Analysis of the genes coding for the BRCA1-interacting proteins, RAP80 and Abraxas (CCDC98), in high-risk, non-BRCA1/2, multiethnic breast cancer cases

Background Around half of familial breast cancer cases are caused by germ-line mutations in genes which are critically involved in the maintenance of genome stability. Mutations in related genes functioning in DNA repair may account for currently unattributed cases. Two such genes, RAP80 and Abraxas, have recently been identified to be in a complex with BRCA1, and are required for the localization of BRCA1 to DNA damage foci. Methods RAP80 and Abraxas variants were screened for in a cohort of 95 high risk, non-BRCA1/2 breast cancer cases of varying ethnicity: those of Ashkenazi Jewish (n = 35), mixed Canadian (n = 34) and Swiss descent (n = 26). Results We have identified four missense variants, four silent SNPs, three SNPs in the UTRs and seven intronic variants in RAP80. Two of the previously reported RAP80 variants were further investigated. In Abraxas, we have identified two missense, nine intronic and two variants in the 3′ UTR. Conclusions Overall, it seems unlikely that moderate to highly penetrant alleles of either RAP80 or Abraxas, confer a significantly high relative risk of breast cancer.     

The spectrum of ATM missense variants and their contribution to contralateral breast cancer

Heterozygous carriers of ATM mutations are at increased risk of breast cancer. In this case-control study, we evaluated the significance of germline ATM missense variants to the risk of contralateral breast cancer (CBC). We have determined the spectrum and frequency of ATM missense variants in 443 breast cancer patients diagnosed before age 50, including 247 patients who subsequently developed CBC. Twenty-one per cent of the women with unilateral breast cancer and 17% of the women with CBC had at least one ATM germline missense variant, indicating no significant difference in variant frequency between these two groups. We have found that carriers of an ATM missense mutation, who were treated with radiotherapy for the first breast tumour, developed their second tumour on average in a 92-month interval compared to a 136-month mean interval for those CBC patients who neither received RT nor carried a germline variant, (p = 0.029). Our results indicate that the presence of ATM variants does not have a major impact on the overall risk of CBC. However, the combination of RT and (certain) ATM missense variants seems to accelerate tumour development.     

RAP80 regulates epithelial–mesenchymal transition related with metastasis and malignancy of cancer

Epithelial–mesenchymal transition (EMT) has been closely related with invasive and metastatic properties of cancer. Recently, the convergence of DNA damage response and EMT in cancer development has received a great amount of scientific attention. Here, we showed that EMT is induced by the downregulation of RAP80, a well‐known regulator for DNA damage response. The knockdown of RAP80 leads to EMT‐like morphological changes and the increase of tumor sphere formation in non‐adhesive culture. Mechanistically, RAP80 controls a reciprocal regulatory axis of ZEB1 (for EMT activation) and miR200c (for EMT inhibition). The downregulation of RAP80 increases ZEB1 protein and decreases miR200c expression to activate EMT signaling in the form of drastic inhibitions of E‐cadherin, p16 and p21 expression. Using in vivo metastasis analysis, RAP80 knockdown cells are shown to dramatically metastasize into the lung and generate more malignant phenotype compared to controls. Interestingly, the expression level of RAP80 was positively correlated with the survival rate in lung adenocarcinoma and breast cancer patients. These findings indicate that RAP80 is a critical gatekeeper in impeding EMT‐induced metastasis and malignant phenotypes of cancer as well as preserving DNA integrity.     

Germline mutations in 40 cancer susceptibility genes among Chinese patients with high hereditary risk breast cancer

Multigene panel testing of breast cancer predisposition genes have been extensively conducted in Europe and America, which is relatively rare in Asia however. In this study, we assessed the frequency of germline mutations in 40 cancer predisposition genes, including BRCA1 and BRCA2, among a large cohort of Chinese patients with high hereditary risk of BC. From 2015 to 2016, consecutive BC patients from 26 centers of China with high hereditary risk were recruited (n = 937). Clinical information was collected and next-generation sequencing (NGS) was performed using blood samples of participants to identify germline mutations. In total, we acquired 223 patients with putative germline mutations, including 159 in BRCA1/2, 61 in 15 other BC susceptibility genes and 3 in both BRCA1/2 and non-BRCA1/2 gene. Major mutant non-BRCA1/2 genes were TP53 (n = 18), PALB2 (n = 11), CHEK2 (n = 6), ATM (n = 6) and BARD1 (n = 5). No factors predicted pathologic mutations in non-BRCA1/2 genes when treated as a whole. TP53 mutations were associated with HER-2 positive BC and younger age at diagnosis; and CHEK2 and PALB2 mutations were enriched in patients with luminal BC. Among high hereditary risk Chinese BC patients, 23.8% contained germline mutations, including 6.8% in non-BRCA1/2 genes. TP53 and PALB2 had a relatively high mutation rate (1.9 and 1.2%). Although no factors predicted for detrimental mutations in non-BRCA1/2 genes, some clinical features were associated with mutations of several particular genes.     

