Physical activity, sedentary time and breast cancer risk: a Mendelian randomisation study.

OBJECTIVES: Physical inactivity and sedentary behaviour are associated with higher breast cancer risk in observational studies, but ascribing causality is difficult. Mendelian randomisation (MR) assesses causality by simulating randomised trial groups using genotype. We assessed whether lifelong physical activity or sedentary time, assessed using genotype, may be causally associated with breast cancer risk overall, pre/post-menopause, and by case-groups defined by tumour characteristics. METHODS: We performed two-sample inverse-variance-weighted MR using individual-level Breast Cancer Association Consortium case-control data from 130 957 European-ancestry women (69 838 invasive cases), and published UK Biobank data (n=91 105-377 234). Genetic instruments were single nucleotide polymorphisms (SNPs) associated in UK Biobank with wrist-worn accelerometer-measured overall physical activity (nsnps=5) or sedentary time (nsnps=6), or accelerometer-measured (nsnps=1) or self-reported (nsnps=5) vigorous physical activity. RESULTS: Greater genetically-predicted overall activity was associated with lower breast cancer overall risk (OR=0.59; 95% confidence interval (CI) 0.42 to 0.83 per-standard deviation (SD;~8 milligravities acceleration)) and for most case-groups. Genetically-predicted vigorous activity was associated with lower risk of pre/perimenopausal breast cancer (OR=0.62; 95% CI 0.45 to 0.87,≥3 vs. 0 self-reported days/week), with consistent estimates for most case-groups. Greater genetically-predicted sedentary time was associated with higher hormone-receptor-negative tumour risk (OR=1.77; 95% CI 1.07 to 2.92 per-SD (~7% time spent sedentary)), with elevated estimates for most case-groups. Results were robust to sensitivity analyses examining pleiotropy (including weighted-median-MR, MR-Egger). CONCLUSION: Our study provides strong evidence that greater overall physical activity, greater vigorous activity, and lower sedentary time are likely to reduce breast cancer risk. More widespread adoption of active lifestyles may reduce the burden from the most common cancer in women.

Mendelian randomisation study of smoking exposure in relation to breast cancer risk.

BACKGROUND: Despite a modest association between tobacco smoking and breast cancer risk reported by recent epidemiological studies, it is still equivocal whether smoking is causally related to breast cancer risk. METHODS: We applied Mendelian randomisation (MR) to evaluate a potential causal effect of cigarette smoking on breast cancer risk. Both individual-level data as well as summary statistics for 164 single-nucleotide polymorphisms (SNPs) reported in genome-wide association studies of lifetime smoking index (LSI) or cigarette per day (CPD) were used to obtain MR effect estimates. Data from 108,420 invasive breast cancer cases and 87,681 controls were used for the LSI analysis and for the CPD analysis conducted among ever-smokers from 26,147 cancer cases and 26,072 controls. Sensitivity analyses were conducted to address pleiotropy. RESULTS: Genetically predicted LSI was associated with increased breast cancer risk (OR 1.18 per SD, 95% CI: 1.07-1.30, P = 0.11 × 10-2), but there was no evidence of association for genetically predicted CPD (OR 1.02, 95% CI: 0.78-1.19, P = 0.85). The sensitivity analyses yielded similar results and showed no strong evidence of pleiotropic effect. CONCLUSION: Our MR study provides supportive evidence for a potential causal association with breast cancer risk for lifetime smoking exposure but not cigarettes per day among smokers.

Gene-Environment Interactions Relevant to Estrogen and Risk of Breast Cancer: Can Gene-Environment Interactions Be Detected Only among Candidate SNPs from Genome-Wide Association Studies?

In this study we aim to examine gene-environment interactions (GxEs) between genes involved with estrogen metabolism and environmental factors related to estrogen exposure. GxE analyses were conducted with 1970 Korean breast cancer cases and 2052 controls in the case-control study, the Seoul Breast Cancer Study (SEBCS). A total of 11,555 SNPs from the 137 candidate genes were included in the GxE analyses with eight established environmental factors. A replication test was conducted by using an independent population from the Breast Cancer Association Consortium (BCAC), with 62,485 Europeans and 9047 Asians. The GxE tests were performed by using two-step methods in GxEScan software. Two interactions were found in the SEBCS. The first interaction was shown between rs13035764 of NCOA1 and age at menarche in the GE|2df model (p-2df = 1.2 × 10-3). The age at menarche before 14 years old was associated with the high risk of breast cancer, and the risk was higher when subjects had homozygous minor allele G. The second GxE was shown between rs851998 near ESR1 and height in the GE|2df model (p-2df = 1.1 × 10-4). Height taller than 160 cm was associated with a high risk of breast cancer, and the risk increased when the minor allele was added. The findings were not replicated in the BCAC. These results would suggest specificity in Koreans for breast cancer risk.

