unit 5. application of biomarkers to disease

chapter 27. Future perspectives on molecular Martyn T. Smith, Pierre Hainaut, Frederica Perera, Paul A. Schulte, Paolo Boffetta, Stephen J. Chanock, and Nathaniel Rothman

The current status of molecular epidemiology

As witnessed in the previous and metabolic disorders. In some remain essentially the same but chapters, since the development cases, educational or regulatory the incidence of disease changes of molecular epidemiology in interventions have been mounted as because of the new environment, the early 1980s (1), the field has a result of these studies. clearly show that non-genetic factors advanced so that large-scale, in- As described throughout this have high attributable risks (2). For depth studies have been performed book, non-genetic environmental some diseases, incidence rates or are ongoing. These studies have factors, broadly defined to include diet, increase or decrease dramatically not only monitored the external lifestyle, infections, stress, ionizing within the first or second generation environment and ascertained radiation, and chemical pollutants of immigrants, with disease patterns clinical disease status, but have in the air, water, food supply and becoming more similar to the also collected data on biomarkers workplace, are important contributors adoptive country and less similar to of exposure, biologically effective to chronic disease. Adverse gene– the country of origin. This highlights dose, preclinical effects, and environment interactions (GxE) the fact that environmental factors susceptibility within population probably influence most chronic (E) can contribute to a large portion studies. Links have been drawn diseases, including neurological of at least some chronic diseases between various environmental disorders and cancer. The genetic (3,4). and nutritional factors and diseases (G) contribution to different diseases Genomic tools arising from the as diverse as childhood asthma, varies, but several lines of evidence, Human Project combined cardiovascular disease, cancer, including classic studies of migrant with bioinformatics have allowed developmental disorders, obesity populations in which the genetics scientists to begin to examine the Unit 5 Chapter 27 Unit 5 • Chapter 27. Future perspectives on molecular epidemiology 493 genetic component of many chronic individual exposures. These conception onwards, as a quantity diseases. Initially, variations in exposures can vary day-to-day as of critical interest to disease etiology candidate genes were examined well as over time, as individuals (12). If we expect to have any in great detail, most notably in age and secular changes occur success at identifying the effects of xenobiotic metabolizing and DNA in a given population. The use of G, E and GxE on chronic diseases, repair genes (5–7). More recently, questionnaires has been the core we must develop 21st century tools genome-wide association studies approach for assessing exposure to measure exposure levels in large (GWAS) have been increasing in studies of chronic diseases in the human populations (11). That is, we in number and scope and have general population that arise, in part, need to quantify the exposome, a provided important insights into the from exposure patterns present over topic we will return to later. roles that particular genes, gene many years. This approach relies on Many lifestyle factors such as regions, regulatory elements, and self-reports, which can be imprecise exercise levels, dietary choices other parts of the genome with and inaccurate. However, they have and stress levels also contribute to function yet to be defined play in been successfully used to identify the environmental component of disease development (8). Thus, a consistent patterns of chronic disease, but are hard to quantify key focus of most current molecular disease risk for several exposures retrospectively and prospectively. epidemiology studies is on the such as tobacco, alcohol, obesity, Modern tools to capture, store and use genome and genetic variation. The components of the reproductive information about physical activity, reason for this focus on genetics, history, air pollution, and some diet and stress levels are needed for even though the environment may aspects of diet (e.g. intake of epidemiological studies. Such tools be more critical, is simply that we cruciferous vegetables). Also, the are being developed. For example, have extremely precise, accurate, increased ability to obtain objective it soon may be possible to perform and global tools to examine the occupational and residential population-scale, longitudinal genome, either measured as histories from study subjects, measurement of physical activity external factors or biologically as linked by sophisticated methods using common cell phones that reflected by the “exposome”. Such to comprehensive exposure include internal accelerometers and tools are not available to examine databases, has allowed advances low-power wireless communication the environment. At the same time, in identifying associations between capabilities (13). Dietary assessment by examining the G component of certain chemical exposures and methods suitable for use in large GxE we may find clues as to where disease risk. At the same time, epidemiologic studies (e.g. dietary to look for E factors (9,10), although methods to measure chemicals recall, food diaries and food success in this regard is very limited in biologic samples have steadily frequency questionnaires) are to date. evolved to measure a wider array subject to considerable inaccuracy. The most productive approach of compounds in smaller amounts More accurate methods (e.g. to assessing the environmental of samples. Nevertheless, these metabolic ward studies and doubly- contribution to disease may be to advances are not comparable to the labelled water) are prohibitively examine environmental exposures quantum leap that has occurred in costly and/or labour-intensive for use agnostically (11). Unfortunately, . in population-based studies. Several compared to genomics, the tools for The Human Genome Project and research groups are assessing assessment of exposures, based at least 20 years of investment in methods that use cell phones to upon measurements of chemicals in genetics are very helpful to molecular capture both voice recordings and air, water, food and the human body, epidemiologists in understanding photographs of dietary intake in real have undergone a more gradual the genetic determinants of time that, along with computerized evolution in the past 30 years and diseases, but we remain much more analysis, may revolutionise have not experienced the same limited when it comes to quantifying nutritional epidemiology studies (14). exponential gains. This is due to human exposures. This disparity in Accumulating evidence is both lack of technological progress current knowledge between genetic also consistent with the role of in the tools available for exposure contributions and environmental psychosocial stress in moderating assessment, as well as the more exposures was recognized by the effects of genetic and other challenging task of obtaining Wild, who defined the exposome, environmental factors on health data on, or estimates of, non- representing all environmental outcomes. Further advances in this fixed exogenous and endogenous exposures and lifestyle factors from area will require the development

