NMR-based metabolomic profiling of overweight adolescents:an elucidation of the effects of inter-/intraindividual differences, gender, and pubertal development

The plasma and urine metabolome of 192 overweight 12–15-year-old adolescents (BMI of 25.4 ± 2.3 kg/m2) were examined in order to elucidate gender, pubertal development measured as Tanner stage, physical activity measured as number of steps taken daily, and intra-/interindividual differences affecting the metabolome detected by proton NMR spectroscopy. Higher urinary excretion of citrate, creatinine, hippurate, and phenylacetylglutamine and higher plasma level of phosphatidylcholine and unsaturated lipid were found for girls compared with boys. The results suggest that gender differences in the metabolome are being commenced already in childhood. The relationship between Tanner stage and the metabolome showed that pubertal development stage was positively related to urinary creatinine excretion and negatively related to urinary citrate content. No relations between physical activity and the metabolome could be identified. The present study for the first time provides comprehensive information about associations between the metabolome and gender, pubertal development, and physical activity in overweight adolescents, which is an important subject group to approach in the prevention of obesity and life-style related diseases. While this study is preliminary, these results may have the potential to translate into clinical applicability upon further investigations; if biomarkers for Tanner stage can be established, these might be used for identification of individuals susceptible to an early pubertal development

Metabolic Effects of Bovine Milk Oligosaccharides on Selected Commensals of the Infant Microbiome - Commensalism and Postbiotic Effects

Oligosaccharides from human or bovine milk selectively stimulate growth or metabolism of bacteria associated with the lower gastrointestinal tract of infants. Results from complex infant-type co-cultures point toward a possible synergistic effect of combining bovine milk oligosaccharides (BMO) and lactose (LAC) on enhancing the metabolism of Bifidobacterium longum subsp. longum and inhibition of Clostridium perfringens. We examine the interaction between B. longum subsp. longum and the commensal Parabacteroides distasonis, by culturing them in mono- and co-culture with different carbohydrates available. To understand the interaction between BMO and lactose on B. longum subsp. longum and test the potential postbiotic effect on C. perfringens growth and/or metabolic activity, we inoculated C. perfringens into fresh media and compared the metabolic changes to C. perfringens in cell-free supernatant from B. longum subsp. longum fermented media. In co-culture, B. longum subsp. longum benefits from P. distasonis (commensalism), especially in a lactose-rich environment. Furthermore, B. longum subsp. longum fermentation of BMO + LAC impaired C. perfringens’ ability to utilize BMO as a carbon source (potential postbiotic effect)

Poison, plants and Palaeolithic hunters. An analytical method to investigate the presence of plant poison on archaeological artefacts

In this paper we present the development of a method for the detection of toxic substances on ancient arrow points. The aim is to go back in time until the Palaeolithic period in order to determine if poisonous substances were used to enhance the hunting weapons. The ethnographic documentation demonstrates that hunters of every latitude poisoned their weapons with toxic substances derived from plants and occasionally from animals. This highlights that often the weapons would be rather ineffective if the tips were not poisoned. The fact that toxic substances were available and the benefits arising from their application on throwing weapons, suggests that this practice could be widespread also among prehistoric hunters. The project reviewed the research of the toxic molecules starting from current information on modern plants and working backwards through the ages with the study of ethnographic and historical weapons. This knowledge was then applied to the archaeological material collected from International museum collections. Results have shown that using this method it is possible to detect traces of toxic molecules with mass spectrometry (MS) and hyphenated chromatographic techniques even on samples older than one hundred years, which we consider a positive incentive to continue studying plant poisons on ancient hunting tools

