Discussion
The aim of our study was to investigate the effect of regulatory genetic variation of the BRCA2 tumor suppressor gene on disease risk. To this end, we identified haplotypes associated with different expression levels of BRCA2 in normal breast tissue (expression haplotypes) and characterized three cis-acting polymorphisms that alter the binding of transcription factors at regulatory sites. We show that these expression haplotypes have an impact on the expression profile of breast cells. Also, we found some evidence of association between a high expression haplotype and lower risk of developing breast cancer among carriers of germline mutations in BRCA2.
The cells of individuals carrying inactivating germline mutations in the
BRCA2 gene rely on the expression of
BRCA2 from the remaining wild-type allele. There is evidence that these cells have a different gene expression profile and ability to repair double-strand DNA breaks compared to cells carrying two wild-type alleles [
21,
22]. We have shown previously that
BRCA2 shows differential allelic expression in normal breast tissue. These observations raised the hypothesis that
cis-regulatory variation could modify the penetrance of germline mutations in
BRCA2, by varying the level of expression of the remaining wild-type allele, if different levels of haploinsufficiency are important. The functional effects of haploinsufficiency at a given locus may vary from tissue to tissue [
22]. Many critical biological processes are context specific and the overlap of
cis-regulatory variants across different tissues is predicted to be between 10% to 80% [
43‐
45], suggesting that the expression haplotypes can vary from tissue to tissue. It is therefore critical to study the relevant tissue when evaluating mechanisms of effect on disease risk. The functional data that we present were obtained from the study of normal breast tissue from control individuals and breast cell lines (normal and cancer).
Haplotypes associated with different levels of gene expression were mapped in unrelated individuals, an approach that has proven powerful to identify
cis-acting loci [
46]. The difference of expression that we observed between the expression haplotypes is small, which suggests that any effect in disease will also be subtle, as expected for common variants. Further functional analysis in breast cells revealed three
cis-acting variants affecting the binding of transcription factors which can explain at least in part the differences in expression level associated with the expression haplotypes. These
cis-acting variants were mapped to the promoter and two intronic regulatory elements of
BRCA2. The regulatory scenario suggested by our findings is complex, with diverse regulatory variants playing a role in different expression haplotypes. The catalogue of genetic variation is improving constantly through studies like the 1000 Genomes Project. The most recent data show more than ten predicted haplotype blocks for the region we studied here. We have analyzed a fraction of the known variation and believe that there may be more
cis-variants regulating the expression of
BRCA2 in breast tissue, as in others.
In a recent report, Bellacosa and colleagues have shown that
BRCA2
+/-
heterozygous cells have an altered gene expression profile compared with normal cells from individuals without mutations and that this effect is tissue-specific [
22]. Even though the expression haplotypes we identified are associated with small differences in the expression of
BRCA2 itself, our analyses revealed that the gene expression level of some of the same genes reported by Bellacosa
et al. could be correlated with the expression haplotypes. These genes were, unsurprisingly, the ones with the most significant differences in the previous report. These findings suggest that even subtle changes in the expression of
BRCA2 can have an impact on the phenotype of normal breast tissue, which can be particularly important when only one expressing allele is present.
In our association study we found some evidence that the minor allele of SNP rs4942440 (corresponding to a high expression haplotype) is associated with a protective effect for the development of breast cancer in mutation carriers. We hypothesize that the higher the expression of BRCA2 from the remaining wild-type allele the better the cells of a mutation carrier individual will carry out DNA repair. The evidence of association in the present study is weak. Mutation phase information for the samples used in the association study was not available. As a consequence, the group of BRCA2 mutation carriers who are heterozygous for this SNP consists of a mixture of individuals who have either the minor or the common allele on the wild type haplotype. We are currently collecting detailed pedigree information and will be able to use phase information in the future, which will strengthen our analysis. Genotyping of additional mutation carriers in the future will further clarify the involvement of this SNP in the penetrance of BRCA2 mutations in breast cancer. The selection of SNPs for the disease association analysis, as well as the gene expression studies, was performed based on our previous data associating these SNPs/haplotypes with different levels of BRCA2 expression. It is difficult therefore to apply an exact multiple testing, but taken together, the gene expression and association results are suggestive for the involvement of this SNP in breast cancer risk for BRCA2 mutation carriers.