Functional annotation of the 2q35 breast cancer risk locus implicates a structural variant in influencing activity of a long-range enhancer element.

A combination of genetic and functional approaches has identified three independent breast cancer risk loci at 2q35. A recent fine-scale mapping analysis to refine these associations resulted in 1 (signal 1), 5 (signal 2), and 42 (signal 3) credible causal variants at these loci. We used publicly available in silico DNase I and ChIP-seq data with in vitro reporter gene and CRISPR assays to annotate signals 2 and 3. We identified putative regulatory elements that enhanced cell-type-specific transcription from the IGFBP5 promoter at both signals (30- to 40-fold increased expression by the putative regulatory element at signal 2, 2- to 3-fold by the putative regulatory element at signal 3). We further identified one of the five credible causal variants at signal 2, a 1.4 kb deletion (esv3594306), as the likely causal variant; the deletion allele of this variant was associated with an average additional increase in IGFBP5 expression of 1.3-fold (MCF-7) and 2.2-fold (T-47D). We propose a model in which the deletion allele of esv3594306 juxtaposes two transcription factor binding regions (annotated by estrogen receptor alpha ChIP-seq peaks) to generate a single extended regulatory element. This regulatory element increases cell-type-specific expression of the tumor suppressor gene IGFBP5 and, thereby, reduces risk of estrogen receptor-positive breast cancer (odds ratio = 0.77, 95% CI 0.74-0.81, p = 3.1 × 10-31).

Author Correction: A case-only study to identify genetic modifiers of breast cancer risk for BRCA1/BRCA2 mutation carriers.

A Correction to this paper has been published: https://doi.org/10.1038/s41467-021-23162-4.

A case-only study to identify genetic modifiers of breast cancer risk for BRCA1/BRCA2 mutation carriers.

Breast cancer (BC) risk for BRCA1 and BRCA2 mutation carriers varies by genetic and familial factors. About 50 common variants have been shown to modify BC risk for mutation carriers. All but three, were identified in general population studies. Other mutation carrier-specific susceptibility variants may exist but studies of mutation carriers have so far been underpowered. We conduct a novel case-only genome-wide association study comparing genotype frequencies between 60,212 general population BC cases and 13,007 cases with BRCA1 or BRCA2 mutations. We identify robust novel associations for 2 variants with BC for BRCA1 and 3 for BRCA2 mutation carriers, P < 10-8, at 5 loci, which are not associated with risk in the general population. They include rs60882887 at 11p11.2 where MADD, SP11 and EIF1, genes previously implicated in BC biology, are predicted as potential targets. These findings will contribute towards customising BC polygenic risk scores for BRCA1 and BRCA2 mutation carriers.

Breast Cancer Risk Genes - Association Analysis in More than 113,000 Women.

BACKGROUND: Genetic testing for breast cancer susceptibility is widely used, but for many genes, evidence of an association with breast cancer is weak, underlying risk estimates are imprecise, and reliable subtype-specific risk estimates are lacking. METHODS: We used a panel of 34 putative susceptibility genes to perform sequencing on samples from 60,466 women with breast cancer and 53,461 controls. In separate analyses for protein-truncating variants and rare missense variants in these genes, we estimated odds ratios for breast cancer overall and tumor subtypes. We evaluated missense-variant associations according to domain and classification of pathogenicity. RESULTS: Protein-truncating variants in 5 genes (ATM, BRCA1, BRCA2, CHEK2, and PALB2) were associated with a risk of breast cancer overall with a P value of less than 0.0001. Protein-truncating variants in 4 other genes (BARD1, RAD51C, RAD51D, and TP53) were associated with a risk of breast cancer overall with a P value of less than 0.05 and a Bayesian false-discovery probability of less than 0.05. For protein-truncating variants in 19 of the remaining 25 genes, the upper limit of the 95% confidence interval of the odds ratio for breast cancer overall was less than 2.0. For protein-truncating variants in ATM and CHEK2, odds ratios were higher for estrogen receptor (ER)-positive disease than for ER-negative disease; for protein-truncating variants in BARD1, BRCA1, BRCA2, PALB2, RAD51C, and RAD51D, odds ratios were higher for ER-negative disease than for ER-positive disease. Rare missense variants (in aggregate) in ATM, CHEK2, and TP53 were associated with a risk of breast cancer overall with a P value of less than 0.001. For BRCA1, BRCA2, and TP53, missense variants (in aggregate) that would be classified as pathogenic according to standard criteria were associated with a risk of breast cancer overall, with the risk being similar to that of protein-truncating variants. CONCLUSIONS: The results of this study define the genes that are most clinically useful for inclusion on panels for the prediction of breast cancer risk, as well as provide estimates of the risks associated with protein-truncating variants, to guide genetic counseling. (Funded by European Union Horizon 2020 programs and others.).

