Developmental validation of a forensic profiling system for black rhinoceros (Diceros bicornis) and extraction of nuclear DNA from historic rhinoceros horns

Demand for rhinoceros horn in the late 20th century led to the population decline of black rhinoceros (Diceros bicornis) by more than 96 %, and the species remains critically endangered. Trade in rhinoceros and their products is prohibited under CITES legislation. Despite this, there has been a recent increase in rhinoceros horn demand for use in traditional Asian medicine (TAM) to treat a range of illnesses including cancer, childhood fevers and as a hangover cure. Consequently, the value of rhinoceros horn in illegal trade has risen dramatically, alongside a dramatic increase in poaching. Large numbers of horns exist in historic collections, and since 2011 there has been numerous thefts in both rhinoceros range states and throughout Europe. Wildlife DNA forensic analysis can be used to individually identify rhinoceros horn, matching a seized horn to a crime scene. This can provide valuable evidence in criminal proceedings. This study therefore aimed to validate a profiling system that could be used for D. bicornis horns and to determine an optimal DNA extraction method for historic horns. A developmental validation of a marker panel consisting of 15 short tandem repeat (STR) and one sexing locus was carried out to determine its reliability and efficacy in singleplex reactions. The markers were not found to deviate from patterns of Mendelian inheritance, and species specificity studies found that only two loci (WR7C and ZF1) amplified human and dog DNA. Alleles could be scored at DNA concentrations of less than 1 ng/μL for all loci except WR7C. The marker panel was found to be highly reproducible with a maximum d-value of 0.545, mean heterozygous balance of 1.30 and stutter ratio of 0.35. The marker panel was also robust to reductions in annealing temperature (TA) and increasing cycle number (up to 43 cycles), although increasing TA reduced amplification success. The markers were then combined into two multiplexes and allele frequency data was generated for 52 D. bicornis michaeli, and two markers (BlRh37D and IR12) were rejected for violating assumptions of Hardy-Weinberg equilibrium and linkage equilibrium. The average probability of identity (PIave) ranged between 1.06 x 10-11 and 1.16 x 10-7 with varying levels of relatedness and population structure. The marker panel was determined suitable for use in forensic casework analysis under the defined conditions, and limitations were described. Previous studies have shown that nuclear DNA (nDNA) can be extracted from modern horns, but how effective those methods would be on historic horns was unknown. Optimisation of a number of aspects of the extraction method was carried out, including sample preparation, chemical breakdown with dithiothreitol (DTT), mass of starting material and extraction kit. Mechanical breakdown of rhinoceros horn material using a mixer mill was determined unnecessary, whilst the addition of DTT was found to improve digestion. Irrespective of extraction kit, 20 mg of horn material yielded sufficient quality and quantity nDNA for forensic profiling. Qiagen extraction kits using the QIAamp Mini columns were found to yield superior DNA quantity compared with QIAamp MinElute columns. Whilst KingFisher Cell and Tissue DNA Kits yielded equivalent extract quantity, mitochondrial DNA contamination between samples was detected. The Qiagen method using QIAamp MinElute columns was therefore considered the most robust extraction method for historic rhinoceros horns. This work describes the conditions and limitations of a forensic profiling system for D. bicornis and an effective extraction methodology for historic rhinoceros horns. This enables this marker panel to be used with confidence in forensic casework analysis of both modern and historic horns.McDowall, Ia

Seroprevalence of HCV, HBV and HIV in two inner-city London Emergency Departments.

