|Year : 2020 | Volume
| Issue : 2 | Page : 140-144
KRAS gene polymorphism (rs61764370) and its impact on breast cancer risk among women in kerala population, South India
MT Mohthash1, Sunil Kumar Shah2, Anand Thirupathi3
1 Department of Biotechnology, Sri Satya Sai University of Technology and Medical Sciences, Pachama, Madhya Pradesh, India
2 College of Pharmacy, Sri Satya Sai University of Technology and Medical Sciences, Pachama, Madhya Pradesh, India
3 Sri Venkateshwara Research Centre, Thanjavur, Tamil Nadu, India
|Date of Submission||21-Jan-2020|
|Date of Decision||26-Feb-2020|
|Date of Acceptance||12-Mar-2020|
|Date of Web Publication||22-Jul-2020|
M T Mohthash
Department of Biotechnology, Sri Satya Sai University of Technology and Medical Sciences, Pachama, Madhya Pradesh
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Background: In association with the risk of developing different types of cancer, several studies have currently reported association of single-nucleotide polymorphisms in the lethal-7 miRNA binding site within the 3'-untranslated region of KRAS gene. The present study was conducted for assessing the role of KRAS gene polymorphism (rs61764370 T >G) and its impact on breast cancer (BC) risk among the Kerala population, South India. Subjects and Methods: A case–control study was conducted at two health-care centers in Kerala, South India, involving 112 BC patients and 112 healthy controls (females). Genetic analysis was performed to detect KRAS polymorphism (rs61764370 T >G) employing polymerase chain reaction–restriction fragment length polymorphism method. Odds ratio (OR) with 95% confidence interval (CI) was used to evaluate the relationship of KRAS (rs61764370) polymorphism with BC susceptibility. Statistical analysis was performed using the Statistical Package for the Social Sciences (SPSS, version 21.0) software and MedCalc software (version 16.4.3). Results: The frequency distribution of KRAS (rs61764370) polymorphism was found to be different between case and control groups significantly indicating that the KRAS gene could play an important role in the pathogenesis of BC in South Indian population. The rs61764370 TG genotype (OR = 1.59; 95% CI = 0.87–2.92; P = 0.02), GG genotype (OR = 3.177; 95% CI = 1.34–7.48; P = 0.008), as well as the G allele (OR = 2.45; 95% CI = 1.32–4.57; P = 0.004) was found to increase the risk of BC among the studied South Indian population. Conclusion: The present study provided evidence regarding the role of KRAS polymorphism (rs61764370) in developing BC among the studied population. The KRAS rs61764370 variant was found to increase the BC risk among the South Indian population (Kerala). Further studies using larger sample sizes in different ethnicities are warranted to confirm the study findings.
Keywords: Breast cancer, KRAS gene polymorphism, rs61764370
|How to cite this article:|
Mohthash M T, Shah SK, Thirupathi A. KRAS gene polymorphism (rs61764370) and its impact on breast cancer risk among women in kerala population, South India. J Nat Sc Biol Med 2020;11:140-4
|How to cite this URL:|
Mohthash M T, Shah SK, Thirupathi A. KRAS gene polymorphism (rs61764370) and its impact on breast cancer risk among women in kerala population, South India. J Nat Sc Biol Med [serial online] 2020 [cited 2020 Oct 27];11:140-4. Available from: http://www.jnsbm.org/text.asp?2020/11/2/140/290486
| Introduction|| |
Breast cancer (BC) is the most frequently occurring female cancers reported globally, and this represents nearly a quarter (25%) of all cancers with 1.67 million newly diagnosed cancer cases. It has been recently reported that there has been an increase in the number of BC cases in developing countries (883000 cases) in comparison to the developed countries (794,000). Although India has a decreased age-adjusted incidence rate of BC (25.8/100,000) when compared to the United Kingdom (95/100,000), the mortality rates between both the countries are almost equal (12.7 vs. 17.1/100,000). In India, BC has acquired the rank of being the most prevalent cancer among females with an age-adjusted rate of 25.8/100,000 women as well as a mortality rate of 12.7/100,000 women. Within the Indian subcontinent, the incidence rate and cancer-associated morbidity, as well as mortality rates, have highly increased over the last few years as it is reported from global and Indian studies.,,,, Previous studies have reported cervical cancer to be the most common cancer in Indian females, but the latest studies (last 5-year studies) mention BC to be more prevalent over cervical cancer, although cervical cancer still