Open Access Highly Accessed Research article

Common breast cancer susceptibility alleles are associated with tumour subtypes in BRCA1 and BRCA2 mutation carriers: results from the Consortium of Investigators of Modifiers of BRCA1/2

Anna Marie Mulligan1, Fergus J Couch2, Daniel Barrowdale3, Susan M Domchek4, Diana Eccles5, Heli Nevanlinna6, Susan J Ramus7, Mark Robson8, Mark Sherman9, Amanda B Spurdle10, Barbara Wappenschmidt11, Andrew Lee3, Lesley McGuffog3, Sue Healey10, Olga M Sinilnikova1213, Ramunas Janavicius14, Thomas vO Hansen15, Finn C Nielsen15, Bent Ejlertsen16, Ana Osorio17, Iván Muñoz-Repeto17, Mercedes Durán18, Javier Godino19, Maroulio Pertesi20, Javier Benítez21, Paolo Peterlongo22, Siranoush Manoukian23, Bernard Peissel23, Daniela Zaffaroni23, Elisa Cattaneo23, Bernardo Bonanni24, Alessandra Viel25, Barbara Pasini26, Laura Papi27, Laura Ottini28, Antonella Savarese29, Loris Bernard30, Paolo Radice22, Ute Hamann31, Martijn Verheus32, Hanne EJ Meijers-Heijboer33, Juul Wijnen34, Encarna B Gómez García35, Marcel R Nelen36, C Marleen Kets37, Caroline Seynaeve38, Madeleine MA Tilanus-Linthorst39, Rob B van der Luijt40, Theo van Os41, Matti Rookus32, Debra Frost3, J Louise Jones42, D Gareth Evans43, Fiona Lalloo43, Ros Eeles44, Louise Izatt45, Julian Adlard46, Rosemarie Davidson47, Jackie Cook48, Alan Donaldson49, Huw Dorkins50, Helen Gregory51, Jacqueline Eason52, Catherine Houghton53, Julian Barwell54, Lucy E Side55, Emma McCann56, Alex Murray57, Susan Peock3, Andrew K Godwin58, Rita K Schmutzler11, Kerstin Rhiem11, Christoph Engel59, Alfons Meindl60, Ina Ruehl61, Norbert Arnold62, Dieter Niederacher63, Christian Sutter64, Helmut Deissler65, Dorothea Gadzicki66, Karin Kast67, Sabine Preisler-Adams68, Raymonda Varon-Mateeva69, Ines Schoenbuchner70, Britta Fiebig71, Wolfram Heinritz72, Dieter Schäfer73, Heidrun Gevensleben74, Virginie Caux-Moncoutier75, Marion Fassy-Colcombet75, François Cornelis76, Sylvie Mazoyer13, Mélanie Léoné12, Nadia Boutry-Kryza12, Agnès Hardouin77, Pascaline Berthet77, Danièle Muller78, Jean-Pierre Fricker78, Isabelle Mortemousque79, Pascal Pujol80, Isabelle Coupier80, Marine Lebrun81, Caroline Kientz81, Michel Longy82, Nicolas Sevenet82, Dominique Stoppa-Lyonnet7583, Claudine Isaacs84, Trinidad Caldes85, Miguel de la Hoya85, Tuomas Heikkinen6, Kristiina Aittomäki86, Ignacio Blanco87, Conxi Lazaro87, Rosa B Barkardottir88, Penny Soucy89, Martine Dumont89, Jacques Simard90, Marco Montagna91, Silvia Tognazzo91, Emma D'Andrea92, Stephen Fox93, Max Yan94, Tim Rebbeck95, Olufunmilayo I Olopade96, Jeffrey N Weitzel97, Henry T Lynch98, Patricia A Ganz99, Gail E Tomlinson100, Xianshu Wang2, Zachary Fredericksen101, Vernon S Pankratz101, Noralane M Lindor102, Csilla Szabo103, Kenneth Offit8, Rita Sakr8, Mia Gaudet104, Jasmine Bhatia8, Noah Kauff8, Christian F Singer105, Muy-Kheng Tea105, Daphne Gschwantler-Kaulich105, Anneliese Fink-Retter105, Phuong L Mai106, Mark H Greene106, Evgeny Imyanitov107, Frances P O'Malley108, Hilmi Ozcelik109, Gordon Glendon110, Amanda E Toland111, Anne-Marie Gerdes112, Mads Thomassen113, Torben A Kruse113, Uffe Birk Jensen114, Anne-Bine Skytte115, Maria A Caligo116, Maria Soller117, Karin Henriksson118, von Anna Wachenfeldt119, Brita Arver119, Marie Stenmark-Askmalm120, Per Karlsson121, Yuan Chun Ding122, Susan L Neuhausen122, Mary Beattie123, Paul DP Pharoah124, Kirsten B Moysich125, Katherine L Nathanson4, Beth Y Karlan126, Jenny Gross126, Esther M John127, Mary B Daly128, Saundra M Buys129, Melissa C Southey130, John L Hopper131, Mary Beth Terry132, Wendy Chung132, Alexander F Miron133, David Goldgar134, Georgia Chenevix-Trench10, Douglas F Easton3, Irene L Andrulis135, Antonis C Antoniou3*, Breast Cancer Family Registry136, EMBRACE3, GEMO Study Collaborators137, HEBON138, kConFab Investigators93, Ontario Cancer Genetics Network139, SWE-BRCA140 and CIMBA

