2005 Annual Report 1.What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? What does it matter? The goals of the Nutrition, Cardiovascular Health and Genomics CRIS are: (a) To identify genetic markers for the assessment of cardiovascular health related to plasma lipoprotein levels, diabetes and obesity and to evaluate the effects of specific gene-gene interactions in common metabolic pathways. (b) To investigate the interactions between genetics and nutrients in the development of cardiovascular disease (CVD), the major age-related disorder affecting life expectancy and quality of life in the United States (US). Emphasis is placed on elucidating mechanisms by which genetic variation interacts with dietary and behavioral factors to regulate the homeostasis of the cardiovascular system. (c) To identify genes newly associated with cardiovascular health and overall longevity and determine their expression response to dietary intervention using animal models of aging. Because the response of the individual to nutrients contains a strong genetic component, our approach aims to uncover sets of genes involved in the dietary response and to describe specific gene-diet interactions. This will be tested, using high throughput genotyping techniques, both in ongoing studies of free-living populations and in intervention studies. Our primary focus is to describe gene-diet interactions affecting/influencing progression of the metabolic syndrome, in particular obesity, often a precursor to CVD and diabetes. Cardiovascular candidate genes, both those previously described in the literature, as well as those we identify through bioinformatics analysis, will be used to examine associations and interactions on various scales. These include genetic variations, disease-related phenotypes and specific nutrients [fatty acids, cholesterol, plant sterols) and behavioral habits (alcohol consumption, smoking, physical (in)activity]. Rigorous statistical analysis will uncover the associations between phenotypes indicative of increased risk of metabolic syndrome and the genes responsible for such. Because CVD and diabetes are traditionally considered diseases of the aged, we will also continue with our investigations to identify genes responsible for healthy aging. The principal approach taken for these studies involves gene expression microarray analysis of fruit fly D. melanogaster populations with a propensity for increased longevity. Candidate aging genes will then be studied in mammalian models as well in human populations. The seriousness of the problem at hand is evident. Cardiovascular diseases (mainly coronary heart disease and stroke) are the leading causes of death in the US, accounting for more than 40 percent of all deaths. About 1 million Americans die of CVD each year, which amounts to approximately two deaths every minute. More than half of all these deaths occur among women. However, the burden of this disease is not fully represented by the mortality figures. About one-fourth of the US population suffers from these diseases and account for 6 million hospitalizations each year. As the population ages the cost of these diseases to health care systems in the US increases. The estimated cost in 2001 - including health care expenditures and lost productivity, was nearly $300 billion. The research program relates to the ARS National Program 107 - Human Nutrition, specifically to component 6: Prevention of Obesity and Disease: Relationship Between Diet, Genetics and Lifestyle. Cardiovascular diseases affect all minorities - some of which, i.e., African Americans and Hispanics, may suffer an even greater burden. Therefore, it is important to understand the specific nutritional needs to prevent disease in general and CVD in particular for all the subgroups represented in the US population. This research impacts on other components of the 107 Program. By addressing the individuality of the nutritional needs based on genetics, this research contributes to the component 4: Nutrient Requirements. By examining the impact of genetic knowledge of the behavioral aspects of the individuals, this work impacts components 3: Nutrition Monitoring, and 7: Health Promoting Intervention Strategies for Targeted Populations. By understanding the molecular mechanisms of nutrients and their interaction with different gene variants, we can contribute to the understanding of component 2: Bioavailability of Nutrients and Food Components. 2.List the milestones (indicators of progress) from your Project Plan. Nutrition and Genomics Laboratory Objective 1 To identify genetic markers for the assessment of cardiovascular health related to plasma lipoprotein levels, diabetes and obesity and to evaluate the effects of specific gene-gene interactions in common metabolic pathways. Objective 2 To investigate the interactions between genetics and nutrients in the development of cardiovascular disease (CVD), the major age-related disorder affecting life expectancy and quality of life in the United States (US). Emphasis is placed on elucidating mechanisms by which genetic variation interacts with dietary and behavioral factors to regulate the homeostasis of the cardiovascular system. Objective 3 To identify genes newly associated with cardiovascular health and overall longevity and determine their expression response to dietary intervention using animal models of aging. Milestones 2005 1. To determine whether obesity modulates the relation between APOE genotype, insulin and glucose. Objective 1. 