Xác định tính đa hình của các gen tp53 và gen mdm2 ở bệnh nhân ung thư phổi tt tiếng anh
- 29 trang
- file .docx
MINISTRY OF EDUCATION AND TRAINING MINISTRY OF HEALTH
HANOI MEDICAL UNIVERSITY
TRAN KHANH CHI
DETERMINATION OF TP53 GENE AND MDM2 GENE
POLYMORPHISMS IN PATIENTS WITH LUNG CANCER
Major : Biochemistry
Code: 62720112
MEDICAL DOCTOR DISSERTATION SUMMARY
HA NOI - 2019
THE DISSERTATION IS COMPLETED AT
HANOI MEDICAL UNIVERSITY
Scientific guidance: 1. Assoc.Prof.PhD. Tran Huy Thinh
2. Assoc.Prof.PhD. Nguyen Thi Ha
Reviewer 1: Assoc.Prof.PhD. Nguyen Nghiem Luat
Reviewer 2: Assoc.Prof.PhD. Phan Quoc Hoan
Reviewer 3: PhD. Tran Thi Chi Mai
The dissertation will be presented to the Board of Ph.D
dissertation at University level at Hanoi Medical University.
At th, , 2019
The dissertation can be found at:
- National Library of Vietnam
- Library of Hanoi Medical University
1
BACKGROUND
1. Urgency of topics
Lung cancer (LC) is one of the most common cancers and has the
highest mortality rate among the current types of cancer. Vietnam is the
country with the highest LC rate among cancers in men and the third
leading cause of cancer among women. Early detection of risk factors
for early diagnosis, follow-up and timely intervention will play a
particularly important role in preventing the onset and development of
cancer while enhancing the effectiveness of medical examination and
treatment.
TP53 and MDM2 are a group of genes in the p53 signaling
pathway that play an important role in maintaining the stability of the
genome under the influence of harmful factors such as DNA damage,
hypoxia, metabolism disorder or enhancement of the activity of
carcinogenic genes. With each change occurring on TP53 or MDM2 can
affect the cell physiological processes and lead to the risk of developing
cancer. TP53 and MDM2 are both polymorphic, many single nucleotide
polymorphisms (SNPs) of these two genes have been found to produce
different genotypes in the community. However, not all SNPs are
capable of promoting the onset and progression of cancer. In fact, some
SNPs of the TP53 and MDM2 have been identified to play a role in the
pathogenesis of some types of cancer, including LC. Identification of
these SNPs plays an important role in assessing the risk of disease and
the ability to respond to treatment individually. Recent years in
Vietnam, there have been a number of studies on the role of TP53 in
LC, but no one have evaluated the polymorphism of TP53 as well as the
role SNPs of MDM2 related to LC.
2. Objectives of the research:
1. Determine the rate the polymorpism of TP53 and MDM2
genotype distribution in patients with lung cancer and the
control group.
2. Evaluate the correlation between TP53, MDM2 genotype
and some risk factors of lung cancer.
3. The meaning of scientific and practical subjects:
Variations in human DNA sequence may affect how the body
develops the disease and responds to pathogens, chemicals, drugs,
vaccines and other agents. SNPs are thought to be potential keys in the
2
implementation of personalized medicine. Their most important role in
medical research, however, is to compare regions of the genome among
groups (possibly between patients and healthy people) in genome-wide
association studies (GWAS). In this study, we investigated the rate of
polymorphic genotypes in patients with LC and control group,
compared two groups and calculated odds ratios to determine the risk of
LC on the subjects. Molecular biology techniques were used to identify
genotypes at single nucleotide polymorphisms of TP53 and MDM2.
Risky genotypes will be able to develop into early screening and
counseling tools for the community, in order to prevent the formation
and development of LC. This is considered a promising new approach,
contributing to the reduction of LC incidence.
4. Thesis structure
The thesis is presented in 116 pages (excluding references and
appendices). The thesis is divided into 7 parts.
+ Introduction: 2 pages
+ Chapter 1: Overview document 36 pages
+ Chapter 2: Objects and methodology 12 pages
+ Chapter 3: Research Results 31 pages
+ Chapter 4: Discussion 32 pages
+ Conclusion: 2 page
+ Propose: 1 page
The thesis consists of 26 tables, 35 figure. Using 192 references,
including Vietnamese, English and some Web pages. The appendix
includes medical studies, lists 220 patients with LC and 230 control and
technical processes.
Chapter 1
OVERVIEW
1. Lung cancer
1.1. Epidemiology
Current epidemiological studies have documented that LC is the
most common cancer and has the highest mortality rates in all types of
cancer. According to global cancer statistics (Globocan 2012), there are
an estimated 1.82 million newly acquired LC and about 1.59 million
deaths related to LC. In the U.S.A , upturned in 2016, LC is the cancer
with the highest mortality and the second highest incidence in both
3
sexes. By 2016, the United States had about 224,390 new LC cases and
about 158,080 deaths, which accounted for 26.5% of all cancer deaths.
Statistics show that LC is more common in men. In developing
countries, male / female ratio is 2.4 / 1 while in developed countries,
male / female ratio is 1.8 / 1. The number of new LC cases for women
is the third in the category of cancer (after breast and colorectal cancer),
but the number of deaths just behind breast cancer.
According to the latest cancer records in Vietnam, after 10 years
from 2000 to 2010, the incidence of LC in women increased by more
than 200% (6.4 / 100,000 in 2000 to 13.9 / 100,000 in 2010), LC is also
one of the five fastest growing types of cancer.
1.2. Molecular pathology of lung cancer
Smoking is considered a major risk factor for LC,
approximately 80-85% of smoking cases are diagnosed with LC in
the world. Risk level depends on factors such as: age of smoking
(the sooner smoking is, the higher risk is), the number cigarette of
years (smoking more, the risk higher), the duration of smoking
(smoking longer, the risk higher). Smokers have a 10-fold increased
risk of LC compare with non-smokers. Studies have also shown that
even people who do not smoke directly, but often exposed to
smokers (passive smoking), also have a high risk of LC. There are
also many factors that are considered risk factors for LC such as air
pollution, ionizing radiation, occupational exposure, virus, diet,
history of bronchopulmonary disease.
Molecular studies show that the development and emergence
of LC occurs over a number of stages, under the influence of risk
factors, genetic susceptibility, and the accumulation of mutations
that occur on oncogenes and tumor suppressor genes. Normally, the
mechanisms of gene regulation that work smoothly and closely. In
the presence of disorders will lead to an abnormal increase or
inhibition of functional genes.
4
Figure 1.1: The molecular signaling pathways in lung cancer pathogenesis
(Pass & et al.).
2. TP53 and MDM2 genes
2.1. TP53, cancer suspressor gene
The TP53 gene is located on the short branch of chromosome 17
(17p13.1). The TP53’s length is 22,000 bp, including 11 exons (Encode
area from E1 to E11, E1 does not encode) and 10 introns. It encodes for
a protein molecules that weigh 53kDa with 393 acid amin and
consisting of 3 functional domains.
The TP53 gene plays an important role in DNA repair, controlling
cell division, and apoptosis. The defective TP53 gene allows abnormal
cell proliferation and leads to cancer formation. When the body is
affected by stimuli (demaged DNA, cellular stress, hypoxia, over
expression of the oncogene), p53 is activated to stop the cell cycle until
the DNA is repaired or induce apoptosis if the demaged DNA does not
repair. Thus, p53 is considered as the guardian of the genome. In
addition, p53 has the ability to activate or inhibit several other genes
2.2 MDM2
5
The MDM2 gene (Murine double minute 2), also known as HDM2
(Human double minute 2), consists of 12 exons and 1 intron on the long
branch of the 12th chromosome, it was first identified in 1980. MDM2
protein molecules are synthesized with 491 amino acids and consisting
5 functional structural domains.