Acknowledgements
The authors would like to thank the women who contributed samples for this study and Dr R Russell, Dr A Hurtado, Ms R Prathalingam, Dr M Wilson, Dr K Meyer and Dr A Lynch for useful discussion and assistance with methodology, Dr E Dicks for assisting with access to data. We would like to acknowledge the support of the Core Genomics team at Cambridge Research Institute, in particular Mr C Bourne. This work was supported by a Breast Cancer Research Foundation grant to ATM and BAJP; the University of Cambridge, Cancer Research UK, Hutchison Whampoa Limited and NIHR Cambridge Biomedical Research Centre. BAJP is the Li Ka Shing Professor of Oncology at the University of Cambridge. ACA is a Cancer Research - UK Senior Cancer Research Fellow. The CIMBA data management and analysis is supported by Cancer Research - UK.
CIMBA Collaborating Centres
University of Pennsylvania (UPENN): This study was supported by the Breast Cancer Research Foundation; Cancer Genetics Network, Marjorie Cohen Foundation.
Epidemiological study of BRCA1 & BRCA2 mutation carriers (EMBRACE): Douglas F. Easton is the Principal Investigator of the study. EMBRACE Collaborating Centers are: Coordinating Centre, Cambridge: Susan Peock, Debra Frost, Steve Ellis, Elena Fineberg, Radka Platte. North of Scotland Regional Genetics Service, Aberdeen: Zosia Miedzybrodzka, Helen Gregory. Northern Ireland Regional Genetics Service, Belfast: Patrick Morrison, Lisa Jeffers. West Midlands Regional Clinical Genetics Service, Birmingham: Trevor Cole, Kai-ren Ong, Jonathan Hoffman. South West Regional Genetics Service, Bristol: Alan Donaldson, Margaret James. East Anglian Regional Genetics Service, Cambridge: Joan Paterson, Sarah Downing, Amy Taylor. Medical Genetics Services for Wales, Cardiff: Alexandra Murray, Mark T. Rogers, Emma McCann. St James's Hospital, Dublin & National Centre for Medical Genetics, Dublin: M. John Kennedy, David Barton. South East of Scotland Regional Genetics Service, Edinburgh: Mary Porteous, Sarah Drummond. Peninsula Clinical Genetics Service, Exeter: Carole Brewer, Emma Kivuva, Anne Searle, Selina Goodman, Kathryn Hill. West of Scotland Regional Genetics Service, Glasgow: Rosemarie Davidson, Victoria Murday, Nicola Bradshaw, Lesley Snadden, Mark Longmuir, Catherine Watt, Sarah Gibson, Eshika Haque, Ed Tobias, Alexis Duncan. South East Thames Regional Genetics Service, Guy's Hospital London: Louise Izatt, Chris Jacobs, Caroline Langman, Anna Whaite. North West Thames Regional Genetics Service, Harrow: Huw Dorkins. Leicestershire Clinical Genetics Service, Leicester: Julian Barwell. Yorkshire Regional Genetics Service, Leeds: Julian Adlard, Carol Chu, Julie Miller. Cheshire & Merseyside Clinical Genetics Service, Liverpool: Ian Ellis, Catherine Houghton. Manchester Regional Genetics Service, Manchester: D Gareth Evans, Fiona Lalloo, Jane Taylor. North East Thames Regional Genetics Service, NE Thames, London: Lucy Side, Alison Male, Cheryl Berlin. Nottingham Centre for Medical Genetics, Nottingham: Jacqueline Eason, Rebecca Collier. Northern Clinical Genetics Service, Newcastle: Fiona Douglas, Oonagh Claber, Irene Jobson. Oxford Regional Genetics Service, Oxford: Lisa Walker, Diane McLeod, Dorothy Halliday, Sarah Durell, Barbara Stayner. The Institute of Cancer Research and Royal Marsden NHS Foundation Trust: Ros Eeles, Susan Shanley, Nazneen Rahman, Richard Houlston, Elizabeth Bancroft, Lucia D'Mello, Elizabeth Page, Audrey Ardern-Jones, Kelly Kohut, Jennifer Wiggins, Elena Castro, Anita Mitra, Lisa Robertson. North Trent Clinical Genetics Service, Sheffield: Jackie Cook, Oliver Quarrell, Cathryn Bardsley. South West Thames Regional Genetics Service, London: Shirley Hodgson, Sheila Goff, Glen Brice, Lizzie Winchester, Charlotte Eddy, Vishakha Tripathi, Virginia Attard. Wessex Clinical Genetics Service, Princess Anne Hospital, Southampton: Diana Eccles, Anneke Lucassen, Gillian Crawford, Donna McBride, Sarah Smalley. EMBRACE is supported by Cancer Research UK Grants C1287/A10118 and C1287/A11990. D. Gareth Evans and Fiona Lalloo are supported by an NIHR grant to the Biomedical Research Centre, Manchester. The Investigators at The Institute of Cancer Research and The Royal Marsden NHS Foundation Trust are supported by an NIHR grant to the Biomedical Research Centre at The Institute of Cancer Research and The Royal Marsden NHS Foundation Trust. Ros Eeles, Elizabeth Bancroft and Lucia D'Mello are also supported by Cancer Research UK Grant C5047/A8385.