Author Affiliations

1 Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, ON M5S 1A8, Canada; Department of Laboratory Medicine, and the Keenan Research Centre of the Li Ka Shing Knowledge Institute, St Michael's Hospital, 30 Bond Street, Toronto, ON M5B 1W8, Canada

2 Department of Laboratory Medicine and Pathology, Mayo Clinic, 200 First Street SW, Rochester, MN 55905, USA

3 Centre for Cancer Genetic Epidemiology, Department of Public Health and Primary Care, University of Cambridge, 2 Worts Causeway, Cambridge, CB1 8RN, UK

4 Department of Medicine, Abramson Cancer Center, Perelman School of Medicine at the University of Pennsylvania, 3400 Civic Center Boulevard, Philadelphia, PA 19104, USA

5 Faculty of Medicine, University of Southampton, University Hospital Southampton NHS Foundation Trust, Tremona Road, Southampton, SO16 6YD, UK

6 Department of Obstetrics and Gynecology, University of Helsinki and Helsinki University Central Hospital, Haartmaninkatu 8, 00290 Helsinki, Finland

7 Department of Preventive Medicine, Keck School of Medicine, University of Southern California, 2001 N Soto Street, Los Angeles, CA 90089-9237, USA

8 Department of Medicine, Memorial Sloan-Kettering Cancer Center and Weill Cornell Medical College, 1275 York Ave, New York, NY 10065, USA

9 National Cancer Institute, Division of Cancer Epidemiology and Genetics, Hormonal and Reproductive Epidemiology Branch, 6120 Executive Blvd., Rockville, MD 20852, USA

10 Queensland Institute of Medical Research, 300 Herston Rd, Herston, Brisbane, QLD 4006, Australia

11 Centre of Familial Breast and Ovarian Cancer, Department of Gynaecology and Obstetrics and Centre for Integrated Oncology (CIO), University Hospital of Cologne, Kerpener Str. 62, Cologne, 50931, Germany

12 Unité Mixte de Génétique Constitutionnelle des Cancers Fréquents, Centre Hospitalier Universitaire de Lyon/Centre Léon Bérard, 28 rue Laënnec, Lyon 69373, France

13 INSERM U1052, CNRS UMR5286, Université Lyon 1, Cancer Research Center of Lyon, 28 rue Laënnec, Lyon 69373, France

14 Department of Molecular and Regenerative Medicine, Hematology, Oncology and Transfusion Medicine Center, Vilnius University Hospital Santariskiu Clinics, Santariskiu st 2, LT-08661 Vilnius and State Research Institute Innovative Medicine Center, Zygimantu st. 9, LT-01102 Vilnius, Lithuania

15 Center for Genomic Medicine, Rigshospitalet, Copenhagen University Hospital, Blegdamsvej 9, DK-2100 Copenhagen, Denmark

16 Department of Oncology, Rigshospitalet Bldg. 4262, Copenhagen University Hospital, Blegdamsvej 9, DK-2100 Copenhagen, Denmark