Summary: In this paper we build on work investigating the feasibility of human immunodeficiency virus (HIV) testing in emergency departments (EDs), estimating the prevalence of hepatitis B, C and HIV infections among persons attending two inner-London EDs, identifying factors associated with testing positive in an ED. We also undertook molecular characterisation to look at the diversity of the viruses circulating in these individuals, and the presence of clinically significant mutations which impact on treatment and control. Blood-borne virus (BBV) testing in non-traditional settings is feasible, with emergency departments (ED) potentially effective at reaching vulnerable and underserved populations. We investigated the feasibility of BBV testing within two inner-London EDs. Residual samples from biochemistry for adults (⩾18 years) attending The Royal Free London Hospital (RFLH) or the University College London Hospital (UCLH) ED between January and June 2015 were tested for human immunodeficiency virus (HIV)Ag/Ab, anti-hepatitis C (HCV) and HBsAg. PCR and sequence analysis were conducted on reactive samples. Sero-prevalence among persons attending RFH and UCLH with residual samples (1287 and 1546), respectively, were 1.1% and 1.0% for HBsAg, 1.6% and 2.3% for anti-HCV, 0.9% and 1.6% for HCV RNA, and 1.3% and 2.2% for HIV. For RFH, HBsAg positivity was more likely among persons of black vs. white ethnicity (odds ratio 9.08; 95% confidence interval 2.72–30), with anti-HCV positivity less likely among females (0.15, 95% CI 0.04–0.50). For UCLH, HBsAg positivity was more likely among non-white ethnicity (13.34, 95% CI 2.20–80.86 (Asian); 8.03, 95% CI 1.12–57.61 (black); and 8.11, 95% CI 1.13–58.18 (other/mixed)). Anti-HCV positivity was more likely among 36–55 year olds vs. ⩾56 years (7.69, 95% CI 2.24–26.41), and less likely among females (0.24, 95% CI 0.09–0.65). Persons positive for HIV-markers were more likely to be of black vs. white ethnicity (4.51, 95% CI 1.63–12.45), and less likely to have one ED attendance (0.39, 95% CI 0.17–0.88), or female (0.12, 95% CI 0.04–0.42). These results indicate that BBV-testing in EDs is feasible, providing a basis for further studies to explore provider and patient acceptability, referral into care and cost-effectiveness

Extinctions, genetic erosion and conservation options for the black rhinoceros (Diceros bicornis)

The black rhinoceros is again on the verge of extinction due to unsustainable poaching in its native range. Despite a wide historic distribution, the black rhinoceros was traditionally thought of as depauperate in genetic variation, and with very little known about its evolutionary history. This knowledge gap has hampered conservation efforts because hunting has dramatically reduced the species’ once continuous distribution, leaving five surviving gene pools of unknown genetic affinity. Here we examined the range-wide genetic structure of historic and modern populations using the largest and most geographically representative sample of black rhinoceroses ever assembled. Using both mitochondrial and nuclear datasets, we described a staggering loss of 69% of the species’ mitochondrial genetic variation, including the most ancestral lineages that are now absent from modern populations. Genetically unique populations in countries such as Nigeria, Cameroon, Chad, Eritrea, Ethiopia, Somalia, Mozambique, Malawi and Angola no longer exist. We found that the historic range of the West African subspecies (D. b. longipes), declared extinct in 2011, extends into southern Kenya, where a handful of individuals survive in the Masai Mara. We also identify conservation units that will help maintain evolutionary potential. Our results suggest a complete re-evaluation of current conservation management paradigms for the black rhinoceros

Genetic diversity of the Nubian ibex in Oman as revealed by mitochondrial DNA

The Nubian ibex (Capra nubiana) is patchily distributed across parts of Africa and Arabia. In Oman, it is one of the few free-ranging wild mammals found in the central and southern regions. Its population is declining due to habitat degradation, human expansion, poaching and fragmentation. Here, we investigated the population's genetic diversity using mitochondrial DNA (D-loop 186 bp and cytochrome b 487 bp). We found that the Nubian ibex in the southern region of Oman was more diverse (D-loop HD; 0.838) compared with the central region (0.511) and gene flow between them was restricted. We compared the genetic profiles of wild Nubian ibex from Oman with captive ibex. A Bayesian phylogenetic tree showed that wild Nubian ibex form a distinct clade independent from captive animals. This divergence was supported by high mean distances (D-loop 0.126, cytochrome b 0.0528) and high FST statistics (D-loop 0.725, cytochrome b 0.968). These results indicate that captive ibex are highly unlikely to have originated from the wild population in Oman and the considerable divergence suggests that the wild population in Oman should be treated as a distinct taxonomic unit. Further nuclear genetic work will be required to fully elucidate the degree of global taxonomic divergence of Nubian ibex populations

