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Association of FGF Receptor-2rs2981578 SNP with breast cancer in women at the University of Gondar Comprehensive Specialized Hospital, Ethiopia

PMCID: PMC12316240

PMID: 40748883

Abstract

Introduction Fibroblast Growth Factor Receptor-2 single nucleotide polymorphisms are implicated in breast cancer development. However, there are inconsistencies and contradictions in the reports. The current study aimed to determine the association between Fibroblast Growth Factor Receptor-2 rs2981578 single nucleotide polymorphism and breast cancer among female breast cancer patients. Methods A case-control study was conducted between August 2023 and April 2024 at University of Gondar Comprehensive Specialized Hospital, Gondar, and Northwest Ethiopia. Semi-structured was adapted to collect socio-demographic character and anthropometric measurement and 5 ml of blood were drawn from each randomly selected participant for molecular analysis. The data was entered into Epi-Data-7.2 and exported to SPSS-25. Binary logistic regression model was used to look into the association between various factors. Statistical significance was regarded as a p-value of < 0.05. Results The Fibroblast Growth Factor Receptor-2 Single Nucleotide Polymorphisms rs2981578 AA genotype and A allele were more frequent in breast cancer patients than in controls (37% vs. 17% and 58% vs 42%; the chi-square p = 0.025 and p = 0.009, respectively). Individuals with the AA genotype and A allele were more prone to breast cancer (OR = 3.3; 95% CI: 1.293–8.538, and OR =1.88; 95% CI: 1.173, 3.029, respectively) than those with the wild type GG genotype. The risk allele correlated with the late TNM stage (COR = 3.41, 95% CI: 1.07–10.88). Conclusion The AA genotype and the A allele of the Fibroblast Growth Factor Receptor-2 Single Nucleotide Polymorphisms rs2981578 gene are more prevalent among women with breast cancer. They are correlated with increased susceptibility to breast cancer and a worsening of its stage.

Full Text

According to the Global Burden of Cancer Study (GLOBOCAN) 2020 report, breast cancer remains the most frequent cancer among women, with 2.3 million (24.5%) cases and 684,996 (15.5%) deaths worldwide. According to the same report, it is the fifth biggest cause of morbidity worldwide. North America, Europe, and Oceania have the highest, whereas Africa and Asia have the lowest incidence rates. However, low and middle-income nations reported an increase in the incidence [1]. It is anticipated that by 2040, there will be over 3 million new cases of breast cancer showing an increase of more than 40% and a 50% increase to 1 million in breast cancer-related deaths [2].Across Africa, there were 186,598 newly reported cases and 85,787 fatalities in 2020, and projected to double by 2040 [3]. Ethiopia’s breast cancer incidence was 32,970 new cases, with a fatality rate of around 16,133 (31.9%) among female cancers [1].
Despite the exact mechanism behind remains unclear, breast cancer is a multifactorial disease, with various sociodemographic, behavioral, genetic, environmental exposures, and reproductive risk factors all contributing to susceptibility [4].Overall, hereditary factors have been identified as the most important characteristics in genetic variants of vulnerable genes in breast cancer patients. To date, various genome-wide association studies have revealed genetic variations that increase the risk of developing breast cancer. Such risk variant mutations possess varying penetrance and produce various malignant phenotypes. High-risk genes and single-nucleotide polymorphisms (SNPs) are associated with up to more than 50% lifetime risk and progression of breast cancer and have a large individual effect, although they are relatively uncommon in the general population [5]. Moderate penetrance genes have 20–50% lifetime breast cancer risk [6,7].In addition, 80 common low penetrance genes including, FGFR2 were identified by genome-wide association studies (GWAS), and smaller studies were also explained as having a 20% lifetime risk of breast cancer [8,9].
The Fibroblast Growth Factor Receptor-2 (FGFR2) gene is located on the longer arm of chromosome 10(10q26.13) and encompasses 120 kb consisting of 26 exons and 38 introns. The gene encodes Fibroblast Growth Factor Receptor 2, which acts as receptor tyrosine kinase tasked with relaying signals from fibroblast growth factors (FGFs). The Association involving the fibroblast growth factor and its corresponding receptor, Fibroblast Growth Factor Receptor-2regulates enormous cellular processes such as growth, survival, cell proliferation, differentiation, motility, and even cell death [10,11]. It suggested to have a role in breast cancer development and progression [12]. FGFR2 rs2981578 is one of the 14 SNP of FGFR2 located in intron 2 [13].FGFR2 rs2981578 was recognized as a genetic variant linked to breast cancer susceptibility in genome-wide studies (GWAS) [8,9]. It is also noted for its significance in dictating the response to the response to chemotherapy protocols incorporating cyclophosphamide, epirubicin, and docetaxel among breast cancer patients [14]. Evidence suggests that the risk allele contributes to an enhancement in FGFR-2 expression. Hence, genetic variations within the FGFR2 gene have pinpointed it as a potential contributor to the onset of breast cancer.
Age (±5 years) and sex-matched case-control study between August2022 to April2023 was conducted at the University of Gondar Comprehensive Specialized Hospital (UoGCSH) in Gondar, Northwest Ethiopia, within the Amhara regional state. The University of Gondar Comprehensive Specialized Hospital is one of the largest hospitals in the country. It is a tertiary teaching hospital serving a catchment area of approximately 7.5 million people. It has both pediatric and adult oncology wards. The adult oncology ward serves more than 1500 cancer patients every year [15]. The molecular analysis was conducted at the molecular biology laboratory of the Institute of Biotechnology at the University of Gondar.
