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ORIGINAL ARTICLE
Year : 2020  |  Volume : 16  |  Issue : 6  |  Page : 1269-1273

Diagnostic value of adiponectin gene polymorphism and serum level in postmenopausal obese patients with breast cancer


1 Department of Clinical and Chemical Pathology, Faculty of Medicine, Cairo University, Cairo, Egypt
2 Department of Clinical and Chemical Pathology, National Cancer Institute, Cairo University, Cairo, Egypt
3 Department of Clinical and Chemical Pathology, Cancer Hospital, Cairo, Egypt

Date of Submission07-Dec-2019
Date of Acceptance12-Apr-2020
Date of Web Publication18-Dec-2020

Correspondence Address:
Nevine F Shafik
Department of Clinical and Chemical Pathology, National Cancer Institute, Fom Elkhalig, Kasr Elaini Street, Cairo
Egypt
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jcrt.JCRT_1091_19

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 > Abstract 


Context: Obesity has been strongly associated with risks and is a common factor in the risk of postmenopausal women with breast cancer (BC). Various single-nucleotide polymorphisms have been identified in the adiponectin gene.
Aims: We aimed in this study to access the diagnostic value of adiponectin gene polymorphism rs 1501299 (G267T) in BC and its association with serum adiponectin level in obese and overweight postmenopausal BC female patients.
Settings and Design: This study was conducted on 90 BC patients divided into two groups according to body mass index (BMI), and 60 apparently healthy females as a control group with matched BMI. Both groups were with BMI >25 (obese or overweight).
Subjects and Methods: All participants were subjected to laboratory investigations (CA 15–3, serum adiponectin) and molecular study of adiponectin gene rs 1501299 (G276T) by polymerase chain reaction-restriction fragment length polymorphism technique.
Results: A statistically significant difference was observed in the polymorphic genotypes (GT and TT) compared to (GG) wild genotype when compared BC patients to control group (P = 0.001). Also on measuring the risk estimate, a significant difference (odd's ratio was 3.76, 95% confidence interval was 1.68–8.4, P = 0.001). While no statistical significant difference in genotype frequency was found between the obese and overweight patients (P > 0.05). Median serum adiponectin level was decreased in BC patients compared to the control group (8.9 vs. 14.6 with P = 0.004).
Conclusions: This study supported the association between adiponectin gene polymorphism, serum level, and BC risk among a group of obese and overweight postmenopausal Egyptian patients.

Keywords: Adiponectin gene polymorphism, breast cancer, obesity


How to cite this article:
Mahmoud EH, Fawzy A, El-Din WM, Shafik NF. Diagnostic value of adiponectin gene polymorphism and serum level in postmenopausal obese patients with breast cancer. J Can Res Ther 2020;16:1269-73

How to cite this URL:
Mahmoud EH, Fawzy A, El-Din WM, Shafik NF. Diagnostic value of adiponectin gene polymorphism and serum level in postmenopausal obese patients with breast cancer. J Can Res Ther [serial online] 2020 [cited 2021 Dec 4];16:1269-73. Available from: https://www.cancerjournal.net/text.asp?2020/16/6/1269/303884




 > Introduction Top


One of the world's dominant causes of cancer-related deaths among females is breast cancer (BC) that is one of the most frequently diagnosed malignancies.[1]

Genetic predisposition play a role in cancer development but aside from it, other factors can play a role in the pathologic process of BC. These factors include obesity which is a major risk issue for breast carcinogenesis and poor disease outcome.[2]

Studies have reported that obesity is greatly linked with increased risk of BC and also its poor outcome.[3] It can promote BC through various processes such as high estrogen production, hyperinsulinemia due to insulin resistance, production of a group of polypeptide growth factors and cytokines including adiponectin and leptin, which may serve as potential biomarkers and therapeutic targets for the management of this aggressive disease.[4],[5]

Adiponectin, a 244-amino acid peptide supermolecule, is one of the most important adipokines, it is produced exclusively from white adipose tissue and encoded by adiponectin gene at 3q27; it contains 3 exons and 2 introns.[6]

