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The Combined Effect of IL7RA Rs6897932 and KRAS Rs61764370 Gene Polymorphism in the Development of Breast Cancer among Women in Kerala

Population: A Follow-Up Study

Mohthash MT^1*, Neelam TRIPATHI², Sunil Kumar SHAH³, Anand THIRUPATHI⁴

1Ph.D. Scholar, Sri Satya Sai University of Technology & Medical Sciences, Madhya Pradesh, India

2Professor University Teaching Department, Sri Satya Sai University of Technology and Medical Sciences, Madhya Pradesh, India.

3Associate Professor. College of Pharmacy, Sri Satya Sai University of Technology & Medical Sciences, Madhya Pradesh, India.

4Sri Venkateshwara Research Centre, Thanjavur, Tamilnadu, India.

Abstract

Interleukin-7 (IL-7) has always been found to play an important role in developing and maintaining lymphoid cells. IL-7 promotes the growth of tumor cells in breast cancer (BC) patients. Several studies have recently identified an association of single nucleotide polymorphisms (SNPs) in the lethal-7 (let-7) miRNA binding site within the 3' untranslated region (3'UTR) of the KRAS gene with the risk of developing various types of cancer. The current research is a continuation of our previous work, which sought to establish whether the IL7RA rs6897932 and KRAS rs61764370 polymorphisms were correlated with BC susceptibility in a diverse population of Kerala, South India. A follow-up study involving 60 breast cancer patients (females) and 60 healthy volunteers was conducted among individual from Kerala, South India.

Genetic analysis was performed to detect IL7RA rs6897932 and KRAS rs61764370 employing the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method.

Odds ratio (OR) with 95% class interval (CI) were used to evaluate the relationship of IL7RA (rs6897932) polymorphism with BC susceptibility. Statistical analysis was performed using Statistical Package for the Social Sciences (SPSS; version 21.0) software and Medcalc software (version 16.4.3). The frequency distribution of the IL7RA rs6897932 and KRAS rs61764370 polymorphisms was found to vary between case and control groups, confirming our previous findings and suggesting that the IL7RA and KRAS genes may play a role in the pathogenesis of BC in the South Indian population. Further advanced and prospective research with larger sample sizes is required to confirm the current study's findings.

Keywords: Breast cancer, IL7RA polymorphism, rs6897932, KRAS polymorphism, rs61764370

Introduction

Breast cancer (BC) is the most common female cancer globally, accounting for nearly a quarter (25%) of all cancer cases, with 1.67 million new cases diagnosed each year. [1] According to recent reports, the number of BC cases in developing countries has increased (883000 cases) concerning developed countries (794 000) [1]. While India has a lower age-adjusted BC incidence rate (25.8 per 100 000) than the United Kingdom (95 per 100 000), the mortality rates are nearly equal in both countries (12.7 vs 17.1 per 100 000) [2]. BC has surpassed breast cancer as the most common cancer among women in India, with an age-adjusted prevalence of 25.8 per 100,000 women and a mortality rate of 12.7 per 100,000 women. According to global and Indian reports, the incidence rate of cancer and cancer-related morbidity and mortality rates have risen dramatically in the Indian subcontinent over the last few years [3–7]. Previous studies have shown that cervical cancer is the most common cancer in Indian women. The most recent studies (the last five years) show that breast cancer is more common than cervical cancer, even though cervical cancer is still the most common cancer in rural India [8]. Currently, multiple genetic studies have been undertaken in different populations to learn more