494 of standardized, psychometrically given common disease. This is not of changes in DNA methylation, sound instruments for quantifying surprising since GWAS interrogate histone modifications and microRNA exposures to psychosocial stress. only common variants, which (miRNA) expression, both in cells Again, progress is being made in this represent only a proportion of genetic and body fluids, have recently been area using, for example, colorimetric variation in the human genome. For published (26–28). ChIP-on-Chip test strips to rapidly detect and example, despite the addition of 10 (chromatin immunoprecipitation quantify salivary α-amylase, a positively-associated SNPs, the with microarray analysis) and ChIP- biomarker of the body’s adrenergic performance of breast cancer risk seq (chromatin immunoprecipitation stress response (15,16). The models only improved modestly; with sequencing) will help in measurement of telomere length the area under the curve of the understanding epigenetic effects is another stress biomarker that is receiver operating curve increased on gene–protein interactions. gaining acceptance (17,18). from 58% to a mere 61.8% (22). Advances in mass spectrometry will Thus far, GWAS have been most soon make it possible to measure The potential contributions successful in identifying regions post-translation modifications of of genomics to molecular that harbour genetic variants that proteins such as histones in small epidemiology are directly associated with risk for a volumes of biological sample, complex disease, such as cancer or adding to our repertoire of epigenetic Genomics is the study of all of the inflammatory bowel disease. For the changes that can be studied in nucleotide sequences, including latter, GWAS have pointed towards human populations. structural genes, regulatory a region on chromosome 2q37.1 Advances in mass spectrometry sequences, and noncoding DNA and identified a novel mechanism and in laboratory-on-a-chip devices segments, in the chromosomes of of autophagy previously not well that use nanotechnology may also an organism. Because of the tools described in the pathogenesis soon permit us to profile all the provided by the Human Genome of inflammatory bowel disease, major protein and DNA adducts in Project, the current focus of many specially as it relates to the genes humans using adductomics. This will molecular epidemiological studies in this pathway (23). Fine mapping allow for the examination of multiple is on genomic variation. GWAS and, of regions together with functional biomarkers in very small sample most recently, examinations of copy work is required to elucidate the volumes, such as a few microlitres number variation have revealed biological underpinnings of the of serum, a drop of blood, or a dried many surprising insights. Results direct association of common blood spot. from these studies show that many variants with complex diseases such These tools are expected common causal variants, each as cancer (24). Certainly, the advent to have great application in of small, additive effect, probably of new technologies, in conjunction molecular epidemiology studies contribute to complex disease risk. with computational resources, will in the near future. There are As of 2010, GWAS had identified enable investigators to use next emerging opportunities to apply over 750 regions in the genome generation sequencing to explore these technologies in molecular strongly associated with more than the contribution of uncommon and epidemiologic studies with banked 125 traits and diseases (http://www. rare variants in the near future. biological samples, including genome.gov/gwastudies) (19). In cross-sectional, case–control, and, chronic complex diseases, such The potential contributions in particular, prospective cohort as type 2 diabetes and Crohn's of molecular epidemiology studies, to study a wide range of disease, over 40 genetic regions in the near future diseases. have been associated with each This should advance the ability disease. For certain heritable traits In the near future, there will also likely of molecular epidemiology to more such as height, recent studies have be a maturing of omic applications broadly explore exposure–disease identified several hundred regions, and the incorporation of systems relationships, to study effect each of which contributes to their biology into molecular epidemiology, modifiers, and to obtain insight heredity (20). In cancer alone there which will produce what some have into the fundamental underlying are over 135 regions associated called systems epidemiology (25). pathogenesis of these conditions. with 21 cancers (21). However, the Studies of epigenetic changes are Further, beyond providing etiologic early GWAS have not sufficiently already coming to the forefront of insights, it is expected that explained the heredity of any molecular epidemiology, and studies molecular epidemiology will be Unit 5 Chapter 27 Unit 5 • Chapter 27. Future perspectives on molecular epidemiology 495 newly positioned to