CYP3A7*1C allele: linking premenopausal oestrone and progesterone levels with risk of hormone receptor-positive breast cancers.

BACKGROUND: Epidemiological studies provide strong evidence for a role of endogenous sex hormones in the aetiology of breast cancer. The aim of this analysis was to identify genetic variants that are associated with urinary sex-hormone levels and breast cancer risk. METHODS: We carried out a genome-wide association study of urinary oestrone-3-glucuronide and pregnanediol-3-glucuronide levels in 560 premenopausal women, with additional analysis of progesterone levels in 298 premenopausal women. To test for the association with breast cancer risk, we carried out follow-up genotyping in 90,916 cases and 89,893 controls from the Breast Cancer Association Consortium. All women were of European ancestry. RESULTS: For pregnanediol-3-glucuronide, there were no genome-wide significant associations; for oestrone-3-glucuronide, we identified a single peak mapping to the CYP3A locus, annotated by rs45446698. The minor rs45446698-C allele was associated with lower oestrone-3-glucuronide (-49.2%, 95% CI -56.1% to -41.1%, P = 3.1 × 10-18); in follow-up analyses, rs45446698-C was also associated with lower progesterone (-26.7%, 95% CI -39.4% to -11.6%, P = 0.001) and reduced risk of oestrogen and progesterone receptor-positive breast cancer (OR = 0.86, 95% CI 0.82-0.91, P = 6.9 × 10-8). CONCLUSIONS: The CYP3A7*1C allele is associated with reduced risk of hormone receptor-positive breast cancer possibly mediated via an effect on the metabolism of endogenous sex hormones in premenopausal women.

The functional ALDH2 polymorphism is associated with breast cancer risk: A pooled analysis from the Breast Cancer Association Consortium.

BACKGROUND: Epidemiological studies consistently indicate that alcohol consumption is an independent risk factor for female breast cancer (BC). Although the aldehyde dehydrogenase 2 (ALDH2) polymorphism (rs671: Glu>Lys) has a strong effect on acetaldehyde metabolism, the association of rs671 with BC risk and its interaction with alcohol intake have not been fully elucidated. We conducted a pooled analysis of 14 case-control studies, with individual data on Asian ancestry women participating in the Breast Cancer Association Consortium. METHODS: We included 12,595 invasive BC cases and 12,884 controls for the analysis of rs671 and BC risk, and 2,849 invasive BC cases and 3,680 controls for the analysis of the gene-environment interaction between rs671 and alcohol intake for BC risk. The pooled odds ratios (OR) with 95% confidence intervals (CI) associated with rs671 and its interaction with alcohol intake for BC risk were estimated using logistic regression models. RESULTS: The Lys/Lys genotype of rs671 was associated with increased BC risk (OR = 1.16, 95% CI 1.03-1.30, p = 0.014). According to tumor characteristics, the Lys/Lys genotype was associated with estrogen receptor (ER)-positive BC (OR = 1.19, 95% CI 1.05-1.36, p = 0.008), progesterone receptor (PR)-positive BC (OR = 1.19, 95% CI 1.03-1.36, p = 0.015), and human epidermal growth factor receptor 2 (HER2)-negative BC (OR = 1.25, 95% CI 1.05-1.48, p = 0.012). No evidence of a gene-environment interaction was observed between rs671 and alcohol intake (p = 0.537). CONCLUSION: This study suggests that the Lys/Lys genotype confers susceptibility to BC risk among women of Asian ancestry, particularly for ER-positive, PR-positive, and HER2-negative tumor types.

Targeted Resequencing of the Coding Sequence of 38 Genes Near Breast Cancer GWAS Loci in a Large Case-Control Study.

