Investigating the association of Angiotensin II Type I Receptor A1166C Polymorphism with Breast Cancer Risk in the Pakistani Population

The polymorphisms of the Renin-Angiotensin System are related to many disorders like diabetes, cardiovascular disease, and different types of cancer. Among all the polymorphisms related to AGTR1, A1166C has been associated with several disorders, including cardiovascular diseases and breast cancer. This study was conducted to discover the association of AGTR1 polymorphism (A1166C) Renin-Angiotensin and its effect on the development and progression of breast cancer in the Pakistani population. One hundred forty participants, including seventy diagnosed breast cancer patients and seventy healthy individuals, were included in this study and genotyped with an allele-specific polymerase chain reaction. The most frequent genotype in healthy participants and breast cancer patients was CC. An insignificant (p value>0.05) risk of breast cancer was found with A1166C polymorphism in codominant (CC vs. AA OR=1.200 [0.256-5.631] and AC vs. AA 0.941 [OR=0.223-3.976]), dominant (OR=1.00 [0.240-4.167]), recessive (OR=1.230 [0.593-2.552]) and additive models (OR=1.028 [0.533-1.983]) of general population genotypes. Nonetheless, when the AA genotype was considered a reference group, a significant association was found between AC and CC genotypes and invasive ductal and ductal carcinoma development in breast cancer patients. In conclusion, this study demonstrated no significant association between AGTR1 (A1166C) polymorphism and breast cancer risk.


Introduction
Breast cancer is a heterogeneous disease characterized by multiple tumor entities with different clinical behavior, biological features, and distinctive histological patterns [1].About a million breast cancer cases are reported Investigating the association of Angiotensin II Type I Receptor A1166C Polymorphism with Breast Cancer Risk in the Pakistani Population (AGTR1) and angiotensin-converting enzyme (ACE) has been reported [4].ACE (17q23) spans ~21kbps in humans, further encoding 25 introns and 25 exons.Zn-dependent dipeptidyl carboxypeptidase cleaves angiotensinogen (synthesized by the liver) into angiotensin I (inactive decapeptide); upon low blood pressure or plasma sodium levels reduction, it is released into the bloodstream.Angiotensin-I-converting enzyme (ACE) further acts on angiotensin I to convert it into active angiotensin II (octapeptide) [5].Angiotensin II increases blood pressure by promoting vasoconstriction.It also increases plasma aldosterone and retains water and electrolytes like sodium [6].Angiotensin II mediates its effect by two G-protein coupled receptor family receptors, AGTR1 and angiotensin II type II receptor (AGTR2).Due to its site of origin and the location of its receptors, Angiotensin II has an important role in the cell proliferation and apoptotic stages of the breast cell cycle [7,8].
The AGTR1 is present on chromosome 3 (3q21-q25) and covers >55kbps region comprising 5 exons.Sharkawy et al. and Ostrosky et al. suggested that AGTR1 (A1166C) single nucleotide polymorphism (SNP) is associated with breast cancer [5,6].This polymorphism is present in the 3' untranslated region and is considered functionally insignificant.However, it was found to be associated with T810A transversion present in the gene's promoter region.Thus, T810A influences transcriptional factor binding and subsequent transcription of mRNA.Moreover, a study observed an elevated AGTR1 mRNA transcription rate in breast cancer patients compared to normal individuals [9].Generally, the AGTR1 signaling pathway involves the induction of cell proliferation, inflammation, and angiogenesis and the inhibition of apoptosis [10,11].AGTR1 receptors are seen to be over-expressed in breast cancer cases, and this increase is about 100 folds more than in normal breast tissues.Additionally, overexpression of AGTR1 due to genetic mutations leads to cellular invasion and tumorigenesis in breast cancer patients [12].
Several studies worldwide have investigated the association of A1166C SNP with the incidence of breast cancer.A study of AGTR1 (A1166C) polymorphism in the Indian population has described the association of AC and CC genotypes and C alleles with the high risk of Breast cancer [3].Moreover, they linked it with severe disease leading to progressive staging and large tumor size.Conversely, in another study conducted on Brazilian women, A1166C polymorphism was not associated with breast cancer risk [13].Similarly, in other populations association of A1166C polymorphism has been investigated with breast cancer risk and its progression.Such as the AC genotype was a riskassociated factor against the AA genotype in Iranian women with higher tumor node metastasis [14].Although many studies have investigated the association of A1166C single nucleotide polymorphism of AGTR1 with the risk of developing disease, the outcomes remain contradictory in different populations.Considering these results, it can be inferred that breast cancer risk association with A1166C polymorphism can be a population-specific phenomenon.Thus, this study aimed to find out the association of AGTR1 (A1166C) polymorphism with breast cancer in the Pakistani population as the proper understanding of this association in Pakistani women could be used as biomarkers for early diagnosis and screening of breast cancer.