2. To elucidate the role of APOE genotype on carotid atherosclerosis in men and women: the Framingham Heart Study. Objective 1. 3. To identify the role of genetic variation at the perilipin (PLIN) locus with obesity-related phenotypes in women. Objective 1. 4. To identify novel genomic regions implicated in HDL levels using Genome-wide linkage analyses and candidate gene fine mapping in the Framingham Study. Objective 1. 5. To determine the effect of polyunsaturated fatty acids and the PPARA-L162V polymorphism on plasma triglyceride and apolipoprotein C-III concentrations in the Framingham Heart Study. Objective 2. 6. Genotype 20 candidate genes for CVD in a large postprandial study (GOLDN). Objective 2. 7. Genotype the PLIN gene to investigate the relation between variants at this gene and response to weight loss diets. Objective 2 8. To carry out bioinformatic analyses of the human genome to identify novel targets to analyze gene-gene and gene-diet interactions. Objective 1. 9. To develop a high throughput genotyping method to map quantitative trait loci affecting lifespan and fat content in Drosophila using microarrays. Objective 3 10. To complete starvation experiments using long and short-lived drosophila lines aimed to confirm candidate genes identified from gene expression analysis. Objective 3 11. To identify 13 mutants in Drosophila for 31 candidate genes identified from gene expression analyses, and characterization of the mutants for lifespan with the identification of one mutant with an extension of lifespan of about 20 days (50% increase). Objective 3 2006 1. To analyze the interaction of the APOE and CETP genes as determinants of plasma lipid levels and CVD risk. Objective 1 2. To determine genotype variants at the Adiponectin and Visfatin genes to establish their association with obesity risk and plasma glucose and lipid levels. Objective 1. 3. Finely map the chromosome 6 region implicated in lipid metabolism to identify the specific genes determining the observed phenotypes. Objective 1. 4. Begin further bioinformatic analyses of the human genome to identify novel targets to analyze novel genes for CVD risk. Objective 1. 5. To determine genetic variants at the adiponectin, visfatin and perilipin genes in association with variability of plasma lipids in response to diet both in the fasting and the fed states. Objective 2. 6. To perform the statistical analyses of 10 of the genes genotyped within the GOLDN study to examine variability in postprandial response. Objective 2. 7. To genotype variants at the TR2 family of genes to determine their influence of dietary preferences and CVD risk markers. Objective 2. 8. Identify, confirm, and characterize genes affecting lifespan in Drosophila by creating transgenic flies. Objective 3 9. Complete the mapping of quantitative trait loci affecting lifespan and fat content in Drosophila using microarray methods. Objective 3 2007 1. Genotype novel genes in chromosome 6 associated with plasma lipids levels. Objective 1. 2. Identify novel targets to analyze novel genes for CVD risk by conducting bioinformatic analyses of the human genome. Objective 1. 3. To perform the statistical analyses of 10 of the genes genotyped within the GOLDN study to examine variability in postprandial response. Objective 2. 4. Use metabonomic variables to determine further insights on genotype-phenotype associations in relation with CVD risk. Objective 1. 2008 1. To identify new genes involved with plasma lipid profiles and obesity by analyzing the data from a 500K SNP gene chip analyses within the Framingham population. Objective 1 2. Bioinformatic work to identify appropriate and informative single nucleotide polymorphisms (SNPs) within genes of interest as identified in milestone 1. Objective 1 3. To determine the potential use of genetic markers at multiple loci and environmental variables to predict CVD risk with greater precision than the traditional biochemical-based approaches by using Bayesian models. Objective 1. 4. To determine new genes found to be associated with CVD risk from milestone 2007-3 for potential interaction with dietary factors modulating plasma lipid phenotypes in the fasting state. Objective 2. 5. Complete experiments involving dietary factors and extension of life expectancy in drosophila. Objective 3 6. Initiate dietary intervention studies in humans aimed to improve quality of life in the elderly by enhancing expression of longevity related genes. Objective 3 2009 1. Genotype new SNPs at loci derived from milestones 2008-1 and 2008-2 to search for associations with CVD risk factors. Objective 1 2. To determine gene-diet interactions for novel SNPs associated with CVD resulting from Milestone 2009-1. Objective 2. 