Romualdo Cardoso, S.
Gillespie, A.
Haider, S.
Fletcher, O.
(2022). Functional annotation of breast cancer risk loci: current progress and future directions. Br j cancer,
Vol.126
(7),
pp. 981-993.
show abstract
full text
Genome-wide association studies coupled with large-scale replication and fine-scale mapping studies have identified more than 150 genomic regions that are associated with breast cancer risk. Here, we review efforts to translate these findings into a greater understanding of disease mechanism. Our review comes in the context of a recently published fine-scale mapping analysis of these regions, which reported 352 independent signals and a total of 13,367 credible causal variants. The vast majority of credible causal variants map to noncoding DNA, implicating regulation of gene expression as the mechanism by which functional variants influence risk. Accordingly, we review methods for defining candidate-regulatory sequences, methods for identifying putative target genes and methods for linking candidate-regulatory sequences to putative target genes. We provide a summary of available data resources and identify gaps in these resources. We conclude that while much work has been done, there is still much to do. There are, however, grounds for optimism; combining statistical data from fine-scale mapping with functional data that are more representative of the normal "at risk" breast, generated using new technologies, should lead to a greater understanding of the mechanisms that influence an individual woman's risk of breast cancer..
Baxter, J.S.
Johnson, N.
Tomczyk, K.
Gillespie, A.
Maguire, S.
Brough, R.
Fachal, L.
Michailidou, K.
Bolla, M.K.
Wang, Q.
Dennis, J.
Ahearn, T.U.
Andrulis, I.L.
Anton-Culver, H.
Antonenkova, N.N.
Arndt, V.
Aronson, K.J.
Augustinsson, A.
Becher, H.
Beckmann, M.W.
Behrens, S.
Benitez, J.
Bermisheva, M.
Bogdanova, N.V.
Bojesen, S.E.
Brenner, H.
Brucker, S.Y.
Cai, Q.
Campa, D.
Canzian, F.
Castelao, J.E.
Chan, T.L.
Chang-Claude, J.
Chanock, S.J.
Chenevix-Trench, G.
Choi, J.-.
Clarke, C.L.
NBCS Collaborators,
Colonna, S.
Conroy, D.M.
Couch, F.J.
Cox, A.
Cross, S.S.
Czene, K.
Daly, M.B.
Devilee, P.
Dörk, T.
Dossus, L.
Dwek, M.
Eccles, D.M.
Ekici, A.B.
Eliassen, A.H.
Engel, C.
Fasching, P.A.
Figueroa, J.
Flyger, H.
Gago-Dominguez, M.
Gao, C.
García-Closas, M.
García-Sáenz, J.A.
Ghoussaini, M.
Giles, G.G.
Goldberg, M.S.
González-Neira, A.
Guénel, P.
Gündert, M.
Haeberle, L.
Hahnen, E.
Haiman, C.A.
Hall, P.
Hamann, U.
Hartman, M.
Hatse, S.
Hauke, J.
Hollestelle, A.
Hoppe, R.
Hopper, J.L.
Hou, M.-.
kConFab Investigators,
ABCTB Investigators,
Ito, H.
Iwasaki, M.
Jager, A.
Jakubowska, A.
Janni, W.
John, E.M.
Joseph, V.
Jung, A.
Kaaks, R.
Kang, D.
Keeman, R.
Khusnutdinova, E.
Kim, S.-.
Kosma, V.-.
Kraft, P.
Kristensen, V.N.
Kubelka-Sabit, K.
Kurian, A.W.
Kwong, A.
Lacey, J.V.
Lambrechts, D.
Larson, N.L.
Larsson, S.C.
Le Marchand, L.
Lejbkowicz, F.
Li, J.
Long, J.
Lophatananon, A.
Lubiński, J.
Mannermaa, A.
Manoochehri, M.
Manoukian, S.
Margolin, S.
Matsuo, K.
Mavroudis, D.
Mayes, R.
Menon, U.
Milne, R.L.
Mohd Taib, N.A.
Muir, K.
Muranen, T.A.
Murphy, R.A.
Nevanlinna, H.
O'Brien, K.M.
Offit, K.
Olson, J.E.
Olsson, H.
Park, S.K.
Park-Simon, T.-.
Patel, A.V.
Peterlongo, P.
Peto, J.
Plaseska-Karanfilska, D.
Presneau, N.
Pylkäs, K.
Rack, B.
Rennert, G.
Romero, A.
Ruebner, M.
Rüdiger, T.
Saloustros, E.
Sandler, D.P.
Sawyer, E.J.
Schmidt, M.K.
Schmutzler, R.K.
Schneeweiss, A.
Schoemaker, M.J.
Shah, M.
Shen, C.-.
Shu, X.-.
Simard, J.
Southey, M.C.
Stone, J.
Surowy, H.
Swerdlow, A.J.
Tamimi, R.M.
Tapper, W.J.
Taylor, J.A.
Teo, S.H.
Teras, L.R.
Terry, M.B.
Toland, A.E.
Tomlinson, I.
Truong, T.
Tseng, C.-.
Untch, M.
Vachon, C.M.
van den Ouweland, A.M.
Wang, S.S.
Weinberg, C.R.
Wendt, C.
Winham, S.J.
Winqvist, R.
Wolk, A.
Wu, A.H.
Yamaji, T.
Zheng, W.
Ziogas, A.
Pharoah, P.D.
Dunning, A.M.
Easton, D.F.
Pettitt, S.J.
Lord, C.J.
Haider, S.
Orr, N.
Fletcher, O.