17 Human Genetics Group, Human Cancer Genetics Programme, Spanish National Cancer Research Centre, C/Melchor Fernández Almagro 3, Madrid, 28029, Spain and the Spanish Network on Rare Diseases (CIBERER)

18 Institute of Biology and Molecular Genetics, Universidad de Valladolid (IBGM-UVA), C/Sanz y Forés, N° 3, Valladolid, 47003, Spain

19 Instituto de investigación sanitaria de Aragón (IIS), Hospital clinico Universitario "Lozano Blesa", San Juan Bosco 15, Zaragoza, 50009, Spain

20 Molecular Diagnostics Laboratory, IRRP, National Center for Scientific Research Demokritos, Patriarchou Gregoriou E' & Neapoleos Str, Ag. Paraskevi 15310, Athens, Greece

21 Human Genetics Group and Genotyping Unit, Human Cancer Genetics Programme, Spanish National Cancer Research Centre, C/Melchor Fernández Almagro 3, Madrid, 28029, Spain and the Spanish Network on Rare Diseases (CIBERER)

22 Unit of Molecular Bases of Genetic Risk and Genetic Testing, Department of Preventive and Predictive Medicine, Fondazione IRCCS Istituto Nazionale Tumouri (INT), via Giacomo Venezian 1, 20133 Milan, Italy; IFOM, Fondazione Istituto FIRC di Oncologia Molecolare, via Adamello 16, 20139 Milan, Italy

23 Unit of Medical Genetics, Department of Preventive and Predictive Medicine, Fondazione IRCCS Istituto Nazionale Tumouri (INT), via Giacomo Venezian 1, Milan, 20133, Italy

24 Division of Cancer Prevention and Genetics, Istituto Europeo di Oncologia, via Ripamonti 435, Milan, 20141, Italy

25 Unit of Experimental Oncology 1, Centro di Riferimento Oncologico, IRCCS, Aviano (PN), Italy

26 Department of Genetics, Biology and BiochemIstry, University of Turin, Turin, Italy

27 Medical Genetics Unit, Department of Clinical Physiopathology, University of Florence, Firenze, Italy

28 Department of Molecular Medicine, "Sapienza" University of Rome, Rome, Italy

29 Division of Medical Oncology, Regina Elena Cancer Institute, Rome, Italy

30 Department of Experimental Oncology, Istituto Europeo di Oncologia. Milan, Italy; Consortium for Genomics Technology (Cogentech), Milan, Italy

31 Molecular Genetics of Breast Cancer, Deutsches Krebsforschungszentrum (DKFZ), Heidelberg, Germany

32 Department of Epidemiology, Netherlands Cancer Institute, Amsterdam, The Netherlands

33 Department of Clinical Genetics, VU Medical Center, Amsterdam, The Netherlands

34 Department of Human Genetics and Department of Clinical Genetics, Leiden University Medical Center, Leiden, The Netherlands

35 Department of Clinical Genetics and GROW, School for Oncology and Developmental Biology, MUMC, Maastricht, The Netherlands

36 Department of Human Genetics 849, Radboud University Nijmegen Medical Centre, P.O. BOX 9101, 6500 HB Nijmegen, The Netherlands

37 Department of Human Genetics 836, Radboud University Nijmegen Medical Centre, P.O. BOX 9101, 6500 HB Nijmegen, The Netherlands

38 Department of Medical Oncology, Family Cancer Clinic, Erasmus University Medical Center, Rotterdam, The Netherlands

39 Department of Surgical Oncology, Family Cancer Clinic, Erasmus University Medical Center, Rotterdam, The Netherlands

40 Department of Medical Genetics, University Medical Center Utrecht, The Netherlands

41 Department of Clinical Genetics, Academic Medical Center, Amsterdam, The Netherlands

42 Barts Cancer Institute, Queen Mary University of London, Centre for Tumour Biology, Charterhouse Square, London, UK

43 Genetic Medicine, Manchester Academic Health Sciences Centre, Central Manchester University Hospitals NHS Foundation Trust, Manchester, UK

