Association of MET gene polymorphisms with breast cancer risk in Nigerian patients: a case-control study.

Introduction
Breast cancer (BC) is a leading cause of cancer-related deaths in females globally [17, 22]. Its occurrence is linked to human development [36]. Therefore, the rate of BC occurrence is higher in more advanced countries. A key risk factor for BC is age, which can be linked to reproductive factors. In developed countries, BC occurs more frequently in females aged > 70 years, whereas in undeveloped countries, it occurs more frequently in females below 50 [3]. Genetic predisposition also plays a role in the etiology of BC. Beyond gender and age, there is still an underlying genetic cause of breast cancer; for instance, the risk of BC doubles if the individual has a close family member diagnosed with the disease. In most cases, 5–10 percent of BC are likened to inherited mutations such as those in the BRCA1 and BRCA2 genes, which substantially enhance BC susceptibility [10]. Despite a family history, a genetic predisposition for BC is still observed in individuals without a family history. Single-nucleotide polymorphisms (SNPs) in specific genes contribute to disease progression, drug resistance, and patient survival. In addition to germline polymorphisms, several oncogenic drivers have been implicated in breast cancer pathogenesis. In approximately 25% of breast cancer (BC) cases, the HER2 oncogene is amplified, contributing to tumor development and progression [11]. The HER2 signaling pathway activates several downstream intracellular pathways, including the phosphoinositide 3-kinase (PI3K) pathway, in which mutations in the PIK3CA gene are known to be oncogenic. Mutations in PIK3CA are among the most frequent genetic alterations in BC, occurring in approximately 35% of cases [37]. Additionally, MYC elevation has been implicated as a potential mechanism contributing to resistance to PI3K-targeted therapies. Both MYC and PIK3CA are well-established oncogenic drivers of breast tumorigenesis. Among these oncogenes, the Mesenchymal Epithelial Transition factor (MET) has garnered attention for its role in tumor progression and metastasis.
MET is located on the long arm of human chromosome 7, band 31 (7q31), and comprises 21 exons demarcated by 20 introns. It encodes c-Met, a receptor tyrosine kinase that was first identified in the early 1980 s [14, 27, 29]. The role of c-Met in promoting tumor progression and metastasis is well-documented [23, 30], making it a cancer treatment target [1, 9]. Genetic alterations such as gene amplification and activating mutations position MET as a key ‘driver’ gene in cancer [25]. These alterations lead to aberrant c-Met signaling, contributing to various cancers, including breast cancer [33]. Stromal cells in mammary tumors release hepatocyte growth factor (HGF), the sole high-affinity ligand for c-Met [4]. The interaction between c-Met and HGF triggers signaling cascades that are crucial for tumor growth and angiogenesis[38]. Cancer cells use the invasive growth caused by c-Met to adapt to adverse conditions and promote resistance to therapies [25]. Targeting c-Met therapeutically is challenging owing to the lack of reliable biomarkers. Recent studies have highlighted MET gene amplification as a potential marker [13]; despite ongoing clinical trials, understanding c-Met biology is vital for developing effective treatments and identifying new therapeutic targets. Polymorphisms in the MET gene can alter the function or expression of the c-Met receptor, influencing cancer cell behavior. SNPs such as rs40239, rs1621, and rs41736 have been identified as potentially significant in solid tumors, including gastric cancer, hepatocellular carcinoma, and papillary thyroid cancer [31, 35].
MET rs40239 is located in the intragenic region of MET and has been linked to gastric cancer. The G allele has been linked to a protective effect in the Japanese population [31]. MET rs41736 is located in the MET exon. Studies have suggested that MET rs41736 could be the sole prognostic factor in individuals with limited-stage small-cell lung cancer (LS-SCLC) because it is linked to shorter progression-free survival and overall survival in patients with LS-SCLC [7]. No study has assessed the association between MET rs41736 and BC risk. Furthermore, MET rs1621, located in the seed-matching sequence (complementary sequence found within the mRNA, usually in the 3’-UTR of the MET gene), has also been associated with hepatocellular carcinoma (HCC) in the South Chinese population [35]. Specifically, females with the AG genotype of this polymorphism have been associated with a higher risk of papillary thyroid cancer, but not in males of the Chinese population, suggesting that it primarily influences disease risk with a notable gender disparity [26]. Despite these insights, there is a paucity of research on MET polymorphisms in the African population. Most studies have focused on Asian and Caucasian cohorts, leaving African populations underrepresented. The current knowledge gap is particularly pronounced in the Nigerian population, as no research has been conducted to examine the association between MET gene polymorphisms and BC risk in this population. The lack of data on MET SNPs in African and Nigerian populations represents a critical gap in current literature. Addressing this gap through targeted research could uncover important genetic risk factors and contribute to improved cancer management strategies, particularly in the era of precision medicine, where genetic profiling increasingly guides therapeutic decision-making and targeted intervention development [6]. In this study, we sought to evaluate the association of rs40239, rs1621, and rs41736 SNPs in the MET with BC risk among Nigerian women via a case–control study of 75 patients with breast cancer and 75 controls. To the best of our knowledge, this study is the first to investigate MET SNPs in Africa and Nigeria.

