Genomics Biomarkers of Environmental Health · neurological and immune diseases Biosamples and data from 3 prospective cohorts 1. Northern Sweden Health and Disease Study 2. EPIC

1 | NEWSLETTER FINAL ISSUE FEBRUARY 2013

Genomics Biomarkers of Environmental Health

NEWSLETTER FINAL ISSUE • FEBRUARY 2013

www.envirogenomarkers.net • An FP7 molecular epidemiology project


What is EnviroGenomarkers?EnviroGenomarkers is a European FP7 research project aiming at the development and ap-plication of a new generation of biomarkers, based on -omics technologies, to study the envi-ronmental aetiology of human disease.

In the project, advanced technologies, including high-density -omics technologies in combina-tion with sophisticated bioinformatics, are being utilised with the following aims:

the discovery and validation of novel biomarkers predictive of increased risks of chronic • diseases in which the environment may play an important role (breast cancer, B-cell lym-phoma, childhood diseases including allergy, neurological and immune diseases),

the exploration of the association of such risk biomarkers with exposure to a number of • high-priority or emerging environmental pollutants with carcinogenic, immunotoxic or hor-mone-like properties, including polychlorinated biphenyls, polycyclic aromatic hydrocar-bons, cadmium, lead, phthalates, brominated flame retardants (polybrominated diphenyl ethers), as well as ambient air pollutants and water treatment byproducts,

the discovery and validation of biomarkers of exposure to the above pollutants.•

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Conceptual basis of the project

Data integration using bioinformatics and systems biology methodologies

exposure

chemicals: PCBs, PAHs, cadmium, phthalates, brominated flame retardants, ambient air pollutants, water disinfection byproducts

biomarkers of exposure intermediate -omicsbiomarkers of early effects

disease

Food frequency • questionnaires

Environmental • monitoring

GIS•

Concentrations • in urine, serum & erythrocytes

Metabonomics• Epigenomics• Proteomics• Transcriptomics•

Breast cancer•

Non-Hodgkin’s • lymphoma

Childhood allergy, • neurological and immune diseases

Biosamples and data from 3 prospective cohorts

Northern Sweden Health and Disease Study1. 2. EPIC Italy 3. Rhea mother-child cohort (Crete, Greece)

Biomarkers of exposureIntermediate biomarkers of disease risk

Exposure-disease risk relationship


Message from the coordinator EnviroGenomarkers is soon coming to the end of its formal duration and the members of the project consortium look back with some pride at the achievements of the past four years. We can say with confidence that the project achieved all its basic objectives and fulfilled to a large extent all rea-sonably optimistic anticipations.

When we set out on the project, early in 2009, we had two main objectives in mind: First, to evaluate the potential for reliable and biologically mean-ingful analysis, by high-density omics technologies, of human biosamples currently stored in biobanks and collected years ago, prior to the advent of omics and without the precautions subsequently recognized as important for their proper preservation, especially in relation to RNA stability. Sec-ond, and assuming that a positive response to the issues just mentioned was achieved, to utilize multiple omics profiling methods (transcriptomics, epigenomics - CpG methylation, serum UPLC/MS/MS metabolomics, wide-target proteomics) in the context of relatively large population-based, mo-lecular epidemiology studies, in a search for new biomarkers of exposure to environmental chemicals and biomarkers of disease risk, i.e. predictive of future disease.

