Ordovas - Nutritional Genomics AnnRevHumGenet

Annu. Rev. Genomics Hum. Genet. 2004. 5:71118 doi: 10.1146/annurev.genom.5.061903.180008 Copyright c 2004 by Annual Reviews.
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NUTRITIONAL GENOMICS
Jose M. Ordovas1 and Dolores Corella1,2
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Annu. Rev. Genom. Human. Genet. 2004.5:71-118. Downloaded from arjournals.annualreviews.org by Universitat de Lleida on 01/18/06. For personal use only.
Nutrition and Genomics Laboratory, Jean MayerU.S. Department of Agriculture, Human Nutrition Research Center on Aging at Tufts University, Boston, Massachusetts; email: jose.ordovas@tufts.edu 2 Genetic and Molecular Epidemiology Unit, School of Medicine, University of Valencia, Valencia, Spain
Key Words nutrigenomics, gene-diet interaction, nutrigenetics Abstract Nutritional genomics has tremendous potential to change the future of dietary guidelines and personal recommendations. Nutrigenetics will provide the basis for personalized dietary recommendations based on the individuals genetic make up. This approach has been used for decades for certain monogenic diseases; however, the challenge is to implement a similar concept for common multifactorial disorders and to develop tools to detect genetic predisposition and to prevent common disorders decades before their manifestation. The preliminary results involving gene-diet interactions for cardiovascular diseases and cancer are promising, but mostly inconclusive. Success in this area will require the integration of different disciplines and investigators working on large population studies designed to adequately investigate gene-environment interactions. Despite the current difculties, preliminary evidence strongly suggests that the concept should work and that we will be able to harness the information contained in our genomes to achieve successful aging using behavioral changes; nutrition will be the cornerstone of this endeavor.
PAST, PRESENT, AND FUTURE OF NUTRITIONAL GENOMICS

The major practical translation of nutrition research to public health consists of dening optimal dietary recommendations aimed to prevent disease and to promote optimal health. For this purpose, and based on the best scientic evidence available at the time, several dietary guidelines have been implemented to improve the health of the general population and of those at high risk for specic diseases [i.e., cardiovascular disease (CVD), cancer, hypertension, and diabetes]. However, past and current dietary guidelines have not considered the dramatic differences in the individuals response to changes in nutrient intake. These differences in response may greatly affect the efcacy of these recommendations at the individual level. The mechanisms responsible for the interindividual differences in dietary response are far from being fully understood. Nevertheless, the presence of a genetic
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component has been proposed for several decades (67), although only recently researchers began examining these nutrient-gene interactions at the molecular level. However, the results of studies aimed to elucidate nutrient-gene interactions for common diseases have been controversial and inconclusive. Even so, these diseases are triggered because of interactions between specic genes and environmental factors (180). These interactions are dynamic, beginning at conception and continuing through adulthood. The concept of environment is complex and broad and it has been frequently associated with tobacco smoking, drug consumption, toxic exposures, education, and socioeconomic status. However, food intake is the environmental factor to which we are all exposed permanently from conception to death. Therefore, dietary habits are the most important environmental factor modulating gene expression during ones life span. The prominent role of diet in the etiology of disease was recognized rst for monogenic diseases and then for multifactorial disorders. Further progress in this area relies on the identication of key genes involved in disease development and on the elucidation of the impact of their variation on health and disease. This knowledge has been facilitated by information generated from the Human Genome Project (17, 88), which is paving the way for gene discovery and for more comprehensive exploration of gene-nutrient or gene-diet interactions. The concept of gene-diet interaction describes the modulation of the effect of a dietary component on a specic phenotype (plasma lipid concentrations, obesity, glucemia, etc.) by a genetic polymorphism. Alternatively, this notion refers to the dietary modication of the effect of a genetic variant on a phenotypic trait. In terms of gene-diet interactions for common, mutifactorial diseases, the fastest development has been in the area of CVD risk, which has easily measured risk factors (i.e., plasma cholesterol concentrations). Some examples of the preliminary gene-diet interactions on lipid metabolism were revised (129) and have been the subject of recent reviews (98, 105, 132). The potential benets of harnessing the power of genomics for dietary prevention of disease are enormous and this approach is considered the future of nutritional research in the postgenomic era (109). Currently, gene modication in humans is neither technically feasible nor ethically admitted (54), so geneticists will use genomics-based knowledge to recommend personalized behavioral changes, which should mean more effective disease prevention and treatment. The genomic revolution has catapulted the development of several new technologies that can be applied in nutritional sciences (25). Genomic, proteomic, metabonomic, and bioinformatic techniques are already beginning to facilitate the study of gene-nutrient interactions at the cell, individual, and population level (28). During this postgenomics era, traditional DNA sequencing and genotyping technologies will shift toward novel approaches using DNA arrays and other highthroughput techniques (115). Transcriptomics are now possible using microarrays that can prole gene expression patterns of thousands of genes or even the entire genome in a single experiment. Proteomics currently enables geneticists to study the complete collection of proteins in a cell or tissue at any given time, and it
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will permit them to determine the role of proteins inside cells and even the role of the molecules with which they interact. Finally, metabonomics will facilitate the investigation of metabolic pathways using noninvasive biomarkers (115). All these techniques can and should be combined to understand both the inuence of specic nutrients and whole dietary patterns on the metabolic behavior of cells, organs, and the whole organism (153). This challenge can be accomplished by using bioinformatics and chemometrics that provide tools for managing the complex data sets provided by genomics, transcriptomics, proteomics, and metabonomics, and constitute what we know as functional genomics, also referred to as systems biology (184). The development of systems biology transformed the concept of gene-nutrient interaction from the traditional reductionism approach of studying the effect of a nutrient over a specic metabolic event into a holistic one, in which a signicant fraction of all regulated genes and metabolites can be quantied concurrently (48). Holistically, the whole is the dynamic interaction of the parts. According to Hoffmann (65), these goals can be accomplished if scientists have: (a) knowledge of the part (nutrients, food, and dietary patterns); (b) valid information: adequate experimental design, dietary assessments, and statistical methods; (c) tools to study and visualize more complex models of interactions; and (d) massive computer power to integrate information, and if they take an interdisciplinary approach by transgressing the boundaries between and beyond disciplines and institutions. Propelled by these technologies and paradigms, nutrition science has introduced the new, and yet undened, term of nutritional genomics or nutrigenomics. One of the rst references to this term in the scientic literature is that of DellaPenna (31), in 1999, who denes nutritional genomics as the general approach to gene discovery that is currently most applicable to compounds of nutritional importance that are synthesized or accumulated by plants and other organisms. This denition, which describes research at the interface of plant biochemistry, genomics, and human nutrition, has the specic goal of dissecting and manipulating micronutrient pathways in plants to improve crop nutritional quality for human health. Two years later, Watkins et al. (192) incorporated the concept of individual genetic variation into agriculture by suggesting that personalized nutrition may dene the added value for the next generation of foods and crops. Currently, developing new foods is one of the many applications of nutritional genomics within the umbrella of its overall goal, which is studying the genome-wide inuences of nutrition. Therefore, nutritional genomics represents the application of systems biology in nutritional research (48, 184), promoting an increased understanding of (a) how nutrition inuences metabolic pathways and homeostatic control, (b) how this regulation is altered in the early phase of a diet-related disease, and (c) to what extent individual sensitizing genotypes contribute to such disease (117). Nutritional genomics studies the functional interaction of food and its components with the genome at the molecular, cellular, and systemic level; the goal is to use diet to prevent or treat disease. In nutritional genomics, two terms are used: nutrigenomics and nutrigenetics. Nutrigenetics examines the effect of genetic
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variation on the interaction between diet and disease. This includes identifying and characterizing gene variants associated or responsible for differential responses to nutrients. The goal of nutrigenetics is to generate recommendations regarding the risks and benets of specic diets or dietary components to the individual. It has been also termed personalized nutrition or individualized nutrition. Nutrigenomics focuses on the effect of nutrients on the genome, proteome, and metabolome. Because it is a new area of knowledge, it is not surprising that this concept has received different denitions, summarized in Table 1 (20, 36, 85, 181, 184). Nutritional genomics has already raised high interest and expectations and some researchers (56) warn that genomic proling and its interaction with environmental
TABLE 1 Concept
Nutritional genomics dened by several authors Denition Reference (36)
Nutritional genomics The application of high-throughput functional genomic technologies in nutrition research. These technologies can be integrated with databases of genomic sequences and interindividual genetic variability, enabling the process of gene expression to be studied for many thousands of genes in parallel. Nutritional genomics The integration of systems biology into nutritional research or nutrigenomics Nutrigenomics Nutrigenomics is the study of molecular relationships between nutritional stimuli and the response of the genes.
(184) (20)
Nutritional genomics Has the potential to assist scientists in interpreting the (85) complex gene-nutrient interaction and the link between genetic abnormalities and disease, to analyze and integrate the vast data sets that these techniques and studies produce, and then to identify new biomarkers. Nutrigenomics The application of high-throughput genomic tools in nutrition research. Applied wisely, it will promote an increased understanding of how nutrition inuences metabolic pathways and homeostatic control, how this regulation is disturbed in the early phase of a diet-related disease, and to what extent individual sensitizing genotypes contribute to such disease. (117)
Nutritional genomics Encompasses the interaction between nutrients and the (181) or nutrigenomics expression of genes harnessing techniques such as DNA microarrays and real-time PCR. It involves the characterization of gene products and the physiological function and interactions of these products; the latter includes how nutrients impact on the production and action of specic gene products and how these proteins in turn affect the response to nutrients
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factors such as diet is not ready for prime time. It is true that evidence supporting health outcome benets based on such testing is lacking, and that before this approach becomes valid and clinically useful, well designed epidemiologic studies and clinical evaluations of recommended interventions based on genotype are required. This paper reviews the current knowledge of nutritional genomics at the population level for both monogenic and multifactorial diseases. Because a major limitation of nutritional genomics is the lack of well designed and conducted epidemiological studies, major emphasis has been placed on applying epidemiological principles to the study of nutritional genomics, not only to interpret the results of published studies, but to provide guidance on the design of new investigations in this area.
METHODOLOGICAL ISSUES
Nutritional genomics is a concept that may revolutionize the prevention and treatment of disease. As indicated above, one goal of nutritional genomics is to nd genetic polymorphisms that reveal signicant gene-diet interaction, thus providing tools for personalized and more successful dietary recommendations. Academic researchers, the public, and industry have a lot of interest in this increasingly popular topic. However, before these tools can be applied to the population, they need to be validated by robust scientic evidence. Unfortunately, the outcome of most of the preliminary studies addressing gene-nutrient interactions in multifactorial diseases has rarely been replicated in follow-up studies and this has started to divide the scientic community among those who believe that nutritional genomics can bring the promised benets to our future and those who express concern or disbelief. To avoid, or at least minimize, the adverse effects on both scientic and public condence that the current confusion may create, one must understand the strengths and limitations of the published evidence. Therefore, it would be useful to apply the principles of evidence-based medicine (55, 136) and epidemiology (56) to nutritional genomics when causality is inferred from the results of association studies. In the past few decades, there has been a dramatic shift in nutrition research from focusing on preventing nutritional deciencies to preventing chronic diseases. This change has given nutritional epidemiology the pivotal role of providing substantial evidence to support global health recommendations, or specic recommendations for selected groups; the latter is a goal of nutritional genomics. Ethical considerations require a high level of evidence of causality for results generated from epidemiological studies prior to their translation into public recommendations. The same level of evidence should apply to nutritional genomics when the outcomes are used to support nutrition recommendations. Below, we review the key points of causality, causal criteria, and types of epidemiological studies, statistical inference, and epidemiological errors.
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Causality in Nutritional Genomics, Types of Studies, and Errors