Metabolomic phenotyping of a cloned pig model

<p>Abstract</p> <p>Background</p> <p>Pigs are widely used as models for human physiological changes in intervention studies, because of the close resemblance between human and porcine physiology and the high degree of experimental control when using an animal model. Cloned animals have, in principle, identical genotypes and possibly also phenotypes and this offer an extra level of experimental control which could possibly make them a desirable tool for intervention studies. Therefore, in the present study, we address how phenotype and phenotypic variation is affected by cloning, through comparison of cloned pigs and normal outbred pigs.</p> <p>Results</p> <p>The metabolic phenotype of cloned pigs (n = 5) was for the first time elucidated by nuclear magnetic resonance (NMR)-based metabolomic analysis of multiple bio-fluids including plasma, bile and urine. The metabolic phenotype of the cloned pigs was compared with normal outbred pigs (n = 6) by multivariate data analysis, which revealed differences in the metabolic phenotypes. Plasma lactate was higher for cloned vs control pigs, while multiple metabolites were altered in the bile. However a lower inter-individual variability for cloned pigs compared with control pigs could not be established.</p> <p>Conclusions</p> <p>From the present study we conclude that cloned and normal outbred pigs are phenotypically different. However, it cannot be concluded that the use of cloned animals will reduce the inter-individual variation in intervention studies, though this is based on a limited number of animals.</p

Metabolomics and bacterial diversity of packaged yellowfin tuna (Thunnus albacares) and salmon (Salmo salar) show fish species-specific spoilage development during chilled storage

Microbial (colony counts, 16S rRNA gene amplification), chemical (pH, 1H NMR spectroscopy) and sensory changes in raw Atlantic Salmon (Salmo salar) and tuna (Thunnus albacares) fillets stored under vacuum at 3 °C were evaluated over a period of 12 days. Both species of fish are globally important and among the ten most consumed fishes in the world. Although the sensory analyses showed a decrease in the quality of both fish species, only the salmon fillets were considered spoiled at the end of the storage period. In salmon, trimethylamine was the main spoilage product and bacterial colony counts reached an average of 7.3 log10 cfu/g. The concentration of glucose decreased and the concentration of organic acids increased during storage revealing glucose fermentation. Photobacterium was the dominating genus in the salmon studied. In the tuna studied, the bacterial colony counts reached only an average of 4.6 log10 cfu/g. The dominating bacteria in tuna were Pseudomonas spp. Glucose levels did not decrease, suggesting that amino acids and lactate most likely acted as carbon sources for bacteria in tuna. In conclusion, the study revealed that salmon was clearly a more perishable fish than tuna.Peer reviewe

Creatine-induced activation of antioxidative defence in myotube cultures revealed by explorative NMR-based metabonomics and proteomics

<p>Abstract</p> <p>Background</p> <p>Creatine is a key intermediate in energy metabolism and supplementation of creatine has been used for increasing muscle mass, strength and endurance. Creatine supplementation has also been reported to trigger the skeletal muscle expression of insulin like growth factor I, to increase the fat-free mass and improve cognition in elderly, and more explorative approaches like transcriptomics has revealed additional information. The aim of the present study was to reveal additional insight into the biochemical effects of creatine supplementation at the protein and metabolite level by integrating the explorative techniques, proteomics and NMR metabonomics, in a systems biology approach.</p> <p>Methods</p> <p>Differentiated mouse myotube cultures (C2C12) were exposed to 5 mM creatine monohydrate (CMH) for 24 hours. For proteomics studies, lysed myotubes were analyzed in single 2-DGE gels where the first dimension of protein separation was pI 5-8 and second dimension was a 12.5% Criterion gel. Differentially expressed protein spots of significance were excised from the gel, desalted and identified by peptide mass fingerprinting using MALDI-TOF MS. For NMR metabonomic studies, chloroform/methanol extractions of the myotubes were subjected to one-dimensional ¹H NMR spectroscopy and the intracellular oxidative status of myotubes was assessed by intracellular DCFH₂oxidation after 24 h pre-incubation with CMH.</p> <p>Results</p> <p>The identified differentially expressed proteins included vimentin, malate dehydrogenase, peroxiredoxin, thioredoxin dependent peroxide reductase, and 75 kDa and 78 kDa glucose regulated protein precursors. After CMH exposure, up-regulated proteomic spots correlated positively with the NMR signals from creatine, while down-regulated proteomic spots were negatively correlated with these NMR signals. The identified differentially regulated proteins were related to energy metabolism, glucose regulated stress, cellular structure and the antioxidative defence system. The suggested improvement of the antioxidative defence was confirmed by a reduced intracellular DCFH₂oxidation with increasing concentrations of CMH in the 24 h pre-incubation medium.</p> <p>Conclusions</p> <p>The explorative approach of this study combined with the determination of a decreased intracellular DCFH₂oxidation revealed an additional stimulation of cellular antioxidative mechanisms when myotubes were exposed to CMH. This may contribute to an increased exercise performance mediated by increased ability to cope with training-induced increases in oxidative stress.</p

