Breast cancer (BC) is an important public health problem worldwide. In Brazil, breast cancer is the most frequently diagnosed tumor and the leading cause of cancer death in women. Hereditary cancer represents approximately 5 to 10 % of BC cases. Even outside the hereditary cancer context, the presence of polymorphisms acting as genetic modifiers may contribute to a better or worse prognosis. Not much is known about the hereditary BC epidemiology in Brazil or about the influence of polymorphisms on hereditary predisposition.BRCA1/2 genes; Group 2: women with variants of uncertain significance in BRCA1/2 and Group 3: women with no mutations in BRCA1/2.This study examined the role of five different polymorphisms in four groups of women with BC: Group 1: women with a germline mutation in the FGFR2 gene); rs3803662 (TNRC9); rs889312 (MAP3K1); rs3817198 (LSP1 gene); and rs13281615 (8q24). The analyses were performed using PCR amplification and bi-directional sequencing.The women included in groups 1, 2 and 3 were patients from the Department of Oncogenetics of the Barretos Cancer Hospital who had undergone genetic testing because of a clinical suspicion of hereditary predisposition syndrome. The constitutive DNA was analyzed for the presence of polymorphisms at rs2981582 .No differences were identified in the frequency of the polymorphisms that were analyzed among the three groups. However, some associations were identified, such as the occurrence of bilateral breast cancer and homozygosity for the G allele in rs13281615 as well as the correlation between the SNPs rs2981582 and rs13281615 and the number of cancer cases in the family. Regarding the G allele of rs13281615, we observed that the proportion of individuals who were homozygous for this allele increased with the number of generations affected by cancer, regardless of the group where the patients were included. Concerning the rs2981582 we could observe that individuals from group 1 and homozygous CC had fewer cancer  cases. Regarding the hormone receptors, we observed an increased frequency in C homozygotes (rs3803662) among estrogen receptor-negative individuals from groups 1 and 3. For rs2981582 (FGFR2), we observed an increased frequency of the T allele in women who were positive for the estrogen and progesterone receptors regardless of the The results presented here provide interesting data on the modifying effect of polymorphisms on a family history of cancer; this may be a variable to consider in the analysis of tumor diversity, and of the family history observed in families with hereditary breast cancer .The online version of this article (doi:10.1186/s13053-015-0042-1) contains supplementary material, which is available to authorized users. BRCA1 and BRCA2 are among the genes associated with hereditary breast cancer [BRCA1 gene show a lifetime cumulative risk (LCR) between 44 and 68 % of developing breast cancer until 70 years of age. Moreover, the LCR for ovarian cancer in these patients is also significantly higher and may reach 60 % by 70 years of age [BRCA2 gene, when altered, is responsible for approximately 30 to 40 % of all cases of hereditary breast cancer. The LCR for breast cancer in women harboring germline mutations in this gene is similar to the risk of carriers of germline mutations in BRCA1 (44 to 68 % until 70 years of age), whereas the risk of ovarian cancer ranges from 11 to 40 % [BRCA1/2 differ in terms of age at diagnosis, the number of family members affected, and tumor prognosis [In Brazil, breast neoplasms are the main cause of death by cancer among women . It is ct cancer . Women hs of age . The BRC to 40 % –8. Familrognosis . Currentrognosis –12.FGFR2, which encodes a tyrosine kinase receptor that acts in the development of the mammary glands; 2) rs3803662 (C > T), localized in a region that includes TNRC9; 3) rs889312 (A > C), localized in a region that includes MAP3K1 in addition to two putative genes, MGC33648 and MIER3; 4) rs3817198 (T > C) in gene LSP1; and 5) rs13281615 (A > G), which resides in a region that does not include any known gene (8q24) but where variants associated with an increased risk of prostate and colorectal cancer have been identified. The authors correlated the presence of the above mentioned polymorphisms with histopathological characteristics such as positivity or negativity for the estrogen and progesterone hormone receptors, stage of tumor development, nodes, size, histology and stage at diagnosis. Among the five SNPs mentioned above, three  were significantly associated with an increased risk of breast cancer in estrogen receptor-positive individuals. Women who were TT homozygous in SNP rs3803662 and who had estrogen-negative tumors exhibited a risk of developing breast cancer that was 1.28-fold (95 % CI = 1.13–1.45) higher than women who were homozygous for the wild-type allele (present in 53 % of the controls). Besides, Hunter and colleagues [In 2007, a large study was conducted by Easton and collaborators  involvinlleagues  identifiStudies involving rs3817198 have found that the least frequent allele (C) leads to an increase of approximately 10 % in the risk of breast cancer in Caucasian women; however, the same allele has a protective effect among women of African ascent , 16., and rs13281615 in women with breast cancer, with or without mutations in the BRCA1 and BRCA2 genes, and to correlate the frequency of the different polymorphisms with the family history of cancer in general and, more specifically with the history of breast and ovarian cancer and with the histopathological features of the tumors.Considering the factors described above and the possible modifying effect conferred by polymorphisms, the objective of this study was to evaluate the frequency of the polymorphisms rs2981582, rs3803662, rs889312, rs3817198BRCA1 and BRCA2 genes and, based on the results, were allocated to three different groups: a) Group 1: 51 women with a personal and family history of breast cancer with a pathogenic germline mutation in the BRCA1 and/or BRCA2 genes; b) Group 2: 53 women with a personal and family history of breast cancer with the presence of a variant of unknown clinical significance (VUS) identified in the BRCA1 and/or BRCA2 genes; c) Group 3: 100 women with a personal and family history of breast cancer without a pathogenic mutation and/or mutation of unknown clinical significance identified in the BRCA1 and/or BRCA2 genes.This project was approved by the Research Ethics Committee of the Barretos Cancer Hospital. All the participants in the study were treated at this institution and signed an informed consent form. The women from the oncogenetics department underwent genetic testing for mutations in the For the polymorphism analysis, DNA was extracted using the QIAamp Blood DNA Mini-Kit (Qiagen) according to the manufacturer's instructions. Genotyping was performed through PCR amplification using the Hot Start Taq enzyme (Qiagen). Following amplification, the products were purified with the ExoSap enzyme (USB products) and sequenced bidirectionally using the BigDye Terminator v3.1 Kit . Electrophoresis was run in the automated sequencer model 3500 .BRCA1 and BRCA2 genes and the subsequent separation of the participants into the three study groups, a multiplex PCR amplification of all coding exons of the BRCA1  and BRCA2  genes and their respective flanking intronic regions was performed, followed by bidirectional sequencing using two platforms (ABI 3500 XL sequencer) and a new generation sequencer . In addition, large rearrangements were investigated using the multiplex ligation-dependent probe amplification (MLPA) technique.For the analysis of mutations in the The program SPSS v.21.0 for Windows  was used for the statistical analysis. The categorical variables were described using absolute frequencies and relative percentage frequencies. The correlations were obtained using the Chi-square and Fisher’s exact tests. The level of significance adopted in all the tests was 5 %.BRCA1 and BRCA2 of all coding regions was performed for the 204 patients. Fifty-one had a pathogenic mutation identified (30 with BRCA1 mutations and 21 with a germline pathogenic mutation on BRCA2 gene), 53 had a VUS identified on BRCA1 and/or BRCA2 genes and 100 women were tested negative for both genes. All 204 women had a personal history of breast cancer (mean age at diagnosis = 38.4 years). In addition all participants had a positive family history of cancer, suggestive of Hereditary Breast and Ovarian Cancer Predisposition Syndrome . The main histological tumor types were invasive ductal carcinomas (91.8 %) and lobular carcinomas (3.9 %). In addition the great majority of the breast tumors (of the probands) were grade II, T1/T2, N0 and M0. Details about the family history of cancer and mutated gene can be found in Additional file Sequencing of FGFR2 , rs3803662 in gene TNRC9 , rs889312 in gene MAP3K1 , rs3817198 in gene LSP1  and rs13281615 at 8q24 .The following polymorphisms were considered in this study: rs2981582 in gene Table i) there was a higher frequency of TT homozygotes for rs3803662 in the groups comprising women without mutations in the BRCA1/BRCA2 genes; ii) regarding rs889312, an increase was observed in the frequency of heterozygous individuals among those without mutations or VUS in BRCA1/2; and iii) the Group3 (WT) had a relatively increased frequency of CC homozygotes for the SNP rs3817198 compared to groups 1 and 2.Although there were no marked differences in the frequency of the polymorphisms studied among the three groups, some findings are noteworthy: The analysis of the family history of cancer according to the different polymorphisms studied is detailed in Table When comparing the genotypes with the family history of cancer, some potential associations were identified, including the occurrence of bilateral breast cancer and homozygosity for the G allele in rs13281615 and “C” in rs889312 in which 85 % of women with bilateral breast cancer had at least one C allele . Likewise, 85 % of the women with bilateral breast cancer had at least one T allele in rs2981582. Furthermore, regarding allele G in rs13281615, we observed that the proportion of individuals who were homozygous for this allele increased with the number of generations affected by cancer. Moreover, we observed that in the case of rs3803662 and rs2981582, heterozygous TC individuals presented a higher number of cancer cases  and a higher number