It is important to remember that statistically signicant associations (P < 0.05) do not imply causality. The concept of causality in nutritional genomics can be viewed from several perspectives. The most pragmatic refers to the burden of proof that is needed to establish a dietary component or a nutritional pattern as a cause of disease, i.e., how much evidence should be collected before practical action is justied. In general, it is well accepted that when interpreting epidemiologic studies, no single study can be considered denitive proof of causality (42). Also, several principles of causality were established in the 1960s (63) and they can be adapted to present and future association and interaction studies. These criteria include consistency and strength of association, as well as dose response and biological plausibility. Consistency is one of the most frequently used measures when practicing causal inference, and it represents the extent to which the association is observed under different circumstances, investigators, study designs, and locations. Consistency is equivalent to replication (193). In terms of strength of association, Hill (63) implied that causal relationships were more likely to demonstrate strong associations than noncausal relationships. However, one should not assume that a strong association alone indicates causality because the presence of other confounders may erroneously lead to a strong association. A dose-response relationship between exposure and outcome provides evidence for a causal relationship. Biological plausibility or coherence is the extent to which an observed association in epidemiological studies is supported (or not) by what is known about the mechanism of action and the underlying disease process. Other relevant criteria of causality are temporality and experimental evidence. Temporality has been suggested as sine qua non for causality. For an exposure to be causal, its presence must precede the development of the outcome. As per the experimental evidence (146), it should be obtained from well-controlled studies, and specially randomized controlled trials. These types of studies can support causality by demonstrating that altering the cause alters the effect (63). However, the limitations imposed by ethics and costs often restrict epidemiologic research to nonexperimental studies. In experimental designs, study conditions, including the degree of subject exposure, are controlled directly by the investigators. This control minimizes the possibility of confounding, a type of error that can distort the results and that is common in nonexperimental studies (observational studies). Thus, epidemiological studies are rst classied according to their level of experimentation and then according to other characteristics (i.e., subjects, selection, and follow-up): (a) observational studies (ecological, cross-sectional, case-control, and cohort studies), and (b) experimental studies (clinical & community trials). The latter group holds the highest level of evidence for causality, obtained from controlled randomized intervention trials (55, 91). In nutritional epidemiology, ecologic analysis (the unit of the analysis is the group) has provided initial results about the association between fat consumption
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and cancer; however, the ecologic fallacy is the main limitation of these studies. In cross-sectional studies, exposure and disease are assessed concurrently in individuals selected from a dened population. In case-control studies, information about diet is obtained from patients with the disease and is compared with controls. This design provides information more quickly than cohort studies, but it is retrospective and subjected to higher error. Prospective cohort studies are the best observational design. In this type of study, geneticists identify a group of individuals and measure their exposures to dietary and other notable risk factors. Assessment of dietary exposures may include both present and past dietary practices. These individuals are then followed over time to identify those who develop disease; the measured exposures are used to determine predictors of disease risk (42, 200). In nutritional genomics, one can measure gene-nutrient interactions using any type of association study in which an individual genotype of a participant can be determined (7). This procedure, which obtains the genotype data, is called the measured genotype approach, and it has been classically attributed to molecular epidemiology. However, genetic epidemiology, through segregation studies, studied gene-diet interactions by the unmeasured genotype approach. The unmeasured genotype approach is based on statistical analysis of the distribution of phenotypes in individuals and families and does not rely on any direct measure of DNA variation. Currently, the measured genotype approach is the standard method for detecting the gene-nutrient interactions. These studies require larger sample sizes to minimize random errors (type I and type II) than traditional association studies. For a more detailed review of confounding random errors and limitations of these studies, see Reference 41. Finally, meta-analysis has been proposed as a potentially viable alternative (5) to evaluate replication of interstudies and to circumvent the problems of small sample sizes.
Dietary Assessment
Dietary assessment plays a crucial role in our ability to detect relationships between dietary exposure and disease causation. Therefore, high-quality dietary information is key to establishing causality in nutritional genomics. The best approach to ascertain true dietary intake is within the context of prospective dietary intervention studies carried out under highly controlled conditions. However, these well-controlled feeding studies have several important logistic limitations (116), including their cost, the small number of participants, and the brief duration of the interventions. Therefore, a considerable proportion of our knowledge relating dietary intake to phenotypes and disease risk comes from population studies using, for the most part, self-reported dietary questionnaires. Diet records, diet-history questionnaires, 24-h recalls, or food-frequency questionnaires (FFQs) are the most common methods to get individual dietary intakes (199). Each method has strengths and weaknesses. For example, due to the large amount of within-person or day-to-day variation in dietary intake, one 24-h
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recall or food record is not considered a valid estimate of an individuals usual intake. To date, the FFQ is the dietary assessment method most commonly used in large-scale studies, primarily because it is easy to administer, it is less expensive than other dietary assessment methods, and it provides a rapid estimate of usual intake. However, recent studies point out the low correlation of this method with others using more direct measures of intake, such as those measuring relevant biomarkers (80), those chemically analyzing true intake during metabolic studies (157), and those using dietary records (79). This information suggests that the impact of measurement error introduced by some dietary assessment instruments on the interpretation of nutritional studies may be much greater than previously estimated. However, the inuence of these errors depends on the specic epidemiologic design and its corresponding hypothesis. Therefore, it is crucial to document the reproducibility and validity of any questionnaire before it is applied to a new study. Recently, some studies began carrying out the calibration procedure (169, 172). Regression calibration is a new technique that uses a calibration substudy to provide information about errors and to correct results from the main study. It corrects attenuation and regression dilution biases in relative risk estimates, depending on the correlation of the FFQ, with different complementary methods to measure the true dietary intake (41). An important question for nutritional epidemiology studies that involve nutritional genomics is: Which type of dietary information is more relevant? Should we use foods, nutrients, or dietary patterns? Foods are directly measured by a dietary instrument. Conversely, nutrients are derived by calculation from food databases. Therefore, appropriate food composition databases with valid nutrient data are required to convert food intake information into nutrient intake data (164). Food preparation and cooking methods can signicantly affect the nal nutrient content in foods. Food items contain thousands of specic chemical compounds, some known and well quantied, some poorly characterized, and others subject to geographical and seasonal variability or still undened. Therefore, apart from the traditional concept of nutrient (a chemical substance obtained from food and needed by the body for tissue growth, maintenance, and repair), foods also contain nonnutritive but bioactive compounds such as natural phytochemicals (avonoids, isoavones, carotenoids, and so on), additives, toxins, and chemicals formed in food processing and cooking (96). If diet is described only in terms of nutrients or food components, it is possible to lose important information hiding in lesser known food components. With the expanding knowledge of the role of nutrients and bioactive compounds in gene expression and cellular response, nutritional genomics needs a new denition of nutrient (48). Young (203) dened nutrient in the postgenomic era as a fully characterized (physical, chemical, physiological) constituent of a diet, natural or designed, that serves as a signicant energy yielding substrate or a precursor for the synthesis of macromolecules or of other components needed for normal cell differentiation, growth, renewal, repair, defense and/or maintenance or a required signaling molecule, cofactor or determinant of normal molecular structure/function and/or a promoter of cell and organ integrity.
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However, describing diet in terms of foods or food groups may lead to new hypotheses enabling the discovery of effects associated with a particular chemical compound. The study of dietary patterns has been carried out by principal component analysis, cluster analysis, and other techniques. Jacobs et al. (71) proposed complementary research methodology in which the study of foods, food patterns, and individual nutrients of food components are considered together. This integrative approach could be useful in nutritional genomics and it is currently being used by ongoing studies, including the Framingham Heart Study, in which dietary intake is measured in terms of foods, nutrients, and dietary patterns to explore the inuence of diet and the possible genetic modulation in metabolic syndrome and CVD. This integrative approach in dietary assessment can be further improved by measuring some biochemical indicators to represent the more objective measures of dietary intake for specic nutrients (14, 122). These biomarkers of dietary intake consist of biochemical measures in blood, urine, fat, or other tissues of compounds that are correlated with intake of specic dietary components (149). However, we still lack reliable biomarkers for many signicant nutrients. The current limitations should be successfully resolved by incorporating new analytical and bioinformatic techniques. One goal for nutritional genomics is to identify biomarkers that will provide better guidance on the relation between nutrition and health. Therefore, applying systems biology in nutritional genomics will provide exciting opportunities to build the required knowledge. Systems biology will facilitate the cross talk of different disciplines and expertise to build models that will integrate information about intake, gene polymorphisms, gene expression, phenotypes, diseases, effect biomarkers, and susceptibility biomarkers.
Genotyping and Quality Control Measures

Nutritional genomics studies must have a good measurement of diet as well as require a good quality control of the genetic measures. Recently, Little et al. (95) proposed a checklist for reporting and appraising studies of genotype prevalence and gene-disease associations, focusing on identifying the study design, selecting study subjects, and noting the characteristics of these subjects (geographical area, gender, age, environmental exposures, time of collection, analytic validity of genotyping, population stratication, confounding and statistical issues). The authors point out that a recent appraisal of 40 studies using molecular genetic techniques demonstrated the need for universal standards for quality control. With the application of high-throughput methods, a number of which are under development, quality control procedures in the laboratory are particularly important. Misclassifying the genotype (i.e., a data set containing less than 95% reproducibility) can bias measure of association between genotype and disease, and largely affect gene-nutrient interactions. Quality control measures, including internal validation, blinding, duplicates, test failure rate, inspection of whether genotype frequencies conform to Hardy-Weinberg equilibrium, and blind data entry, must be reported in the methodology section.
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Another development is the use of haplotypes (27), instead of individual polymorphisms, for genomic analysis. Various statistical algorithms have been developed to estimate haplotypes from genotypic data. Because these statistical methods typically apply to nonrelated individuals, such data often consist of unphased genotypes that result in haplotype ambiguity and yield different results (173). Therefore, proper standardization will be required to perform valid comparisons across different studies involving haplotype analyses and nutritional genomics. Similar concern applies to the use of microarrays in nutrigenomics. However, we emphasize the need for standardization, data quality control, and data analysis to generate valid and comparable information. Page et al. (138) and Potter (147) reviewed the crucial aspects of microarray experimentation in nutritional genomics that must be considered before and during research, including experimental design, sample size, statistical analysis, data verication, data handling, and experimental interpretation. Increased availability of genomic, transcriptomic, proteomic, and metabonomic techniques will foster their application in nutritional genomics, but the complexity and quantity of information generated by these approaches, and the need to share databases among multiple investigators, will require the implementation of quality control and validation measures several order of magnitude more complex than those currently used in more conventional nutritional research.
GENE-NUTRIENT INTERACTIONS IN HEALTH AND DISEASE

Here we aim to summarize the evidence currently available about the role of gene-nutrient interactions in human disease. Evidence from monogenic diseases is far more convincing than that from multifactorial diseases. However, despite the limited number of studies and the shortcomings of their experimental designs, the preliminary evidence about gene-diet interactions for CVD and cancer is both revealing and promising, and it is anticipated that within this decade we will achieve the highest level of evidence. Currently, this area of research focuses rst on identifying the genes responsible for these interaction effects, and second on characterizing the mechanisms responsible for those gene-nutrient interactions.
Levels of Interaction
As indicated above, it is important to consider the dynamic nature of these interactions through the life span. First, fetal development and the in uterus conditions would be essential to produce the rst gene-nutrient interactions. Second, in some conditions, as in the case of the inborn errors of metabolism, nutrition in the rst years of life is a key determinant of health or disease status. Third, for multifactorial diseases such as atherosclerosis and cancer, a long period of exposure to the
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same dietary pattern would be necessary to develop the disease phenotype (92). The hormonal environment could also be a major determinant of the interaction, and this is especially important in womens health and could be the basis for future gender- and age-specic recommendations based on genetic make up.
Monogenic Versus Multifactorial Diseases