The body weight and height were measured using a portable digital scale and portable stadiometer, respectively. Standing barefoot, the height was measured and reported in meters. The calculation of body mass index (BMI) involved dividing weight (in kilograms) by the square of height (in meters). Participants’ BMI was then used to classify them into different groups; underweight (BMI < 18.5 kg/m2), healthy weight (18.5–25 kg/m2), overweight (25.0–29.9 kg/m2) or obese (≥ 30 kg/m2) [16]. Data collectors were oriented for one day on procedures for measuring the weight and height of the study participants and were also made familiar with the questionnaire.
The DNA isolation was done using non-enzymatic salting-out approach [17] by taking EDTA anti-coagulated blood of both patients and healthy controls and transferring it to a sterilized 1.5 ml Eppendorf tube, then red blood cells were removed by lysing them with a buffer solution (TKM 1 containing Tris-KCl-MgCl2, and 1x Triton-X). similarly, white blood cells were lysed using a nuclear lysis buffer solution containing TKM 2 and 10% sodium dodecyl sulfate(SDS) was utilized. Then, a highly concentrated salt solution of 6M NaCl was used to precipitate and eliminate proteins. Subsequently, the DNA was precipitated by freezing with isopropanol and washed with 70% ice-cold ethanol. The genomic DNA was dissolved using Tris-EDTA (TE) buffer and stored at −20 °C till use Verification of both the quality and quantity of the isolated genomic DNA was conducted by examining it on 1.5% agarose gel under UV light, utilizing a gel documentation system (JY04S-3C, UK) as well as by measuring with 2 microliters (2μl) of Ethidium bromide and a Nanodrop reader [18].
THE (PCR-RFLP) method was employed to determine the FGFR2 rs2981578 genotypes, utilizing specific forward (5’ AATGCTGCTTTGGAGGATTG-3’) and reverse (5’-CCAGAGGACTGAAACCCACA-3’) primers. Within a 25 μl reaction mixture, amplification was undertaken using 12.5 μl of 5.0 X Firepolmaster mix (which includes 12.5mM MgCl2, 1mM dNTPs, 0.4M Tris-HCl, 0.1M (NH4)2SO4, 0.1% w/v Tween-20, and DNA polymerase), together with 1 μl forward primer, 1 μl reverse primer, 2 μl of each sample, and 8.5 μl PCR grade water, ensuring the total volume was achieved. Reactions were carried out in a TC 412 PCR thermocycler (Eppendorf, United Kingdom). The PCR condition was modified according to the master mix condition and primer melting point. At the outset of amplification, there was a 4-minute denaturation phase at 95 °C, succeeded by 35 cycles. In each cycle, denaturation took place for 30 seconds at 95 °C, followed by primer hybridization for 35 seconds at 56.8°C, elongation for 40 seconds at 72 °C, and finally, a 5-minute elongation step at 72 °C concluded the process [19].Two separate PCRs were carried out for every individual DNA sample. The results are summarized in Table 1.
The PCR products, amplified to a volume of 12 μL, were combined with 3 μL of PCR loading dye (Medax®) and then carefully loaded into wells on an agarose gel. Electrophoresis was subsequently performed in 1X tris acetate EDTA (TAE) buffer, leading to the separation of components through electrophoretic action for a total of 50 minutes at 100V on a 2% Agarose gel. After electrophoresis, 173 band-size fragments were observed by UV transilluminator gel documentation system (JY04S-3C, UK) using Gelpro analyzer version 6.3.To digest the PCR products, a mixture of 10 μL was prepared, comprising 5 μL of PCR product, 1 μL of orange buffer (#B19), 3.5 μL of distilled water, and 1 μL of the SsiI (AciI, #ER1791) restriction enzyme from Thermo Fisher Scientific, U.S. The FGFR2 rs2981578 fragment, amplified to 173 bp, underwent enzymatic digestion at 37°C for 3 hours before being stored at −20°C for 20 minutes. The digestion products underwent electrophoretic separation on a 2.5% agarose gel at 120 volts for 35 minutes. When subjected to digestion, the wild GG genotype breaks into two segments measuring 84 and 89 base pairs. In contrast, the AA genotype, with its mutated form, remains undigested. The AG genotype, representing a heterozygous state, produces three fragments measuring 173, 84, and 89 base pairs [20].Recognition by AciI occurs with the 4-base non-palindromic motif 5’ CCGC 3’, but if a substitution of the “G” nucleotide with an “A” (G/A) happens, the AciI recognition site is lost [21]. To gauge genotyping quality, a portion of 10% of randomly chosen samples was re-genotyped, revealing no disparities.
The study included 140 participants, divided equally into two groups: 70 diagnosed with breast cancer and 70 non-cancer controls, matched for age (±5 years) and sex. The age group of 41–50 years constituted the largest segment of participants, making up 32.1% of the total. For cases and controls, it was roughly 50% and 56% of them that were unemployed. For the cases and controls, the mean BMI was 21.93 ± 3.09 kg/m2 and 21.86 ± 3.40 kg/m2, respectively. See Table 2 for Sociodemographic details.
The majority of cases (70%) experienced menarche after the age of 12 and had three or more children. Among the 63 cases with a history of breastfeeding, 57.1% revealed that their children had been breastfed for more than twenty-four months on average. Of the parous cases, 60.3% had their first child between the ages of 20 and 30.Out of the 41 postmenopausal cases, 70.3% of them had an early onset of menopause, occurring at age forty-five or younger. Between cases and controls, none of the reproductive traits differed significantly (p. value >0.005).See Table 3 for detail Reproductive characteristics.
Conventional PCR-RFLP techniques were employed to analyze DNA extracted from blood samples of 140 individuals, comprising 70 cases and 70 controls. DNA presence was verified through electrophoresis on a 1.5% agarose gel stained with Ethidium bromideas shown in Fig 1. The pattern of amplification of PCR products of FGFR2 (rs2981578) with 173 base pairs was shown in a 2% agarose gel as shown in Fig 2. Restriction endonuclease (SsiI/AciI)-digested and undigested products were shown in a 2.5% agarose gel.