of breast cancer cases.BRCA1 and BRCA2 gene mutation status are found in Additional file Detailed data on the frequency of the different genotypes versus the family history of cancer with the women subgrouped according to their BRCA1 to those with mutations in BRCA2. Given the limited sample size of patients with a deleterious mutation identified in these genes, the group stratification per mutated gene limited the statistical analyses that were conducted. However, some trends could be observed, for example, all of the women with mutations in the BRCA2 gene with bilateral breast cancer were homozygous for the G allele in rs132281615, and among those with mutations in BRCA1 and with bilateral breast cancer, 86 % had at least one G allele. Moreover, in regard to rs3817198, all of the women with mutations in BRCA1 and with ovarian cancer had at least one T allele (with 88.9 % being TT homozygotes); however, the same pattern was not observed for the women with mutations in BRCA2 and a personal history of ovarian cancer (p = 0.006). To reach a conclusion on the association of these polymorphisms with the BRCA1/2 mutational status and with a family history of cancer, more patients with these characteristics must be genotyped.In addition, the frequency of the different genotypes was compared to the family history by separately considering the women with a mutation in The data correlating the genotypes of the five polymorphisms that were analyzed with the status of the hormone receptors  are shown in Table When the different polymorphisms were analyzed with the hormone receptors, we observed an increased frequency of heterozygotes for rs3803662 in individuals with estrogen receptor (ER)-positive tumors, in individuals with progesterone receptor (PR)- positive tumors and in individuals who were negative for the HER2 receptor. Regarding rs2981582, individuals with at least one T allele more frequently exhibited positivity for the three receptors . We also observed an increased frequency of the WT allele among ER-negative individuals. For the SNPs rs13281615, rs889312 and rs3817198, no association was found between their frequencies and the hormone receptors considered.FGFR2, we observed an association between group 2 (with VUS in the BRCA1/2 genes) with the ER in which the presence of TT homozygotes was not observed, and TC heterozygosity was present in 73.7 % of the ER- negative cases , with 50 % of individuals being AG heterozygotes vs. 25 % and 28.6 % of those from groups one and two respectively , in which 50.0 % of the ER-positive cases were heterozygous (TC) .An analysis of genotypes versus hormone receptors was conducted by separately considering the individuals with The association between the genotypes in the five SNPs considered and the age at diagnosis was evaluated. Although no significant association was identified, these data are shown in Additional file Genetic polymorphisms are being widely evaluated as modifiers of the genetic risk of disease by several research groups. A study conducted by Garcia-Closas and collaborators  evaluateBRCA1 and BRCA2 genes, women with VUS identified in one of those genes, and women WT for both genes. The genotype data from the five polymorphisms were then correlated with the family history of cancer and the histopathological features of the tumors, always considering a possible influence of the mutational status of BRCA1/2. A similar work was performed by Antoniou and collaborators in 2008,[FGFR2 (rs2981582), TNRC9 (rs3803662), and MAP3K1 (rs889312) SNPs in a sample of 10,358 mutation carriers from 23 studies.In this study, we compared the genotype distribution of the five polymorphisms that were previously reported in the literature to be associated with the risk of breast cancer , 17, 18  in 2008, where thTNRC9, there was an increased frequency of CC homozygotes among ER-negative individuals ; this increase in frequency among individuals positive for the hormone receptors (ER and PR) did not depend on the group to which the women belonged. The same association with the rs2981582 and positivity for estrogen and progesterone receptors was described by Garcia-Closas and collaborators in an analysis of the association among breast cancer risk, FGFR2 rs2981582 and clinical and pathological characteristics and they found that the effect of rs2981582 was stronger in ER/PR-positive patients [FGFR2 variants with high levels of endogenous sex hormones (FGFR2 is upregulated in ERα-positive breast tumors) [When we compared the genotypes of the polymorphisms analyzed with the pathological characteristics of the tumors, we noted that, in the case of the SNP rs3803662 in gene borators  in 2007)patients . Additio tumors) , especiaCASP8, TNRC9 and ESR1 genes. The authors also reported an association between survival and eight SNPs in six genes  and another SNP in a gene-free region in 8q24. For the SNPs in genes MMP7, TOX3 and MAP3K1, the survival effects did not depend on the known main clinical prognosis factors. The effect of the SNP in ESR1 on survival was more significant when only ER-positive tumors were evaluated. When the cases were stratified according to tumor features, it was observed that the SNPs in FGFR2 and TOX3 were associated with the disease. Finally, the study showed that several SNPs were associated with survival. In some cases, this association may occur because of an effect on the tumor characteristics that has an impact on prognosis; in other cases, the effect appears to be independent of these prognostic factors [There are several studies evaluating the effects of polymorphisms on the risk of cancer and their association with tumor features. According to the study published by Tapper and collaborators  in 2008, factors .TNRC9, FGFR2, MAP3K1, H19 and LSP1. The most strongly associated SNP is localized in gene FGFR2, which was overexpressed in 5-10 % of the cases of breast cancer. The SNP in gene TNRC9 showed a stronger association with breast cancer and appeared to be correlated with the presence of bone metastases and positivity for the estrogen receptor. The SNP rs889312 in gene MAP3K1 showed sensitivity only in individuals harboring mutations in BRCA2, and it was not associated with an increased risk in carriers of BRCA1 mutations. Several SNPs in LSP1 and H19 were most likely associated with the risk of developing cancer. Therefore, the study concluded that the identification of risk-modifying polymorphisms may lead to a better understanding of the biological mechanism of breast cancer, thus improving the prevention, detection and early treatment of the disease.Results published by Fanale and collaborators  in 2012 The results of the analyses of the presence and frequency of the five polymorphisms considered here were compared to the family history of cancer. Several associations were identified in that comparison, either with the number of cancer cases in the family (associated with the SNPs rs2981582 and rs13281615), with the presence of bilateral breast cancer (SNP rs13281615) or with the number of generations affected by cancer (SNP rs13281615). Moreover, regarding allele G of rs13281615, we observed that the proportion of individuals who were homozygous for this allele increased as the number of generations affected by cancer increased. Many of the findings described here corroborate data in the literature, such as the association found between SNP rs13281615 and a positive family history of cancer, which was a correlation previously reported by Gorodnova and collaborators in 2010 . BesidesFGFR2 and the average number of first- and second-degree relatives with breast and/or ovarian cancers (P = 0.05). The authors also found an association with the age at diagnosis, in which individuals who were heterozygous or homozygous for the least frequent allele for rs3803662 had a higher probability of having the cancer diagnosed at an age younger than 60 years.The effect of polymorphisms on the family history of cancer was also reported by Huijts and collaborators , who shoAn association with age at diagnosis was not observed in this study for any of the five polymorphisms considered. However, we should reinforce that these findings should be regarded with caution, given the relatively small size of our study, which is the main limitation of our study. For that reason the obtained data and the possible associations identified with the tumor pathological features and with the family history of cancer must be validated using a larger sample size. Nonetheless, if these associations are confirmed, we will have additional data to understand the epidemiology of hereditary and familial cancer in our population. In addition, the results contributed to the continued research efforts in the search for common variants for breast cancer that could help to explain the high variability observed among family history of families with hereditary susceptibility to breast cancer.BRCA1/BRCA2 has been identified, the influence of polymorphisms acting as genetic modifiers of cancer was observed, especially for the SNP rs13281615 whose presence the G allele was associated with the occurrence of bilateral breast cancer, as well as with an increased number of cancer cases in the family and with an increasing number of generations affected by cancer and, this association was, in general, independent of the mutational status of the BRCA1 or BRCA2 genes.Thus, although no significant association between the presence of the 5 analyzed polymorphisms and the occurrence of pathogenic mutation in Some potential associations were found between the five polymorphisms analyzed in this study previously reported in the literature as being associated and an increase in the risk of breast cancer, with an emphasis placed on the effect of these polymorphisms on the family history of cancer and on hormone receptor positivity/negativity. These findings must be interpreted with caution because of the limited sample size in this study. The data must be validated with a larger number of cases and, if confirmed, the clinical relevance of the associations identified and their applicability in medical practice must be considered carefully. However, the results presented here provide interesting information on the modifying effect of polymorphisms on the family history of cancer and may be a variable to consider in the analysis of tumor diversity, and of the family history observed in families with hereditary breast cancer .