Familial breast cancer: double heterozygosity for BRCA1 and BRCA2 mutations with differing phenotypes

The co-existence of mutations in the BRCA1 and BRCA2 genes is unusual, and to date almost all cases reported have had at least one of the Ashkenazi founder mutations. We report on a family in whom individuals are double heterozygotes for a mutation in BRCA1 and a novel splice site mutation in BRCA2. The phenotypes are discordant, where one sister has had multiple cancers in the BRCA spectrum, while the other is unaffected at 65 years of age. The utility of testing is discussed, and the completion of diagnostic testing despite the finding of a potentially causal mutation is validated.     

Genetic susceptibility to breast cancer

Genetic and lifestyle/environmental factors are implicated in the aetiology of breast cancer. This review summarizes the current state of knowledge on rare high penetrance mutations, as well as moderate and low‐penetrance genetic variants implicated in breast cancer aetiology. We summarize recent discoveries from large collaborative efforts to combine data from candidate gene studies, and to conduct genome‐wide association studies (GWAS), primarily in breast cancers in the general population. These findings are compared with results from collaborative efforts aiming to identify genetic modifiers in BRCA1 and BRCA2 carriers. Breast cancer is a heterogeneous disease, and tumours from BRCA1 and BRCA2 carriers display distinct pathological characteristics when compared with tumours unselected for family history. The relationship between genetic variants and pathological subtypes of breast cancer, and the implication of discoveries of novel genetic variants to risk prediction in BRCA1/2 mutation carriers and in populations unselected for mutation carrier status, are discussed.     

Survival in familial and non-familial breast cancer by age and stage at diagnosis

We aimed to compare the survival in familial and sporadic breast cancer (BC) patients who were diagnosed at an identical age and TNM stage. The Nationwide Swedish Family-Cancer Database including all Swedes born after 1931 and their biological parents, totalling >14.7 million individuals, was used. Hazard ratios (HRs) were calculated for women with BC in a first-degree relative (FDR) versus BC patients without positive family history. There was no difference in survival of familial BC patients who were diagnosed at higher TNM status or older age (>40) compared to sporadic BC cases diagnosed at the same late TNM stage. Young BC patients (age <40) in early stages had the worst survival when their FDR was diagnosed with single (HR: 2.0–3.7) or multiple (HR: 2.4–7.1) BC at any age. We concluded that there is no difference in survival of familial and non-familial BC patients who are diagnosed at higher TNM status or older ages (>40). Young familial BC patients (age <40), diagnosed at early stage, have the poorer survival compared to sporadic cases. Our results urge the need for identifying the underling genetic component for such a difference in survival of familial BC.     

Novel BRCA1 and BRCA2 germline mutations and assessment of mutation spectrum and prevalence in Italian breast and/or ovarian cancer families

Familial aggregations of breast/ovarian cancer cases frequently depend on BRCA1/2 pathogenic mutations. Here we counselled 120 Italian breast/ovarian cancer families and selected 73 probands for BRCA1/2 mutation screening. Through this analysis we defined the prevalence of BRCA1/2 pathogenic mutations occurring in Italian breast/ovarian cancer families, enlarged the spectrum of Italian BRCA1/2 mutations by 15% and report on the identification of 13 novel variants, including two deleterious truncating mutations and two potentially pathogenic missense mutations, on the BRCA1 and BRCA2 genes. Finally in hereditary breast cancer families with three or more female breast cancer cases we observed a low mutation prevalence and a significant association with BRCA2 mutations.     