To date, the most known important role of MDM2 has been to
regulate the activity of the TP53 gene in the p53 signaling pathway.
Under normal conditions, MDM2 binds to the p53-activated region,
which controls the distribution and degradation of the p53 protein. In
contrast, activated p53 promotes MDM2 replication so that the
expression of p53 and MDM2 in the cell is always maintained in
equilibrium through the reversal of MDM2 and p53. When stimulatory
factors (demaged DNA, cellular stress, hypoxia, over expression of the
oncogene) occur, MDM2 will be phosphorylated and exposed to the p53
activation region, triggering the p53 function.
3. TP53 and MDM2 gene polymorphisms in lung
cancer
Single nucleotide polymorphism (SNP) is the difference in DNA
sequence in the genome between individual persons or between
chromosomes of a person. This is a common phenomenon. It is the
result of mutation points that replace a pair of nucleotides. According to
the published studies, many SNPs were found in the TP53 gene and
dozens one on the MDM2 gene. These single nucleotide polymorphisms
create many different TP53 and MDM2 genotypes in the community.
The genotypes of some of these SNPs are involved in the onset of
development many type of cancers including LC. They are risk factors
to be considered.
The SNPs we analyzed in this study may change coding sequences
or not but they are all located in the key functional areas of TP53.
Theoretically, these areas can affect to the control tumor ability of
TP53. First of all, a polymorphism caused by the addition of 16 base
pairs in the intron-3 region of TP53. Those who carry these genotypes
express low levels of p53 in the cell and have increased risk for certain
cancers including lung, breast and colorectal cancer. It proves that SNPs
are capable of altering mRNA completion. In addition, although SNPs
on p53 coding regions 21 (GAC → GAT), 34 (CCC → CCA) and 36
(CCG → CCT) not change the amino acid sequence but reducing the
expression of p53 protein. Studies have shown that the SNPs in the
6
TP53 N-terminal activation region where contained an interactive
position with MDM2 and they can reduced the translation of TP53.
On the other hand, SNPs on the coding region altering the amino
acid sequence can lead to a change in the p53 binding ability to the
specific sequence in the target gene, in mRNA completment and
stability of the protein as well as alter the interactions of p53 with
intracellular proteins. These are SNPs located in codenamed 47 (P47S),
72 (R72P), 217 (V217M) and 360 (G360A). Under normal conditions,
with the action of p38 and homeodomain-interacting protein kinase 2
(HIPK2), p53 is phosphorylated at position S46 leading to increased
replication of genes involved in programmed death (appotosis). And
when the p53-P47 allele was replaced by the p53-S47 allele, the
phosphorylation at site S46 reduced activity on the target genes of
phagocytosis and increased the probability of cancer.
Similarly, polymorphism in the triple coding 72 (R72P) produced
two genotypes: p53-R72 and p53-P72. Studies by Boldrine et al. Show
that p53-P72 homozygotes have a higher risk of lung cancer [48]. At the
same time, the p53-P72 genotype and MDM2 G/G genotype are also
common in patients with lung cancer who has smoked over the long
term. For the other two forms of SNPs, V217M is located on the DNA
binding domain of the p53, which may reduce p53 activity and directly
affected genes including CDKN1A, BAX and PMAIP1. Functional
studies have shown that the p53-M217 genotype have a higher
expression of the p53-V217. Thus, the p53-M217 genotype is capable
of protecting cells against carcinogens better than p53-V217. However,
the molecular mechanism of this phenomenon is not yet clear. SNPs
G360A is located at the junction of p53. These SNPs affect the
expression of BAX and MDM2, which are important genes in the p53
signaling pathway.
The single nucleotid polymorphism of the MDM2 gene is located at
the first intron, rs2279744 (MDM2 - SNP309), with the change from T
to G (MDM2 - SNP309 T>G) increased the affinity of SP1 (Stimulatory
protein 1) with MDM2, results in increased expression of MDM2
leading to inhibitory TP53 gene as a condition for cancer formation and
progression.
Many international epidemiological studies have been conducted to
find a link between single nucleotide polymorphism of TP53, MDM2
and lung cancer. The published results are still unanimous, but one
7
thing in common is that all R72P gene polymorphism TP53 and
309T>G MDM2 genes are the two most commonly SNPs associated
with lung cancer. The differences among studies that can be explained
by the differences in sample size or racial and environmental factors of
the study population.
The fact that cancer is the result of a complex process in which
there are interactions of many factors such as genotype, biological
characteristics as well as habitat. Therefore, when studying SNPs in
lung cancer, relevant analyzes with biological characteristics or
smoking status and environmental pollution should be conducted in
order to assess in a comprehensive manner and recommend valuable
information for lung cancer prevention strategies.
Chapter 2
SUBJECTS AND METHODS
2.1. Research Subjects
The study was conducted on 220 patients with primary lung cancer
diagnosed at the Respiratory Center, the Nuclear Medicine and
Oncology Center - Bach Mai Hospital and 230 controls from October
2013 to December 2017.
2.1.1. Criteria for selecting patients
- 220 patients were diagnosed with primary lung cancer at the
Respiratory Center and the Nuclear Medicine and Oncology Center -
Bach Mai Hospital with histopathological results.
- Agree to participate in research.
2.1.2. Exclusion criteria
- Secondary lung cancer.
- Lung cancer combine with other cancers.
- Do not agree to participate in research.
2.1.3. Control group
- 230 controls were selected from those who came to the medical
examination at Bach Mai Hospital. Clinical examinations, laboratory
tests, pulmonary X-rays, ultrasonography and conclusions without LC
or any other cancer.
- Corresponding age and gender to lung cancer patients.
2.1.4. Genotypic polymorphisms were analyzed
- TP53 gene
8
+ Add 16 pairs of base pairs at intron 3 (dup 16).
+ SNP P34P, at 34 codon, exon 4 (CCC → CCA), Prolin coding.
+ SNP P36P, at codon 36, exon 4 (CCG → CCA), Prolin coding.
+ SNP P47S, at codon 47, exon 4, (CCG or TCG), encoding Prolin
or Serin.
+ SNP R72P at codon 72, exon 4, (CGC or CCC), encoding
Arginine or Prolin.
+ SNP V217M, at codon 217, exon 6, (GTG or ATG), encoding
Valin or Methionine.
+ SNP G360A at codon 360, exon 10, (GGG or GCG), encoding
Glycin or Alanin.
- MDM2 gene: SNP locates at nucleotide position 309, intron 1,
promoter region.
2.2. Research Methodology:
Using cross-sectional descriptive study with control.
2.3. Study time and place
Time from 10/2013 to 10/2017.
Research site: the Respiratory Center, the Nuclear Medicine and
Oncology Center - Bach Mai Hospital. Department of Biochemistry and
Center for Gene Research - Protein, Hanoi Medical University.
2.4. Thread adhere research ethics in medicine
This study was approved by the ethics committee of Hanoi
Medical University (Dicision no. 188/HĐĐĐĐHYHN, 31/1/2013).
2.5. Funds for the study
Our study got the funding support of a national level project
“Evaluate the genotype distribution of several genes involved in lung and
liver cancer” belong to study "Evaluating Vietnamese Genetic
Characteristics".