Because caspase-8 is an important protein involved in receptor-mediated apoptosis whose activity is affected by estrogen, we hypothesized that additional SNPs in CASP8 could be associated with breast cancer risk, perhaps in a subtype-specific manner.The ability of tamoxifen and raloxifene to decrease breast cancer risk varies among different breast cancer subtypes. It is important to determine one’s subtype-specific breast cancer risk when considering chemoprevention. A number of single nucleotide polymorphisms (SNPs), including one in CASP8 were analyzed in a nested case control study  of non-Hispanic white women participating in the California Teachers Study. Odds ratios (ORs) and 95 % confidence intervals (CIs) were calculated for each SNP using all, estrogen receptor (ER)-positive, ER-negative, human epidermal growth factor receptor 2 (HER2)-positive, and HER2-negative breast cancers as separate outcomes.Twelve tagging SNPs of p < 0.0008), only rs2293554 was statistically significantly associated with HER2-positive breast cancer .Several SNPs were associated with all, ER-positive, and HER2-positive breast cancers; however, after correcting for multiple comparisons  contains supplementary material, which is available to authorized users. Breast cancer risk factors include a woman’s age, family history, reproductive and gynecologic factors, and lifestyle factors including alcohol consumption and lack of physical activity . When trBRCA1 and BRCA2 markedly increase one’s risk of developing breast cancer  [The 12 tagging SNPs included in this analysis were selected to capture all common linkage disequilibrium tagging SNPs [minor allele frequency (MAF) of at least 5 %], 20 kb upstream of the 5' untranslated region (UTR) and 10 kb downstream of the 3' UTR, in individuals of European ancestry with minimum pairwise ih.gov)] .CASP8 were included and genotyped. Samples with genotype call rates <90 % were excluded. Among the remaining samples, 160 randomly selected duplicates exhibited a genotype concordance rate of 99.9 %. Additional details were described previously  , and frociations , our stuCASP8, which involved >30,000 invasive breast tumors, showed that rs1045485 was most strongly related with the risk of PR-negative tumors [Our results for rs1045485 were not consistent with those from two meta-analyses, which reported inverse associations with breast cancer, with pooled ORs of 0.87 (95 % CI 0.83-0.92)  and 0.79e tumors , but an e tumors . BecauseCASP8 sites identified in this study are all intronic, it is possible that they may affect expression of the protein or RNA splicing, which may affect protein-protein interactions and other functions. rs6723097 and rs6736233 were found to have features consistent with involvement in gene transcription regulation according to the Variant Effect Predictor (VEP) on the Ensembl website (http://uswest.ensembl.org/Homo_sapiens/Tools/VEP) [http://gvs.gs.washington.edu/GVS144/). Also, rs1861270 is in strong LD with the SNP rs3769823, which is also a missense. Neither rs13006529 nor rs3769823 have been reported to be associated with breast cancer risk. The remaining SNPs on our panel are not in LD with other SNPs with known functions.While the polymorphic CASP8 SNP, rs2293554, is nominally statistically significantly associated with HER2-positive breast cancer risk in non-Hispanic white women, even after stringent correction for multiple comparisons. Other CASP8 SNPs were also associated with overall, ER-positive, and HER2-negative breast cancer risk but the associations were not statistically significant after correction. While our results should be validated in other cohorts with subtype-specific information, this study contributes to our understanding of CASP8 SNPs and subtype-specific breast cancer risk. The mechanistic and functional consequences of CASP8 SNPs in breast cancer development and their relevance in women of other racial/ethnic groups remain to be investigated.We conclude that the


MTHFR), methionine synthesis reductase (MTRR) and methionine synthase (MTR) genes and breast cancer risk. A case-control study of 570 consecutively recruited breast cancer cases and 576 controls was conducted in Guangzhou, China. Multifactor dimensionality reduction and logistic regression approach were used to evaluate gene-gene interaction. The covariates were chosen based on comparison of baseline characteristics of cases and controls. Folate intake was found to be inversely associated with breast cancer risk. The MTRRrs162036 GG genotype was associated with a decreased risk of breast cancer . Compared with the wild-type group (MTRRrs162036 AA with MTRrs1805087 AA) MTRRrs162036 AA with MTRrs1805087 GA + GG was associated with a decreased risk . With the combined MTHFRrs1801131 TT and MTHFRrs1801133 GG genotypes as a reference, MTHFRrs1801131 TT with MTHFRrs1801133 GA + AA was associated with a decreased risk  and MTHFRrs1801131 GT + GG with MTHFRrs1801133 GA + AA was associated with an increased risk . The joint impact of MTRRrs162036 and MTRrs1805087, MTHFRrs1801131 and MTHFRrs1801133, folate and MTHFRrs1801133 may contribute to breast cancer risk.This study aimed to examine the joint effects of folate intake, polymorphisms of 5,10- methylenetetrahydrofolate reductase ( MTHFR) is a key enzyme in folate metabolism, irreversibly catalyzing the conversion of 5,10-methylene tetrahydrofolate  to 5-methyltetrahydrofolate (5-methyl THF), the primary circulatory form of folate and a carbon donor for remethylation of homocysteine to methionine. The latter is the precursor for S-adenosylmethionine, which is the universal methyl donorMTR) and methionine synthesis reductase (MTRR), are critical enzymes responsible for the biosynthesis of methionine, the precursor for methylation reactions, and the regeneration of THF for nucleotide biosynthesisFolate in the one-carbon metabolism pathway can influence DNA synthesis, repair, and methylation, which played a critical role in carcinogenesisMTHFR and MTR polymorphisms and found that variant genotypes of MTHFRrs1801133 GA + AAMTHFRrs1801131GGMTRrs1805087 GAMTHFRrs1801131 or MTHFRrs1801133 and folate intake in breast cancer risk8Given the relevance of these mechanisms to carcinogenesis, dietary folate intake and genetic polymorphisms of related genes in the one-carbon metabolism pathway may be associated with the development of breast cancer67MTHFRrs1801131, MTHFRrs1801133, MTRRrs162036, MTRRrs1801394 and MTRrs1805087 genotype with breast cancer risk, and further investigate the joint effects of folate intake and one-carbon-metabolizing genetic polymorphisms on breast cancer risk among Chinese women.Our previous publicationThe sociodemographic and established breast cancer risk factors of cases and controls are shown in Ptrend < 0.01). No deviation from the Hardy-Weinberg equilibrium was observed in the controls. MTHFRrs1801131 had low linkage disequilibrium with MTHFRrs1801133 (r2 = 0.261) and MTRRrs1801394 had low linkage disequilibrium with MTRRrs162036 (r2 = 0.104). The frequencies of MTRRrs162036 AA, GA and GG genotypes were 63.2, 34.9 and 1.9% in cases and 64.4, 30.2 and 5.4% in controls, respectively. Genotypes of MTRRrs162036 were not equally distributed in breast cancer cases and controls (P = 0.003). With the MTRRrs162036 AA genotype as a reference, the GG genotype was associated with a decreased risk of breast cancer . There were no significant associations for other four SNPs.Data for the associations between dietary folate intake, genotypes of related enzymes and breast cancer risk are presented in MTRRrs162036, MTRrs1805087, MTHFRrs1801133 and MTHFRrs1801131. The interaction dendrogram showed that there were synergistic interactions between MTHFRrs1801131 and MTHFRrs1801133, and between MTRRrs162036 and MTRrs1805087 MTRRrs1801394 with a vertical line suggested an additive effect (e effect .The associations between four-locus genotype combinations and the risk of breast cancer are shown in . There wMTRRrs162036 and MTRrs1805087 in breast cancer (Pinteraction = 0.02). Likewise, there was an interaction between MTHFRrs1801131 and MTHFRrs1801133 in breast cancer risk (Pinteraction = 0.02). However, neither of the Bonferroni correction was statistically significant. When compared with the reference group (MTRRrs162036 AA with MTRrs1805087 AA genotypes), MTRRrs162036 AA with MTRrs1805087 GA + GG was associated with a decreased risk of breast cancer  and MTRRrs162036 GA + GG with MTRrs1805087 GA + GG was associated with an increased risk of breast cancer . With the combined MTHFRrs1801131 TT and MTHFRrs1801133 GG genotypes as a reference, MTHFRrs1801131 TT with MTHFRrs1801133 GA + AA was associated with a decreased the risk of breast cancer . Besides, MTHFRrs1801131 GT + GG with MTHFRrs1801133 GA + AA was associated with an increased risk of breast cancer . All of these ORs were borderline statistically significant.As shown in MTHFR, MTRR and MTR polymorphisms and dietary folate intake on breast cancer risk are shown in MTHFRrs1801133 polymorphism, even after the Bonferroni correction. (Pinteraction = 0.01 and Bonferroni Pinteraction = 0.05). Compared with women with MTHFRrs1801133 GG and folate intake less than 214.7 μg/d, those having the MTHFRrs1801133 GA + AA genotype and folate intake more than 214.7 μg/d was associated with a decreased risk of breast cancer . However, women having MTHFRrs1801133 GA + AA genotype and folate intake less than 214.7 μg/d was associated with an increased risk of breast cancer . No significant interaction was found between other genetic polymorphisms and folate consumption in breast cancer risk.The joint effects of Because household income and educational level were not well-balanced between cases and controls in the present study, a stratification analysis was conducted by these two socioeconomic factors. The results showed that no interaction was observed between socioeconomic status and folate intake and five SNPs (data not shown).MTRRrs162036 GG genotype and breast cancer risk. Both MDR approach and logistic regression analysis supported that there were gene-gene interactions between MTRRrs162036 and MTRrs1805087 and between MTHFRrs1801131 and MTHFRrs1801133 in breast cancer risk. Interaction was also observed between consumption of folate and MTHFRrs1801133.In this study, we confirmed that dietary folate intake was inversely associated with breast cancer risk. We observed an inverse association between the MTHFRrs1801131814MTHFRrs18011337111415MTRRrs1801394214MTRrs1805087MTHFRrs1801131, MTHFRrs1801133, MTRRrs1801394 and MTRrs1805087 genotypes, which was consistent with previous studies. Some studies showed that MTRRrs162036 GG polymorphism was not associated with the incidence of colorectal cancerMTRRrs162036 AA genotype as a reference. However, the present study, the first study assessing the relationship between MTRRrs162036 polymorphism and breast cancer risk, showed that the homozygous GG genotype was a protective factor for breast cancer. The possible mechanism might be that the function of MTRR is essential for providing methyl groups. It is highly likely that enzymatic variants due to functional polymorphisms may alter DNA methylation, which would greatly affect carcinogenesisIt has been suggested that genetic polymorphisms in folate metabolic enzyme genes could influence breast cancer riskMTRRrs162036, MTHFRrs1801131, MTHFRrs1801133 and MTRrs1805087 was the best four factor model with the maximum test balance accuracy and cross-validation consistency compared with other models. However, the other models were not likely to generalize to independent datasets due to the lower test balance accuracy and cross-validation consistency. Meanwhile, the interaction dendrogram showed that there were interactions between MTHFRrs1801131 and MTHFRrs1801133 and between MTRRrs162036 and MTRrs1805087. The logistic regression analysis further validated the results of MDR analysis. To the best of our knowledge, no previous studies have investigated the interactions between genetic polymorphisms in breast cancer risk using both MDR analysis and logistic regression analysis. However, in terms of bladder cancer, a case-control study conducted in America showed that applying these two methods might facilitate the identification of