Germline mutations in the breast cancer susceptibility gene PTEN are rare in high-risk non-BRCA1/2 French Canadian breast cancer families

Cowden syndrome is a disease associated with an increase in breast cancer susceptibility. Alleles in PTEN and other breast cancer susceptibility genes would be responsible for ∼25% of the familial component of breast cancer risk, BRCA1 and BRCA2 being the two major genes responsible for this inherited risk. In order to evaluate the proportion of high-risk French Canadian non-BRCA1/BRCA2 breast/ovarian cancer families potentially harboring a PTEN germline mutation, the whole coding and flanking intronic sequences were analyzed in a series of 98 breast cancer cases. Although no germline mutation has been identified in the coding region, our study led to the identification of four intronic variants. Further investigations were performed to analyze the effect of these variants, alone and/or in combination, on splicing and PTEN protein levels. Despite suggestive evidence emerging from in silico analyses, the presence of these intronic variants do not seem to alter RNA splicing or PTEN protein levels. In addition, as loss of PTEN or part of it has been reported, Western blot analysis has also been performed. No major deletion could be identified in our cohort. Therefore, assuming a Poisson distribution for the frequency of deleterious mutation in our cohort, if the frequency of such deleterious mutation was 2%, we would have had a 90% or greater chance of observing at least one such mutation. These results suggest that PTEN germline mutations are rare and are unlikely to account for a significant proportion of familial breast cancer cases in the French Canadian population. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. Other members of INHERIT BRCAs involved in this study are listed in Appendix 1.     

Germline TP53 mutations in BRCA1 and BRCA2 mutation-negative French Canadian breast cancer families

About 40% of French Canadian breast and/or ovarian cancer families harbor germline BRCA1 or BRCA1 mutations where common mutations account for about 84% of all mutations identified in cancer families. Within a series of BRCA1 and BRCA2 mutation-negative families, a germline TP53 13398 G>A (Arg213Gln) mutation was identified, which was selected for mutation analysis in this gene because of a family history consistent with Li–Fraumeni syndrome (LFS). Given the founder effects in this population, the 13398 G>A mutation was screened in series of 52 BRCA1 and BRCA2 mutation-negative cancer families, and a mutation-positive family was identified. However, pedigree inspection and expansion of mutation-positive families with the same mutation revealed that they were closely related to each other. To further characterize the contribution of TP53 in cancer families, mutation analysis was performed in the remaining BRCA1 and BRCA2 mutation-negative cancer families. Thirty sequence variants were identified, the majority of which occur in intronic sequences and are not predicted to affect the functionality of TP53. However, the 14538 G>A (Arg290His) mutation was identified in a family which did not exhibit features consistent with LFS or Li–Fraumeni-like (LFL) syndrome. Neither of the TP53 mutations was detected in 381 French Canadian women with breast cancer diagnosed before 50 years of age not selected for family history of cancer. In all, germline TP53 mutations were identified in two of 52 (3.8%) cancer families, suggesting that TP53 is not a major contributor to BRCA1 and BRCA2 mutation-negative breast and/or ovarian cancer families of French Canadian descent.     

Prevalance of BRCA1 and BRCA2 mutations in familial breast cancer patients in Lebanon

Breast cancer is the most prevalent malignancy in women in Western countries, currently accounting for one third of all female cancers. Familial aggregation is thought to account for 5–10 % of all BC cases, and germline mutations in BRCA1 and BRCA2 account for less of the half of these inherited cases. In Lebanon, breast cancer represents the principal death-causing malignancy among women, with 50 % of the cases diagnosed before the age of 50 years.In order to study BRCA1/2 mutation spectra in the Lebanese population, 72 unrelated patients with a reported family history of breast and/or ovarian cancers or with an early onset breast cancer were tested. Fluorescent direct sequencing of the entire coding region and intronic sequences flanking each exon was performed.A total of 38 BRCA1 and 40 BRCA2 sequence variants were found. Seventeen of them were novel. Seven confirmed deleterious mutations were identified in 9 subjects providing a frequency of mutations of 12.5 %. Fifteen variants were considered of unknown clinical significance according to BIC and UMD-BRCA1/BRCA2 databases.In conclusion, this study represents the first evaluation of the deleterious and unclassified genetic variants in the BRCA1/2 genes found in a Lebanese population with a relatively high risk of breast cancer.     

