nucleotide polymorphisms (SNPs) between
members of the same species [7].
It is important to highlight the difference between mutation and polymorphism words.
A mutation is any change in a DNA sequence away from normal. In contrast, a polymorphism is a
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DNA sequence variation which is common in the main population. So in this case no single allele is
taken as the standard sequence. Instead there are two or more equally acceptable alternatives.
SNPs are biological markers which can help in determining the location of the genes
associated with disease. SNPs can affect gene function when they occur within the gene or in a
regulatory region for gene. So SNPs is believed to play a more direct role in disease [8].
SNPs may help in predicting an individual’s response to drugs, environmental factors
such as toxins, and risk of developing certain diseases. Researcher can use SNPs to determine the
inheritance of disease genes within families [3].
The increasing need for large-scale genotyping applications of single nucleotide
polymorphisms (SNPs) requires the development of low-cost technologies. Standard ARMS
(amplification refractory mutation system) is one of the low cost technology used for the SNPs [9].
The amplification refractory mutation system (ARMS) or PCR amplification of
specific alleles (PASA)) (i.e., known as Allele-specific polymerase chain reaction (AS-PCR)), is the
PCR-based method that can be employed for detecting the known SNPs [10].
The concept of AS-PCR was initially given by Newton et al., [11], approximately six
years after the invention of PCR.
Conventional or standard ARMS use two PCR complementary reactions with 3
primers. 1st primer is common for the two alleles, 2nd primer is specific for the wild or the major
allele in the population, and 3rd primer is specific to the minor allele (not to use mutant word) in the
population. The summary of this method protocol is to make two separate PCR reactions. The 1 st
reaction contains DNA template, PCR master mix, common primer, and the primer specific for the
major allele. While the 2nd reaction contain DNA template, PCR master mix, common primer, and
the primer specific for the minor allele. The PCR products are visualized after mixing with Ethidium
Bromide on U.V lamp using gel electrophoresis technique. If bands appear in both two reactions, the
sample is heterozygote. If band appear in only in 1st reaction, the sample is homozygote for the major
allele. If band appear in only in 2nd reaction, the sample is homozygote for the minor allele [12].
(rs1801133) polymorphism plays a great role in the susceptibility to breast cancer. It
is used in this work as an example for using standard ARMS-PCR genotyping method. It plays a great
role in the susceptibility to breast cancer [13]
In this article the authors are trying to present a guide for standard ARMS with the
small details that can be hidden for the beginners in this field. Actually, most studies depend on
presenting the results without making a zoom on the details that can give accurate result with less
effort.
2. Subjects and Methods
2.1 Subjects
The present analysis included the one hundred five Egyptian women with
histologically proven diagnosis of the breast cancer between Jun-2012 and Dec-2014 from oncology
clinics of Mansoura University Hospital and Zagazig University Hospital of Egypt. The patients were
evaluated according to the staging system of the American Joint Committee on Cancer (AJCC) TNM
(tumor, node, and metastasis) system and histological diagnosis was performed according to world
health organization (WHO). The control group was 42 age-matched women with no signs or
symptoms of malignancy; they were randomly selected from various clinics at zagazig and mansoura,
Egypt. An informed consent for the experimental use of specimens was obtained from all participants.
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Fig (1): The steps of the work plan for both preparatory stage and working stage.
ST
2.2 1
STAGE: Preparatory Stage
2.2.1 Patients Data Collection
The clinical archive files of the breast cancer patients were available as a source for
their data. Also, the participants completed a questionnaire about their medical and reproductive
histories in particular. Breast cancer cases filled in the questionnaire at the time of clinic appointment
whereas controls were interviewed at the medical center at the time of enrollment. The data related to
reproductive history (including parity, gravidity, history of breastfeeding, age at first-full term
pregnancy, age at menarche and menopause, hormone replacement therapy, menstrual cycle
regularity), use of oral contraceptives (OC) ("Ever use" of OC was observed as at least 3 months of
use), age at first OC use and duration of OC, anthropometric characteristics (height, waist and hip
measurements weight, weight at the age of 20 yrs.), the smoking status ("ever smoker" was observed
as at least 1 year of the smoking) were collected.