Many studies reported the anti-carcinogenic effects of adiponectin through decreasing angiogenesis, anti-proliferative role, pro-apoptotic, and anti-inflammatory effect that propose it as a potent tumor suppressor protein.[7],[8] The role of adiponectin in BC needs more studies, it has reported that adiponectin stimulates the sensitivity of peripheral tissue to insulin, and minimized levels of adiponectin are related to enhanced serum insulin levels, that accompany insulin resistance. Insulin has been revealed to stimulate the proliferation of breast cancerous cells through its binding to insulin and insulin-like growth factor I (IGF-I) receptors. Besides, insulin may synergize with the mitogenic effects of estrogen. It may also upregulate the vascular endothelial growth factor expression, a potent angiogenic agent that is secreted by BC cells.[9] Adiponectin level has been inversely related with estrogen levels.

Several polymorphisms have been identified in humans and studied by many authors for their possible association with insulin resistance indexes and circulating adiponectin levels.[10],[11] One polymorphism with T to G substitution in exon 2 (T45G) and another one with G to T substitution (G276T) at intron 2 were frequently encountered in all population tests, for that most researches study the effect of these single-nucleotide polymorphisms (SNPs) in different diseases including cancer.[12]

We aimed to study the influence of (G 276 T) polymorphism in the adiponectin gene on serum adiponectin level and its association with BC in a group of postmenopausal obese females with BC.


 > Subjects and Methods Top


This study was conducted on a total number of 150 participants, 90 newly diagnosed patients with BC admitted to the outpatient clinic of National Cancer Institute (NCI) from April 2014 to May 2016, Cairo University. Moreover, 60 healthy control volunteers. The study was approved by the ethical committee of NCI and informed consent was obtained from the participant before enrollment in the study. Premenopausal females and females with body mass index (BMI) <25 kg/m2 were excluded from the study.

Anthropometric measurement

BMI (kg/m2) was calculated according to the following formula:

BMI = Weight (kg)/Height.

Sampling

From each patient, 10 ml venous blood was withdrawn; 3 mL was collected in a sterile EDTA for vacationers for DNA extraction and analysis for adiponectin G276T polymorphism using polymerase chain reaction (PCR)-restriction fragment length polymorphism and 5 ml in plain tubes, sera were separated for assay of serum CA15-3 level and serum adiponectin level by enzyme-linked immunosorbent assay kit supplied by SunRed Biological co.(http://www.sunredbio.com).

Imaging techniques and radiological examination

Breast mammography, MRI for cancer patients were done. Chest X-ray, PET scan and bone scan were done for cancer patients to exclude metastasis.

Histopathological study and immunohistochemical assay

IHC analysis of paraffin sections is routinely performed for the evaluation of estrogen receptor (ER), progesterone receptor (PR), and Her-2/neu (HER2) status for molecular characterization of breast cancer

Methodology

Extraction of genomic DNA

Genomic DNA was isolated from peripheral blood leucocytes using the Biospin Whole Blood Genomic DNA Extraction kit (Bioflux Corporation, Arrow Plaza, Tokyo, Japan). Enzymatic amplification was performed by PCR using Master Taq polymerase enzyme supplied by Fermentas (LT-02241 Vilnius, Lithuania) and thermal cycler (Promega Corporation, Madison, USA).

Amplification of adiponectin gene polymorphism rs 1501299 (G276 T)

Amplification of adiponectin gene polymorphism rs 1501299 (G276 T) was performed using two sets of primers provided by Operon Biotechnologies GmbH (Germany), forward primer (sense primer) 5' GGC CTC TTT CAT CAC AGA CC 3', Reverse primers (anti sense primer) 5' AGA TGC AGC AAA GCC AAA GT 3'. The PCR reaction mixture (20 μL) contained 10 μL 2 x PCR Master Mix [10 x PCR buffer, 4 mmol/L MgCl2, 0.5 units Taq DNA polymerase/μL, and 400 μmol/L of each deoxyribonucleotide triphosphate (dNTP) (dATP, dCTP, dGTP, dTTP)], 1 μL of each primer, 3 μL of genomic DNA, and 5 μL sterilized nuclease-free water.

