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Published in final edited form as: Breast Cancer Res Treat. 2010 Jul 30;128(1):79–84. doi: 10.1007/s10549-010-1080-z

Prevalence of the variant allele rs61764370 T>G in the 3′UTR of KRAS among Dutch BRCA1, BRCA2 and non-BRCA1/BRCA2 breast cancer families

Antoinette Hollestelle 1,✉, Cory Pelletier 2, Maartje Hooning 3, Ellen Crepin 4, Mieke Schutte 5, Maxime Look 6, J Margriet Collee 7, Anja Nieuwlaat 8, Lambert C J Dorssers 9, Caroline Seynaeve 10, Yurii S Aulchenko 11, John W M Martens 12, Ans M W van den Ouweland 13, Joanne B Weidhaas 14,✉
PMCID: PMC3735357  NIHMSID: NIHMS484807  PMID: 20676756

Abstract

Recently, a variant allele in the 3′UTR of the KRAS gene (rs61764370 T>G) was shown to be associated with an increased risk for developing non-small cell lung cancer, as well as ovarian cancer, and was most enriched in ovarian cancer patients from hereditary breast and ovarian cancer families. This functional variant has been shown to disrupt a let-7 miRNA binding site leading to increased expression of KRAS in vitro. In the current study, we have genotyped this KRAS-variant in breast cancer index cases from 268 BRCA1 families, 89 BRCA2 families, 685 non-BRCA1/BRCA2 families, and 797 geographically matched controls. The allele frequency of the KRAS-variant was found to be increased among patients with breast cancer from BRCA1, but not BRCA2 or non-BRCA1/BRCA2 families as compared to controls. As BRCA1 carriers mostly develop ER-negative breast cancers, we also examined the variant allele frequency among indexes from non-BRCA1/BRCA2 families with ER-negative breast cancer. The prevalence of the KRAS-variant was, however, not significantly increased as compared to controls, suggesting that the variant allele not just simply associates with ER-negative breast cancer. Subsequent expansion of the number of BRCA1 carriers with breast cancer by including other family members in addition to the index cases resulted in loss of significance for the association between the variant allele and mutant BRCA1 breast cancer. In this same cohort, the KRAS-variant did not appear to modify breast cancer risk for BRCA1 carriers. Importantly, results from the current study suggest that KRAS-variant frequencies might be increased among BRCA1 carriers, but solid proof requires confirmation in a larger cohort of BRCA1 carriers.

Keywords: KRAS-variant, Let-7, Breast cancer susceptibility, Association, BRCA1

Introduction

Breast cancer is the most frequently diagnosed type of cancer in women from western countries and an appreciable fraction is attributable to genetic predisposition to the disease. Disease-causing mutations in the high-risk breast cancer susceptibility genes BRCA1 and BRCA2 together explain about 20% of the familial clustering of breast cancer. Population-based studies have estimated the cumulative risk of breast cancer to be 65 and 45% for BRCA1 and BRCA2 mutation carriers, respectively [1]. The penetrance of BRCA1 and BRCA2 mutations, however, is generally higher in women from families with multiple affected individuals, suggesting the presence of other risk-modifying factors in these families [2–4]. Indeed, it was shown that low-risk breast cancer susceptibility alleles exist that may act multiplicatively on the breast cancer risk for BRCA1 and BRCA2 mutation carriers [5, 6].

Besides BRCA1 and BRCA2, three other high-risk breast cancer susceptibility genes have been recognized: p53, PTEN, and STK11. However, these account for only a small fraction (less than 1%) of the familial breast cancer risk and are rarely observed outside the context of the cancer syndromes they cause (e.g., Li-Fraumeni syndrome, Cowden syndrome, and Peutz-Jeghers syndrome, respectively). Another 5% of the familial breast cancer risk is explained by mutations in moderate-risk genes ATM, BRIP1, CHEK2, NBS1, and PALB2 and low-risk alleles such as those in or near the FGFR2, TNRC9/TOX3, and MAP3K1 genes [7, 8]. This means that so far three quarters of the familial clustering of breast cancer remains unexplained. Importantly, twin studies suggested that a significant proportion of the remaining familial breast cancer risk is most probably due to genetic factors [9, 10].

