TP53 (rs1042522, rs28934571) and TP21 (rs1801270, rs1059234) Polymorphisms and Risk of Breast Cancer among Rural Women of Maharashtra: Findings from a Hospital Based Case- Control Study

Background: Various studies all around the world depicted the relationship of polymorphisms in tumor suppressor genes with risk of various cancers, but there are unambiguous conclusions on this association. A hospital based case-control study was designed to review the association of polymorphism of tumor suppressor genes p21 and p53 with breast cancer risk in women residing in rural Maharashtra. Methods: Two single nucleotide polymorphisms (SNPs) a C>A transversion (Ser>Arg) at codon 31 of exon 2 (rs1801270), C>T transition occurring 20bp upstream from stop codon of exon 3 (rs1059234) in p21 gene and G>C (Arg>Pro) transition at codon 72 of exon 4 (rs1042522), G>T (Arg>Ser) transition at codon 249 in exon 7 (rs28934571) in p53 gene were studied. To precise the quantitative assessment, we enrolled 800 subjects sorted into 400 clinically confirmed breast cancer patients and 400 healthy women from a tertiary care hospital (Krishna Hospital and Medical Research Centre) of south-western Maharashtra. The genetic polymorphisms in p21 and p53 genes was studied by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method using blood genomic DNA isolated from breast cancer patients and controls. The level of association of polymorphisms was assessed using Odds ratio (OR) with 95% confidence interval and p-value identified using logistic regression model. Results: After the analysis of SNPs (rs1801270, rs1059234) of p21 and (rs1042522, rs28934571) in p53 gene our analysis suggested that heterozygote Ser/Arg genotype with OR=0.66; 95% CI: 0.47- 0.91; p=0.0003 and homozygote variant Arg/Arg genotype with OR=0.23; 95% CI: 0.13- 0.40; p<0.0001of rs1801270 of p21 was negatively associated with risk of breast cancer in studied population. Conclusion: The findings from this study supported that rs1801270 SNP of p21 was inversely associated with breast cancer risk in the studied rural women population.


Introduction
Breast cancer (BC) is the most common malignancy among women worldwide estimated with 2261, 419 (24.5%) new cases of all ages females and 684, 994 deaths accounting 15.5% of all cancers in women in year 2020 (GLOBOCAN 2020). Global Cancer Observatory reported BC as the largest cause of cancer causing deaths in India where 178, 361 (26.3%) of new cases and 90, 408 (10.6%) deaths reported in 2020 (Sung et al., 2021). Current trend indicated that the incidence of BC occurring in Indian women at younger age likely to begin in early thirties and peak ages of 50-64 years as compared to western countries (National Cancer Registry Programme genetic factors are thought of key determinants in determining host susceptibility towards developing BC along with combination of lifestyle and environmental factors (Collins and Politopoulos 2011;Cobain et al 2016). Studies all around the globe pointed out functional association of polymorphism of several genes with increased risk of developing cancers in different population from different countries. It was previously reported that single nucleotide polymorphisms (SNPs) of the tumor suppressor protein p53 (TP53) and tumor suppressor protein p21 (TP21) might be involved in determining the individual's susceptibility to multiple cancers including BC. The TP53 is a crucial gene involved in critical cellular processes including cell cycle progression, DNA repair and apoptosis. Somatic alterations in the gene lead to an altered expression of various associated genes which are directly or indirectly under the transcriptional control of TP53 (Ali and Kamath 2022;Ghosh et al., 2022;Ali 2023).
To date, several literature information on case-control and other epidemiological studies have identified the association of the p53 and p21 SNPs with susceptibility to cervical ( Datkhile et al., 2019;Ratre et al., 2019;Yu et al., 2022), ovarian (Alqumber et al., 2014), lung (Matikodu et al., 2003), head and neck (Bau et al., 2007) bladder (Zang et al., 2018) and gastric (Song et al., 2011) cancer risk, while others pointed out contrast observations where these SNPs did not show any association of either p53 or p21 polymorphism with development of oral (Lin et al., 2018) ovarian (Alqumber et al., 2014), colorectal (Asadi et al., 2017), lung (Nicholas et al., 2019), prostate (Sivonova et al., 2015;Song et al., 2017) and cervical cancer risk (Martinez-Nava et al., 2016). Thus, it is not clear from the earlier evidences that the polymorphisms in tumor suppressor molecules may play a role in genetic susceptibility of variety of cancers and the findings appeared conflicting. Previous studies conferred the association of p53 and p21 polymorphisms with increased risk of BC (Ayobi et al., 2018;Diakite et al., 2021), but others disagree to prove an association with BC risk (Hou et al., 2013, Habyarimana et al., 2018Zhao et al., 2022). Thus, for determining the role of p53 and p21 SNPs in epidemiology and pathogenesis of BC, most of the studies were conducted in American, European and West and East Asian countries, but limited studies are available from South Asia, especially from India. Findings from Indian case-control and meta-analysis studies declared significant association between p53 polymorphism and increased BC risk in Indian population Akhter et al 2018). Some other Indian studies concluded no association of the SNPs of p53 and p21 with breast cancer risk (Surekha et al., 2011;Vijayaraman et al., 2012). To the best of our knowledge, there have been no such studies from rural population of India on the association between polymorphisms of the above mentioned genes and risk of BC. The present study was intended to identify the risk association of p53 and p21 gene polymorphisms with breast cancer. We conducted a case-control study in order to explore the possible association of (rs1042522, rs28934571) SNPs of p53 and (rs1801270, rs1059234) SNPs of p21 with BC risk in women residing in the rural areas of south-western Maharashtra from India.