make important Additional profoundly important and complicated protocols as contributions to translating these steps taking place in molecular sample sizes are generally small (a findings into primary, secondary and epidemiology are the increased few hundred subjects). Often they tertiary prevention strategies. This size of studies and the formation of can have very detailed assessment would begin with broad public health dozens of international consortia, of the target exposure, evaluate practices that could include removal including those that focus on specific potential confounders, modifiers, or substantial reduction of exposure diseases as well as those that are and other contributors to endpoints to hazardous environmental based on study design (e.g. various under study in greater detail (e.g. compounds, making available cohort consortia). There are now a nutritional, genetic, psychosocial healthier food in schools and better large number of prospective cohort factors) (33), and arrange for education on lifestyle risk factors. studies in North America, Europe, samples to be transported and At the same time, molecular Asia and Australia that have enrolled processed very quickly, allowing epidemiology is likely to play an or are continuing to enrol several specialty assays to be carried out. important role in the upcoming million study subjects. These cohort They can incorporate both state-of- revolution in personalized studies have millions of samples the-art omics platforms as well as (29). At present, identifying individual of DNA, serum, blood cells, and in-depth hypothesis-testing (34). genetic risk is at the forefront of this other biological material stored at personalization of health care. But low temperatures. Some studies Future use of omic given the limited role for genetics are tracking individuals in utero technologies in molecular in comparison to the environment through adolescence, providing an epidemiology in causing disease, the focus must opportunity to assess the earliest eventually shift to include individual determinants of disease. These The field of molecular epidemiology environmental risk factors, again samples are precious as well as is entering an exciting new phase in broadly defined to include toxic numerous. Efficient, high-throughput which the innovative tools of omics, exposures, lifestyle, diet, drugs, methods that work on minute such as microarrays and metabolic etc. This could help bring about not amounts of sample are needed to and peptide profiling, are being only lifestyle modification to prevent analyse nested case–control or applied along with novel laboratory- disease and improve drug treatment, case–cohort studies carried out on-a-chip microdevices that can act but it could also help individuals within them. The combination of as biosensors of everything from gain an understanding of their prior new nanotechnology/laboratory- glucose levels to protein adducts and current chemical exposures on-a-chip methodologies with large (Figure 27.1). The term omics has and other risk factors, leading to prospective cohort studies holds come to mean any field of study personalized risk assessment. great promise for new research in biology in which the totality of Molecular epidemiologists may findings. At the same time, there something is studied, beginning with be able to identify not only broad will still be a need for focused, genomics which surveys across the subgroups of the population with hypothesis-testing studies carried genome. The tools of genomics, a higher probability of developing out within these cohorts, in addition developed as a consequence disease given genetic and other to the application of discovery of the Human Genome Project, risk factors, but also move to technologies. Such studies can often include microarrays allowing the further develop predictive models be carried out on smaller sample examination of gene variation and that can be applied to individuals sizes, as they generally do not expression and high-throughput by preventive and clinical medicine need to contend with the low prior sequencing. The latter is now being practitioners. An example of this is hypothesis/multiple testing problem. used not only to sequence DNA but the Gail Model for predicting breast In addition, there is still a need the RNA transcriptome to give a cancer, which is based on all known for focused, cross-sectional studies more complete picture of gene and risk factors including BRCA1 and of well-defined populations with siRNA expression. Transcriptomics BRCA2 mutations (30). Genetics is particular exposure or lifestyle is the study of all forms of RNA now poised to augment this model patterns of interest, such as new that are transcribed from the DNA and provide even greater sensitivity exposures recently introduced into and includes mRNA and miRNA and specificity, but as mentioned the environment, high or low levels expression. previously, success to date using of exposures, etc. (e.g. (31,32)). GWAS data is limited. These studies can use extensive