MicroRNAs (miRNAs) are a class of small non-coding RNA molecules that negatively regulate gene expression by binding partially complementary sites in the 3′ untranslated region (UTR) of their target mRNAs. The importance of miRNAs in cancer is stressed by their widespread deregulation in virtually all cancer types [11, 12]. Recently, a variant allele in the 3′UTR of the KRAS gene (rs61764370 T>G) was shown to disrupt a let-7 miRNA binding site leading to increased expression of KRAS in vitro and lower let-7 miRNA levels in vivo [13]. Consistent with the oncogenic nature of KRAS, the variant allele was shown to confer an increased risk for developing non-small cell lung cancer (NSCLC), as well as ovarian cancer ([13, 14]), although the results in NSCLC were not replicated [15]. In head and neck cancers, the KRAS-variant was shown to be a genetic marker of poor outcome as well [16]. As the frequency of the KRAS-variant was found to be increased among unselected ovarian cancer cases as well as within the context of hereditary breast and ovarian cancer (HBOC) families, we wondered whether the variant allele frequency might also be increased in breast cancer families. Therefore, we genotyped the KRAS-variant in breast cancer index cases from 268 BRCA1 families, 89 BRCA2 families, and 685 non-BRCA1/BRCA2 families and compared its prevalence with 797 geographically matched controls. We also investigated the association of the KRAS-variant with specific characteristics of BRCA1 families.

Materials and methods

Study population

Families with clustering of breast cancer were selected from the database of the Rotterdam Family Cancer Clinic at Erasmus University Medical Center—Daniel den Hoed Cancer Center in Rotterdam, representing the Southwestern part of the Netherlands. Selected families included at least two cases of female breast cancer or at least one case of female breast cancer and one case of ovarian cancer in first-or second-degree relatives. For each family, the youngest breast cancer patient who had been tested for BRCA1 and BRCA2 was assigned to be the index case. The control population was geographically matched and included 797 females from cystic fibrosis families who were either spouses of individuals at risk of being carrier of a CFTR mutation or individuals who were tested negative for a CFTR mutation. This unmatched case–control study was approved by the local ethical committee and all individuals gave full informed consent to search for susceptibility alleles.

Genotyping

DNA isolated from peripheral blood was amplified using a custom-made Taqman genotyping assay (Applied Biosystems, Foster City, CA) designed specifically to identify the T or variant G allele of the KRAS-variant (rs61764370). Forward primer: 5′-GCCAGGCTGGTCTCGAA-3′, reverse primer: 5′-CTGAATAAATGAGTTCTGCAAAACAGGT T-3′, VIC reporter probe: 5′-CTCAAGTGATTCACCCA C-3′, and FAM reporter probe: 5′-CAAGTGATTCACC-CAC-3′. Because of the low frequency of homozygotes for the variant allele, patient samples that were either heterozygous (TG) or homozygous (GG) for the variant allele were considered positive for the KRAS-variant.

Statistical analyses

Associations between the KRAS-variant and BRCA1-, BRCA2-, or non-BRCA1/BRCA2-related familial breast cancer, as well as associations between the KRAS-variant and specific characteristics of BRCA1 families were tested for significance by a χ2-test. Case–control odds ratios and their confidence intervals were calculated using Woolf approximations. The difference in mean age of onset between BRCA1 index cases carrying the variant allele and BRCA1 index cases not carrying the variant allele was determined by a t-test. The result of the Shapiro–Wilk test showed that the assumption of a normal distribution for the age of onset variable was valid. KRAS-variant frequencies among affected and unaffected BRCA1 carriers were compared using a Cox proportional hazard model. For this purpose, BRCA1 carriers were censored at either age of breast cancer diagnosis or age at last follow-up. Only BRCA1 carriers that were censored at breast cancer diagnosis were considered to be affected. Hazard ratios for the TG and GG genotypes combined were estimated using TT homozygotes as a baseline. P values of 0.05 or smaller were considered statistically significant. All statistical analyses were performed with STATA statistical package, release 11.0 (STATA Corp, College Station, TX).

Results

To establish whether the KRAS-variant (rs61764370 T>G) is implicated in breast cancer susceptibility, we have evaluated the prevalence of this variant in patients with breast cancer from BRCA1, BRCA2, and non-BRCA1/BRCA2 families as compared to controls. The variant allele was detected in 108 of 685 (15.8%) of indexes with breast cancer from non-BRCA1/BRCA2 families and 12 of 89 (13.5%) indexes with breast cancer from BRCA2 families. These frequencies were not significantly different from the 17.3% (138 of 797) prevalence among the control individuals. In contrast, the variant allele was present in 63 of 268 (23.5%) indexes with breast cancer from BRCA1 families, which was significantly different from the prevalence in controls (Table 1; P= 0.025; OR= 1.47 (1.05–2.06)). However, after adjustment for multiple testing (Bonferroni correction for three tests) this difference was not significant anymore (P = 0.073).