2.2.2 Blood Sampling
Five ml of blood sample was taken by all the participants under full aseptic condition
and was divided into 2 parts; 2 ml of whole blood was collected in sterile EDTA containing tubes for
DNA extraction, and the rest was left for 30-60 minutes for spontaneous clotting at room temperature
then centrifuged at 3000 rpm for 10 minutes. Serum samples were separated into another set of tubes
and kept frozen at -20 °C.
2.2.3 Polymorphic Site Data Collection
Choosing the polymorphic site for the research is very important matter. MTHFR
(C667T) (Ala222Val) (rs1801133) has been proven to have a relation with other disease. This
polymorphism is one of MTHFR gene polymorphisms that have a clinical relevance. It falls in the
coding sequence of the MTHFR gene and represents a missense substitution. We made access to this
polymorphism site in NCBI (http://www.ncbi.nlm.nih.gov/snp/?term=rs1801133). The gene code in
gene database in NCBI is (4524). The polymorphism ID in SNP database in NCBI is rs1801133. It is
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published in 357 study indexed in pubmed database with uncertain significance. According to the
provided data in all these databases; this polymorphism is a substitution in the amino acid alanine
(GCA, GCC, GCG, and GCT) to the valine (GTA, GTC, GTG, GTT) in the nucleotide number 2. So
the single nucleotide substitution (SNP) is from C (major allele) and T (minor allele).
2.2.4 Genotyping Method Selection
The genotyping method used is a standard ARMS- PCR.
2.2.5 Primer Design
The primer used was according to that previous published (ref) using t-ARMS-PCR.
We checked the specificity using blast program in NCBI
(http://www.ncbi.nlm.nih.gov/tools/primer-blast/index.cgi?LINK_LOC=BlastHome). In addition, we
used standard ARMS-PCR with 2 pairs of primers in two separate reactions.
Fig (2): A photo taken for the primers result for C allele and the expected PCR product size of the
chosen polymorphic site (rs1801133) using BLAST software in NCBI database.
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Fig (3): A photo taken for the primers result for T allele and the expected PCR product size of the
chosen polymorphic site (rs1801133) using BLAST software in NCBI database.
2.2.6 Annealing Temperature Calculation
Annealing temp (Ta= ±5 Tm) used is 57 C. it was chosen after several PCR gradient
trials to the calculated Tm by online calculators as from the sources.
(http://www6.appliedbiosystems.com/support/techtools/calc/)
(http://www.promega.com/a/apps/biomath/index.html?calc=tm)
(http://www.biophp.org/minitools/melting_temperature/demo.php)
2.2.7 Amplicon Size Calculation (PCR product calculation)
Amplicon size was calculated according to blast program during checking primer
specificity.it is the difference in base pair number between start of forward primer and the end of
reverse primer
For C allele (according to figure ) = 505-259=272 bp
For T allele (according to figure) =640-476=189bp
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2.3 2ND STAGE: Working stage
2.3.1 DNA Isolation
Genomic DNA was extracted from the EDTA whole blood using genomic DNA
Purification Kit (FERMENTAS). The quality of genomic DNA was tested using agarose gel
electrophoresis. DNA was stored at the temperature of 20 °C till the time of use.
2.3.2 Standard ARMS-PCR
Samples were genotyped for detecting SNP of MTHFR (C667T) in genomic DNA.
Each sample has been done in two separated PCR reactions (one for common C allele and the other is
for minor allele T). each PCR reaction was done in a final volume of 25µl containing 100 mg of
genomic DNA, 1× PCR mix (Taq PCR Master Mix Kit, QIAGEN, GmbH, Hilden, Germany) that
contains (20 mM Tris–HCl, pH 8.3, 2U Taq DNA polymerase, 1.5 mM MgCl2, 50 mM KCl, 0.3 mM
dNTPs) and 0.2 µM of each primer.
The amplification was carried out by using a PTC-100 thermal cycler (MJ Research,
Inc., Watertown, Massachusetts, USA). The thermal cycling was performed as given:
The initial activation at the temperature of 94°C during 5 min of the time interval, followed by 33
amplification cycles having of denaturation at 94 °C for 25s, annealing at the temperature of 57 °C for
30s and extension at the temperature of 72 °C for 25s and the final extension at the temperature of
72 °C for 10 min of duration.
2.3.4 Gel Electrophoresis
The total amount of 15ul from PCR product were electrophoresed on 2% standard agarose gels at 100
V for the duration of 15 min.