(2021). Functional annotation of the 2q35 breast cancer risk locus implicates a structural variant in influencing activity of a long-range enhancer element. Am j hum genet,
Vol.108
(7),
pp. 1190-1203.
show abstract
full text
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)..
Holgersen, E.M.
Gillespie, A.
Leavy, O.C.
Baxter, J.S.
Zvereva, A.
Muirhead, G.
Johnson, N.
Sipos, O.
Dryden, N.H.
Broome, L.R.
Chen, Y.
Kozin, I.
Dudbridge, F.
Fletcher, O.
Haider, S.
(2021). Identifying high-confidence capture Hi-C interactions using CHiCANE. Nat protoc,
Vol.16
(4),
pp. 2257-2285.
show abstract
The ability to identify regulatory interactions that mediate gene expression changes through distal elements, such as risk loci, is transforming our understanding of how genomes are spatially organized and regulated. Capture Hi-C (CHi-C) is a powerful tool to delineate such regulatory interactions. However, primary analysis and downstream interpretation of CHi-C profiles remains challenging and relies on disparate tools with ad-hoc input/output formats and specific assumptions for statistical modeling. Here we present a data processing and interaction calling toolkit (CHiCANE), specialized for the analysis and meaningful interpretation of CHi-C assays. In this protocol, we demonstrate applications of CHiCANE to region capture Hi-C (rCHi-C) and promoter capture Hi-C (pCHi-C) libraries, followed by quality assessment of interaction peaks, as well as downstream analysis specific to rCHi-C and pCHi-C to aid functional interpretation. For a typical rCHi-C/pCHi-C dataset this protocol takes up to 3 d for users with a moderate understanding of R programming and statistical concepts, although this is dependent on dataset size and compute power available. CHiCANE is freely available at https://cran.r-project.org/web/packages/chicane ..
Maguire, S.
Perraki, E.
Tomczyk, K.
Jones, M.E.
Fletcher, O.
Pugh, M.
Winter, T.
Thompson, K.
Cooke, R.
kConFab Consortium,
Trainer, A.
James, P.
Bojesen, S.
Flyger, H.
Nevanlinna, H.
Mattson, J.
Friedman, E.
Laitman, Y.
Palli, D.
Masala, G.
Zanna, I.
Ottini, L.
Silvestri, V.
Hollestelle, A.
Hooning, M.J.
Novaković, S.
Krajc, M.
Gago-Dominguez, M.
Castelao, J.E.
Olsson, H.
Hedenfalk, I.
Saloustros, E.
Georgoulias, V.
Easton, D.F.
Pharoah, P.
Dunning, A.M.
Bishop, D.T.
Neuhausen, S.L.
Steele, L.
Ashworth, A.
Garcia Closas, M.
Houlston, R.
Swerdlow, A.
Orr, N.
(2021). Common Susceptibility Loci for Male Breast Cancer. J natl cancer inst,
Vol.113
(4),
pp. 453-461.
show abstract
full text
BACKGROUND: The etiology of male breast cancer (MBC) is poorly understood. In particular, the extent to which the genetic basis of MBC differs from female breast cancer (FBC) is unknown. A previous genome-wide association study of MBC identified 2 predisposition loci for the disease, both of which were also associated with risk of FBC. METHODS: We performed genome-wide single nucleotide polymorphism genotyping of European ancestry MBC case subjects and controls in 3 stages. Associations between directly genotyped and imputed single nucleotide polymorphisms with MBC were assessed using fixed-effects meta-analysis of 1380 cases and 3620 controls. Replication genotyping of 810 cases and 1026 controls was used to validate variants with P values less than 1 × 10-06. Genetic correlation with FBC was evaluated using linkage disequilibrium score regression, by comprehensively examining the associations of published FBC risk loci with risk of MBC and by assessing associations between a FBC polygenic risk score and MBC. All statistical tests were 2-sided. RESULTS: The genome-wide association study identified 3 novel MBC susceptibility loci that attained genome-wide statistical significance (P < 5 × 10-08). Genetic correlation analysis revealed a strong shared genetic basis with estrogen receptor-positive FBC. Men in the top quintile of genetic risk had a fourfold increased risk of breast cancer relative to those in the bottom quintile (odds ratio = 3.86, 95% confidence interval = 3.07 to 4.87, P = 2.08 × 10-30). CONCLUSIONS: These findings advance our understanding of the genetic basis of MBC, providing support for an overlapping genetic etiology with FBC and identifying a fourfold high-risk group of susceptible men..
Baxter, J.S.
Leavy, O.C.
Dryden, N.H.
Maguire, S.
Johnson, N.
Fedele, V.
Simigdala, N.
Martin, L.-.
Andrews, S.
Wingett, S.W.
Assiotis, I.
Fenwick, K.
Chauhan, R.
Rust, A.G.
Orr, N.
Dudbridge, F.
Haider, S.
Fletcher, O.
(2018). Capture Hi-C identifies putative target genes at 33 breast cancer risk loci. Nat commun,
Vol.9
(1),
p. 1028.
show abstract
full text
Genome-wide association studies (GWAS) have identified approximately 100 breast cancer risk loci. Translating these findings into a greater understanding of the mechanisms that influence disease risk requires identification of the genes or non-coding RNAs that mediate these associations. Here, we use Capture Hi-C (CHi-C) to annotate 63 loci; we identify 110 putative target genes at 33 loci. To assess the support for these target genes in other data sources we test for associations between levels of expression and SNP genotype (eQTLs), disease-specific survival (DSS), and compare them with somatically mutated cancer genes. 22 putative target genes are eQTLs, 32 are associated with DSS and 14 are somatically mutated in breast, or other, cancers. Identifying the target genes at GWAS risk loci will lead to a greater understanding of the mechanisms that influence breast cancer risk and prognosis..