Table 1.

KRAS 3′UTR SNP genotype frequencies among indexes from BRCA1, BRCA2, and non-BRCA1/BRCA2 families

Population Genotype Frequency OR 95% CI P value
Controls GG+TG 138/797 (17.3%) 1.00
TT 659/797 (82.7%)
BRCA1 carriers GG+TG 63/268 (23.5%) 1.47 1.05–2.06 0.025*
TT 205/268 (76.5%)
BRCA2 carriers GG+TG 12/89 (13.5%) 0.74 0.39–1.40 0.36
TT 77/89 (86.5%)
Non-BRCA1/BRCA2 carriers GG+TG 108/685 (15.8%) 0.89 0.68–1.18 0.42
TT 577/685 (84.2%)
 ER-positive GG+TG 63/356 (17.7%) 1.03 0.74–1.43 0.87
TT 293/356 (82.3%)
 ER-negative GG+TG 18/127 (14.2%) 0.79 0.46–1.34 0.38
TT 109/127 (85.8%)
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Differences in KRAS-variant allele frequencies between cases and controls were tested for significance by a χ2-test. Case–control odds ratios and their confidence intervals were calculated using Woolf approximations

*

After adjustment for multiple testing (Bonferroni correction for three tests) the P-value between BRCA1 carriers and controls was not statistically significant anymore (P = 0.073)

As BRCA1 carriers mostly develop ER-negative breast cancers and BRCA2 carriers mostly develop ER-positive breast cancers, we wondered whether the KRAS-variant was associated with ER-negative breast cancer rather than mutant BRCA1 breast cancer. We had information on ER-status of indexes from 483 non-BRCA1/BRCA2 families of whom 127 were ER-negative and 356 were ER-positive. The variant allele was present in 18 (14.2%) indexes with ER-negative breast cancer and 63 (17.7%) indexes with ER-positive breast cancer which was not significantly different from the prevalence in the controls (Table 1). These results suggest that the KRAS-variant not just simply associates with ER-negative breast cancer.

To evaluate whether the variant allele associates with a particular phenotype in BRCA1 families, we compared family characteristics of BRCA1 families with a KRAS-variant positive index case to BRCA1 families with a KRAS-variant negative index case (Table 2). There was no evidence for the variant to be associated with the presence of either one or more ovarian cancer cases or bilateral breast cancer cases in the family. We did find a higher frequency of the KRAS-variant in families with three or more breast cancer cases younger than 60 years compared with families with two or less breast cancer cases younger than 60 years (20.4 vs. 27.9%), however, this was not statistically significant (P = 0.16). Additionally, we found no difference in mean age of onset between BRCA1 index cases carrying the variant allele and BRCA1 index cases not carrying the variant allele (39.8 vs. 39.0 years, respectively; P = 0.55).

Table 2.

Association of the KRAS-variant with specific characteristics of BRCA1 families

Family characteristic Frequency of the GG+TG genotype OR 95% CI P value
No OvC in family 38/152 (25.0%)
One or more OvC in family 22/96 (22.9%) 0.89 0.49–1.63 0.71
No Bil BrC in family 36/147 (24.5%)
One or more Bil BrC in family 24/106 (22.6%) 0.90 0.50–1.63 0.73
Less than 3 BrC < 60 years in family 29/142 (20.4%)
Three or more BrC < 60 years in family 31/111 (27.9%) 1.51 0.84–2.70 0.16
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Association of the KRAS-variant with specific characteristics of BRCA1 families was tested for significance by a χ2-test. Case–control odds ratios and their confidence intervals were calculated using Woolf approximations

OvC ovarian cancer, Bil bilateral, BrC breast cancer

In order to gain more statistical power for the analysis of association between the KRAS-variant and mutant BRCA1 breast cancer, we expanded this analysis by including other family members in addition to the index cases. In total, we had DNA from 390 BRCA1 carriers with breast cancer available for genotyping among the 268 BRCA1 families. However, the KRAS-variant was present in only 20.3% (79 of 390) of affected BRCA1 carriers (Table 3), which is lower than the initial 23.5% we found among the index cases (Table 1). Consequently, the prevalence of the KRAS-variant among BRCA1 carriers was not significantly different from the controls (P = 0.22; OR= 1.21 (0.89–1.65)), despite expansion of the number of BRCA1 carriers with breast cancer.

Table 3.