Traditionally, genetic diseases have been classied as monogenic when they are determined by a single gene, or multifactorial when their expression is determined by several genes in combination with other nongenetic factors. However, this classication is an oversimplication (160), and the reality is far from clear cut. This is evident from the dramatic phenotypic diversity of the so-called classical monogenic diseases reecting the heterogeneity of mutations at the major locus, the action of some secondary and tertiary modiers, and the inuence of a wide range of environmental factors. Therefore, most monogenic traits share some of the features found in the multifactorial diseases. Diet may be the most inuential environmental factor modulating the phenotypes for both monogenic and multifactorial diseases. Therefore, nutritional genomics provides the tools and the evidence to modulate the phenotypic expression of these diseases. Pragmatically, the goals of nutritional genomics will be easier to accomplish in monogenic than in polygenic diseases. Therefore, understanding the genetic interactions that determine the phenotype for traditional monogenic diseases should help us gain further insight into the more complex interactions between several genes and environmental factors involved in the phenotypic expression of multifactorial diseases. In this review, we rst select a set of classical monogenic diseases in which diet plays a determinant role in the nal phenotype (such is the case of phenylketonuria, galactosemia, lactose intolerance, celiac disease and familial hypercholesterolemia) to illustrate the key role of nutrition in the clinical manifestation of disease. Some of these diseases are relatively rare compared to other diseases, but may affect millions of people worldwide. We describe each disease and its general characteristics and then we focus on specic gene-nutrient interactions. Subsequently, we present the current evidence for gene-diet interactions for the most common multifactorial diseases, such as CVD and cancer.
CLASSIC MONOGENIC DISEASES
As discussed above, the classical concept of monogenic diseases may be an oversimplication of the biological reality. First, some of them may involve more than one gene, and second, there may be signicant environmental inuences modulating the expression of the phenotype. However, from a didactic point of view it is convenient to maintain this notion. Phenylketunuria (PKU), galactosemia, lactose intolerance, and celiac disease are conditions that can be used as classical examples of gene-diet interactions. In all of them, diet plays a major role in phenotypic expression and dietary modication has been largely used to prevent the development of the disease. Although in
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these diseases the genetic component is recognized, for some of them the specic gene(s) and/or the particular mutations have not been well characterized. Nevertheless, clinical and biochemical procedures have indirectly diagnosed the genetic susceptibility for many years. In affected subjects, restricting the harmful nutrient was based on conclusive evidence from interventional trials. However, nutritional genomics will be crucial to improving the prevention or treatment of these diseases by the early identication of specic mutations or haplotype combinations that modulate dietary response in affected subjects. PKU was the rst genetic disease in which a gene-diet interaction was described. Still, some issues remain to be resolved and nutritional genomics may supply the information needed to provide a more individualized and specic dietary restriction for this disease. Phenylketonuria PKU is an autosomal recessive disorder resulting from phenylalanine (Phe) hydroxylase (PAH, EC 1.14.16.1) deciency and characterized by mental retardation. PKU is one of the rst treatable genetic disorders (160). It is considered one of the best examples of gene-diet interactions because the mental retardation in subjects with mutations in the PAH gene is easily preventable with dietary modication. PAH is the rate-controlling enzyme of Phe homeostasis. In the liver, PAH, which requires 6(R)-erythro-5,6,7,8-tetrahydrobiopterin (BH4) as a cofactor, converts Phe, an essential amino acid, to tyrosine. PKU is the most common inborn error of amino acid metabolism in Caucasians, with an average incidence of 1/10,000 (see 18 for review). This condition can be detected by measuring serum Phe levels, and the detection of hyperphenylalaninemia (HPA) is included in the newborn screening programs of most Western countries. Testing for PKU is generally done with a screening blood test collected from a heel stick onto lter paper within 48 hours of birth, as Guthrie & Susi reported (52). Babies who test positive on the initial screening test are re-evaluated with a more specic test for PKU. In untreated classical PKU, blood levels can be as high as 2400 microM/liter (23), which is about 20 times the upper limit for normality in newborns. PAH deciency is a highly heterogeneous trait that shows a broad spectrum of phenotypes. Complete or near-complete deciency of PAH activity is designated classic PKU. The continuum of milder forms has been subdivided into arbitrary categories, generally termed moderate PKU, mild PKU, and mild hyperphenylalaninemia (MHP) (51, 160). Phe is toxic to the brain, and if untreated, severe mental retardation occurs. Signs and symptoms of PKU may include abnormal movements, decreased muscle tone, a head that is smaller than normal in size, learning disabilities, and a musty odor due to skin excretion of phenylacetic acid (24). A Phe-restricted diet has been the mainstay of treatment for PKU since its development by Bickel et al. (13) in 1953. Currently, this intervention has the highest level of experimental evidence. Phe is an essential amino acid found in all protein foods, and current treatment of PKU consists of a Phe-restricted diet (well-adjusted to the tolerance) supplemented with a tyrosine-, vitamin-, and oligoelement-enriched amino acid mixture or with a specic formulation high in
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all the other amino acids necessary for protein synthesis (18). For patients with classical PKU, adequate restriction of Phe intake ensures normal development in patients with milder forms of PKU, whereas in general, no dietary restriction is required in subjects with MHP (160). Implementing a Phe-restricted diet after birth reduces blood Phe concentrations and avoids the severe consequences of untreated PKU, such as mental retardation. The need for this restricted diet in infancy and early childhood is clear; however, controversy remains about its continuation for extended periods, mainly because of the many reports describing very poor adherence over time, especially during adolescence (190). Following the work of Horner et al. in 1962 (68), which suggests terminating the diet in adulthood, most dietary recommendations in the 1960s and 1970s were consistent with the discontinuation of the Phe restriction. To evaluate the effects of discontinuing the Phe-restricted diet and the benets of the late dietary intervention, Koch et al. (83) studied 211 newborn infants identied as having PKU in a newborn screening program during 19671983. The infants were treated with a Phe-restricted diet until age 6 and then were randomly selected to either continue the diet or discontinue dietary treatment altogether. Of the 211 children, 135 were then followed until 10 years of age. This study revealed that the subjects who maintained a Phe-restricted diet reported fewer problems than the diet discontinuers, who had an increased rate of eczema, asthma, mental disorders, headache, hyperactivity, and hypoactivity. Psychological data showed that lower intellectual and achievement test scores were associated with dietary discontinuation and with higher childhood and adult blood Phe concentrations. They concluded that early dietary discontinuation for subjects with PKU is associated with poorer outcomes. The drawbacks of discontinuation are especially important in women because they have a high risk of having babies with mental retardation. If the mother is not on a low-Phe diet, the fetus is exposed to toxic levels of Phe that may result in severe mental retardation or death (30). Based on the evidence, a recent National Institutes of Health Consensus statement (121) recommends a lifetime Phe restriction for individuals with PKU. Discovering the PAH gene provided an excellent opportunity to study the molecular basis of the phenotypic variability of this disease. The PAH gene is located at 12q22q24.1, includes about 90,000 base pairs, and contains 13 exons (202). To date, more than 400 different mutations have been identied at this locus, showing an enormous genetic heterogeneity. Ledley et al. (89) studied two families in which one member had PKU and other members had MHP. They identied restriction fragment length polymorphisms (RFLPs) that differentiated the PAH alleles and concluded that there were multiple mutations in the PAH gene with different levels of severity. Subsequent worldwide genetic analyses reported additional mutations with different prevalence depending on the geographical origin. In Europe, common mutations include R408W on a haplotype 2 background in eastern Europe, IVS1011G > A in the Mediterranean, IVS12 + 1G > A in Denmark and England, Y414C in Scandinavia, I65T in western Europe, and R408W on haplotype 1 in the United Kingdom (207). One can nd a complete description of mutations in the PAH gene at http://www.pahdb.mcgill.ca/, a Web site created by the curators
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of a PKU Consortium (88 investigators, 28 countries) (124). This database has been revised and improved using bioinformatic tools (159). Most (63%) of the pathogenic PAH mutations are missense, few of which have a primary effect on PAH enzyme kinetics and may exert their effect through misfolding, aggregation, and intracellular degradation of the protein. Others are point mutations that create new splice sites. Data on expression analysis of 81 PAH mutations in multiple in vitro systems is summarized in tabular form on this Web site and is and reviewed by Walter (191). The European Multicenter Study of PAH deciency (51) has published a classication of 105 mutations in the PAH gene on the four phenotypes (PKU, moderate PKU, mild PKU, and MHP) to provide a general system for genotype-based prediction of metabolic phenotype. These authors conclude that the PAH genotype is the main determinant of metabolic phenotype in most patients with PAH deciency. Pey et al. (142) elucidated the structural consequences for 18 PKU mutations in a combined approach of expression in eukaryotic cells and coexpression of chaperonins in a prokaryotic system. Four mutations abolish the specic activity in all conditions. Two are catalytic mutations (Y277D and E280K) and two are severe structural defects (IVS1011G > A and L311P). The remaining mutations (D59Y, I65T, E76G, P122Q, R158Q, G218V, R243Q, P244L, R252W, R261Q, A309V, R408Q, R408W, and Y414C) are folding defects that cause reduced stability and accelerated degradation. The authors demonstrated that the amount of mutant protein and residual activity could be modulated by in vitro experimental conditions, providing an explanation for the inconsistencies for some patients with similar genotypes. G ttler & Gulberg (53) reported that mutation analysis of the FHA u gene may be useful for rening diagnosis and anticipating dietary requirements. They also suggested that the genotype determines cognitive development if dietary therapy is discontinued at 6 years of age. In the last two years, there has been a renewed interest in the use of BH4 to treat HPA. Although it had previously been suggested that some patients with PAH deciency responded to BH4 loading (145), not much attention was paid until the work of Kure et al. (86) conrmed these results. In these patients, treatment with BH4 increased Phe tolerance. Similar results were recently reported by some independent groups (9, 118, 195). Muntau et al. (118) prospectively studied 38 children with various classes of HPA: 10 had MHP, 21 had mild PKU, and 7 had classic PKU. To accomplish the short-term Phe loading, patients consumed a meal containing 100 mg of Phe per kilogram of body weight. One hour after, the patients ingested 20 mg of BH4 per kilogram. Muntau et al. also carried out a long-term assay by replacing dietary Phe restriction with the oral administration of BH4 in ve children with mild PKU. They found that BH4 led to normal or nearly normal blood Phe concentrations in most patients (87%) with MHP or mild PKU. Long-term treatment with BH4 increased daily Phe tolerance, allowing these patients to discontinue their restricted diet. None of the seven patients with classic PKU responded to BH4. Muntau et al. identied seven mutations (P314S, Y417H, V177M, V245A, A300S, E390G, and IVS45C G) as probably associated with responsiveness
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to BH4. These mutations were mostly located in the catalytic domain of the PAH. Despite the importance of these ndings, the authors pointed out several obstacles that must be overcome before BH4 treatment can be used routinely. If these results are further conrmed by other interventional studies, the early detection of this group of patients has signicant implications for developing a new treatment using BH4 instead of a Phe-restricted diet, which would be expected to signicantly improve their quality of life. In the next few years, the holistic approach of systems biology applied to the nutritional genomics of PKU will provide the appropriate biomarkers and information to provide successful personalized therapeutic recommendations at different stages of life for the affected individual. Galactosemia Clinical galactosemia is a complex trait in which multiple developmental and metabolic pathways are involved. As an inborn error of metabolism, it is dened as an autosomically inherited disorder of galactose metabolism, which occurs because of a deciency of one of three principal enzymes involved in the metabolism of galactose, through its conversion to glucose (123). These enzymes are galactose-1-phosphate uridyltransferase (GALT; EC2.7.7.10), galactokinase (GALK; EC2.7.1.6), and uridine-diphosphate galactose-4 epimerase (GALE; EC 5.1.3.2). The most common deciency in all communities is that of the transferase enzyme, and this is the enzyme deciency associated with classical galactosemia. Incidence of classical galactosemia is approximately 1:40,000 live births. GALT catalyzes the second step in the Leloir pathway of galactose metabolism, converting UDP-glucose and galactose-1-P to glucose-1-P and UDP-galactose. This condition usually presents in the neonatal period with failure to thrive, feeding difculties, and prolonged conjugated hyperbilirubinemia. It can be fatal if a lactose-/galactose-restricted diet is not introduced. The therapeutic effects of this galactose-restricted diet in children with GALT deciency constitute an example of gene-nutrient interaction with the highest level of evidence and with long tradition and widespread use in clinical practice. Thus, as a general recommendation, as soon as there is evidence of galactosemia (either biochemical, genetic, or clinical), galactose must be excluded from the diet (189) to prevent the lethal consequences of this defect. This means a lifelong avoidance of normal milk or dairy products. Breast-feeding and cows milk formula are contraindicated and galactosefree products should be used instead. However, this GALT deciency remains an enigma. Instituting a galactose-restricted diet, which has been the therapy used for more than six decades, alleviates the neonatal toxicity syndrome but fails to prevent long-term complications. Presentation later in life may involve hepatic cirrhosis, cataracts, ataxia, speech defects, mental retardation, and premature ovarian failure. Measurements of red cell galactose-1-phosphate monitor classic galactosemia. Raised concentrations may indicate continuous and serious deviations from the diet but some patients have unusually high values despite careful dietary compliance. Currently, long-term monitoring of galactose-1-phosphate concentrations is not recommended. Unlike dietary intervention in PKU, in galactosemia, neurological complications, poor growth, and reduced fertility frequently occur in affected
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individuals, despite early diagnosis and treatment and adherence to a lactose-free diet (93). The etiology of these dietary independent complications has yet to be elucidated. Recently, application of novel analytical techniques that have elucidated alternate metabolic pathways involving galactitol and galactonate formation, use of stable isotopes of galactose to assess in vivo galactose oxidative capacity, and endogenous galactose formation have greatly enhanced our knowledge about the disorder and the potential molecular causes of these long-term effects. Further elucidation and quantication of these processes and their regulation may aid in formulating new strategies for clinically managing the galactosemia patients. In addition, the recent unraveling of the molecular genetics of galactosemia and of the genetic variants related to different phenotypes as well as to a different response to diet will be fundamentally important in terms of dietary intervention to prevent its long-term complications. Allelic variation at the GALT gene undoubtedly plays a role in dening the biochemical and clinical phenotype. In this regard, the cloning and sequencing of the GALT gene has facilitated the identication of more than 170 mutations associated with GALT deciency. The GALT database lists most of these mutations. Q188R is the most common mutation in northern European populations and those predominantly of European descent. K285N is much rarer, but in some eastern and central European populations it is the second most common mutation (182). In Europe, these two mutations can be found on 60% to 80% of mutant chromosomes. Conversely, the S135L mutation is found almost exclusively in galactosemic individuals of African origin. In the African-American population, S135L accounts for 62% of the alleles causing galactosemia and is associated with mild outcomes. However, both Q188R and K285N mutations are associated with a complete loss in enzyme activity and, thus, a more severe biochemical phenotype. The allelic heterogeneity of this disorder as genetic modulator of the dietary response in determining the clinical phenotype remains to be investigated in adequately designed intervention trials. These results, as well as the additional discovery of the metabolic routes participating in the endogenous galactose toxicity, may provide the proper approach for controlling the subsequent complications of galactosemia, which may be beyond the control of a single gene. Lactose intolerance Lactose intolerance is a disorder with a very high prevalence worldwide. The digestive system of lactose-intolerant individuals cannot break down the lactose. This inability results from a shortage of the enzyme lactase, which is produced by the cells that line the small intestine (hence, the term lactase deciency). This disorder presents dramatic geographical differences. It is least common among people of northern European descent, where lactase deciency is present in no more than 15% of the adult population. The prevalence increases up to 80% in African-Americans and Hispanics, and even higher (95%) in American Indians and Asians. However, in most cases, lactase deciency develops naturally over time when, after about the age of 2, the body begins to produce less lactase. Most people do not experience symptoms until they reach an older age. Another
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term that refers to this syndrome is lactase persistence, which indicates that intestinal lactase activity persists at childhood levels into adulthood. A less common form is the so-called congenital lactose intolerance, a severe disorder that manifests with vomiting, failure to thrive, dehydration, disacchariduria including lactosuria, renal tubular acidosis, aminoaciduria, and liver damage. Its incidence is very low. Symptoms of lactose intolerance in adults are caused by lactose fermentation. Because lactose is not broken into glucose, and hence left unabsorbed by the body, the conditions found in the intestines help the lactose to ferment, leading to the formation of gases. A particular gas, methane, is usually the cause of painful aggressive atulence. Common symptoms include nausea, atulence, meteorism, and diarrhea when signicant quantities of fresh milk are consumed. The enzyme responsible for this condition, the intestinal lactase-phlorizin hydrolase (LPH), has been clearly identied. However, sequence analyses of the coding and promoter regions of LCT, the gene encoding LPH, located on 2q21, has revealed no DNA variations correlating with lactose intolerance in most studies (175). Although the molecular basis of this phenotypic polymorphism is not known, lactase persistence is associated with a 70-kb haplotype in Europeans, and the locus is cis-acting and has been proposed as an example of selective advantage (66). Hollox et al. (66) suggested that genetic drift was important in shaping the general pattern of non-African haplotype diversity, with recent directional selection in northern Europeans for the haplotype associated with lactase persistence. They studied 11-site haplotype in 1338 chromosomes from 11 populations differing in lactase persistence frequency. This showed that haplotype diversity was generated by both point mutations and recombinations, and it identied four common haplotypes (A, B, C, and U) that were not closely related and had different distributions. Recently, in Finnish families, an association was observed between biochemically veried lactose intolerance and a C/T(13,910) polymorphism roughly 14-kb upstream from the LCT, with the C allele associated with lactase deciency (38). Because of this gene-nutrient interaction, subjects with specic mutations in the gene(s) involved in LPH deciency should avoid lactose-containing foods. The degree of lactose intolerance varies greatly among patients with lactose intolerance, which suggests an important allelic effect of some particular mutations. However, to date, there are not results from studies examining this specic gene-nutrient interaction at the molecular level. On the other hand, some lactose-intolerant individuals are at greater risk of osteoporosis because such individuals often restrict dairy and calcium intake. Thus, Segal et al. (162) demonstrated a decrease in quantitative bone parameters in the appendicular skeleton in lactose-intolerant individuals compared to age-matched controls. This decrease was higher in men and in postmenopausal women. Celiac disease Celiac disease, also known as gluten-sensitive enteropathy (32), is a more complex disease resulting from a permanent intolerance to ingested gluten and related cereal prolamins in genetically predisposed individuals. This disease
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results in immunological-mediated inammatory damage to the small-intestinal mucosa. It is characterized by malabsorption that results in a wide spectrum of both pathophysiological changes in the intestine and clinical syndromes, many of which remain undiagnosed (for review, see 119). The traditional signs are diarrhea, steatorrhea, and loss of weight or failure to thrive (39). More common but more difcult to recognize are the other presentations of celiac disease that include abdominal pain, atulence, secondary lactose intolerance, dyspepsia, deciencies of single micronutrients, osteoporosis, and iron deciency anemia (119). The actual prevalence of this syndrome is not known because it depends on the various criteria used to dene this disorder (4, 39). The current estimates indicate that celiac disease is relatively common, affecting 1 of every 120 to 300 subjects in both Europe and North America (74). Celiac disease is considered a multifactorial disease with a strong genetic component. As a heritable condition, familial aggregation is common, and it is more prevalent in Caucasians. Celiac disease has one of the strongest associations with human leukocyte antigen (HLA) class II markers of the known HLA-linked diseases (139). Howell et al. (69) demonstrated that this disease is associated with a subset of HLA-DR3, DQw2 haplotypes characterized by HLA-DP alpha- and beta-chain gene RFLPs. It has been proposed that celiac disease results from the interaction of two loci: one locus linked to HLA and associated with dominant inheritance, and the other, a non-HLA-linked gluten intolerance-associated B-cell alloantigen, exhibiting recessive inheritance. However, the results from Greenberg et al. (50) using affected sib-pair data supported a recessive model consistent with a HLA-linked disease allele inherited in a recessive manner. Subsequent whole-genome screens to identify non-HLA loci for celiac disease have produced inconsistent results suggesting genetic heterogeneity (32). Complementary studies by Sollid (170) provided an attractive pathogenic scheme explaining the interplay between the environmental triggering antigen, the main genetic risk factor represented by HLA-class II molecules DQ2 or DQ8, and tissue transglutaminase, the target of CD-specic auto antibodies. His work assigns a central role to adaptive immunity, and suggests that intestinal inammation is orchestrated by gliadin-specic CD4+ T lymphocytes. The most important dietary factor in celiac disease is gluten. The deleterious proteins are gliadins (wheat), hordeins (barley), secalins (rye), and possibly avidins (oats). It is now clear that some gluten sequences can bind to HLA-DQ2/8 and induce Tcell responses. In addition, modication of gluten peptides by the enzyme tissue transglutaminase results in high-afnity HLA-DQ2/8 binding peptides that can induce T-cell responses (84). Removing gluten from the diet can result in a total recovery of gut function and a correction of most other consequences. Therefore, based on decades of experimental results (165), the treatment of choice for this gene-nutrient interaction requires a gluten-free diet excluding all cereals. However, this measure is very hard to adhere to and ongoing research aims to design easier-to-adopt nongluten-free diets (8). Nutritional genomics will evaluate the feasibility, efcacy, and risks of any dietary approach tailored for treating celiac disease.
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Familial hypercholesterolemia Familial hypercholesterolemia (FH) is a common inherited disorder of lipoprotein metabolism and it is not included among the classical inborn errors of metabolism; however, it may provide another example of monogenic disease in which dietary and environmental factors can strongly modulate manifestation. FH is caused by one of multiple mutations in the gene that encodes the low-density lipoprotein (LDL) receptor (LDLR) localized on chromosome 19. These mutations lead to a reduced number of functional LDL receptors, resulting in defective uptake of plasma LDL and dramatic elevation in plasma LDL cholesterol levels (49). It is estimated that FH affects 1 of every 500 people. Although this appears to be a small number in relative terms, in absolute terms it represents an important global health problem: in the United States alone more than half a million people are affected by this genetic disorder. Patients with FH are clinically characterized by the presence of tendon xanthomas, xanthelasmata, or arcus lipoides corneae. However, much more important is that in most patients there is also an excessive deposition of cholesterol in arterial walls, which leads to accelerated atherosclerosis and premature CVD. Currently, if untreated, 75% of male FH patients suffer from coronary artery disease (CAD) before age 60 (12). The mean age of onset for CVD is between 40 and 45 years in male FH patients and is 10 years later for female FH patients. Although FH is monogenic, the phenotypic expression varies considerably in terms of onset and severity of atherosclerotic disease. One explanation is that this variability may be due to the severity of the specic defect within the LDLR gene coding the LDL receptor, a 160-kDa transmembrane glycoprotein that is pivotal in the receptor-mediated endocytosis of LDL particles. It is a multifunctional protein with several structural domains. To date, more than 700 different mutations have been described in the LDLR gene (see http://www.gene.ucl.ac.uk/ideas/fh.htm). Mutations have been categorized into ve different classes. The so-called nullalleles fail to produce any protein, whereas other mutations lead to impairments in binding capacity, in post-translational processing, or in recycling. Scientists suggest that the class of receptor mutation and resulting defective protein impact clinical phenotype. The effects of mutation type on lipoprotein levels and on the risk for CVD have been studied extensively. Several investigators have shown signicant differences in lipid levels between various types of mutations (10, 19, 34). Although some have found an increased CAD risk in patients with receptor-defective mutations as compared with receptor-negative mutations, others could not conrm this. The clinical presentation differs substantially even among individuals who share an identical gene defect (166). Therefore, additional factors inuence the course of FH and lead to the dramatic variation in clinical manifestations. In support of this concept, once a person reaches age 6070 the excess risk fades and approaches that of the general population (167). This might be due to survival bias because patients who are most susceptible to the ravaging effects of this genetic disease may have died at a younger age, which suggests that there may be gene-environment and/or gene-gene interactions protecting some of these less susceptible FH subjects from early disease. Evidence
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from the United States (201), Europe (166), and Asia (144, 174) supports this notion. Williams et al. (201) carried out studies within the large Utah families to produce this evidence. They screened 1134 individuals from 18 pedigrees. In most pedigrees, serum cholesterol was a polygenic trait with 54% heritability. However, in four pedigrees with FH, male heterozygotes had a mean serum cholesterol level of 352 mg/dl, myocardial infarction at an average age of 42 years, and coronary death at an average age of 45 years. However, the four ancestral males born before 1880 who were the known founders of the LDLR mutations for each pedigree, survived to ages 62, 68, 72, and 81 years. This suggests that some healthy lifestyle factors protected these men against the expression of a mutation that has led to coronary disease by age 45 years in all of their heterozygous great-grandsons. Specically, the previous generations of these families had a much more active lifestyle and consumed diets that were lower in total, especially saturated, fat than their contemporary descendants. One heterozygote showed a drop in serum cholesterol level from 426 to 248 mg/dl with strict adherence to a low-fat diet without drugs. These observations are important because they demonstrate that, even when the life expectancy was much shorter than it is now, there were disease-susceptible ancestors who had improved longevity without the benet of medications and who relied exclusively on a prudent and active lifestyle. These ndings were corroborated by another study in the Netherlands that used a similar experimental approach (167). The goal of the study was to estimate all causes of mortality from untreated FH on a large Dutch pedigree traced back to a single pair of ancestors in the nineteenth century. A total of 70 deaths occurred among the 250 people analyzed. Mortality did not increase; it slightly decreased in carriers of the mutation during the nineteenth and early twentieth century. Mortality rose after 1915, reached its maximum between 1935 and 1964, and fell slightly thereafter. In addition to the observed time effect, mortality also differed signicantly between two branches of the pedigree that may have been exposed to different environmental factors or different gene-gene interactions, adding to or reducing the predisposition provided by the LDLR gene. These two studies show that the risk of death varies signicantly among FH patients. This large variability over time and between branches on these pedigrees points to a strong interaction with environmental factors, even for this monogenic and highly penetrant disease. A different type of experimental approach was used in Asian populations, producing similar ndings (144, 174). FH heterozygotes are not detected in the general population and obligate heterozygotes are often not hypercholesterolemic by Western standards, meaning that Chinese may carry milder LDLR mutations than those present in other ethnic groups. However, this is not true because many LDLR mutations cause a receptor-negative phenotype (174). Therefore, the lack of clinical expression in obligate Chinese FH heterozygotes living a traditional lifestyle is not due to unusually mild mutations in the LDL-receptor gene, but probably to environmental factors, such as those predicted for the Utah and Dutch pedigrees. Investigators in Canada tested this hypothesis on Chinese FH heterozygotes living
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in Canada who were screened for mutations that had been previously described in FH patients living in China (144). These investigators found signicantly higher LDL cholesterol concentrations in FH heterozygotes with dened mutations living in Canada than in those living in China. About 40% of FH heterozygotes residing in Canada had evidence of tendon xanthomata, and 25% had a history of premature CAD, whereas none of those in China had tendon xanthomata or CAD. Therefore, Chinese FH heterozygotes living in Canada exhibit a phenotype similar to that of other FH patients in Western societies. The difference between patients living in Canada and those living in China could be ascribed to differences in dietary fat consumption and physical activity, again showing that environmental factors such as diet play a signicant role in modulating the phenotype of heterozygous FH. In summary, patients with FH have an increased risk for CVD; it can be about 100 times higher early in life than that of normal individuals. Age, sex, LDL cholesterol levels, and a positive family history for premature atherosclerosis are the most important determinants for early and severe events. However, the clinical phenotype is highly modiable by environmental factors, the type of LDL-receptor mutation, and coinheritance of other genetic factors. The variability in clinical phenotype of FH demonstrates that environmental and other genetic factors play equally important roles, even in a monogenic common disorder. Therefore, FH provides an excellent model for future studies on complex gene-gene and geneenvironment interactions. In addition, there are other instances in which monogenic predisposition to CHD is inuenced by the environment (58). This combined evidence provides a strong basis to propose that the effect of the environment should be even stronger in subjects whose phenotypic risk factors and/or disease predisposition are due to the combination of mild contributions from several genetic and nongenetic factors, as illustrated below. Other There are numerous other examples of gene-diet interactions among the classically known inborn errors of metabolism, which tend to be found in very low prevalence (161). Early identication and treatment of these genetic diseases require prompt diagnosis and correction of metabolic abnormalities by dietary restriction of the offending substances and, in most cases, the consumption of various special formulas to meet the nutritional requirements (150). Congenital hyperhomocystinemia, cystinuria, hyperuricemia, and hemochromatosis are some examples of these inborn errors. Dietary modications such as folate intake, uid intake, a diet low in purine, and an iron-restricted diet, respectively, may modulate the phenotypical expression of these diseases (37, 40, 143, 148).
MULTIFACTORIAL CHRONIC/AGE-RELATED DISEASES
Multifactorial diseases such as CVD, cancer, osteoporosis, and neurological diseases are usually associated with the aging process. Therefore, they are currently the major health problems in a world that is growing older. About 19% of the people in developed countries are over 60 years old, whereas only 50 years ago this gure was just 8%, and the
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current predictions estimate that by the year 2050, the over 60 population will more than double the current gures (179). We consider physiological decline the normal path to old age. However, the increasing frailty that we call senescence may not be the obligated fate of the aging human (81). Although utopia is the complete elimination of age-related decline, we hope to make signicant strides in reducing the gap between normal and ideal aging. To do so, action is needed early in life, and solid scientic evidence must be presented to support these actions. There is little doubt that the best approach to achieve the goal of healthy aging is disease prevention, which for a large proportion of the population could be achieved with dietary and other behavioral changes. Cardiovascular disease and the metabolic syndrome CVD is the major cause of morbidity and mortality in the United States. Like all age-related diseases, the incidence of CVD has experienced a dramatic evolution during the last 100 years. It was this sudden emergence of CVD as a major public health concern during the 1940s that fostered research to understand the factors determining this disease. Projects like the Framingham Heart Study were launched to identify the triggers of CVD. Initially, the focus of this research was placed on the identication of biochemical, environmental, and behavioral risk factors. As a result, several CVD risk factors were well established and are commonly used to detect and treat subjects at risk. However, due to the complexity of this disorder, we are still far from reaching complete knowledge about its risks and how to prevent it. CVD represents the paradigm of multifactorial disorders encompassing multiple genetic and modiable risk factors. The current recommendations aim to reduce the classical modiable risk factors, and much emphasis has been placed on controlling high-plasma cholesterol levels. However, this is just one of a constellation of risk factors associated with CVD. The most common cluster of these risks is the Metabolic Syndrome, which is characterized by the concurrence of obesity, dyslipidemia, hyperglycemia, and hypertension. The dramatic increase in CVD risk associated with this syndrome has brought to it a graphic and self-explanatory denition: The Deadly Quartet (16, 103, 120). The most current estimates indicate that 24% of the population 20 years and older are affected by metabolic syndrome. However, underneath this global statistic hide even more alarming gures. First, there is considerable heterogeneity among ethnic groups, with Hispanic populations withstanding the worst of the problem, probably due to a combination of gene and environmental interactions. The impact on the elderly is even more terrifying, with 40% of people older than 60 years of age affected by this disorder. If we are to make signicant strides against this major killer, we need to understand the molecular mechanisms responsible for the metabolic abnormalities and how these four apparently distinct conditions (obesity, dyslipidemia, hyperglycemia, and hypertension) may arise from a common pathophysiologic process. In other words, we need to identify who is the elusive conductor of the Deadly Quartet (103).
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Below, we summarize our knowledge about the interactions between genetic and dietary factors that modulate the expression of this syndrome and the clinical manifestation of CVD. We place major emphasis on the dyslipidemia component of the metabolic syndrome.
PLASMA LIPIDS: THE FIRST OF MANY CVD RISK FACTORS