gene-gene interactionsAs more and more studies evaluate risk associated with multiple genes factors, it has become clear that traditional logistic regression analysis could not deal with the dimensionality problem very effectively and is not adequate for modeling complex multi-factor interactionsMTHFRrs1801131 and MTHFRrs1801133 by logistic regression analysis, but have yielded controversial results. Three studies2324MTHFRrs1801131 and MTHFRrs1801133 in breast cancer risk. On the other hand, two studies10MTHFRrs1801131 GG and/or MTHFRrs1801133 AA was associated with a decreased risk of breast cancer compared with compound wild type subjects, whereas three studies1127MTHFRrs1801131 GT + GG and MTHFRrs1801133 GA + AA in breast cancer risk. The mechanism might be that the combined effect of MTHFRrs1801131 GT + GG and MTHFRrs1801133 GA + AA have lowered MTHFR enzyme activity, elevated homocysteine, decreased plasma folate levels and increased the risk of cancerSome studies have analyzed the interaction between MTRRrs162036 and MTRrs1805087 in any cancer. Our study found that the combination of MTRRrs162036 GA + GG and MTRrs1805087 GA + GG was associated with an increased risk of breast cancer compared with MTRRrs162036 AA with MTRrs1805087 AA. Although the Bonferroni-corrected Pinteraction were above 0.05, Bonferroni method is often considered to be overly conservativeMTRrs1805087 affected the enzymatic activity of MTR and may affect DNA methylation. It was reported that MTRrs1805087 GA + GG was associated with both global genomic hypomethylation and low level of hypermethylated CpG islands within promoters of tumor suppressor genes in individuals with kinds of cancersMTRRrs162036 GA + GG might lead to the decreased activity of MTR because MTRR serves to maintain MTR in its active stateNo previous studies have examined the interaction between MTHFRrs1801133 polymorphism in breast cancer risk (P < 0.05). Our study also found an interaction between folate consumption and MTHFRrs1801133 polymorphism in breast cancer risk. MTHFRrs1801133 GA + AA genotype was associated with an increased risk of breast cancer among women with folate intake less than 214.7 μg/d and a decreased risk of breast cancer among women with folate intake more than 214.7 μg/d. It is well known that MTHFR is a key enzyme in folate metabolism, irreversibly catalyzing the conversion of 5,10-methylene THF to 5-methyl THF, the primary circulatory form of folate. The 5,10-methylene THF reduces the incorrect inclusion of uracil in the DNA, leading to double-strand breakages during excision of uracil and increased chromosome instability, which is a phenomenon usually associated to carcinogenesisMTHFRrs1801131, MTRRrs162036, MTRRrs1801394 and MTRrs1805087 in the risk of breast cancer.It is biologically plausible that folate-related gene–nutrient interactions might play a role in breast cancer risk. The studies in BrazilMTRR polymorphisms in modulating the susceptibility to breast cancer. Moreover, this is the first epidemiological study that investigated the interaction between genetic polymorphisms of related enzymes in breast cancer risk by MDR approach. Furthermore, a validated FFQ was used to assess the frequency and portion size of dietary folate intake. We had a wide range of potential confounders including dietary and non-dietary factors and were able to adjust for them in the analyses.The present study had some methodological strength. This is the first study simultaneously investigating the role of two Our study had some limitations. First, the results obtained in this study could be affected by sources of selection bias that commonly emerged in case-control study. However, the genotype frequencies of the SNPs among our controls were consistent with those derived from the Hardy-Weinberg equilibrium. Moreover, the high participation rate  also helped to minimize this bias. Second, recall bias is also of concern in case-control studies. Controls did not have a malignant problem and the recall of their past food intake might be different from the cases. To reduce this bias, we tried to interview the cases as soon as the diagnosis was made and food photographs were provided as visual aids. Third, misclassification in genotyping status is unavoidable. However, cases and controls were analyzed in the same plate by laboratory personnel who were blinded to case-control status and there was no discrepancy between repeated analyses. Moreover, the genotyping call rate for each SNP was 100%. Therefore, any influence of genotyping misclassification on the present findings was likely to be minimal. Fourth, it was difficult to interpret the MDR model. A consistent trend of high risk or low risk cells across a series of rows or columns may indicate that a particular locus has a main effectMTRRrs162036 GG genotype was associated with a decreased risk of breast cancer. Significant gene-gene interactions were found between MTRRrs162036 and MTRrs1805087, MTHFRrs1801131 and MTHFRrs1801133. And there was an interaction between folate intake and MTHFR rs1801133 gene variants in relation to risk for breast cancer. Breast cancer is a multi-factorial complex disease. More emphasis should be placed on the detection and characterization of multiple genetic and environmental interactions of breast cancer to predict high-risk individuals for the personalized prevention and treatment of breast cancer.In conclusion, our study showed that the This is an ongoing case-control study beginning in September 2011. Potential cases were recruited among patients who admitted to the surgical units of three teaching and general hospitals in Guangzhou from September 2011 to September 2014. Inclusion criteria were female subjects aged 25–70 years and natives of the province of Guangdong or having lived in Guangdong for at least 5 years, with incident, primary, histological confirmed breast cancer diagnosed no more than 3 months before the interview. Women were excluded if they could not understand or speak Mandarin/Cantonese or with prior history of breast cancer or other cancers. A total of 613 eligible cases were identified and 570 were interviewed, yielding a participation rate of 93%.Controls were patients with no history of cancer and admitted to the same hospitals during the same time period as the cases. They were frequency matched by age  to the case patients. They were selected from the departments of Ophthalmology, Plastic and Reconstructive Surgery, Vascular Surgery, Ear-Nose-Throat, and Orthopedics and Microsurgery. In total, 576 controls out of 613 eligible controls were successfully interviewed (94% participation rate).The present study was approved by the ethical committee of School of Public Health of Sun Yat-sen University. Written informed consent was obtained from all participants prior to the interview and the methods were carried out in accordance with the approved guidelines.e.g. active and passive smoking, alcohol drinking, and physical activity), menopausal status, reproductive history and family history of cancer. In this study, regular smokers were defined as someone smoking at least one cigarette a day for more than 6 consecutive months. Passive smoking meant to be exposed to others’ tobacco smoke for at least 5 min/day in the previous 5 years. Regular drinking was defined as alcohol drinking at least once per week in the past year. Postmenopausal status was defined as at least 12 months since the last menstrual cycle. Body mass index (BMI) was calculated by dividing weight (kg) by height squared (m2). The socioeconomic status (SES) was obtained by accumulating the total scores of education, income and occupation and divided into lower, middle and upper groups according to the manual of socioeconomic status scaleTrained interviewers conducted face-to-face interviews using a structured questionnaire to collect information on sociodemographic factors, body weight and height, lifestyle factors (Dietary information was obtained from an 81-item interviewer-administered food frequency questionnaire (FFQ) covering the habitual diet of participants during the previous year. Food photographs were used to help participants quantify the portions consumed. Information on frequency of intake and portion size was used to calculate the amount of each food item consumed on average (g/d). Daily nutrients intakes were estimated using the China Food Composition TableMTHFR, MTRR and MTR). We chose SNPs with an expected minor allele frequency >5% that showed previous evidence of associations with cancer risk or had possible functional significance. Genotyping of 5 polymorphisms in MTHFRrs1801131, MTHFRrs1801133, MTRRrs162036, MTRRrs1801394 and MTRrs1805087 was performed using a custom-by-design 48-Plex SNPscan Kit  as previously describedBlood samples were collected immediately after admission to the hospital for cases or after the interview for controls and were stored at −80 °C until analysis. Genomic DNA samples were extracted from the peripheral blood using a TIANamp Genomic DNA Kit  according to the manufacturer’s instructions. We selected five single nucleotide polymorphisms (SNPs) in three genes , 2 (Q2-Q3), 3 (Q3-Q4) and 4 (above Q4). When the intake was categorized into two groups, it was coded as 1 (below median) and 2 (above median). To calculate the P values for the trend of each SNP, the genotypes was regarded as a continuous variable after coding as 1 (wild-type), 2 (hybrid-type) and 3 (mutant-type)Dietary folate intake was adjusted for total energy intake using the residual methodHo) and expected heterozygosity (He) using exact tests implemented by Microsoft Excel. To increase statistical power, respective genotypes were combined into two groups when the polymorphisms were less frequent. GT and GG genotype groups of MTHFRrs1801131, GA and AA genotype groups of MTHFRrs1801133, GA and GG genotype groups of MTRRrs162036, GA and GG genotype groups of MTRRrs1801394 and GA and GG genotype groups of MTRrs1805087 were combined, respectively. Homozygous genotypes of MTHFRrs1801131 TT, MTHFRrs1801133 GG, MTRRrs162036 AA, MTRRrs1801394 AA and MTRrs1805087 AA were considered as the reference group. Linkage disequilibrium between the two MTHFR polymorphisms and the two MTRR polymorphisms were obtained with Haploview 4.2  and tight linkage disequilibrium was defined by r2 > 0.8Hardy-Weinberg equilibrium (HWE) was used to assess whether genotypes fell within a standard distribution. Deviation from the HWE in genotype frequencies was assessed with chi-square test. To test for deviation from HWE, comparisons were made between observed heterozygosity (e.g. linkage disequilibrium). Statistical significance of the model was evaluated using a 1000-fold permutation test. Meanwhile, logistic regression approach was used to evaluate the interaction between MTHFR, MTRR or MTR genotype in breast cancer risk. The combined effects of MTHFR, MTRR or MTR polymorphisms were assessed by adding the multiplicative interaction product (genotype × genotype) into the final model as an indicator variable. Similarly, the multiplicative interaction product [dietary folate intake values below and above the median (<214.7 versus ≥ 214.7 μg/d) × genotype] was added into the final model to identify the joint effects of dietary folate intake and each SNPs. Stratified analyses by socioeconomic factors  were also conducted. In this study, all P values are two sided and statistical significance was determined at the P value less than 0.05 level. The Bonferroni method was used to adjust the P value for multiple comparisons.Multi-factor Dimensionality Reduction (MDR) analysis was performed to evaluate the interactions between genetic polymorphisms and create the interaction dendrogram and graph by MDR 3.0.2 software. The software gives number of output parameters like cross-validation consistency, test balance accuracy for different interactions and single best model is identified as interaction that had maximum consistency and accuracy. In the interaction dendrogram, a black or diagonal line connecting two polymorphisms suggests a synergistic or nonadditive relationship while a vertical line indicates independence or additivity. A spotted or white line suggests a loss of information which can be interpreted as redundancy or correlation .