Genotype frequencies for FGFR2 rs2981578 in cases and controls conformed to Hardy-Weinberg equilibrium (p = 0.399, p = 0.993) as shown in Table 5.
A significant variation (p < 0.05) was observed in the distribution of the FGFR2 rs2981578 genotype polymorphism between the two groups The distribution of FGFR2 rs2981578 genotypes among cases and controls revealed notable differences. Among the cases, 21.4% carried the “GG” genotype, 41.4% had the “AG” genotype, and 37.1% exhibited the “AA” genotype. In contrast, the control group showed a genotype distribution of 32.9% for “GG”, 50.0% for “AG”, and 17.1% for “AA”. Using the “GG” wild type genotype as the reference, the “AG” genotype was associated with a non-significant increase in breast cancer risk (COR = 1.27; 95% CI: 0.562–2.872; p = 0.565). However, individuals with the “AA” genotype demonstrated a statistically significant association with increased breast cancer risk (COR = 3.30; 95% CI: 1.293–8.538; p = 0.013). When the variant genotypes AG and AA were combined (AG + AA), they were found in 78.6% of cases and 67.1% of controls, with a non-significant association with an increased risk of breast cancer (COR = 1.79; 95% CI: 0.841–3.829, p = 0.131). At the allele level, the “A” allele was significantly more frequent among cases (58%) compared to controls (42%), and was associated with an increased risk of breast cancer (COR = 1.88; 95% CI: 1.173–3.029; p = 0.004), whereas the “G” allele was more prevalent in the control group (58%) and was used as the reference. See Table 6 for detail Genotype Characteristics of cases and controls.
The association between various FGFR2 rs2981578 genotypes and the stratified nature of tumors demonstrated a statistically insignificant (p > 0.05) variation in the occurrence of GG and AG + AA within both histopathological and clinical characteristics of breast cancer (COR = 3.41, 95% CI: 1.07–10.88, p = 0.038) as shown in Table 7.
Women who have at least one risk allele A did not display any variation in the distribution of behavioral characteristics between cases and controls, as indicated by a p-value>0.05See Table 9 for more details.
The study assessed how variations in the FGFR2 rs2981578 gene influence the likelihood of developing breast cancer among women with varying reproductive traits. The findings revealed that women carrying at least one risk allele A showed no difference in the distribution of reproductive traits between cases and controls (p-value>0.05) as shown in Table 10.
The findings are in harmony with a meticulous genotype mapping study involving 1253 African American invasive breast cancer individuals and 1245 controls. This investigation demonstrated a notable correlation between the prevalence of the minor allele A and the risk of breast cancer for rs2981578 (per-allele COR, 1.20: 95% CI, 1.03–1.41: ptrend = 0.02) [22], demonstrating odds ratio estimates similar to those seen European [8]and Asian population [23], akin to the findings of GWAS research. Similarly, a meta-analysis encompassing three Asian studies, comprising 833 cases and 1012 controls, also demonstrated a notable correlation across various genetic models. For instance, in the Allele model, the odds ratio (OR) stood at 1.29 with a 95% confidence interval (CI) of 1.13–1.47, and a p-value of 0.0002. Moreover, under the Dominant model, the OR was 1.71, with a 95% CI of 1.32–2.21, and a p-value of less than 0.0001 [24]. In a study conducted in North India, an association was found between rs2981578SNP and breast cancer susceptibility. The odds ratios (OR) were reported as 1.661 (95% CI: 1.108–2.489) and 1.613 (95% CI: 1.124–2.314) for co-dominant and dominant models, respectively, with corresponding p-values of 0.014 and 0.009 [18].
One potential explanation for the link between the FGFR2 rs2981578 AA genotype and breast cancer, both in our investigation and in studies conducted on different populations, might be attributed to differences in FGFR2 expression. Several studies have done the mRNA or protein expression of FGFR2 in breast cancer. The expression level varies and is influenced by genetic, epigenetic, and molecular factors. Elevated mRNA or protein expression of FGFR2 depends was seen in SUM-52PE cell line [25] and associates with poor prognosis [26]. On the other hand, some of them reported lower FGFR2 expression levels in tumor tissues compared to adjacent normal breast ducts. This could be due to loss of heterozygosity or methylation affecting the FGFR2 locus [27]. These discrepancies highlight the complexity of FGFR2’s role in breast cancer.
Studies focusing on biochemical and structural aspects indicate a marked increase in FGFR2 messenger RNA (mRNA) expression within breast cancer tissues compared to their normal counter parts. The AA genotype in estrogen receptor-positive tumors led to changes in the DNA binding capability of transcription factors such as Oct-1/Runx2 and C/EBPb [28–30]. The binding of heterozygous alleles to FOXA1 and ERα is mirrored by allele-specific changes in chromatin accessibility and the recruitment of RNA polymerase II to a region believed to be an enhancer [31,32].However, even with the proposed potential pathways connecting FGFR2 rs2981578 to the risk of breast cancer, the precise mechanism underlying the development of breast cancer associated with this genetic variant remains unclear.