The link between serum cholesterol and the development of atherosclerosis was established a few decades ago and is now widely accepted. The National Cholesterol Education Program (NCEP) Adult Treatment Panel (ATP) publishes updated guidelines for treating lipid disorders. The last version is the ATPIII. These guidelines consider dietary modication the cornerstone of primary prevention, with emphasis on reducing the high-saturated fat atherogenic diet, as well as controlling other behavioral factors such as sedentary lifestyle (178). However, despite these and other guidelines, we are still far from accurately predicting disease and from knowing how many individuals can achieve the recommended goals using the proposed global approaches. The latter problem exists because the relationships between dietary changes and serum lipid changes are predictable for groups; however, due to the striking variability in the interindividual response of serum cholesterol to diet, we cannot predict individual response. There is evidence that the dramatic variation in individual plasma lipids in response to changes in dietary fat and cholesterol has a genetic component. So far, the identication of these genetic factors has been limited to the candidate gene approach, and the obvious targets of this search are those genes with products involved in lipoprotein metabolism. Lipoproteins are macromolecular complexes of lipids and proteins that originate mainly in the liver and intestine and are involved in transporting and redistributing lipids in the body. Lipid and lipoprotein metabolism comprises complex biological pathways that contain multiple steps. Lipid homeostasis is achieved by the coordinated action of numerous nuclear factors, binding proteins, apolipoproteins, enzymes, and receptors. Lipid metabolism is also closely linked with energy metabolism and is subject to many hormonal controls essential for adjusting to environmental and internal conditions. Genetic variability exists in humans for most of these components, and some of these mutations result in abnormal lipid metabolism and plasma lipoprotein proles that may contribute to the pathogenesis of atherosclerosis. Many of these genes have been explored in terms of gene-diet interactions. A detailed account of each of these studies and their interpretation would exceed the space available for this work, but they were the subject of recent comprehensive reviews (98, 105, 156). We highlight some of the loci, specically APOE, APOA4, APOA1, and APOB, that provide the most relevant examples for different types of interactions with specic dietary components. Information about other genes that show signicant gene-diet interaction in the modulation of plasma lipid are detailed in a recent systematic review by Masson et al. (105).
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Apolipoprotein E (APOE)
The Apolipoprotein E (APOE) gene has been the locus most intensively examined in terms of its potential to determine the individual variability in its LDL cholesterol response to diet interventions. This interest is obvious, considering apoEs pivotal role in lipoprotein metabolism. ApoE in serum is associated with chylomicrons, VLDL, and HDL, and serves as a ligand for multiple lipoprotein receptors (102). Genetic variation at the apoE locus results from three common alleles in the population, E 4, E 3, and E 2, with frequencies in Caucasian populations of approximately 0.15, 0.77, and 0.08, respectively (29). In addition, several other genetic variants have been described at the apoE locus (2). Population studies show that plasma cholesterol, LDL cholesterol, and apoB levels are highest in subjects carrying the apoE4, intermediate in those with the apoE3, and lowest in those with the apoE2 isoform (128, 158). ApoE allelic variation may account for up to 7% of the variation in total and LDL-cholesterol levels in the population (29). An initial observation was that the association of the apoE4 isoform with elevated serum cholesterol levels was greater in populations consuming diets rich in saturated fat and cholesterol than in other populations. These epidemiological data indicate that the higher LDL cholesterol levels observed in subjects carrying the apoE4 isoform were manifested primarily in the presence of an atherogenic diet characteristic of certain societies, and that the response to dietary saturated fat and cholesterol could differ among individuals carrying different apoE alleles. Previous ndings related to this locus have been extensively reviewed (130 133, 156). Note that despite the numerous studies examining the relation between APOE genetic variability and LDL-cholesterol response to diet intervention, there is considerable inconsistency regarding the magnitude and signicance of the reported associations, and this locus continues to be the subject of intense research. Rubins revision (156) includes 29 intervention studies that examine APOE-diet interactions. A total of 3224 subjects participated in these studies, ranging from 16 to 420 subjects per study. Of the 29 studies, 12 demonstrated no signicant APOE-diet interactions, 15 reported signicant interactions (E4 was usually associated with increased dietary response), and 2 were undened. Using the same available literature, but different selection criteria, Masson et al. (105) reviewed 62 dietary intervention periods, including 3223 subject-by-diet interventions. Again, the range of the studies varied between 8 and 210 subjects per dietary intervention. According to this review, 42 of the diet interventions did not demonstrate signicant APOE-diet interactions, and only 19 provided evidence for signicant interactions, clearly demonstrating the diversity of the results presented in the original papers as well as those obtained from review papers. The important messages to extract from the available information are: Significant dietapoE gene interactions occurred in male-only studies. In studies including men and women, signicant effects were noted only in men, suggesting a signicant gene-sex interaction. Another difference between the negative studies
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and those reporting signicant apoE genediet interactions relates to the baseline lipid levels of the subjects. Studies reporting signicant associations often included subjects who were moderately hypercholesterolemic and/or had signicant differences in base total cholesterol and LDL-cholesterol among the APOE genotype groups. This suggests that the signicant gene-diet interaction is apparent only in subjects susceptible to hypercholesterolemia. Concerning differences in dietary interventions, there were signicant interactions in studies in which total dietary fat and cholesterol were modied. Dietary cholesterol might play a signicant role in genedietary fat interaction. This combination of dietary factors needed to elicit signicant gene-diet interactions was also evidence for another locus (APOA4), which we discuss below. Several mechanisms are proposed to explain these apoE-related differences in individual response to dietary therapy. Some studies show that intestinal cholesterol absorption is related to APOE phenotype, with APOE4 carriers absorbing more cholesterol than non-APOE4 carriers. Other mechanisms such as different distribution of apoE on the lipoprotein fractions, LDL apoB production, bile acid, and cholesterol synthesis, and postprandial lipoprotein clearance may also be involved. Although the obvious dietary factors implicated in gene-diet interactions affecting plasma lipid levels are dietary fats and cholesterol, other dietary components have revealed signicant interactions. This is the case for alcohol intake. Although the raising effect of alcohol consumption on high-density lipoprotein (HDL)-cholesterol levels is well established, the effect on LDL-cholesterol is still unclear. It is possible that the reported variability will be due to interactions between genetic factors and alcohol consumption. Using cross-sectional analysis, we examined whether variation at the APOE locus modulates the association between alcohol consumption and LDL-cholesterol levels in a healthy populationbased sample of 1014 male and 1133 female participants in the Framingham Offspring Study (26). In male nondrinkers, LDL-cholesterol levels were not different across APOE groups; however, in male drinkers, there were differences in LDL-cholesterol, with APOE2 subjects displaying the lowest levels. When LDLcholesterol levels were compared among the APOE subgroups by drinking status, LDL-cholesterol levels in APOE2 male drinkers were lower than in APOE2 nondrinkers. Conversely, in APOE4 males, LDL-cholesterol was higher in drinkers than in nondrinkers. This APOE-alcohol interaction remained signicant after controlling for age, BMI, smoking, fat, and energy intake. In women, the expected effect of APOE alleles on LDL-cholesterol levels was present in both drinkers and nondrinkers. Multiple linear regression models showed a negative association between alcohol and LDL-cholesterol levels in APOE2 men, with alcohol intake a continuous variable. Conversely, in APOE4 men, this association was positive. There were no statistically signicant associations in either APOE3 men or in women. These data suggest that in men variability at the APOE locus partially modulates the effects of consuming alcoholic beverages on LDL-cholesterol levels.
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Apolipoprotein A-IV (APOA4)