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about the genetic factors associated with breast cancer that could assist in early detection in addition to traditional screening techniques such as breast ultrasound, magnetic resonance imaging (MRI), diagnostic mammogram, and biopsy in the quest for new methods for screening different cancers. Despite the fact that many epidemiologic studies have been conducted in cancers such as prostate, oral gastric, and BC, [9-12] studies that concentrate on the genetic factors associated with BC in South Indian ethnicity remain scarce. The genes BRCA1 and BRCA2 are the most widely studied for BC with inherited breast and/or ovarian cancer syndrome susceptibility [2, 3]. However, due to the polygenic nature of BC, a number of large-scale studies have recently documented the value of multi-gene testing and the polygenic nature of BC, which would require screening of non-BRCA genes that are often overlooked in standard genetic screening procedures [13]. In BC development, the interaction of growth factors, extracellular matrix, cytokines, fibroblasts with tumor cells, and immune and tumor cells was discovered to be extremely significant. [13]. Interleukin-7 (IL-7) has recently been discovered to play a key role in BC pathogenesis, promoting tumor cell growth and survival. The expression of its signaling pathway molecules has been linked to the worst prognosis in human BC samples [14].

Furthermore, BC cancer patients' peripheral blood mononuclear cells have lower expression of the IL-7 receptor alpha chain (IL-7RA; CD127) and impaired IL-7 response and cytokine production compared to healthy controls [15], meaning that IL-7 deficiencies are related to BC. IL-7 is a type 1 cytokine that belongs to the hematopoietin family. These are primarily formed by non-immune stromal cells, which play an important role in immune system advancement and homeostasis by encouraging lymphoid cell growth and survival. The IL-7 receptor (IL-7R) [composed of the cytokine receptor gamma chain (c; CD132) and the IL-7RA] activates Janu Kinases 1 and 3, encouraging STAT5 activity and gene expression regulation, as well as phosphatidylinositol 3- kinase (PI3K), resulting in pro-survival and angiogenesis [16]. The IL7RA gene is located on chromosome 5 at 5p13.2 [17]. The SNP (rs6897932, Thr244Ile) occurs at exon 6, which results in the conversion of cytosine (C) into thymine (T) at codon 244 (ACC > ATC). This further leads to a change of threonine into isoleucine (Thr > Ile) at the border between the transmembrane and extracellular regions. In individuals with multiple sclerosis, T (Ile) allele for Thr244Ile polymorphism is a marker allele for a protective haplotype [18]. It has also been associated with various other immune-related disorders [19]. Moreover, it is also shown in detailed studies that the T allele is associated with decreased expression of soluble IL-7RA (sIL-7RA) isoform [13].

Despite this evidence demonstrating the influence of IL-7 in various cancers, including BC [13], and the documented functional implications of IL7RA Thr244Ile polymorphism in autoimmune diseases and immune function [13], no other study is available in the literature which had investigated this polymorphism on BC among South Indian society. Therefore, our group has carried out a study earlier to investigate the association of IL7RA Thr244Ile (rs6897932) polymorphism with BC's susceptibility among ethnicity of Kerala [20].

The V-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog is a well-known effector molecule (KRAS). It is involved in various signal transduction pathways, including the Ral (Ras- Like) guanine nucleotide exchange factor, mitogen-activated protein kinase, and phosphatidylinositol 3-kinase. KRAS is a known let-7 target with numerous complementary sites in the mRNA's untranslated (3'UTR) region. A germ-line and functional single-nucleotide polymorphism (SNP) in the KRAS 3'UTR region (rs61764370 T>G) located on the let-7 complementary site 6 has been discovered in recent studies (LCS-6). Further study has discovered that LCS-6 decreases the affinity of let-7 for KRAS. This resulted in enhanced tumour growth and decreased KRAS inhibition [21-22]. Several recent studies have looked into the effect of the KRAS rs61764370 T>G polymorphism on the risk of developing different cancers. In inherited BC and ovarian cancer syndrome families, the LCS6 variant allele has been shown to cause non- small cell lung cancer (NSCLC) in moderate smokers, [23] triple-negative BC in premenopausal women [24], and ovarian cancer in BRCA negative female individuals [25]. In light of this, our group had earlier looked into the possible connection between BC danger and the KRAS variant

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(rs61764370 T>G) in the women of Kerala, South India [26]. Here we did a follow-up study involving 60 more individuals from both BC and control groups to validate our previous results and perform a combined statistical analysis.