Conversely, a broader study involving 7,800 postmenopausal African American women, of whom 316 were diagnosed with invasive breast cancer, found that rs2981579, a SNP closely related to rs2981578, displayed a statistically insignificant negative correlation (HR for the G allele 0.99, 95% CI: 0.83–1.17, p = 0.87) among women aged 50–79 [33]. Moreover, findings from a population-based case-control study, which included 5,761 unrelated African-American women from 11 epidemiological studies, also showed consistent results [34]. Among 2,594 African-American women examined through a meta-analysis, which encompassed 810 cases and 1,784 controls, a mutation proximal to rs2981578 (G > A), known as rs2981578 (C > T), exhibited a potential decrease in breast cancer risk, particularly among those with the TT genotype. The odds ratio (OR) for TT versus CC/CT was 0.55 with a 95% confidence interval (CI) of 0.38–0.79, 0.51 (95% CI: 0.35–0.76) for TT versus CC, and 0.58 (95% CI: 0.40–0.85) for TT versus CT. This trend was notably more prominent in the Asian population [35].Compared to the results of the present study, other studies conducted in populations from Taiwan [20], Japan [36], and China [37] failed to uncover any notable correlation between the G/A polymorphism of the FGFR2rs2981578 gene and the risk of breast cancer. Inconsistent findings concerning the influence of FGFR2 gene G/A polymorphisms on breast cancer risk may be attributed to the heterogeneity of populations studied, variations in ethnic backgrounds and geographic locations, and potential sampling biases. Various lifestyle factors, including diet and exercise, are also linked to changes in the epigenetic state. Hence, the disparities noted in correlation analyses regarding the FGFR2 G/A polymorphism and breast cancer across various ethnic groups could arise from the interconnections between epigenetic modifications and polymorphism, thereby elucidating the intricate nature of genetic composition [38].
Hinting at the possibility that the FGFR2 rs2981578 polymorphism may heighten FGFR2’s transcriptional activity or expression as breast cancer advances, underscoring the potential prognostic value of this genetic variation in breast cancer prognosis [28,39]. This contradicts findings that presented more compelling evidence for increasing susceptibility to low-grade subtypes [18,40].Discrepancies in the prevalence of allele A could explain the inconsistency in the Association term observed in both studies.
The present study also examines the Association of FGFR2 rs2981578G/A Genotypes with socio-demographic, behavioral, and reproductive risk factors. Stratified analysis showed a statistically non-significant Association between those factors and rs2981578 polymorphism. Among individuals carrying at least one risk allele (AA + AG genotype) and exhibiting diverse risk factors, the contrast between cases and controls did not reach statistical significance (p > 0.05).The study found no notable divergence in how each locus impacted the risk of breast cancer between pre and postmenopausal women in Japan, as evidenced by the analysis of 697 female breast cancer patients and matched 1,394 controls [36]. Another study in India also reported a non-significant Association of rs2981578 and menopausal status with OR =1.848; 95%CI, (0.846–4.035); p = 0.123 [18]. The exact mechanisms by which FGFR2 rs2981578 polymorphism jointly with demographic and lifestyle factors influence breast cancer development are complex and not yet fully understood. However, several studies have proposed possible mechanisms that may explain the relationship [41]. Therefore, additional investigation is warranted to authenticate and substantiate this finding.

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"[{\"pmc\": \"PMC12316240\", \"pmid\": \"40748883\", \"reference_ids\": [\"pone.0327034.ref001\", \"pone.0327034.ref002\", \"pone.0327034.ref003\", \"pone.0327034.ref001\"], \"section\": \"Introduction\", \"text\": \"According to the Global Burden of Cancer Study (GLOBOCAN) 2020 report, breast cancer remains the most frequent cancer among women, with 2.3 million (24.5%) cases and 684,996 (15.5%) deaths worldwide. According to the same report, it is the fifth biggest cause of morbidity worldwide. North America, Europe, and Oceania have the highest, whereas Africa and Asia have the lowest incidence rates. However, low and middle-income nations reported an increase in the incidence [1]. It is anticipated that by 2040, there will be over 3 million new cases of breast cancer showing an increase of more than 40% and a 50% increase to 1 million in breast cancer-related deaths [2].Across Africa, there were 186,598 newly reported cases and 85,787 fatalities in 2020, and projected to double by 2040 [3]. Ethiopia\\u2019s breast cancer incidence was 32,970 new cases, with a fatality rate of around 16,133 (31.9%) among female cancers [1].\"}, {\"pmc\": \"PMC12316240\", \"pmid\": \"40748883\", \"reference_ids\": [\"pone.0327034.ref004\", \"pone.0327034.ref005\", \"pone.0327034.ref006\", \"pone.0327034.ref007\", \"pone.0327034.ref008\", \"pone.0327034.ref009\"], \"section\": \"Introduction\", \"text\": \"Despite the exact mechanism behind remains unclear, breast cancer is a multifactorial disease, with various sociodemographic, behavioral, genetic, environmental exposures, and reproductive risk factors all contributing to susceptibility [4].Overall, hereditary factors have been identified as the most important characteristics in genetic variants of vulnerable genes in breast cancer patients. To date, various genome-wide association studies have revealed genetic variations that increase the risk of developing breast cancer. Such risk variant mutations possess varying penetrance and produce various malignant phenotypes. High-risk genes and single-nucleotide polymorphisms (SNPs) are associated with up to more than 50% lifetime risk and progression of breast cancer and have a large individual effect, although they are relatively uncommon in the general population [5]. Moderate penetrance genes have 20\\u201350% lifetime breast cancer risk [6,7].In addition, 80 common low penetrance genes including, FGFR2 were identified by genome-wide association studies (GWAS), and smaller studies were also explained as having a 20% lifetime risk of breast cancer [8,9].