44 Oncogenetics Team, The Institute of Cancer Research and Royal Marsden NHS Foundation Trust, UK

45 Clinical Genetics, Guy's and St. Thomas' NHS Foundation Trust, London, UK

46 Yorkshire Regional Genetics Service, Leeds, UK

47 Ferguson-Smith Centre for Clinical Genetics, Yorkhill Hospitals, Glasgow, UK

48 Sheffield Clinical Genetics Service, Sheffield Children's Hospital, Sheffield, UK

49 Clinical Genetics Department, St Michael's Hospital, Bristol, UK

50 North West Thames Regional Genetics Service, Kennedy-Galton Centre, Harrow, UK

51 North of Scotland Regional Genetics Service, NHS Grampian and University of Aberdeen, Foresterhill, Aberdeen, UK

52 Nottingham Clinical Genetics Service, Nottingham University Hospitals NHS Trust, UK

53 Cheshire and Merseyside Clinical Genetics Service, Liverpool Women's NHS Foundation Trust, Liverpool, UK

54 Leicestershire Clinical Genetics Service, University Hospitals of Leicester NHS Trust, UK

55 North East Thames Regional Genetics Service, Great Ormond Street Hospital for Children NHS Trust, London, UK

56 All Wales Medical Genetics Service, Glan Clwyd Hospital, Rhyl, UK

57 All Wales Medical Genetics Services, Singleton Hospital, Swansea, UK

58 Department of Pathology and Laboratory Medicine, University of Kansas Medical Center, Kansas City, KS, USA

59 Institute for Medical Informatics, Statistics and Epidemiology, University of Leipzig, Leipzig, Germany

60 Department of Gynaecology and Obstetrics, Division of Tumour Genetics, Klinikum rechts der Isar, Technical University, Munich, Germany

61 Department of Gynaecology and Obstetrics, Ludwig-Maximillians University, Munich, Germany

62 Department of Gynaecology and Obstetrics, University Hospital of Schleswig-Holstein (UKSH), Campus Kiel, Christian-Albrechts University, Kiel, Germany

63 Department of Gynaecology and Obstetrics, University Hospital Düsseldorf, Heinrich-Heine University, Düsseldorf, Germany

64 Institute of Human Genetics, Department of Human Genetics, Heidelberg University Hospital, Heidelberg, Germany

65 Department of Gynaecology and Obstetrics, University Hospital, Ulm, Germany

66 Institute of Cell and Molecular Pathology, Hannover Medical School, Hannover, Germany

67 Department of Gynaecology and Obstetrics, University Hospital Carl Gustav Carus, Technical University, Dresden, Germany

68 Institute of Human Genetics, University of Münster, Münster, Germany

69 Institute of Human Genetics, Campus Virchov Klinikum, Charite Berlin, Germany

70 Centre of Familial Breast and Ovarian Cancer, Department of Medical Genetics, Institute of Human Genetics, University Würzburg, Germany

71 Institute of Human Genetics, University Regensburg, Germany

72 Institute of Human Genetics, University Leipzig, Germany

73 Institute of Human Genetics, University Hospital, Frankfurt a.M., Germany

74 Breakthrough Breast Cancer Research Centre, Institute of Cancer Research, UK

75 Service de Génétique Oncologique, Institut Curie, Paris, France

76 Genetic Unit, Avicenne Hospital, Assitance Publique-Hôpitaux de Paris, Paris, France; Sud-Francilien Hospital, Evry-Corbeil, France; University Hospital, Clermont-Ferrand, France

77 Centre François Baclesse, Caen, France

78 Unité d'Oncogénétique, CLCC Paul Strauss, Strasbourg, France

79 Service de Génétique, Centre Hospitalier Universitaire Bretonneau, Tours, France

80 Unité d'Oncogénétique, CHU Arnaud de Villeneuve, Montpellier, France

81 Service de Génétique Clinique Chromosomique et Moléculaire, Centre Hospitalier Universitaire de St Etienne, St Etienne, France

82 Cancer Genetics Unit, INSERM U916, Institut Bergonié, Université de Bordeaux, Bordeaux, France

83 Unité INSERM U830, Institut Curie, Paris, France; Université Paris Descartes, Faculté de Médecine, Paris, France

84 Lombardi Comprehensive Cancer Center, Georgetown University, Washington DC, USA

85 Molecular Oncology Laboratory, Hospital Clinico San Carlos, Madrid, Spain

86 Department of Clinical Genetics, Helsinki University Central Hospital, Meilahdentie 2, 00290 Helsinki, Finland

87 Hereditary Cancer Program, Institut Català d'Oncologia, Hospital Duran i Reynals - Bellvitge Biomedical Research Institute (IDIBELL), L'Hospitalet de Llobregat, Barcelona, Spain