Apo A-IV is a 46-Kd plasma glycoprotein (197) that is synthesized by intestinal enterocytes during lipid absorption and is incorporated into nascent chylomicrons. Apo A-IV enters circulation on lymph chylomicrons, but then dissociates from their surface and circulates primarily as a lipid-free protein. Several genetically determined isoforms of apoA-IV have been detected; amino acid positions 360 and 347 of the mature protein are the most common (15). The polymorphism at position 360 is due to a CAG CAT substitution at codon 360 in the APOA4 gene and encodes a Q360H (Gln His) substitution in the carboxyl terminus (97), and generates an isoform, originally known as apo A-IV-2, one charge unit more basic than the common isoform, apo A-IV-1 (110). For Caucasians, the apoA-IV-2 isoform has an allele frequency ranging from 0.05 to 0.12. In some population studies the apoA-IV-2 allele is associated with higher levels of HDL-cholesterol and or apoA-I (90, 111, 140) and/or lower triglyceride (TG) levels (111), as well as lower LDL-cholesterol (35), lower Lp(a) (186), and higher fasting glucose and insulin levels (76), but no associations have been observed in other studies. The other common mutation (Thr347 Ser) is due to an ACT TCT substitution at codon 347 in the human apo A-IV gene (97); it is found within subjects with the apoA-IV-1 isoform. Several population studies note that carriers of the 347S allele have lower plasma, total cholesterol, LDL-cholesterol, apoB levels (186), and Lp(a) levels (140) than 347T/T homozygotes. Interest in this locus originated from the original reports showing that male carriers of the less common allele at the Gln360His polymorphism were less responsive to changes in dietary fat and cholesterol or cholesterol alone (107, 108). Since then, several studies have focused on the interaction between the APOA4 locus and dietary factors, both in the fasting and postprandial states (59, 64, 134, 135, 188, 194, 196). Similar to the ndings for other genes, the data are conicting when it comes to the effect of apo A-IV polymorphisms on the LDL response to dietary cholesterol (for reviews, see 105 and 198). However, according to Weinberg (198), the results from different studies can be partially reconciled if one assumes that the dietary fatty acid effects dominate over the allele effects. Therefore, if dietary cholesterol intake is the principal variable, and total fat intake is moderate and constant, Q/H subjects display an attenuated response of LDL-cholesterol. However, when dietary cholesterol intake is changed in the setting of a higher baseline dietary fat intake or with a change in fat saturation, the fatty acid effects on LDL levels predominate and overrule the allele effect. The impact of the Q360H polymorphism on cholesterol absorption may be greater on a high polyunsaturated fatty acids (PUFAs) intake. However, dietary PUFA counteract the effect of dietary cholesterol on the expression of hepatic LDL receptors (196). Thus, the nal effect of apo AIV alleles on the LDL response to dietary cholesterol may be determined by the relative amounts of cholesterol, saturated fatty acids (SAFAs), and PUFAs in the diet.
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There is more consistency and probably less complexity regarding the impact of apo A-IV polymorphisms on HDL-cholesterol: When total fat intake is raised or lowered, Q/H subjects have an exaggerated, and Threonine/Serine (T/S) subjects an attenuated, response in plasma HDL levels. It has been suggested (188), and Weinberg (196) demonstrated, that a high-PUFA intake may amplify this effect. Given the relationships between plasma TG and plasma HDL-cholesterol levels, it is possible that the response of plasma HDL-cholesterol levels to changes in dietary fat is mediated by apo A-IV allele effects on postprandial triglyceride-rich lipoprotein metabolism. These studies clearly illustrate the extreme complexity associated with the interpretation of results from studies involving gene-diet interactions. Orchestrating the different dietary components (cholesterol, amount of fat, and type of fat) can change the direction of the outcomes. The current evidence supports strong interactions with gender. Finally, alleles at other loci, or even within the same locus as demonstrated for the Q360H and T347S at the APOA4 locus, need to be considered to explain the results of one study and to place them in the context of the current knowledge of nutritional genomics.
Apolipoprotein A-I (APOA1)

