A younger age at menarche and an older age at menopause are well established risk factors for breast cancer. Recent genome-wide association studies have identified several novel genetic loci associated with these two traits. However, the association between these loci and breast cancer risk is unknown.In this study, we investigated 19 and 17 newly identified single nucleotide polymorphisms (SNPs) from the ReproGen Consortium that have been associated with age at menarche and age at natural menopause, respectively, and assessed their associations with breast cancer risk in 6 population-based studies among up to 3,683 breast cancer cases and 34,174 controls in white women of European ancestry. In addition, we used these SNPs to calculate genetic risk scores (GRSs) based on their associations with each trait.th and 5th highest quintiles of the younger age at menarche GRS had odds ratios of 1.14  and 1.13 , respectively, compared to the lowest quintile.After adjusting for age and potential population stratification, two age at menarche associated SNPs (rs1079866 and rs7821178) and one age at natural menopause associated SNP (rs2517388) were associated with breast cancer risk . The odds ratios for breast cancer corresponding to per-risk-allele were 1.14 , 1.08  and 1.10 , respectively, and were in the direction predicted by their associations with age at menarche or age at natural menopause. These associations did not appear to be attenuated by further controlling for self-reported age at menarche, age at natural menopause, or known breast cancer susceptibility loci. Although we did not observe a statistically significant association between any GRS for reproductive aging and breast cancer risk, the 4Our study suggests that three genetic variants, independent of their associations with age at menarche or age at natural menopause, were associated with breast cancer risk and may contribute modestly to breast cancer risk prediction; however, the combination of the 19 age at menarche or the 17 age at natural menopause associated SNPs did not appear to be useful for identifying a high risk subgroup for breast cancer. A younger age at menarche and an older age at menopause are well-established risk factors for the development of breast cancer . In the LIN28B and 9q31.2 [Recently, genome-wide association studies (GWAS) have identified several new common genetic loci associated with either age at menarche or age at natural menopause. Four independent GWAS of age at menarche have identified two novel loci at d 9q31.2 -8, and td 9q31.2 ,9. Most d 9q31.2  and age d 9q31.2  and repod 9q31.2 -16, we aWe therefore conducted a meta-analysis of six population-based studies to investigate the association between genetic loci associated with age at menarche or age at natural menopause and breast cancer risk. We assessed 19 and 17 single-nucleotide polymorphisms (SNPs) that have been previously reported to be linked to age at menarche  and age The ReproGen Consortium was formed by more than 30 studies in the US and Europe to investigate the genetics of reproductive aging traits ,11. Our P value of less than 1 × 10-8) [P value threshold used to declare genome-wide significance . As positive controls, 10 SNPs with consistently reported associations with breast cancer as shown in recent GWAS were included [We analyzed genotypes for 19 and 17 independent SNPs with reported associations with age at menarche and age at natural menopause, respectively, in the ReproGen Consortium, in which all SNPs achieved genome-wide significance in the meta-analysis of each trait (combined stage 1 and replication  × 10-8) . None ofincluded . CompletThe six studies from the ReproGen Consortium provided information on one or more of the following risk factors for breast cancer: age , age at menarche , age at natural menopause , age at first live birth , family history of breast cancer in first-degree relatives (yes/no), alcohol consumption , parity , menopausal hormone therapy (ever/never), oral contraceptive (OC) use (ever/never), and adult body mass index (BMI) (continuous).The GRS was calculated on the basis of the 19 and 17 independent SNPs identified in previous studies as being associated with age at menarche and age at natural menopause, respectively ,11. As aIn each of the six studies, we performed logistic regression to evaluate the association with breast cancer for each of the 46 candidate SNPs, assuming an additive genetic model. Logistic regression was also used to analyze the association between GRS and breast cancer by including both GRSs for age at menarche and age at natural menopause in the model as the main effects. The GRSs were modeled as continuous variables or categorized into quintiles, and the cutoff points for quintiles were based on the WGHS population, which is the largest prospective cohort population among all participating studies. This approach was applied to each of the six participating studies. Odds ratios (ORs) and 95% confidence intervals (CIs) were estimated from logistic regression. To control for potential confounding by population stratification, we adjusted for the top principal components of genetic variation chosen for each study. We adjusted for age in the main model. To examine whether the genetic association of each of the candidate SNPs or GRSs with breast cancer is mediated through the onset of menarche or natural menopause, we then adjusted for self-reported age at menarche and age at natural menopause in the main model. Other conventional risk factors for breast cancer - including age at first live birth, family history of breast cancer in first-degree relatives, alcohol consumption, parity, menopausal hormone therapy, OC use, and adult BMI - were further included in the model to control for potential confounding in studies which had such data available. To examine whether these genetic associations differ by breast cancer subtypes, in each of the two studies that provided information on ER status, we then investigated the genetic association of each of the candidate SNPs or GRSs with breast cancer in subgroup analysis by ER histological status (positive or negative).Forest plots were used to present study-specific ORs and 95% CIs. We then performed meta-analyses by using the fixed-effects model to estimate summary ORs from study-specific estimatesthat were weighted by the inverse of the variance of each study. As the meta-analyses restricted to prospective cohort studies or case-control studies yielded similar results, we present results from only the meta-analysis of all six participating studies. We also tested the heterogeneity of associations across studies as well as across different tumor subtypes by using the Q test .P values were based on two-sided tests and were considered statistically significant if less than 0.05. Because SNPs were selected on the basis of an a priori hypothesis, adjustments for multiple comparison tests were not performed.All statistical analyses were performed by using SAS version 9.1 software . Power calculations were carried out by using Quanto . All The six participating studies contributed 3,683 breast cancer cases and 34,174 controls of self-reported white women of European ancestry . The allele frequency for each SNP in the controls was similar to those reported for populations of European descent [P value = 0.003; P for heterogeneity = 0.37) and 1.08 , respectively. The SNP rs1079866 is located about 250 kb away from the INHBA gene on chromosome 7, whereas SNP rs7821178 is about 181 kb away from the PXMP3 gene  on chromosome 8. The strongest GWAS hit for age at menarche, rs7759938 at LIN28B on chromosome 6, was not found to be associated with breast cancer risk . Of the 17 candidate SNPs associated with an older age at natural menopause, one SNP, rs2517388, was significantly associated with breast cancer risk with a per-risk-allele OR of 1.10 . This SNP is an intronic SNP in the ASH2L gene on chromosome 8. The study-specific and summary ORs for the three associated SNPs are shown in Figure Table  descent -23. AfteP for trend = 0.06). Compared with women in the lowest quintile, women in the fourth and fifth quintiles had ORs for breast cancer of 1.14 (95% CI = 1.01 to 1.28) and 1.13 (95% CI = 1.00 to 1.27), respectively. Results were similar when analyses were performed by using weighted GRS. Overall, we did not observe statistically significant associations between breast cancer risk and age at natural menopause when either count or weighted GRS was used.To evaluate the combined effect of candidate SNPs on breast cancer risk, we calculated a GRS for each trait by using either a count GRS or a weighted GRS approach. The mean values of count and weighted GRSs were 20.41 and 20.03, respectively, for age at menarche and 16.21 and 14.32, respectively, for age at natural menopause , although rs1079866 appeared to have a stronger association with ER+ tumors  than with ER- tumors . Of note, one SNP that had a reported association with age at menarche, rs17188434, had a significantly stronger association with ER- tumors  than with ER+ tumors . Another SNP that had a reported association with age at menarche, rs17268785, was associated with a decreased risk of ER- tumors  but an increased risk of ER+ tumors . For the SNP that had a reported association with age at natural menopause and that was associated with overall breast cancer risk, we observed a stronger association with ER- tumors  than ER+ tumors ; however, the test for heterogeneity was not statistically significant (P for heterogeneity = 0.12). When the count GRS for age at menarche or age at natural menopause was applied to ER+ and ER- breast cancer separately, the trend in the OR for ER+ tumors was very similar to that for overall breast cancer. The ER- tumor data suggested a somewhat different pattern, although the statistical power was limited for this subtype  with per-risk-allele ORs of 1.26 (95% CI = 1.16 to 1.37) and 0.98 (95% CI = 0.82 to 1.16) for ER+ and ER- tumors, respectively  than ER+ tumors , and rs3817198 was associated with a decreased risk of ER- tumors  but an increased risk of ER+ tumors .Of the 