BACKGROUND: Genes regulated by breast cancer risk alleles identified through genome-wide association studies (GWAS) may harbor rare coding risk alleles. METHODS: We sequenced the coding regions for 38 genes within 500 kb of 38 lead GWAS SNPs in 13,538 breast cancer cases and 5,518 controls. RESULTS: Truncating variants in these genes were rare, and were not associated with breast cancer risk. Burden testing of rare missense variants highlighted 5 genes with some suggestion of an association with breast cancer, although none met the multiple testing thresholds: MKL1, FTO, NEK10, MDM4, and COX11. Six common alleles in COX11, MAP3K1 (two), and NEK10 (three) were associated at the P < 0.0001 significance level, but these likely reflect linkage disequilibrium with causal regulatory variants. CONCLUSIONS: There was no evidence that rare coding variants in these genes confer substantial breast cancer risks. However, more modest effect sizes could not be ruled out. IMPACT: We tested the hypothesis that rare variants in 38 genes near breast cancer GWAS loci may mediate risk. These variants do not appear to play a major role in breast cancer heritability.

The BRCA2 c.68-7T > A variant is not pathogenic: A model for clinical calibration of spliceogenicity.

Although the spliceogenic nature of the BRCA2 c.68-7T > A variant has been demonstrated, its association with cancer risk remains controversial. In this study, we accurately quantified by real-time PCR and digital PCR (dPCR), the BRCA2 isoforms retaining or missing exon 3. In addition, the combined odds ratio for causality of the variant was estimated using genetic and clinical data, and its associated cancer risk was estimated by case-control analysis in 83,636 individuals. Co-occurrence in trans with pathogenic BRCA2 variants was assessed in 5,382 families. Exon 3 exclusion rate was 4.5-fold higher in variant carriers (13%) than controls (3%), indicating an exclusion rate for the c.68-7T > A allele of approximately 20%. The posterior probability of pathogenicity was 7.44 × 10-115 . There was neither evidence for increased risk of breast cancer (OR 1.03; 95% CI 0.86-1.24) nor for a deleterious effect of the variant when co-occurring with pathogenic variants. Our data provide for the first time robust evidence of the nonpathogenicity of the BRCA2 c.68-7T > A. Genetic and quantitative transcript analyses together inform the threshold for the ratio between functional and altered BRCA2 isoforms compatible with normal cell function. These findings might be exploited to assess the relevance for cancer risk of other BRCA2 spliceogenic variants.

Inherited mutations in BRCA1 and BRCA2 in an unselected multiethnic cohort of Asian patients with breast cancer and healthy controls from Malaysia.

BACKGROUND: Genetic testing for BRCA1 and BRCA2 is offered typically to selected women based on age of onset and family history of cancer. However, current internationally accepted genetic testing referral guidelines are built mostly on data from cancer genetics clinics in women of European descent. To evaluate the appropriateness of such guidelines in Asians, we have determined the prevalence of germ line variants in an unselected cohort of Asian patients with breast cancer and healthy controls. METHODS: Germ line DNA from a hospital-based study of 2575 unselected patients with breast cancer and 2809 healthy controls were subjected to amplicon-based targeted sequencing of exonic and proximal splice site junction regions of BRCA1 and BRCA2 using the Fluidigm Access Array system, with sequencing conducted on a Illumina HiSeq2500 platform. Variant calling was performed with GATK UnifiedGenotyper and were validated by Sanger sequencing. RESULTS: Fifty-five (2.1%) BRCA1 and 66 (2.6%) BRCA2 deleterious mutations were identified among patients with breast cancer and five (0.18%) BRCA1 and six (0.21%) BRCA2 mutations among controls. One thousand one hundred and eighty-six (46%) patients and 97 (80%) carriers fulfilled the National Comprehensive Cancer Network guidelines for genetic testing. CONCLUSION: Five per cent of unselected Asian patients with breast cancer carry deleterious variants in BRCA1 or BRCA2. While current referral guidelines identified the majority of carriers, one in two patients would be referred for genetic services. Given that such services are largely unavailable in majority of low-resource settings in Asia, our study highlights the need for more efficient guidelines to identify at-risk individuals in Asia.

Rare, protein-truncating variants in ATM, CHEK2 and PALB2, but not XRCC2, are associated with increased breast cancer risks.