The first of these objectives was achieved by conducting a pilot study using freshly collected samples as well as samples already in storage for up to 17 years. By experimenting with the conditions of sample collection, handling and storage, we were able to define cut-off criteria for ensuring adequate sample quality and minimal alteration of omics profiles. A particularly im-portant achievement was the clear demonstration that, by using appropri-ate methodology, transcriptomics-quality RNA can be prepared from buffy coats prepared in the absence of RNA stabilizer and kept in cold storage for many years, something that until now many people thought impossible. Subsequent analysis, on different omics platforms, of RNA, DNA and sera from biobanked samples and careful evaluation of the data obtained did not reveal any systematic influence of storage time on the omics profiles. Of course the possibility that the long storage time may have altered to a certain degree the profiles observed cannot be excluded with complete cer-tainty, and the reliability of any biomarkers derived from such data will have to be confirmed through validation studies. However we believe that our findings `[recently been published, Hebels et al., Envir. Hlth Perspect. 121 (2013) 480–487] provide a positive message as regards the prospect of ex-

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ploitation of the power of omics profiling of samples from existing biobanks to look for new biomarkers of exposure and disease risk.

Encouraged by these results, we went on to analyse many hundreds of samples collected 13-17 years ago from individuals healthy at the time and stored in the EPIC-Italy and Northern Sweden Health and Disease Study biobanks. These samples were matched between individuals who subse-quently developed disease (breast cancer or B-cell lymphoma) and indi-viduals who have remained healthy, and the omic profiles of cases and controls have been compared. Corresponding statistical analyses of the same omic profiles to look for markers of environmental exposures were also carried out.

In the case of the breast cancer study, where samples from approx. 100 case/control pairs were analysed, although some signals associating with disease risk or exposure appeared to emerge, they did not satisfy the strin-gent statistical criteria of genome-wide significance. On the other hand, in the lymphoma study, where we analysed samples from nearly 300 case/control pairs, large numbers of statistically very significant signals associat-ing with disease risk, especially risk of particular lymphoma sub-types, were found in the transcriptomics, proteomics and epigenomics profiles. Very im-portantly, the observed signals make strong biological sense in that they are closely related to immune system functions. Analogous signals were also found associating with some of the chemical-specific exposure biomarkers. These results triumphantly confirm the conclusions of the pilot study and open the way for the wider application of modern profiling technologies on samples stored in existing biobanks in the context of prospective studies of the environmental causes of disease. Analogous results were obtained when cord blood or blood collected at age 4 years of children from the Rhea mother-child cohort were examined and the genomic profiles obtained eval-uated against either in utero exposures or clinical end-points related to birth outcomes or neurodevelopment.

The results of the EnviroGenomarkers study show that omics profiling us-ing multiple platforms in the context of relatively large population samples (samples from more than 1,000 subjects were analysed within the project) can be realistically conducted and amply demonstrate the ability of these technologies to help discover novel biomarkers of exposure to environmen-tal toxins and of disease risk and provide biological rationale for etiologi-cal links between the two. In combination with the demonstration of the potential for use for the same purpose of samples from existing biobanks, the project can be considered as having made a valuable contribution to population-based environmental health research.

S.A. Kyrtopoulos, Athens, February 2013


EnviroGenomarkers is a case-control study nested within 3 prospective cohorts:

The Northern Sweden Health and Disease • Study (NSHDS, Umea, Sweden)

EPIC Italy •

The Rhea mother-child birth cohort (Crete, Greece)•

These cohorts contain information (mainly on dietary habits) as well as blood fractions (white cells, plasma or serum, erythrocytes) stored in freezers.

i) The Northern Sweden Health and Disease Study biobank contains samples and data from more than 100,000 individuals, collected in the context of various studies. In the largest among these, the Västerbotten Intervention program, blood samples and lifestyle and environmental exposure information have been collected since 1985 from all individuals of the county aged 40, 50 and 60 years, and currently more than 100,000 samples from 80,000 unique individuals are available. Since 1995, a second blood sample (and questionnaire) is taken with a 10-year interval of the individuals within the cohort, an important asset which is exploited in the Envi-roGenomarkers project.

ii) EPIC-Italy is part of the continent-wide European Investigation into Cancer and Nutrition (EPIC) study in the context of which, since 1992, subjects without known disease, aged 35-70 years, were invited to provide blood samples and information on diet and lifestyle. EPIC-Italy contains samples and information from approximately 47,700 subjects in 5 different areas of the country.

iii) The Rhea mother-child birth cohort, set up in 2007, has enrolled approx. 1,700 pregnant women in the Heraklion region of Crete at the 3rd month of pregnancy and has followed them up until birth and after birth together with their children. Urine and blood samples were col-lected from the mothers at different times during pregnancy and at birth, along with cord blood, as well as information on dietary habits and lifestyle.