2.6. Procedures and techniques used in the study
The techniques used in the study included: Interview and turn
up medical treatment documents to identify risk factors exposuring.
DNA extraction technique from peripheral blood samples. PCR
technique detects the genotype of dup16 polymorphism of TP53 gene.
Using restriction fragment length polymorphism technique for
genotyping R72P SNP of TP53 gene and 309T>G SNP of MDM2 gene.
Sequencing technique to identify genotypes of SNPs: P34P, P36P,
9
P47S, V217M, G360A of TP53 gene. Research process follow as below
diagram.
PROCESS DIAGRAM
Chapter 3
RESULTS
3.1. Characteristics of the study subjects
Table 3.1. Distribution of study subjects
Patients Controls
Characteristic (220) (230) p
n % n %
59,89 ± 60,67 ±
Age (year) X SD 9,432 9,335
0,379
Male 163 74,1 157 68,3
Sex 0,173
Female 57 25,9 73 31,7
Yes 94 42,7 68 29,6
0,004
No 126 57,3 162 70,4
Smoking history
< 20 pack-year 43 45,7 33 48,5
0,726
> 20 pack-year 51 54,3 35 51,5
Histopa NSCLC Adenocarcinoma 161 73,2
10
Carcinoma 13 5,9
-thology Other carcinoma 25 11,4
SCLC 21 9,5
Comment:
- Adenocarcinoma accounts for the highest proportion of
histopathology.
- The smoking rate in the patients group was 42.7% higher and
statistically significant than the control group (p = 0.004). The
proportion of LC patients who have smoked > 20 pack-year was 54.3%,
had no difference with the proportion of LC patients who have smoked
<20 pack-year (45.7% (p = 0.726)).
- No smoking in women was found in our study.
3.2 Results of TP53 genotyping
3.2.1. Adding 16 base pairs at intron 3 (dup16)
The fragment carrying intron 3 of the TP53 gene was amplified
by PCR reaction with specific primers, PCR product was
electrophoresed on 3% agarose gel.
Figure 3.1. Electrophoresis of PCR product amplified the fragment
carrying intron 3 of the TP53 gene on agarose gel 3%
Samples K114, K115, K117, K118, C96÷C100: genotype A1A1; sample
K116: genotype A1A2; M: Ladder 100bp, (-): Nagative
Comment: A1A1 genotype had single band with 119bp size. A1A2
genotype had 2 band with 119bp and 135bp size. DNA band had clear
without additional banding, ensure that 16bp polymorphic genotypes
are identified at the intron 3 of the TP53 gene.
11
Table 3.2 Genetic analysis results of dup16 SNP of TP53 gene between
patient and control group
Patient Control
SNP (n=220) (n=230) OR
n % n %
A1A1 212 96,4 226 98,3 1,0
A1A2 8 3,6 4 1,7 2,13 (0,633 - 7,184)
Comment: A1A2 genotype was 3.6% that the lung cancer group was
higher than the control group (1.7%), but the difference was not
statistically significant.
3.2.2. SNP R72P gen TP53
Results of genotyping at SNP R72P by PCR-RFLP method.
Figure 3.2: Electrophoresis of the cutting product of gene fragment
contains R72P SNP by BstUI enzyme on research samples.
M: Ladder 100bp; (-): Negative; (+): Positive. Samples K60, K61, C7 :
Genotype CC (Pro/Pro). Samples K69, K73, C8: Genotype GG (Arg/Arg).
Samples K46, K48, C13: Genotype GC (Arg/Pro).
Comment:
The cutting product of gene fragment contains R72P SNP by BstUI
enzyme including DNA fragment of different sizes, in accordance with the
theoretical calculations. The GG (Arg / Arg) genotype consists of two DNA
fragments of 165 bp and 231 bp (K69, K73, C8). CC (Pro / Pro) hybridization
when only one DNA band of 396bp (K60, K61, C7) appears. The GC
homologation (Arg / Pro) consists of three bands with dimensions of 396bp,
231bp and 165bp (K46, K48, C13).
12
Checking the results of genotyping of SNP R72P by sequencing.
Figure 3.3: Sequencing results of exon 4 on TP53 containing SNP R72P are
corresponding to genotype: GC (Arg/Pro), CC (Pro/Pro), GG (Arg/Arg).
Comment: The DNA sequence of these sample was completely
matched to its PCR-RFLP analysis.
Table 3.3: R72P SNP genotype of TP53 gene and risk
of lung cancer
Patient Control
SNP (n=220) (n=230) OR, 95%CI
n % n %
G 219 49,8 248 53,9 1,0
Allel
C 221 50,2 212 46,1 1,18 (0,91 – 1,53)
G/G 57 25,9 77 33,5 1,0
Genotype
G/C 105 47,7 94 40,9 1,51 (0,97 - 2,35)
C/C 58 26,4 59 25,7 1,33 (0,81 - 2,19)
Combination G/
162 73,6 171 74,3 1,0
of recessive G+G/C
genes C/C 58 26,4 59 25,7 1,04 (0,68 - 1,58)
Combination G/G 57 25,9 77 33,5 1,0
of recessive G/C +
163 74,1 153 66,5 1,44 (0,96 - 2,16)
genes C/C
Comment: G/C genotype of codon 72 of TP53 gene was the highest in both
patient and control group. G/C and C/C genotype of codon 72 of TP53 gene
may be possible to increase the risk of lung cancer, but the association is not
statistically significant.
3.2.3. Polymorphism in SNPs: P34P, P36P, P47S,
V217M, G360A
Using the sequencing techniques to analyze genotypes at these
SNPs of TP53 gene.
13
Table 3.4: Genotypes of SNPs: P34P, P36P, P47S, V217M, G360A
of TP53 gene
Mutant
Wild homozygous Heteropathic
homozygous
Genotype genotype genotypes
genotypes
n % n % n %
C/C C/A A/A
P34P(C>A)
450 100 0 0 0 0
G/G G/A A/A
P36P(G>A)
450 100 0 0 0 0
* *
C/C (P47P) C/T (P47S) T/T (S47S)*
P47S(C>T)
450 100 0 0 0 0
G/G (V217V)* G/A (V217M)* A/A (M217M)*
V217M(G>A)
450 100 0 0 0 0
G/G (G360G)* G/C (G360A)* C/C (A360A)*
G360A(G>C)
450 100 0 0 0 0
(*) The amino acids are encoded by that change the nucleotid sequence.
Comment: No mutation genotype was found in our study.
3.3. Results of SNP309 genotype of MDM2 analysis
Using restriction fragment length polymorphism technique
(RFLP) for genotyping 309T>G SNP of MDM2 gene.
Hình 3.4: Electrophoresis of the cutting product of gene fragment
contains SNP309 of MDM2 gene by MspA1i enzyme on research samples.
Samples K17, C7: Homozygous genotype T/T. Samples K16, K23, C16:
Homozygous genotype G/G. Samples K7, K13, C18, C21: Heterozygous
14
genotype T/G. M: Ladder 100bp; (-): Negative; (+): Positive.
Comment:
The cutting product of gene include DNA fragment of different sizes, in
accordance with the theoretical calculations. The samples with T/T genotype
consist of only one DNA band of 157 bp (K17, C7). The samples with G/G
genotype consist of two DNA bands of 109 bp and 48 bp (K16, K23, C16) CC
(Pro / Pro) hybridization when only one DNA band of 396bp (K60, K61, C7)
appears. The samples with heterozygous TG consists of three bands with
dimensions of 157bp, 109 bp và 48 bp (K7, K13, C18, C21).