BRCA1基因和RAP80 mRNA表达水平与胃癌铂类化疗预后的相关性研究

目的探讨肿瘤石蜡组织中BRCA1基因和RAP80 mRNA表达水平与接受铂类药物化疗的胃癌患者生存时间之间的关系。方法提取福尔马林固定-石蜡包埋胃癌组织RNA,采用逆转录聚合酶链反应（RT-PCR）检测BRCA1基因和RAP80 mRNA的表达水平,比较其表达水平与接受铂类化疗患者的临床病理与生存时间之间的关系。结果 74例胃癌患者中,BRCA1 mRNA低表达患者生存时间为19.3个月,高表达患者生存时间为13.3个月（P=0.17）。RAP80 mRNA低表达患者生存时间为18.2个月,高表达患者生存时间为10.9个月（P=0.07）。BRCA1和RAP80均低表达的患者生存时间为20.3个月,均高表达的患者生存时间为9.1个月（P=0.015）。结论 BRCA1和RAP80 mRNA的表达水平可以作为以铂类为基础药物化疗的胃癌患者的生存时间的预测分子,结论需更大样本的临床研究进一步验证。     

Tubulin, BRCA1, ERCC1, Abraxas, RAP80 mRNA expression, p53/p21 immunohistochemistry and clinical outcome in patients with advanced non small-cell lung cancer receiving first-line platinum-gemcitabine chemotherapy

Background

The aim of this study was to assess the predictive value of tumor expression of nine genes on clinical outcome in patients with advanced NSCLC receiving platinum-gemcitabine chemotherapy.

Methods

Quantitative PCR or immunohistochemistry were used to analyze the expression of β-tubuline IIA (TUBB2A), β-tubuline III (TUBB3), BRCA1, ERCC1, Abraxas (ABRX) and RAP80 in mRNA isolated from paraffin-embedded tumor biopsies of 45 NSCLC patients treated as part of a larger observational trial. All patients received first-line platinum-gemcitabine chemotherapy for stage IIIB or IV NSCLC.

Results

Median progression-free survival (PFS) was 7 months, overall survival (OS) 12 months. A partial treatment response was found in 14 patients (33%). Patients with low ERCC1 or ABRX expression had a significantly better response to chemotherapy (R = −0.45, p < 0.01 for ERCC1; R = −0.40, p = 0.016 for ABRX). A significant correlation was found between the individual time for PFS and the expression of both ERCC1 (R = −0.36, p = 0.015) and ABRX (R = −0.46, p = 0.001). Patients with low ERCC1 expression had a longer OS as compared to patients with high ERCC1 expression (HR = 0.26, log-rank p = 0.02).

Conclusions

The study confirms tumor expression of ERCC1 as a predictor for clinical outcome in patients with advanced NSCLC receiving platinum-based chemotherapy, and found ABRX expression to be similarly predictive of clinical outcome. Prospective validation is warranted and - if confirmed - non platinum-containing chemotherapy should be explored as the preferred treatment in patients with high ERCC1 or ABRX expression and no activating mutations of EGFR.     

Prognostic value of BRCA1 mutations in familial breast cancer patients affected by a second primary cancer

The aim of this prospective study is to assess the prognostic value of BRCA1 mutations in familial breast cancer patients affected by a second primary cancer. The study group comprised 19 women having multiple primary breast cancers (breast-breast, breast-other primary) who were either BRCA1 mutation carriers, or not. Appearance of a second primary cancer was recognised as the event and survival and second primary free cancer survival was calculated from the date of diagnosis to the secondary primary cancer. The results of this study show that the event free survival of women with familial breast cancer affected by a second primary cancer, who are BRCA1 mutation carriers is better, compared with women from the general population with breast cancer selected for second primary cancer sites and all second primary sites—P = 0.009 and P = 0.0078 respectively. In contrast, the event free survival of women with breast cancer affected by a second primary cancer, without a breast cancer family history, who are not BRCA1 mutation carriers is the same, as for women from the general population with breast cancer selected for second primary cancer sites and all second primary sites—P = 0.6417 and P = 0.4859 respectively. The median time from diagnosis of the first to second primary cancer in the mutation carrying, and non-carrying, groups was 8,7 and 1,9 years respectively. In the study group, the highest event free survival rates had been observed among those carrying the said mutations—66.7% at 5 years, and 33.3% at 10 years—in contrast with those not carrying the mutations, with rates of 30.8% and 15.4% respectively.     