Materials and methods

Study subjects: The present case-control study was carried in a health care hospital in Kerala, India. The study involved 60 BC patients and 60 age-adjusted healthy controls. The inclusion criteria for the study were (1) women with age ≥35 years;(2) All study subjects should have undergone breast cancer screening: mammography (i.e., film, digital, tom synthesis) or with other modality; other screening modality (i.e., MRI, ultrasound) and have been confirmed of BC. The exclusion criteria included: women with pre-existing other types of cancer or Li-Fraumeni syndrome, Cowden syndrome, hereditary diffuse gastric cancer, or other familial cancer syndromes. The clinicopathological characteristics, including age, tumor size, nodal metastasis, grade, and BC patients' stages, were collected and documented. Written informed consent was acquired from all the study subjects. The Ethics Committee approved the present study's procedures, Mar Baselios College, Kerala, India(Ref no: IEC/17/2017/MBDC).

Genome DNA extraction, IL7RA rs6897932 and KRAS rs61764370 genotyping: A 5 ml venous blood was collected from the participants during their hospital visit. Genomic DNA was extracted using QIAamp DNA Mini Kit (Qiagen, Germany). The high-quality DNA isolated from whole blood samples were checked for its quality and quantity using NanoDropTM 2000 UV-Vis Spectrophotometer (Thermo Fisher Scientific, United States). The samples' purity was checked based on 260/280nm and 260/230nm ratios. DNA samples' quality was also cross-checked on Agarose gel electrophoresis by preparing 1% gel to avoid any major contaminations. Extracted DNA samples were stored at -20oC for future use. The genotyping has been performed for IL7RA rs6897932, and KRAS rs61764370 gene polymorphism as previously reported [20,26]. In both groups, deviations from Hardy–Weinberg equilibrium (HWE) were evaluated using the Chi- square (2 test). Using MedCalc Statistical Tools, the relative risk associated with genotype and alleles was measured as an odds ratio (OR) with a 95 percent confidence interval (CI) (version 16.4.3). A p-value of less than 0.05 was considered statistically significant..

Results and discussion

The demographic data (age) and clinical details of the case and control groups are shown in Table 1. The present study group included 60 BC patients with a mean age of 45.1 ±10.3 years and 60 healthy women with a mean age of 48.9 ±10.1 years. The PCR-RFLP results showed that KRAS (rs61764370) T>G polymorphism contained three genotypes: TT as a wild-type, TG as a heterozygous mutant and GG as a homozygous mutant. The genotype and allele frequencies for the KRAS (rs61764370) polymorphism in all studied groups is illustrated in Figure 1. We analyzed KRAS (rs61764370) polymorphism in 60 individuals with BC and 60 healthy controls with no BC in the present study. The differences between genotypes and alleles frequencies of KRAS (rs61764370) were remarkable between the case and control group. The genotype distribution of all three genotypes was significantly different with p-value <0.05 between cases (TT = 64%, TG = 31.3% and GG=21.6%) vs controls (TT = 70.4%, TG =21.4% and GG=6.6%) respectively. The genotype frequency of both homozygous mutations (GG) and heterozygous mutations (TG) was higher in the case group when compared with the control group with statistical significance. The corresponding allele frequencies showed a difference in the levels as well; cases had T = 66.6% and G = 33.4%, whereas controls had T = 84.9% and G = 18% with statistical significance (p=.004 for both T and G allele). Individuals with the TG and GG genotype had a significantly elevated risk of developing BC compared with TT. In addition, we also found that the minor allele frequency for the G allele in the case group was higher than that of the