Sample Collection
The Institutional Review Board of Kinnaird College approved this case-control analytical study for women in Lahore.In this study, 140 individuals were enrolled, including 70 with breast cancer and 70 healthy women without disease and any other disease.The breast cancer case subjects with other chronic diseases like cardiovascular disorder, cancer (apart from breast cancer), diabetes, kidney disease, and hypertension were excluded from the study.All the samples were procured from INMOL Cancer Hospital, Lahore, while the genotypic analysis was performed at the Biochemistry Laboratory of Kinnaird College for Women, Lahore.The present study was performed according to the principles of the Declaration of Helsinki, and ethical approval was obtained from the institutional board committee of Kinnaird College for Women, Lahore (KC/ORIC/ERC/2015/002).Informed consent was obtained from all participants of the study.A structured questionnaire was used to obtain data from each participant regarding the demographics (age and body mass index) and clinical and pathological details.The data include participant age, body mass index, family history of the disease, menstrual cycle status, tumor type, and location, etc. 140 blood samples were collected, 70 from healthy Pakistani women (control group) and 70 from Pakistani women diagnosed with breast cancer (case group).

DNA Extraction
Each participant's blood sample of 2-3ml was collected in EDTA vacutainers (Fisher Scientific, Franklin Lakes, NJ, USA).750µl of peripheral blood of each participant was used to extract DNA by standard phenol-chloroform method with the two-day protocol (Sambrook and Russell, 2006).The quality of DNA was checked by running the samples on 1% agarose gel (Thermo Fisher Scientific, Baltic, UAB).

Genotyping
The genotype of the samples was determined using allele-specific polymerase chain reaction (PCR).Two reaction mixtures were prepared for each sample (Ugozzoli and Wallace, 1992).Two PCRs and 3 primers amplified the AGTR1 polymorphic sequence.For the first PCR following primers were used: forward allele specific A primer 5'-GCACTTCACTACCAAATGAGCA-3' ( s e n s e ) a n d n o r m a l r e v e r s e p r i m e r 5'-AGGGAGATTGCATTTCTGTCAGT-3' (antisense).For 20µl reaction mixture of PCR contains 2.5U Taq polymerase (Thermo Fisher Scientific, Baltic, Lithuania), 25ng of genomic DNA, 50pmol of each primer, 2mM MgCl2 (Thermo Fisher Scientific, Baltic, Lithuania), 0.2mM dNTPs (Thermo Scientific, Foster City, California, USA), 1X PCR buffer (Thermo Fisher Scientific, Baltic, Lithuania).The second reaction for the same sample contains all the reagents mentioned above but different primers, i.e., 50pmol of allele-specific C primer 5'-GCACTTCACTACCAAATGAGCC-3' ( s e n s e ) a n d n o r m a l r e v e r s e p r i m e r 5'-AGGGAGATTGCATTTCTGTCAGT-3' (antisense).PCR was run through a thermocycler (Bio-Rad Laboratories, Singapore) at conditions as follows: an initial denaturation at 94ºC for 4mins, followed by 30 cycles of denaturation at 94ºC for 30sec, annealing at 67ºC for 30 sec, extension at 72ºC for 30 sec with a final extension at 72ºC for 5 mins.The results of the PCR were analyzed using 1.5% agarose gel.