WHO draft guidelines on dietary saturated and trans fatty acids: time for a new approach?

The 2018 WHO draft guidelines on dietary saturated fatty acids and trans fatty acids recommend reducing total intake of saturated fat and replacing it with polyunsaturated and monounsaturated fatty acids. The recommendations fail to take into account considerable evidence that the health effects of saturated fat varies depending on the specific fatty acid and on the specific food source. Maintaining general advice to reduce total saturated fatty acids will work against the intentions of the guidelines and weaken their effect on chronic disease incidence and mortality. A food based translation of the recommendations for saturated fat intake would avoid unnecessary reduction or exclusion of foods that are key sources of important nutrients

The Serum Metabolite Response to Diet Intervention with Probiotic Acidified Milk in Irritable Bowel Syndrome Patients Is Indistinguishable from that of Non-Probiotic Acidified Milk by 1H NMR-Based Metabonomic Analysis

The effects of a probiotic acidified milk product on the blood serum metabolite profile of patients suffering from Irritable Bowel Syndrome (IBS) compared to a non-probiotic acidified milk product was investigated using 1H NMR metabonomics. For eight weeks, IBS patients consumed 0.4 L per day of a probiotic fermented milk product or non-probiotic acidified milk. Both diets resulted in elevated levels of blood serum L-lactate and 3-hydroxybutyrate. Our results showed identical effects of acidified milk consumption independent of probiotic addition. A similar result was previously obtained in a questionnaire-based evaluation of symptom relief. A specific probiotic effect is thus absent both in the patient subjective symptom evaluations and at the blood serum metabolite level. However, there was no correspondence between symptom relief and metabolite response on the patient level

Identification and characterization of novel associations in the CASP8/ALS2CR12 region on chromosome 2 with breast cancer risk.