Apolipoprotein A-I (apoA-I) is the major apolipoprotein of HDL, constituting about 70% to 80% of HDL protein mass, and is the main activator of the enzyme lecithin cholesterol acyl transferase (LCAT). Therefore, the APOA1 locus is a prime candidate for studying genetic variability in HDL levels. APOA1 maps to the long arm of chromosome 11, clustered with the structural loci for apoC-III and apoA-IV (78). Its gene product apoA-I plays a central role in lipid metabolism and CVD risk (163). Pedigree studies have reported associations between genetic variation at the APOA1 locus and plasma lipid and lipoprotein levels. A common G to A transition (G/A) located 75-bp upstream from the transcription start site of the APOA1 gene has been extensively studied. In 1990, it was reported that this polymorphism was associated with apoA-I and HDL-C (73) concentrations, and individuals carrying the A-allele presented with the highest levels, compared with subjects homozygotes for the G allele (G/G) (137). Subsequent studies examining this association have shown contradictory results. Juo et al. (75) used a meta analysis approach to show this lack of consistency between the less common A-allele and higher HDL-cholesterol concentrations. The inuence of environmental factors that modulate the effect of the genetic polymorphism may explain these diaparities. Although some of these studies investigated the possible interaction with tobacco smoking (177), none assessed the inuence of diet on the observed associations. In dietary intervention studies we addressed this issue by investigating this potential interaction (99, 106). Our data was consistent with a signicant gene-diet interaction modulating the response of plasma lipids to dietary modication. In view of the signicant gene-diet interaction observed for those intervention studies, we examined whether these results could be extrapolated to a free living population, consisting of about 1600
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Framingham Offspring Study participants (127). The frequency for the A-allele in this population was 0.165, similar to previous reports in Caucasian populations. The results from the straightforward association between genotype and phenotype were disappointing and suggested that the G/A polymorphism was not associated with HDL-cholesterol, apoA-I concentrations, nor with any other anthropometrical or plasma lipid variable examined. To examine the potential modifying effect of dietary fat on these associations, we tted multivariate linear regression models, including interaction terms for fat intake [total, SAFA, monounsaturated (MUFA), and PUFA fat]. No signicant interactions were observed between the G/A polymorphism, total, SAFA, and MUFA fat intakes. However, in women, HDL-cholesterol concentrations were associated with a signicant interaction between PUFA intake and APOA1 genotype (p = 0.005). Using PUFA as a dichotomous variable, our data show that G/G women consuming <6% PUFA/day had higher HDL-cholesterol (1.48 0.40 mmol/L) than A-carriers (1.43 0.40 mmol/L). Conversely, when consuming 6% PUFA/day, G/G had lower HDL-cholesterol concentrations (1.44 0.39 mmol/L) than A-carriers (1.49 0.39 mmol/L). In men, the situation was more complex because the effects were observed using three-way interactions, including smoking and alcohol consumption, in the analyses. As illustrated above, incorporating environment (dietary habits) into these analyses uncovered an interesting observation. The most evident application of these results may be to help us make more efcacious dietary recommendations based on genetic prole. It is clear that subjects with the A-allele at this APOA175(G/A) polymorphism will benet from diets containing a high percentage (it is important to underscore that we are talking about percent in the diet and not about total amounts) of PUFA (i.e., vegetable oils, sh, nuts, and so on). According to our data, this should result in higher HDL-cholesterol concentrations, which in turn should lower CVD risk. These ndings suggest that the expression of the APOA1 gene may be regulated by PUFA, which may interact with transcription factors, which may bind to a region within or close to the polymorphic site we examine.
Hepatic Lipase (LIPC)