88 Department of Pathology, Landspitali University Hospital, Reykjavik, Iceland, Faculty of Medicine, University of Iceland, Reykjavik, Iceland

89 Cancer Genomics Laboratory, Centre Hospitalier Universitaire de Québec, 2705 Laurier Boulevard, T3-57, Quebec City, QC, Canada

90 Cancer Genomics Laboratory, Centre Hospitalier Universitaire de Québec, 2705 Laurier Boulevard, T3-57, Quebec City, QC, Canada; Canada Research Chair in Oncogenetics, Department of Molecular Medicine, Faculty of Medicine, Laval University, QC, Canada

91 Immunology and Molecular Oncology Unit, Istituto Oncologico Veneto IOV - IRCCS, Via Gattamelata 64, 35128 Padua, Italy

92 Department of Oncology and Surgical Sciences, University of Padua and Istituto Oncologico Veneto IOV - IRCCS, Via Gattamelata 64, 35128 Padua, Italy

93 Peter MacCallum Cancer Centre, Melbourne, VIC 3052, Australia

94 Department of Anatomical Pathology, Prince of Wales Hospital, Randwick, NSW 2031, Australia

95 Abramson Cancer Center and University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, USA

96 University of Chicago, Chicago, IL, USA

97 City of Hope Comprehensive Cancer Center and Department of Population Sciences, Beckman Research Institute, City of Hope, Duarte, CA, USA

98 Departments of Medicine, and Preventive Medicine and Public Health, Creighton University, Omaha, NE, USA

99 Jonsson Comprehensive Cancer Center at the University of California, Los Angeles, CA, USA

100 Department of Internal Medicine and Harold C. Simmons Comprehensive Cancer Center, University of Texas, Southwestern Medical Center, Dallas, TX; USA; Department of Pediatrics, University of Texas Health Science Center at San Antonio, San Antonio, TX, USA

101 Department of Health Sciences Research, Mayo Clinic, Rochester, MN, USA

102 Department of Medical Genetics, Mayo Clinic, Rochester, MN, USA

103 University of Delaware, Newark, DE, USA

104 Epidemiology Research Program, American Cancer Society, Atlanta, GA, USA

105 Department of Obstetrics/Gynaecology and Comprehensive Cancer Center, Medical University of Vienna, Vienna, Austria

106 Clinical Genetics Branch, Division of Cancer Epidemiology and Genetics, US National Cancer Institute, Rockville, MD, USA

107 Laboratory of Molecular Oncology, N.N. Petrov Institute of Oncology, St.-Petersburg, Russia

108 Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, ON, Canada; Department of Laboratory Medicine, and the Keenan Research Centre of the Li Ka Shing Knowledge Institute, St Michael's Hospital, Toronto, ON, Canada

109 Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto; Department of Laboratory Medicine and Pathobiology, University of Toronto, ON, Canada

110 Cancer Care Ontario, ON, Canada

111 Departments of Molecular Virology, Immunology and Medical Genetics and Internal Medicine, The Ohio State University Comprehensive Cancer Center, Columbus, OH, USA

112 Clinical Genetics, Rigshospital, Copenhagen University, Copenhagen, Denmark

113 Clinical Genetics, Odense University Hospital, Odense, Denmark

114 Department of Clinical Genetics, Aarhus University Hospital, Aarhus, Denmark

115 Clinical Genetics, Vejle Hospital, Denmark

116 Section of Genetic Oncology, Dept. of Laboratory Medicine, University and University Hospital of Pisa, Pisa, Italy

117 Department of Clinical Genetics, Lund University Hospital, Lund, Sweden

118 Oncological Centre, Lund University Hospital, Lund, Sweden

119 Department of Oncology, Karolinska University Hospital, Stockholm, Sweden

120 Division of Clinical Genetics, Department of Clinical and Experimental Medicine, Linköping University, Linköping, Sweden