Previous studies have suggested that polymorphisms in CASP8 on chromosome 2 are associated with breast cancer risk. To clarify the role of CASP8 in breast cancer susceptibility, we carried out dense genotyping of this region in the Breast Cancer Association Consortium (BCAC). Single-nucleotide polymorphisms (SNPs) spanning a 1 Mb region around CASP8 were genotyped in 46 450 breast cancer cases and 42 600 controls of European origin from 41 studies participating in the BCAC as part of a custom genotyping array experiment (iCOGS). Missing genotypes and SNPs were imputed and, after quality exclusions, 501 typed and 1232 imputed SNPs were included in logistic regression models adjusting for study and ancestry principal components. The SNPs retained in the final model were investigated further in data from nine genome-wide association studies (GWAS) comprising in total 10 052 case and 12 575 control subjects. The most significant association signal observed in European subjects was for the imputed intronic SNP rs1830298 in ALS2CR12 (telomeric to CASP8), with per allele odds ratio and 95% confidence interval [OR (95% confidence interval, CI)] for the minor allele of 1.05 (1.03-1.07), P = 1 × 10(-5). Three additional independent signals from intronic SNPs were identified, in CASP8 (rs36043647), ALS2CR11 (rs59278883) and CFLAR (rs7558475). The association with rs1830298 was replicated in the imputed results from the combined GWAS (P = 3 × 10(-6)), yielding a combined OR (95% CI) of 1.06 (1.04-1.08), P = 1 × 10(-9). Analyses of gene expression associations in peripheral blood and normal breast tissue indicate that CASP8 might be the target gene, suggesting a mechanism involving apoptosis.Part of this work was supported by the European Community´s Seventh Framework Programme under grant agreement number 223175 (grant number HEALTH-F2-2009-223175) (COGS). Funding for the iCOGS infrastructure came from: the European Community's Seventh Framework Programme under grant agreement n° 223175 (HEALTH-F2-2009-223175) (COGS), Cancer Research UK (C1287/A10118, C1287/A 10710, C12292/A11174, C1281/A12014, C5047/A8384, C5047/A15007, C5047/A10692), the National Institutes of Health (CA128978) and Post-Cancer GWAS initiative (1U19 CA148537, 1U19 CA148065 and 1U19 CA148112 - the GAME-ON initiative), the Department of Defence (W81XWH-10-1-0341), the Canadian Institutes of Health Research (CIHR) for the CIHR Team in Familial Risks of Breast Cancer, Komen Foundation for the Cure, the Breast Cancer Research Foundation, and the Ovarian Cancer Research Fund. The ABCFS, NC-BCFR and OFBCR work was supported by the United States National Cancer Institute, National Institutes of Health (NIH) under RFA-CA-06-503 and through cooperative agreements with members of the Breast Cancer Family Registry (BCFR) and Principal Investigators, including Cancer Care Ontario (U01 CA69467), Northern California Cancer Center (U01 CA69417), University of Melbourne (U01 CA69638). Samples from the NC-BCFR were processed and distributed by the Coriell Institute for Medical Research. The content of this manuscript does not necessarily reflect the views or policies of the National Cancer Institute or any of the collaborating centers in the BCFR, nor does mention of trade names, commercial products, or organizations imply endorsement by the US Government or the BCFR. The ABCFS was also supported by the National Health and Medical Research Council of Australia, the New South Wales Cancer Council, the Victorian Health Promotion Foundation (Australia) and the Victorian Breast Cancer Research Consortium. J.L.H. is a National Health and Medical Research Council (NHMRC) Australia Fellow and a Victorian Breast Cancer Research Consortium Group Leader. M.C.S. is a NHMRC Senior Research Fellow and a Victorian Breast Cancer Research Consortium Group Leader. The ABCS was supported by the Dutch Cancer Society [grants NKI 2007-3839; 2009 4363]; BBMRI-NL, which is a Research Infrastructure financed by the Dutch government (NWO 184.021.007); and the Dutch National Genomics Initiative. The ACP study is funded by the Breast Cancer Research Trust, UK. The work of the BBCC was partly funded by ELAN-Fond of the University Hospital of Erlangen. The BBCS is funded by Cancer Research UK and Breakthrough Breast Cancer and acknowledges NHS funding to the NIHR Biomedical