Genome-Wide Association Study in BRCA1 Mutation Carriers Identifies Novel Loci Associated with Breast and Ovarian Cancer Risk

BRCA1-associated breast and ovarian cancer risks can be modified by common genetic variants. To identify further cancer risk-modifying loci, we performed a multi-stage GWAS of 11,705 BRCA1 carriers (of whom 5,920 were diagnosed with breast and 1,839 were diagnosed with ovarian cancer), with a further replication in an additional sample of 2,646 BRCA1 carriers. We identified a novel breast cancer risk modifier locus at 1q32 for BRCA1 carriers (rs2290854, P = 2.7×10-8, HR = 1.14, 95% CI: 1.09-1.20). In addition, we identified two novel ovarian cancer risk modifier loci: 17q21.31 (rs17631303, P = 1.4×10-8, HR = 1.27, 95% CI: 1.17-1.38) and 4q32.3 (rs4691139, P = 3.4×10-8, HR = 1.20, 95% CI: 1.17-1.38). The 4q32.3 locus was not associated with ovarian cancer risk in the general population or BRCA2 carriers, suggesting a BRCA1-specific associat

Fine-Scale Mapping of the 4q24 Locus Identifies Two Independent Loci Associated with Breast Cancer Risk

Background: A recent association study identified a common variant (rs9790517) at 4q24 to be associated with breast cancer risk. Independent association signals and potential functional variants in this locus have not been explored. Methods: We conducted a fine-mapping analysis in 55,540 breast cancer cases and 51,168 controls from the Breast Cancer Association Consortium. Results: Conditional analyses identified two independent association signals among women of European ancestry, represented by rs9790517 [conditional P = 2.51 × 10−4; OR, 1.04; 95% confidence interval (CI), 1.02–1.07] and rs77928427 (P = 1.86 × 10−4; OR, 1.04; 95% CI, 1.02–1.07). Functional annotation using data from the Encyclopedia of DNA Elements (ENCODE) project revealed two putative functional variants, rs62331150 and rs73838678 in linkage disequilibrium (LD) with rs9790517 (r2 ≥ 0.90) residing in the active promoter or enhancer, respectively, of the nearest gene, TET2. Both variants are located in DNase I hypersensitivity and transcription factor–binding sites. Using data from both The Cancer Genome Atlas (TCGA) and Molecular Taxonomy of Breast Cancer International Consortium (METABRIC), we showed that rs62331150 was associated with level of expression of TET2 in breast normal and tumor tissue. Conclusion: Our study identified two independent association signals at 4q24 in relation to breast cancer risk and suggested that observed association in this locus may be mediated through the regulation of TET2. Impact: Fine-mapping study with large sample size warranted for identification of independent loci for breast cancer risk

Common non-synonymous SNPs associated with breast cancer susceptibility: findings from the Breast Cancer Association Consortium.