KRAS 3′UTR SNP genotype frequencies among affected and unaffected BRCA1 carriers

Genotype Frequency in unaffected carriers Frequency in carriers with breast cancer HR 95% CI P value
TT 215/260 (82.7%) 311/390 (79.7%) 1.00
GG+TG 45/260 (17.3%) 79/390 (20.3%) 1.07 0.88–1.30 0.50
Open in a new tab

Differences in KRAS-variant allele frequencies between affected and unaffected BRCA1 carriers were tested for significance by a Cox proportional hazards model. Mean age at censure was 41.0 years for affected BRCA1 carriers and 45.5 years for unaffected BRCA1 carriers. Hazard ratios for the TG+GG genotype were estimated using TT homozygotes as a baseline

To determine whether or not the KRAS-variant modifies BRCA1 mutant breast cancer risk, all affected and unaffected BRCA1 carriers among the 268 BRCA1 families were genotyped for the variant allele. Mean age at censure was 41.0 years for affected BRCA1 carriers and 45.5 years for unaffected BRCA1 carriers. Similar to the frequency in the controls, the KRAS-variant was present in 17.3% (45 of 260) unaffected BRCA1 carriers (Table 3). Importantly, there was no evidence of association between the KRAS-variant and breast cancer risk among BRCA1 carriers (Table 3; P = 0.50; HR= 1.07 (0.88–1.30)).

Discussion

In the current study we have found an increase in the frequency of the KRAS-variant (rs61764370 T>G) in breast cancer index cases from BRCA1 families as compared to geographically matched controls, whereas the frequency of this variant allele was not significantly different among breast cancer index cases from BRCA2 or non-BRCA1/BRCA2 families. However, expansion of the number of BRCA1 carriers with breast cancer by including other family members in addition to the index cases did not improve significance for the association between the variant allele and mutant BRCA1 breast cancer. Additionally, the KRAS-variant did not appear to modify breast cancer risk for BRCA1 carriers. Still the question remains whether the breast cancer risk conferred by the KRAS-variant is not too small to be detected in our case–control study. Considering the relatively high prevalence in the general population, the variant allele would not be expected to confer a dramatically increased breast cancer risk. Also, the breast cancer risk conferred by the KRAS-variant may be larger in GG homozygotes than TG heterozygotes. Therefore, we feel that this trend toward association between the KRAS-variant and mutant BRCA1 breast cancer requires validation in a larger cohort of BRCA1 carriers, providing more statistical power and enabling the analysis of TG heterozygotes and GG homozygotes separately.

Interestingly, stratification of breast cancer patients from non-BRCA1/BRCA2 families according to their tumor’s ER-status did not reveal an increased KRAS-variant frequency among ER-negative breast cancers, suggesting that the variant is not just simply associated with ER-negative breast cancer. This may seem unexpected as the KRAS-variant has been shown to lead to increased KRAS expression in vitro and hyperactivation of the MAPK pathway has been shown to downregulate ERα expression in vitro [13, 17]. However, one may wonder whether the level of increased KRAS expression as a result of the KRAS-variant is similar to that of an oncogenic RAS or RAF mutation. This may thus be a matter of dosage and therefore MAPK pathway activation should be assessed in tumors from patients that carry the KRAS-variant. Unfortunately, due to missing ERBB2 status of the vast majority of non-BRCA1/BRCA2 cases we have not been able to assess variant allele frequencies in index cases with triple (ER, PR, and ERBB2) negative breast cancers, preventing us to study whether the KRAS-variant frequency is increased specifically in BRCA1 carriers or more generally in patients with triple negative or basal-like breast cancer.

If the KRAS-variant were to be specifically associated with mutant BRCA1 breast cancer, this would suggest a biological interaction between BRCA1 and KRAS or between BRCA1 and let-7 miRNA. Many human cancers, including breast cancers, display altered expression of the let-7 miRNA family and let-7 has been shown to act as a tumor suppressor regulating the expression of RAS, HMGA2, LIN28, and PEBP1 [11, 18, 19]. Recently, let-7 was shown to regulate stem cell-like properties in breast tumor initiating cells as downregulation of let-7 increased proliferation and self-renewal, whereas expression of let-7 enhanced differentiation [20]. As the KRAS-variant allele was associated with lower let-7 levels in NSCLC patients as compared to patients without the variant allele, the KRAS-variant may therefore act in tumorigenesis through maintaining a proliferative cell state [13]. Interestingly, knockdown of BRCA1 in primary breast epithelial cells was shown to increase the number of cells displaying a stem/progenitor phenotype [21]. The presence of the KRAS-variant in the germline of a BRCA1 carrier may thus further facilitate this stem cell phenotype imposed by BRCA1. If the KRAS-variant would eventually be conclusively associated with mutant BRCA1 breast cancer, it might be interesting to study this hypothesis in future research.