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control group, which indicated the risk for BC to be increased by 2.42 folds in the presence of G allele. The IL7RA (rs6897932) T>C polymorphism contained three genotypes: TT as a wild-type, TC as a heterozygous mutant and CC as a homozygous mutant. The genotype and allele frequencies for the IL7RA (rs6897932) polymorphism in all studied groups are illustrated in Figure 1. The differences between genotypes and alleles frequencies of IL7RA (rs6897932) were remarkable between the case and control group. The genotype distribution of two of the genotypes was significantly different with p-value <0.05 between cases (TT = 46.6% and CC=26.6%) vs controls (TT = 49.1%, and CC=10.71) respectively. However, the differences between genotype TC IL7RA (rs6897932) between the case and control remained insignificant (p>0.05). The genotype frequency of homozygous mutations (CC) was higher in the case group when compared with the control group with statistical significance. The corresponding allele frequencies were also showing a difference in the levels as well; cases had T = 59.8% and C = 40.1% whereas controls had T= 79.4% and C = 20.5% with statistical significance (p=0.001 for both T and C allele).

Individuals with CC genotype had a significantly elevated risk of developing BC than TT, with an OR (95%CI) of 3.04 vs 0.38, respectively. In addition, we also found that the minor allele frequency for the C allele in the case group was higher than that of the control group, which indicated the risk for BC to be increased by 2.5 folds in the presence of C allele.

The current study found a close correlation between KRAS (rs61764370) polymorphisms and the risk of developing BC in Kerala, South India. According to our results, the rs61764370 TG vs TT and G vs T allele was the risk factors for BC. Johnson et al., 2007 and Chung et al., 2014, respectively, were the first to record a germline and functional SNP in the KRAS 3'UTR region (rs61764370 T>G) located in the let-7 complementary site 6 (LCS-6) [21-22]. LCS-6 disrupts the let-7 binding affinity for KRAS, resulting in decreased KRAS inhibition and increased tumor development, according to further research [24-25]. Many other studies have recently been effective in determining the effect of the KRAS (rs61764370) polymorphism on the risk of various other cancers, including non-small cell lung cancer (NSCLC) in moderate [23], triple-negative breast cancer in premenopausal women [24], and ovarian cancer in BRCA negative females from hereditary breast cancer and ovarian cancer syndrome families [25]. A recent study looked into KRAS gene polymorphisms and the risk of developing breast cancer in the Iranian population and discovered a clear correlation between the KRAS gene (rs61764370) mutation and an increased risk breast cancer [27]. This study's findings are consistent with those of a previous study that linked the KRAS rs61764370 polymorphism to the risk of double primary breast and ovarian cancer [28]. The findings of this study are consistent with those of a previous study that linked the KRAS rs61764370 polymorphism to the risk of double primary breast and ovarian cancer [25]

Furthermore, a study by Paranjape et al. [24] found that the KRAS variant is a risk factor for triple-negative BC in premenopausal women, emphasizing its importance as a genetic marker for BC. Uvirova et al. [29] found no correlation between the KRAS rs61764370 variant and the risk of BC, but they did indicate that the KRAS rs61764370 TG genotype could affect HER2 gene expression profile. Cerne et al. [30] found no evidence of a correlation between the KRAS rs61764370 variant and the risk of sporadic and familial BC. The results of these studies [29-30]

support the findings of a meta-analysis published in 2016 [31] that concluded the KRAS genotype GT/GG of rs61764370 has no connection with the risk of breast, ovarian, NSCLC, colorectal, or head-neck cancer. Let-7 is thought to be essential in the progression of BC. Recent evidence also indicates that the let-7 miRNA family plays a key role in the development of BC by altering the expression of KRAS, an unusual target of activating mutations in breast tumors [32]. In the KRAS 3'-UTR mRNA, LCS6 induced an increase in KRAS expression in vitro and decreased inlet-7 levels in vivo [23]. Another study [33] found that targeted knock-in of the polymorphism rs61764370 did not reduce let-7 expression but did not affect KRAS levels. According to recent research, the KRAS LCS6 variant is inconsistent, uncommon in East Asians and Native Americans, and highly rare in Africans, with a minor allele frequency of 7% in European populations 19. In the present study, the KRAS rs61764370 variant allele was 34.8% in BC

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patients and 17.8% in healthy women.