\"}, {\"pmc\": \"PMC12316240\", \"pmid\": \"40748883\", \"reference_ids\": [\"pone.0327034.ref010\", \"pone.0327034.ref011\", \"pone.0327034.ref012\", \"pone.0327034.ref013\", \"pone.0327034.ref008\", \"pone.0327034.ref009\", \"pone.0327034.ref014\"], \"section\": \"Introduction\", \"text\": \"The Fibroblast Growth Factor Receptor-2 (FGFR2) gene is located on the longer arm of chromosome 10(10q26.13) and encompasses 120\\u2009kb consisting of 26 exons and 38 introns. The gene encodes Fibroblast Growth Factor Receptor 2, which acts as receptor tyrosine kinase tasked with relaying signals from fibroblast growth factors (FGFs). The Association involving the fibroblast growth factor and its corresponding receptor, Fibroblast Growth Factor Receptor-2regulates enormous cellular processes such as growth, survival, cell proliferation, differentiation, motility, and even cell death [10,11]. It suggested to have a role in breast cancer development and progression [12]. FGFR2 rs2981578 is one of the 14 SNP of FGFR2 located in intron 2 [13].FGFR2 rs2981578 was recognized as a genetic variant linked to breast cancer susceptibility in genome-wide studies (GWAS) [8,9]. It is also noted for its significance in dictating the response to the response to chemotherapy protocols incorporating cyclophosphamide, epirubicin, and docetaxel among breast cancer patients [14]. Evidence suggests that the risk allele contributes to an enhancement in FGFR-2 expression. Hence, genetic variations within the FGFR2 gene have pinpointed it as a potential contributor to the onset of breast cancer.\"}, {\"pmc\": \"PMC12316240\", \"pmid\": \"40748883\", \"reference_ids\": [\"pone.0327034.ref015\"], \"section\": \"Study setting, design\", \"text\": \"Age (\\u00b15 years) and sex-matched case-control study between August2022 to April2023 was conducted at the University of Gondar Comprehensive Specialized Hospital (UoGCSH) in Gondar, Northwest Ethiopia, within the Amhara regional state. The University of Gondar Comprehensive Specialized Hospital is one of the largest hospitals in the country. It is a tertiary teaching hospital serving a catchment area of approximately 7.5 million people. It has both pediatric and adult oncology wards. The adult oncology ward serves more than 1500 cancer patients every year [15]. The molecular analysis was conducted at the molecular biology laboratory of the Institute of Biotechnology at the University of Gondar.\"}, {\"pmc\": \"PMC12316240\", \"pmid\": \"40748883\", \"reference_ids\": [\"pone.0327034.ref016\"], \"section\": \"Anthropometric measurements\", \"text\": \"The body weight and height were measured using a portable digital scale and portable stadiometer, respectively. Standing barefoot, the height was measured and reported in meters. The calculation of body mass index (BMI) involved dividing weight (in kilograms) by the square of height (in meters). Participants\\u2019 BMI was then used to classify them into different groups; underweight (BMI\\u2009<\\u200918.5\\u2009kg/m2), healthy weight (18.5\\u201325\\u2009kg/m2), overweight (25.0\\u201329.9\\u2009kg/m2) or obese (\\u2265 30\\u2009kg/m2) [16]. Data collectors were oriented for one day on procedures for measuring the weight and height of the study participants and were also made familiar with the questionnaire.\"}, {\"pmc\": \"PMC12316240\", \"pmid\": \"40748883\", \"reference_ids\": [\"pone.0327034.ref017\", \"pone.0327034.ref018\"], \"section\": \"Genomic DNA analysis.\", \"text\": \"The DNA isolation was done using non-enzymatic salting-out approach [17] by taking EDTA anti-coagulated blood of both patients and healthy controls and transferring it to a sterilized 1.5\\u2009ml Eppendorf tube, then red blood cells were removed by lysing them with a buffer solution (TKM 1 containing Tris-KCl-MgCl2, and 1x Triton-X). similarly, white blood cells were lysed using a nuclear lysis buffer solution containing TKM 2 and 10% sodium dodecyl sulfate(SDS) was utilized. Then, a highly concentrated salt solution of 6M NaCl was used to precipitate and eliminate proteins. Subsequently, the DNA was precipitated by freezing with isopropanol and washed with 70% ice-cold ethanol. The genomic DNA was dissolved using Tris-EDTA (TE) buffer and stored at \\u221220 \\u00b0C till use Verification of both the quality and quantity of the isolated genomic DNA was conducted by examining it on 1.5% agarose gel under UV light, utilizing a gel documentation system (JY04S-3C, UK) as well as by measuring with 2 microliters (2\\u03bcl) of Ethidium bromide and a Nanodrop reader [18].\"}, {\"pmc\": \"PMC12316240\", \"pmid\": \"40748883\", \"reference_ids\": [\"pone.0327034.ref019\", \"pone.0327034.t001\"], \"section\": \"Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) Technique.