Checking the results of genotyping of SNP 309T>G of MDM2 gene
by sequencing.
Figure 3.5: Sequencing results of gene fragment contains SNP 309T>G
of MDM2 corresponding to T/T, T/G, G/G genotypes
Comment: The DNA sequence of these sample was completely
matched to its PCR-RFLP analysis.
Table 3.5: SNP309T>G genotype of MDM2 gene and risk
of lung cancer
Patient Control
OR, OR*,
SNP (n=220) (n=230)
95%CI 95%CI
n % n %
21 49, 24 52,
T 1,0
7 3 1 4
Allel 1,13
22 50, 21 47,
G (0,87 –
3 7 9 6
1,47)
Genotype 27, 23,
TT 60 55 1,0 1,0
3 9
TG 97 44, 13 57, 0,68 0,65
1 1 0 (0,43 – (0,41 –
1,07) 1,03)
15
1,31 1,10
28, 19,
GG 63 44 (0,77 – (0,84 –
6 1
2,32) 1,44)
TT+T 15 71, 18 80,
Combinati 1,0 1,0
G 7 4 6 9
on of
recessive
1,7 1,61
28, 19,
genes GG 63 44 (1,09 – (1,03 –
6 1
2,63) 2,51)
72, 23,
Combinati TT 60 55 1,0 1,0
7 9
on of
recessive
0,84 0,78
TG + 16 27, 17 76,
genes (0,55 – (0,51 –
GG 0 3 5 1
1,28) 1,20)
OR* is adjusted from the variables: age, gender, smoking habit by
multivariate logistic regression model.
Nhận xét:
The heterozygosity of SNP 309TG was highest in both disease and
control groups. The SNP 309GG homozygous genotype increased the risk of LC
by 1,7-fold in the recessive gene model (OR = 1,7; 95% CI = 1,09-2,63). When
corrected for the variables age, gender and smoking status in the multivariate
logistic regression model still showed homozygous SNP 309GG genotype
increased the risk of 1,61-fold as the model of recessive genes (OR = 1,61; 95%
CI = 1,03 – 2,51).
3.4 Correlation between polymorphism of TP53, MDM2 and risk of
lung cancer.
3.4.2. Relationship between polymorphism SNP309T> G of MDM2 gene and
the risk of lung cancer by clinical and subclinical characteristics in lung
cancer.
3.4.2.1. Relationship between polymorphism SNP309T> G of MDM2 gene and
the risk of lung cancer by gender.
Table 3.6: Relationship between polymorphism SNP309T> G of MDM2
gene and the risk of lung cancer by gender
OR GG/TT OR TG/TT OR GG/TG+TT OR TG+GG/TT
Group
(95%CI) (95%CI) (95%CI) (95%CI)
Male 1,31 0,69 1,66 0,87
16
(0,71 – 2,43) (0,41 – 1,18) (1,01 - 2,76) (0,526 - 1,43)
1,26 0,67 1,67 0,79
Female
(0,43 – 3,67) (0,29 – 1,56) (0,68 – 4,06) (0,35 – 1,77)
Comment: The homozygous SNP 309GG genotype increases the risk of LC in
males by 1,66-fold as the model of recessive genes ( OR-1,66; 95%CI=1,01-
2,76).
17
3.4.2.2. Relationship between polymorphism SNP309T> G of MDM2 gene and
the risk of lung cancer by histopathology.
Table 3.7: Relationship between polymorphism SNP309T> G of MDM2
gene and the risk of lung cancer by histopathology
OR GG/TT OR TG/TT OR GG/TG+TT OR TG+GG/TT
Group
(95%CI) (95%CI) (95%CI) (95%CI)
1,37 0,72 1,71 0,88
NSCLC
(0,79 – 2,37) (0,45 – 1,14) (1,09 – 2,68) (0,57 – 1,37)
0,94 0,42 1,59 0,55
SCLC
(0,30 – 2,90) (0,15 – 1,18) (0,59 – 4,28) (0,22 – 1,38)
Adenocarcinom 1,40 0,76 1,69 0,92
a (0,79 – 2,50) (0,46 – 1,24) (1,05 – 2,72) (0,58 – 1,47)
2,50 1,47 1,88 1,73
Carcinoma
(0,44 – 14,29) (2,97 – 7,30) (0,55 – 6,38) (0,37 – 8,04)
Comment: The homozygous SNP 309GG genotype increases the risk of
NSCLC by 1,71-fold (OR=1,71; 95% CI= 1,09-2,68) and Adenocarcinoma by
1,69-fold (OR=1,69; 95%CI= 1,05-2,72) as the model of recessive genes in
smoking group.
3.4.2.3. Relationship between polymorphism SNP309T> G of MDM2 gene and
the risk of lung cancer by smoking status
Table 3.8: Relationship between polymorphism SNP309T> G of MDM2
gene and the risk of lung cancer by smoking status
OR GG/TT OR TG/TT OR GG/TG+TT OR TG+GG/TT
Group
(95%CI) (95%CI) (95%CI) (95%CI)
1,86 0,85 2,09 1,12
Smoking
(0,74 – 4,68) (0,38 – 1,90) (1,01 – 4,31) (0,52 – 2,40)
0,98 0,58 1,38 0,68
No smoking
(0,50 – 1,90) (0,34 – 1,01) (0,78 – 2,43) (0,41 – 1,14)
Smoking <20 1,86 0,92 1,99 1,18
pack-year (0,48 – 7,26) (0,28 – 3,04) (0,70 – 5,65) (0,37 – 3,66)
Smoking >20 1,87 0,80 2,18 1,08
pack-year (0,53 – 6,60) (0,27 – 2,39) (0,80 – 5,98) (0,38 – 3,03)
18
Comment: The homozygous SNP 309GG genotype increases the risk of LC in
smoker group by 2,09-fold as the recessive gene model (OR= 2,09; 95%CI=
1,01-4,31).
3.4.3. Risk of lung cancer in combination of R72P SNP of TP53 gene and
SNP309T> G of MDM2 gene
3.4.3.1. Risk of lung cancer in combination of R72P SNP of TP53 gene and
SNP309T> G of MDM2 gene with smoking
Table 3.9: Risk of lung cancer in combination of R72P SNP of TP53 gene
and SNP309T> G of MDM2 gene with smoking
Patient Control
Characteristics OR
n % n %
Smoking 126 57,3 162 70,4 1,00
1,78
No smoking 94 42,7 68 29,6
(1,20 - 2,62)
Smoking <20 pack- 1,68 (1,01 –
43 45,7 33 48,5
year 2,79)
Smoking >20 pack- 1,87 (1,15 –
51 54,3 35 51,5
year 3,06)
GG of SNP R72P
of TP53 gene and 32 57,1 53 80,3 1,00
no smoking
CC of SNP R72P
3,06
of TP53 gene and 24 42,9 13 19,7
(1,37 - 6,84)
smoking
TT of
SNP309T>G of
41 55,4 40 74,1 1,00
MDM2 gene and
no smoking
GG of 2,30
SNP309T>G of (1,07 - 4,93)
33 44,6 14 25,9
MDM2 gene and
smoking
Comment:
- Smoking increased the risk of LC by 1,78-fold (OR = 1,78; 95% CI = 1,20-
2,62).
- Smoking > 20 pack-year increased the risk of LC by 1,87-fold (OR = 1,87;
95% CI = 1,15-3,06) was higher than the risk of LC when smoking <20
pack-year (OR = 1.68, 95% CI = 1.01 - 2.79).