10 candidate SNPs with consistently reported associations with breast cancer risk, five SNPs  appeared to have a stronger association with ERP values were less than 0.001)  in our study, although the direction of the associations was consistent with that of previous reports [+ than for ER- tumors (P for heterogeneity < 0.001), and the OR across quintiles was no longer monotonic in ER- tumors  Table . We did  reports ,22. We aIn this large meta-analysis of six population-based studies, we investigated whether 19 loci linked with age at menarche and 17 loci linked with age at natural menopause were associated with breast cancer risk among up to 3,683 breast cancer cases and 34,174 controls. We found that two SNPs with reported associations with age at menarche and one SNP with a reported association with age at natural menopause were significantly associated with breast cancer risk. However, no statistically significant associations were found for GRSs that combined all 19 or 17 loci associated with each trait, although the association for age-at-menarche GRS was marginally statistically significant. We confirmed most of the candidate loci for breast cancer which were identified in previous GWAS. Some of these associations appeared to differ by tumor subtypes defined by ER status.In our analyses, most of the candidate SNPs, including the strongest GWAS hit for age at menarche or age at natural menopause, were not found to be associated with breast cancer risk. This is not necessarily surprising given that age at menarche and age at natural menopause are relatively weak risk factors ,3, and aFGFR2-rs2981582, TNRC9-rs3803662, 1p-rs11249433, 5p-rs7716600, 2q35-rs13387042, MAP3K1-rs889312, 8q24-rs13281615, and RAD51L1- rs999737. The direction and magnitude of these associations were consistent with those of previous reports [LSP1-rs3817198 or CASP8-rs1045485. However, these two SNPs had relatively small reported effects that our study might not have been able to detect. When the 10 candidate SNPs were combined by using a polygenic risk score, the relative risk for women in the highest quintile was about twice that in the lowest quintile, and this is in accordance with other published results [We found statistically significant evidence of association with breast cancer for eight of the 10 breast cancer susceptibility loci examined:  reports ,18,22-25 results . In this results .post hoc and exploratory analysis, we created a polygenetic risk score by including only the three candidate loci associated with either age at menarche or age at natural menopause and with breast cancer risk and found that each risk allele increment was associated with an approximately 17% increased risk for breast cancer. Women with four or more risk alleles had an approximately 60% increased risk for breast cancer in comparison with those with two risk alleles or less. When we further combined the three associated SNPs with the 10 breast cancer susceptibility loci to create a polygenetic risk score, each risk allele increment was associated with an approximately 18% increased risk for breast cancer. For women with 14 or more risk alleles (the highest quintile), the risk for breast cancer increased threefold in comparison with those with 10 or less (the lowest quintile). Because the former group constitutes approximately 20% of the study population, the GRS that combines the three candidate SNPs for age at menarche and age at natural menopause and the identified breast cancer susceptibility loci might be useful for identifying a subgroup of women with a high genetic risk for breast cancer. Further research is needed to confirm this finding.Given that most of the candidate loci for age at menarche and age at natural menopause were not associated with breast cancer risk, it is not surprising that there were no statistically significant associations for the polygenetic risk scores that combined all candidate loci for each trait. To conduct a + breast cancer is positively associated with a woman's cumulative lifetime exposure to endogenous ovarian hormones [+ than ER- tumors. In this report, we found that candidate loci for age at menarche and age at natural menopause may also be differentially associated with tumor subtypes defined by ER status. Of the three candidate loci that were found to be associated with overall breast cancer risk, rs1079866 was more strongly associated with ER+ tumors, rs7821178 was equally associated with both, whereas rs2517388 was more strongly associated with ER- tumors, although differences were not statistically significant. Importantly, two candidate loci for age at menarche, rs17188434 and rs17268785, had significantly different associations with ER+ and ER- tumors. Whereas both SNPs were not significantly associated with overall and ER+ breast cancer, the former showed a statistically significant positive association with ER- tumors, whereas the latter showed a statistically significant inverse association with ER- tumor. These findings provide further support for the notion that ER+ and ER- tumors are the result of different etiologic pathways [It has been hypothesized that the risk of ERhormones . A younghormones ,15,29 anhormones  have beepathways .+ tumors was less adequately powered, as the ER status was not available for all cases, and the study had limited statistical power for ER- tumors. One limitation in our study is the multiple comparisons that could lead to false-positive results. Although none of the candidate SNPs with a reported association with age at menarche or age at natural menopause survived Bonferroni correction in the test of breast cancer association, this correction is considered to be overly conservative given that the candidates were chosen on the basis of promising hypotheses. Another potential limitation of our study comes from differences in the study population and designs and methods of collecting risk factors and genetic marker data across studies. However, the findings were generally consistent across studies, arguing for the robustness of our results. Finally, as our analyses were restricted to women of European ancestry, results from this study may not be generalizable to other ethnic groups.Although common genetic variants that influence the intermediate phenotypes or risk factors have been hypothesized to subsequently affect disease risk, few studies have assessed the association between these genetic variants and disease risk or, furthermore, whether these associations are mediated through the intermediate phenotypes. Chen and colleagues  investigIn summary, in this large analysis of the association of several novel candidate loci for age at menarche and age at natural menopause with breast cancer risk, we observed that three loci - two for age at menarche and one for age at natural menopause - were significantly associated with breast cancer risk independently of their associations with each trait and independently of known breast cancer susceptibility loci. These associations may differ by tumor subtypes defined by ER status. A combination of all 19 loci associated with age at menarche or 17 loci associated with age at natural menopause did not appear to be helpful for identifying a high-risk subgroup for breast cancer.ARIC: Atherosclerosis Risk in Communities Study; BMI: body mass index; CI: confidence interval; ER: estrogen receptor; FHS: Framingham Heart Study; GRS: genetic risk score; GWAS: genome-wide association studies; MAF: minor allele frequency; NHS: Nurses' Health Study; OC: oral contraceptive; OR: odds ratio; RSI+II: Rotterdam Study I and II; SardiNIA: SardiNIA Breast Cancer Study; SNP: single-nucleotide polymorphism; WGHS: Women's Genome Health Study.The authors declare that they have no competing interests.CH performed the meta-analysis, wrote the manuscript, helped to conceive and design the experiments, helped to perform the primary statistical analyses in each study, shared responsibility for the interpretation of results and critical revision of the manuscript, and contributed to a critical revision of the manuscript for important intellectual content. DIC helped to conceive and design the experiments, helped to perform the primary statistical analyses in each study, shared responsibility for the interpretation of results and critical revision of the manuscript, and contributed to a critical revision of the manuscript for important intellectual content. DJH helped to conceive and design the experiments, contributed to a critical revision of the manuscript for important intellectual content, and participated in the original design, subject recruitment, acquisition of data, biospecimen collection for the studies, and the genotyping and quality control of genotype and other data. JD, SJH, RR, and SS helped to perform the primary statistical analyses in each study and shared responsibility for the interpretation of results and critical revision of the manuscript. SJC, LC, EWD, JMM, PMR, and BHS contributed to a critical revision of the manuscript for important intellectual content and participated in the original design, subject recruitment, acquisition of data, biospecimen collection for the studies, and the genotyping and quality control of genotype and other data. JEB, LF-R, NF, SEH, AH, KLL, GP, EP, FR, LMR, GLS, LS, and AGU participated in the original design, subject recruitment, acquisition of data, biospecimen collection for the studies, and the genotyping and quality control of genotype and other data. All authors read and approved the final manuscript.Supplementary methods for study population.Click here for fileTable S1: Information on the 46 candidate SNP loci identified in previous genome-wide association studies for age at menarche, age at natural menopause and breast cancer.Click here for fileTable S2: Characteristics of non-genetic risk factors for breast cancer in each participating study.Click here for file