Sood, D.
Johnson, N.
Jain, P.
Siskos, A.P.
Bennett, M.
Gilham, C.
Busana, M.C.
Peto, J.
Dos-Santos-Silva, I.
Keun, H.C.
Fletcher, O.
(2017). CYP3A7*1C allele is associated with reduced levels of 2-hydroxylation pathway oestrogen metabolites. Br j cancer,
Vol.116
(3),
pp. 382-388.
show abstract
full text
BACKGROUND: Endogenous sex hormones are well-established risk factors for breast cancer; the contribution of specific oestrogen metabolites (EMs) and/or ratios of specific EMs is less clear. We have previously identified a CYP3A7*1C allele that is associated with lower urinary oestrone (E1) levels in premenopausal women. The purpose of this analysis was to determine whether this allele was associated with specific pathway EMs. METHODS: We measured successfully 12 EMs in mid-follicular phase urine samples from 30 CYP3A7*1C carriers and 30 non-carriers using HPLC-MS/MS. RESULTS: In addition to having lower urinary E1 levels, CYP3A7*1C carriers had significantly lower levels of four of the 2-hydroxylation pathway EMs that we measured (2-hydroxyestrone, P=1.1 × 10-12; 2-hydroxyestradiol, P=2.7 × 10-7; 2-methoxyestrone, P=1.9 × 10-12; and 2-methoxyestradiol, P=0.0009). By contrast, 16α-hydroxylation pathway EMs were slightly higher in carriers and significantly so for 17-epiestriol (P=0.002). CONCLUSIONS: The CYP3A7*1C allele is associated with a lower urinary E1 levels, a more pronounced reduction in 2-hydroxylation pathway EMs and a lower ratio of 2-hydroxylation:16α-hydroxylation EMs in premenopausal women. To further characterise the association between parent oestrogens, EMs and subsequent risk of breast cancer, characterisation of additional genetic variants that influence oestrogen metabolism and large prospective studies of a broad spectrum of EMs will be required..
Johnson, N.
De Ieso, P.
Migliorini, G.
Orr, N.
Broderick, P.
Catovsky, D.
Matakidou, A.
Eisen, T.
Goldsmith, C.
Dudbridge, F.
Peto, J.
Dos-Santos-Silva, I.
Ashworth, A.
Ross, G.
Houlston, R.S.
Fletcher, O.
(2016). Cytochrome P450 Allele CYP3A7*1C Associates with Adverse Outcomes in Chronic Lymphocytic Leukemia, Breast, and Lung Cancer. Cancer res,
Vol.76
(6),
pp. 1485-1493.
show abstract
CYP3A enzymes metabolize endogenous hormones and chemotherapeutic agents used to treat cancer, thereby potentially affecting drug effectiveness. Here, we refined the genetic basis underlying the functional effects of a CYP3A haplotype on urinary estrone glucuronide (E1G) levels and tested for an association between CYP3A genotype and outcome in patients with chronic lymphocytic leukemia (CLL), breast, or lung cancers. The most significantly associated SNP was rs45446698, an SNP that tags the CYP3A7*1C allele; this SNP was associated with a 54% decrease in urinary E1G levels. Genotyping this SNP in 1,008 breast cancer, 1,128 lung cancer, and 347 CLL patients, we found that rs45446698 was associated with breast cancer mortality (HR, 1.74; P = 0.03), all-cause mortality in lung cancer patients (HR, 1.43; P = 0.009), and CLL progression (HR, 1.62; P = 0.03). We also found borderline evidence of a statistical interaction between the CYP3A7*1C allele, treatment of patients with a cytotoxic agent that is a CYP3A substrate, and clinical outcome (Pinteraction = 0.06). The CYP3A7*1C allele, which results in adult expression of the fetal CYP3A7 gene, is likely to be the functional allele influencing levels of circulating endogenous sex hormones and outcome in these various malignancies. Further studies confirming these associations and determining the mechanism by which CYP3A7*1C influences outcome are required. One possibility is that standard chemotherapy regimens that include CYP3A substrates may not be optimal for the approximately 8% of cancer patients who are CYP3A7*1C carriers..
Orr, N.
Dudbridge, F.
Dryden, N.
Maguire, S.
Novo, D.
Perrakis, E.
Johnson, N.
Ghoussaini, M.
Hopper, J.L.
Southey, M.C.
Apicella, C.
Stone, J.
Schmidt, M.K.
Broeks, A.
Van't Veer, L.J.
Hogervorst, F.B.
Fasching, P.A.
Haeberle, L.
Ekici, A.B.
Beckmann, M.W.
Gibson, L.
Aitken, Z.
Warren, H.
Sawyer, E.
Tomlinson, I.
Kerin, M.J.
Miller, N.
Burwinkel, B.
Marme, F.
Schneeweiss, A.
Sohn, C.
Guénel, P.
Truong, T.
Cordina-Duverger, E.
Sanchez, M.
Bojesen, S.E.
Nordestgaard, B.G.
Nielsen, S.F.
Flyger, H.
Benitez, J.
Zamora, M.P.
Arias Perez, J.I.
Menéndez, P.
Anton-Culver, H.
Neuhausen, S.L.
Brenner, H.
Dieffenbach, A.K.
Arndt, V.
Stegmaier, C.
Hamann, U.