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NSHDS - the Northern Sweden Health & Disease Study

EPIC - ItalyRhea mother - child cohort

Crete

The EnviroGenomarkers cohorts


Diseases and environmental chemicals investigated

Breast cancer is a leading cause of death for women aged from their late 30s to their early • 50s, with more than 270,000 women being diagnosed with breast cancer each year in the EU. The large geographic variation in its frequency suggests that environmental factors (nutrition, lifestyle, environmental chemicals) may contribute to its causation. There is evi-dence that breast cancer risk may be increased by exposure to polychlorinated biphenyls (PCBs, pollutants with carcino genic, immunotoxic and hormone-like activities, which accumulate in food and in human fatty tissues), polycyclic aromatic hydrocarbons (PAHs, carcinogens found mainly in charcoaled foods and the atmospheric air polluted with ex-haust gases) and cadmium (found in food as a result of its presence in fertilisers).

B-Cell lymphoma is one of the two major sub-types of non-Hodgkin lymphoma, the fifth • most common malignancy in the Western world, with more than 50,000 new cases being diagnosed in the European Union each year. Its frequency has risen dramatically in the lat-ter half of the 20th century, exceeding 4% annually between 1985 and 1992 in some of Eu-ropean countries. Little is known about its causes. Several recent studies have implicated PCBs in the causation of the disease.

Birth outcomes (e.g. preterm birth) and neurodevelopment and their relationship to in utero • exposure. During recent years, there has been growing awareness of an emerging risk related to the exposure during pregnancy or early life to environmental chemicals which give rise to health effects during childhood persisting into adulthood. Such effects include persistent effects on behaviour and cognitive function (altered play behaviour pattern, over-all cognitive performance, greater impulsivity, poorer concentration, and poorer verbal, pictorial, and auditory working memory), deficient immune function leading to long-term effects (into adulthood) including allergies and asthma. There is growing epidemiological and experimental evidence that environmental chemicals like PCBs, phthalates (widely used as plasticisers) and brominated flame retardants can cause such effects owing to their hormone-like properties.

Diseases and environmental chemicals

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Disease

Breast cancer •

Non-Hodgkins lymphoma •

Chronic neurological, • immune and allergy effects of childhood origin

Chemical

PCBs, PAHs, Cd•

PCBs•

PCBs, phthalates, • polybrominated diphenyl ethers


Biomarkers in EnviroGenomarkers

Biomarkers of exposure, as well as intermediate biomarkers based on -omics technologies, are being measured in biosamples coming from matched pairs of subjects with or without disease and collected prior to the appearance of the disease

Biomarkers of exposure

plasma concentrations of POPs •

erythrocyte cadmium and lead concentrations •

urine concentrations of phthalate metabolites•

Intermediate, - omics biomarkers

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Why -omics?“-omics” technologies make possible the simultaneous examination and measure-ment of all, or a large fraction of, features of a given type in the cell (see below). This provides unprecedented opportunities for biomarker discovery since one can search for biomarkers of interest among an enormous number of observations, without being limited by prior hypotheses.

Metabonomics: analysis, • using advanced liquid chromatography-mass spectrometry, of hundreds of low-molecular weight substances present in serum

Epigenomics: • Measurement, using mi-croarray technology, of the levels of 5-methylcytosine at more than 450,000 of CpG dinucleotides in DNA scattered throughout the genome

Transcriptomics: • Measurement, using microarray technology, of the level of expression of all genes in the cell

Proteomics: Analysis of the • levels of 47 inflammation-related proteins in serum


EnviroGenomarkers pilot study: Criteria and procedures for conducting omics profiling of human biosamples in long-term storage in existing biobanks*

An important question examined by EnviroGenomarkers is whether samples from existing (“old”) biobanks, which were collected prior to the advent of omics technologies and without the precautions subsequently considered as vital for the preservation of sample quality (espe-cially RNA integrity) can be meaningfully analysed using omics technologies. To address this question, initially we used fresh blood samples collected, handled and stored in different ways reflecting those typically found among samples collected in previous decades and currently stored biobanks.