There has been a sharp increase in the incidence of breast cancer in China in recent years. A number of female physical characteristics, such as age at menarche, menopause, first birth and the duration of breastfeeding, have been linked to breast cancer, yet data on these factors in Chinese women is largely missing both for aggregate and age-specific data. Thus, the objective of this study was to explore changes in female menstrual and childbearing characteristics as a possible explanation for increasing rates of breast cancer in this country.From July to September 2008, a population based cross-sectional breast cancer survey covering 124,758 females from 4 provinces or cities in Eastern China was carried out, using multi-stage and cluster methods. In-person interviews based on a self-designed structured questionnaire were performed, in which female physiological and reproductive factors, such as age at menarche and menopause, menstrual cycle history, childbearing history, breastfeeding methods, abortions or miscarriage, were included. For every 10-year age category, the subjects were divided, and across those age groups, all the above factors were compared respectively and changes in physical and childbearing characteristics were evaluated. ANOVA was used to compare the differences across the groups.A total of 122058 subjects were included in the final analysis. The mean age at menarche was 15.39 years, the mean number of full-term pregnancies was 1.58, the mean duration of breastfeeding was 22.68 months, the mean age at first birth was 23.75 years, the mean frequency of miscarriage was 0.36, and the mean age at menopause was 48.63 years. Significant differences across the several age groups were noted for the age at menarche, number of full-term pregnancies, accumulated duration of breastfeeding, age at first birth, number of miscarriages, and age at menopause. These data clearly showed a gradual shift towards an earlier age at menarche, fewer pregnancies and shorter breastfeeding lengths.Significant changes in female physical and childbearing characteristics across a number of different age ranges were detected. These changes may be related to the increasing trend of breast cancer in China. Breast cancer is one of the most prevalent malignancies in women worldwide. In 2008, 1.38 million people worldwide, mostly from Europe and the USA, were diagnosed with breast cancer . TraditiRandom samples were obtained through multi-stage stratified cluster sampling. The target population included 25 to 70-year-old females of the Han ethnic group who at the time of survey had been registered for over two years at a local residence and had lived at least six months in that local residence. Long-term migrant workers were excluded. The provinces in Eastern China where the Han ethnic group mainly resides, include Shandong, Jiangsu, Hebei and Tianjin, were selected as the survey provinces. Subsequently, counties or regions, villages or communities were successively randomly selected. Women who met the study requirements were selected for the survey. A schematic outlining the sampling procedure is shown in Figure This study was a cross-sectional epidemiological survey, including face-to face interviews and breast examinations. Data were collected through in-person interviews based on a self-designed structured questionnaire, in which female physiological and reproductive factors, such as age at menarche, age at menopause, menstrual cycle history, childbearing history, breastfeeding methods, history of abortions or miscarriage, contraceptive methods and the use of contraceptive medications were included. For all of the variables covered by the questionnaire, the answers were defined by strict criteria. For example, menopausal status was divided into premenopausal and postmenopausal refer to the criteria the National Comprehensive Cancer Network (NCCN) in 2008 . If the As a cross-sectional study, all interviews were completed in 2 months: from July 15 to Sept 15, 2008. And before the survey began, this study was approved by the ethics committees of the second hospital of Shandong University and those at the collaborating institutions in each region  and informed consent was obtained from all of the study subjects. Surveyors and physicians were recruited in each survey region before the implementation of this study. A total of 77 physicians and 248 surveyors participated in the on-site survey process.All study subjects were strictly selected according to random sampling methods, and the final sampling concordance rate was 97.8%. During the survey process, all interviews were under strict quality control. Firstly, a supervisor in each region oversaw the surveyors and sampled their work for inspection. Further, 10% of the questionnaires completed at the end of each day were randomly sampled to inspect for their completeness, accuracy and standardization. After the quality assessment was completed, all identified errors and missed survey items were promptly corrected. If important information was found to be missing in the questionnaire, the interviewer would collect the missing information from the original survey site the next day. Finally, after the completion of the survey, sampled re-screening was performed for each survey site, and the results showed a consistency of 92.19%.Epidata 3.1  was used to establish the database. Analysis of variance was used to compare differences across different age groups with different variables. The liner regression analysis was used to performe the trend test in age groups and Age at menarche, Number of full-term gestations, Accumulative breastfeeding periods . SPSS 16.0  was used to carry out the data analysis. Tests of significance were evaluated against the null hypothesis of no association, using P < 0.01 as a cut-off for significance.In this survey, 147,538 women were initially selected, and of these women, 124,758 were approached to undertake the survey, giving a 15.44% rate of loss. A total of 124,758 women from Shandong, Hebei, Jiangsu province and the Tianjin metropolitan area were registered and interviewed. Of these 1037 subjects were younger than 25 years, 1629 subjects were older than 70 years. A further 34 were excluded due to a loss of important information. Thus, the final total study population was 122,058 (sampling concordance rate: 97.84%). Among these women, 61,104 women were come from Shandong province, amounting to the 0.64% of Shandong province’s total population and 1.29% of Shandong province’s total female population; 19,741 women were from Hebei province, amounting to 0.27% of Hebei’s total population and 0.56% of it’s female population; 20,921 women were from Jiangsu province, representing 0.27% of Jiangsu province’s total population and 0.5% of its female population; 20,292 women were from the Tianjin metropolitan, amounting to1.56% of Tianjin’s total population and 3.33% of its female population [The mean age for all subjects was 44.2 years . Of the total cohort, 85,755 (70.26%) of the subjects were premenopausal, while 36,303 (29.74%) were postmenopausal, with a mean age of 48.76 years  at menopause. The mean age at menarche was 15.39 years (SD = 2.02) for all subjects. Overall, 118,623 subjects had given birth at least once, and 95,434 (78.6% of 118 623) had breastfed for at least 12 months. There were 88,632 subjects from rural areas and 33,696 from urban areas, giving an urban/rural proportion of 0.38, which is similar to that found for China as a whole. Characteristics of the population, both in total and for each age group, are shown in Table p < 0.001). Trend test showed the age at menarche bring forward 0.479 years every 10 years young (p < 0.001), clearly indicating a gradual change towards an earlier age at menarche.After excluding 162 women due to a loss of age data and 52 women due to a loss of menarche data, 121,844 subjects remained in the study. As shown in Table p < 0.001) were observed, trend test showed the number of full-term gestations decrease 0.441 every 10 years young(p < 0.001),clearly indicating a gradual shift to fewer gestations.After excluding 162 women loss of age data, 121,896 women were left to be analyzed. As shown in Table p < 0.001) were noted, trend test showed the accumulative breastfeeding periods decrease 4.50 months every 10 years young(p < 0.001), indicating gradual shift to shorter breastfeeding periods.After excluding 162 women loss of age data and 2,601 women due to a loss of breastfeeding data, 119,295 women remained. As shown in Table p < 0.001) was also noted.Of the 121,896 women to be analyzed, as shown in Table p < 0.001) was also observed.After excluding 162 women due to a loss of age data and 168 women due to a loss of miscarriage data, there were 121,730 women left to be analyzed (2 women were excluded due to a loss of both age and miscarriage data). As shown in Table p < 0.001) were detected.A small number of women in the 25-34 and 35-44 age groups had gone through menopausal as a result of medical conditions or procedures and were excluded from the final analysis, leaving a total population of 34,797. As shown in Table In 2008, 1.38 million women across the world were diagnosed with breast cancer, resulting in 458 000 deaths. Of this number, 170,000 cases were in China, resulting in 44,000 deaths. Globally, 23% of cancer in women is due to breast cancer, making it the most common type of cancer in women. Data published by the WHO shows that the standardized incidence  of Chinese female breast cancer in 2008 was 21.6, which is much higher the figure of 16.4 observed in 2000. This data indicates that there has been a continuous increase in the incidence of breast cancer in China [The mean age at menarche in this study was 15.39 years. A number of statistically significant differences across the several age groups were observed, indicating a gradual shift in the age of menarche, towards an earlier age. In their study, Tianlin et al., studied age of menarche in a cohort of female Chinese students from 1985-2000, and found that approximately every 10 years the age of menarche was brought forward by about 2.5 months for urban women and 4.6 months for rural women. In their assessment of their findings, the shift to an earlier age at menarche was mainly attributed to economic development and increasing incomes . HoweverOur study also showed