HANOI MEDICAL UNIVERSITY
TRAN KHANH CHI
DETERMINATION OF TP53 GENE AND MDM2 GENE
POLYMORPHISMS IN PATIENTS WITH LUNG CANCER
Major : Biochemistry
Code: 62720112
MEDICAL DOCTOR DISSERTATION SUMMARY
HA NOI - 2019
THE DISSERTATION IS COMPLETED AT
HANOI MEDICAL UNIVERSITY
Scientific guidance: 1. Assoc.Prof.PhD. Tran Huy Thinh
2. Assoc.Prof.PhD. Nguyen Thi Ha
Reviewer 1: Assoc.Prof.PhD. Nguyen Nghiem Luat
Reviewer 2: Assoc.Prof.PhD. Phan Quoc Hoan
Reviewer 3: PhD. Tran Thi Chi Mai
The dissertation will be presented to the Board of Ph.D
dissertation at University level at Hanoi Medical University.
At th, , 2019
The dissertation can be found at:
- National Library of Vietnam
- Library of Hanoi Medical University
1
BACKGROUND
1. Urgency of topics
Lung cancer (LC) is one of the most common cancers and has the
highest mortality rate among the current types of cancer. Vietnam is the
country with the highest LC rate among cancers in men and the third
leading cause of cancer among women. Early detection of risk factors
for early diagnosis, follow-up and timely intervention will play a
particularly important role in preventing the onset and development of
cancer while enhancing the effectiveness of medical examination and
treatment.
TP53 and MDM2 are a group of genes in the p53 signaling
pathway that play an important role in maintaining the stability of the
genome under the influence of harmful factors such as DNA damage,
hypoxia, metabolism disorder or enhancement of the activity of
carcinogenic genes. With each change occurring on TP53 or MDM2 can
affect the cell physiological processes and lead to the risk of developing
cancer. TP53 and MDM2 are both polymorphic, many single nucleotide
polymorphisms (SNPs) of these two genes have been found to produce
different genotypes in the community. However, not all SNPs are
capable of promoting the onset and progression of cancer. In fact, some
SNPs of the TP53 and MDM2 have been identified to play a role in the
pathogenesis of some types of cancer, including LC. Identification of
these SNPs plays an important role in assessing the risk of disease and
the ability to respond to treatment individually. Recent years in
Vietnam, there have been a number of studies on the role of TP53 in
LC, but no one have evaluated the polymorphism of TP53 as well as the
role SNPs of MDM2 related to LC.
2. Objectives of the research:
1. Determine the rate the polymorpism of TP53 and MDM2
genotype distribution in patients with lung cancer and the
control group.
2. Evaluate the correlation between TP53, MDM2 genotype
and some risk factors of lung cancer.
3. The meaning of scientific and practical subjects:
Variations in human DNA sequence may affect how the body
develops the disease and responds to pathogens, chemicals, drugs,
vaccines and other agents. SNPs are thought to be potential keys in the
2
implementation of personalized medicine. Their most important role in
medical research, however, is to compare regions of the genome among
groups (possibly between patients and healthy people) in genome-wide
association studies (GWAS). In this study, we investigated the rate of
polymorphic genotypes in patients with LC and control group,
compared two groups and calculated odds ratios to determine the risk of
LC on the subjects. Molecular biology techniques were used to identify
genotypes at single nucleotide polymorphisms of TP53 and MDM2.
Risky genotypes will be able to develop into early screening and
counseling tools for the community, in order to prevent the formation
and development of LC. This is considered a promising new approach,
contributing to the reduction of LC incidence.
4. Thesis structure
The thesis is presented in 116 pages (excluding references and
appendices). The thesis is divided into 7 parts.
+ Introduction: 2 pages
+ Chapter 1: Overview document 36 pages
+ Chapter 2: Objects and methodology 12 pages
+ Chapter 3: Research Results 31 pages
+ Chapter 4: Discussion 32 pages
+ Conclusion: 2 page
+ Propose: 1 page
The thesis consists of 26 tables, 35 figure. Using 192 references,
including Vietnamese, English and some Web pages. The appendix
includes medical studies, lists 220 patients with LC and 230 control and
technical processes.
Chapter 1
OVERVIEW
1. Lung cancer
1.1. Epidemiology
Current epidemiological studies have documented that LC is the
most common cancer and has the highest mortality rates in all types of
cancer. According to global cancer statistics (Globocan 2012), there are
an estimated 1.82 million newly acquired LC and about 1.59 million
deaths related to LC. In the U.S.A , upturned in 2016, LC is the cancer
with the highest mortality and the second highest incidence in both
3
sexes. By 2016, the United States had about 224,390 new LC cases and
about 158,080 deaths, which accounted for 26.5% of all cancer deaths.
Statistics show that LC is more common in men. In developing
countries, male / female ratio is 2.4 / 1 while in developed countries,
male / female ratio is 1.8 / 1. The number of new LC cases for women
is the third in the category of cancer (after breast and colorectal cancer),
but the number of deaths just behind breast cancer.
According to the latest cancer records in Vietnam, after 10 years
from 2000 to 2010, the incidence of LC in women increased by more
than 200% (6.4 / 100,000 in 2000 to 13.9 / 100,000 in 2010), LC is also
one of the five fastest growing types of cancer.
1.2. Molecular pathology of lung cancer
Smoking is considered a major risk factor for LC,
approximately 80-85% of smoking cases are diagnosed with LC in
the world. Risk level depends on factors such as: age of smoking
(the sooner smoking is, the higher risk is), the number cigarette of
years (smoking more, the risk higher), the duration of smoking
(smoking longer, the risk higher). Smokers have a 10-fold increased
risk of LC compare with non-smokers. Studies have also shown that
even people who do not smoke directly, but often exposed to
smokers (passive smoking), also have a high risk of LC. There are
also many factors that are considered risk factors for LC such as air
pollution, ionizing radiation, occupational exposure, virus, diet,
history of bronchopulmonary disease.
Molecular studies show that the development and emergence
of LC occurs over a number of stages, under the influence of risk
factors, genetic susceptibility, and the accumulation of mutations
that occur on oncogenes and tumor suppressor genes. Normally, the
mechanisms of gene regulation that work smoothly and closely. In
the presence of disorders will lead to an abnormal increase or
inhibition of functional genes.
4
Figure 1.1: The molecular signaling pathways in lung cancer pathogenesis
(Pass & et al.).
2. TP53 and MDM2 genes
2.1. TP53, cancer suspressor gene
The TP53 gene is located on the short branch of chromosome 17
(17p13.1). The TP53’s length is 22,000 bp, including 11 exons (Encode
area from E1 to E11, E1 does not encode) and 10 introns. It encodes for
a protein molecules that weigh 53kDa with 393 acid amin and
consisting of 3 functional domains.
The TP53 gene plays an important role in DNA repair, controlling
cell division, and apoptosis. The defective TP53 gene allows abnormal
cell proliferation and leads to cancer formation. When the body is
affected by stimuli (demaged DNA, cellular stress, hypoxia, over
expression of the oncogene), p53 is activated to stop the cell cycle until
the DNA is repaired or induce apoptosis if the demaged DNA does not
repair. Thus, p53 is considered as the guardian of the genome. In
addition, p53 has the ability to activate or inhibit several other genes
2.2 MDM2
5
The MDM2 gene (Murine double minute 2), also known as HDM2
(Human double minute 2), consists of 12 exons and 1 intron on the long
branch of the 12th chromosome, it was first identified in 1980. MDM2
protein molecules are synthesized with 491 amino acids and consisting
5 functional structural domains.