Statistical Analysis
Data was analyzed using SPSS version 26.0 for Microsoft Windows (SPSS, Chicago, IL, USA).The data for the continuous variables such as age and BMI was presented mean±standard deviation, while the categorical data was presented as frequencies (percentages).The data was categorized into different groups for each variable, and their risk factor was analyzed through logistic regression.The p-value less than 0.05 was considered significant.and showed no association with the disease.Furthermore, alleles A/C were also compared between control and case groups but then again, the insignificant results were obtained OR=1.028 [0.533-1.983](p-value 0.933).

Association of AGTR1 Polymorphism with Tumor Type and Location
The genotypes were then compared with three tumor types (Invasive ductal carcinoma, Lobular carcinoma, and Ductal carcinoma) and tumor locations (left, right, and both) (Table 3).By considering the AA genotype as a reference group, there was a significant association found between the CC genotype with invasive ductal carcinoma (OR=11.400[1.068-121.70])(p-value 0.04) and ductal carcinoma (OR=0.116[0.013-1.016])(p-value 0.052).The AC genotype was also found to be associated with the significant risk of breast tumors occurring as invasive ductal carcinoma (OR=15.00[1.136-198.039])(p-value 0.04) and ductal carcinoma (OR=0.059[0.004-0.979])(p-value 0.048).Lastly, no association was found between the location of breast tumor and lobular carcinoma (type of tumor) (p-value>0.05).

Association of AGTR1 Polymorphism with Baseline Characteristics
Moreover, the association of baseline characteristics of the study participants was compared with the genotypic and allelic frequencies.No significant association was found in genotypes and alleles with age groups, BMI groups, number of children, miscarriages, marital status, family history, and menstrual irregularities (Figure 1).

Discussion
Breast cancer constitutes a major health problem in women, with over a million cases diagnosed annually.There is increasing evidence that the local reninangiotensin system may impact angiogenesis, apoptosis, cell proliferation, and inflammation [15].Consequently, it may provide an adjuvant therapy by the blockade of RAS that can be implemented as an alternative to previous ones for managing breast tumors.Polymorphism in RAS is involved in the proliferation of cells during different kinds of malignancies [6].If Angiotensin II binds with AGTR1, it facilitates the proliferation of cells.The A1166C polymorphism of AGTR1 is the most studied polymorphism for various diseases, such as cardiovascular diseases and multiple cancers, including breast cancer.
The results of this study have shown that the mean age of the control group was 40.14±15.72 while the mean age of cases was 46.49±10.93.Similarly, Body Mass Index was also observed; the mean BMI of controls was 24.33±4.47,and that of cases was 25.58±4.76.An Independent t-test was applied to check the difference in the means of BMI and ages of controls and patients suffering from breast cancer.The p-value showed no difference between the mean BMI and the ages of controls and patients suffering from breast cancer, which is important for examining the other risk factors.These results were supported by previous studies by different previous studies [16,17], while marital status did not seem to correlate with the The association of AGTR1 polymorphisms (A1166C) genotype/allele with the risk of breast cancer development was calculated by odds ratio (OR) at 95% confidence interval (95% CI) using codominant (AC vs AA and CC vs AA), dominant (AC+CC vs AA), recessive (CC vs AC+AA), and heterozygous (AC vs AA+CC) models.The risk analysis of breast tumor type and location against genotypic data of breast cancer patients was also recorded as OR [95% CI].The baseline characteristics of both case and control groups were analyzed against AGTR1 A1166C polymorphisms through regression analysis.

Demographic and Clinical Characteristics of Study Participants
140 (70 healthy individuals and 70 breast cancer patients) participated in this study.The data regarding the patient's physical fitness, family history, and history related to cancer was obtained and analyzed by determining the p-value for each variable, thus defining the risk factors for breast cancer.Table 1 summarizes the demographic and clinical characteristics of the study population groups (controls and cases).The mean ages of controls and breast cancer patients were calculated as 40.14±10.93and 46.49±15.72 (p-value 0.006), respectively.In the subgroup analysis of variables, including age, BMI, marital status, number of children, history of menstrual irregularities, and breast cancer, a significant difference (p-value<0.05) was found in the data distribution amid the case and control population.An insignificant difference was found in the distribution of data for miscarriages between both groups (p-value 0.052).According to risk analysis, patients aged between 31-45 years (OR=14.00[4.145-47.291])and greater than 46 years (OR=9.333[2.925-29.785])showed higher risk for developing breast cancer about 16-30 years.Likewise, overweight patients (OR=3.022[1.379-6.626])have more chances of developing breast cancer than normal subjects.Married women (OR=1.296[1.141-1.473])were also likely to develop breast cancer 1.3 times more than unmarried women having 1-3 (OR=9.913[2.634-37.306])and 4-6 (OR=8.636[2.270-32.856])children.Moreover, it was found that there were more than 3.5 times the chances of women getting breast cancer who has gone through menopause as compared to women with oligomenorrhea, amenorrhea, or with no irregularities at all.