that fewer Chinese women were reaching full-term gestation, as revealed by our finding that more women in the 65-70 age reached full-time gestation than those in the 25-34 age group by an average of 1.94. This finding maybe related to the one child policy of China. The relationship between gestation and breast cancer is controversial, with most studies supporting the view that increasing birth number has a positive effects on breast cancer. Some studies have also shown that every pregnancy could reduce the risk of breast cancer by 7% . A studyIn this study we also showed that accumulative breastfeeding periods were shorter in the younger age group, and this decreasing trend was linked to changes in full-term pregnancy, suggesting that the decreased length of breastfeeding periods was because women were, on average, having fewer pregnancies. Most studies support the view that breastfeeding is a protective factors for breast cancer. A study covering 150,000 females in 30 countries conducted by the Breast Cancer Estrogen Intergroup showed that for breastfeeding (in excess of 12 months) could reduce the risk of breast cancer by 4.3%, and that breastfeeding is an independent risk factor for breast cancer, and is not affected by other confounders such as age at first birth, parturition times, or race . StudiesOur study showed that the mean age at first birth was 23.75 years old. Of this, women from the 34-54 age group were the oldest at the time of first birth, followed by those from the 55-age group and then the 25-34 age group. This finding may be a reflection of policy changes. The Chinese government began implementing laws related to family planning, which advocated for late marriage and late childbirth, in late 1970s. Under this policy, all married women, were not be permitted to have a child without a baby birth permit. Thus, women aged between 34 and 54 may have delayed first birth. However, in the last 10 years, with the baby birth permit system nearly repealed and the easing of Family Planning Policy, women below 34 began to have their first birth at a much earlier age. Given that many studies have shown that an earlier age at first birth may reduce the risk of breast cancer, this demographic change has important implications. For instance, research has shown that white American women are at a 25% increased risk of breast cancer for every 4 years of delayed first pregnancy , but thaThis study showed that Chinese women suffered an average of 0.36 miscarriages, of whom women from the 35-54 age group experienced the most miscarriages. Given that numerous studies have shown that neither spontaneous nor artificial abortion is related to breast cancer , it is pWith respect to menopause, our study showed in Chinese women, the mean age at menopause was 48.63 years, and there were statistically significant differences across different age groups, among which women from 55-64 age group underwent menopause the latest. It is generally understood that delaying menopause increases the risk of breast cancer suggesting that women who underwent menopause below the age of 45 would have a decreased relative risk (by about 30-50%) than those who underwent breast cancer after 50 . Work byThe findings of this study clearly showed a gradual shift to an earlier age at menarche, decreased duration of pregnancy and shorter breastfeeding periods. On the basis that the above determinants are traditional risk factors of breast cancer, we therefore concluded that these shifts in female physiology and reproductive characteristics are probably linked to the increasing incidence of breast cancer in China. This study was the first large-scale epidemiological investigation in China in the last 30 years, and such findings are important for further research on the treatment and prevention of breast cancer in this country, and contribute significantly to public health.The authors declare that they have no competing interests, including financial and non-financial interests.QZ participated in the study design, population survey, data interpretation and draft the manuscript. LYL participated in the study design, population survey and statistical analysis. FW participated in the population survey, data collection and helped to draft the manuscript. KM participated in the population survey and data collection. ZGYwas the chief designer and executor of the programme and contributed the most to the data interpretation. ZGY had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2458/12/368/prepub


Breast cancer-preventive behaviors are critical for community and women’s health. Although many studies have addressed women’s knowledge and attitudes toward breast cancer, little information is available about their experiences of breast cancer preventive behaviors. This study aimed to explore the experiences of Iranian women regarding preventive behaviors.This was a qualitative study. A sample of Iranian women aged 30 years and over was selected purposefully. Data collected through focus group and semi-structured audiotaped interviews and were analyzed by conventional content analysis.The following five main themes emerged from the analysis: attitude toward breast cancer and preventive behaviors, stress management, healthy lifestyle, perceived social support and individual/environmental barriers. The findings showed that women were highly motivated to preventive behaviors of breast cancer but faced considerable challenges.The findings indicated that increased awareness, positive attitudes, stronger motivational factors, and fewer barriers toward preventive behaviors are most important parameters that might encourage women to practice breast cancer-preventive behaviors. Breast cancer is the most common cancer among women and is the leading cause of female deaths due to cancer . In IranThere are several risk factors for breast cancer and are numerous, but some life style and behavioral factors may have an important role on increased risk for developing breast cancer including dietary habits ,5, low pIt has been suggested that the appropriate method for breast cancer prevention and early detection is performing preventive behaviors and these might include lifestyle modification and screening behaviors .Lifestyle modification can help to reduce risk of breast cancer to a large scale . PreviouScreening behaviors including mammography, clinical breast examination and breast self-examination are also considered to be effective for early detection and reduced mortality due to breast cancer -24, althAt present there is no a national population-based screening mammography program in Iran, although it is estimated that there are about 200 diagnostic mammography centers in the country . If women complain about her breasts usually physicians may ask women to come back with a diagnostic mammogram. The data for diagnostic mammography in Iran varies from 1.3% to 30% ,35. UnfoTo this end one might conclude that lifestyle modifications and screening behaviors are important issues in breast cancer prevention programs. However, it is important to consider that none of the behaviors discussed could be regarded as the best behavior for breast cancer prevention. Thus, this study aimed to explore Iranian women’s experiences of breast cancer- preventive behaviors, and provide more insight on the topic. It was hoped this piece of work could contribute to the existing literature from a country where lifestyle modification interventions and screening programs are lacking and women live in a cross road of tradition, culture, religion and modernity.This was a qualitative study. Participants for this study were recruited from a health care center affiliated to Tehran University of Medical Sciences. In order to gain a different perspective we used purposive sampling method to insure that women with varying levels of socioeconomic status, educational level, age, occupation and marital status are present in the study. In addition a team of experts were asked to participate in the study and comment on the topic.The data were collected using two approaches: focus group discussions, and in-depth interviews with laywomen.i. Focus group discussionsWe conducted two focus group discussions each comprising of eight women in order to explore the issue. Sessions were held in locations that were convenient and accessible for participants. Each session lasted for three and half hours.ii. Semi structured interviewsFollowing focus group discussions, in order to get deeper information, 16 semi-structured, open-ended interviews were conducted with laywomen. At the beginning of each interview, the women were asked about demographic characteristics. The interviewer followed a guide designed to encourage women to explore and discuss their knowledge, perception and action about breast cancer. We also asked about social, cultural and environmental factors that might have influenced their behaviors. Then, focusing on each of the expressed behaviors, we asked some questions such as ‘What do you do to prevent breast cancer?’ What are behaviors that might prevent breast cancer? What factors are associated with behaviors that might prevent breast cancer? How do you practice preventive behaviors? Following each interviews women were asked to explain more about what they have said. For instance they were asked ‘What do you mean?’ ‘Explain more’ in order to have deeper understanding of experiences of women. The interviews were carried out at participants’ home or at a public place convenient for the respondent. Each interviews lasted for approximately 1.5–2 hours.A content analysis with a conventional approach was carried out to analyze the data and to identify key themes as well as comparing trends and patterns occurred across the various groups. As such the main investigator read the interviews several times and discussed the best approach about coding. During the period of analysis, researcher continually checked similarities and differences in the data and manuscripts, and selected main concepts and formed analytical categories by using comparison of each part of data with other data. We imported the transcripts into the MAXQDA software version 10.The ethics committee of Tehran University of Medical Sciences approved the study and all participants completed informed written consent.In all 24 women aged 30 and over without breast cancer took part in the study. The characteristics of the participants are reported in Table One of the elicited themes in the present study was attitudes toward breast cancer and preventive behaviors including three different categories namely beliefs, fears and worries, and individual responsibility. Women also expressed different views about breast cancer.Most participants linked breast cancer to death. Some women believed that even talking about breast cancer might put one at risk of getting breast cancer:‘I don’t even want to talk about breast cancer… talking about breast cancer brings bad luck....’ (Participant 14)Many participants described the negative effects of breast cancer on women’s physical appearance. Some believed that breast cancer is equal to loss of femininity. A woman said:‘I hate to hear the word breast cancer. In my view, for a woman, breast cancer is the loss of identity and charm of feminine. I think after that, woman feels unattractive. It is equal with death. It is the end of good life. I am very scared of cancer’ (Participant 2)A woman indicated that leaving without breasts is meaningless. She said:‘I know the breasts are very important to woman. Because of their maternal role, they need to breastfeed their babies. Also women with breast look beautiful. A woman is never complete and beautiful without breast. I would prefer to die than to live without my breasts’ (Participant 7).There were mixed beliefs linked with abilities of women about breast cancer-preventive behaviors including healthy lifestyle and screening behaviors: A woman said:‘I think if women care for themselves, they must be pay attention to their own breast health by healthy lifestyle, breast check-up and screening’. (Participant 4)‘I do not go for mammography; I really scare in case it will become breast cancer, I have these things in my mind’. (Participant 9)In contrast a woman stated: The majority of women expressed fears and worries over their future life. They believed that breast cancer has some social implications such as divorce. They were also worried about their children’s fate. A participant explained that:‘I think developing breast cancer might lead to divorce. It might be the end of a married life’ (Participant.11) or it was said that:‘The most important thing is my children’s future and happiness. I said to myself I should stay healthy for my children’. (Participant 6)Desire to perform preventive behaviors is an important step for prevention of this disease influenced by several factors, including individual responsibility, ability to deal with negative elements of life, effort to maintain normal life, and deal with health barriers. The majority of participants believed that they were able to do preventive behaviors such as regular breast examinations. One participant mentioned that:Everyone has an important role in own health. If I am not responsible to my health, others cannot help me. I am aware of my abilities. I always try to deal with the negative factors affected my health especially, about an awful disease like breast cancer.’ (Participant.7)‘Another participant said:I think for better and healthier future. I will do everything that has a positive effect on my physical and mental health such as healthy diet, regular exercise and breast exam. Because I love myself, and my health is important to me. I am responsible for my health.’ (Participant.4)‘In summary we found that beliefs, fears and worries, and personal responsibility were the most important components of women’s attitudes toward breast cancer.Since many women believed that stress could cause breast cancer, thus they used different techniques including relaxation, positive thinking and prayer to control their stress and in fact fight against breast cancer. According to one participant:Yuga helps me to become relax and calm. I believe this makes me strong enough to fight breast cancer’ (Participant.1)‘I go to meditation classes once a week, because it makes me relaxed and will prevent me from getting breast cancer’ (Participant.4)Another woman said: ‘Women also expressed other ways to manage their stress to safe guard themselves against breast cancer. For instance the use of prayer was evident in women’s sayings. For many participants, believe in God and prayer, played an important role to prevent cancer. A participant pointed out‘I believe that prayer is a source of comfort and composure. Praying and trusting in God get me a peace of mind to care my health. I think it helps me in a spiritual sense and in making me more positive. I believe that healing can be received from God.’ (Participant. 4)A positive thinking was believed to be helpful to the final outcome of the breast cancer prevention as expressed by a woman:‘It’s very important to have a balance between physical, spiritual, and mental health. This involves working out, eating, believing spirituality, setting life goals, etc.’ (Participant.7)The majority of participants agreed that breast cancer could be prevented by a healthy lifestyle including having healthy diets, doing sufficient exercise, and attending breast check up. Some women made improvements in dietary fat reduction. One of the women mentioned:‘I think the best way against breast cancer is healthy lifestyle. I eat healthy foods such as milk, vegetables, low fat, and fruits in my diet. Also, I exercise at least for one hour two or three times weekly’. (Participant. 3)‘I really changed my eating habits and tried to use low-fat foods…’ (Participant. 9)Another woman said: Some participants put more value on their health and paid more attention to their self-care in order to gain a perfect life. To have clinical breast checkup pointed out by some women:‘I went to the doctor for a breast exam. I was so happy, and I forgot all of my worries when the doctor reassured me about my breast health.’ (Participant. 6)Other participant said:‘If I feel something is wrong with my breasts I just go to the doctor, I don’t hesitate going to see a doctor at all even a male doctor’. (Participant. 11)Most women mentioned they want living life to the full, thus they care about their breasts. One participant stated:‘I realized that I have so many reasons to be healthy and live longer. I care about my breasts so that could enjoy my life.’ (Participant. 1)Both financial and emotional aspects of social support were perceived as fundamental elements of the preventive process for women. Although many participants acknowledged fear and doubts upon hearing the word breast cancer and screening, social support from family and friends served as a source of motivation, enhanced confidence, lessened fears or having someone to encourage them for performing preventive behaviors. One woman said:“We’re a happy family. When I go for doing mammography, my husband supports me continuously. He encourages me to do it. He tells: I don’t like that you lose your life due to the breast cancer, I would like to see you safe and healthy.” (Participant.9)A few women stated that they received emotional support from friends. They could share their experiences about mammography and BSE. These women felt encouragement for performing preventive behaviors by friends. Peers also provided a means of receiving valuable information regarding breast cancer prevention that they said they did not receive from physicians. A woman pointed out:‘I’m willing to do mammography because many of my friends do regularly and they encourage me to do so. Encouragments by friends is very worthwhile…’ (Participant.5)Also, media  could positively affect on encouraging women for performing preventive behaviors. It is important to be aware of how women may interact with or be exposed to these types of media. Our understanding of these and our knowledge of where to go for help may prevent a breast cancer. Media can also be used to increase awareness and provide information on breast cancer prevention programs. One woman said that:‘I feel more courage getting mammography after watching TV about woman suffering from breast cancer. I know that the mammography is necessary for early detection and prevention of breast cancer’. (Participant.8)Another participant said:‘I read an article about proper diet and exercise in Internet that is effective for breast cancer prevention. So, I regularly use vegetable, fruit and low-fat diet. That’s what I have been doing’ (Participant.10)Having health care insurance, and good financial position are some of the effective factors which can encourage women to follow healthy behaviors and also can give them peace of mind. One woman said that‘They can offer mammography for free in the government clinics, right?’ (Participant.16)“I saw some women that they didn’t receive mammography due to poor financial position and lack of health care insurance. But I did it on time. It is very awful to lose my breasts because of money.” (Participant.9)Participants demonstrated that there were many barriers to achieve effective prevention of breast cancer and performing preventive behaviors. These ranged from educational and behavioral aspects to cultural and economic concerns including lack of knowledge and skill, misinformation, doubts, fears, shame, bad experiences and ignorance about risks. Many women reported that they were reluctant to visit doctor, and that were hesitant, or even delayed mammography, because they embarrassed to be examined by a male physicians. They preferred female physicians for breast exams. Although, they was uneasy about breast exams by female physicians. A participant stated that:‘I’m willing to do regular breast self-examination, but I don’t know how to do it. I don’t like being seen visiting a male physicians. I feel embarrassed when I have to show my breasts in front of someone especially male doctors…’ (Participant.2)One woman said that she would never go for a breast exam because she felt so ashamed the last time she went.