Research Centre, and the National Cancer Research Network (NCRN). The BBCS GWAS received funding from The Institut National de Cancer. ES is supported by NIHR Comprehensive Biomedical Research Centre, Guy's & St. Thomas' NHS Foundation Trust in partnership with King's College London, United Kingdom. IT is supported by the Oxford Biomedical Research Centre. The BSUCH study was supported by the Dietmar-Hopp Foundation, the Helmholtz Society and the German Cancer Research Center (DKFZ). The CECILE study was funded by Fondation de France, Institut National du Cancer (INCa), Ligue Nationale contre le Cancer, Ligue contre le Cancer Grand Ouest, Agence Nationale de Sécurité Sanitaire (ANSES), Agence Nationale de la Recherche (ANR). The was supported by the Chief Physician Johan Boserup and Lise Boserup Fund, the Danish Medical Research Council and Herlev Hospital. The CNIO-BCS was supported by the Genome Spain Foundation, the Red Temática de Investigación Cooperativa en Cáncer and grants from the Asociación Española Contra el Cáncer and the Fondo de Investigación Sanitario (PI11/00923 and PI081120). The Human Genotyping-CEGEN Unit (CNIO) is supported by the Instituto de Salud Carlos III. The CTS was supported by the California Breast Cancer Act of 1993; National Institutes of Health (grants R01 CA77398 and the Lon V Smith Foundation [LVS39420].); the California Breast Cancer Research Fund (contract 97-10500). Collection of cancer incidence data used in this study was supported by the California Department of Public Health as part of the statewide cancer reporting program mandated by California Health and Safety Code Section 103885. The ESTHER study was supported by a grant from the Baden Württemberg Ministry of Science, Research and Arts. Additional cases were recruited in the context of the VERDI study, which was supported by a grant from the German Cancer Aid (Deutsche Krebshilfe). The GC-HBOC was supported by Deutsche Krebshilfe (107 352). The GENICA was funded by the Federal Ministry of Education and Research (BMBF) Germany grants 01KW9975/5, 01KW9976/8, 01KW9977/0 and 01KW0114, the Robert Bosch Foundation, Stuttgart, Deutsches Krebsforschungszentrum (DKFZ), Heidelberg, Institute for Prevention and Occupational Medicine of the German Social Accident Insurance, Institute of the Ruhr University Bochum (IPA), Bochum, Germany, as well as the Department of Internal Medicine, Evangelische Kliniken Bonn gGmbH, Johanniter Krankenhaus, Bonn, Germany. The HEBCS was financially supported by the Helsinki University Central Hospital Research Fund, Academy of Finland (266528), the Finnish Cancer Society, The Nordic Cancer Union and the Sigrid Juselius Foundation. The GWS population allele and genotype frequencies were obtained from the data source funded by the Nordic Center of Excellence in Disease Genetics based on samples regionally selected from Finland, Sweden and Denmark. The HERPACC was supported by a Grant-in-Aid for Scientific Research on Priority Areas from the Ministry of Education, Science, Sports, Culture and Technology of Japan, by a Grant-in-Aid for the Third Term Comprehensive 10-Year Strategy for Cancer Control from Ministry Health, Labour and Welfare of Japan, by Health and Labour Sciences Research Grants for Research on Applying Health Technology from Ministry Health, Labour and Welfare of Japan and by National Cancer Center Research and Development Fund. The HMBCS was supported by a grant from the Friends of Hannover Medical School and by the Rudolf Bartling Foundation. Financial support for KARBAC was provided through the regional agreement on medical training and clinical research (ALF) between Stockholm County Council and Karolinska Institutet, The Swedish Cancer Society and the Gustav V Jubilee foundation. The KBCP was financially supported by the special Government Funding (EVO) of Kuopio University Hospital grants, Cancer Fund of North Savo, the Finnish Cancer Organizations, the Academy of Finland and by the strategic funding of the University of Eastern Finland. kConFab is supported by grants from the National Breast Cancer Foundation, the NHMRC, the Queensland Cancer Fund, the Cancer Councils of New South Wales, Victoria, Tasmania and South Australia and the Cancer Foundation of Western Australia. The kConFab Clinical Follow Up Study was funded by the NHMRC [145684, 288704, 