\", \"text\": \"THE (PCR-RFLP) method was employed to determine the FGFR2 rs2981578 genotypes, utilizing specific forward (5\\u2019 AATGCTGCTTTGGAGGATTG-3\\u2019) and reverse (5\\u2019-CCAGAGGACTGAAACCCACA-3\\u2019) primers. Within a 25 \\u03bcl reaction mixture, amplification was undertaken using 12.5 \\u03bcl of 5.0 X Firepolmaster mix (which includes 12.5mM MgCl2, 1mM dNTPs, 0.4M Tris-HCl, 0.1M (NH4)2SO4, 0.1% w/v Tween-20, and DNA polymerase), together with 1 \\u03bcl forward primer, 1 \\u03bcl reverse primer, 2 \\u03bcl of each sample, and 8.5 \\u03bcl PCR grade water, ensuring the total volume was achieved. Reactions were carried out in a TC 412 PCR thermocycler (Eppendorf, United Kingdom). The PCR condition was modified according to the master mix condition and primer melting point. At the outset of amplification, there was a 4-minute denaturation phase at 95 \\u00b0C, succeeded by 35 cycles. In each cycle, denaturation took place for 30 seconds at 95 \\u00b0C, followed by primer hybridization for 35 seconds at 56.8\\u00b0C, elongation for 40 seconds at 72 \\u00b0C, and finally, a 5-minute elongation step at 72 \\u00b0C concluded the process [19].Two separate PCRs were carried out for every individual DNA sample. The results are summarized in Table 1.\"}, {\"pmc\": \"PMC12316240\", \"pmid\": \"40748883\", \"reference_ids\": [\"pone.0327034.ref020\", \"pone.0327034.ref021\"], \"section\": \"Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) Technique.\", \"text\": \"The PCR products, amplified to a volume of 12 \\u03bcL, were combined with 3 \\u03bcL of PCR loading dye (Medax\\u00ae) and then carefully loaded into wells on an agarose gel. Electrophoresis was subsequently performed in 1X tris acetate EDTA (TAE) buffer, leading to the separation of components through electrophoretic action for a total of 50 minutes at 100V on a 2% Agarose gel. After electrophoresis, 173 band-size fragments were observed by UV transilluminator gel documentation system (JY04S-3C, UK) using Gelpro analyzer version 6.3.To digest the PCR products, a mixture of 10 \\u03bcL was prepared, comprising 5 \\u03bcL of PCR product, 1 \\u03bcL of orange buffer (#B19), 3.5 \\u03bcL of distilled water, and 1 \\u03bcL of the SsiI (AciI, #ER1791) restriction enzyme from Thermo Fisher Scientific, U.S. The FGFR2 rs2981578 fragment, amplified to 173\\u2009bp, underwent enzymatic digestion at 37\\u00b0C for 3 hours before being stored at \\u221220\\u00b0C for 20 minutes. The digestion products underwent electrophoretic separation on a 2.5% agarose gel at 120 volts for 35 minutes. When subjected to digestion, the wild GG genotype breaks into two segments measuring 84 and 89 base pairs. In contrast, the AA genotype, with its mutated form, remains undigested. The AG genotype, representing a heterozygous state, produces three fragments measuring 173, 84, and 89 base pairs [20].Recognition by AciI occurs with the 4-base non-palindromic motif 5\\u2019 CCGC 3\\u2019, but if a substitution of the \\u201cG\\u201d nucleotide with an \\u201cA\\u201d (G/A) happens, the AciI recognition site is lost [21]. To gauge genotyping quality, a portion of 10% of randomly chosen samples was re-genotyped, revealing no disparities.\"}, {\"pmc\": \"PMC12316240\", \"pmid\": \"40748883\", \"reference_ids\": [\"pone.0327034.t002\"], \"section\": \"Sociodemographic profiles of study participants\", \"text\": \"The study included 140 participants, divided equally into two groups: 70 diagnosed with breast cancer and 70 non-cancer controls, matched for age (\\u00b15 years) and sex. The age group of 41\\u201350 years constituted the largest segment of participants, making up 32.1% of the total. For cases and controls, it was roughly 50% and 56% of them that were unemployed. For the cases and controls, the mean BMI was 21.93\\u2009\\u00b1\\u20093.09\\u2009kg/m2 and 21.86\\u2009\\u00b1\\u20093.40\\u2009kg/m2, respectively. See Table 2 for Sociodemographic details.\"}, {\"pmc\": \"PMC12316240\", \"pmid\": \"40748883\", \"reference_ids\": [\"pone.0327034.t003\"], \"section\": \"Reproductive characteristics of study participants\", \"text\": \"The majority of cases (70%) experienced menarche after the age of 12 and had three or more children. Among the 63 cases with a history of breastfeeding, 57.1% revealed that their children had been breastfed for more than twenty-four months on average. Of the parous cases, 60.3% had their first child between the ages of 20 and 30.Out of the 41 postmenopausal cases, 70.3% of them had an early onset of menopause, occurring at age forty-five or younger. Between cases and controls, none of the reproductive traits differed significantly (p. value >0.005).See Table 3 for detail Reproductive characteristics.\"}, {\"pmc\": \"PMC12316240\", \"pmid\": \"40748883\", \"reference_ids\": [\"pone.0327034.g001\", \"pone.0327034.g002\"], \"section\": \"FGFR2 rs2981578 genotypes of study participants\", \"text\": \"Conventional PCR-RFLP techniques were employed to analyze DNA extracted from blood samples of 140 individuals, comprising 70 cases and 70 controls. DNA presence was verified through electrophoresis on a 1.5% agarose gel stained with Ethidium bromideas shown in Fig 1. The pattern of amplification of PCR products of FGFR2 (rs2981578) with 173 base pairs was shown in a 2% agarose gel as shown in Fig 2. Restriction endonuclease (SsiI/AciI)-digested and undigested products were shown in a 2.5% agarose gel.\"}, {\"pmc\": \"PMC12316240\", \"pmid\": \"40748883\", \"reference_ids\": [\"pone.0327034.t005\"], \"section\": \"FGFR2 rs2981578 genotypes of study participants\", \"text\": \"Genotype frequencies for FGFR2 rs2981578 in cases and controls conformed to Hardy-Weinberg equilibrium (p\\u2009=\\u20090.399, p\\u2009=\\u20090.993) as shown in Table 5.