To date, the most known important role of MDM2 has been to
regulate the activity of the TP53 gene in the p53 signaling pathway.
Under normal conditions, MDM2 binds to the p53-activated region,
which controls the distribution and degradation of the p53 protein. In
contrast, activated p53 promotes MDM2 replication so that the
expression of p53 and MDM2 in the cell is always maintained in
equilibrium through the reversal of MDM2 and p53. When stimulatory
factors (demaged DNA, cellular stress, hypoxia, over expression of the
oncogene) occur, MDM2 will be phosphorylated and exposed to the p53
activation region, triggering the p53 function.
3. TP53 and MDM2 gene polymorphisms in lung
cancer
Single nucleotide polymorphism (SNP) is the difference in DNA
sequence in the genome between individual persons or between
chromosomes of a person. This is a common phenomenon. It is the
result of mutation points that replace a pair of nucleotides. According to
the published studies, many SNPs were found in the TP53 gene and
dozens one on the MDM2 gene. These single nucleotide polymorphisms
create many different TP53 and MDM2 genotypes in the community.
The genotypes of some of these SNPs are involved in the onset of
development many type of cancers including LC. They are risk factors
to be considered.
The SNPs we analyzed in this study may change coding sequences
or not but they are all located in the key functional areas of TP53.
Theoretically, these areas can affect to the control tumor ability of
TP53. First of all, a polymorphism caused by the addition of 16 base
pairs in the intron-3 region of TP53. Those who carry these genotypes
express low levels of p53 in the cell and have increased risk for certain
cancers including lung, breast and colorectal cancer. It proves that SNPs
are capable of altering mRNA completion. In addition, although SNPs
on p53 coding regions 21 (GAC → GAT), 34 (CCC → CCA) and 36
(CCG → CCT) not change the amino acid sequence but reducing the
expression of p53 protein. Studies have shown that the SNPs in the
6
TP53 N-terminal activation region where contained an interactive
position with MDM2 and they can reduced the translation of TP53.
On the other hand, SNPs on the coding region altering the amino
acid sequence can lead to a change in the p53 binding ability to the
specific sequence in the target gene, in mRNA completment and
stability of the protein as well as alter the interactions of p53 with
intracellular proteins. These are SNPs located in codenamed 47 (P47S),
72 (R72P), 217 (V217M) and 360 (G360A). Under normal conditions,
with the action of p38 and homeodomain-interacting protein kinase 2
(HIPK2), p53 is phosphorylated at position S46 leading to increased
replication of genes involved in programmed death (appotosis). And
when the p53-P47 allele was replaced by the p53-S47 allele, the
phosphorylation at site S46 reduced activity on the target genes of
phagocytosis and increased the probability of cancer.
Similarly, polymorphism in the triple coding 72 (R72P) produced
two genotypes: p53-R72 and p53-P72. Studies by Boldrine et al. Show
that p53-P72 homozygotes have a higher risk of lung cancer [48]. At the
same time, the p53-P72 genotype and MDM2 G/G genotype are also
common in patients with lung cancer who has smoked over the long
term. For the other two forms of SNPs, V217M is located on the DNA
binding domain of the p53, which may reduce p53 activity and directly
affected genes including CDKN1A, BAX and PMAIP1. Functional
studies have shown that the p53-M217 genotype have a higher
expression of the p53-V217. Thus, the p53-M217 genotype is capable
of protecting cells against carcinogens better than p53-V217. However,
the molecular mechanism of this phenomenon is not yet clear. SNPs
G360A is located at the junction of p53. These SNPs affect the
expression of BAX and MDM2, which are important genes in the p53
signaling pathway.
The single nucleotid polymorphism of the MDM2 gene is located at
the first intron, rs2279744 (MDM2 - SNP309), with the change from T
to G (MDM2 - SNP309 T>G) increased the affinity of SP1 (Stimulatory
protein 1) with MDM2, results in increased expression of MDM2
leading to inhibitory TP53 gene as a condition for cancer formation and
progression.
Many international epidemiological studies have been conducted to
find a link between single nucleotide polymorphism of TP53, MDM2
and lung cancer. The published results are still unanimous, but one
7
thing in common is that all R72P gene polymorphism TP53 and
309T>G MDM2 genes are the two most commonly SNPs associated
with lung cancer. The differences among studies that can be explained
by the differences in sample size or racial and environmental factors of
the study population.
The fact that cancer is the result of a complex process in which
there are interactions of many factors such as genotype, biological
characteristics as well as habitat. Therefore, when studying SNPs in
lung cancer, relevant analyzes with biological characteristics or
smoking status and environmental pollution should be conducted in
order to assess in a comprehensive manner and recommend valuable
information for lung cancer prevention strategies.
Chapter 2
SUBJECTS AND METHODS
2.1. Research Subjects
The study was conducted on 220 patients with primary lung cancer
diagnosed at the Respiratory Center, the Nuclear Medicine and
Oncology Center - Bach Mai Hospital and 230 controls from October
2013 to December 2017.
2.1.1. Criteria for selecting patients
- 220 patients were diagnosed with primary lung cancer at the
Respiratory Center and the Nuclear Medicine and Oncology Center -
Bach Mai Hospital with histopathological results.
- Agree to participate in research.
2.1.2. Exclusion criteria
- Secondary lung cancer.
- Lung cancer combine with other cancers.
- Do not agree to participate in research.
2.1.3. Control group
- 230 controls were selected from those who came to the medical
examination at Bach Mai Hospital. Clinical examinations, laboratory
tests, pulmonary X-rays, ultrasonography and conclusions without LC
or any other cancer.
- Corresponding age and gender to lung cancer patients.
2.1.4. Genotypic polymorphisms were analyzed
- TP53 gene
8
+ Add 16 pairs of base pairs at intron 3 (dup 16).
+ SNP P34P, at 34 codon, exon 4 (CCC → CCA), Prolin coding.
+ SNP P36P, at codon 36, exon 4 (CCG → CCA), Prolin coding.
+ SNP P47S, at codon 47, exon 4, (CCG or TCG), encoding Prolin
or Serin.
+ SNP R72P at codon 72, exon 4, (CGC or CCC), encoding
Arginine or Prolin.
+ SNP V217M, at codon 217, exon 6, (GTG or ATG), encoding
Valin or Methionine.
+ SNP G360A at codon 360, exon 10, (GGG or GCG), encoding
Glycin or Alanin.
- MDM2 gene: SNP locates at nucleotide position 309, intron 1,
promoter region.
2.2. Research Methodology:
Using cross-sectional descriptive study with control.
2.3. Study time and place
Time from 10/2013 to 10/2017.
Research site: the Respiratory Center, the Nuclear Medicine and
Oncology Center - Bach Mai Hospital. Department of Biochemistry and
Center for Gene Research - Protein, Hanoi Medical University.
2.4. Thread adhere research ethics in medicine
This study was approved by the ethics committee of Hanoi
Medical University (Dicision no. 188/HĐĐĐĐHYHN, 31/1/2013).
2.5. Funds for the study
Our study got the funding support of a national level project
“Evaluate the genotype distribution of several genes involved in lung and
liver cancer” belong to study "Evaluating Vietnamese Genetic
Characteristics".
2.6. Procedures and techniques used in the study
The techniques used in the study included: Interview and turn
up medical treatment documents to identify risk factors exposuring.