BACKGROUND: Breast cancer (BC) is the most common malignancy in women and has a major heritable component. The risks associated with most rare susceptibility variants are not well estimated. To better characterise the contribution of variants in ATM, CHEK2, PALB2 and XRCC2, we sequenced their coding regions in 13 087 BC cases and 5488 controls from East Anglia, UK. METHODS: Gene coding regions were enriched via PCR, sequenced, variant called and filtered for quality. ORs for BC risk were estimated separately for carriers of truncating variants and of rare missense variants, which were further subdivided by functional domain and pathogenicity as predicted by four in silico algorithms. RESULTS: Truncating variants in PALB2 (OR=4.69, 95% CI 2.27 to 9.68), ATM (OR=3.26; 95% CI 1.82 to 6.46) and CHEK2 (OR=3.11; 95% CI 2.15 to 4.69), but not XRCC2 (OR=0.94; 95% CI 0.26 to 4.19) were associated with increased BC risk. Truncating variants in ATM and CHEK2 were more strongly associated with risk of oestrogen receptor (ER)-positive than ER-negative disease, while those in PALB2 were associated with similar risks for both subtypes. There was also some evidence that missense variants in ATM, CHEK2 and PALB2 may contribute to BC risk, but larger studies are necessary to quantify the magnitude of this effect. CONCLUSIONS: Truncating variants in PALB2 are associated with a higher risk of BC than those in ATM or CHEK2. A substantial risk of BC due to truncating XRCC2 variants can be excluded.

Fine scale mapping of the 17q22 breast cancer locus using dense SNPs, genotyped within the Collaborative Oncological Gene-Environment Study (COGs).

Genome-wide association studies have found SNPs at 17q22 to be associated with breast cancer risk. To identify potential causal variants related to breast cancer risk, we performed a high resolution fine-mapping analysis that involved genotyping 517 SNPs using a custom Illumina iSelect array (iCOGS) followed by imputation of genotypes for 3,134 SNPs in more than 89,000 participants of European ancestry from the Breast Cancer Association Consortium (BCAC). We identified 28 highly correlated common variants, in a 53 Kb region spanning two introns of the STXBP4 gene, that are strong candidates for driving breast cancer risk (lead SNP rs2787486 (OR = 0.92; CI 0.90-0.94; P = 8.96 × 10(-15))) and are correlated with two previously reported risk-associated variants at this locus, SNPs rs6504950 (OR = 0.94, P = 2.04 × 10(-09), r(2) = 0.73 with lead SNP) and rs1156287 (OR = 0.93, P = 3.41 × 10(-11), r(2) = 0.83 with lead SNP). Analyses indicate only one causal SNP in the region and several enhancer elements targeting STXBP4 are located within the 53 kb association signal. Expression studies in breast tumor tissues found SNP rs2787486 to be associated with increased STXBP4 expression, suggesting this may be a target gene of this locus.

No evidence that protein truncating variants in BRIP1 are associated with breast cancer risk: implications for gene panel testing.

BACKGROUND: BRCA1 interacting protein C-terminal helicase 1 (BRIP1) is one of the Fanconi Anaemia Complementation (FANC) group family of DNA repair proteins. Biallelic mutations in BRIP1 are responsible for FANC group J, and previous studies have also suggested that rare protein truncating variants in BRIP1 are associated with an increased risk of breast cancer. These studies have led to inclusion of BRIP1 on targeted sequencing panels for breast cancer risk prediction. METHODS: We evaluated a truncating variant, p.Arg798Ter (rs137852986), and 10 missense variants of BRIP1, in 48 144 cases and 43 607 controls of European origin, drawn from 41 studies participating in the Breast Cancer Association Consortium (BCAC). Additionally, we sequenced the coding regions of BRIP1 in 13 213 cases and 5242 controls from the UK, 1313 cases and 1123 controls from three population-based studies as part of the Breast Cancer Family Registry, and 1853 familial cases and 2001 controls from Australia. RESULTS: The rare truncating allele of rs137852986 was observed in 23 cases and 18 controls in Europeans in BCAC (OR 1.09, 95% CI 0.58 to 2.03, p=0.79). Truncating variants were found in the sequencing studies in 34 cases (0.21%) and 19 controls (0.23%) (combined OR 0.90, 95% CI 0.48 to 1.70, p=0.75). CONCLUSIONS: These results suggest that truncating variants in BRIP1, and in particular p.Arg798Ter, are not associated with a substantial increase in breast cancer risk. Such observations have important implications for the reporting of results from breast cancer screening panels.