Brauch, H.
Justenhoven, C.
Brüning, T.
Ko, Y.-.
Nevanlinna, H.
Aittomäki, K.
Blomqvist, C.
Khan, S.
Bogdanova, N.
Dörk, T.
Lindblom, A.
Margolin, S.
Mannermaa, A.
Kataja, V.
Kosma, V.-.
Hartikainen, J.M.
Chenevix-Trench, G.
Beesley, J.
Lambrechts, D.
Moisse, M.
Floris, G.
Beuselinck, B.
Chang-Claude, J.
Rudolph, A.
Seibold, P.
Flesch-Janys, D.
Radice, P.
Peterlongo, P.
Peissel, B.
Pensotti, V.
Couch, F.J.
Olson, J.E.
Slettedahl, S.
Vachon, C.
Giles, G.G.
Milne, R.L.
McLean, C.
Haiman, C.A.
Henderson, B.E.
Schumacher, F.
Le Marchand, L.
Simard, J.
Goldberg, M.S.
Labrèche, F.
Dumont, M.
Kristensen, V.
Alnæs, G.G.
Nord, S.
Borresen-Dale, A.-.
Zheng, W.
Deming-Halverson, S.
Shrubsole, M.
Long, J.
Winqvist, R.
Pylkäs, K.
Jukkola-Vuorinen, A.
Grip, M.
Andrulis, I.L.
Knight, J.A.
Glendon, G.
Tchatchou, S.
Devilee, P.
Tollenaar, R.A.
Seynaeve, C.M.
Van Asperen, C.J.
Garcia-Closas, M.
Figueroa, J.
Chanock, S.J.
Lissowska, J.
Czene, K.
Darabi, H.
Eriksson, M.
Klevebring, D.
Hooning, M.J.
Hollestelle, A.
van Deurzen, C.H.
Kriege, M.
Hall, P.
Li, J.
Liu, J.
Humphreys, K.
Cox, A.
Cross, S.S.
Reed, M.W.
Pharoah, P.D.
Dunning, A.M.
Shah, M.
Perkins, B.J.
Jakubowska, A.
Lubinski, J.
Jaworska-Bieniek, K.
Durda, K.
Ashworth, A.
Swerdlow, A.
Jones, M.
Schoemaker, M.J.
Meindl, A.
Schmutzler, R.K.
Olswold, C.
Slager, S.
Toland, A.E.
Yannoukakos, D.
Muir, K.
Lophatananon, A.
Stewart-Brown, S.
Siriwanarangsan, P.
Matsuo, K.
Ito, H.
Iwata, H.
Ishiguro, J.
Wu, A.H.
Tseng, C.-.
Van Den Berg, D.
Stram, D.O.
Teo, S.H.
Yip, C.H.
Kang, P.
Ikram, M.K.
Shu, X.-.
Lu, W.
Gao, Y.-.
Cai, H.
Kang, D.
Choi, J.-.
Park, S.K.
Noh, D.-.
Hartman, M.
Miao, H.
Lim, W.Y.
Lee, S.C.
Sangrajrang, S.
Gaborieau, V.
Brennan, P.
Mckay, J.
Wu, P.-.
Hou, M.-.
Yu, J.-.
Shen, C.-.
Blot, W.
Cai, Q.
Signorello, L.B.
Luccarini, C.
Bayes, C.
Ahmed, S.
Maranian, M.
Healey, C.S.
González-Neira, A.
Pita, G.
Alonso, M.R.
Álvarez, N.
Herrero, D.
Tessier, D.C.
Vincent, D.
Bacot, F.
Hunter, D.J.
Lindstrom, S.
Dennis, J.
Michailidou, K.
Bolla, M.K.
Easton, D.F.
dos Santos Silva, I.
Fletcher, O.
Peto, J.
GENICA Network,
kConFab Investigators,
Australian Ovarian Cancer Study Group,
(2015). Fine-mapping identifies two additional breast cancer susceptibility loci at 9q31 2. Hum mol genet,
Vol.24
(10),
pp. 2966-2984.
show abstract
We recently identified a novel susceptibility variant, rs865686, for estrogen-receptor positive breast cancer at 9q31.2. Here, we report a fine-mapping analysis of the 9q31.2 susceptibility locus using 43 160 cases and 42 600 controls of European ancestry ascertained from 52 studies and a further 5795 cases and 6624 controls of Asian ancestry from nine studies. Single nucleotide polymorphism (SNP) rs676256 was most strongly associated with risk in Europeans (odds ratios [OR] = 0.90 [0.88-0.92]; P-value = 1.58 × 10(-25)). This SNP is one of a cluster of highly correlated variants, including rs865686, that spans ∼14.5 kb. We identified two additional independent association signals demarcated by SNPs rs10816625 (OR = 1.12 [1.08-1.17]; P-value = 7.89 × 10(-09)) and rs13294895 (OR = 1.09 [1.06-1.12]; P-value = 2.97 × 10(-11)). SNP rs10816625, but not rs13294895, was also associated with risk of breast cancer in Asian individuals (OR = 1.12 [1.06-1.18]; P-value = 2.77 × 10(-05)). Functional genomic annotation using data derived from breast cancer cell-line models indicates that these SNPs localise to putative enhancer elements that bind known drivers of hormone-dependent breast cancer, including ER-α, FOXA1 and GATA-3. In vitro analyses indicate that rs10816625 and rs13294895 have allele-specific effects on enhancer activity and suggest chromatin interactions with the KLF4 gene locus. These results demonstrate the power of dense genotyping in large studies to identify independent susceptibility variants. Analysis of associations using subjects with different ancestry, combined with bioinformatic and genomic characterisation, can provide strong evidence for the likely causative alleles and their functional basis..