The fragments were analyzed by the ethidium bromide on the UV transilluminator.
Three genotypes were detected: CC 273 bp, CT (307+ 190 bp) and TT (190 bp)
These gel patterns appeared because of substitution in the amino acid alanine (GCA,
GCC, GCG, GCT) to the valine (GTA, GTC, GTG, GTT) in the nucleotide number 2. So the single
nucleotide substitution (SNP) is from C (major allele) and T (minor allele).
2.3.5 Gel Analysis
For analyzing the photo taken to the samples after gel electrophoresis, we used free
software gel analyzer program version 2010 (http://www.gelanalyzer.com/download.html). The photo
analyzing showed that the higher band is 273 bp while the lower band is 190 bp according to 100 bp
marker used in the work.
Each sample has two lanes; the 1st lane for common allele, the 2nd for minor allele. A
band in the 1 lane at 273bp means homozygote common genotype (CC). A band in the 2nd lane at
190 bp means homozygot minor genotype (TT). Bands in both two lanes at 273 +190 bp mean
heterozygote genotype (CT).
st
3. Statistical analysis
Statistical analysis was done using the Statistical Package for Social Sciences version 14 (SPSS Inc.,
Chicago, USA). For categorical variables (nominal variables), the significance of association between
the observed and expected number of the genotypes for a population was analyzed using hardyWeinberg equilibrium equation. The differences of genotype and allele frequencies between the cases
and controls were observed using the chi-square test. So that the odds ratios (ORs) and 95%
confidence intervals (CIs) were analyzed and observed as the measure of association of polymorphic
site with the breast cancer.
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MTHFR (C667T) Polymorphism in breast cancer patients
1a
1b
2a
2b
M
3a
3b
4a
4b
273
190
Fig (9): Representative Agarose gel electrophoresis results for MTHFR (C667T) Polymorphism in
breast cancer patients by the standard ARMS-PCR method. Lane M marker (100bp), lanes 1a +1b is
for first sample and lanes 2a+2b is foe 2nd sample. Lanes 1, 2, are heterozygous (CT) genotype
because bands in both a +b appear. Lanes 3a+3b is for 3rd sample, lanes 4a+4b is for 4th sample .lanes
3,4 are wild homozygot (CC) genotype because only one band appear in only a lane.
4. Results
4.1 Genotypes and alleles frequency for MTHFR (C667T) polymorphism
Frequency Test
The genotype frequencies of major homozygous (CC), heterozygous (CT), and minor
homozygous (TT) were 6, 83, and 11% in patients with breast, respectively; and 26, 67, and 7% in
controls, respectively.
The major allele C represents 60% and 47% in controls and patients respectively. The minor allele T
represents 40% and 53% in controls and patients respectively (Table 1).
4.2 The association between observed and expected number of the genotypes
Hardy-Weinberg Equilibrium = Chi-square test (goodness of fit)
There is no significant and meaningful association between observed and expected
number of genotypes in both patients and healthy female persons.
Both breast cancer patients (p=0.000) and female normal donors (p=0.0129) show
deviation from HWE for polymorphic site. This test was performed using Chi-square test (goodness
of fit) and was confirmed by online HWE calculators as
(http://www.had2know.com/academics/hardy-weinberg-equilibrium-calculator-2-alleles.html) or
(http://www.oege.org/software/hwe-mr-calc.shtml) or with Michael H. Court’s (2005–2008).
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4.3 Association between genotypes frequencies between patients and non patients Chi-square test
(test for independence)
Generally, there was a significant difference in genotypes frequencies of the MTHFR
(C667T) polymorphism between control and breast cancer patients (P=0.002). The frequency of T
allele was 53% in the breast cancer patients and the 40% in controls.
Table 1: Distribution of (rs1801133) alleles and genotypes frequencies in breast cancer patients
(n=105) and the control subjects (n=42)
Polymorphism
Breast cancer patients n
Controls subjects
(%)
Genotypes
CC
6 (6)
11 (26)
CT
87 (83)
28 (67)
TT
12 (11)
3 (7)
Alleles
C
99 (47)
50 (60)
T
111 (53)
34 (40)
a
X2
Pa
12.42
0.002
3.68
0.07
n (%)
Calculated by chi-square test (for independence)...Significant at P<0.05
4.4 Association between the MTHFR (C667T) polymorphism and risk of breast cancer Odds
Ratio Test
Regarding the risk of the development of breast cancer, the CC common genotype and C common
allele were taken as references. The test was performed under different genotypic and allelic models.