The IL7RA (rs6897932) Thr244Ile polymorphism was correlated with BC susceptibility in South Indian ethnicity (Kerala population) in the current research. Previous research has identified a connection between the IL7RA (rs6897932) gene and autoimmune disorders [34]. The IL-7RA gene (IL7R) is located on the 5th chromosome at 5p13.2, and it has multiple single nucleotide polymorphisms (SNPs) [17]. IL-7 has been studied as a pleiotropic cytokine that helps immune function by promoting lymphoid cell growth and homeostasis. IL-7 activates JAK1 and 3, which causes phosphorylation of the signal transducers STAT5 and PI3K pathway, triggering pro- survival responses in lymphoid cells, upon ligation with its receptor complex, which includes the typical cytokine-receptor chain (CD132) and the IL-7RA (CD127). The ability of IL-7 to promote cell growth and survival has been demonstrated in cell culture experiments with BC cells, and the process is based on both STAT5 and PI3K activation [36]. IL-7 has been shown to function on tumor cells in addition to its role in the immune system [36]. Exon 6 contains the IL7RA SNP (rs6897932), which causes a threonine to isoleucine conversion (Thr > Ile) on the extracellular- transmembrane boundary. This polymorphism has also been linked to multiple sclerosis [18-20]

and several other immune-related disorders [19]. Despite evidence that IL-7 plays a role in a variety of cancers, including breast cancer and functional effects of the IL7RA rs6897932 polymorphism on immune function and autoimmune diseases [36-37], there are only a few studies that look into this polymorphism's impact on breast cancer. Two recent Brazilian studies looked into the relationship between this IL7RA rs6897932 and the sensitivity and clinicopathological parameters of breast cancer subgroups in the Brazilian population [35,38].

Compared to other studies conducted around the world [39], our results on the T allele frequency in the stable control group (79.4%) were higher than the majority of Asian and Admixed American ethnicity studies. Our earlier findings [20], on the other hand, are in line with a recent study in the Brazilian population that found a strong link between the IL7RA rs6897932 (Thr244Ile) polymorphism and BC growth.

The current research has a few drawbacks, including the following: (1) The current study only looked at one SNP in the IL7RA and KRAS gene; (2) the study lacked patient information on established risk factors for BC (e.g., parity, use of oral contraceptives or hormone therapy, breastfeeding, smoking, and alcohol intake); (3) The research did not look into the relationship between clinical characteristics of BC patients such as age, tumor size, nodal metastasis, grade, stage, and histology; (4) Also the study was carried out only at one health care centre. As a result, the study subjects would not fully reflect the Kerala, South Indian population. For better results, the authors suggest larger studies. (Times New Roman, 10 pt, Tab 0.8 cm)

Conclusions

Overall, the results of this follow-up study confirmed the role of IL7RA rs6897932 and KRAS rs61764370 in the development of breast carcinoma in the Kerala population in South India.

These gene polymorphisms may be used as a genetic marker to assess breast cancer risk in high- risk women. More research with larger sample size and a larger number of BC patients is required.

Acknowledgements

We thank all research staff who has helped us during the study.

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Table 1: The clinicopathological characteristics of BC patients

Characteristics Cases n (%)

Age

Mean±SD 45.1 ±10.3 (48.9 ±10.1 healthy controls)

Tumor Size (cm)

≤2 28(25.0%)

>2 63 (56.25%)

Unknown 21(18.7%)

Nodal metastasis

No 23 (21.2%)

Yes 71(68.9%)

Unknown 12 (11.3%)

Grade

I 19 (16 .8%)

II 52 (46..1%)

III 15(13.2%)

IV 2 (1.7%)

Unknown 25(22.1%)

Stage

I 10(10.2%)

II 32(32.6%)

III 23(23.4%)

IV 24(24.4%)

Unknown 9 (9.1%)

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Figure 1: Distribution IL7RA rs6897932 and KRAS rs61764370 gene polymorphism in BC patients (cases) and BC controls