3. To determine more complex gene-gene-diet interactions using genes involved in specific metabolic pathways (i.e., PPARA, LPL, APOC3 and PUFA intake). Objective 2. 4. To identify genes affecting lifespan and the rate of aging in mammals and understand how the affected metabolic processes can be regulated by dietary factors. Objective 3. 4a.What was the single most significant accomplishment this past year? Nutrition and Genomics Laboratory A Global Genetic Marker for Obesity in Women: Obesity has been identified “one of today’s most blatantly visible – yet most neglected – public health problems.” This rising epidemic of overweight and obesity has been called by some as “globesity” to clearly reflect that is a global problem and that, unless action is taken, billions will suffer from debilitating conditions associated with this disorder. Overeating and sedentary lifestyle are major environmental factors determining the current globesity; however, genetic factors are also important to predispose some people to obesity, especially in some minority groups. We know very little about the genetic component of obesity. We have examined the gene for perilipin (PLIN), a protein that coats intracellular lipid droplets and modulates adipocyte lipolysis. We have evaluated the association between several polymorphisms at the PLIN gene and obesity-related phenotypes in 1,589 white subjects randomly selected from a general population. In 801 women the less common alleles of two of the polymorphisms were significantly associated with lower body mass index (BMI) as well as lower waist-to-hip ratio, plasma glucose, and triacylglycerol concentrations. No significant associations with these obesity-related phenotypes were found in men. Moreover, women carrying the polymorphism had half the risk of being obese. These seminal findings were investigated in two other populations to assess its replication and the public health relevance of these findings. For the first replication we evaluated the association between the same polymorphisms at the PLIN gene and obesity-related phenotypes in 734 white subjects (373 men and 361 women) attending a residential lifestyle intervention program. Our analysis demonstrated that, again, in women, polymorphisms at this gene were significantly associated with percent body fat and waist circumference and obesity risk. Consistent with our initial data, no significant associations between PLIN variations and obesity were found in men. Finally, we evaluated the extension of our findings to Asian subjects by examining the PLIN gene and obesity-related phenotypes in 4,134 Asian subjects participating in a National Health Survey launched in Singapore. Our analysis demonstrated that, in women, polymorphisms at this gene were significantly associated with BMI in Indians and Malays. No significant associations between PLIN variations and obesity were found in men of any of the races investigated or in Chinese men and women. Our findings show for the first time that common mutations at the PLIN gene modulate body weight in humans and more so in women. The impact of these findings are expected to be highly relevant in view of their unusual consistency, the relevance of the problem and the preliminary data showing that this gene may also predict the success of dietary therapies design to loose weight in obese subjects. These findings have received much interest by the private sector to create kits aimed to identify predisposition to obesity. A patent has been filed on these major accomplishments. 4b.List other significant accomplishments, if any. Nutrition and Genomics Laboratory A Transcription Factor Modulates the Response of Plasma Lipids to Dietary Habits: There is an increased interest to understand the factors that regulate the expression of our genes. Some of them are known nuclear transcription factors and are involved in the expression of genes following their activation by external factors such as nutrients. The gene coding for the transcription factor peroxisome proliferator-activated receptor alpha (PPARA) contains several mutations associated with different cardiovascular risk factors including plasma lipids, obesity and diabetes status. We investigated one of these mutations to ascertain the potential interaction between this gene, plasma lipids and dietary factors in the population enrolled in the Framingham study (1003 men and 1103 women). Our data clearly demonstrate that the effect of a common variant at the PPARA gene known as the L162V polymorphism, is associated with plasma triglyceride concentrations in a manner that is modulated by the intake of polyunsaturated fatty acids, with high intake of these fatty acids triggering lower triglycerides only in carriers of the 162V form of this gene. Our results support the relevance of knowing the genetic make up in order to provide more precise and successful dietary recommendations for disease prevention. Novel Genes Involved in the Metabolism of High-density Lipoproteins (HDL): Lipoproteins are particles carrying cholesterol and fats in the bloodstream. Their atherogenicity may be modulated by their serum levels and these are determined in part by genetic factors. One of