Single nucleotide polymorphisms in leptin gene may increase the risk of breast cancer. The aim of this study was to investigate the association of leptin G-2548A gene polymorphism with breast cancer risk in an Iranian population. In a case-control study, 98 subjects including 45 women with breast cancer and 53 healthy women were recruited. The genotypes of G-2548A polymorphism were detected by polymerase chain reaction-restriction fragment length polymorphism. Our data revealed that there is a significant association between AG homozygote genotype and breast cancer risk (OR= 2.41, 95CI= 1.05-5.52, p= 0.037). Also, we observed that G allele marginally increases the breast cancer risk (OR= 1.92, 95CI= 1.00-3.70, p= 0.049). Based on our preliminary study, leptin G-2548A gene polymorphism may be a genetic risk factor for breast cancer and further studies with larger sample size are required to obtain more accurate results. Â© 2019, Indian Journal of Forensic Medicine and Toxicology. All rights reserved

Arab-Yarmohammadi, V.

Sharifi, M.

English

kashan university of medical sciences

Correlation of A G to A Nucleotide Substitution in Upstream 
Region of Leptin with Breast Cancer Susceptibility
 Vahid Arab-Yarmohammadi1, Mehran Sharifi2
1Student Research Committee, School of Nursing and Midwifery, Shahroud University of Medical Sciences, 
Shahroud, Iran, 2Department of Internal Medicine‚ Faculty of Medicine‚ Kashan University of Medical Sciences, 
Kashan, Iran
Abstract
Single nucleotide polymorphisms in leptin gene may increase the risk of breast cancer. The aim of this 
study was to investigate the association of leptin G-2548A gene polymorphism with breast cancer risk in 
an Iranian population. In a case-control study, 98 subjects including 45 women with breast cancer and 53 
healthy women were recruited. The genotypes of G-2548A polymorphism were detected by polymerase chain 
reaction-restriction fragment length polymorphism. Our data revealed that there is a significant association 
between AG homozygote genotype and breast cancer risk (OR= 2.41, 95%CI= 1.05-5.52, p= 0.037). Also, 
we observed that G allele marginally increases the breast cancer risk (OR= 1.92, 95%CI= 1.00-3.70, p= 
0.049). Based on our preliminary study, leptin G-2548A gene polymorphism may be a genetic risk factor for 
breast cancer and further studies with larger sample size are required to obtain more accurate results.
Keywords: Breast cancer: Leptin gene; G-2548A variety.
Introduction
Breast cancer is one of the common malignancy 
among women around the world1, 2. The breast cancer is a 
heterogenic disease and many environmental and genetic 
factors are involved in the development of it3, 4. Single 
nucleotide polymorphisms (SNPs) as genetic variations 
could be involved in susceptibility and development 
of diseases including breast cancer5, 6, 7. Genes such as 
BRCA-1, BRCA-2, and P53 are most well-known genes 
in breast cancer. Women with mutations in these genes 
are diagnosed with breast cancer during their lifetimes 
which most of them can be recognized at a young age 
before menopause8, 9. Leptin gene is another gene which 
may have an important role in breast cancer risk10.
Leptin as an adipocytokine, encodes by the human 
LEP gene and it secrets by adipocytes11. Some studies 
revealed that this protein and its receptor have an 
increased expression in breast cancer, particularly 
in high-grade tumors. Also, it is associated with 
development of breast cancer12. Leptin is essential for 
the growth of tumor cells and its effects arises from 
discerning binding to the receptor. The receptor of 
this protein is expressed in various tissues, such as the 
mammary gland13.
The LEP gene is located on chromosome 7 (7q32.1) 
with three exons. There are many polymorphisms in 
this gene that the rs7799039 is one of the common 
polymorphisms of this gene. This polymorphism is 
located at the upstream of LEP gene, and it results in 
G to A nucleotide at -2548 position (G-2548A). The 
aim of this study was to investigate the association of 
aforementioned polymorphism with breast cancer risk 