The reaction was carried out with the following cycles: 94oC for 5 min; 35 cycles of 30 s denaturation at 94oC, 30 s annealing at 55oC and 1 min extension at 72oC; and a 10-min final extension at 72oC after completion of the cycles.

The presence of amplified 196 bp on 1.5% agarose gel containing ethidium bromide was detected by performing E/P on the gel electrophoresis apparatus and visualized by UV transillumination (Promega, USA). Then, 7 μL of the amplified products were digested with 5 units of Fast Digest BsmI restriction enzyme at 65oC for 10 min (supplied by Fermentas, Vilnius, Lithuania). The digested products were then detected on 3.5% agarose gel electrophoresis containing ethidium bromide: GG wild pattern appeared as a single band of 196 bp, GT heterozygous pattern appeared as three bands of 196, 148, and 48 bp, and TT homozygous pattern appeared as two bands of 148 and 48 bp [Figure 1].
Figure 1: Results of polymerase chain reaction.based restriction fragment length polymorphism analysis of G276T adiponectin polymorphism digested by BsmI. The band of 48 was too small

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Statistical analysis

The data were coded and entered using the statistical package SPSS version 15 (SPSS Inc., Chicago, IL, USA). The data were summarized using descriptive statistics: median, minimal, and maximum values for quantitative variables, and number and percentage for qualitative values. Genotype frequencies fulfilled Hardy–Weinberg equilibrium. Statistical differences between groups were tested using the Chi-square test for qualitative variables, independent sample t-test, and ANOVA with post hoc test for quantitative normally distributed variables, and the nonparametric Mann–Whitney test and Kruskal–Wallis test were used for quantitative variables which are not normally distributed. Correlations were performed to test for linear relations between variables. P < 0.05 was considered statistically significant.


 > Results Top


Age, laboratory markers of the breast cancer and control groups, and tumor characteristics in breast cancer group

We observed no statistical difference between ages on comparing the BC group to control group. There was a statistical significant decrease in the median level of serum adiponectin in cancer group compared to control, P = 0.004. Data are summarized in [Table 1]. The different characters of the cancer group are demonstrated in [Table 2].
Table 1: Age and laboratory investigations of the breast cancer and control groups

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Table 2: The different characters of the cancer group

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Immunohistochemical and immunostaining

The immunohistochemical study of breast specimen showed 86 out of 90 cases (95.6%) were positive for both estrogen and progesterone receptors, and 4 cases were negative.

Immunostaining of the cancer breast tissue revealed 88/90 (97.8%) cases were negative to HER-2/neu including 36 cases of score (0) and 52 cases of score (1) and 2/90 (2.2%) were positive (+++).

Genotype and allelic frequency of adiponectin gene polymorphism (G276T) of the breast cancer and control groups

The frequency of polymorphic adiponectin genotypes and allelic frequency was higher in cancer group compared to control group. Data are summarized in [Table 3].
Table 3: Genotype and allelic frequency of adiponectin gene polymorphism (G276T) of the breast cancer and control groups

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Estimated risk factor of adiponectin gene polymorphism in the breast cancer and control groups

On measuring the risk estimate of the variant genotypes (GT and TT) versus wild genotype (GG), odd's ratio was 3.76 (95% confidence interval [CI] was 1.68–8.4) with P = 0.001. We observed that patients carrying (T) allele are susceptible 1.68 to develop cancer breast than those patients carrying (G) allele, P = 0.04.

Relation of obesity and genotypes frequency of the adiponectin gene

We subdivided the BC group patients into two groups: the first include patients with BMI =30 kg/m2 they represent obese group (56/90) and the second group with BMI <30 kg/m2 includes (34/90) cases as overweight group.

On comparing the both genotype (GT and TT) versus the wild genotype (GG), a significant difference was observed between control and obese group with P = 0.002. Data are illustrated in [Table 4].
Table 4: Association of obesity and genotypes frequency

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Logistic regression

Forward stepwise logistic regression was run to select the minimum combination of variables that maximally discriminates between patients prone to BC and those who are not.