454508]. Financial support for the AOCS was provided by the United States Army Medical Research and Materiel Command [DAMD17-01-1-0729], the Cancer Council of Tasmania and Cancer Foundation of Western Australia and the NHMRC [199600]. G.C.T. and P.W. are supported by the NHMRC. LAABC is supported by grants (1RB-0287, 3PB-0102, 5PB-0018, 10PB-0098) from the California Breast Cancer Research Program. Incident breast cancer cases were collected by the USC Cancer Surveillance Program (CSP), which is supported under subcontract by the California Department of Health. The CSP is also part of the National Cancer Institute's Division of Cancer Prevention and Control Surveillance, Epidemiology, and End Results Program, under contract number N01CN25403. LMBC is supported by the 'Stichting tegen Kanker' (232-2008 and 196-2010). Diether Lambrechts is supported by the FWO and the KULPFV/10/016-SymBioSysII. The MARIE study was supported by the Deutsche Krebshilfe e.V. [70-2892-BR I], the Hamburg Cancer Society, the German Cancer Research Center and the Federal Ministry of Education and Research (BMBF) Germany [01KH0402, 01KH0408, 01KH0409]. MBCSG is supported by grants from the Italian Association for Cancer Research (AIRC) and by funds from the Italian citizens who allocated the 5/1000 share of their tax payment in support of the Fondazione IRCCS Istituto Nazionale Tumori, according to Italian laws (INT-Institutional strategic projects “5x1000”). The MCBCS was supported by the NIH grant CA128978, an NIH Specialized Program of Research Excellence (SPORE) in Breast Cancer [CA116201], the Breast Cancer Research Foundation, a generous gift from the David F. and Margaret T. Grohne Family Foundation and the Ting Tsung and Wei Fong Chao Foundation. MCCS cohort recruitment was funded by VicHealth and Cancer Council Victoria. The MCCS was further supported by Australian NHMRC grants 209057, 251553 and 504711 and by infrastructure provided by Cancer Council Victoria. The MEC was support by NIH grants CA63464, CA54281, CA098758 and CA132839. The work of MTLGEBCS was supported by the Quebec Breast Cancer Foundation, the Canadian Institutes of Health Research for the “CIHR Team in Familial Risks of Breast Cancer” program – grant # CRN-87521 and the Ministry of Economic Development, Innovation and Export Trade – grant # PSR-SIIRI-701. MYBRCA is funded by research grants from the Malaysian Ministry of Science, Technology and Innovation (MOSTI), Malaysian Ministry of Higher Education (UM.C/HlR/MOHE/06) and Cancer Research Initiatives Foundation (CARIF). Additional controls were recruited by the Singapore Eye Research Institute, which was supported by a grant from the Biomedical Research Council (BMRC08/1/35/19/550), Singapore and the National medical Research Council, Singapore (NMRC/CG/SERI/2010). The NBCS was supported by grants from the Norwegian Research council, 155218/V40, 175240/S10 to ALBD, FUGE-NFR 181600/V11 to VNK and a Swizz Bridge Award to ALBD. The NBCS was supported by grants from the Norwegian Research council, 155218/V40, 175240/S10 to ALBD, FUGE-NFR 181600/V11 to VNK and a Swizz Bridge Award to ALBD. The NBHS was supported by NIH grant R01CA100374. Biological sample preparation was conducted the Survey and Biospecimen Shared Resource, which is supported by P30 CA68485. The OBCS was supported by research grants from the Finnish Cancer Foundation, the Academy of Finland Centre of Excellence grant 251314, the Sigrid Juselius Foundation, the University of Oulu, and the Oulu University Hospital special Govermental EVO Research Funds. The OFBCR work was supported by grant UM1 CA164920 from the National Cancer Institute. The content of this manuscript does not necessarily reflect the views or policies of the National Cancer Institute or any of the collaborating centers in the Breast Cancer Family Registry (BCFR), nor does mention of trade names, commercial products, or organizations imply endorsement by the US Government or the BCFR. The ORIGO study was supported by the Dutch Cancer Society (RUL 1997-1505) and the Biobanking and Biomolecular Resources Research Infrastructure (BBMRI-NL CP16). The PBCS was funded by Intramural Research Funds of the National Cancer Institute, Department of Health and Human Services, USA. The pKARMA study was supported by Märit and Hans Rausings Initiative Against Breast