These samples were collected without RNA preservative in different anticoagulants commonly used by different biobanks (heparin, EDTA or citrate) and held at room temperature for up to 24hr to reflect the delay (“bench-time”) usually encountered in the field prior to their final pro-cessing and placement in cold storage. Subsequently the samples were fractionated into buffy coat, erythrocytes and plasma and freezing at -80oC or in liquid nitrogen. We also developed methodology for RNA isolation from buffy coats collected and frozen in the absence of RNA preservative. We found that microarray-quality RNA could be isolated from buffy coats (includ-ing most biobank samples) frozen within 8hr of blood collection, by thawing in RNA preserva-tive (for a video of the procedure see http://ehp.niehs.nih.gov/pdf-files/2013/Feb/ehp.1205657.s002.mp4).

Following these findings, we conducted omic analyses on these samples. We found that dif-ferent anticoagulants influenced the metabolomic, proteomic and, to a lesser extent, transcrip-tomic profiles. The transcriptomic profiles were affected by as little as 2hr delay prior to blood fractionation, while analogous effects were found for metabolomic and proteomic profiles after a bench-time of 8hr or more. Storage temperature had a small effect only on the transcriptomic profile, while none of the variables examined significantly influenced the epigenomic profiles.

Finally we selected from the EPIC Italy and NSHDS biobanks samples satisfying the criterion of having been fractionated and placed in cold storage within 2 hours of collection and subjected them to omic profiling. These samples had been in storage for 13-17 years.

The profiles obtained did not exhibit any obvious irregularity, relative to freshly collected sam-ples, and we could not detect any systematic influence of time-in-storage.

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* see Hebels DG, Georgiadis P, Keun HC, Athersuch TJ, Vineis P, Vermeulen R, Portengen L, Bergdahl IA, Hallmans G, Palli D, Bendinelli B, Krogh V, Tumino R, Sacerdote C, Panico S, Kleinjans JC, de Kok TM, Smith MT, Kyrtopoulos SA. Performance in Omics Analyses of Blood Samples in Long-Term Storage: Opportunities for the Exploitation of Existing Biobanks in Environmental Health Research. Environ Health Perspect. 121 (2013) 480–487, doi:10.1289/ehp.1205657


Principal component plots illustrating the impact of subjects (different colours) and bench-time (num-bers) on transcriptomics (left), metabolomics (middle) and proteomic (right) profiles

Principal component plots illustrating the absence of significant impact of time in storage (13-17 years) on genomic profiles (left to right: transcriptomics, metabolomics, epigenomics, proteomics) of samples in biobanks

Conclusions from pilot study

A large fraction of biosamples derived from human blood and stored in biorepositories constructed in previous decades can be meaning-fully analysed using omics technologies, including transcriptomics provided that a) they had been frozen within 2 hours of blood collec-tion and b) special precautions are taken during RNA isolation from buffy coats


Numbers of transcriptomics signals associating with exposures or NHL (LY) risk at different statistical significance levels

Conclusions from main study in adult cohorts

Omic profiles derived from biosamples isolated from circulat-• ing blood and kept in long-term storage provide biomarkers of exposure to environmental chemicals or lymphoma risk

Numerous transcriptomics, proteomics, metabolomics and • epigenomics signals correlate with exposure to polychlori-nated hydrocarbons as estimated through their concentra-tions in serum, even when very stringent statistical criteria are applied

Numerous transcriptomics, proteomics and epigenomics sig-• nals correlate with furture risk of lymphoma, especially B-cell lympocytic leukemia. Many of these signals are associated with pathways and processes related to immune function and lymphocyte signalling, thus providing the outcome of agnostic statistical analyses of the omic profiles with strong biological plausibility.