Study subjects
We conducted a case-control study in a consecutive sample of 400 histopathologically confirmed BC cases. All BC cases ranged in age from 23-85 years (Mean ± SD) (52.43 ±12.37) were enrolled immediately after diagnosis during the year 2018-2021. The cases already receiving treatment for malignancy were excluded. Equal numbers (400) of healthy women volunteers without history of any disorders and history of hysterectomy or mastectomy were randomly selected from a group of women visiting to Krishna Hospital & Medical Research Centre (KH&MRC) for blood donation and other purposes. Controls were frequency matched to cases by the age group at enrolment (42.37 ± 13.90). The control group and the study group resided in the same geographical location (Western Maharashtra region). Trained interviewers used a structured questionnaire to collect demographic and clinical data from the participants. The study protocol was approved by Institutional Ethics Committee for the utilization of human subjects in the research.

SNP Selection and Genotyping
Genomic DNA was isolated from venous blood of BC cancer patients and normal controls by a modified method where red blood cells are processed with red cell lysis buffer (10mM Tris-HCl pH 7.6, 320 mM sucrose, 5mM MgCl2, 1% Triton X-100, pH 7.6), thereafter treated with nucleic lysis buffer (10mM Tris-HC1, 11.4 mM sodium citrate, 1 mM EDTA, 1 % SDS, pH 8.0). After treatment with 100 μg/mL concentration of proteinase K at 55 degree celsius and subsequently RNase A (100 μg/mL) at 370C, precipitated and purified DNA was checked on 1% agarose gel for its quality as well as quantity. The DNA samples were then genotyped at p21 codon 31 and p53 codon 72, codon 249 loci by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). The polymorphisms of both p21 and p53 were chosen for this study based on previously described studies (Ressiniotis et al., 2005;Vijayaraman et al 2012). The primers, PCR and RFLP conditions for the studied polymorphisms were selected from earlier publications by Ressiniotis et al., 2005. The primers and PCR-RFLP conditions optimized for amplification of codon 72 of exon 4 (309 bp) and codon 249 of exon 7 (286 bp) of p53 gene were selected from earlier prescribed by Vijayaraman et al., 2012.

Correlation between p21 and p53 genotypes and clinicopathologic characteristics among breast cancer cases
When we analyzed correlation of both p21 and p53 genotypes with clinicopathologic characterstics among 400 breast cancer cases, we observed that there were no significant correlations between the p21 codon 31 Arg or Ser alleles (Table 5) and p53 codon 72 alleles (Pro or Arg) or codon 249 alleles (Ser or Arg) and patients' clinical parameters (Table 6). We observed that all the studied clinicopathological characterstics showed no significant differences in breast cancer patients.