in an Iranian population.
Materials and method
Subjects
In a case-control study, we employed 98 subjects 
(including 45 women with breast cancer and 53 age-
matched healthy women) from Shahid Beheshti Hospital 
(Kashan, Iran). The breast cancer was approved by 
pathology tests. The control subjects were healthy 
women without any familial history of malignancy. After 
obtaining signed informed consent form, we collected 
2 ml peripheral blood samples from each subjects into 
sterile tubes containing EDTA anticoagulant agent.
DOI Number: 10.5958/0973-9130.2019.00146.4 
Indian Journal of Forensic Medicine & Toxicology,  April-June 2019, Vol. 13, No. 2        365 
DNA extraction and SNP genotyping
The genomic DNA was extracted from blood samples 
by a general salting out procedure14. The G-2548A SNP 
genotyping was performed by polymerase chain reaction-
restriction fragment length polymorphism (PCR-RFLP) 
method. The primers sequences were obtained from 
previous study15. For primer checking, at first the 
whole genomic sequence of LEP gene was deduced 
from national center for biotechnology information 
(NCBI) databank. The primer sequences were analyzed 
around the G-2548A polymorphism position by Oligo7 
software. The forward and reverse primers sequences 
were 5’-TTTCCTGTAATTTTCCCATGAG-3’ 
and 5’-AAAGCAAAGACAGGCATAAAA-3’, 
respectively. PCR was carried out in 25µl total volume 
containing 2.5μl 10X PCR buffer, 0.75μl of each primer 
(10pm/μl), 0.75μl MgCl2 (50 mM), 0.5 μl dNTP (10 
mM), 0.3U Taq DNA polymerase, and 60 ng template 
DNA. The PCR was performed in a Peqlab thermal cycler 
system with the following program: initial denaturation 
at 95°C for 5 min, followed by 35 repetitive cycles 
including denaturation at 95°C for 45 secs, annealing 
at 51°C for 45 secs, and extension at 72°C for 1min 
and a final extension at 72°C for 10 min. The 242bp 
amplified fragments were treated by HhaI restriction 
enzyme (Fermentas CO., Germany) according to the 
manufacturer protocol. The digested fragments were 
separated by 3% agarose gel electrophoresis which 
stained by ethidium bromide and visualized in UV light. 
It should be noted that the presence of G nucleotide at 
G-2548A position created HhaI restriction site.
Statistical analysis
The Hardy-Weinberg equilibrium (HWE) was 
evaluated by Chi-square test in both case and control 
groups. Also, the similar test was used to assess the 
differences in the distribution of alleles and genotypes 
between case and control groups. The association of 
G-2548A polymorphism with breast cancer risk was 
also was evaluated by odds ratios (ORs) with 95% 
confidence intervals (CIs). A p value less than 0.05 
was considered statistically significant. SPSS software 
version 19.0 (SPSS, Inc., Chicago IL, USA) was used 
for the all statistical analyses.
Results
The distribution of allele and genotype frequencies 
is presented in table 1. The genotype distribution 
showed that control group was at Hardy-Weinberg 
equilibrium (p> 0.05). Our data revealed that there is 
a significant association between AG genotype and 
breast cancer risk (OR= 2.41, 95%CI= 1.05-5.52, p= 
0.037), whereas there was no significant association 
between GG genotype and breast cancer risk (OR= 
3.67, 95%CI= 0.31-43.27, p= 0.302). Also, carriers of 
G allele (AG+GG) were at a high risk for breast cancer 
risk (OR= 2.48, 95%CI= 1.10-5.59, p= 0.029). Allele 
analysis showed that the frequency of A allele is 67.78% 
and 80.19% for case and control groups, respectively. 
Therefore, the frequency of G allele was 32.22% and 
19.81% for cases and controls, respectively. Thereby, 
we observed a marginal significant association between 
G allele and breast cancer risk (OR= 1.92, 95%CI= 
1.00-3.70, p= 0.049). 
Table 1. Genotype and allele frequencies of G-2548A in cases and controls
Genotype
No. and  Percentage
OR
(95% CI)
p-value
Control  (n=53) Case(n=45)
AA
33
(62.26%)
18
(40.00%)
- -
AG
19
(35.85%)
25
(55.56%)
2.41 
(1.05-5.52)
0.037
GG
1
(01.89%)
2
(04.44%)
3.67 
(0.31-43.27)
0.302
AG+GG
20
(37.74%)
27
(60.00%)
2.48 
(1.10-5.59)