496 Figure 27.1 induces gene expression changes in the human airway epithelium (43) with some genes modulated by miRNA (44). Expression profiling analyses have revealed characteristic miRNA signatures in certain human cancers (45) and other diseases. The study of miRNA in molecular epidemiology will likely explode in the near future as new tools become available and the biology is better understood.

Applications of proteomics

While toxicogenomics studies using global transcriptional analysis have enormous potential, the transcriptome does not always reflect the functional proteome. Further, proteins may be subject to post-translational modification and Recent and near-future was shown to distinguish individuals translocation. However, proteomics, contributions exposed to cigarette smoke (CS) the analysis of the total protein of transcriptomics from unexposed individuals (38). An output encoded by the genome association between CS-induced using techniques such as mass Distinctive blood transcriptional gene expression and DNA adduct spectrometry and antibody arrays, is profiles have been demonstrated formation was later shown in a study more challenging and less amenable for over 35 human diseases (35). of monozygotic twin pairs (39). The to application in a high-throughput As more data become available impact of air pollution on children capacity due to differences in protein on global gene expression in at the transcriptional level in blood properties, location and abundance. the blood of humans following cells was investigated by comparing Recently, a multilaboratory study exposure, it will become easier to children from urban and rural regions has attempted to dispel some of identify molecular mechanisms by of the Czech Republic (40). Several the notions of the irreproducibility which environmental chemicals genes were differentially expressed of mass spectrometry-based promote/cause human disease. and a correlation with micronuclei proteomics by pinpointing where the Initiatives such as the Comparative frequencies was shown. Further, methodological problems are and Toxicogenomics Database (http:// the effects on children and adults where challenges remain (46). By ctd.mdibl.org/) (36) have been at the transcriptional level differed addressing these methodological developed towards this goal. (41). A small study of children in issues researchers hope to bring A broad array of environmental New York City found that a gene- proteomics to the forefront of exposures including specific methylation change in biomarker research. pharmaceuticals, pesticides, air umbilical cord white blood cell DNA pollutants, industrial chemicals, was associated both with prenatal Applications of heavy metals, hormones, nutrition exposure to PAH air pollutants and and behaviour can change gene with reported asthma in the children Metabolomics is defined as the expression through several gene by age 5 years (42). study of metabolic profiles in easily regulatory mechanisms (37). The More recently, many groups have collected biological samples such potential of toxicogenomics in the begun profiling miRNA expression. as urine, saliva or plasma. The discovery of biomarkers of complex A role for miRNAs in mediating metabolome is highly variable environmental exposures was the response to environmental and time-dependent, and consists illustrated by a study in which gene exposures has been demonstrated of several thousand chemical expression profiling of leukocytes by a study showing that smoking structures. Since it is sensitive to Unit 5 Chapter 27 Unit 5 • Chapter 27. Future perspectives on molecular epidemiology 497 age, gut microbial composition, Importantly for epidemiologists, mechanisms (50). Given the and lifestyle, metabolomics is ideal metabolomics is relatively easy sensitivity of omic analyses, low- for the characterization of dietary to apply in large-scale human dose adverse effects can also be and therapeutic interventions, studies. For example, a large- observed and distinguished from metabolism and metabolism-related scale exploratory analytical high-dose phenomena, if exposure disorders (47). While successfully approach investigated metabolic is accurately assessed, allowing for established in the screening phenotype variation across and dose–response data from molecular of inborn errors in neonates, within four human populations epidemiology studies to be metabolomics is being increasingly using 1H NMR spectroscopy (49). incorporated into risk assessments. applied to several diseases. For Metabolites discriminating across For additional public health many years specific metabolites populations were then linked impact, molecular epidemiology must have been measured in body fluids to data for individuals on blood continue to expand its contributions to to diagnose particular diseases pressure. Spectra were analysed surveillance, mechanistic research, such as diabetes, by measurement from two 24-hour urine specimens efficacy trials, translational research of glucose, and vascular diseases, for each of 4630 participants from and health policy. We must assemble by determination of cholesterol. the INTERMAP epidemiological and communicate information to Metabolomics, with its ever- study, which involved 17 population decision-makers, medical and health increasing coverage of endogenous samples in China, Japan, the United professionals, and the public. If compounds and its high-throughput Kingdom, and the USA. It was shown molecular epidemiology is to make capacity, now provides a much that urinary metabolite excretion a major impact on population health, more comprehensive assessment patterns for East Asian and western it must be preventive and have a of health status and can be used in population samples, with contrasting global as well as a local focus. A life- the identification, qualification, and diets, diet-related major risk factors, course approach is also important in development of biomarkers. and coronary heart disease/stroke establishing the earliest causes of An important challenge in rates, were significantly differentiated diseases both in children and adults. metabolomics is the acquisition (P < 10(−16)). Among discriminatory We must expand our horizons to of qualitative and quantitative metabolites, four were quantified develop affordable population- information concerning the and showed associations with blood wide tools for combating common metabolites that occur under normal pressure. diseases. circumstances to be able to detect Serving as the linking hub perturbations in the complement Potential impact of molecular for laboratory and population, of metabolites as a result of epidemiology on public problem and solution, molecular changes in environmental factors. health and regulatory policy epidemiology can help translate Technologies that rely on UPLC- research to practice. To do this, there MS/MS, FT-ICR-MS, Orbitrap, A bioinformatics database could will be a need to continue current and asymmetric waveform ion be built of the human response to trends in the discipline and establish mobility analysers are emerging different chemical exposures and new ones. Continuation of the trend as dominant analytical methods associated chronic diseases. This towards large-scale consortia and for metabolomic studies because database may well be useful in biobanks, use of bioinformatics, and of the accuracy, high throughput many ways for risk assessment. attention to individual and collective and coverage (>1000 unique For example, by comparing the ethical issues will serve to move metabolites) that can be achieved molecular effects of newly tested the field forward, as will in-depth (48). However, even though these chemicals to those of established hypothesis-driven studies of at-risk methods provide accurate mass carcinogens, we could identify populations. Powerful impacts will be values that may reduce the number potential carcinogens (hazard achieved by incorporating epigenetic of potential molecular formulas identification) and establish modes and biological systems theory in down to a few candidates, further of action by studying the effects of research and by expanding skill development is needed to provide the same chemicals in experimental sets and professional knowledge complete structural information. The animals and on human cells in to complement translation research exchange of chemical and analytical vitro. This would allow for better and risk communication and to information must be encouraged for prediction of human carcinogenicity foster public health perspectives. metabolomics to expand. and assessment of carcinogenic A broad population-wide vision for