Correlation of p21 and p53 gene polymorphisms with confounding factors associated with breast cancer risk
The association of p21 and p53 with the risk of BC was further examined after stratification of confounding factors such as age of cancer occurence, age at first pregnancy and tobacco habit status. The genotype distributions of the selected p21 and p53 gene polymorphisms in cases and controls and their associations with CC risk are summarized in Table 7. The logistic regression analysis showed that none of the polymorphisms except Ser/ Arg genotype of p21 codon 31 of exon 2 was negatively associated with BC risk (OR=0.46; 95% CI: 0.32-0.67 p=0.0001) after being adjusted for age >40 years and earlier age at first pregnancy (OR=0.40; 95% CI: 0.26-0.60 p< 0.0001) with development of BC. In Maharashtrian patients, the age of BC occurred at 23 years which was considerably lesser than reported in other reports. Also, the association of BC with first delivery age was reviewed in this study which showed that 15-20 yrs age of first delivery, considerably associated with increased BC risk. When we considered the association of p53 codon 72 and codon 249, there was no evidence of any association of Arg/Pro alleles of codon 72 of exon 4 (OR=1.11; 95% CI: 0.74-1.67 p=0.59) and Arg/Ser of codon 249 of exon 7 (OR=1.28; 95% CI: 0.82-1.99 p=0.72) with subgroups defined by age >40 yrs. Similarly the arginine/proline alleles of codon 72 (OR=1.11; 95% CI: 0.71-1.74, p=0.63) and Arg/Ser allele of codon 249 were (OR=1.09; 95% CI: 0.67 -1.78, p=0.72) not associated with breast cancer development in women with early age of first pregnancy.

C o m p a r i s o n o f s e l e c t e d d e m o g r a p h i c a n d clinicopathological characteristics of study subjects
In this case-control study p21 (rs1801270, rs1059234) and p53 (rs1042522, rs28934571) SNPs were analyzed by PCR-RFLP in 400 cases of BC and equal number of control women from a tertiary care hospital in Karad city. The age range of BC patients were 23-85 years (Mean ± SD: 52.43 ±12.40; Median age, 50yrs) whereas healthy control females ranged from 24-81 yrs (Mean ± SD: 42.37 ±13.90; Median age 40yrs). The demographic variables of BC cases and controls (age at cancer occurrence, age at first pregnancy, diet, tobacco habit, education and economic status, family history) and clinicopathological characteristics of BC patients including hormone status, histological subtypes, histological grade, tumor localization, tumor size were recorded ( Table 1). Occurrence of BC frequency was high among the individuals more than 40 years of age (80.50%) than those of patients less than 40 years (19.50%). Significant difference was observed in BC cases when compared for their age of cancer occurrence (OR 4.42; 95%CI, 3.22-6.07.20; p<0.0001). There was no significant difference in diet (OR 1.57; 95%CI, 1.14-2.17; p=0.05) and economic status (OR 0.74; 95%CI, 0.55-1.01; p=0.06) were considered for the relationship with BC development but surprisingly, when we checked for tobacco chewing status we observed significant association with BC (OR 3.07; 95%CI, 2.29-4.12; p<0.0001) in women of rural population.

Comparative analysis of genotypic polymorphism of p21 and p53 gene in breast cancer cases and controls
The genotypic distribution of p21 and p53 genes determined in BC cases and healthy age and sex matched controls is summarized in Table 2. When we studied frequency distribution of serine and arginine in codon 31 of exon 2, the Ser/Ser genotype of patients (71.75 %) and controls (56.50%) where Arg/Arg genotype in p21 showed negative association for the development of BC (OR=0.23, 95% CI: 0.13-0.40; p<0.0001). Similarly when we studied polymorphisms in C/T and T/T genotypes of exon 3 of p21 we noted, neither of heterozygous C/T genotype (OR=1.50, 95% CI: 1.08-2.08; p=0.01) nor homozygous T/T genotype (OR=1.11, 95% CI: 0.38-3.19; p=0.85) showed relationship with BC development. The distribution of codon 72 and codon 249 genotypes of p53 in patients and control did not deviate from the Hardy-Weinberg equilibrium. The genotype frequencies in cases and controls are presented in Table 2    Neither of p21 nor p53 gene SNPs showed positive or negative association with breast cancer development when confounding factor tobacco habit was considered.