Candidate variant association studies have been largely unsuccessful in identifying common breast cancer susceptibility variants, although most studies have been underpowered to detect associations of a realistic magnitude. We assessed 41 common non-synonymous single-nucleotide polymorphisms (nsSNPs) for which evidence of association with breast cancer risk had been previously reported. Case-control data were combined from 38 studies of white European women (46 450 cases and 42 600 controls) and analyzed using unconditional logistic regression. Strong evidence of association was observed for three nsSNPs: ATXN7-K264R at 3p21 [rs1053338, per allele OR = 1.07, 95% confidence interval (CI) = 1.04-1.10, P = 2.9 × 10(-6)], AKAP9-M463I at 7q21 (rs6964587, OR = 1.05, 95% CI = 1.03-1.07, P = 1.7 × 10(-6)) and NEK10-L513S at 3p24 (rs10510592, OR = 1.10, 95% CI = 1.07-1.12, P = 5.1 × 10(-17)). The first two associations reached genome-wide statistical significance in a combined analysis of available data, including independent data from nine genome-wide association studies (GWASs): for ATXN7-K264R, OR = 1.07 (95% CI = 1.05-1.10, P = 1.0 × 10(-8)); for AKAP9-M463I, OR = 1.05 (95% CI = 1.04-1.07, P = 2.0 × 10(-10)). Further analysis of other common variants in these two regions suggested that intronic SNPs nearby are more strongly associated with disease risk. We have thus identified a novel susceptibility locus at 3p21, and confirmed previous suggestive evidence that rs6964587 at 7q21 is associated with risk. The third locus, rs10510592, is located in an established breast cancer susceptibility region; the association was substantially attenuated after adjustment for the known GWAS hit. Thus, each of the associated nsSNPs is likely to be a marker for another, non-coding, variant causally related to breast cancer risk. Further fine-mapping and functional studies are required to identify the underlying risk-modifying variants and the genes through which they act.BCAC is funded by Cancer Research UK (C1287/A10118, C1287/A12014) and by the European Community’s Seventh Framework Programme under grant agreement n8 223175 (HEALTH-F2–2009-223175) (COGS). Meetings of the BCAC have been funded by the European Union COST programme (BM0606). Genotyping of the iCOGS array was funded by the European Union (HEALTH-F2-2009-223175), Cancer Research UK (C1287/A10710), the Canadian Institutes of Health Research for the ‘CIHR Team in Familial Risks of Breast Cancer’ program and the Ministry of Economic Development, Innovation and Export Trade of Quebec (PSR-SIIRI-701). Additional support for the iCOGS infrastructure was provided by 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), Komen Foundation for the Cure, the Breast Cancer Research Foundation, and the Ovarian Cancer Research Fund. The ABCFS and OFBCR work was supported by grant UM1 CA164920 from the National Cancer Institute (USA). 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 t 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) Senior Principal Research Fellow and M.C.S. is a NHMRC Senior Research Fellow. The OFBCR work was also supported by the Canadian Institutes of Health Research ‘CIHR Team in Familial Risks of Breast Cancer’ program. The ABCS was funded by the Dutch Cancer Society Grant no. NKI2007-3839 and NKI2009-4363. The ACP study is funded by the Breast Cancer Research Trust, UK. The work of the BBCC was partly funded by ELAN-Programme 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). E.S. is supported by NIHR Comprehensive Biomedical Research Centre, Guy’s & St. Thomas’ NHS Foundation Trust in partnership with King’s College London, UK. Core funding to the Wellcome Trust Centre for Human Genetics was provided by the Wellcome Trust (090532/Z/09/Z). I.T. 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 the Fondation de France, the French National Institute of Cancer (INCa), The National League against Cancer, the National Agency for Environmental l and Occupational Health and Food Safety (ANSES), the National Agency for Research (ANR), and the Association for Research against Cancer (ARC). The CGPS 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. D.A. was supported by a Fellowship from the Michael Manzella Foundation (MMF) and was a participant in the CNIO Summer Training Program. The CTS was initially supported by the California Breast Cancer Act of 1993 and the California Breast Cancer Research Fund (contract 97-10500) and is currently funded through the National Institutes of Health (R01 CA77398). Collection of cancer incidence e data 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. HAC receives support from the Lon V Smith Foundation (LVS39420). 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 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), as well as the Department of Internal Medicine , Evangelische Kliniken Bonn gGmbH, Johanniter Krankenhaus Bonn, Germany. The HEBCS was supported by the Helsinki University Central Hospital Research Fund, Academy of Finland (132473), the Finnish Cancer Society, The Nordic Cancer Union and the Sigrid Juselius Foundation. 