miRNAs make up one of the most abundant classes of regulatory genes, with 30% of the human genes harboring miRNA target sites [22, 23]. Thus far, many miRNAs have already been implicated in cancer as either oncogenes or tumor suppressor genes [11, 18]. This makes both the miRNA genes themselves as well as the miRNA binding sites in the 3′UTR of well-known cancer genes attractive candidate breast cancer susceptibility alleles. Although several key targets of many miRNAs have been identified, also many target genes remain elusive. A significant portion of these miRNA target genes include well-known breast cancer oncogenes and tumor suppressor genes, such as ERBB2, CCND1, MYC, and PTEN, but also breast cancer susceptibility genes such as ATM [18, 24]. The KRAS-variant was the first single nucleotide variant in a miRNA binding site shown to be implicated in cancer risk, but many more are likely to be discovered. Screening of miRNA genes and their target sequences in the 3′UTR of genes known to be implicated in breast cancer may thus provide a productive strategy for defining part of the excess familial breast cancer risk.

Acknowledgments

We wish to thank all members of the breast cancer families that have participated in this study. We also thank Reneé Foekens, Bahar Özturk, Marc Kribbe, and Rahena van Kampen-Binda for assistance with collecting DNA samples. Financial support was provided by the Netherlands Genomics Initiative (NGI)/Netherlands Organization for Scientific Research (NWO), a K08 grant from the NIH [CA124484] and a CTSA Grant, Number UL1 RR024139 from the National Center for Research Resources (NCRR), a component of the National Institutes of Health (NIH), and NIH roadmap for Medical Research.

Footnotes

Conflict of interest Joanne B. Weidhaas has founded a company that has licensed IP discussed in this article.

Contributor Information

Antoinette Hollestelle, Email: a.hollestelle@erasmusmc.nl, Department of Medical Oncology, Josephine Nefkens Institute and Daniel den Hoed Cancer Center, Erasmus University Medical Center, P.O. Box 2040, 3000 CA Rotterdam, The Netherlands. Cancer Genomics Centre, Rotterdam, The Netherlands.

Cory Pelletier, Department of Genetics, Yale University, New Haven, USA. Department of Therapeutic Radiology, Yale University, 333 Cedar Street, New Haven, CT 06520, USA.

Maartje Hooning, Department of Medical Oncology, Josephine Nefkens Institute and Daniel den Hoed Cancer Center, Erasmus University Medical Center, P.O. Box 2040, 3000 CA Rotterdam, The Netherlands.

Ellen Crepin, Department of Medical Oncology, Josephine Nefkens Institute and Daniel den Hoed Cancer Center, Erasmus University Medical Center, P.O. Box 2040, 3000 CA Rotterdam, The Netherlands.

Mieke Schutte, Department of Medical Oncology, Josephine Nefkens Institute and Daniel den Hoed Cancer Center, Erasmus University Medical Center, P.O. Box 2040, 3000 CA Rotterdam, The Netherlands.

Maxime Look, Department of Medical Oncology, Josephine Nefkens Institute and Daniel den Hoed Cancer Center, Erasmus University Medical Center, P.O. Box 2040, 3000 CA Rotterdam, The Netherlands.

J. Margriet Collee, Department of Clinical Genetics, Erasmus University Medical Center, Rotterdam, The Netherlands.

Anja Nieuwlaat, Department of Clinical Genetics, Erasmus University Medical Center, Rotterdam, The Netherlands.

Lambert C. J. Dorssers, Department of Pathology, Josephine Nefkens Institute, Erasmus University Medical Center, Rotterdam, The Netherlands

Caroline Seynaeve, Department of Medical Oncology, Josephine Nefkens Institute and Daniel den Hoed Cancer Center, Erasmus University Medical Center, P.O. Box 2040, 3000 CA Rotterdam, The Netherlands.

Yurii S. Aulchenko, Department of Epidemiology, Erasmus University Medical Center, Rotterdam, The Netherlands

John W. M. Martens, Department of Medical Oncology, Josephine Nefkens Institute and Daniel den Hoed Cancer Center, Erasmus University Medical Center, P.O. Box 2040, 3000 CA Rotterdam, The Netherlands

Ans M. W. van den Ouweland, Department of Clinical Genetics, Erasmus University Medical Center, Rotterdam, The Netherlands

Joanne B. Weidhaas, Email: joanne.weidhaas@yale.edu, Department of Therapeutic Radiology, Yale University, 333 Cedar Street, New Haven, CT 06520, USA

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