Women with an increased lifetime risk of ovarian cancer are advised to undergo risk-reducing salpingo-oophorectomy (RRSO) to reduce risk of adnexal cancer. We investigated the uptake of RRSO and evaluated the influence of personal medical history of (breast) cancer, risk-reducing mastectomy (RRM) and family history of ovarian and/or breast cancer on the RRSO decision. This single center retrospective observational cohort study was performed in a tertiary multidisciplinary clinic for hereditary cancer of the University Medical Centre Utrecht, the Netherlands. Women ≥35 years old with an estimated lifetime risk of ovarian cancer ≥10 %, who had completed childbearing, were eligible for RRSO. Uptake and timing of RRSO were analyzed. Influence of personal medical history and family history on RRSO decision making, were evaluated with logistic regression. The study population consisted of 218 women  with 87.2 % RRSO uptake. The median age at RRSO was 44.5 (range 28–73) years. Of the women undergoing RRSO, 78.3 % needed ≤3 consultations to reach this decision. Multivariable analysis showed a significant difference in RRSO uptake for women with a history of RRM [OR 3.66 95 % CI (1.12–11.98)], but no significant difference in women with a history of breast cancer [OR 1.38 95 % CI (0.50–3.79)], nor with a family history of ovarian and/or breast cancer [OR 1.10 95 % CI (0.44–2.76)]. We conclude that RRSO counseling, without the alternative of screening, is effective. The uptake is increased in women with a history of RRM. Women carrying a BRCA1 or BRCA2 gene mutation have a life time risk of developing ovarian cancer of 31–58.9 and 6–34.5 % respectively . A largeScreening for ovarian cancer by determining CA125 protein serum levels and performing transvaginal ultrasonography, has proven to be neither reliable, nor beneficial in reducing the risk of ovarian cancer, nor does it improve survival –19. TherThe Multidisciplinary Hereditary Cancer Clinic (MHCC) of the UMC Utrecht aims to counsel and treat women at increased lifetime risk of developing ovarian cancer. This encompasses BRCA1/2 mutation carriers and women with familial susceptibility to ovarian cancer, which is determined by having a pedigree-based estimated lifetime risk of ≥10 % . When ovReported RRSO uptake in the literature varies from 10.9 to 75 % –34. WithThere are demographic, medical and psychosocial factors that affect the decision making to undergo risk-reducing surgery, including age, parity, menopausal status, ethnicity, education level, idea of perceived risk, level of anxiety or distress, etcetera , 32–35. Here, we explore the effect of a personal medical history of breast cancer and risk-reducing mastectomy (RRM) as well as family history of ovarian and/or breast cancer in the decision making of undergoing RRSO for women with an increased lifetime risk of ovarian cancer. Secondly, we determined how many consultations were needed for women to decide to undergo RRSO. This insight is of importance, to provide personally appropriate counseling to every patient.In this single center retrospective cohort study, we identified all women who visited the MHCC from January 2011 through June 2013. Genetic testing and pedigree-based risk assessment were performed by the medical genetics department. Carriers of a BRCA1 mutation are explained to have a lifetime risk of ovarian cancer of 30–60 % and BRCA2 mutation carriers of 5–20 %. RRSO is recommended from age 35 years onwards (BRCA1 carrier) or from 40 years onwards (BRCA2 carrier). Women with a positive family history, but in whom genetic testing in an affected relative could not be performed, or could not identify a BRCA mutation (e.g. due to uninformative test result), were defined as women with a familial susceptibility to ovarian cancer. They are advised to undergo RRSO when the estimated lifetime risk is ≥10 %.In adherence to the Dutch guideline for treatment of hereditary ovarian cancer, women were considered eligible for RRSO with an estimated lifetime risk ≥10 % and if they were ≥35 years of age and had completed childbearing . They reThe study population consists of all women who received counseling for RRSO, based on the criteria described previously. Women who had completed childbearing at <35 years of age and had received counseling for RRSO were included too.Women previously diagnosed with ovarian cancer, carriers of a mutation in one of the MMR genes (predisposing to Lynch syndrome), women with increased risk of breast cancer or other non-breast and ovarian related hereditary tumors only, were excluded.U test was used for count data and continuous variables that were not normally distributed. A p value of <0.05 was considered statistically significant.Statistical analyses were carried out using the ‘Statistical Package for the Social Sciences’ version 21.0 . Comparison of categorical variables was performed by Chi square testing. The Mann–Whitney Uni- and multivariable logistic regression, producing odds ratios (OR), were used to model the explanatory variables against the binary RRSO decision. Explanatory variables of interest included the effect of personal medical history of breast cancer, any malignancy  and RRM. Furthermore, family history of ovarian and/or breast cancer were analyzed according to the first or second/third degree in which they occurred. Multiple imputations (m = 10) were used to correct randomly missing values.During the multivariable analysis, explanatory variables were adjusted for mutation status, age, parity and menopausal status, since these have been identified as significant factors of influence in previous studies , 32–34.We identified 218 women who were considered eligible for undergoing RRSO. As shown in Table In total, 190 women opted for RRSO, resulting in an uptake of 87.2 %. Uptake was higher in BRCA1/2 mutation carriers than in women with familial susceptibility to ovarian cancer (90.6 and 78.0 % respectively).Median age at RRSO was 44.5 (range 28–73). Median ages at RRSO for BRCA1 mutation carriers, BRCA2 mutation carriers and women with familial susceptibility were 42 (range 30–63), 47 (range 28–73) and 48 years (range 31–65) respectively.We registered the number of consultations women had had with the gynecologic oncologist before they decided to undergo RRSO. It showed that 57.4 % of the women who eventually underwent RRSO, had decided so at the first consultation with the gynecologic oncologist or the specialized nurse. An additional 11.1 % had decided so by the second consultation and an additional 5.3 % had made this decision by the third consultation. The remaining 26.2 % needed four or more consultations to make the decision.p 0.01) with 1.51 (95 % CI 0.32–2.70) less consultations before the RRSO decision.Women with 1st degree relatives who had breast cancer needed significantly less consultations to decide for RRSO, this in contrast to having 1st degree relatives with ovarian cancer. See Table Table Women who had previously been diagnosed with breast cancer or any other type of cancer , did not have significantly increased odds of undergoing a RRSO . Women who had undergone RRM were more likely to undergo RRSO, with an odds ratio of 3.66; 95 % CI (1.12–11.98) in multivariable analysis.No significant increased odds of undergoing a RRSO were identified if there was a first or a second/third degree relative with a history of ovarian cancer . This also accounted for women with a first or second/third degree relative with a history of breast cancer . Family history of ovarian and/or breast cancer evaluated altogether also showed no significant increased odds for undergoing RRSO .We show that RRSO uptake is high after counseling and that only few consultations at the gynecologist are needed, to decide to undergo RRSO.Secondly, women with a personal medical history of breast cancer do not seem to be more likely to undergo RRSO, than those without. This also accounts for women with a family history of ovarian and/or breast cancer.Reported RRSO uptake in the literature varies from 10.9 to 75 % –34. In clower in women with a medical history of breast cancer [It has been published that RRSO uptake is higher in women with a personal medical history of breast cancer , 27, 29.t cancer , 32.In several studies, RRM in personal medical history also seemed to have an influence, showing positive odds ratios in univariable analyses , 23, 28.Family history of ovarian and/or breast cancer were thought to be factors of influence, as some studies conclude , 30, 33,A limitation of this study is that information bias might have occurred because of the study design. Although not all possible factors of influence in the decision making of undergoing RRSO (i.e. demographic factors) could be evaluated, for lack of available information. Detailed information on family history was not present for every patient, but it was collected as thoroughly as possible by standardized questioning by the experts.Our findings are likely influenced by the method of counseling performed and particularly the fact that screening was not offered. Therefore, results of this study may not be extended towards situations where alternative strategies such as screening are offered. However, our results do indicate that a protocol based solely on RRSO counseling, without the alternative option of screening, is effective in establishing a high uptake of RRSO in women with an increased lifetime risk of ovarian cancer. This protocol can be useful in a variety of health-care systems given that most women who ultimately decide for RRSO needed 3 or less counseling sessions. We consider a multidisciplinary approach by a gynecologic oncologist, clinical geneticist and nurse specialist, specialized in familial cancer care, pivotal in this counseling practice.Future studies may focus on validating the current results, as well as investigating other possible factors of influence , possibly within the context of a qualitative study. Especially the subgroup who needed >3 consultations in the RRSO decision making process is of interest with respect to their considerations. Such an analysis may identify opportunities for improving and individualizing the counseling practice.In conclusion, the majority (87.2 %) of the women carrying a BRCA mutation or having familial susceptibility to ovarian cancer, who visited our clinic, where screening is not offered, opted for RRSO. This decision is made relatively quick; the majority decided after the first consultation with the gynecologist. This approach is therefore likely to be effective in reducing ovarian cancer related mortality in this high risk population. Personal medical history of (breast) cancer was not found to significantly affect the decision to undergo RRSO, though women who had undergone RRM were more likely to opt for RRSO. Also, patients with 1st degree relatives who had breast cancer needed significantly less consultations to decide for RRSO.