Lemnrau, A.
Brook, M.N.
Fletcher, O.
Coulson, P.
Tomczyk, K.
Jones, M.
Ashworth, A.
Swerdlow, A.
Orr, N.
Garcia-Closas, M.
(2015). Mitochondrial DNA Copy Number in Peripheral Blood Cells and Risk of Developing Breast Cancer. Cancer res,
Vol.75
(14),
pp. 2844-2850.
show abstract
Increased mitochondrial DNA (mtDNA) copy number in peripheral blood cells (PBC) has been associated with the risk of developing several tumor types. Here we evaluate sources of variation of this biomarker and its association with breast cancer risk in a prospective cohort study. mtDNA copy number was measured using quantitative real-time PCR on PBC DNA samples from participants in the UK-based Breakthrough Generations Study. Temporal and assay variation was evaluated in a serial study of 91 women, with two blood samples collected approximately 6-years apart. Then, associations with breast cancer risk factors and risk were evaluated in 1,108 cases and 1,099 controls using a nested case-control design. In the serial study, mtDNA copy number showed low assay variation but large temporal variation [assay intraclass correlation coefficient (ICC), 79.3%-87.9%; temporal ICC, 38.3%). Higher mtDNA copy number was significantly associated with younger age at blood collection, being premenopausal, having an older age at menopause, and never taking HRT, both in cases and controls. Based on measurements in a single blood sample taken on average 6 years before diagnosis, higher mtDNA copy number was associated with increased breast cancer risk [OR (95% CI) for highest versus lowest quartile, 1.37 (1.02-1.83); P trend = 0.007]. In conclusion, mtDNA copy number is associated with breast cancer risk and represents a promising biomarker for risk assessment. The relatively large temporal variation should be taken into account in future analyses..
Dudbridge, F.
Fletcher, O.
(2014). Gene-environment dependence creates spurious gene-environment interaction. Am j hum genet,
Vol.95
(3),
pp. 301-307.
show abstract
full text
Gene-environment interactions have the potential to shed light on biological processes leading to disease and to improve the accuracy of epidemiological risk models. However, relatively few such interactions have yet been confirmed. In part this is because genetic markers such as tag SNPs are usually studied, rather than the causal variants themselves. Previous work has shown that this leads to substantial loss of power and increased sample size when gene and environment are independent. However, dependence between gene and environment can arise in several ways including mediation, pleiotropy, and confounding, and several examples of gene-environment interaction under gene-environment dependence have recently been published. Here we show that under gene-environment dependence, a statistical interaction can be present between a marker and environment even if there is no interaction between the causal variant and the environment. We give simple conditions under which there is no marker-environment interaction and note that they do not hold in general when there is gene-environment dependence. Furthermore, the gene-environment dependence applies to the causal variant and cannot be assessed from marker data. Gene-gene interactions are susceptible to the same problem if two causal variants are in linkage disequilibrium. In addition to existing concerns about mechanistic interpretations, we suggest further caution in reporting interactions for genetic markers..
Dryden, N.H.
Broome, L.R.
Dudbridge, F.
Johnson, N.
Orr, N.
Schoenfelder, S.
Nagano, T.
Andrews, S.
Wingett, S.
Kozarewa, I.
Assiotis, I.
Fenwick, K.
Maguire, S.L.
Campbell, J.
Natrajan, R.
Lambros, M.
Perrakis, E.
Ashworth, A.
Fraser, P.
Fletcher, O.
(2014). Unbiased analysis of potential targets of breast cancer susceptibility loci by Capture Hi-C. Genome res,
Vol.24
(11),
pp. 1854-1868.
show abstract
Genome-wide association studies have identified more than 70 common variants that are associated with breast cancer risk. Most of these variants map to non-protein-coding regions and several map to gene deserts, regions of several hundred kilobases lacking protein-coding genes. We hypothesized that gene deserts harbor long-range regulatory elements that can physically interact with target genes to influence their expression. To test this, we developed Capture Hi-C (CHi-C), which, by incorporating a sequence capture step into a Hi-C protocol, allows high-resolution analysis of targeted regions of the genome. We used CHi-C to investigate long-range interactions at three breast cancer gene deserts mapping to 2q35, 8q24.21, and 9q31.2. We identified interaction peaks between putative regulatory elements ("bait fragments") within the captured regions and "targets" that included both protein-coding genes and long noncoding (lnc) RNAs over distances of 6.6 kb to 2.6 Mb. Target protein-coding genes were IGFBP5, KLF4, NSMCE2, and MYC; and target lncRNAs included DIRC3, PVT1, and CCDC26. For one gene desert, we were able to define two SNPs (rs12613955 and rs4442975) that were highly correlated with the published risk variant and that mapped within the bait end of an interaction peak. In vivo ChIP-qPCR data show that one of these, rs4442975, affects the binding of FOXA1 and implicate this SNP as a putative functional variant..
Fletcher, O.
Dudbridge, F.
(2014). Candidate gene-environment interactions in breast cancer. Bmc medicine,
Vol.12.
full text
Johnson, N.
Dudbridge, F.
Orr, N.
Gibson, L.
Jones, M.E.
Schoemaker, M.J.
Folkerd, E.J.
Haynes, B.P.
Hopper, J.L.
Southey, M.C.
Dite, G.S.
Apicella, C.
Schmidt, M.K.
Broeks, A.
Van't Veer, L.J.