DNA extraction technique from peripheral blood samples. PCR
technique detects the genotype of dup16 polymorphism of TP53 gene.
Using restriction fragment length polymorphism technique for
genotyping R72P SNP of TP53 gene and 309T>G SNP of MDM2 gene.
Sequencing technique to identify genotypes of SNPs: P34P, P36P,
9
P47S, V217M, G360A of TP53 gene. Research process follow as below
diagram.
PROCESS DIAGRAM
Chapter 3
RESULTS
3.1. Characteristics of the study subjects
Table 3.1. Distribution of study subjects
Patients Controls
Characteristic (220) (230) p
n % n %
59,89 ± 60,67 ±
Age (year) X SD 9,432 9,335
0,379
Male 163 74,1 157 68,3
Sex 0,173
Female 57 25,9 73 31,7
Yes 94 42,7 68 29,6
0,004
No 126 57,3 162 70,4
Smoking history
< 20 pack-year 43 45,7 33 48,5
0,726
> 20 pack-year 51 54,3 35 51,5
Histopa NSCLC Adenocarcinoma 161 73,2
10
Carcinoma 13 5,9
-thology Other carcinoma 25 11,4
SCLC 21 9,5
Comment:
- Adenocarcinoma accounts for the highest proportion of
histopathology.
- The smoking rate in the patients group was 42.7% higher and
statistically significant than the control group (p = 0.004). The
proportion of LC patients who have smoked > 20 pack-year was 54.3%,
had no difference with the proportion of LC patients who have smoked
<20 pack-year (45.7% (p = 0.726)).
- No smoking in women was found in our study.
3.2 Results of TP53 genotyping
3.2.1. Adding 16 base pairs at intron 3 (dup16)
The fragment carrying intron 3 of the TP53 gene was amplified
by PCR reaction with specific primers, PCR product was
electrophoresed on 3% agarose gel.
Figure 3.1. Electrophoresis of PCR product amplified the fragment
carrying intron 3 of the TP53 gene on agarose gel 3%
Samples K114, K115, K117, K118, C96÷C100: genotype A1A1; sample
K116: genotype A1A2; M: Ladder 100bp, (-): Nagative
Comment: A1A1 genotype had single band with 119bp size. A1A2
genotype had 2 band with 119bp and 135bp size. DNA band had clear
without additional banding, ensure that 16bp polymorphic genotypes
are identified at the intron 3 of the TP53 gene.
11
Table 3.2 Genetic analysis results of dup16 SNP of TP53 gene between
patient and control group
Patient Control
SNP (n=220) (n=230) OR
n % n %
A1A1 212 96,4 226 98,3 1,0
A1A2 8 3,6 4 1,7 2,13 (0,633 - 7,184)
Comment: A1A2 genotype was 3.6% that the lung cancer group was
higher than the control group (1.7%), but the difference was not
statistically significant.
3.2.2. SNP R72P gen TP53
Results of genotyping at SNP R72P by PCR-RFLP method.
Figure 3.2: Electrophoresis of the cutting product of gene fragment
contains R72P SNP by BstUI enzyme on research samples.
M: Ladder 100bp; (-): Negative; (+): Positive. Samples K60, K61, C7 :
Genotype CC (Pro/Pro). Samples K69, K73, C8: Genotype GG (Arg/Arg).
Samples K46, K48, C13: Genotype GC (Arg/Pro).
Comment:
The cutting product of gene fragment contains R72P SNP by BstUI
enzyme including DNA fragment of different sizes, in accordance with the
theoretical calculations. The GG (Arg / Arg) genotype consists of two DNA
fragments of 165 bp and 231 bp (K69, K73, C8). CC (Pro / Pro) hybridization
when only one DNA band of 396bp (K60, K61, C7) appears. The GC
homologation (Arg / Pro) consists of three bands with dimensions of 396bp,
231bp and 165bp (K46, K48, C13).
12
Checking the results of genotyping of SNP R72P by sequencing.
Figure 3.3: Sequencing results of exon 4 on TP53 containing SNP R72P are
corresponding to genotype: GC (Arg/Pro), CC (Pro/Pro), GG (Arg/Arg).
Comment: The DNA sequence of these sample was completely
matched to its PCR-RFLP analysis.
Table 3.3: R72P SNP genotype of TP53 gene and risk
of lung cancer
Patient Control
SNP (n=220) (n=230) OR, 95%CI
n % n %
G 219 49,8 248 53,9 1,0
Allel
C 221 50,2 212 46,1 1,18 (0,91 – 1,53)
G/G 57 25,9 77 33,5 1,0
Genotype
G/C 105 47,7 94 40,9 1,51 (0,97 - 2,35)
C/C 58 26,4 59 25,7 1,33 (0,81 - 2,19)
Combination G/
162 73,6 171 74,3 1,0
of recessive G+G/C
genes C/C 58 26,4 59 25,7 1,04 (0,68 - 1,58)
Combination G/G 57 25,9 77 33,5 1,0
of recessive G/C +
163 74,1 153 66,5 1,44 (0,96 - 2,16)
genes C/C
Comment: G/C genotype of codon 72 of TP53 gene was the highest in both
patient and control group. G/C and C/C genotype of codon 72 of TP53 gene
may be possible to increase the risk of lung cancer, but the association is not
statistically significant.
3.2.3. Polymorphism in SNPs: P34P, P36P, P47S,
V217M, G360A
Using the sequencing techniques to analyze genotypes at these
SNPs of TP53 gene.
13
Table 3.4: Genotypes of SNPs: P34P, P36P, P47S, V217M, G360A
of TP53 gene
Mutant
Wild homozygous Heteropathic
homozygous
Genotype genotype genotypes
genotypes
n % n % n %
C/C C/A A/A
P34P(C>A)
450 100 0 0 0 0
G/G G/A A/A
P36P(G>A)
450 100 0 0 0 0
* *
C/C (P47P) C/T (P47S) T/T (S47S)*
P47S(C>T)
450 100 0 0 0 0
G/G (V217V)* G/A (V217M)* A/A (M217M)*
V217M(G>A)
450 100 0 0 0 0
G/G (G360G)* G/C (G360A)* C/C (A360A)*
G360A(G>C)
450 100 0 0 0 0
(*) The amino acids are encoded by that change the nucleotid sequence.
Comment: No mutation genotype was found in our study.
3.3. Results of SNP309 genotype of MDM2 analysis
Using restriction fragment length polymorphism technique
(RFLP) for genotyping 309T>G SNP of MDM2 gene.
Hình 3.4: Electrophoresis of the cutting product of gene fragment
contains SNP309 of MDM2 gene by MspA1i enzyme on research samples.
Samples K17, C7: Homozygous genotype T/T. Samples K16, K23, C16:
Homozygous genotype G/G. Samples K7, K13, C18, C21: Heterozygous
14
genotype T/G. M: Ladder 100bp; (-): Negative; (+): Positive.
Comment:
The cutting product of gene include DNA fragment of different sizes, in
accordance with the theoretical calculations. The samples with T/T genotype
consist of only one DNA band of 157 bp (K17, C7). The samples with G/G
genotype consist of two DNA bands of 109 bp and 48 bp (K16, K23, C16) CC
(Pro / Pro) hybridization when only one DNA band of 396bp (K60, K61, C7)
appears. The samples with heterozygous TG consists of three bands with
dimensions of 157bp, 109 bp và 48 bp (K7, K13, C18, C21).