‘I had a breast exam about 3 years ago. When he was doing the breast exam, I felt really ashamed. He did not talk anything when he was doing the breast exams. After the exams, he said that everything is fine. I really do not want to go through the exams (like that) again’ (Participant.4)One woman mentioned that she didn’t breast exam due to the lack of knowledge about it.‘I think, there’s really not enough information out there. My doctor said that you are the second case during thirty years of my medical practice that seeks clinical breast examination’. (Participant.11)A few women said that they did not get screening for breast cancer because they believed that it was carcinogenic, and others felt that they were not scared about breast cancer them because they did not have any symptoms or any family history of breast cancer. They had little risk of developing breast cancer. They thought that breast cancer would happen only in older women or those with a family history of breast cancer. A woman said‘I have not done mammography because it is carcinogenic. It seems to me that breast cancer is for old women. I don’t think I am likely to get breast cancer. In my family no one has history of breast cancer, so I don’t have any risk of breast cancer’. (Participant.15)A woman considered herself as risk free and said:I am not at all at risk of the breast cancer so why I should visit doctor. Nobody in my family has breast cancer. So, I never thought about breast cancer. I don’t think that it would happen to me.‘I do not want to do it. When I am health, why I make problem for myself. I imagine it is going to hurt me” (Participant.5)Also, since there is no national screening program for breast cancer in Iran, some women mentioned that this has negative effect on their behaviors. They stated that the government could make annual mammography for free through cooperation with insurance agencies. Some women proposed free mammogram offered by government at a convenient time. A lady with higher education pointed out:As far as I know, unfortunately, there is no national screening program of mammography for breast cancer in Iran’. (Participant.1)‘“In my opinion, the government and ministry of health in cooperation with insurance companies can help women to cover the charge of periodic mammography. They can offer mammography for free in the government clinics, right?” (Participant.4)Another woman said, Having financial support had positive psychological effect on participants for attending for annual mammography. In some cases, high cost of mammography was a barrier. One woman mentioned:“I’m house wife and my husband has not a good job. Our income is not enough. My doctor recommended doing mammography, but I cannot afford it. If we had enough income I could care of my health much better.” (Participant.5)Another participant stated,“Although I need to have the breast check-up, I cannot do it because my insurance company does not cover the cost of mammography. I think mammography is very expensive’ (Participant.11)This study was designed to explore Iranian women’s experiences of breast cancer-preventive behaviors. As a result, several core concepts including attitudes toward breast cancer, preventive behaviors, stress management, healthy lifestyle, perceived social support and individual/environmental barriers emerged. However, one might argue that the analysis did not focus only on preventive behaviors but addressed attitudes, and mammography (which is early detection and screening and prevention). There is evidence that having strong interest and positive attitude to maintain breast health may encourage women for performing preventive action against breast cancer . Thus weThe findings highlighted that social, cultural and religious attitudes affected breast cancer-preventive behaviors, and there were numerous misconceptions. Many participants stated that they were afraid or scared of the word breast cancer and some believed that even talking about it might cause breast cancer. Similar findings reported by previous studies found that there were lack of knowledge, fear, misconceptions, and fatalistic beliefs among women . The facAs same as other studies some women had physical/emotional concerns toward breast cancer ,40. TheyThere have been mixed results about stress and breast cancer. As same as previous studies -49, our Most women noted that healthy lifestyle including having healthy diet, exercise and weight control helped them to fight against breast cancer. We thought these are good indications that highlight the fact that women are becoming more alert about factors that jeopardize their life and that they are prepared to change their habits if health care workers recognize such important potentials for behavior changes among women.Using different levels of emotional, financial, and informational supports motivated women to do preventive behaviors due to increased self-confidence. Previous literature also showed that encouragement by family members, physicians and friends leads to participation in performing preventive behaviors -53. One According to our findings, lack of knowledge, doubt, fear, shame, hesitancy, busy lifestyle, and fatalism were some barriers to perform preventive behaviors. Similarly, several studies have shown that women did not follow preventive behaviors due to fear of the results, embarrassment, pain, discomfort, and doubt about the efficacy of mammography, high cost of mammography, lack of health care service, and lack of insurance service -56. As sLimitations include the potential that we studied women living in Tehran  and thus their views may not adequately reflect the views of women living in other settings. Further qualitative and quantitative investigations are needed to examine the experiences and needs of women from other regions and cultures.The findings indicated that positive attitudes; individual’s ability to control stress, healthy lifestyle, social support, and fewer barriers are most important parameters that might encourage women to perform breast cancer preventive behaviors.The authors declare that they have no competing interests.MK was the main investigator and involved in the study design, data collection, analysis and drafting of the manuscript. AM was involved in the study design, data analysis, and providing the final draft. FM contributed to the idea, study design, data analysis and, drafting of the manuscript. AR was involved in the study design, and data analysis. SN and DS participated in the data analysis and, drafting of the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1472-6874/14/41/prepub


Recent discussions on research priorities in the United States have revealed a widespread assumption that known risk factors entirely explain the historic international differences in rates of breast cancer. In fact, formal investigations of this question, both by modelling between-country differences and studies of migrants, indicate that an appreciable amount of the international differences in this disease remains unexplained. If this is not recognised, opportunities for research on breast cancer aetiology may be lost. Adrian L Harris and Leo KinlenMisconceptions and ill-founded theories can arise in all areas of science. However, the apparent accessibility of many epidemiology findings and popular interest in the subject can lead to additional misunderstandings. The article below is the fifth in an occasional series of short editorials highlighting some current misinterpretations of epidemiological findings. Invited authors will be given wide scope in judging the prevalence of the misconception under discussion. We hope that this series will prove instructive to cancer researchers in other disciplines as well as to students of epidemiology.Since the pioneering work of Segi  and HaenThis might have been expected to renew interest in what is unexplained, but this has not happened. Indeed, there are signs in research and funding circles  that many now assume that the international differences are adequately explained, particularly by reproductive factors. Exactly why this change in attitude has occurred is not clear, although the declines in the international variation may have appeared to reduce what needs to be explained, overlooking the relevance of past differences. Also, the impressive results of large collaborative studies of breast cancer risk factors  etc haveThere have been several quantitative attempts to assess the impact of these risk factors on geographic differences in risk, either by modelling or by studies of migrant groups. In an early example of the former, applied to a Japanese population with a breast cancer rate five times lower than in the US, this difference was reduced to over 2.5 times in one model and 1.6 in another . The valBreast cancer in Asian-American migrants was the subject of a population-based case–control study of breast cancer in Hawaii, Los Angeles and San Francisco, conducted in 1983–87 . Asians There are concerns about the robustness of any of the above methods to assess adequately the impact of controlling for recognised risk factors. The modelling exercises are based on assumptions about underlying baseline rates and relative risks for two very different populations (This issue aside, findings to date suggest that an appreciable amount of the international differences in breast cancer are not explained by known risk factors or mechanisms. With respect to mechanisms, attempts to find a hormonal basis for reproductive risk factors have on the whole been disappointing, although in the present context the report that the level of endogenous oestradiol was about one-third higher in US Whites than in rural Asian women held promise (It is important to recognise that known risk factors have not been found to explain the entirety of breast cancer epidemiology. To assume otherwise may limit the opportunities for aetiological research and preventive interventions.