remains most common in rural parts of India. At present, in the search of novel strategies for screening different cancers, various genetic studies have been performed in different population to know in details about the genetic factors associated with BC which could help in early detection in comparison to conventional screening techniques that includes breast ultrasound, magnetic resonance imaging (MRI), diagnostic mammogram, and biopsy. Although a large number of epidemiologic studies have been previously performed in cancers including prostate, oral gastric, and BC,,,, studies that focus on the genetic factors associated with BC in ethnicity of South India remain very limited. BRCA1 and BRCA2 are the genes that are most frequently studied for BC with susceptibility to hereditary breast and/or ovarian cancer syndrome., Recently, however, a number of large-scale studies reported the importance of multigene testing and the polygenic nature of BC which would include a screening of non-BRCA genes that are often missed in the conventional genetic screening procedure. Micro-ribonucleic acids (miRNAs) are small, noncoding, sequences of nucleotides that have the capability of controlling gene expression by attaching to complementary sites in the 3'-untranslated region (3'UTR) of target messenger ribonucleic acids (mRNAs). It is reported over the years that nearly 2000 miRNAs are found in humans by various studies, and almost every study group has highlighted their importance in potent cellular processes which include cell propagation, differentiation, carcinogenesis, response to treatment, and tumor progression. The lethal-7 (let-7) family members are considered to be the most studied miRNAs in human cancers. They behave as tumor suppressors by suppressing oncogenes that are responsible for the regulation of the intracellular signaling cascades or the cell cycle. A well-known effector molecule is the V-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS). It plays a very vital part in a number of signal potent transduction pathways such as Ral (Ras-Like) guanine nucleotide exchange factor as well as mitogen-activated protein kinase and phosphatidylinositol 3-kinase pathways. KRAS is a recognized target of let-7 containing numerous complementary sites at untranslated (3'UTR) region of the mRNA. Present studies have reported a germline and functional single-nucleotide polymorphism (SNP) in the KRAS 3'UTR (rs61764370 T >G) positioned on the let-7 complementary site 6 (LCS-6). Additional studies have found that LCS-6 impairs the let-7 binding affinity for KRAS. This results in enhanced tumor growth and decreased KRAS inhibition., Numerous studies in recent times have delved into the details of the impact of KRAS rs61764370 T >G polymorphism on the risk of growing various cancers. The LCS-6 variant allele was shown to affect non-small cell lung cancer (NSCLC) in moderate smokers, triple-negative BC in premenopausal women, and ovarian cancer in BRCA-negative female individuals among inherited BC and ovarian cancer syndrome families. In accordance with that, the current study was aimed at investigating the potential relationship between BC risk and KRAS variant (rs61764370 T >G) among the women population of Kerala, South India.
| Subjects and Methods|| |
The present case–control study was carried out in two health-care hospitals in Kerala, India. The study involved 112 BC patients and 112 age-adjusted healthy controls. The inclusion criteria for the study were (1) women with age ≥35 years and (2) all study participants should have undergone BC screening either mammography (i.e., film, digital, tom synthesis) or other screening modality (i.e., MRI and ultrasound) for confirmation of BC. The exclusion criteria included: women with preexisting 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, stage, and histology of BC patients were collected and documented. Written informed consent was acquired from all the study participants. All procedures in the present study were approved by the Ethics Committee, Mar Baselios College, Kerala, India (Ref no: IEC/17/2017/MBDC).
Genome DNA extraction
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 was checked for its quality and quantity using NanoDrop™ 2000 UV-Vis spectrophotometer (Thermo Fisher Scientific, United States). The purity of the samples was checked based on 260/280 nm and 260/230 nm ratios. The quality of DNA samples was also cross-checked on agarose gel electrophoresis by preparing 1% gel to avoid any major contaminations [Figure 1]. Extracted DNA samples were stored at −20°C for future use.