Checking the results of genotyping of SNP 309T>G of MDM2 gene
by sequencing.
Figure 3.5: Sequencing results of gene fragment contains SNP 309T>G
of MDM2 corresponding to T/T, T/G, G/G genotypes
Comment: The DNA sequence of these sample was completely
matched to its PCR-RFLP analysis.
Table 3.5: SNP309T>G genotype of MDM2 gene and risk
of lung cancer
Patient Control
OR, OR*,
SNP (n=220) (n=230)
95%CI 95%CI
n % n %
21 49, 24 52,
T 1,0
7 3 1 4
Allel 1,13
22 50, 21 47,
G (0,87 –
3 7 9 6
1,47)
Genotype 27, 23,
TT 60 55 1,0 1,0
3 9
TG 97 44, 13 57, 0,68 0,65
1 1 0 (0,43 – (0,41 –
1,07) 1,03)
15
1,31 1,10
28, 19,
GG 63 44 (0,77 – (0,84 –
6 1
2,32) 1,44)
TT+T 15 71, 18 80,
Combinati 1,0 1,0
G 7 4 6 9
on of
recessive
1,7 1,61
28, 19,
genes GG 63 44 (1,09 – (1,03 –
6 1
2,63) 2,51)
72, 23,
Combinati TT 60 55 1,0 1,0
7 9
on of
recessive
0,84 0,78
TG + 16 27, 17 76,
genes (0,55 – (0,51 –
GG 0 3 5 1
1,28) 1,20)
OR* is adjusted from the variables: age, gender, smoking habit by
multivariate logistic regression model.
Nhận xét:
The heterozygosity of SNP 309TG was highest in both disease and
control groups. The SNP 309GG homozygous genotype increased the risk of LC
by 1,7-fold in the recessive gene model (OR = 1,7; 95% CI = 1,09-2,63). When
corrected for the variables age, gender and smoking status in the multivariate
logistic regression model still showed homozygous SNP 309GG genotype
increased the risk of 1,61-fold as the model of recessive genes (OR = 1,61; 95%
CI = 1,03 – 2,51).
3.4 Correlation between polymorphism of TP53, MDM2 and risk of
lung cancer.
3.4.2. Relationship between polymorphism SNP309T> G of MDM2 gene and
the risk of lung cancer by clinical and subclinical characteristics in lung
cancer.
3.4.2.1. Relationship between polymorphism SNP309T> G of MDM2 gene and
the risk of lung cancer by gender.
Table 3.6: Relationship between polymorphism SNP309T> G of MDM2
gene and the risk of lung cancer by gender
OR GG/TT OR TG/TT OR GG/TG+TT OR TG+GG/TT
Group
(95%CI) (95%CI) (95%CI) (95%CI)
Male 1,31 0,69 1,66 0,87
16
(0,71 – 2,43) (0,41 – 1,18) (1,01 - 2,76) (0,526 - 1,43)
1,26 0,67 1,67 0,79
Female
(0,43 – 3,67) (0,29 – 1,56) (0,68 – 4,06) (0,35 – 1,77)
Comment: The homozygous SNP 309GG genotype increases the risk of LC in
males by 1,66-fold as the model of recessive genes ( OR-1,66; 95%CI=1,01-
2,76).
17
3.4.2.2. Relationship between polymorphism SNP309T> G of MDM2 gene and
the risk of lung cancer by histopathology.
Table 3.7: Relationship between polymorphism SNP309T> G of MDM2
gene and the risk of lung cancer by histopathology
OR GG/TT OR TG/TT OR GG/TG+TT OR TG+GG/TT
Group
(95%CI) (95%CI) (95%CI) (95%CI)
1,37 0,72 1,71 0,88
NSCLC
(0,79 – 2,37) (0,45 – 1,14) (1,09 – 2,68) (0,57 – 1,37)
0,94 0,42 1,59 0,55
SCLC
(0,30 – 2,90) (0,15 – 1,18) (0,59 – 4,28) (0,22 – 1,38)
Adenocarcinom 1,40 0,76 1,69 0,92
a (0,79 – 2,50) (0,46 – 1,24) (1,05 – 2,72) (0,58 – 1,47)
2,50 1,47 1,88 1,73
Carcinoma
(0,44 – 14,29) (2,97 – 7,30) (0,55 – 6,38) (0,37 – 8,04)
Comment: The homozygous SNP 309GG genotype increases the risk of
NSCLC by 1,71-fold (OR=1,71; 95% CI= 1,09-2,68) and Adenocarcinoma by
1,69-fold (OR=1,69; 95%CI= 1,05-2,72) as the model of recessive genes in
smoking group.
3.4.2.3. Relationship between polymorphism SNP309T> G of MDM2 gene and
the risk of lung cancer by smoking status
Table 3.8: Relationship between polymorphism SNP309T> G of MDM2
gene and the risk of lung cancer by smoking status
OR GG/TT OR TG/TT OR GG/TG+TT OR TG+GG/TT
Group
(95%CI) (95%CI) (95%CI) (95%CI)
1,86 0,85 2,09 1,12
Smoking
(0,74 – 4,68) (0,38 – 1,90) (1,01 – 4,31) (0,52 – 2,40)
0,98 0,58 1,38 0,68
No smoking
(0,50 – 1,90) (0,34 – 1,01) (0,78 – 2,43) (0,41 – 1,14)
Smoking <20 1,86 0,92 1,99 1,18
pack-year (0,48 – 7,26) (0,28 – 3,04) (0,70 – 5,65) (0,37 – 3,66)
Smoking >20 1,87 0,80 2,18 1,08
pack-year (0,53 – 6,60) (0,27 – 2,39) (0,80 – 5,98) (0,38 – 3,03)
18
Comment: The homozygous SNP 309GG genotype increases the risk of LC in
smoker group by 2,09-fold as the recessive gene model (OR= 2,09; 95%CI=
1,01-4,31).
3.4.3. Risk of lung cancer in combination of R72P SNP of TP53 gene and
SNP309T> G of MDM2 gene
3.4.3.1. Risk of lung cancer in combination of R72P SNP of TP53 gene and
SNP309T> G of MDM2 gene with smoking
Table 3.9: Risk of lung cancer in combination of R72P SNP of TP53 gene
and SNP309T> G of MDM2 gene with smoking
Patient Control
Characteristics OR
n % n %
Smoking 126 57,3 162 70,4 1,00
1,78
No smoking 94 42,7 68 29,6
(1,20 - 2,62)
Smoking <20 pack- 1,68 (1,01 –
43 45,7 33 48,5
year 2,79)
Smoking >20 pack- 1,87 (1,15 –
51 54,3 35 51,5
year 3,06)
GG of SNP R72P
of TP53 gene and 32 57,1 53 80,3 1,00
no smoking
CC of SNP R72P
3,06
of TP53 gene and 24 42,9 13 19,7
(1,37 - 6,84)
smoking
TT of
SNP309T>G of
41 55,4 40 74,1 1,00
MDM2 gene and
no smoking
GG of 2,30
SNP309T>G of (1,07 - 4,93)
33 44,6 14 25,9
MDM2 gene and
smoking
Comment:
- Smoking increased the risk of LC by 1,78-fold (OR = 1,78; 95% CI = 1,20-
2,62).
- Smoking > 20 pack-year increased the risk of LC by 1,87-fold (OR = 1,87;
95% CI = 1,15-3,06) was higher than the risk of LC when smoking <20
pack-year (OR = 1.68, 95% CI = 1.01 - 2.79).