Materials and methods

Study participants
Seventy-five (75) patients with histologically confirmed BC, regardless of treatment regimen, were recruited, along with 75 population-based normal controls, for this case–control study. Patients were recruited from the Lagos State Teaching Hospital (LASUTH) and Lagos University Teaching Hospital (LUTH) Oncology Centers between August 2023 and May 2024. Healthy individuals were recruited from the National Hospital, Abuja, during the same period.
Inclusion criteria: Nigerian women diagnosed with BC during the study period were included as cases. Healthy Nigerian women with normal mammogram results and no history of breast cancer, matched to cases by age and ethnicity, were included as controls. The exclusion criteria were: individuals who did not provide informed consent; non-Nigerians; and patients with a history of prior malignancy other than BC. Demographic data for all participants were collected through interviews, and information on clinical parameters was retrieved from patient files. This study is in accordance with the Helsinki Declaration and was approved by the Covenant Health Research Ethics Committee (CHREC) at Covenant University, Ota, Nigeria (Approval No: CHREC/445/2024). All participants in the study gave informed consent prior to sample and data collection.

DNA extraction
The Aidlab Blood and Tissue Mini Kit (Beijing, China) was used to extract DNA from the buffy coat following the manufacturer’s instructions. The isolated DNA concentration(ηg/μl) and purity (A260/280 ratio) were measured using a NanoDrop 2000/2000c spectrophotometer (Thermo Fisher Scientific Inc., USA). The DNA was preserved at −20 °C until used for genotyping.

SNP genotyping
Three SNPs of the MET gene, rs40239, rs1621, and rs41736, were selected for evaluation based on the NCBI dbSNP database (http://www.ncbi.nlm.nih.gov/projects/SNP) and publication. SNP rs40239 in the intragenic axis of the MET gene can impact mRNA splicing and lncRNA binding. The SNP rs41736 in the exon region can inactivate the enhancer of exon splicing, causing exon skipping. SNP rs1621 in the 3’-UTR may disrupt miR-199a activity, thereby increasing MET protein levels. Each PCR mixture had a final volume of 10 μL, consisting of TaqMan Universal PCR Master Mix, specific TaqMan SNP Genotyping Assays from Applied Biosystems, nuclease-free water (7 μL), and 3 μL of genomic DNA. The cycling protocol for TaqMan PCR involved an initial phase at 95 °C for 10 min, followed by 40 cycles at 92 °C for 15 s and 60 °C for 1 min. This protocol was applied to amplify DNA samples of the rs40239, rs1621, and rs41736 SNPs.
using the QuantStudio™ 5 Real-Time PCR System. The TaqMan genotyping assay software, version 1.7.1, automatically called all results.