Cancer. The RBCS was funded by the Dutch Cancer Society (DDHK 2004-3124, DDHK 2009-4318). The SASBAC study was supported by funding from the Agency for Science, Technology and Research of Singapore (A*STAR), the US National Institute of Health (NIH) and the Susan G. Komen Breast Cancer Foundation. The SBCGS was supported primarily by NIH grants R01CA64277, R01CA148667, and R37CA70867. Biological sample preparation was conducted the Survey and Biospecimen Shared Resource, which is supported by P30 CA68485. The SBCS was supported by Yorkshire Cancer Research awards S295, S299, S305PA, and by the Sheffield Experimental Cancer Medicine Centre Network. NJC was supported by NCI grant R01 CA163353 and The Avon Foundation (02-2009-080). The SCCS is supported by a grant from the National Institutes of Health (R01 CA092447). Data on SCCS cancer cases used in this publication were provided by the Alabama Statewide Cancer Registry; Kentucky Cancer Registry, Lexington, KY; Tennessee Department of Health, Office of Cancer Surveillance; Florida Cancer Data System; North Carolina Central Cancer Registry, North Carolina Division of Public Health; Georgia Comprehensive Cancer Registry; Louisiana Tumor Registry; Mississippi Cancer Registry; South Carolina Central Cancer Registry; Virginia Department of Health, Virginia Cancer Registry; Arkansas Department of Health, Cancer Registry, 4815 W. Markham, Little Rock, AR 72205. The Arkansas Central Cancer Registry is fully funded by a grant from National Program of Cancer Registries, Centers for Disease Control and Prevention (CDC). Data on SCCS cancer cases from Mississippi were collected by the Mississippi Cancer Registry which participates in the National Program of Cancer Registries (NPCR) of the Centers for Disease Control and Prevention (CDC). The contents of this publication are solely the responsibility of the authors and do not necessarily represent the official views of the CDC or the Mississippi Cancer Registry. SEARCH is funded by programme grants from Cancer Research UK [C490/A10124, C490/A16561] and supported by the UK National Institute for Health Research Biomedical Research Centre at the University of Cambridge. The SEBCS was supported by the Korea Health 21 R&D Project [AO30001], Ministry of Health and Welfare, Republic of Korea. SGBCC is funded by the National Medical Research Council start-up Grant and Centre Grant (NMRC/CG/NCIS /2010). Additional controls were recruited by the Singapore Consortium of Cohort Studies-Multi-ethnic cohort (SCCS-MEC), which was funded by the Biomedical Research Council, grant number: 05/1/21/19/425. SKKDKFZS is supported by the DKFZ. The SZBCS was supported by Grant PBZ_KBN_122/P05/2004. The TBCS was funded by The National Cancer Institute Thailand. The TNBCC was supported by: MCBCS (National Institutes of Health Grants CA122340 and a Specialized Program of Research Excellence (SPORE) in Breast Cancer (CA116201), a generous gift from the David F. and Margaret T. Grohne Family Foundation and the Ting Tsung and Wei Fong Chao Foundation. This research has been partly financed by the European Union (European Social Fund – ESF) and Greek national funds through the Operational Program "Education and Lifelong Learning" of the National Strategic Reference Framework (NSRF) - Research Funding Program of the General Secretariat for Research & Technology: ARISTEIA. Investing in knowledge society through the European Social Fund; and the Stefanie Spielman Breast Fund and the Ohio State University Comprehensive Cancer Center. The TWBCS is supported by the Taiwan Biobank project of the Institute of Biomedical Sciences, Academia Sinica, Taiwan. The UKBGS is funded by Breakthrough Breast Cancer and the Institute of Cancer Research (ICR). ICR acknowledges NHS funding to the NIHR Biomedical Research Centre. The Nurses’ Health Studies (CGEMS) are supported by NIH grants CA 65725, CA87969, CA49449, CA67262, CA50385 and 5UO1CA098233. The UK2 GWAS was funded by Wellcome Trust and Cancer Research UK. It included samples collected through the FBCS study, which is funded by Cancer Research UK [C8620/A8372]. It included control data obtained through the WTCCC which was funded by the Wellcome Trust. The DFBBCS GWAS was funded by The Netherlands Organisation for Scientific Research (NWO) as part of a ZonMw/VIDI grant number 91756341. Control