498 using biological markers is required programs are urgently needed to Conclusion to leverage the power of molecular assist in the analysis. scale insight to give beneficial Exposure assessment must Molecular epidemiology is poised macro-scale impacts on public also be able to address low-level to make ever-greater contributions health. exposure to complex mixtures. The to understanding the genetic and current cost of analysis for most environmental causes of human Future challenges: chemicals in blood and other fluids disease. Both agnostic and Dealing with complexity is prohibitive if one wishes to assess hypothesis-driven approaches to and lack of resources multiple compounds. New analytical both categories of risk factors could chemical approaches are needed to lead to leaps in our understanding. A major challenge to many of the assess the thousands of chemicals Investment in new methods and novel approaches described above and their metabolites to which we approaches will be needed, is the size and complexity of the are exposed. however. Strong links between data generated. Currently, it is a One method to overcome population scientists, bench major biostatistical undertaking to resource difficulties may be to pool scientists, bioinformaticians and analyse terabytes of data, and the samples. Recently, this approach engineers must also be forged if emerging results require extensive has been used with considerable progress is to be made. further analysis by bioinformatics. success in GWAS and in studies of Efforts must be made to simplify the the plasma proteome (51,52). Disclaimer: The findings and conclusions in this chapter are those of the author analysis and reduce the data. New and do not necessarily represent the statistical approaches and computer views of the Centers for Disease Control and Prevention. References

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