Atsma, F.
Muir, K.
Lophatananon, A.
Fasching, P.A.
Beckmann, M.W.
Ekici, A.B.
Renner, S.P.
Sawyer, E.
Tomlinson, I.
Kerin, M.
Miller, N.
Burwinkel, B.
Marme, F.
Schneeweiss, A.
Sohn, C.
Guenel, P.
Truong, T.
Cordina, E.
Menegaux, F.
Bojesen, S.E.
Nordestgaard, B.G.
Flyger, H.
Milne, R.
Zamora, M.P.
Arias Perez, J.I.
Benitez, J.
Bernstein, L.
Anton-Culver, H.
Ziogas, A.
Clarke Dur, C.
Brenner, H.
Muller, H.
Arndt, V.
Dieffenbach, A.K.
Meindl, A.
Heil, J.
Bartram, C.R.
Schmutzler, R.K.
Brauch, H.
Justenhoven, C.
Ko, Y.D.
Network, G.
Nevanlinna, H.
Muranen, T.A.
Aittomaki, K.
Blomqvist, C.
Matsuo, K.
Dork, T.
Bogdanova, N.V.
Antonenkova, N.N.
Lindblom, A.
Mannermaa, A.
Kataja, V.
Kosma, V.M.
Hartikainen, J.M.
Chenevix-Trench, G.
Beesley, J.
kConFab, I.
Australian Ovarian Cancer Study, G.
Wu, A.H.
Van den Berg, D.
Tseng, C.C.
Lambrechts, D.
Smeets, D.
Neven, P.
Wildiers, H.
Chang-Claude, J.
Rudolph, A.
Nickels, S.
Flesch-Janys, D.
Radice, P.
Peterlongo, P.
Bonanni, B.
Pensotti, V.
Couch, F.J.
Olson, J.E.
Wang, X.
Fredericksen, Z.
Pankratz, V.S.
Giles, G.G.
Severi, G.
Baglietto, L.
Haiman, C.
Simard, J.
Goldberg, M.S.
Labreche, F.
Dumont, M.
Soucy, P.
Teo, S.
Yip, C.H.
Phuah, S.Y.
Cornes, B.K.
Kristensen, V.N.
Grenaker Alnaes, G.
Borresen-Dale, A.L.
Zheng, W.
Winqvist, R.
Pylkas, K.
Jukkola-Vuorinen, A.
Grip, M.
Andrulis, I.L.
Knight, J.A.
Glendon, G.
Mulligan, A.M.
Devillee, P.
Figueroa, J.
Chanock, S.J.
Lissowska, J.
Sherman, M.E.
Hall, P.
Schoof, N.
Hooning, M.
Hollestelle, A.
Oldenburg, R.A.
Tilanus-Linthorst, M.
Liu, J.
Cox, A.
Brock, I.W.
Reed, M.W.
Cross, S.S.
Blot, W.
Signorello, L.B.
Pharoah, P.D.
Dunning, A.M.
Shah, M.
Kang, D.
Noh, D.Y.
Park, S.K.
Choi, J.Y.
Hartman, M.
Miao, H.
Lim, W.Y.
Tang, A.
Hamann, U.
Forsti, A.
Rudiger, T.
Ulmer, H.U.
Jakubowska, A.
Lubinski, J.
Jaworska-Bieniek, K.
Durda, K.
Sangrajrang, S.
Gaborieau, V.
Brennan, P.
McKay, J.
Slager, S.
Toland, A.E.
Vachon, C.
Yannoukakos, D.
Shen, C.Y.
Yu, J.C.
Huang, C.S.
Hou, M.F.
Gonzalez-Neira, A.
Tessier, D.C.
Vincent, D.
Bacot, F.
Luccarini, C.
Dennis, J.
Michailidou, K.
Bolla, M.K.
Wang, J.
Easton, D.F.
Garcia-Closas, M.
Dowsett, M.
Ashworth, A.
Swerdlow, A.J.
Peto, J.
dos Santos Silva, I.
Fletcher, O.
(2014). Genetic variation at CYP3A is associated with age at menarche and breast cancer risk: a case-control study. Breast cancer res,
Vol.16,
p. R51.
show abstract
full text
INTRODUCTION: We have previously shown that a tag single nucleotide polymorphism (rs10235235), which maps to the CYP3A locus (7q22.1), was associated with a reduction in premenopausal urinary estrone glucuronide levels and a modest reduction in risk of breast cancer in women age /=15 years (OR(het) = 0.84, 95% CI 0.75, 0.94; OR(hom) = 0.81, 95% CI 0.51, 1.30; P(trend) = 0.002) but not for those who had their menarche age
Johnson, N.
Walker, K.
Gibson, L.J.
Orr, N.
Folkerd, E.
Haynes, B.
Palles, C.
Coupland, B.
Schoemaker, M.
Jones, M.
Broderick, P.
Sawyer, E.
Kerin, M.
Tomlinson, I.P.
Zvelebil, M.
Chilcott-Burns, S.
Tomczyk, K.
Simpson, G.
Williamson, J.
Hillier, S.G.
Ross, G.
Houlston, R.S.
Swerdlow, A.
Ashworth, A.
Dowsett, M.
Peto, J.
Dos Santos Silva, I.
Fletcher, O.