GWA genotype data from the Rotterdam Study were funded by NWO Groot Investments (project nr. 175.010.2005.011). We thank all the individuals who took part in these studies and all the researchers, clinicians, technicians and administrative staff who have enabled this work to be carried out. This study would not have been possible without the contributions of the following: Andrew Berchuck (OCAC), Rosalind A. Eeles, Ali Amin Al Olama, Zsofia Kote-Jarai, Sara Benlloch (PRACTICAL), Antonis Antoniou, Lesley McGuffog, Ken Offit (CIMBA), Andrew Lee, and Ed Dicks, and the staff of the Centre for Genetic Epidemiology Laboratory, the staff of the CNIO genotyping unit, Sylvie LaBoissière and Frederic Robidoux and the staff of the McGill University and Génome Québec Innovation Centre, the staff of the Copenhagen DNA laboratory, and Julie M. Cunningham, Sharon A. Windebank, Christopher A. Hilker, Jeffrey Meyer and the staff of Mayo Clinic Genotyping Core Facility. We also thank Maggie Angelakos, Judi Maskiell, Gillian Dite (ABCFS), and extend our thanks to the many women and their families that generously participated in the Australian Breast Cancer Family Study and consented to us accessing their pathology material. JLH is a National Health and Medical Research Council Australia Fellow. MCS is a National Health and Medical Research Council Senior Research Fellow. JLH and MCS are both group leaders of the Victoria Breast Cancer Research Consortium. We thank Sten Cornelissen, Richard van Hien, Linde Braaf, Frans Hogervorst, Senno Verhoef, Ellen van der Schoot, Femke Atsma (ABCS). The ACP study wishes to thank the participants in the Thai Breast Cancer study. Special Thanks also go to the Thai Ministry of Public Health (MOPH), doctors and nurses who helped with the data collection process. Finally, the study would like to thank Dr Prat Boonyawongviroj, the former Permanent Secretary of MOPH and Dr Pornthep Siriwanarungsan, the Department Director-General of Disease Control who have supported the study throughout. We thank Eileen Williams, Elaine Ryder-Mills, Kara Sargus (BBCS), Niall McInerney, Gabrielle Colleran, Andrew Rowan, Angela Jones (BIGGS), Peter Bugert, and Medical Faculty Mannheim (BSUCH). We thank the staff and participants of the Copenhagen General Population Study, and for excellent technical assistance: Dorthe Uldall Andersen, Maria Birna Arnadottir, Anne Bank, and Dorthe Kjeldgård Hansen. The Danish Breast Cancer Group (DBCG) is acknowledged for the tumor information. We thank Guillermo Pita, Charo Alonso, Daniel Herrero, Nuria Álvarez, Pilar Zamora, Primitiva Menendez, the Human Genotyping-CEGEN Unit (CNIO), Hartwig Ziegler, Sonja Wolf, and Volker Hermann (ESTHER). We thank Heide Hellebrand, Stefanie Engert (GC-HBOC). GC-HBOC would like to thank the following persons for providing additional informations and samples: Prof. Dr. Norbert Arnold, Dr. Sabine Preissler-Adams, Dr. Monika Mareeva-Varon, Dr. Dieter Niederacher, Prof. Dr. Brigitte Schlegelberger, Dr. Clemens Mül. The GENICA Network: Dr. Margarete Fischer-Bosch-Institute of Clinical Pharmacology, Stuttgart, and University of Tübingen, Germany; [HB, Wing-Yee Lo, Christina Justenhoven], German Cancer Consortium (DKTK) and German Cancer Research Center (DKFZ) [HB], Department of Internal Medicine, Evangelische Kliniken Bonn gGmbH, Johanniter Krankenhaus, Bonn, Germany [YDK, Christian Baisch], Institute of Pathology, University of Bonn, Germany [Hans-Peter Fischer], Molecular Genetics of Breast Cancer, Deutsches Krebsforschungszentrum (DKFZ), Heidelberg, Germany [Ute Hamann] and Institute for Prevention and Occupational Medicine of the German Social Accident Insurance, Institute of the Ruhr University Bochum (IPA), Bochum, Germany [TB, Beate Pesch, Sylvia Rabstein, Anne Lotz]; Institute of Occupational Medicine and Maritime Medicine, University Medical Center Hamburg-Eppendorf, Germany [Volker Harth]. HEBCS thanks Kirsimari Aaltonen, Tuomas Heikkinen, and Dr. Karl von Smitten and RN Irja Erkkilä for their help with the HEBCS data and samples. We thank Peter Hillemanns, Hans Christiansen and Johann H. Karstens (HMBCS), Eija Myöhänen, Helena Kemiläinen (KBCP). kConFab thanks Heather Thorne, Eveline Niedermayr, the AOCS Management Group (D Bowtell, G Chenevix-Trench, A deFazio, D Gertig, A Green, P Webb), the ACS Management Group (A