Hepatic lipase (HL) is a plasma lipolytic enzyme that participates in metabolizing intermediate-density lipoprotein and large LDL into smaller, denser LDL particles, and in converting HDL2 to HDL3 during reverse cholesterol transport. HL has also been suggested to act as a ligand for cell-surface proteoglycans in the uptake of lipoproteins by cell-surface receptors. HL deciency is characterized by mildly elevated concentrations of triglyceride-rich LDL and HDL particles, as well as impaired metabolism of postprandial triglyceride-rich lipoproteins, which may result in premature atherosclerosis. Conversely, increased HL activity is associated with increased small, dense LDL particles and decreased HDL2 concentrations, which may also result in increased CAD risk.
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Four common single nucleotide polymorphisms (SNPs) on the 5 -anking region of the HL gene (LIPC) [763(A/G), 710(T/C), 514(C/T), and 250(G/A)] are in total linkage disequilibrium and dene a unique haplotype that is associated with variation in HL activity and HDL-cholesterol levels (72). The less common A-allele of the SNP at position 250 is associated with lower HL activity and buoyant LDL particles. Normal and CAD subjects heterozygous for the A-allele have lower HL activity and signicantly more buoyant LDL particles. Homozygosity for this allele (AA) is associated with an even lower HL activity. The A-allele is associated with higher HDL2-cholesterol (205). Given the wide spectrum of effects that HL exerts on lipoprotein metabolism, and the signicance of the promoter variant(s), it is reasonable to hypothesize that genetic variation at this locus may also be involved in variability in the response to dietary therapy. We began to address this issue using the wealth of phenotypic and dietary information collected from Framingham Heart Study participants (126). Our data shows that subjects carrying the CC genotype (the most common among Caucasian subjects) react to higher contents of fat in their diets by increasing the concentrations of HDL-cholesterol, which could be interpreted as a defense mechanism to maintain the homeostasis of lipoprotein metabolism. Conversely, carriers of the TT genotype cannot compensate, and experience decreases on the HDL-cholesterol levels. These data could identify a segment of the population especially susceptible to diet-induced atherosclerosis. Considering the higher frequency of the T allele among certain ethnic groups (i.e., African-Americans), these data could shed some light on the impaired ability of certain ethnic groups to adapt to new nutritional environments, as clearly seen for Native Americans and Asian Indians. In this regard, we replicated the signicant gene-diet interaction demonstrated in the Caucasian population of Framingham in another multiethnic cohort that consisted of Chinese, Malays, and Indians representing the population of Singapore (176). In addition to the signicant gene-diet interactions reported in these papers, our data provides clues about the reasons why genotype-phenotype association studies fail to show consistent results. In theory, this polymorphism at the hepatic lipase gene will show dramatically different outcomes in association studies depending on the dietary environment of the population studies. The impact of these interactions will be magnied in populations with a high prevalence of the T-allele, as it is with Asians and African-Americans. Although we do not discuss pharmacogenetics here, some genes showing significant nutrigenetic effects also present signicant gene-drug interactions. Zambon et al. (204) explored this notion for the hepatic lipase gene in the Familial Atherosclerosis Treatment Study (FATS) trial. Following intensive lipid-lowering therapy, subjects with the CC genotype at the 514(C/T) polymorphism, which is equivalent to the A-allele for the 250(G/A) polymorphism (described above), had a greater decrease in HL activity compared with carriers of the T-allele. Consistent with this effect on HL, these subjects also experienced a greater improvement in their LDL-subclass distribution, as well as increases in HDL2-cholesterol concentrations. In addition to the greater improvement in their lipid prole, CC subjects also
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demonstrated the greatest coronary stenosis regression, as determined by quantitative angiography. Therefore, there appears to be consistency for gene by therapy interaction for the LIPC locus. However, these data need further replication by other investigators as well as the elucidation of the molecular mechanisms driving these interactions.
CANCER
In 1975, Armstrong & Doll (3) analyzed the correlation between incidence rates and diet for 27 cancers in 23 countries and mortality rates for 14 cancers in 32 countries. They concluded that dietary variables were strongly correlated with several types of cancer, particularly colon and breast cancers. Later, Doll & Peto (33) estimated that dietary factors contributed to 35% of cancer deaths in the United States. The authors stated that this estimation was highly speculative and proposed a condence interval from 10% to 70%, with gastric cancer the most related to diet. Over the past three decades, numerous epidemiological studies (ecological, case-control, and follow-up) have reported associations between diet and cancer. The idea that diet is an important cause of cancer led various health organizations to formulate dietary guidelines to reduce cancer risk at the population level. These guidelines generally propose reducing fat intake, particularly saturated fat, including a variety of vegetables and fruits in the daily diet, being physically active and maintaining a healthy weight, consuming alcoholic beverages in moderation, and minimizing intake of salt-cured, salt-pickled, or smoked foods. Although these general recommendations have been partially adopted, the food or food components that provide the highest protection remain largely obscure (200). There is concern about the fact that few reported associations between nutrients and cancer are consistently and convincingly replicated (85). One example is the association between dietary ber and colon cancer. Although ecological and various case-control studies have reported an inverse association between ber intake and colon cancer (45, 112), results from cohort studies did not support these ndings (44, 46). One thing that might contribute to the discrepancy between studies is the measurement error of dietary components and the confounding from other factors, including other environmental and genetic factors. Although observational evidence from ecological, case-control, and cohort studies strongly supports an inverse relationship between cancer risk and intake of vegetables and fruits (152, 187), the specic protective food or food component has not been characterized. Carotenoids are ubiquitous in the plant kingdom, and as many as 1000 naturally occurring variants have been identied. Carotenoids are among the easiest molecules to measure in plasma and tissue samples, and they are considered a marker for a healthy diet rich in fruits and vegetables (104). Results from in vitro, animal, and some observational studies that estimate carotenoid intake suggest that a high intake of carotenoids is related to a lower cancer risk (11, 171). Therefore, it was hypothesized that -carotene would decrease cancer incidence, especially lung cancer, and three interventional studies were launched to test that hypothesis (1, 62, 125). Unexpectedly, the
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results were the reverse of the hypothesis in two of three studies (1, 125), and no association was found in the other (62). Several reasons were proposed to explain these results: One is that it is possible that -carotene is simply a marker for the truly protective substance present in vegetables and fruits. Another is that most of the participants were smokers, and cigarette smoke was later found to degrade carotenoids, yielding toxic carotenoid metabolites in the lung (57). Another reason that has not been contemplated relates to a potential gene-diet interaction with a polymorphism in metabolic enzymes potentially involved in bioactivating procarcinogens in smokers. All these ndings indicate that the relationship between diet and cancer is more complex than originally thought. In addition to the well-known specic methodological difculties associated with the epidemiological studiesthe most important being the complexity of measuring usual long-term diet at the individual levelthere are other confounders, including behavioral factors (such as tobacco smoking) and genetic factors that modify the effect. These caveats need to be considered when designing new investigations in this eld. It is important to remember that gene-nutrient interactions can occur at any time during the process of multistage carcinogenesis: preventing the initial mutation; blocking promotion to premalignant tumors; stopping progression from the premalignant state to in situ carcinomas; or preventing invasion or metastasis. Another area of concern is dening the relevant time frame over which to measure diet. For cancer, it is generally hypothesized that the effect of diet may occur many years before diagnosis; thus, the ability to recall diet in the remote past is of considerable interest. An accurate measure of intake, but which covers an irrelevant time frame, is not valuable. This is the main limitation that is pointed out when interpreting the results of clinical trials with dietary interventions involving short periods. It is currently well accepted that the need for cohort studies involving a considerable number of cases and using quantitative dietary assessment, biomarkers of food intake, and genetic measures is necessary to gain further understanding of the etiologic roles of dietary factors in the causation of cancer before making specic dietary recommendations (185). The European Prospective Investigation into Cancer and Nutrition (EPIC) is one study that was designed with these considerations in mind. As indicated in the description of this study (151), the EPIC is a multicenter prospective cohort study designed to investigate the relationship between nutrition and cancer; it currently includes 519,978 participants in 23 centers in 10 European countries. This cohort will be followed for cancer incidence and causespecic mortality for several decades. EPIC represents the largest single resource available today for prospective investigations that can integrate data on lifestyle and diet, biomarkers of diet and of endogenous metabolism, and genetic polymorphisms to investigate gene-diet interactions. In September 2002, in the conference entitled Nutritional Genomics and Proteomics in Cancer Prevention, some general directions of future investigations in the nutritional genomics of cancer were proposed. Milner (113) reiterated that the major research needs identied by the panel participants were: (a) Identication and validation of biomarkers for cancer;
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(b) Investigation of the exposure/temporal relationship between nutrients (bioactive food components) and cancer prevention; (c) Examination of possible tissue specicity in response to food components; (d) Denition of interactions among food components as determinants of response; (e) Mechanisms of action (targets) of food components. More information about the outcomes of the conference is available online (http://www3.cancer.gov/prevention/ngpcp2002/index.html). The results of such investigations will be extremely useful in the further investigation of gene-nutrient interactions at the population level. Considering the above limitations, it is clear that much remains to be learned about diet and cancer, in particular about gene-diet interactions, and that the level of evidence provided by the few studies in the area are not adequate to advise dietary modications. However, during the past decade some pioneer studies revealed interesting and promising gene-nutrient interactions that are guiding current investigations. Some examples of the most relevant gene-nutrient interactions in cancer research are presented below.
Interaction Between the Methylenetetrahydrofolate Reductase (MTHFR) Gene and Folate Intake in Cancer Risk
Methylenetetrahydrofolate reductase (MTHFR) catalyzes the reduction of 5,10methylenetetrahydrofolate to 5-methyltetrathydrofolate, the predominant circulatory form of folate and methyl donor for the remethylation of homocysteine to methionine. Kang et al. (77) identied a thermolabile form of MTHFR that is associated with a reduced enzyme activity and with elevated levels of plasma total homocysteine. The MTHFR gene is located on chromosome 1 at 1p36.3, and the molecular basis of the thermolabile variant is a cytosine (C) to a thymine (T) substitution at nucleotide 677 (C677 > T), which converts an alanine residue to a valine (43). This mutation is relatively common and results in an enzyme that has decreased stability and specic activity. Homozygosity (TT) for this polymorphism is associated with hyperhomocysteinemia and higher cardiovascular risk, particularly among those with low folate intake (82, 101). Paradoxically, the T allele is associated with a lower risk of cancer (21, 100). Folate may reduce carcinogenesis through various mechanisms, including maintenance of normal DNA synthesis and DNA methylation. 5-methyltetrathydrofolate is essential for DNA methylation, and both hypo- and hypermethylation of DNA, which can cause either over- or underexpression of genes, respectively, may contribute to carcinogenesis (reviewed in 47). Reduced activity of MTHFR in T carriers may increase the likelihood of sufcient methylation of deoxyuridine monophosphate (dUMP) to deoxythymidine monophosphate (dTMP), resulting in less incorporation of uracil into DNA, fewer DNA breaks, and a decreased risk for cancer. However, the lower risk of carriers of the T allele was only noted when folate intake was normal. This gene-diet interaction was specially found for colorectal adenomas (an intermediate end point of colon cancer) and colon cancer risk. Slattery et al. (168) found that colon cancer risk was reduced 30% to 40% in TT individuals who consumed
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adequate dietary intakes of folate, vitamin B12, and vitamin B6. The inverse association (a higher risk of cancer) was stronger in individuals over 60 years old with a low nutrient intake. Replicating these results, Ulrich et al. (183) reported that low folate and low intake of vitamins B12 and B6 were associated with an increased risk of colorectal adenomas in individuals with the TT genotype compared with the CC genotype. The increased risk was especially high in the >60 age group. Folate status in individuals who chronically consume moderate amounts of alcohol may be impaired because alcohol causes malabsorption of folate, increased excretion, and abnormal folate metabolism (6). Data from the Physicians Health Study (100) conrmed that individuals with the TT genotype and with a lower intake of folate are especially sensitive to the carcinogenic effect of alcohol in colorectal cancer. This gene-nutrient interaction may have a potential application in tailored prevention, which suggests consuming excess amounts of alcohol may be particularly counterindicated for TT homozygotes in terms of the risk of developing an adenoma or carcinoma, especially if folate intake is low. Corroborating this interaction is the work of Zang et al. (206) in the Nurses Health Study, which shows that alcohol consumption increased the risk for breast cancer in all quintiles of folate intake except in the highest.
Interaction with the NAT Genotypes

In the last decade, there has been increasing evidence that common polymorphisms in genes involved in the metabolism and detoxication of dietary carcinogens may be related to different activities for the corresponding enzymes as well as with cancer risk. This has been observed in the P450 genes for the cytochrome P450 phase I enzymes as well as in genes for the phase II enzymes that detoxify carcinogenic metabolites by producing readily excreted, hydrophilic conjugation molecules (141). N-acetyltransferase (NAT) is a phase II enzyme that is found in two isoforms (NAT1 and NAT2) and may be involved in the acetylation of aromatic and heterocyclic amine carcinogens such as those found in cooked proteins. The NAT2 polymorphism was discovered more than 40 years ago following differences observed in tuberculosis patients and isoniazid toxicity. The classical isoniazid slow acetylator phenotype(s) is due to a reduced NAT2 protein with a frequency that is approximately 30% to 50% in Caucasian populations but has striking geographical differences. This polymorphism is important in clinical pharmacology and toxicology because of its primary role in the activation or deactivation of many drugs (for review, see 60). The NAT1 isoenzyme was initially described as monomorphic; however, subsequent studies conrmed its polymorphism. NAT1 and NAT2 share 87% nucleotide homology in the coding region, yielding 55 amino acid differences. Several polymorphisms have been characterized in NAT1 and NAT2, and a consensus NAT nomenclature was published following the directions of the First International Workshop on the Arylamine N-Acetyltransferases held in Cairns, Australia in 1998 (60), and a Web page (http://www.louisville.edu/medschool/pharmacology/NAT) lists the NAT1
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and NAT2 alleles. Some of these DNA polymorphisms are related to NAT activity that has complex interactions with other environmental factors such as tobacco smoking. Based on polymorphisms in the NAT1 and NAT2 genes, individuals may be classied into one of the three categories: fast, slow, and intermediate rate metabolizers. Several studies relate these categories to different risks for bladder, colon, lung, breast, and prostate cancer, producing very controversial results, because for some cancers, the slow metabolizer phenotype was associated with higher risk, whereas for others the risk was associated with the fast metabolizer genotypes. Attempting to explain the controversy, Hein suggested (61) that for cancers in which N-acetylation is a detoxication step (such as aromatic aminerelated urinary bladder cancer), NAT2 slow acetylator phenotype is at higher risk. In contrast, for cancers in which N-acetylation is negligible and O-acetylation is an activation step (such as for heterocyclic amine-related colon cancer), NAT2 rapid acetylator phenotype is at higher risk. The data for NAT1 is less abundant and equally controversial. NAT1 has been suggested to be of greater importance in breast cancer, because NAT1, but not NAT2, activity is present in mammary cells. Despite the current confusion about the role of NAT polymorphism in cancer risk, one gene-nutrient interaction has been consistently reported in some epidemiological studies. Subjects carrying the rapid NAT2 acetylator allele have a higher risk of colon cancer when consuming larger amounts of meat. This reects the greater ability of rapid acetylators to activate heterocyclic aromatic amines to carcinogenic derivatives within the colon mucosa. This higher risk was reported in the rst case-control studies in Australia (154), as well as in prospective cohort studies in the United States (22). Recently, Huang et al. (70) replicated these results in hepatocellular carcinoma (HCC) in a case-control study. They did not nd signicant associations between the susceptibility of HCC and the overall NAT2 genotypes. However, there was a trend of increased HCC risk in rapid acetylators from low to intermediate, and there was high red meat intake (P = 0.016), even when adjusted for family history of HCC and habitual alcohol drinking. They also specically tested the interaction between red meat intake and the NAT2 acetylator status, obtaining a signicant P value (P = 0.007).
Interaction with the Glutatione-S-Transferases (GSTs) Polymorphisms