To our knowledge, the Alberta Moving Beyond Breast Cancer (AMBER) Study is the first and only prospective cohort study of breast cancer survivors that includes objectively-measured physical activity (PA), sedentary behavior, health-related fitness (HRF), and biologic mechanisms focused on understanding breast cancer outcomes. The purpose of the present study was to report on the feasibility of recruitment, baseline measurement completion, and the representativeness of the first 500 participants.AMBER is enrolling newly diagnosed stage I (≥T1c) to IIIc breast cancer survivors in Alberta, Canada. Baseline assessments are completed soon after diagnosis and include cardiorespiratory fitness, musculoskeletal fitness, body composition, objective and self-reported PA and sedentary behavior, lymphedema, and blood collection.Between July 2012 and November 2014, AMBER recruited its first 500 participants from a pool of 1,447 (35 %) eligible breast cancer survivors. Baseline HRF assessments were completed on ≥85 % of participants with the exception of upper body strength. Collection of ≥4 days/week of monitoring for the Actigraph GT3X® and ActivPAL® were obtained from 90 % of participants. Completion rates were also high for blood (99 %), lymphedema (98 %), and questionnaires (95 %) including patient-reported outcomes and correlates of exercise. The first 500 participants in AMBER are an average age of 56 years, 60 % are overweight or obese, and 58 % have disease stage II or III.Despite the modest recruitment rate and younger age, AMBER has demonstrated that many newly diagnosed breast cancer survivors are willing and able to complete a wide array of sophisticated and physically demanding HRF and PA assessments soon after diagnosis. AMBER is a unique breast cancer survivor cohort that may inform future randomized controlled trials on lifestyle and breast cancer outcomes as well as PA behavior change in breast cancer survivors. Moreover, AMBER may also inform guidelines on PA, sedentary behavior, and HRF for improving breast cancer outcomes and survivorship. Breast cancer is a major disease in Canada with 25,000 women expected to be diagnosed in 2015 and 5,100 expected to die from the disease [Unfortunately, surviving breast cancer often requires difficult and prolonged medical treatments that can last from several months to many years. Multimodal therapy for breast cancer often includes surgery, radiation therapy, chemotherapy, hormone therapy, and/or biologic therapy. Not surprisingly, breast cancer and its treatments have a negative effect on the health and well-being of breast cancer survivors, especially the physical and functional aspects of quality of life . For exaPhysical activity (PA) and health-related fitness (HRF) improve many outcomes for breast cancer survivors, however, limited research has focused on recurrence and survival. A recent systematic review  identifiDespite these promising findings, these studies are limited because few were originally designed as breast cancer survivor cohorts and none have focused on PA and HRF. Consequently, these studies have relied on basic self-reported measures of PA and simple self-reported or objective measures of height and weight. The studies lack objective measures of PA and sedentary behavior, objective assessments of key HRF parameters , biomarkers, and/or standardized assessment time points.We are currently conducting the Alberta Moving Beyond Breast Cancer (AMBER) Study which, to our knowledge, is the first and only prospective cohort study designed specifically to examine the role of PA, sedentary behavior, and HRF in breast cancer survivorship from the time of diagnosis and for the balance of life . The oveAMBER was approved by the Health Research Ethics Board of Alberta: Cancer Committee and all participants provide written informed consent. AMBER is a prospective cohort study of newly diagnosed breast cancer survivors in Alberta, Canada. Assessments are made at baseline, 1, 3, and 5 years follow-up and include clinic-based and patient-reported measures. The goal is to complete all baseline assessments before any neoadjuvant or adjuvant therapy, however, women may complete baseline assessments after receiving up to 1 cycle of chemotherapy or 2 weeks (10 fractions) of radiation therapy. For women receiving surgery as their first treatment, baseline assessments are generally completed within 90 days of surgery. The baseline blood draw is generally performed pre-surgically in Calgary because of an existing biospecimen banking program and post-surgically in Edmonton.Eligibility for AMBER includes: (1) histologically-confirmed stage 1 (≥T1c) to stage IIIc breast cancer, (2) females between 18 and 80 years old, (3) completion of the revised Physical Activity Readiness Questionnaire for Everyone (rPAR-Q+)  and the In Calgary, Alberta Health Services has developed the comprehensive biospecimen rapid ascertainment method (CoBRA) in conjunction with the Alberta Cancer Research Biobank (ACRB). This method uses a population-based sampling approach to identify all breast cancer cases prior to surgery. All newly diagnosed women are contacted by the ACRB to provide a pre-surgical blood and tumour tissue sample. If consent is obtained, their blood and tissue samples are stored for future research purposes. At the same time, they are asked if they consent to be contacted about future research. If this consent is obtained, an AMBER recruiter contacts the individual by telephone to recruit them prior to their treatment consultation. Those women who agree to participate are emailed a letter of invitation, information brochure, and consent form. The rPAR-Q+ is then administered by telephone by a Canadian Society for Exercise Physiology Certified Exercise Physiologist® (CSEP-CEP) prior to fitness testing.In Edmonton, breast cancer survivors are recruited in person from the Cross Cancer Institute. All newly diagnosed breast cancer survivors are reviewed for AMBER eligibility during the New Patient Breast Clinic. If eligible, they are approached about AMBER by their treating oncologist during their treatment consultation. If interested, an AMBER recruiter then meets with prospective participants to explain the study and provide them with an information brochure. Participation is confirmed during a follow-up telephone call and the consent form and rPAR-Q+ are completed during the first day of testing.Edmonton participants are assessed at the Cross Cancer Institute and Behavioral Medicine Fitness Center at the University of Alberta. Calgary participants are assessed at the Human Performance Laboratory and the REACH Center in the Faculty of Kinesiology at the University of Calgary. The assessments are scheduled for 1 or 2 clinic visits depending on participant preference and logistical issues. Single day clinic visits are split into morning and afternoon assessments to avoid undue fatigue. The assessments include: (1) questionnaires, (2) completion of HRF testing including DXA scans, (3) lymphedema/upper body function measurements, (4) blood draw, and (5) training in the use of the accelerometers and activity monitor logs to complete a week-long assessment of objective PA and sedentary behavior. The measures in the AMBER Study have been described in detail previously  and are The baseline blood draw is completed after an overnight fast of at least eight hours although unfasted bloods are permitted in some circumstances. A 60 ml sample is taken at baseline and 30 ml samples at 1 and 3 years follow-up. The samples are processed with 5 aliquots (2 buffy coat and 3 serum) stored for the ACRB and the remaining aliquots available for our study for the biomarker assays. For this study, we are storing 20 aliquots per person per blood draw . A complete blood collection, processing, shipping and storage protocol has been developed to ensure standardization of the procedures for the bloods at the collection sites in Calgary and Edmonton. The aliquoted blood samples are stored in our–86 °C freezers and long-term storage is in Calgary.Baseline Health Questionnaire which includes demographic characteristics, menopausal status, menstrual and reproductive history, exogenous hormone use history, personal health history, medication history, vitamin and supplement history, family history of cancer, and smoking and alcohol drinking histories; (2) the Past Year Physical Activity Questionnaire [Diet History Questionnaire [General Health Questionnaire that includes health-related quality of life, symptoms, psychosocial outcomes, the theoretical determinants of PA, and a sedentary behavior questionnaire.Participants complete 4 questionnaire packages consisting of: (1) a ionnaire , (3) theionnaire , and (4)PA is measured objectively using the Actigraph GT3X® . This small and light weight device is a highly sensitive instrument that records acceleration using a tri-axial accelerometer. Participants wear the monitor on their right hip attached by an elastic belt during all waking hours for 7 consecutive days. Participants also record the time they put on and take off the monitor each day. The participants are given these accelerometers after their HRF assessments and are trained in their use as well as in the use of the daily recording log.Sitting time is assessed by the activPAL® inclinometer . The activPal® classifies free-living activity into time spent sitting, standing and walking. This information can be used to estimate daily energy expenditure and changes in the free-living activity profile. The activPAL® is worn on the anterior thigh. It provides accurate information about posture  and transitions between postures as well as raw accelerometer count information . For the2peak and recovery heart rate); and upper and lower body muscular strength and endurance (chest and leg press predicted 1 repetition maximum and multiple repetition maximum). Adequate recovery time, nutrition and hydration is provided between tests. The upper and lower body strength measures are performed either on the same day after approximately 2 h of full recovery or on a separate day. The total HRF assessment time is approximately 2.5 to 3 h split over the morning and afternoon or over 2 separate days.The HRF assessments are performed by CSEP-CEPs using standardized testing protocols and the same equipment at both sites. Complete description of the HRF assessment protocols has been previously published . The assArm volume is measured objectively using the Perometer® . The measurement of shoulder range of motion includes active and passive shoulder movements and measurements of forward flexion, abduction, internal rotation, external rotation and horizontal abduction movements. Arm function is assessed using the Disabilities of the Arm, Shoulder and Hand scale (DASH) [Lymphedema is assessed both by self-report and by clinical examination. e (DASH) . The pren = 1,960; 37 %), incorrect disease stage , and medical/age issues . Of the 1,447 eligible, 500 (35 %) were recruited to the study and completed at least some baseline assessments. The most common reasons for refusal were being overwhelmed/too sick , not interested , and out of town/distance . Quarterly accrual to the study overall and by center is depicted in Fig. Flow of the first 500 participants through the baseline assessment in AMBER is presented in Fig. sick n = 97; 21%, 2max. The primary reasons for not completing the cardiorespiratory fitness test were safety issues such as the presence of cardiovascular risk factors  or musculoskeletal problems such as knee pain or ankle injury . All other secondary HRF exposures were completed by over 85 % of participants with the exception of upper body strength and endurance. The HRF assessment completion rates were comparable between the 2 centers with the exception of the muscular strength and endurance measures, which were completed at a lower rate in Calgary.Table The baseline assessment completion rates for lymphedema, upper arm function, peripheral neuropathy, and blood collection were over 98 % and were comparable between centers. The relative proportion of blood draws that were performed pre-surgically versus post-surgically differed by center because of the difference in recruitment methods used within each center. The questionnaires were completed by approximately 95 % of participants. Useable data of ≥4 days/week of monitoring for the Actigraph GT3X® and activPAL® were obtained from approximately 90 % of participants with slightly lower rates in Calgary.Table AMBER is a unique breast cancer survivor cohort study focused on the role of PA, sedentary behavior, and HRF in breast cancer survivorship. The eligibility rate for AMBER has been 27 %, largely because of challenges in screening women through the ACRB in Calgary, restrictions on disease stage, and medical/age issues. Some of the challenges of screening through the ACRB in Calgary include women declining to donate blood, opting out of being contacted for future