BRCA1 and 2,020 BRCA2 carriers from 11 centers in the Consortium of Investigators of Modifiers of BRCA1/2 (CIMBA). Based on the BCAC data from women of European ancestry, we found evidence for a weak association with breast cancer risk for rs2180341  = 1.03, 95% CI 1.00–1.06, p = 0.023). There was evidence for heterogeneity in the ORs among studies . In CIMBA, we observed an inverse association with the minor allele of rs2180341 and breast cancer risk in BRCA1 mutation carriers , indicating a potential protective effect of this allele. These data suggest that that 6q22.33 confers a weak effect on breast cancer risk.Recently, a locus on chromosome 6q22.33 (rs2180341) was reported to be associated with increased breast cancer risk in the Ashkenazi Jewish (AJ) population, and this association was also observed in populations of non-AJ European ancestry. In the present study, we performed a large replication analysis of rs2180341 using data from 31,428 invasive breast cancer cases and 34,700 controls collected from 25 studies in the Breast Cancer Association Consortium (BCAC). In addition, we evaluated whether rs2180341 modifies breast cancer risk in 3,361  Genome-wide association analyses have recently identified multiple loci conferring genetic susceptibility to breast cancer BRCA mutation, followed by phase 2 analysis of 979 AJ controls and 950 AJ breast cancer cases ECHDC1 and RNF146, mapping to this locus. In a follow-up study, an association was observed in an independent analysis of 1,953 breast cancer cases and 1,467 controls of non-AJ, predominantly European ancestry  with some evidence of a stronger association for ER+ than ER- tumors Recently, a putative breast cancer susceptibility locus at chromosome 6q22.33 (tagged by rs2180341) was identified by a two-stage genome wide-association study (GWAS) based on a phase 1 analysis of 299 Ashkenazi Jewish (AJ) controls and 249 AJ kindreds with family history of breast cancer and no known BRCA1 or BRCA2 mutation carriers, by genotyping 5,381 mutation carriers from 11 studies in the Consortium of Investigators on Modifiers of BRCA1/2 (CIMBA).Our objective in the current analysis was to further investigate the association of the 6q22.33 locus with breast cancer risk. To this end, we genotyped rs2180341 in 27,950 invasive breast cancer cases and 32,219 controls from 23 case-control studies of primarily European ancestry and 2 studies of Asians included in the Breast Cancer Association Consortium (BCAC). We also evaluated whether rs2180341 was associated with breast cancer risk in Ethics committee approval was obtained for the collection and genetic analysis of all samples, and an informed written consent was obtained from all participants. For detailed description, see Twenty-five case-control studies (described in in situ diagnoses (736 cases). Final analyses included 27,950 invasive breast cancer cases and 32,219 controls of European ancestry, as well as 2,836 invasive breast cancer cases and 2,149 controls of Asian ancestry. All studies received approval from their institutional review committees and participants provided informed consent or were analyzed under specific coding procedures (ABCS).In the current dataset, we excluded breast cancer cases with BRCA1 and/or BRCA2. Data were available on year of birth, age at study recruitment, age at cancer diagnosis, age of bilateral prophylactic mastectomy, BRCA1 and BRCA2 mutation description, and ethnicity. Final analyses included 2,776 invasive breast cancer cases and 2,605 unaffected mutation carriers.Eleven studies  and 95% confidence intervals (CI). The percent of between-study heterogeneity was estimated using the IBRCA1 and BRCA2 mutation carriers. We used a robust variance approach to allow for the dependence among related mutation carriers. We also adjusted for study, country, ethnicity, and year of birth. Relative risk estimates were calculated separately for BRCA1 and BRCA2 mutation carriers.Hazard ratios (HRs) and 95% CIs were estimated using a weighted Cox regression approach as described in detail elsewhere BRCA1/2 mutation carriers beyond age 80.Mutation carriers were censored at the first breast or ovarian cancer or bilateral prophylactic mastectomy. Carriers who developed either cancer were censored at the time of bilateral prophylactic mastectomy only if it occurred more than a year prior to the cancer diagnosis . The remaining carriers were censored at the age of last observation. This was defined either by the age at interview or age at follow-up depending on the information provided by the participating center. Carriers censored at diagnosis of breast cancer were considered affected in the analysis. Carriers with a censoring/last follow-up age older than age 80 were censored at age 80 because there are no reliable cancer incidence rates for All analyses were performed with STATA (Version 10.0).The mean (±SD) age was 53.1 (±13.1) years for invasive cases, 52.7 (±11.8) years for controls. A total of 88.9% of invasive cases and 92.9% of controls were of European-descent. Other women were of Asian ancestry (9.0% cases and 6.2% controls) or unknown ancestry .Among controls of European descent, the minor allele frequency (MAF) of rs2180341 ranged from 22.6% to 28.7% , which was mainly attributable to the strong inverse associations for kConFab/AOCS and HMBCS, and a strong positive association for MSKCC and SBCS. Exclusion of these studies did not alter the overall magnitude of the relative risk estimate and there was no longer evidence of between-study heterogeneity .There was some evidence for an association between the G allele and breast cancer risk .Among women of Asian ancestry, we did not observe an association with overall breast cancer risk and the 6q22.33 locus , but theWe examined the association of rs2180341 with breast cancer by ER status, which was available from 19 studies that were conducted predominantly among women of European ancestry. There was no significant difference in the per-allele OR for rs2180341 in the risk of ER+ and ER- disease , BRCA1 unaffected carriers at age 43.1 (±12.7 SD), BRCA2 affected carriers at age 44.3 (±13.5 SD), and BRCA2 unaffected carriers at age 45.1 (±13.5 SD). All study subjects were of European-descent. Genotype frequencies were in HWE for all studies. Among unaffected carriers, the MAF of rs2180341 ranged from 19.2% to 33.3%  or BRCA2 (p = 0.19) mutation carriers , with the between study heterogeneity of p = 0.005.We have investigated if other factors also contribute to the heterogeneity of risk estimates observed in this large combined study. As illustrated in the The initial reports of this locus suggested a stronger association for rs2180341 with ER-positive tumors than ER-negative tumors BRCA1 mutations. In the 3,361 BRCA1 mutation carriers in CIMBA, we observed a statistically significant inverse association with breast cancer risk  and BRCA1 may act in conjunction to regulate ER-mediated pathways in breast cancer tumorigenesis RNF146 to be a critical player in Wnt signaling pathway, providing an evidence for novel biochemical properties of the enzyme in ubiquitination of axin, a critical protein involved in stabilization of beta-catenin RNF146 in other types of common cancer.Lastly, our study provides the first estimate of the potential breast cancer modifying effect of 6q22.33 in carriers of RNF146 with the dosage of high-risk allele of rs2180341. While sequencing of the subset of breast tumors did not identify any coding SNPs in RNF146 associated with the risk allele RNF146. In order to provide further biological insight, the more systematic analysis would be needed to test the correlation of RNF146 expression with identified genetic variants in larger subset of breast tumors.Hence, further functional analysis is needed to link rs2180341 with tumorigenesis. In our original discovery study, we have demonstrated that rs2180341 tags relatively conserved region of ∼100 kb In conclusion, this large study found evidence for a weak overall association between the 6q22.33 locus and sporadic breast cancer risk. Relative risk estimates for rs2180341 were lower in non-AJ Europeans as compared to AJ populations. The study illustrates the difficulties inherent in the reliable estimation of low risk susceptibility alleles – even with a study as large as the current one, in which the overall effect was only marginally significant. It is likely there are many such variants, conferring ORs<1.1, and characterizing such associations with common diseases disease presents a major challenge. It is possible that comprehensive sequencing across the region may identify the true causal variant(s) with stronger effects. If the heterogeneity among studies is due to differences in linkage disequilibrium (LD), fine-scale mapping might also allow identification of more strongly associated variants. The combined effect of these common variants and other as-yet-undiscovered rare variants, together with lifestyle risk factors, could provide the basis for risk algorithms for the preventive management of breast cancer.Funding S1The description of sources of funding for the study in detail.(DOCX)Click here for additional data file.Supporting Information S1Table A: Summary of the 25 breast cancer case studies used in the BCAC analyses Table B: Genotype frequency among Caucasian BCAC case and controls, minor allele frequencies (MAF), and Hardy-Weinberg Equilibrium (HWE) by study Table C: Summary of the 11 breast cancer case studies used in the CIMBA analyses Table D: Genotype frequency among CIMBA case and controls, minor allele frequencies (MAF), and Hardy-Weinberg Equilibrium (HWE) by study Table E: Ethics committee approvals  Table F: SNPs from 6q22.33 with the highest functional impact and highly correlated with rs2180341. Correlated proxies of rs2180341 (r2>0.8) were extracted from latest release of 1000 genomes project on ~300 individuals of European ancestry. The functional impact of all correlated SNPs was assessed using the pipelines of ANNOVAR suite. The functional impact (FI) of each SNP for each of 3 selected categories is defined by FI score. TF binding site prediction also includes DNAI hypersensitivity data. Conserved elements were assessed using placental 46way analysis. r-square values are relative to rs2180341.(DOC)Click here for additional data file.