Glutatione-S-transferases (GSTs) are a major family of cytosolic enzymes that detoxify reactive electrophiles, and they are very active in the detoxication of many toxins and carcinogens. The GSTs are divided into four major classes: alpha (GSTA), pi (GSTP), mu (GSTM), and theta (GSTT), and within each class, several isoenzymes exist (reviewed in 155). In relation to cancer risk, the most studied GSTs are the genetic polymorphisms of GSTM1, GSTT1, and GSTP1 genes. Thus, it has been hypothesized that GST induction results in overall decreased susceptibility, and that, conversely, impaired detoxication by GST will confer increased cancer risk. For GSTM1 and GSTT1, inherited homozygous deletions
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result in deciencies of these two isoenzymes. Both GSTM1- and GSTT1-null genotypes, either alone or in combination, confer a high risk of several types of cancer, particularly among subgroups exposed to tobacco smoking or to other sources of carcinogens. Dietary modulation of GSTs has been reported in animal studies as well as in some controlled feeding studies in humans (155). Numerous compounds, including those in cruciferous vegetables, increase GSTs in rats. In humans, it is suggested that the cancer-protective effects of these vegetables may depend on the ability to induce high levels of GSTs. Subsequently, the hypothesis of the interaction between the GST genotype and dietary patterns on cancer risk has been addressed by some case-control and cohort studies. In agreement with the animal results, Lin et al. (94) found a statistically signicant interaction (P = 0.01) when testing the hypothesis that the protection of cruciferous vegetable intake on colon adenoma risk depends on GST genotypes. Thus, a protective effect of the intake of cruciferous vegetables occurred only in individuals who were GSTM1null. This single observation requires conrmation, and subsequent studies have revealed additional complexity. Lampe et al. (87) recently outlined the metabolism and targets of cruciferous vegetable constituents, and discussed and summarized relevant human intervention studies relating to the effect of genetic polymorphisms, cruciferous consumption, and cancer risk. These authors highlighted the several layers of complexity that affect the study of gene-diet interactions and cancer risk in humans. They specically focused on the variation in content of bioactive compounds of cruciferous vegetables and the methods used to prepare these foods. In addition to these examples of gene-nutrient interaction on cancer risk, there are many other candidate genes, and only one paper has been published that studied their DNA variation and the modication of their effects by dietary components. A review of some of these state-of-the-art interactions was recently published (98). Two excellent reviews (48, 114) about the effects of new genomic technologies on the simultaneous elucidation of the biological effects of dietary constituents on cell function and global gene expression will be useful for understanding the new research framework of nutritional genomics in cancer research in the postgenomic era.
CONCLUSIONS
Nutritional genomics is a fast-developing research area with tremendous potential to yield results that could change the way dietary guidelines and personal recommendations are established and carried out in the future. The notion is that nutrigenetics will provide the basis for personalized dietary recommendations based on the individuals genetic make up and information from other environmental factors. This will probably require individual ascertainment of all informative SNPs or, as forecast by others, complete sequencing of the genome. Geneticists will use this data to forecast future genetic predisposition for disease, and it will guide the implementation of the proper preventive measures. For several decades,
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a very simplied version of this concept has been implemented in many countries. Through established programs to detect inborn errors of metabolism, millions of babies have been analyzed for the presence of rare monogenic disorders and, based on the results, many of those affected have been spared of the sometimes lethal consequences of their genetic defect. In many cases, the solution was as simple as providing them with the right dietary mix. From a genetics viewpoint, there is still a lot of work to be done, even for these relatively simple diseases,, but there is excellent proof that the concept works. From the conceptual point of view, the situation with multifactorial disorders is more complex. However, the range and complexity are vastly different; the goal of nutrigenetics aims to detect predisposition for all diseases with a genetic component, and to provide the tools for its prevention decades before they could be manifested, instead of detecting and preventing monogenic disorders with very rare prevalence. Whether this is feasible remains to be seen. For now, this knowledge is developed from multiple small intervention studies that provide a body of observational ndings that generally show little consistency. Nutrigenetics needs to move forward with nutrigenomics to translate observational ndings into molecular mechanisms. To achieve these ambitious goals, it will be necessary to move toward strategies that will yield ndings that are more robust, some of which we present below. 1. We need more and better phenotypes. Many observational studies rely on a single measure, and most phenotypes (i.e., lipid levels, blood pressure) experience signicant biological variation daily as well as methodological variation inherent to the instrument or technique used for its measurement. The solution is simple but costly. Intervention and observational studies that attempt to examine gene-diet interactions need to include repeated sampling and measurement to provide an accurate measure of the phenotypes. We need more biomarkers that are informative; some may come from developing research areas such as metabonomics and lipomics. 2. In addition to the phenotypes, there are two obvious key pieces of information to determine when examining gene-diet interactions: the accurate measurement of gene variants and dietary intake. The rst is easy, although historically not enough attention has been placed on quality control issues in genetic research. Assessing dietary intake and/or habits is more complex. We need more complete databases that reect updated nutrient information and local foodstuffs, as well as instruments that faithfully capture long-term dietary habits. This has been a major Achilles heel in nutritional research, especially for observational studies. 3. Most of the effort is devoted to identifying genetic variants in nuclear DNA, but mutations involving mitochondrial DNA may also impact age-related diseases. Another concept captivating the attention of many investigators, but also adding another layer of complexity, is epigenetics, which are the subtle modications to the genome that do not alter its DNA sequence.
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The most well-known modications are DNA methylation and chromatin remodeling, which are modulate gene expression throughout the genome and may be modulated by dietary factors. Therefore, studies involving nutritional genomics should not ignore these potentially important mechanisms of regulating gene expression modulated by nutritional factors. 4. In the past, the cost of genotyping was a severe limitation on performing genetic studies in populations. With the availability of high-throughput techniques, and the lowering cost of genotyping, the limitations have shifted back to accurately phenotyping large population samples. To elucidate geneenvironment interactions, and specically gene-diet interactions, we need population sizes several order of magnitude larger than those currently used for common multifactorial diseases. This will require similar nationwide efforts to those already put in place in Iceland and the United Kingdom, and those in the planning stages in the United States. A better option would be to create international consortiums built along the models of the EPIC study or the Human Genome Project. This does not mean that smaller studies do not have a future in nutritional genomics; they can be tailored to answer specic questions or to generate hypotheses to be examined in more detail in other studies. 5. These consortiums will be able to coordinate cross-cultural/ethnic studies and will be extremely useful in dening gene-environment interactions. The current hypothesis is that the dramatic increase in morbidity and mortality due to CVD and other age-related diseases that the world population has been experiencing during recent years is due in part to the higher frequency of deleterious alleles that predispose certain ethnic groups to be especially sensitive to the inuence of environmental CVD risk factors, such as diet and a sedentary lifestyle. Therefore, elucidating such ethnic-specic genetic markers will be important for efcacious prevention of chronic disorders in countries undergoing Westernization of lifestyles. 6. Observational ndings will need to be followed up on with in vitro or in vivo experiments, which will lead to the molecular mechanisms responsible for the observed interactions. This will fall within the scope of nutrigenomics and will involve in vitro, in vivo, and in silico experimentation. This will be wrapped up within the notion of systems biology or functional genomics. 7. Complex phenotype and genotype interactions require analysis of their combined effects. The current statistical tools are limited in their ability to deal with this complexity. Therefore, developing appropriate statistical tools will be indispensable for analyzing and understanding the effects of variations in multiple genes, in combination with environmental and phenotypic information. The information will need to be incorporated into predictive models that can be used clinically to improve disease assessment and prevention. This will be probably happen within the umbrella of bioinformatics or computational biology.
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In summary, nutritional genomics will be the driving force of future nutritional research, and it has the potential to change dietary disease prevention and therapy and have a major impact on public health. However, the complexity of the goals set for nutritional genomics is tremendous and their accomplishment will require breaking many of the molds of traditional research and seeking integration of multiple disciplines and laboratories working coordinately. Despite the difculties described, preliminary evidence strongly suggests that the concept will work and that by using behavioral tools founded on nutrition, we will be able to harness the information contained in our genomes to achieve successful aging. ACKNOWLEDGMENTS Supported by NIH/NHLBI grant no. HL54776, contracts 53-K06-510 and 58 1950-9001 from the U.S. Department of Agriculture Research Service, and grants CTIDIB/2002/197 from the Ocina de Ciencia y Tecnologa, Generalitat Valen ciana, Spain and G03/140 from the Instituto de Salud Carlos III, Spain.
The Annual Review of Genomics and Human Genetics is online at http://genom.annualreviews.org
LITERATURE CITED
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Annual Review of Genomics and Human Genetics Volume 5, 2004
CONTENTS
GENETIC TESTING IN PRIMARY CARE, Wylie Burke COMPARATIVE GENOMICS, Webb Miller, Kateryna D. Makova, Anton Nekrutenko, and Ross C. Hardison GENETIC SCREENING: CARRIERS AND AFFECTED INDIVIDUALS, Linda L. McCabe and Edward R.B. McCabe NUTRITIONAL GENOMICS, Jose M. Ordovas and Dolores Corella AFRICANS AND ASIANS ABROAD: GENETIC DIVERSITY IN EUROPE, Guido Barbujani and David B. Goldstein FINDING PROSTATE CANCER SUSCEPTIBILITY GENES, Elaine A. Ostrander, Kyriacos Markianos, and Janet L. Stanford MOLECULAR NETWORKS IN MODEL SYSTEMS, Timothy Galitski GENETICS OF ATHEROSCLEROSIS, Aldons J. Lusis, Rebecca Mar, and P ivi Pajukanta a MEDICAL GENETICS IN DEVELOPING COUNTRIES, Arnold Christianson and Bernadette Modell PROTEOMICS, Carmen L. de Hoog and Matthias Mann POPULATION GENETICS, HISTORY, AND HEALTH PATTERNS IN NATIVE AMERICANS, Connie J. Mulligan, Keith Hunley, Suzanne Cole, and Jeffrey C. Long VARIATION IN HUMAN MEIOTIC RECOMBINATION, Audrey Lynn, Terry Ashley, and Terry Hassold COMPARATIVE PRIMATE GENOMICS, Wolfgang Enard and Svante P abo a AUTISM AS A PARADIGMATIC COMPLEX GENETIC DISORDER, Jeremy Veenstra-VanderWeele, Susan L. Christian, and Edwin H. Cook, Jr. MAMMALIAN CIRCADIAN BIOLOGY: ELUCIDATING GENOME-WIDE LEVELS OF TEMPORAL ORGANIZATION, Phillip L. Lowrey and Joseph S. Takahashi PLANT GENOMICS: THE THIRD WAVE, Justin O. Borevitz and Joseph R. Ecker EPIGENETICS AND HUMAN DISEASE, Yong-hui Jiang, Jan Bressler, and Arthur L. Beaudet
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295 317 351 379 407 443 479
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CONTENTS
INDEXES Subject Index Cumulative Index of Contributing Authors, Volumes 15 Cumulative Index of Chapter Titles, Volumes 15 ERRATA An online log of corrections to Annual Review of Genomics and Human Genetics chapters (if any) may be found at http://genom.annualreviews.org/
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Annu. Rev. Genomics Hum. Genet. 2004. 5:71118 doi: 10.1146/annurev.genom.5.061903.180008 Copyright c 2004 by Annual Reviews.

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PAST, PRESENT, AND FUTURE OF NUTRITIONAL GENOMICS

Nutritional genomics dened by several authors Denition Reference (36)

Causality in Nutritional Genomics, Types of Studies, and Errors

Genotyping and Quality Control Measures

GENE-NUTRIENT INTERACTIONS IN HEALTH AND DISEASE

Monogenic Versus Multifactorial Diseases

PLASMA LIPIDS: THE FIRST OF MANY CVD RISK FACTORS

Apolipoprotein A-IV (APOA4)

Apolipoprotein A-I (APOA1)

Hepatic Lipase (LIPC)

Interaction with the NAT Genotypes

Interaction with the Glutatione-S-Transferases (GSTs) Polymorphisms

36. 37. 38.

Annual Review of Genomics and Human Genetics Volume 5, 2004

1 15 57 71 119 151 177 189 219 267

295 317 351 379 407 443 479

511 537 539

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