research, or not giving consent for their blood to be used. AMBER is benefiting from an existing infrastructure in Calgary for obtaining pre-surgical blood and tissue samples, however, this infrastructure also restricts the number of eligible participants since only women who have consented to be contacted for future research after they have donated a blood sample are eligible for AMBER. Large numbers of women were also ineligible because of our disease stage restriction to ≥ T1c, however, we felt this restriction was necessary given our focus on recurrence and survival. Finally, women in poor health or over the age of 80 were often ineligible because of the requirement of maximal fitness testing. The only change in eligibility we have made in AMBER since its inception is to remove the restriction of a previous cancer.The first 500 participants in AMBER were recruited over a 29 month period with a stable accrual rate of approximately 20/month. The overall recruitment rate is 35 %, which is comparable to other breast cancer survivor cohort studies focused on lifestyle, health, and cancer outcomes. For example, the Pathways Study  recruiteThe baseline assessment completion rates in AMBER are excellent given the nature and timing of the assessments. For our primary exposure of cardiorespiratory fitness, we have evaluable data on 86.2 % of participants. The main reasons for missed or incomplete cardiorespiratory testing are safety/medical issues and musculoskeletal injuries, which are difficult to circumvent. All other HRF assessments were completed by over 85 % of participants with the exception of upper body strength and endurance. Body composition assessed by DXA scan has been completed by over 98 % of participants, highlighting the differences between passive  versus physically demanding  assessments. We are unaware of any other breast cancer survivor cohort studies that are including HRF assessments beyond BMI and waist-hip circumference. In AMBER, we have obtained over 98 % baseline completion for BMI compared to 92 % in Pathways , 100% inAs noted, the lowest baseline assessment completion rate is for upper body strength and endurance, which was completed by just under 80 %. The main reason for the missed assessments was safety issues  based on the primary surgery and/or immediate reconstructive surgery. These restrictions occurred more often in Calgary than Edmonton because of the different recruitment protocols in the 2 centers. Specifically, recruitment and testing was occurring earlier in Calgary, resulting in less recovery time after surgery. To standardize the timing of the baseline HRF assessments between the 2 centers, the protocol has been modified to stipulate that strength testing should occur at least 6 weeks after surgery.The baseline assessment completion rates for lymphedema, upper arm function, peripheral neuropathy, and blood draws were all over 98 % and did not differ by center. Few breast cancer survivor cohort studies have even attempted to collect such data. Pathways  collecteIn terms of accelerometry, we have obtained useable data of ≥4 days/week of monitoring for the Actigraph GT3X® and activPAL® from approximately 90 % of participants with slightly lower rates in Calgary. We are unaware of any large population-based cancer survivor cohort studies that have attempted to collect objectively assessed PA and sedentary behavior (including sitting time), however, our rate of useable data ≥4 days/week compares favorably to the 80-85 % reported for women over 40 years old in the National Health and Nutritional Examination Survey . The colWe can compare our sample to the broader breast cancer survivor population in Alberta and to other breast cancer survivor cohorts in the United States. Fisher et al.  reportedAlthough not exactly representative of the stage distribution of breast cancer survivors in Alberta, the stage distribution in AMBER compares favorably to other breast cancer survivor cohorts in the United States. For example, LACE  includesIn terms of the age distribution, the Alberta Cancer Registry data  showed tObesity is emerging as a critical issue in cancer treatment and care . AMBER iAMBER has many important strengths but also some emerging limitations. The strengths of AMBER include the prospective cohort design; restriction to ≥ stage 1 (T1c) breast cancer; recruitment soon after diagnosis; the comprehensive objective assessments of PA, sedentary behavior, and HRF using gold standard measures; the multiple standardized assessment time points; and the high rate of completion for many baseline assessments. The emerging limitations of AMBER include the modest eligibility and recruitment rates; the lack of data on those women who did not consent for research with the tumor bank or provide a presurgical blood sample; the lack of data on those women who refused participation in AMBER; differences between the 2 centers in the timing and completion rates of some baseline assessments; and the under-representation of older and stage III breast cancer survivors. Efforts are ongoing to improve the eligibility and recruitment rates, standardize the timing and completion rates of the baseline assessments between centers, and recruit larger proportions of older and advanced stage breast cancer survivors.Despite some emerging limitations, AMBER is establishing a cohort that will provide the most comprehensive and rigorous evaluation of the role of PA, sedentary behavior, and HRF in breast cancer survivorship. Once completed, AMBER will allow for a sophisticated and detailed analyses of the outcomes, determinants, biologic mechanisms, and moderators of PA, sedentary behavior, and HRF for breast cancer outcomes and survivorship. The data generated may inform phase III randomized controlled trials on lifestyle and breast cancer outcomes; and may also inform PA, sedentary behavior, and HRF guidelines to optimize breast cancer outcomes and quality of life.2max, maximal oxygen consumption; VO2peak, peak oxygen consumption.ACRB, Alberta Cancer Research Biobank; AMBER, The Alberta Moving Beyond Breast Cancer Study; BMI, body mass index; CoBRA, comprehensive biospecimen rapid ascertainment method; CSEP-CEP, Canadian Society for Exercise Physiology Certified Exercise Physiologist®; DASH, Disabilities of the Arm, Shoulder and Hand scale; DXA, dual x-ray absorptiometry; ePARmed-X+, electronic Physical Activity Readiness Medical Examination questionnaire; HEAL, the Health, Eating, Activity and Lifestyle Study; HRF, heath-related fitness; LACE, the Life After Cancer Epidemiology Study; PA, physical activity; rPAR-Q+, revised Physical Activity Readiness Questionnaire for Everyone; VO


Increased survival rates among breast cancer patients have drawn significant attention to consequences of both the presence of cancer, and the subsequent treatment-related impact on the brain. The incidence of breast cancer and the effects of treatment often result in alterations in the microstructure of white matter and impaired cognitive functioning. However, physical activity is proving to be a successful modifiable lifestyle factor in many studies that could prove beneficial to breast cancer survivors. This study investigates the link between white matter lesion volume, moderate physical activity, and cognition in breast cancer survivors following treatment compared to non-cancer age-matched controls. Results revealed that brain structure significantly predicted cognitive function via mediation of physical activity in breast cancer survivors. Overall, the study provided preliminary evidence suggesting moderate physical activity may help reduce the treatment related risks associated with breast cancer, including changes to WM integrity and cognitive impairment. Breast cancer is the leading cancer type in women; in 2015, 29% of new cancer cases in the United States are estimated to be breast cancer related . AlthougAlthough some studies find no differences following breast cancer treatment , many reDe Ruiter and colleagues reported cognitive impairments, reduced WM integrity, increased axonal injury and reduced gray matter (GM) volume in a small group of C+ patients when compared to C- group up to 10 years after treatment . They suThere are well-documented changes in brain structure and function with aging –34. GiveWhile breast cancer may result in accelerated aging of the brain, and aging brains benefit greatly from increased PA, considerably less is understood about the link between PA, cognition and breast cancer. PA levels often change after a diagnosis of breast cancer; with up to an 11% decrease in PA within a year of diagnosis, though these decreases are considerably higher in women treated with radiation and chemotherapy . The danWe were interested in examining the role that PA plays in the structure and function of the brain following treatment for breast cancer. Our primary objectives were: (a) to determine whether BCS and non-cancer age-matched controls differed in memory recall, WM lesion volume and PA and (b) to examine the relations among these measures and the extent to which PA mediated the relations between WM lesion volume and memory recall.BCS were primarily recruited through an oncology clinic, though some were recruited, along with the non-cancer age-matched controls, through local advertisements. After expressing initial interest, women were contacted by phone and provided a full study description. During the initial contact, interested individuals completed a demographics questionnaire and a personal medical history questionnaire, including self-reported information on breast cancer diagnosis and treatment history (BCS only). Of the 141 total contacts, 73 consented, and 11 women withdrew after consenting owing to schedule conflicts (n = 2), no longer interested (n = 3), or unable to contact (n = 6). Furthermore, some women only underwent surgery (n = 3) and were excluded from these analyses, and an additional participant was identified as an outlier (see below). This resulted in a final sample of 58. Participants were female BCS who had undergone surgery for breast cancer and had completed primary treatment  within the past 3 years (n = 30), or age-matched controls with no prior diagnosis of cancer (n = 28).nd Edition (MMSE-2), and able to walk on a treadmill unaided. All participants provided written informed consent and were required to provide written consent from a primary care physician or oncologist indicating they were cleared to participate in the cardiorespiratory fitness, magnetic resonance imaging (MRI) and cognitive testing sessions. Participants completed an initial visit for cardiorespiratory fitness assessment and 7 days of accelerometer monitoring. During a second visit participants completed cognitive testing followed by a third visit to complete a series of MRI scans. The study was approved by the University of Illinois Urbana Champaign Institutional Review Board.Given the likely interaction between time since cessation of therapy and age at onset of disease, we recruited BCS within 3 years of completion of treatment and aged between 18–70 years. Inclusion criteria for all included English speaking, normal/corrected to normal vision, no current use of brain training games , no history of stroke, transient ischemic attack, or surgery that involved removal of brain tissue, not currently pregnant, a score of ≥ 26 on the modified Mini-Mental Status Exam– 2Physical activity (PA) was determined using 7-day accelerometer monitoring . Participants were asked to wear the accelerometer during waking hours and wear time was recorded on an accelerometer log. Data were downloaded and digitally converted to “activity counts” per minute , and processed using MeterPlus 4.2 software . Only days with at least 10 valid hours of wear time were included in the analysis, and hours with greater than 60 min of consecutive zeros were considered invalid . Activity counts were summed and averaged across the total number of valid days for a total daily activity score. Average daily moderate PA was calculated by summing the total valid moderate activity counts, then dividing by the number of valid days.2.Height and weight were measured using a Seca electronic scale and stadiometer . Participants were measured while wearing light clothing and without shoes. BMI was calculated using the standard formula of weight (kg) / height (m)The standard version of the MMSE-2 which allows for a brief standardized assessment of cognitive status was used to screen for signs of cognitive impairment . It examThe expanded version of the MMSE-2 includes an immediate story recall–participants were read a short story aloud and asked to recall the story using the same words. There are 25 elements to the story, and a credit was given for each one successfully recalled. The story memory was designed to evaluate verbal explicit learning and verbal free recall .All images were acquired during a single session on a 3 T Siemens Trio Tim system . High-resolution structural MR scans were acquired using a 3D magnetization prepared rapid acquisition gradient echo (MPRAGE) T1-weighted sequence . While the lesion volumes were acquired using a 3D, variable flip angle turbo spin echo sequence with fluid-attenuated inversion recovery (FLAIR) .We used previously developed open-source software based on the image processing toolkit FSL  Software library) to perform automated computation of lesion masks and volumes ,59. We aAll analyses were performed using IBM SPSS version 22 , and data is provided in Given the small sample size, analyses were conducted across all BCS in comparison to non-cancer age-matched controls. To investigate these differences multivariate ANCOVAs were conducted on background demographics, lesion volume, moderate PA and story memory recall. Effects sizes, partial Eta squared, were calculated and reported. We explored associations between lesion volume, PA and story memory using partial correlations, controlling for age and years of education. Further analyses in the BCS group also included time since cessation of treatment.Where significant relationships were found, a simple mediation analysis was conducted using ordinary least squares path analysis , controlModerate physical activity significantly mediates the relationship between lesion volume and story memory recall in breast cancer survivors.There were no significant differences between the groups (n = 58) on background characteristics such as age, years of education and BMI, nor were there significant differences on lesion volume or moderate PA; however, there were differences on levels of story recall , with BCS remembering significantly fewer details . SpecifiIn BCS, those who were more engaged in moderate PA had smaller WM lesion volumes ; however, there was no relationship evident in the non-cancer age-matched controls . However, lesion volume also indirectly influenced story memory recall through its effect on moderate PA. BCS with lower lesion volume had higher levels of moderate PA (a = -6.14), and those with higher levels of moderate PA had greater recall of the story (b = 0.19). A bias-corrected bootstrap confidence interval for the indirect effect based on 10,000 bootstrap samples was entirely above zero (-0.0966 to -2.7069), verifying that moderate PA significantly mediated the impact of WM lesion volume on story memory recall in BCS .While there were no differences between the treatment types in time since treatment completion for the BCS , we wantAfter re-running the mediation analysis controlling for age, years of education and months since end of treatment, we again confirmed that moderate PA significantly mediated the relationship between lesion volume and story memory recall in BCS. Lesion volume influenced story memory recall independent of its effect on moderate PA (c’ = -2.21). Lesion volume also indirectly influenced story memory recall through its effect on moderate PA. BCS with lower lesion volume had higher levels of moderate PA (a = -6.89), and those with higher levels of moderate PA had greater recall of the story (b = 0.19). A bias-corrected bootstrap confidence interval for the indirect effect based on 10,000 bootstrap samples was entirely above zero (-0.1821 to -3.2297). Despite the range in time since treatment (2–33 months), moderate PA significantly mediated the influence of WM lesion on story memory recall.Previous studies have suggested that diagnosis and treatment for breast cancer results in cognitive impairments and structural changes in the brain, which may reflect a pattern of accelerated aging ,9,12,38.This study is the first to examine the link between WM lesions and PA in any cancer group. While there were no differences in WM lesion volume or moderate PA measures between the groups, we found that WM lesion volume was associated with moderate PA in BCS–those who were more active had fewer WM lesions. There was no relationship between these variables in the non-cancer age-matched controls. These results are particularly noteworthy given that we controlled for age, years of education, and in an additional analysis, time since end of treatment. Taken together, these suggest that moderate PA could play an important role in WM integrity following treatment for breast cancer. Previous work in older adults indicated that self-reported measures of PA predicted structural integrity, including WM lesion volume 3 years later . They foAlthough the time since the end of treatment was relatively short in the current study, and the results may differ with a longer time span, they do indicate that treatment for breast cancer impacts the relationship between the structure and function of the brain. Several studies have investigated the link between changes in WM integrity and cognition following treatment for breast cancer ,24,25,65Cognitive function can be improved with increased PA across the lifespan ,32,33; hWhile previous studies have found that gentle exercise and meditation improves self-reported cognitive function in cancer patients , and thaOne of the strengths of the current study is the use of objective measurements of PA, WM lesion volume and memory recall. However, the cross sectional nature of our study did not allow us to determine the impact of change in one or all of these variables; for example, would increasing PA by taking part in an exercise intervention, result in decreased WM lesions and increased memory recall. Yet, previous studies in healthy adults offer great promise–following a one-year walking intervention, Voss and colleagues reported that increased fitness in older adults who had completed an aerobic exercise program was significantly related to improved WM integrity . This waWhile several studies have indicated that there are differences following diagnosis of breast cancer but before treatment ,69, our There are modifiable risk factors associated with treatment related changes in brain structure and function, and while there is evidence indicating change in cognitive function, WM integrity and PA in BCS, there are no studies that have directly compared all three variables in this group. Our novel findings suggest that continued physical activity might be particularly important in BCS as brain structure significantly predicted cognitive function via mediation of physical activity. Continually active lifestyles may help reduce the treatment related vulnerabilities associated with breast cancer, including reduced WM integrity and cognitive impairment. Collectively, these findings suggest that PA plays an important role in both brain structure and function, warranting further investigation in larger PA intervention studies.S1 DatasetDemographic information, moderate physical activity, lesion volume, and story memory recall data for each subject included in the analysis (n = 58). Data set also includes disease and treatment characteristics of breast cancer survivors (n = 30).(XLSX)Click here for additional data file.


BRCA2, has a large number of genetic variants of unknown effect. The variant rs11571833, an A > T transversion in the final exon of the gene that leads to the creation of a stop codon 93 amino acids early (K3326*), is reported as a neutral polymorphism but there is some evidence to suggest an association with an increased risk of breast cancer. We assessed whether this variant was enriched in a cohort of breast cancer cases ascertained through familial cancer clinics compared to population-based non-cancer controls using a targeted sequencing approach. We identified the variant in 66/2634 (2.5%) cases and 33/1996 (1.65%) controls, indicating an enrichment in the breast cancer cases . This data is consistent with recent iCOGs data suggesting that this variant is not neutral with respect to breast cancer risk. rs11571833 may need to be included in SNP panels for evaluating breast cancer risk.The breast cancer predisposition gene,  BRCA2 was established as a high-penetrance breast cancer predisposition gene following its identification in 19951BRCA2 are still of unknown clinical significance. For the most part these are missense variants, but the 3′ end of the gene can be affected by truncating mutations that appear to have little impact on breast cancer risk or protein function. One such variant is the c.9976A > T transversion (rs11571833), which leads to a nonsense mutation (K3326*) and loss of the final 93 amino acids. This variant was first assumed to be pathogenic5in vitro functional data failed to find a difference from wild-type of K3326* with respect to cell viability, protein localization, homologous recombination repair and response to DNA damaging agents7n rs1157133, whichNonetheless, one study reported an enrichment of rs11571833 in multi-case pancreatic cancer families512BRCA2, in 2634 breast and/or ovarian cancer cases attending familial cancer clinics in Australia in comparison with ~2000 population-based non-cancer controls.In this study we re-evaluate the role of rs11571833 in breast and ovarian cancer predisposition using a panel mutation screen including all coding regions and intron/exon boundaries of BRCA2 variant rs11571833 in 2634 cases and 1996 controls, which had previously been filtered to remove cases with pathogenic mutations in BRCA1 and BRCA2, including the BRCA2 c.6503delTT pathogenic mutation linked to rs11571833 (seen in one control). We detected rs11571833 in 66 cases (2.5% carrier frequency) and 33 controls (1.65%) .As part of a panel screening approach in breast cancer cases ascertained through familial cancer clinics in Australia, we evaluated the incidence of the The carriers of the variant with a diagnosis of breast (n = 64) or ovarian cancer (n = 2) had a mean age of diagnosis of 43.2 ± 9.6 (range 28–67) , no diffFor the controls, 14/33 (42%) carriers reported having a first or second degree relative with breast/ovarian cancer, and 11 with a history of only other cancer types in first degree relatives . This waOne control individual was homozygous for rs11571833, which was confirmed by Sanger sequencing (data not shown). This individual was 49 years of age and had not developed cancer of any type, although her mother had two breast cancers aged approximately 60 and 70. The control individual was diagnosed with an unspecified benign condition aged 41.http://evs.gs.washington.edu/EVS/, Oct. 2014) and 1.1% .We searched the literature using the terms “K3326X” and “rs1157833” in Google Scholar, and identified 23 studies of breast and/or ovarian cancer that reported the number of cases with the variant . K3326* BRCA2 mutations so the possible effect of linkage disequilibrium with the 6503delTT or other mutations on the reported OR must be considered. There was no genome-wide level association with ovarian cancer, either in iCOGs or an earlier study where 2.5% of cases and 2% of controls carried the variantThere have also been a number of genome-wide association studies (GWAS) reporting on this variant: one US Caucasian study  or sensitivity to DNA damaging agents7BRCA2 truncating mutations located more 5′ in the gene, its impact on modifying breast and/or ovarian cancer is not negligible. This variant may need to be included in panels testing all low-penetrance predisposition SNPs once robust risk estimates are obtained.In conclusion, our results indicate that although K3326* does not have the same clinical significance as other et al.BRCA1 and BRCA2, was previously performed in all cases and individuals with pathogenic mutations were not included in this study.Breast-cancer affected, unrelated index cases were ascertained from two locations: 998 individuals from the Hunter Area Pathology Service (“HAPS”), Newcastle, Australiawww.lifepool.org) who were cancer-free at the time of enrollment. This study aimed to recruit women over 40 years in age through the population-based mammographic screening program in the state of Victoria, Australia (BreastScreen Victoria). All participants provided epidemiological and mammographic data and were followed up through the Victorian Cancer Registry to detect cancer incidence. The average age of the participants was 58.84 ± 9.9 years (range 19–91).The control cohort was 1996 participants in the LifePool study  and an Agilent Bravo Automated Liquid Handling System according to the manufacturer’s protocol. Indexed, 96-sample pooled libraries were analysed on a HiSeq2500 Genome Analyzer (Illumina) to generate paired-end 100 or 150 bp sequencing reads. Sequencing alignment was performed using BWAwww.hgvs.org/mutnomen).Germline DNA 225 ng) mostly from blood  or saliva (2.6% of controls) was analysed using two methods. A subset of ViP study cases (n = 646) were specifically genotyped for the rs11571833 variant using a Fluidigm sequencing assay ng mostlHow to cite this article: Thompson, E. R. et al. Reevaluation of the BRCA2 truncating allele c.9976A > T (p.Lys3326Ter) in a familial breast cancer context. Sci. Rep. 5, 14800; doi: 10.1038/srep14800 (2015).