Participants were used as (binary) dependent variable; while age, serum adiponectin, adiponectin gene were entered as independent (predictive values). Adiponectin gene was the only predictor of cancer breast odd's ratio = 4.74, (95% CI was 1.98–11.33). Data are illustrated in [Table 5].
Table 5: Adjusted odd's ratio

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Receiver operating characteristic curve

Receiver operating characteristic (ROC) curve analyses were applied to assess the diagnostic value of serum adiponectin in cancer breast patients versus normal control group. At a cut off value 4.35 μl/ml, area under the curve was 0.64, specificity was 75%, sensitivity was 79%, positive predictive value was77.2%, negative predictive value was 67.2%, and accuracy was 73.3%.


 > Discussion Top


BC is a major health problem among females with a high mortality rate.[13] obesity is also a growing problem in many developing countries.[14]

For many years, adipose tissues were considered as fat storage of energy, it is now described as an endocrine organ releasing many biologically active molecules, named adipokines.[15] Several authors reported that the low level of adiponectin encountered in obese person is related to cancer development, such as breast,[16] prostate,[17] and colon.[18] This agrees with our findings, where serum adiponectin was reduced in BC group compared to the control group. We further subdivide the cancer group into two subgroups according to BMI, serum adiponectin was lower in obese cancer patients compared to the other groups, with P = 0.001. They suggested that low adiponectin produces insulin resistance and compensatory hyperinsulinemia which results in the reduction of synthesis of IGFBP-1 and IGFBP-2. These result in increasing IGF that favors tumor growth. Another author explained that low adiponectin upregulates a key lipogenic enzyme (fatty acid synthase) that has been associated with cancers.[19]

Up to 80% of BC are hormone-receptor positive either estrogen or progesterone.[20] A strong correlation was reported between menopausal status and hormonal receptors (HRs), about 75% of postmenopausal female with BC were HR-positive and 50% in premenopausal women.[21] In postmenopausal period, an increase in body mass is connected with unfavorable redistribution of fatty tissue with a rise in the amount of visceral fat, this was one of the causes that direct us to choose postmenopausal BC patients. One explanation for the association between obesity and cancer breast is the increase in peripheral aromatization of androgens in the adipose tissue in postmenopausal obese female an enhance proliferation of hormonal dependent tumors.

SNP 276G > T has been studied in different populations, with marked discrepancy in its results among patient with BC. In the present study, the frequency of variant genotypes was statistically significantly elevated in cancer females (obese and overweight) when compared to control group, P value was 0.001, also the frequency of T allele is significantly elevated in cancer group compared to the control group, P value was 0.04. These were in accordance with several studies.[12],[22] On the other hand, other authors reported no significant difference between rs 1501299 and the risk of BC,[23],[24] endometrial cancer.[25] They referred these discrepancies to ethnic variation.

On measuring risk estimate of these variants we found that patients carrying GT and TT genotypes are susceptible 3.76 times to develop BC than those patients carrying GG genotypes. This may highlight the role of this gene in increasing the risk of BC patients.

ROC curve was constructed to detect performance of serum adiponectin in diagnosing cancer breast. We selected a cut off value 4.35 μl/ml for serum adiponectin, at this point, sensitivity was 79% and specificity was 75%, positive predictive value was 77.2%, negative predictive value was 67.2%, accuracy 73.3% was with P = 0.04.

Finally, in this study, we observed a statistically significant increase in the frequency of variant genotypes to wild genotype when comparing cancer and control groups. The median level of serum adiponectin was lower in BC group compared to control group with P = 0.004. Furthermore, we observed increasing the BMI considered a risk factor of BC. Moreover, no significant difference was observed in genotype frequency on comparing obese and overweight cases groups. This may be explained by what was previously reported about obesity, menopausal state, and BC development. Obesity was shown to decrease concentrations of sex hormone–binding globulin and adiponectin level and leads to increase level of pro-inflammatory cytokines and estrogen levels, also increase the insulin which affects the signal of IGFR.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
 > References Top

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    Figures

  [Figure 1]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5]



 

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