The results show significant association between breast cancer development and the minor genotype
TT under homozygote and heterozygote models (OR =7.33; CI 95% (1.46-36.66); P=0.011) and (OR
=5.69; CI 95% (1.93-16.8); P= 0.001) respectively. A surprising result confirm the highly association
between breast cancer risk and TT genotype under dominant model (OR =5.85; CI 95% (2.00-17.12);
P=0.00).
Table (2): Association between the (rs1801133) polymorphism and risk of breast cancer
Models
OR
X2
P
(CI 95%)
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Allelic (T vs. C)
1.64 (0.98-2.75)
3.68
0.055
homozygote (TT vs. CC )
7.33 (1.46-36.66)
6.47
0.011
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Heterozygote (CT vs. CC)
5.69(1.93-16.8)
11.58
0.001
Dominant (TT+CT vs. CC)
5.85(2.00-17.12)
12.29
0.00
Recessive (TT vs. CT+CC)
1.67(0.44-6.27)
0.6
0.43
Calculated by OR Test …significant at P<0.05
5. Discussion
The breast cancer is the first cancer type that threatens the women all over the world
[20]. Many genes are reported to be associated with the breast cancer incidence [14-21] etc. The
MTHFR gene is responsible for the variation in MTHFR enzyme activity that may influence the
balance between DNA 3 important processes (synthesis, repair, and methylation) [22-25].
Most of the studies reported the association between (MTHFR) gene polymorphisms
and breast cancer risk. But results were greatly different and underpowered. Several Meta analyses
tried to give more precise estimation [26, 27]. So it is still under considerations due to several
limitations as non homogeneity and sample size between the controls.
One of the most focused polymorphisms of MTHFR is C667T polymorphism that
had been reported by different studies from different population as a disposing molecular factor on the
breast cancer susceptibility. This polymorphism is never been investigated as the risk factor for breast
cancer in the Egyptian women.
Our results showed the significant association between this polymorphism and breast
cancer susceptibility under three models (homozygote, heterozygote and dominat).
Qi X and coworkers [27] agreed by their Meta analysis conclusion with our results.
They reported that MTHFR C677T polymorphism was significantly associated with breast cancer risk
in the overall and the population of East Asians, whereas no association was shown in the Caucasian
and the menopausal status-based populations.
The results of Liang H and colleagues [28] disagreed with us in the significant
association with breast cancer risk under allelic and recessive models but they agreed with us in
association in homozygote models three models (T vs. C: OR = 1.12, 95 % CI = 1.02-1.23, P = 0.015;
TT vs. CC: OR = 1.35, 95 % CI = 1.10-1.67, P = 0.005; TT vs.CC/CT: OR = 1.37, 95 % CI = 1.111.70, P = 0.004).
Zhang, J. et al. [29] showed the equal results as they reported elevated breast cancer
risk with TT variant genotype in various dominant genetic model and homozygote comparison when
all the studies were pooled into the meta-analysis (CC vs. TT: OR = 1.11, 95% CI = 1.01-1.23;
dominant model: OR = 1.04, 95% CI = 1.00-1.09).
The Lin, W.Y. et al. [30] reported that there is not numerical association between the
risk of breast cancer and the MTHFR C677T genotype. So that a significantly elevated risk of the
breast cancer predisposed by the MTHFR 677T variant genotype (CT and TT) can be analyzed in the
women with prolonged exposure to the estrogens prior to FFTP (adjusted OR = 4.98, 95% CI = 2.0012.43).
In conclusion, our study has a limitation point for the sample size. However, it has
two power points; it is the first Egyptian study for the relation between C667T polymorphism of the
MTHFR gene and the breast cancer susceptibility in Egyptian women, secondly we tried to make a
focus on every step, we made.
6. Conflict of Interest
The authors declare that they have no financial and personal relationships with other
people or organizations that could inappropriately influence (bias) this work.
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7. Acknowledgments: The authors thank the patients for their willingness to cooperate with our
study.
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