Experimental data suggest a protective effect of vitamin D on breast cancer; however, epidemiologic results remain inclusive. With a Chinese population-based case-control study and meta-analysis of the observational studies, we here systematically evaluated the association of blood 25(OH)D level and breast cancer risk. With 593 breast cancer cases and 580 cancer-free controls from Shanghai, China, we found that 80% of the normal women had severe vitamin D deficiency (less than 20 ng/mL) and 15.2% had mild deficiency (20 to 30 ng/mL) and only 4.8% of women had sufficient vitamin D level (>30 ng/mL) while the proportion was 96.1%, 3.2% and 0.7% respectively for the breast cancer patients. Compared to those with the lowest quartile of plasma 25(OH)D level, women with highest quartile 25(OH)D level showed a significant decreased breast cancer risk  and every 1 ng/ml increment of plasma 25(OH)D level led to a 16% lower odds of breast cancer . From the meta-analysis of the observational studies, we found that women with highest quantile of blood 25(OH)D level was associated with a significantly reduced breast cancer risk compared to those with lowest quantile of blood 25(OH)D level for the 11 nested case-control and retrospective studies  and 10 case-control studies . These results suggest that vitamin D may have a chemo-preventive effect against breast cancer. The patients were genetically unrelated ethnic Han Chinese from Shanghai and the surrounding regions. All patients had been histopathologically diagnosed with primary breast cancer and not yet treated. A 5-ml blood sample was provided by the patients before any treatment after the written informed consent was obtained. Patients were excluded if they had received any treatment for the breast cancer, or were not Chinese Han population, or with any other cancer disease.The 580 unrelated female control samples were randomly extracted from the women who have participated in the Breast Cancer Screening Project of the same hospital during the year 2005 to 2008 at Minhang district of Shanghai, and the controls reported no previous cancer history and were further confirmed with no evidence of breast cancer, or any suspicious precancerous lesions of the breast by the physicians. The controls were Chinese Han population and between 25 to 85 years old. A 5-ml of blood sample was also collected for the controls and the date was recorded. The controls were matched with the breast cases with ±5 years old and the time of blood collection (±3 months).All the participants were personally interviewed with the pre-trained interviewers to complete the structured questionnaire that evaluated the potential breast cancer factors for women, including age, age at menarche, age at first birth, marital status, and the first-degree relatives’ history of breast cancer. For the menopausal status, women were classified as postmenopausal if they affirmed that their menstruations had ceased or had no menstrual history during the past 12 months or more. Those with normal menstrual cycle or still with menstrual history during the past 12 months were assessed as pre-menopausal/peri-menopausal status as defined This project has been approved by the Scientific and Ethical Committee of the Cancer Hospital of Fudan University. All of the samples were collected with a written informed consent provided by the participants.After collection, all the blood samples were centrifuged at 3000g for 15 minutes and the plasma were immediately storage at −20°C in aliquots until use. The plasma 25(OH)D level was determined using a radioimmunoassay kit  according to the standard procedure. All of the coefficients of variation for within-assay and between-assay were less than 10%.We systematically searched the PubMed and MEDLINE databases for eligibility studies that have been published online up to September 2012. We used the term ‘‘breast cancer’’ in combination with ‘‘vitamin D,’’ or ‘‘25-hydroxyvitamin D” and “plasma” or “serum” to identify the studies that have evaluated the blood 25(OH)D level and breast cancer risk. References of the included studies and related reviews were checked to identify any missing data in the database search. Only studies reported in English were included in our meta-analysis.Eligibility studies should have reported blood 25(OH)D level in quantiles and the corresponding risk estimates and their 95% confidential intervals (CI), or provided the estimates for the highest quantile in contrast with the lowest quantile of blood 25(OH)D, or provided sufficient data that could be used to calculate the risk estimate and its 95% CI between the quantiles. If overlapping populations were identified between the studies, only the most complete data were included in the meta-analysis. Descriptive characteristics about each study, when available, were extracted from the published reports: first author’s last name, the year of publication, country of the study performed, study design, the sample size, the age range of the subjects, blood 25(OH)D level, the risk estimates that reflected the greatest degree of control for potential confounders with their corresponding confidence intervals, and the adjusted covariates.2 test  and Student’s t-test (for continuous variables) were used to evaluate the differences in demographic characteristics and selected variables. To assess the seasonal distribution of vitamin D level in the cases and controls, the sample collect date was categorized into spring (March to May), summer (June to August), autumn (September to November) and winter . Comparisons of the vitamin D level between cases and controls and in different seasons were done using the nonparametric Wilcoxon tests. To determine the association of vitamin D status and breast cancer risk, we firstly divided the 25(OH)D level into quartiles according to its distribution in the control group. The multivariable conditional logistic regression was used to calculated the odds ratios (ORs) and their 95% confidential interval (95% CI) for quartiles Q2 through Q4 compared with the lowest quartile Q1 with or without the adjustment of the co-variants of age, age at first birth, age at menarche, menopausal status, first-degree family history of breast cancer and use of oral contraception. We also assessed the 25(OH)D level as continuous variants (per 1 ng/ml increment) to evaluate its association with breast cancer risk.The χ2 values (significance set at I2>25%). Publication bias was represented as funnel plot and further assessed using the Egger’s test www.r-project.com).For the meta-analysis, the risk estimate OR and its 95% CI was extracted or calculated from each study. To establish the appropriate weighting for each study, the SE for each logarithm OR was calculated and each study was weighted using the generic inverse variance approach. The DerSimonian and Laird random-effects method which considers the variability within and between studies was used to calculate the pooled estimate The baseline characteristics of the participants are shown in In total, the mean 25(OH)D level for the normal women was 15.67 ng/ml and 11.31 ng/ml for the breast cancer patients . Among the normal women, 80.0% showed vitamin D severe deficiency (less than 20 ng/ml), 15.2% have vitamin D deficiency status (20–30 ng/ml) and only 4.8% have sufficiency vitamin D in their blood (>30 ng/ml). However, the situation of vitamin D status for the breast cancer patients is worse with 96.1% of severe vitamin D deficiency, 3.2% of vitamin D deficiency and only 0.7% have sufficiency vitamin D in their blood sample (P<0.001). The mean 25(OH)D level was different among the seasons, with the highest 25(OH)D level was found in summer (17.76 ng/ml) and lowest in winter (10.75 ng/ml) for the normal women. The mean circulating 25(OH)D level was 15.67 ng/ml and 16.25 ng/ml in spring and autumn respectively for the normal women. For the breast cancer patients, the mean 25(OH)D was highest level in autumn (14.38 ng/ml) and lowest in the spring (10.16 ng/ml) with the mean 25(OH)D level was 11.24 ng/ml and 11.27 ng/ml in summer and winter. The seasonal 25(OH)D level was significantly lower for the breast cancer patients compared to normal women except the winter .We found a significantly inverse association between plasma concentration of 25(OH)D and breast cancer risk. Compared with the lowest quartile (≤10.4 ng/ml), women with higher 25(OH)D level showed a significantly reduced breast cancer risk with the adjusted OR was 0.43 (95% CI = 0.31–0.60) for Q2 (10.4–13.4 ng/ml), 0.24 (95% CI = 0.17–0.35) for Q3 (13.4–17.9 ng/ml) and 0.10 (95% CI = 0.06–0.15) for Q4 (≥17.9 ng/ml) and the p-trend value was less than 0.001 . Additio2 = 40%). The summary estimates for the population based case-control studies  and hospital based case-control studies  also suggested that women with higher blood 25(OH)D level showed a reduced breast cancer risk. Statistically significant heterogeneity among the subgroup studies was found D concentration was less than 30 ng/ml. A dual-centered study conducted by Woo et al. in China found that more than 90% of young women in Beijing and Hong Kong had circulating 25(OH)D level less than 50 nmol/L  2D could inhibit cell proliferation, promote cell differentiation, inhibit the tumor cell metastasis and angiogenesis, suggests an anticancer activity for the vitamin D metabolite 2D in the kidney nephrons and possibly also in breast tissue. Many epidemiological studies have been conducted to evaluate the association for the blood 25(OH)D and/or 1α,25-(OH)2D level and the risk for breast cancer. Our previous systematic review suggested that no significant association between blood 1α,25-(OH)2D level and breast cancer risk 2D is under tight regulation by 1a-hydroxylase which is under the control of the parathyroid hormone (PTH) in response to serum calcium. For blood 25(OH)D level, 7 population based case-control studies and 3 hospital based case-control studies consistently reported an inverse relationship between blood 25(OH)D level and breast cancer risk; however, the blood sample for the breast cancer patients was collected after the diagnosis. It not clear that whether cancer diagnosis may have influenced circulating 25(OH)D through treatment or behavioral changes; however, the blood samples for the patients were usually collected shortly after the diagnosis and prior to any treatment. We cannot exclude the possibility that the presenting of breast cancer cells may influence the blood 25(OH)D level. From the systematic literature search, we have identified 10 prospective nested case-control studies have evaluated the pre-diagnostic blood 25(OH)D level and breast cancer risk. Rejnmark et al. observed a 48%  reduced breast cancer risk for those with a plasma 25(OH)D level over 84 nM compared to those less than 60 nM for the Danish women; however, significant reduced breast cancer was only found for the premenopausal women  but not for the postmenopausal women  in the stratification study Experimental studies have revealed that 1α,25-(OH)Vitamin D deficiency is common for breast cancer patients and it may be correlated with poorer prognosis. Breast cancer patients with early-stage have higher vitamin D level than those of advanced stage disease We acknowledged there are several limitations for the current study. Firstly, the design of our population based case-control study is more prone to a variety of biases such as selection bias and recall bias, and the results could not excluded the possibility that lower vitamin D level for the patients was affected by the presenting of cancer cells. Although the data from the nested case-control studies suggested an anticancer activity for the higher blood 25(OH)D level, we also could not directly come to the conclusion that high vitamin D supplementation would have reduced the breast cancer risk. It should be confirmed by other well designed observational studies such as cohort studies as well as by randomized clinical trials. A reduced of overall cancer risk was found when vitamin D  and calcium  were simultaneously administered to postmenopausal women in a clinical trial conducted by Lappe et al., although only a few breast cancers occurred in either subgroup In conclusion, our study suggested that higher blood vitamin D level was associated with a significantly reduced risk of breast cancer. Considering the vitamin D deficiency is common in populations, women will likely to be benefited by taking sufficient vitamin D with the goal of reducing the breast cancer risk; however, more studies should be conducted before we could achieve the goals.