(2012). CYP3A variation, premenopausal estrone levels, and breast cancer risk. J natl cancer inst,
Vol.104
(9),
pp. 657-669.
show abstract
BACKGROUND: Epidemiological studies have provided strong evidence for a role of endogenous sex steroids in the etiology of breast cancer. Our aim was to identify common variants in genes involved in sex steroid synthesis or metabolism that are associated with hormone levels and the risk of breast cancer in premenopausal women. METHODS: We measured urinary levels of estrone glucuronide (E1G) using a protocol specifically developed to account for cyclic variation in hormone levels during the menstrual cycle in 729 healthy premenopausal women. We genotyped 642 single-nucleotide polymorphisms (SNPs) in these women; a single SNP, rs10273424, was further tested for association with the risk of breast cancer using data from 10 551 breast cancer case patients and 17 535 control subjects. All statistical tests were two-sided. RESULTS: rs10273424, which maps approximately 50 kb centromeric to the cytochrome P450 3A (CYP3A) gene cluster at chromosome 7q22.1, was associated with a 21.8% reduction in E1G levels (95% confidence interval [CI] = 27.8% to 15.3% reduction; P = 2.7 × 10(-9)) and a modest reduction in the risk of breast cancer in case patients who were diagnosed at or before age 50 years (odds ratio [OR] = 0.91, 95% CI = 0.83 to 0.99; P = .03) but not in those diagnosed after age 50 years (OR = 1.01, 95% CI = 0.93 to 1.10; P = .82). CONCLUSIONS: Genetic variation in noncoding sequences flanking the CYP3A locus contributes to variance in premenopausal E1G levels and is associated with the risk of breast cancer in younger patients. This association may have wider implications given that the most predominantly expressed CYP3A gene, CYP3A4, is responsible for metabolism of endogenous and exogenous hormones and hormonal agents used in the treatment of breast cancer..
Fletcher, O.
Johnson, N.
Orr, N.
Hosking, F.J.
Gibson, L.J.
Walker, K.
Zelenika, D.
Gut, I.
Heath, S.
Palles, C.
Coupland, B.
Broderick, P.
Schoemaker, M.
Jones, M.
Williamson, J.
Chilcott-Burns, S.
Tomczyk, K.
Simpson, G.
Jacobs, K.B.
Chanock, S.J.
Hunter, D.J.
Tomlinson, I.P.
Swerdlow, A.
Ashworth, A.
Ross, G.
dos Santos Silva, I.
Lathrop, M.
Houlston, R.S.
Peto, J.
(2011). Novel breast cancer susceptibility locus at 9q31 2: results of a genome-wide association study. J natl cancer inst,
Vol.103
(5),
pp. 425-435.
show abstract
BACKGROUND: Genome-wide association studies have identified several common genetic variants associated with breast cancer risk. It is likely, however, that a substantial proportion of such loci have not yet been discovered. METHODS: We compared 296,114 tagging single-nucleotide polymorphisms in 1694 breast cancer case subjects (92% with two primary cancers or at least two affected first-degree relatives) and 2365 control subjects, with validation in three independent series totaling 11,880 case subjects and 12,487 control subjects. Odds ratios (ORs) and associated 95% confidence intervals (CIs) in each stage and all stages combined were calculated using unconditional logistic regression. Heterogeneity was evaluated with Cochran Q and I(2) statistics. All statistical tests were two-sided. RESULTS: We identified a novel risk locus for breast cancer at 9q31.2 (rs865686: OR = 0.89, 95% CI = 0.85 to 0.92, P = 1.75 × 10(-10)). This single-nucleotide polymorphism maps to a gene desert, the nearest genes being Kruppel-like factor 4 (KLF4, 636 kb centromeric), RAD23 homolog B (RAD23B, 794 kb centromeric), and actin-like 7A (ACTL7A, 736 kb telomeric). We also identified two variants (rs3734805 and rs9383938) mapping to 6q25.1 estrogen receptor 1 (ESR1), which were associated with breast cancer in subjects of northern European ancestry (rs3734805: OR = 1.19, 95% CI = 1.11 to 1.27, P = 1.35 × 10(-7); rs9383938: OR = 1.18, 95% CI = 1.11 to 1.26, P = 1.41 × 10(-7)). A variant mapping to 10q26.13, approximately 300 kb telomeric to the established risk locus within the second intron of FGFR2, was also associated with breast cancer risk, although not at genome-wide statistical significance (rs10510102: OR = 1.12, 95% CI = 1.07 to 1.17, P = 1.58 × 10(-6)). CONCLUSIONS: These findings provide further evidence on the role of genetic variation in the etiology of breast cancer. Fine mapping will be needed to identify causal variants and to determine their functional effects..
Fletcher, O.
Houlston, R.S.
(2010). Architecture of inherited susceptibility to common cancer. Nat rev cancer,
Vol.10
(5),
pp. 353-361.
show abstract
This Timeline article looks back at 40 years of research into the inherited genetic basis of cancer and the insights these studies have yielded. Early epidemiological research provided evidence for the 'two-hit' model of cancer predisposition. During the 1980s and 1990s linkage and positional cloning analyses led to the identification of high-penetrance cancer susceptibility genes. The past decade has seen a shift from models of predisposition based on single-gene causative mutations to multigenic models. These models suggest that a high proportion of cancers may arise in a genetically susceptible minority as a consequence of the combined effects of common low-penetrance alleles and rare disease-causing variants that confer moderate cancer risks..
Walker, K.
Fletcher, O.
Johnson, N.
Coupland, B.
McCormack, V.A.
Folkerd, E.
Gibson, L.
Hillier, S.G.
Holly, J.M.
Moss, S.
Dowsett, M.
Peto, J.
dos Santos Silva, I.