Statistical analysis
Categorical variables were generated in this study. To compare categorical variables. The data generated during this study were analyzed using Microsoft Excel and SPSS 20.0. The threshold for statistical significance was set at p < 0.05. Allelic frequencies for MET were obtained using the Hardy–Weinberg formula. The chi-squared test was used to analyze the association between the observed distribution of genotypes and the categories being compared (cases vs. controls). The Odds ratio (OR) for BC risk was examined using a 95% CI. Statistical significance was set at P < 0.05. The post hoc power analysis was performed using G*Power (version 3.1.9.7) at α = 0.05 to estimate the achieved power for chi-square tests based on the observed effect sizes and the total sample size (n = 150).

Results

Demographic profile
The demographic characteristics of our case–control study included 61 patients and 60 controls. The mean ages of the patients and control subjects were 49 years (49.02 ± 9.67) and 44 years (44.26 ± 6.48), respectively. This indicates that breast cancer patients are primarily at risk during the premenopausal age.

rs40239 Polymorphism in BC
The rs40239 polymorphism was genotyped in all subjects. The frequency of the allelic distribution is shown in Fig. 1, and the genotype distribution is shown in Table 1. Partition of the mutant allele G was not predominant in the cancer population, with a frequency of 17% compared to 55% in healthy controls. The frequencies of the A/A and A/G genotypes of rs40239 A > G were 60% and 3% in controls, and 17% and 4%, respectively. The AA genotype was more frequent in normal controls (60%) than in cases (17%), with an odds ratio (OR) of 4.706, suggesting a potential association with BC risk; however, the result was not statistically significant (p = 0.062). Therefore, no significant association was observed between rs40239 and the risk of BC.

rs1621 Polymorphism in BC
TaqMan genotyping of the rs1621 polymorphism in cases and controls showed that the frequencies of A/A, A/G, and G/G genotypes of rs1621 were 12%, 25%, and 14% in cases and 19%, 32%, and 20% in controls, respectively. The distribution of genotypes was similar between the groups, with an OR of 1.188, indicating no significant association (p = 0.426). Analyses of rs1621 allele frequencies revealed that the partition of the mutant allele G was not predominant in the cancer population. The frequency was 32% in cases compared to 43% in the control group, as shown in Fig. 2.

rs41736 Polymorphism in BC
The C/C genotype or rs41736 polymorphism was more common in controls (62%) than in cases (36%), and the C/T genotype was 3% in controls and absent in cases. These data indicate no significant association between this SNP and BC risk (OR = 0.954, p = 0.262). The allele frequencies for C and T were 36% and 32% in cases and 65% and 3% in controls, respectively (Fig. 3).
When the association of mutant alleles for the rs40239, rs1621, and rs41736 polymorphisms with tumor grade was studied in a subset of 27 patients whose tumor grade was recorded alongside 60 control subjects, our results indicated no statistically significant association between the mutant allele rs1621 and tumor grade (OR = 0.475, p = 0.090, 95% CI: −1.606–0.116).