\"}, {\"pmc\": \"PMC12316240\", \"pmid\": \"40748883\", \"reference_ids\": [\"pone.0327034.t006\"], \"section\": \"FGFR2 rs2981578 genotypes of study participants\", \"text\": \"A significant variation (p\\u2009<\\u20090.05) was observed in the distribution of the FGFR2 rs2981578 genotype polymorphism between the two groups The distribution of FGFR2 rs2981578 genotypes among cases and controls revealed notable differences. Among the cases, 21.4% carried the \\u201cGG\\u201d genotype, 41.4% had the \\u201cAG\\u201d genotype, and 37.1% exhibited the \\u201cAA\\u201d genotype. In contrast, the control group showed a genotype distribution of 32.9% for \\u201cGG\\u201d, 50.0% for \\u201cAG\\u201d, and 17.1% for \\u201cAA\\u201d. Using the \\u201cGG\\u201d wild type genotype as the reference, the \\u201cAG\\u201d genotype was associated with a non-significant increase in breast cancer risk (COR\\u2009=\\u20091.27; 95% CI: 0.562\\u20132.872; p\\u2009=\\u20090.565). However, individuals with the \\u201cAA\\u201d genotype demonstrated a statistically significant association with increased breast cancer risk (COR\\u2009=\\u20093.30; 95% CI: 1.293\\u20138.538; p\\u2009=\\u20090.013). When the variant genotypes AG and AA were combined (AG\\u2009+\\u2009AA), they were found in 78.6% of cases and 67.1% of controls, with a non-significant association with an increased risk of breast cancer (COR\\u2009=\\u20091.79; 95% CI: 0.841\\u20133.829, p\\u2009=\\u20090.131). At the allele level, the \\u201cA\\u201d allele was significantly more frequent among cases (58%) compared to controls (42%), and was associated with an increased risk of breast cancer (COR\\u2009=\\u20091.88; 95% CI: 1.173\\u20133.029; p\\u2009=\\u20090.004), whereas the \\u201cG\\u201d allele was more prevalent in the control group (58%) and was used as the reference. See Table 6 for detail Genotype Characteristics of cases and controls.\"}, {\"pmc\": \"PMC12316240\", \"pmid\": \"40748883\", \"reference_ids\": [\"pone.0327034.t007\"], \"section\": \"Association FGFR2 rs2981578 and clinicopathological characteristics of breast cancer patients\", \"text\": \"The association between various FGFR2 rs2981578 genotypes and the stratified nature of tumors demonstrated a statistically insignificant (p\\u2009>\\u20090.05) variation in the occurrence of GG and AG\\u2009+\\u2009AA within both histopathological and clinical characteristics of breast cancer (COR\\u2009=\\u20093.41, 95% CI: 1.07\\u201310.88, p\\u2009=\\u20090.038) as shown in Table 7.\"}, {\"pmc\": \"PMC12316240\", \"pmid\": \"40748883\", \"reference_ids\": [\"pone.0327034.t009\"], \"section\": \"Association of FGFR2 rs2981578 G/A genotypes with behavioral characteristics\", \"text\": \"Women who have at least one risk allele A did not display any variation in the distribution of behavioral characteristics between cases and controls, as indicated by a p-value>0.05See Table 9 for more details.\"}, {\"pmc\": \"PMC12316240\", \"pmid\": \"40748883\", \"reference_ids\": [\"pone.0327034.t010\"], \"section\": \"Association of FGFR2 rs2981578 G/A genotypes with reproductive characteristics\", \"text\": \"The study assessed how variations in the FGFR2 rs2981578 gene influence the likelihood of developing breast cancer among women with varying reproductive traits. The findings revealed that women carrying at least one risk allele A showed no difference in the distribution of reproductive traits between cases and controls (p-value>0.05) as shown in Table 10.\"}, {\"pmc\": \"PMC12316240\", \"pmid\": \"40748883\", \"reference_ids\": [\"pone.0327034.ref022\", \"pone.0327034.ref008\", \"pone.0327034.ref023\", \"pone.0327034.ref024\", \"pone.0327034.ref018\"], \"section\": \"Discussion\", \"text\": \"The findings are in harmony with a meticulous genotype mapping study involving 1253 African American invasive breast cancer individuals and 1245 controls. This investigation demonstrated a notable correlation between the prevalence of the minor allele A and the risk of breast cancer for rs2981578 (per-allele COR, 1.20: 95% CI, 1.03\\u20131.41: ptrend\\u2009=\\u20090.02) [22], demonstrating odds ratio estimates similar to those seen European [8]and Asian population [23], akin to the findings of GWAS research. Similarly, a meta-analysis encompassing three Asian studies, comprising 833 cases and 1012 controls, also demonstrated a notable correlation across various genetic models. For instance, in the Allele model, the odds ratio (OR) stood at 1.29 with a 95% confidence interval (CI) of 1.13\\u20131.47, and a p-value of 0.0002. Moreover, under the Dominant model, the OR was 1.71, with a 95% CI of 1.32\\u20132.21, and a p-value of less than 0.0001 [24]. In a study conducted in North India, an association was found between rs2981578SNP and breast cancer susceptibility. The odds ratios (OR) were reported as 1.661 (95% CI: 1.108\\u20132.489) and 1.613 (95% CI: 1.124\\u20132.314) for co-dominant and dominant models, respectively, with corresponding p-values of 0.014 and 0.009 [18].\"}, {\"pmc\": \"PMC12316240\", \"pmid\": \"40748883\", \"reference_ids\": [\"pone.0327034.ref025\", \"pone.0327034.ref026\", \"pone.0327034.ref027\"], \"section\": \"Discussion\", \"text\": \"One potential explanation for the link between the FGFR2 rs2981578 AA genotype and breast cancer, both in our investigation and in studies conducted on different populations, might be attributed to differences in FGFR2 expression. Several studies have done the mRNA or protein expression of FGFR2 in breast cancer. The expression level varies and is influenced by genetic, epigenetic, and molecular factors. Elevated mRNA or protein expression of FGFR2 depends was seen in SUM-52PE cell line [25] and associates with poor prognosis [26]. On the other hand, some of them reported lower FGFR2 expression levels in tumor tissues compared to adjacent normal breast ducts. This could be due to loss of heterozygosity or methylation affecting the FGFR2 locus [27]. These discrepancies highlight the complexity of FGFR2\\u2019s role in breast cancer.