Genetic Modifiers of cancer risk in BRCA1/2 mutation carriers (GEMO): We wish to thank all the GEMO collaborating Cancer Genetics Network 'Groupe Génétique et Cancer' and Fédération Nationale des Centres de Lutte Contre le Cancer, France. The study was supported by the Ligue National Contre le Cancer; Association for International Cancer Research Grant (AICR-07-0454); and the Association "Le cancer du sein, parlons-en!" Award. We wish to thank all the GEMO collaborating groups for their contribution to this study. GEMO Collaborating Centers are: Coordinating Centres, Unité Mixte de Génétique Constitutionnelle des Cancers Fréquents, Centre Hospitalier Universitaire de Lyon/Centre Léon Bérard, & Equipe «Génétique du cancer du sein», Centre de Recherche en Cancérologie de Lyon: Olga Sinilnikova, Sylvie Mazoyer, Laure Barjhoux, Carole Verny-Pierre, Sophie Giraud, Mélanie Léone; and Service de Génétique Oncologique, Institut Curie, Paris: Dominique Stoppa-Lyonnet, Marion Gauthier-Villars, Bruno Buecher, Claude Houdayer, Virginie Moncoutier, Muriel Belotti, Carole Tirapo, Antoine de Pauw. Institut Gustave Roussy, Villejuif: Brigitte Bressac-de-Paillerets, Audrey Remenieras, Véronique Byrde, Olivier Caron, Gilbert Lenoir. Centre Jean Perrin, Clermont-Ferrand: Yves-Jean Bignon, Nancy Uhrhammer. Centre Léon Bérard, Lyon: Christine Lasset, Valérie Bonadona. Centre François Baclesse, Caen: Agnès Hardouin, Pascaline Berthet. Institut Paoli Calmettes, Marseille: Hagay Sobol, Violaine Bourdon, Tetsuro Noguchi, François Eisinger. Groupe Hospitalier Pitié-Salpétrière, Paris: Florence Coulet, Chrystelle Colas, Florent Soubrier. CHU de Arnaud-de-Villeneuve, Montpellier: Isabelle Coupier, Pascal Pujol. Centre Oscar Lambret, Lille: Jean-Philippe Peyrat, Joëlle Fournier, Françoise Révillion, Philippe Vennin, Claude Adenis. Hôpital René Huguenin/Institut Curie, St Cloud: Etienne Rouleau, Rosette Lidereau, Liliane Demange, Catherine Nogues. Centre Paul Strauss, Strasbourg: Danièle Muller, Jean-Pierre Fricker. Institut Bergonié, Bordeaux: Emmanuelle Barouk-Simonet, Françoise Bonnet, Virginie Bubien, Nicolas Sevenet, Michel Longy. Institut Claudius Regaud, Toulouse: Christine Toulas, Rosine Guimbaud, Laurence Gladieff, Viviane Feillel. CHU de Grenoble: Dominique Leroux, Hélène Dreyfus, Christine Rebischung, Magalie Peysselon. CHU de Dijon: Fanny Coron, Laurence Faivre. CHU de St-Etienne: Fabienne Prieur, Marine Lebrun, Caroline Kientz. Hôtel Dieu Centre Hospitalier, Chambéry: Sandra Fert Ferrer. Centre Antoine Lacassagne, Nice: Marc Frénay. CHU de Limoges: Laurence Vénat-Bouvet. CHU de Nantes: Capucine Delnatte. CHU Bretonneau, Tours: Isabelle Mortemousque. Creighton University, Omaha, USA: Henry T.Lynch, Carrie L.Snyder.