Discussion
BC is a highly prevalent malignancy in females, with single-nucleotide polymorphisms as one of its genetic risk factors. SNPs in MET have been proven to be associated with cancer risk. However, there is a lack of exploration of specific MET polymorphisms in black populations, particularly in Nigeria. It is imperative to note that this is the first study to examine these polymorphisms (rs40239, rs1621, and rs41736) as potential risk factors for BC in the Nigerian population. In the current study, a greater percentage of the AA genotype was observed in controls than in cases of the rs40239 polymorphism, suggesting that this genotype is more frequent in healthy individuals than in those with BC. This large difference in distribution can lead to a high OR (OR = 4.706), indicating a potential association between the AA genotype and a lower risk of breast cancer. This finding indicates a potential trend that could be explored further using larger sample sizes or additional research. Our study found no significant association between the rs40239 SNP and BC risk. This finding aligns with those of previous studies by Kalapanida et al., who observed no significant association between the rs40239 SNP and the risk of Triple-negative breast cancer (TNBC) in a Greek population [19]. However, this result contrasts with the study by Sunakawa et al. [31], which identified a significant association between the rs40239 SNP and gastric cancer risk in the Japanese population. Specifically, Sunakawa et al. found that the G allele (A/G or G/G) is associated with longer disease-free survival and overall survival, suggesting a protective or favorable effect on cancer prognosis[31]. Interestingly, we did not observe any G/G genotype carriers in the Nigerian population. This absence might explain the lack of a protective effect of the AG genotype on BC, as the G allele is considered a minor allele in African populations according to the 1000 Genomes Project. These disparities underscore significant population-specific differences in the association of the rs40239 SNP with the risk of cancer and prognosis. Consequently, the role of the rs40239 SNP may vary significantly depending on the genetic background, environmental factors, and the type of cancer (e.g., breast vs. gastric cancer). This highlights the importance of considering ethnic and genetic diversity to understand the genetic underpinnings of cancer.
For the rs41736 SNP, our study found no association with breast cancer risk. This lack of association may be attributed to the absence of the mutant genotype (TT) in the population studied, as well as the absence of heterozygote carriers (CT) in the cancer-positive group. Notably, this study is the first to examine the association between rs41736 and the risk of BC in an African population. There is no prior research on the association of this SNP with BC risk in African, Asian, or Caucasian populations, highlighting the novelty of our findings. This limitation underscores the need for further studies across diverse ethnic groups to fully elucidate the role of rs41736 in BC risk. Previous studies have linked rs41736 to clinical outcomes in other cancers such as LS-SCLC, indicating its potential role as a prognostic factor [7]. However, it is worth noting that rs41736 has been classified as a SILENT mutation in Taiwanese patients [15]. This classification implies that although the mutation does not alter the protein sequence, its potential impact on gene expression or other regulatory mechanisms in cancer warrants further investigation. This study emphasizes the importance of investigating genetic variants in diverse populations to identify potential population-specific genetic risk factors for cancer.
Furthermore, the presence of adenosine (A) at the rs1621 SNP is implicated in increasing BC risk. However, our study found no significant association between the rs1621 SNP and the risk of BC in the Nigerian population. This result aligns with previous work by Tchatchou et al., who similarly reported no association between rs1621 and the risk of BC in the German population [34]. The strength of their study lies in the extensive sample size of familial breast cancer cases, enhancing sensitivity to detect rare alleles that could influence cancer risk. In contrast, studies by Wang et al. and Ning et al. have reported significant associations between rs1621 and HCC and PTC in the Chinese population [26, 35]. Ning et al. further noted that while rs1621 was significantly associated with a high risk of PTC in female patients, no such association was observed in male patients. These disparities underscore the importance of considering ethnic and genetic diversity when evaluating the association of SNPs with cancer risk. While rs1621 appears to influence cancer risk in certain populations and cancer types, its role in breast cancer risk in Nigerians differs from that in other ethnic groups and cancer types. Therefore, identifying MET SNPs as significant genetic risk factors in Nigerian BC patients could have implications for future genetic screening and personalized treatment strategies.