Discussion
The present study was intended to investigate whether   Table 6. Association between p53 codon 72 of exon 4 and codon 249 of exon 7 Genotypes and Clinicopathologic Characteristics among Breast Cancer Cases the SNPs of tumor suppressor genes p21 and p53 are associated with breast cancer risk in Maharashtra women.
In this hospital based case-control study, most studied polymorphisms of p21 (rs1801270 C/A, rs1059234 C/T) and p53 (rs1042522 G/C, rs28934571 G/T) and the risk of breast cancer in women residing in the rural areas of Maharashtra was studied. To the best of our knowledge, we have demonstrated these SNPs in relation with BC in rural population of Maharashtra for the first time, as these SNPs are not studied earlier in relation with BC risk from the rural areas of Maharashtra. The association of the SNPs in p21 and p53 with risk of BC was evaluated by Crude and Adjusted Odds ratio with 95% CI estimated for homozygous and heterozygous genotypes. The rs1801270, rs1059234 SNPs of p21 are not much studied however; p53 is studied for its SNPs (rs1042522, rs28934571) of codon 72 and codon 249 and their association with variety of cancers, but deficit in the literature for their role in breast carcinogenesis. Our results revealed that the C>A polymorphism of p21 gene at codon 31 showed significant association with BC development in women of studied population. The minor variant A allele was significantly (OR=0.23; 95% CI: 0.13-0.40 p<0.0001) associated with decreased risk of BC development. It is evidenced from earlier studies that the polymorphisms in tumor suppressor genes may play an imperative role in genetic susceptibility towards carcinogenesis. Few studies on the polymorphisms in p21 and p53 have been studied and some of them were evidenced to be associated with cancer risk with significance. A polymorphism of p21 i.e., Serine to Arginine transversion at codon 31 of exon . The rs1042522, SNP of p53 at codon 72 with a substitution of arginine to proline and rs1801270 SNP of p21 at codon 31 with serine to arginine substitute have been widely studied in diverse group of population for their association with carcinogenesis (Huang et al., 2019;Yu et al 2022), but others reported conflicting results with no association with cancer development (Song et al., 2017;Lin et al., 2018;Nicholas et al., 2019). Similar type of epidemiological studies reported from India for those SNPs and their association with cancer development (Lakshmi et al., 2012;Saikia et al., 2014;Khan et al., 2020) whereas other objected for their association with carcinogenesis in different ethnic population (Pillai and Nasir 2016;Bansal et al., 2016). Various investigations reported some associations of the studied genes with breast cancer development (Ayobi et al., 2018;Diakite et al., 2021) whereas others suggested no association between the polymorphisms either of p21 or p53 genes and the breast carcinogenesis (Habyarimana et al., 2018;Aceto et al., 2019;Zhao et al., 2022). The major attention of this study was to identify the polymorphisms in p53 gene in rural women of Maharashtra and demonstration of their association with risk of BC in studied population. Interestingly, we observed no significant differences in either codon 72 or codon 249 of the p53 gene of controls  and cases when statistically analyzed for their association with BC development. Our results are in accordance with earlier observations noted by the researchers in South Indian populations (Suresh et al., 2011;Vijayaraman et al., 2012) and North Indian population (Mandal et al., 2014). When we studied the polymorphism of p21 (codon 31 of exon 2 and C>T transition occurring 20bp upstream from stop codon of exon 3, the results indicated that the homozygous variant Arg/Arg genotype of codon 31 of p21 was negatively associated with BC susceptibility. In conclusion, the results derived from this study stated that the functional SNP rs1801270 of p21 gene showed significant association with decreased risk of BC in studied rural population of south-western Maharashtra which was not reported earlier from this part of the world. The findings obtained from this study also indicated that neither of p53 Arg72Pro nor Arg249Ser was associated with BC risk among studied Maharashtra women