|Figure 1: Extraction of DNA samples from the study participants (1% agarose gel)|
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KRAS (rs61764370) polymorphism genotyping
The information for genotyping KRAS (rs61764370 T >G) polymorphism was obtained from a previously reported study. The details of the primers, restriction enzymes, and the length of digested fragments used in genotyping were as follows: forward: 5'-GTGTCAGAGTCTCGCTCTTGTC-3' and reverse: 5'-AGACCACACTAGCACTACCTAAGGA-3'. The primers for the targeted amplification of KRAS (rs61764370) polymorphism were designed as per our request from Eurofins Genomics, United States. Primers were diluted to a stock solution of 100 μM then to a working solution of 10 μM before setting up the reaction. The polymerase chain reaction (PCR) reactions were performed in a final volume of 25 μl, containing approximately 50 ng genomic DNA, 10 μM each primer, AmpliTaq Gold™ 360 Master Mix (Thermo Fisher Scientific, United States), and nuclease-free water. All reactions were performed using Applied Biosystems® GeneAmp® PCR System 9700. The PCR conditions were standardized as follows: 5 min preheating at 95°C, 30 cycles of 95°C for the 30 s, 58°C for 30 s, and 72°C for 30 s, followed by a final extension step for 10 min at 72°C. The PCR products (296 bp) were further run on 2% agarose gel with a 50-bp ladder from Promega (Wisconsin, the United States) to check the product size and its quality. For genotyping KRAS rs61764370 T >G polymorphism, the PCR product (10 μl) was digested using HinfI restriction enzyme (Thermo Fisher Scientific, United States). The manufacturer's instruction was strictly followed while performing the reaction and digestion following it. The digested product was run on 2% agarose gel to check for restriction digestion, as shown in [Figure 2]. The digested T allele produced 80-, 135-, and 161-bp fragments, whereas the G allele produced 296- and 80-bp amplicons [Figure 2]. Results for each reaction product were recorded in table format for statistical analysis. Demographic details of both cases and controls were matched using IBM SPSS Windows software (Version 21.0). (SPSS Inc., Chicago, Ill., USA). After the genotypes of each individual were determined, genotype and allele frequencies were calculated by direct counting. Deviations from Hardy–Weinberg equilibrium were tested using the Chi-square test in both the groups. The relative risk associated with genotype and alleles estimated as an odds ratio (OR) with a 95% confidence interval (CI) were obtained using MedCalc statistical software (version 16.4.3). Statistical significance was defined as P < 0.05.
|Figure 2: Polymerase chain reaction–restriction fragment length polymorphism genotyping of KRAS rs61764370 T>G polymorphism: Polymerase chain reaction digestion for KRAS rs61764370 polymorphism using HinfI, resulting in digested products (run on 2% agarose gel), M-DNA marker (50 bp); S1 and S2 show participants with TG genotype with 4 digested products (296, 161, 135, and 80 bp); S3 shows participants with TT genotype with 3 digested products (161,135, and 80 bp) and S4 shows participants with GG genotype with 2 digested products (296 and 80 bp)|
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| Results|| |
The demographic data (age) and clinical details of the case and control groups are shown in [Table 1]. The present study group included 112 BC patients with a mean age of 47.1 ± 11.3 years and 112 healthy women with a mean age of 47.9 ± 11.1 years. The results of PCR-restriction fragment length polymorphism 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 are illustrated in [Table 2]. In the present study, we analyzed KRAS ( rs61764370) polymorphism in 112 individuals with BC and 112 healthy controls with no BC. The differences between genotype and allele frequencies of KRAS (rs61764370) were remarkable between the case and control groups. The genotype distribution of all three genotypes was significantly different with P < 0.05 between cases (TT = 50%, TG = 30.3%, and GG = 19.6%) and controls (TT = 71.4%, TG = 21.4%, and GG = 7.1), respectively. The genotype frequency of both homozygous mutations (GG) and heterozygous mutations (TG) was found to be 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 = 65.1% and G = 34.8%, whereas controls had T = 82.1% and G = 17.8% with statistical significance (P = 0045 for both T and G alleles). Individuals with the TG and GG genotypes had a significantly elevated risk of developing BC compared with TT, with an OR (95% CI) of 1.59 and 3.17 versus 0.4, respectively. In addition, we also found that the minor allele frequency for G allele in the case group was higher than that of the control group, which indicated the risk for BC to be increased by 2.45 folds in the presence of G allele.