Helsinki Breast Cancer Study (HBECS): HEBCS would like to thank Dr. Kristiina Aittomäki and Tuomas Heikkinen and research nurse Irja Erkkilä for their help with the patient data and samples. The HEBCS study has been financially supported by the Helsinki University Central Hospital Research Fund, Academy of Finland (132473), the Finnish Cancer Society, and the Sigrid Juselius Foundation.
Mayo Clinic (MAYO): The Mayo Study was supported by NIH grants CA128978 and a Breast Cancer Specialized Program of Research Excellence (SPORE), CA116167, the Komen Foundation for the Cure and the Breast Cancer Research Foundation.
Fox Chase Cancer Center (FCCC): A.K.G. was funded by U01CA69631, 5U01CA113916, and the Eileen Stein Jacoby Fund. The author acknowledges support from The University of Kansas Cancer Center and the Kansas Bioscience Authority Eminent Scholar Program. A.K.G. is the Chancellors Distinguished Chair in Biomedical Sciences endowed Professor.
The Pisa Breast Cancer Study (PBCS): We would like to acknowledge the 'Tumour Tuscany Institute' for a grant (n. AOOGRT/0011780/Q.30.11) to Maria Adelaide Caligo.
The Swedish BRCA1 and BRCA2 Study (SWE-BRCA): Per Karlsson, Margareta Nordling, Annika Bergman and Zakaria Einbeigi, Gothenburg, Sahlgrenska University Hospital; Marie Stenmark-Askmalm and Sigrun Liedgren, Linköping University Hospital; Åke Borg, Niklas Loman, Håkan Olsson, Ulf Kristoffersson, Helena Jernström, Katja Harbst and Karin Henriksson, Lund University Hospital; Annika Lindblom, Brita Arver, Anna von Wachenfeldt, Annelie Liljegren, Gisela Barbany-Bustinza and Johanna Rantala, Stockholm, Karolinska University Hospital; Beatrice Melin, Henrik Grönberg, Eva-Lena Stattin and Monica Emanuelsson, Umeå University Hospital; Hans Ehrencrona, Richard Rosenquist Brandell and Niklas Dahl, Uppsala University Hospital.
Iceland Landspitali--University Hospital (ILUH): The ILUH group was supported by the Icelandic Association 'Walking for Breast Cancer Research' and by the Landspitali University Hospital Research Fund.
Kathleen Cunningham Foundation Consortium for Research into Familial Breast Cancer (kConFab): We wish to thank Heather Thorne, Eveline Niedermayr, all the kConFab research nurses and staff, the heads and staff of the Family Cancer Clinics, and the Clinical Follow Up Study (funded 2001-2009 by NHMRC and currently by the National Breast Cancer Foundation and Cancer Australia #628333) for their contributions to this resource, and the many families who contribute to kConFab. kConFab is supported by grants from the National Breast Cancer Foundation, the National Health and Medical Research Council (NHMRC) and by the Queensland Cancer Fund, the Cancer Councils of New South Wales, Victoria, Tasmania and South Australia, and the Cancer Foundation of Western Australia.
Authors' contributions
The project was conceived by ATM and BAJP. Functional analysis was performed by ATM and MOR. Gene expression experiments were conceived, performed and analyzed by ATM, SFC, MD, CNC and CC. The study of CIMBA carriers was coordinated by GC-T. The CIMBA data management is coordinated by ACA and LM. Genotyping of CIMBA carriers was performed by GC-T and OMS. Patient DNA material and clinical data of carriers was provided by SMD, EMBRACE, DFE, SP, DF, DGE, RE, LI, JA, DE, GEMO, OMS, SM, DSL, MGV, LF, LVB, CD, HN, FJC, AKG, MAC, SWE-BRCA, RBB, KConFab, XC, JB, SH and GCT. Data were analyzed by ATM, ACA, SS, MD, CK and CNC. The manuscript was drafted by ATM, ACA and BAJP. All authors read, critically revised and approved the final manuscript.