0.029
Allele
A
85
(80.19%)
61
(67.78%)
- -
G
21
(19.81%)
29
(32.22%)
1.92 
(1.00-3.70)
0.049
366        Indian Journal of Forensic Medicine & Toxicology, April-June 2019, Vol. 13, No. 2
OR: odds ratio, CI: confidence interval
Significant differences between the case and control 
groups are bolded
Discussion
In this study we evaluated the association of LEP 
G-2548A gene transition with risk of breast cancer in 
an Iranian population. Our data revealed that there are 
significant associations between AG genotype and G 
allele and risk of breast cancer. Also, we observed that 
carriers of G allele (AG+GG) were at a high risk for breast 
cancer. Some previous studies showed similar results. 
For example15 reported that AG, GG and G varieties are 
associated with risk of breast cancer in population of 
south of Iran. In addition16 reported that GG genotype 
and G allele are associated with breast cancer risk in 
north of Iran. While17 reported that there is no significant 
association between LEP G-2548A polymorphism and 
risk of breast cancer in a population from center of Iran. 
The different results in aforementioned studies may 
result in environmental and ethnic factors18. 
Leptin is considered as an important protein for the 
human mammary gland growth, as far as latest studies 
have revealed that the protein might play a pivotal role 
in breast cancer19. Expression of leptin gene in cell 
lines of breast cancer, solid tumors, and normal breast 
tissue have been reported. Overexpression of leptin 
in the most breast cancers was reported whereas this 
overexpression has not been reported in cell of healthy 
Moreover, leptin can cause breast cancer cells to become 
malignant forms20, 21. Also, it is reported that leptin 
and leptin receptor overexpress tumorous tissues19. 
Leptin plays some roles such as growth of cells, cell 
differentiation, and angiogenesis. Also, it could increase 
some products of endothelial cells including nitric oxide 
and overexpression of VEGFR-2, FGF2, and VEGF22. 
In some cells such as malignant or normal epithelial 
cells, the leptin can act as a migration and mitogenic 
factor23. Leptin also could induce cancer cells growth 
in breast cancer epithelium via aromatase expression 
and estrogen production or activation24. Evidences show 
overexpression of leptin and leptin receptor in initial 
breast cancer with metastases25.
Single nucleotide polymorphisms are a group of 
genetic variations that could happen in every region 
of genes. The influence of SNPs is dependent on the 
location of them on genes26, 27. SNPs in promoter 
region of genes could affect the gene expression28. 
LEP G-2548A transition as an upstream polymorphism 
may alter gene expression. In silico analysis is a useful 
tool for evaluation of effects of SNPs29, 30, 31. Therefore, 
it is suggested that further studies are focused on the 
molecular effects of LEP G-2548A by computational 
analysis.
Finally, there are some limitations in our study that 
we should be mentioned in this section. At first, we don’t 
evaluate gene-gene and gene-environment interactions. 
Also, the sample size of our study was very small.
Conclusion
Leptin G-2548A gene transition may be a genetic 
risk factor for breast cancer, but further studies with 
larger sample size with regard to mentioned interactions 
are need to obtain more accurate results. 
Ethical Clearance- All the participants’ informed 
written consent and this study confirmed by the principles 
outlined in the Declaration of Helsinki and approved by 
the Shahid Beheshti Hospital’s Ethics Committee.
Source of Funding- This study was performed by 
a personal expense. 
Conflict of Interest- The authors report no conflicts 
of interest.
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Correlation of A G to A nucleotide substitution in upstream region of leptin with breast cancer susceptibility

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Correlation of A G to A nucleotide substitution in upstream region of leptin with breast cancer susceptibility

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