RAP80 and RNF8, key players in the recruitment of repair proteins to DNA damage sites

Chromosomal double-strand breaks (DSBs) in eukaryotes provoke a rapid, extensive modification in chromatin flanking the breaks. The DNA damage response (DDR) coordinates activation of cell cycle checkpoints, apoptosis, and DNA repair networks, to ensure accurate repair and genomic integrity. The checkpoint kinase ATM plays a critical role in the initiation of DDR in response to DSBs. The early ATM-mediated phosphorylation of the histone variant H2AX proteins near DSBs leads to the subsequent binding of MDC1, which functions as a scaffold for the recruitment and assembly of many DDR mediators and effectors, including BRCA1. Recent studies have provided new insights into the mechanism by which BRCA1 and associated proteins are recruited to DNA damage foci and revealed key roles for the receptor-associated protein 80 (RAP80) and the E3 ligase RNF8 in this process. RAP80 is an ubiquitin-interaction motif (UIM) containing protein that is associated with a BRCA1/BARD1 complex through its interaction with CCDC98 (Abraxas). The UIMs of RAP80 are critical for targeting this protein complex to DSB sites. Additional studies revealed that after binding gamma-H2AX, ATM-phosphorylated MDC1 is recognized by the FHA domain of RNF8, which subsequently binds the E2 conjugating enzyme UBC13. This complex catalyzes K63-linked polyubiquitination of histones H2A and gamma-H2AX, which are then recognized by the UIMs of RAP80, thereby facilitating the recruitment of the BRCA1/BARD1/CCDC98/RAP80 protein complex to DSB sites. Depletion of RAP80 or RNF8 impairs the translocation of BRCA1 to DNA damage sites and results in defective cell cycle checkpoint control and DSB repair. In this review, we discuss this cascade of protein phosphorylation and ubiquitination and the role it plays in the control of cellular responses to genotoxic stress by regulating the interactions, localization, and function of DDR proteins.     

Germline mutations in E-cadherin do not explain association of hereditary prostate cancer, gastric cancer and breast cancer

Somatic mutations in the E-cadherin (CDH1) gene have frequently been reported in cases with diffuse gastric and lobular breast cancers. Recently, germline mutations have been identified in families with diffuse gastric cancers. In families with hereditary prostate cancer (HPC), a significant association of prostate cancer, gastric and/or breast cancer has been observed in epidemiological studies. The aim of this study was to investigate if germline mutations in CDH1 could explain the risk for cancer in HPC families with an excess of gastric and breast cancer. In total, 17 members from 13 HPC families and 3 members from 3 families with hereditary gastric cancer (HGC) were screened for germline CDH1 sequence alterations using PCR/Denaturing HPLC for initial screening of nucleotide variants followed by confirmatory direct sequencing analysis. The frequency of identified novel germline mutations were tested for in 136 cases with hereditary prostate cancer and 215 cases of sporadic prostate cancer with 422 age matched controls in an allelic discrimination assay. In total, 8 sequence variants were detected in 20 samples tested. In the HPC families, we found 2 missense mutations, A592T in exon 12 and a novel D777N in exon 15 and a mutation in intron 5, 687+92T>A. A previously known polymorphism in exon 13 and 3 sequence variations in introns and untranslated regions were also found, of which the significance is unknown. In HGC-023 with early onset diffuse gastric cancer a truncating mutation, R335X, was identified in exon 7. None of the missense mutations or 687+92T>A were found in the extended HPC material or in the sporadic prostate cancer cases with age-matched controls in the allelic discrimination assay. We found several germline mutations of unknown clinical significance in the CDH1 gene that probably do not explain the association of prostate, gastric and/or breast cancers in the HPC-families. Two missense mutations and a mutation in intron 5 were identified that do not influence the risk of hereditary or sporadic prostate cancer in general and are considered to be pedigree specific. In a family with hereditary gastric cancer of the diffuse type, we identified the first truncating germline mutation in a Scandinavian family.     

Germline alterations in the 53BP1 gene in breast and ovarian cancer families

The conserved TP53-binding protein 1 (53BP1) is a central mediator of the DNA damage checkpoint and appears to be one of the sensors of DNA double-strand breaks (DSBs). Improper processing of DSBs can result in loss or rearrangement of genetic information, leading to cell death or tumorigenesis. 53BP1 interacts with both TP53 and ATM, key proteins involved in the monitoring of genomic integrity and regulation of apoptosis. 53BP1 is also required for the formation of BRCA1 foci and the C-terminal part of these two proteins display significant homology. Based on its biological function, the 53BP1 gene is a good candidate for being involved in cancer susceptibility. Consequently, in the current study patients belonging to 126 breast and/or ovarian cancer families were screened for germline mutations in the entire coding region of the 53BP1 gene. A number of sequence variants were found, but none of them appeared to associate with cancer predisposition. To our knowledge this is the first comprehensive screening of 53BP1 mutations in familial breast and ovarian cancer cases.     