Numbers of epigenomics signals associating with risk of different types of NHL – note the great pre-dominance of signals associating with BCLL (BCLL: B-cell chronic lymphocytic leukemia; DLBL: difuse large B-cell lymphoma; LPL: lymphoplasmacytic malignant lymphoma)

Principal component plots of metabolomics profiles of sera obtained from children with normal and pre-term births (cord blood and blood collected at age 4 years) as well as their mothers during pregnancy

Conclusions from main study with children

Omic profiles of biosamples collected from children (cord blood or blood collected at age 4 year) or their mothers at the time of preg-nancy provide biomarkers of in utero exposure to environmental chemicals and biomarkers predictive of birth outcomes and devel-opmental effects


Final project meeting

The final meeting of EnviroGenomarkers was held at the National Hellenic Research Foundation, Athens, on 20-21 February 2013.

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Project partners

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Partner Principal investigator

National Hellenic Research Foundation, Athens, Greece

S.A. Kyrtopoulos (co-ordinator) [email protected]

University of Maastricht, Netherlands

J. Kleinjans ([email protected])

Imperial College London, United Kingdom

P. Vineis ([email protected])

Umeå University, Sweden

I. Bergdahl ([email protected])

Istituto per lo Studio e la Prevenzione Oncologica, Florence, Italy

D. Palli ([email protected])

University of Crete, Greece

E. Stephanou ([email protected])

University of Utrecht, Netherlands

R. Vermeulen ([email protected])

Istituto Superiore di Sanita, Rome, Italy

P. Comba ([email protected])

National Institute for Health and Welfare, Kuopio, Finland

H. Kiviranta ([email protected])

University of Leeds, United Kingdom

M. Gilthorpe ([email protected])

Lund University, Sweden

B.G. Jönsson ([email protected])

National Taiwan University K-L. Chien ([email protected])


Project identity:

FP7 medium-scale collaborative project; priority area “Environment (including climatic change)”, sub-activity “Environment and health”; Grant Agreement 226756 Co-ordinator: S.A. Kyrtopoulos, National Hellenic Research Foundation, Athens, GreeceProject duration: March 2009 – February 2013Budget (EC contribution): 3.5 M euro

Project co-ordinator: Dr. Soterios A. Kyrtopoulos, National Hellenic Research Foundation, Institute of Biology, Medicinal Chemistry & Biotechnology, 48 Vassileos Constantinou Avenue, Athens 11635, Greece, Tel.: 30-210-7273740, Fax: 30-210-7273677, email: [email protected]

Project Manager:

Dr. Maria Botsivali, National Hellenic Research Foundation, 48 Vassileos Constantinou Avenue, Athens 11635, Greece, Tel: +30-210-7273784, Fax: +30-210-7273677, email: [email protected]

EC Project Officer:

Dr. Tuomo Karjalainen, European Commission, Research Directorate - General Directorate I (Environment), Unit I.5 (Climate Change and Environmental Risks), B-1049 Brussels, BelgiumTel: +32-2-2984660, Fax: +32-2-2994249, email: [email protected]


Envirogenomarkers NEWSLETTER details:

Editor / Contact Point:• Dissemination officer M. Botsivali, National Hellenic Research Foundation, Tel. +210 7273784, e-mail: [email protected] Designer :• Dimitra Pelekanou e-mail: [email protected], Tel. +6974820031

FINAL ISSUE • FEBRUARY 2013

www.envirogenomarkers.net

Documents

Genomics Biomarkers of Environmental Health · neurological and immune diseases Biosamples and data from 3 prospective cohorts 1. Northern Sweden Health and Disease Study 2. EPIC