Accumulating evidence suggests that certain MET SNPs could have translational significance in the management of BC. For example, the germline MET-T1010I variant has been implicated in tumor invasion and aggressiveness in BC and proposed as a promising biomarker for stratifying patients in MET-targeted clinical trials [21]. Such findings highlight how specific MET variants could serve as molecular markers to pinpoint patients with more aggressive subtypes, including TNBC, thus informing precision-based treatment approaches. Furthermore, several deleterious nonsynonymous SNPs (nsSNPs) in the MET genes have been linked to resistance mechanisms of MET kinase inhibitors. These variants may alter protein structure and signaling dynamics, therefore impacting treatment responsiveness [20]. Collectively, these findings highlight the potential of MET genetic profiling to contribute to the development of biomarkers, treatment stratification, and improvement of targeted therapies. Although this current study did not find significant associations between the examined SNPs and breast cancer risk, however; continuous assessment of functionally impactful MET variants remains crucial for advancing precision oncology in African populations that remain underrepresented in cancer genomic research.
Furthermore, despite the fact that the HGF/c-MET signaling pathway has been widely implicated in breast cancer progression, the findings from this present study revealed no significant association between the investigated MET polymorphisms and breast cancer risk in this cohort. A possible biological explanation is that MET-driven tumorigenesis may be predominantly mediated by somatic genomic alterations rather than inherited variants. Previous studies have demonstrated that MET gene amplification results in increased receptor expression and is associated with high-grade tumors and enhanced metastatic spread [5, 8]. Similarly, exon 14 skipping mutations eliminate the Y1003 residue required for CBL-mediated ubiquitination, leading to impaired receptor degradation, sustained c-MET accumulation, and prolonged downstream signaling [18]. Activating missense mutations within the kinase and ATP-binding domains have also been shown to promote constitutive phosphorylation and enhanced kinase activity. Collectively, these alterations directly increase receptor activation and oncogenic signaling. In contrast, germline SNPs often exert modest functional effects and may not substantially alter receptor structure, degradation, or catalytic activity. Therefore, the absence of association in this study may reflect the possibility that MET contributes to breast cancer progression primarily through acquired genomic alterations and pathway activation rather than inherited genetic susceptibility.
This case–control study has several limitations that should be acknowledged, with the hope that it will serve as a foundation for future research. First, a limitation of this study is the modest sample size, which restricts statistical power, particularly for low-frequency alleles, and increases the risk of Type II error. Post-hoc power analysis indicated that the study had sufficient power (98.8%) to detect moderate effects for the MET SNP rs40239, but limited power (39.8%) for rs41736 and very low power (5.3%) for rs1621, suggesting that small effects may have been missed. Future studies with larger sample sizes are warranted to confirm potential associations. Second, MET signaling demonstrates differential biological relevance across breast cancer subtypes. Elevated MET expression has been reported in basal-like and ERBB2-positive tumors compared to luminal subtypes and has been associated with poorer prognosis [12, 28]. In TNBC, particularly in tumors with mesenchymal signatures, increased MET activity has been linked to reduced relapse-free survival and enhanced tumor aggressiveness [32]. Additionally, MET overexpression has been observed in Luminal B tumors, although gene amplification is not consistently detected in this subtype [24]. Since the present study evaluated the association between MET SNPs and overall breast cancer risk without stratifying cases by molecular subtype, the interpretation of subtype-specific associations remains limited. It is plausible that the impact of these polymorphisms differs across distinct molecular subgroups. Future studies incorporating molecular classification are therefore needed to determine whether MET variants exert subtype-dependent effects in breast cancer. Another key limitation is the study design: as a case–control study, causal inferences cannot be drawn. In addition, because the study population consisted solely of Nigerian women, the findings may not be generalizable to other populations with different genetic backgrounds and allele frequencies. Cancer development is also influenced by both genetic and epigenetic factors [2]. While this study focused on germline polymorphisms, it did not evaluate epigenetic modifications, such as DNA methylation, histone modifications, or RNA dysregulation, which can also regulate oncogene expression and tumor suppressor activity [16]. The absence of these data may limit our understanding of the functional biological mechanisms underlying MET-related breast cancer risk. However, this case–control study benefited from allele frequencies of MET SNPs (rs40239, rs1621, and rs41736) determined with high precision using the TaqMan SNP genotyping assay (minimum 96%, maximum 99%). TaqMan Genotyper Software confirmed that all frequencies were in Hardy–Weinberg Equilibrium (HWE) (p > 0.05), ensuring robust data quality. This finding supports the validity of the study by accurately reflecting the general population in the control group. Deviations from the HWE could introduce bias, genotyping errors, or population stratification, potentially compromising study integrity.