Association of AGTR1 Polymorphism with Breast Cancer
The AGTR1 (A1166C) polymorphism genotypes/ alleles association with breast cancer was found using logistic regression analysis and chi-square test (Table 2).As it had been shown in the table, frequencies of AA, AC, and CC were 4 (5.7%), 15 (21.45%), and 51 (72.85%) in controls and 4 (5.7%),18 (25.72%),and 48 (68.58%) in cases, respectively.Nonetheless, no association was found between the genotypes and the risk of breast cancer.Considering this, the combined effect of genotypes was assessed for risk analysis as dominant (AC+CC vs AA), recessive (CC vs AC+AA), and heterozygous (AC vs CC+AA) models.Yet, all the results were insignificant  [18].AGTR1 (A1166C) polymorphism genotypic results were evaluated in different modes to assess its association with breast cancer.This study found no significant association between breast cancer cases and genotypes CC and AC concerning AA.The same results were observed in a study on Brazilian women diagnosed with breast cancer (p-value 0.114) [13].However, another study conducted in the North Indian population presented contradictory results.A significant association was found between AC   and CC genotype/C allele and the risk of developing a more aggressive disease with large tumor size at advanced stages.
In this study, CC genotype frequency was higher in the case group than the AC genotype.CC homozygous and AC heterozygous genotype was found to be significantly associated with a higher risk of breast cancer patients developing invasive ductal carcinoma than ductal carcinoma in comparison to AA homozygous.These results aligned with a study on the Iranian population where a stronger significant association was found between the homogenous groups of 61 breast cancer patients with A1166G AGTR1 polymorphism [14].Although, they didn't find any specific association between gene polymorphism and breast cancer occurrence risk.
Due to the availability of less data on the expression of AGTR1 concerning the effect of AGTR1 (A1166C) polymorphism, additional studies are suggested to assess the risk of (A1166C) polymorphism with breast cancer.An insignificant association of genotypes may be due to its relatively small sample size.If a large sample of data is obtained, it can be easier to highlight the risk association of polymorphism with breast cancer.Moreover, the main reason for the significant association of invasive ductal and ductal carcinoma with (A1166C) polymorphism can be identified, possibly due to overexpression of AGTR1.
As elucidated in a research study, A1166C polymorphism can lead to renin-angiotensin system activation and overexpression of AGTR1 [19].As mentioned, AGTR1 mediates tumorigenic actions such as inflammation, angiogenesis, and cell proliferation.These findings can lead to proper diagnosis and prognosis of breast cancer [20].
In conclusion, the present study found that AGTR1 (A1166C) polymorphism was not associated with the risk of breast cancer in the Pakistani population.Further analysis of a large population should be performed to validate the results of this study.

Figure 1 .
Figure 1.Regression Analysis of the (a) genotype (AA/AC+CC) and (b) alleles (A/C) with breast cancer risk and associated baseline characteristics of participants

Table 1 .
Demographic and Clinical Data Distribution along with Their Risk Analysis between Controls and Cases *, The p-value <0.05 was considered statistically significant; N, number; %, frequency; Ref, reference; OR 95% CI, Odd Ratios at 95% confidence interval

Table 2 .
Risk Analysis of A1166C Polymorphism of AGTR1 Gene as a Function of the Inheritance Model in Controls and Breast Cancer Patients

Table 3 .
Risk Analysis of AGTR1 Polymorphism (A1166C) in Relation to Breast Tumor Type and Location among Breast Cancer Patients *The p-value <0.05 was considered statistically significant, N, number; %, frequency; Ref, reference; ꭓ 2 , Chi-square analysis; OR 95% CI, Odd Ratios at 95%