|Table 1: The clinicopathological characteristics of breast cancer patients|
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|Table 2: Distribution of V-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog rs61764370 alleles in breast cancer patients (cases) and breast cancer controls|
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| Discussion|| |
The current study found a strong association of KRAS (rs61764370) polymorphisms with the risk of BC development in Kerala, South Indian population. According to our results, the rs61764370 TG versus TT and G versus T allele were the risk factors for BC. Study groups Johnson et al., 2007, and Chung et al., 2014, were the first to report a germline and functional SNP in the KRAS 3'UTR (rs61764370 T >G) located in the let-7 complementary site 6 (LCS-6), respectively., Further studies found that that LCS-6 disrupts the let-7 binding affinity for KRAS, and result in lower KRAS inhibition and increased tumor growth., Recently, many other studies have been also successful in studying the impact of KRAS(rs61764370) polymorphism on the risk of various other cancers including NSCLC in moderate smokers, triple-negative BC in premenopausal women, and ovarian cancer in BRCA-negative females belonging to inherited BC and ovarian cancer syndrome families. A study recently explored KRAS gene polymorphisms and risk of developing BC among Iranian population and found a strong association of KRAS gene (rs61764370) mutation with the increased risk of BC. The present study is in strong agreement with a previous study which indicated the KRAS rs61764370 polymorphism to be associated with the risk of double primary breast and ovarian cancer. Further, a study from Paranjape et al. reported the KRAS variant to be a risk factor for triple-negative BC in premenopausal women highlighting its role as a genetic marker for BC. On the other hand, Uvirova et al. reported no association to exist between KRAS rs61764370 variant and risk of BC, although they suggested that KRAS rs61764370 TG genotype could affect the HER2 gene expression profile. Similar results were provided by Cerne et al. stating no proof of relationship of KRAS rs61764370 variant with risk of sporadic and familial BC, and the findings of these studies, further support the findings of a meta-analysis published in 2016 which summarized the KRAS genotype GT/GG of rs61764370 to have no relation with the risk of breast, ovarian, NSCLC, colorectal, or head–neck carcinoma among Caucasian population. Let-7 has been reported to play a key role in the BC advancement. Recent proof also suggests a central role for the let-7 miRNA family in the progression of BC through altered expression of KRAS, an infrequent target of activating mutations in breast tumors. LCS-6 in the KRAS 3'-UTR mRNA was found to cause an increase in expression of KRAS in vitro and a reduction in let-7 levels in vivo. In contrast, another study indicated that targeted knock-in of the polymorphism rs61764370 was not associated with KRAS levels but decreased let-7 expression. Recent studies also report the KRAS LCS-6 variant to be inconsistent, and extremely rare in East Asians and in Native Americans, and so uncommon in Africans with a minor allele frequency of 7% in the European population. In the present study, the KRAS rs61764370 variant allele was 34.8% in BC patients and 17.8% in healthy women. There exist few limitations for the present study including: (1) the sample size for the current study is less as it has been undertaken as a pilot study considering that there has been no previous study carried in studying the KRAS gene polymorphism (rs61764370 T >G) and its impact on BC risk among the Kerala population, South India, to best of our knowledge. (2) The current study assessed only one SNP of the KRAS gene. (3) The study lacked patient details concerning the known risk factors for BC (e.g., family history, parity, oral contraceptive or hormone therapy use, breastfeeding, smoking, and alcohol intake). (4) The present study only involved two health-care centers for sample collection as a reason the study participants would not completely represent the Kerala population, South Indian. (5) The study did not look into the relationship between clinical characteristics of BC patients including age, tumor size, nodal metastasis, grade, stage, and histology. The authors recommend larger studies for better conclusive results.
| Conclusion|| |
Overall, the present study provided evidence regarding the implication of KRAS polymorphism rs61764370 in developing breast carcinoma among the Kerala population, South India. The present study revealed significant differences in the frequencies of KRAS rs61764370 gene genotypes and alleles between the studied BC case group and the healthy controls with no BC. The rs61764370 gene polymorphism could be considered as a useful genetic marker to evaluate the susceptibility to BC among high-risk women. Further studies with larger sample size and on a wider scale of BC patients are needed to confirm this preliminary conclusion. Thus, developing such early genetic screening techniques could help BC patients who are at high risk to be identified at the early stages of disease development, whereby they could benefit from more targeted prevention programs.
Consent for publication
Informed consent was obtained from all the participants.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2]
[Table 1], [Table 2]