(2009). Premenopausal mammographic density in relation to cyclic variations in endogenous sex hormone levels, prolactin, and insulin-like growth factors. Cancer res,
Vol.69
(16),
pp. 6490-6499.
show abstract
Mammographic density is strongly associated with breast cancer risk, and endogenous hormones, which are risk factors for breast cancer, may be involved in the mechanism. This cross-sectional study of 494 premenopausal women is the first to account for cyclic variations in estrogen levels, by measuring urinary estrone glucuronide (E1G) in the periovulatory and luteal phases of the menstrual cycle, and to assess the role of androgens. Computer-assisted density readings were obtained from digitized mammograms. Mean ovulatory E1G level and daily E1G load were both positively associated with percent density before adjustment for body mass index (BMI), with women in the top fourth having 10.2% (95% CI: 2.9%, 18.1%) and 8.9% (1.7%, 16.7%), respectively, higher density than those in the bottom fourth (Ptrend before/after BMI adjustment=0.006/0.11 and 0.01/0.13, respectively). Neither the peak nor luteal E1G levels were predictive of density after adjustment for E1G levels at other points in the cycle. The plasma androgens testosterone, androstenedione, and dehydroepiandrosterone sulfate were negatively associated with density. In mutually adjusted analyses, density was positively associated with insulin-like growth factor (IGF)-I and negatively with IGF-II (Ptrend=0.006 for both) but not with IGF binding protein-3. There was also weak evidence of a positive association of prolactin with density. The study supports the hypothesis that endogenous hormones affect density in premenopausal women; in particular, it shows a positive association between estrogen levels and density and suggests that the mean level throughout the cycle is the most biologically relevant measure. Most of these hormone-density associations were attenuated with further adjustment for BMI..
Palles, C.
Johnson, N.
Coupland, B.
Taylor, C.
Carvajal, J.
Holly, J.
Fentiman, I.S.
Silva, I.D.
Ashworth, A.
Peto, J.
Fletcher, O.
(2008). Identification of genetic variants that influence circulating IGF1 levels: a targeted search strategy. Hum mol genet,
Vol.17
(10),
pp. 1457-1464.
show abstract
An important class of genetic variants that affect disease susceptibility may lie within regulatory elements that influence gene expression. Regulatory sequences are difficult to identify and may be distant from the genes they regulate, but many lie within evolutionarily conserved regions (ECRs). We used comparative genomics to identify 12 ECRs up to 75 kb 5' to and within introns of IGF1. These were screened by high-resolution melting curve analysis, and 18 single-nucleotide polymorphisms (SNPs) were identified, including five novel variants. We analysed two large population-based series of healthy women to test the nine SNPs with minor allele frequency (MAF) >1% within ECRs. Three of the nine SNPs within ECRs (rs35455143, rs35765817 and rs3839984) were significantly associated with circulating IGF1 levels in a multivariate analysis (P 70 kb 5' of IGF1. Two (rs35455143 and rs35765817) are in strong LD with each other and appear to have opposite effects on circulating IGF1. Our results on a subset of other SNPs in or near IGF1 were consistent with previously reported associations with IGF1 levels, although only one (rs35767: P = 0.05) was statistically significant. We believe that this is the first systematic study of an association between a phenotype and SNPs within ECRs extending over a large region adjacent to a gene. Targeting ECRs appears to be a useful strategy for identifying a subset of potentially functional non-coding regulatory SNPs..
Johnson, N.
Fletcher, O.
Palles, C.
Rudd, M.
Webb, E.
Sellick, G.
dos Santos Silva, I.
McCormack, V.
Gibson, L.
Fraser, A.
Leonard, A.
Gilham, C.
Tavtigian, S.V.
Ashworth, A.
Houlston, R.
Peto, J.
(2007). Counting potentially functional variants in BRCA1, BRCA2 and ATM predicts breast cancer susceptibility. Hum mol genet,
Vol.16
(9),
pp. 1051-1057.
show abstract
Rare inactivating mutations in BRCA1, BRCA2, ATM, TP53 and CHEK2 confer relative risks for breast cancer between about 2 and more than 10, but more common variants in these genes are generally considered of little or no clinical significance. Under the polygenic model for breast cancer carriers of multiple low-penetrance alleles are at high risk, but few such alleles have been reliably identified. We analysed 1037 potentially functional single nucleotide polymorphisms (SNPs) in candidate cancer genes in 473 women with two primary breast cancers and 2463 controls. Twenty-five of these SNPs were in BRCA1, BRCA2, ATM, TP53 and CHEK2. Among the 1037 SNPs there were a few significant findings, but hardly more than would be expected in this large experiment. There was, however, a significant trend in risk with increasing numbers of variant alleles for the 25 SNPs in BRCA1, BRCA2, ATM, TP53 and CHEK2 (P(trend) = 0.005). For the 21 of these with minor allele frequency <10% this trend was highly significant (P(trend) = 0.00004, odds ratio for 3 or more SNPs = 2.90, 95% CI 1.69-4.97). The individual effects of most of these risk alleles were undetectably small even in this well powered study, but the risk conferred by multiple variants is readily detectable and makes a substantial contribution to susceptibility. A risk score incorporating a suitably weighted sum of all potentially functional variants in these and a few other candidate genes may provide clinically useful identification of women at high genetic risk..
Johnson, N.
Fletcher, O.
Naceur-Lombardelli, C.
Silva, I.D.
Ashworth, A.
Peto, J.
Interaction between CHEK2*1100delC and other low-penetrance breast-cancer susceptibility genes:: a familial study. Lancet,
Vol.366
(9496),
pp. 1554-4.