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 a research grant from Takeda Science Foundation , 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 short-term fellowships from the German Academic Exchange Program (to N.B), and the Friends of Hannover Medical School (to N.B.). 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 Stockholm Cancer Foundation and the Swedish Cancer Society. 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 and 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). The MARIE study was supported by the Deutsche Krebshilfe e.V. (70-2892-BR I), the Federal Ministry of Education Research (BMBF) Germany (01KH0402), the Hamburg Cancer Society and the German Cancer Research Center (DKFZ). MBCSG is supported by grants from the Italian Association ciation for Cancer Research (AIRC) and by funds from the Italian citizens who allocated a 5/1000 share of their tax payment in support of the Fondazione IRCCS Istituto Nazionale Tumori, according to Italian laws (INT-Institutional strategic projects ‘5 × 1000’). The MCBCS was supported by the NIH grants (CA122340, CA128978) and a Specialized Program of Research Excellence (SPORE) in Breast Cancer (CA116201), the Breast Cancer Research Foundation and 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 supported 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 (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,tel:08/1/35/19./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 A.L.B.D., FUGE-NFR 181600/ V11 to V.N.K. and a Swizz Bridge Award to A.L.B.D.). 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 Sigrid Juselius Foundation, the Academy of Finland, the University of Oulu, and the Oulu University Hospital. The ORIGO study was supported by the Dutch Cancer Society (RUL 1997-1505) and the Biobanking and Biomolecular Resources Research Infrastructure (BBMRI-NLCP16). The PBCS was funded by Intramural Research Funds of the National Cancer Institute, Department of Health and Human Services, USA. pKARMA is a combination of the KARMA and LIBRO-1 studies. KARMA was supported by Ma¨rit and Hans Rausings Initiative Against Breast Cancer. KARMA and LIBRO-1 were supported the Cancer Risk Prediction Center (CRisP; www.crispcenter.org), a Linnaeus Centre (Contract ID 70867902) financed by the Swedish Research Council. The RBCS was funded by the Dutch Cancer Society (DDHK 2004-3124, DDHK 2009-4318). SASBAC 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 KC was financed by the Swedish Cancer Society (5128-B07-01PAF). 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 S305PA, S299 and S295. Funding for the SCCS was provided by NIH grant R01 CA092447. 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 a programme grant from Cancer Research UK (C490/A10124) and supported by the UK National Institute for Health Research Biomedical Research Centre at the University of Cambridge. The SEBCS was supported by the BRL (Basic Research Laboratory) program through the National Research Foundation of Korea funded by the Ministry of Education, Science and Technology (2012-0000347). SGBCC is funded by the National Medical Research Council Start-up Grant and Centre Grant (NMRC/CG/NCIS /2010). The recruitment of controls by the Singapore Consortium of Cohort Studies-Multi-ethnic cohort (SCCS-MEC) 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. K. J. is a fellow of International PhD program, Postgraduate School of Molecular Medicine, Warsaw Medical University, supported by the Polish Foundation of Science. The TNBCC was supported by the NIH grant (CA128978), the Breast Cancer Research Foundation , Komen Foundation for the Cure, the Ohio State University Comprehensive Cancer Center, the Stefanie Spielman Fund for Breast Cancer Research and a generous gift from the David F. and Margaret T. Grohne Family Foundation and the Ting Tsung and Wei Fong Chao Foundation. Part of the TNBCC (DEMOKRITOS) has been co-financed by the European Union (European Social Fund – ESF) and Greek National Funds through the Operational Program ‘Education and Life-long Learning’ of the National Strategic Reference Framework (NSRF)—Research Funding Program of the General Secretariat for Research & Technology: ARISTEIA. The TWBCS is supported by the Institute of Biomedical Sciences, Academia Sinica and the National Science Council, 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. Funding to pay the Open Access publication charges for this article was provided by the Wellcome Trust.This is the advanced access published version distributed under a Creative Commons Attribution License 2.0, which can also be viewed on the publisher's webstie at: http://hmg.oxfordjournals.org/content/early/2014/07/04/hmg.ddu311.full.pdf+htm