121 Department of Oncology, Sahlgrenska University Hospital, Gothenburg, Sweden

122 Department of Population Sciences, the Beckman Research Institute of the City of Hope, Duarte, CA, USA

123 UCSF Cancer Risk Program and Departments of Medicine and Epidemiology and Biostatistics, University of California San Francisco, San Francisco, CA, USA

124 Department of Oncology, University of Cambridge, Cambridge, UK

125 Department of Cancer Prevention and Control, Roswell Park Cancer Institute, Buffalo, NY, USA

126 Women's Cancer Program at the Samuel Oschin Comprehensive Cancer Institute, Cedars-Sinai Medical Center, Los Angeles, CA, USA

127 Department of Epidemiology, Cancer Prevention Institute of California, Fremont, CA, USA

128 Fox Chase Cancer Center, Philadelphia, PA, USA

129 Department of Oncological Sciences, Huntsman Cancer Institute, University of Utah, Salt Lake City, UT, USA

130 Genetic Epidemiology Laboratory, Department of Pathology, University of Melbourne, Melbourne, VIC, Australia

131 Centre for Molecular, Environmental, Genetic and Analytic Epidemiology, University of Melbourne, Melbourne, VIC, Australia

132 Department of Epidemiology, Columbia University, New York, NY, USA

133 Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, MA, USA

134 Department of Dermatology, University of Utah School of Medicine, Salt Lake City, UT, USA

135 Samuel Lunenfeld Research Institute, Mount Sinai Hospital, 600 University Avenue, Toronto, ON, M5G 1X5, Canada; Cancer Care Ontario, Departments of Molecular Genetics and Laboratory Medicine and Pathobiology, University of Toronto, ON, Canada

136 Breast Cancer Family Registry

137 Cancer Genetics Network "Groupe Génétique et Cancer", Fédération Nationale des Centres de Lutte Contre le Cancer

138 Netherlands Cancer Institute, Amsterdam, The Netherlands

139 Ontario Cancer Genetics Network, Cancer Care Ontario, 620 University Avenue, Toronto, ON M5G 2L7, Canada

140 Karolinska Institute, Stockholm, Sweden

For all author emails, please log on.

Breast Cancer Research 2011, 13:R110  doi:10.1186/bcr3052

Published: 2 November 2011

Abstract

Introduction

Previous studies have demonstrated that common breast cancer susceptibility alleles are differentially associated with breast cancer risk for BRCA1 and/or BRCA2 mutation carriers. It is currently unknown how these alleles are associated with different breast cancer subtypes in BRCA1 and BRCA2 mutation carriers defined by estrogen (ER) or progesterone receptor (PR) status of the tumour.

Methods

We used genotype data on up to 11,421 BRCA1 and 7,080 BRCA2 carriers, of whom 4,310 had been affected with breast cancer and had information on either ER or PR status of the tumour, to assess the associations of 12 loci with breast cancer tumour characteristics. Associations were evaluated using a retrospective cohort approach.

Results

The results suggested stronger associations with ER-positive breast cancer than ER-negative for 11 loci in both BRCA1 and BRCA2 carriers. Among BRCA1 carriers, single nucleotide polymorphism (SNP) rs2981582 (FGFR2) exhibited the biggest difference based on ER status (per-allele hazard ratio (HR) for ER-positive = 1.35, 95% CI: 1.17 to 1.56 vs HR = 0.91, 95% CI: 0.85 to 0.98 for ER-negative, P-heterogeneity = 6.5 × 10-6). In contrast, SNP rs2046210 at 6q25.1 near ESR1 was primarily associated with ER-negative breast cancer risk for both BRCA1 and BRCA2 carriers. In BRCA2 carriers, SNPs in FGFR2, TOX3, LSP1, SLC4A7/NEK10, 5p12, 2q35, and 1p11.2 were significantly associated with ER-positive but not ER-negative disease. Similar results were observed when differentiating breast cancer cases by PR status.

Conclusions

The associations of the 12 SNPs with risk for BRCA1 and BRCA2 carriers differ by ER-positive or ER-negative breast cancer status. The apparent differences in SNP associations between BRCA1 and BRCA2 carriers, and non-carriers, may be explicable by differences in the prevalence of tumour subtypes. As more risk modifying variants are identified, incorporating these associations into breast cancer subtype-specific risk models may improve clinical management for mutation carriers.