\"}, {\"pmc\": \"PMC12316240\", \"pmid\": \"40748883\", \"reference_ids\": [\"pone.0327034.ref028\", \"pone.0327034.ref030\", \"pone.0327034.ref031\", \"pone.0327034.ref032\"], \"section\": \"Discussion\", \"text\": \"Studies focusing on biochemical and structural aspects indicate a marked increase in FGFR2 messenger RNA (mRNA) expression within breast cancer tissues compared to their normal counter parts. The AA genotype in estrogen receptor-positive tumors led to changes in the DNA binding capability of transcription factors such as Oct-1/Runx2 and C/EBPb [28\\u201330]. The binding of heterozygous alleles to FOXA1 and ER\\u03b1 is mirrored by allele-specific changes in chromatin accessibility and the recruitment of RNA polymerase II to a region believed to be an enhancer [31,32].However, even with the proposed potential pathways connecting FGFR2 rs2981578 to the risk of breast cancer, the precise mechanism underlying the development of breast cancer associated with this genetic variant remains unclear.\"}, {\"pmc\": \"PMC12316240\", \"pmid\": \"40748883\", \"reference_ids\": [\"pone.0327034.ref033\", \"pone.0327034.ref034\", \"pone.0327034.ref035\", \"pone.0327034.ref020\", \"pone.0327034.ref036\", \"pone.0327034.ref037\", \"pone.0327034.ref038\"], \"section\": \"Discussion\", \"text\": \"Conversely, a broader study involving 7,800 postmenopausal African American women, of whom 316 were diagnosed with invasive breast cancer, found that rs2981579, a SNP closely related to rs2981578, displayed a statistically insignificant negative correlation (HR for the G allele 0.99, 95% CI: 0.83\\u20131.17, p\\u2009=\\u20090.87) among women aged 50\\u201379 [33]. Moreover, findings from a population-based case-control study, which included 5,761 unrelated African-American women from 11 epidemiological studies, also showed consistent results [34]. Among 2,594 African-American women examined through a meta-analysis, which encompassed 810 cases and 1,784 controls, a mutation proximal to rs2981578 (G\\u2009>\\u2009A), known as rs2981578 (C\\u2009>\\u2009T), exhibited a potential decrease in breast cancer risk, particularly among those with the TT genotype. The odds ratio (OR) for TT versus CC/CT was 0.55 with a 95% confidence interval (CI) of 0.38\\u20130.79, 0.51 (95% CI: 0.35\\u20130.76) for TT versus CC, and 0.58 (95% CI: 0.40\\u20130.85) for TT versus CT. This trend was notably more prominent in the Asian population [35].Compared to the results of the present study, other studies conducted in populations from Taiwan [20], Japan [36], and China [37] failed to uncover any notable correlation between the G/A polymorphism of the FGFR2rs2981578 gene and the risk of breast cancer. Inconsistent findings concerning the influence of FGFR2 gene G/A polymorphisms on breast cancer risk may be attributed to the heterogeneity of populations studied, variations in ethnic backgrounds and geographic locations, and potential sampling biases. Various lifestyle factors, including diet and exercise, are also linked to changes in the epigenetic state. Hence, the disparities noted in correlation analyses regarding the FGFR2 G/A polymorphism and breast cancer across various ethnic groups could arise from the interconnections between epigenetic modifications and polymorphism, thereby elucidating the intricate nature of genetic composition [38].\"}, {\"pmc\": \"PMC12316240\", \"pmid\": \"40748883\", \"reference_ids\": [\"pone.0327034.ref028\", \"pone.0327034.ref039\", \"pone.0327034.ref018\", \"pone.0327034.ref040\"], \"section\": \"Discussion\", \"text\": \"Hinting at the possibility that the FGFR2 rs2981578 polymorphism may heighten FGFR2\\u2019s transcriptional activity or expression as breast cancer advances, underscoring the potential prognostic value of this genetic variation in breast cancer prognosis [28,39]. This contradicts findings that presented more compelling evidence for increasing susceptibility to low-grade subtypes [18,40].Discrepancies in the prevalence of allele A could explain the inconsistency in the Association term observed in both studies.\"}, {\"pmc\": \"PMC12316240\", \"pmid\": \"40748883\", \"reference_ids\": [\"pone.0327034.ref036\", \"pone.0327034.ref018\", \"pone.0327034.ref041\"], \"section\": \"Discussion\", \"text\": \"The present study also examines the Association of FGFR2 rs2981578G/A Genotypes with socio-demographic, behavioral, and reproductive risk factors. Stratified analysis showed a statistically non-significant Association between those factors and rs2981578 polymorphism. Among individuals carrying at least one risk allele (AA\\u2009+\\u2009AG genotype) and exhibiting diverse risk factors, the contrast between cases and controls did not reach statistical significance (p\\u2009>\\u20090.05).The study found no notable divergence in how each locus impacted the risk of breast cancer between pre and postmenopausal women in Japan, as evidenced by the analysis of 697 female breast cancer patients and matched 1,394 controls [36]. Another study in India also reported a non-significant Association of rs2981578 and menopausal status with OR =1.848; 95%CI, (0.846\\u20134.035); p\\u2009=\\u20090.123 [18]. The exact mechanisms by which FGFR2 rs2981578 polymorphism jointly with demographic and lifestyle factors influence breast cancer development are complex and not yet fully understood. However, several studies have proposed possible mechanisms that may explain the relationship [41]. Therefore, additional investigation is warranted to authenticate and substantiate this finding.\"}]"

Metadata

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