Non-founder BRCA1 mutations in Russian breast cancer patients

A few founder BRCA1 mutations (5382insC, 4154delA, 185delAG) account for up to 15% of high-risk (young-onset or familial or bilateral) breast cancer (BC) cases in Russia. The impact of non-founder BRCA1 mutations in this country is less studied; in particular, there are no reports analyzing gross rearrangements of this gene in the Russian patient series. We selected for the study 95 founder mutation negative high-risk BC cases. Combination of high-resolution melting (HRM) and sequencing revealed six presumably BC-associated alleles (2080delA, 4808C > G, 5214C > T, 5236G > A, 5460G > T, 5622C > T) and one variant of an unknown significance (4885G > A). The pathogenic role of the 5236G > A mutation leading to G1706E substitution was further confirmed by the loss of heterozygosity analysis of the corresponding tumor tissue. Multiplex ligation-dependent probe amplification (MLPA) revealed two additional BRCA1 heterozygotes, which carried BRCA1 deletions involving exons 1–2 and 3–7, respectively. Based on the results of this investigation and the review of prior Russian studies, three BRCA1 mutations (2080delA, 3819del5, 3875del4) were considered with respect to their possible founder effect and tested in the additional series of 210 high-risk BC patients; two BRCA heterozygotes (2080delA and 3819del5) were revealed. We conclude that the non-founder mutations constitute the minority of BRCA1 defects in Russia.     

219例中国汉族遗传性乳腺癌患者BRCA1和BRCA2突变的研究

  目的  分析中国新疆多民族地区的高风险遗传性乳腺癌BRCA1/2基因突变位点情况。  方法  以2009年1月到2010年12月新疆医科大学附属肿瘤医院收治的来自新疆地区的68例符合高风险遗传性乳腺癌标准的患者为研究对象,其中HBC 12例,HBOC 4例,E-BC 25例,BI-BC 10例,TNB 17例。通过外周静脉血提取基因组DNA,对BRCA1/2基因的全部编码序列进行扩增。用高效液相色谱分析（DHPLC）进行突变分析预筛,结果经DNA测序验证。  结果  BRCA1/2致病性突变在新疆地区高风险遗传性乳腺癌的突变率为8.8%（6/68）。其中BRCA1的突变率为4.4%（3/68）,BRCA2的突变率为4.4%（3/68）。不同民族之间BRCA1/2突变率无统计学差异。  结论  中国新疆多民族地区的高风险遗传性乳腺癌患者部分病例具有与内地汉族人群不同的BRCA基因突变谱。BRCA1 2073delA,BRCA2 6873del CTCC及BRCA2 9481del A可能是新疆遗传性乳腺癌特有的突变位点。     

Estrogen receptor alpha haplotypes and breast cancer risk in older Caucasian women

Life-long exposure to estrogen is an established risk factor for breast cancer development. The underlying mechanism has been suggested to be the binding of estrogen-to-estrogen receptors in mammary tissue, which in turn promotes the proliferation and differentiation of breast tissue. Polymorphisms and haplotypes in estrogen receptor alpha (ESR1) have been reportedly associated with breast cancer risk; however, the results are not fully consistent. In this study, we investigated breast cancer risk associated with genotypes and haplotypes resulting from four ESR1 single nucleotide polymorphisms (SNPs), rs746432, rs2234693, rs9340799, and rs1801132. Genotyping has been performed on 393 breast cancer cases and 790 randomly selected controls in 1,183 Caucasian women over age 65 from the Study of Osteoporotic Fractures (SOF). We observed an allelic protective effect for SNP rs9340799 with an estimated odds ratio (OR) of 0.82 (95% CI = 0.68–1.00; P = 0.04) after adjustment for age, BMI and hip BMD. A protective effect of this SNP has been reported before in several different studies. We did not replicate the previously reported C–C–A–G haplotype association to breast cancer—the C–C–A–G haplotype from these SNPs was rare in this study (estimated frequency below 0.001% in cases and controls). No other statistically significant associations were observed between ESR1 haplotypes from the same four SNPs and the risk of breast cancer in older Caucasian women.