the major lipid-related risk factors is determined by the levels of HDL. The levels of these macromolecules in plasma are determined by a host of environmental and genetic factors, the most relevant being diet, smoking, alcohol and physical activity. Although, several genes have been identified and associated with HDL-C levels, they explain a very small percent of the total genetic contribution. Therefore, it is paramount to identify more gene targets to help to understand the metabolism of HDL and define new therapeutic approaches to increase HDL and to decrease CVD risk. In this research, we have searched for new genes for HDL in the whole human genome of participants in the Framingham Heart Study and narrowed down our search to a region of chromosome 6 that may hold additional clues to the metabolism of HDL. We have preliminary data suggesting that several genes within that region may be responsible for the observed variation in plasma HDL levels in the population. These findings could open new therapeutic avenues to our fight against CVD. Moreover, it could be used to differentiate among individuals according to their genetic cardiovascular risk and to target those subjects at higher risk for more aggressive dietary therapy. 4c.List any significant activities that support special target populations. None. 4d.Progress report. None. 5.Describe the major accomplishments over the life of the project, including their predicted or actual impact. Nutrition and Genomics Laboratory This is a relatively new research project. Nevertheless, the findings from this CRIS have been instrumental to establish the concept of Nutrigenetics for CVD prevention on a more solid basis. The National Institutes of Health released a Request for Applications (RFA) program to study gene-diet interactions using as example the major accomplishments derived from this CRIS. Moreover, personal care, food and biotechnology companies are setting up research programs based largely on the findings and concepts developed by this CRIS. The current recommendations to lower cardiovascular risk are based on therapeutic lifestyle changes, including diet modification, smoking and physical activity. However, the benefits of these universal recommendations fall short of the expectations for a significant proportion of the US population. Scientists in this CRIS, in collaboration with scientists at the Framingham Heart Study conducted several studies to determine whether levels of the protective lipoprotein circulating in blood, namely, HDL-C are determined by the interaction between genes and diet. One of the major goals is to achieve reductions in blood low-density lipoproteins cholesterol (LDL-C) levels. However, in addition to LDL-C, the levels of HDL-C are a major risk factor for CVDs worldwide. Circulating HDL-C levels are regulated by sex hormones, genetics and other behavioral factors including dietary habits and physical activity. Although we know that the use of polyunsaturated fatty acids (PUFA) decreases LDL-C, some scientists have warned about the use of those fats present in vegetable oils such as corn and soybean oils because they also decrease the levels of the protective HDL. However, many intervention studies have shown a dramatic range in HDL-C response to the consumption of PUFA. Scientists in this CRIS, in collaboration with the Framingham Heart Study clearly demonstrated that the levels of HDL-C are determined by the interaction between genes and diet. Framingham Heart Study subjects with a particular mutation in the gene for apolipoprotein A-I (about 20% of the US population) who consumed more polyunsaturated forms of fat had higher HDL-C levels - which are protective against heart disease - than those with the same gene who ate less polyunsaturated fat. Those subjects who did not have this gene mutation had lower HDL-C levels as their intake of polyunsaturated fat increased. A related study focused on a polymorphism present in one of the genes, (LIPC) involved on the removal of excess cholesterol from the body by regulating the protective HDL fraction. This polymorphism is common in Caucasians and even more common in African Americans, Hispanics and other minorities. Our data shows that subjects carrying the CC form of the gene “react” to higher contents of fat in their diets by increasing the concentrations of HDL-C, which could be interpreted as a “defense mechanism” against atherosclerosis and subsequently CVD. Conversely, subjects carrying the TT form of the gene are not able to “compensate” for the nutritional stress and they experience decreases on the HDL-C levels. These data contribute to the identification of a segment of the population especially susceptible to diet-induced atherosclerosis. Considering the higher frequency of the TT form of the gene among African Americans and Hispanics, our results provide crucial information about the impaired ability of these minorities to adapt to new dietary environments. These findings underscore the importance of dietary recommendations tailored to specific population groups or to individuals based on their genetic makeup and provides strength to the future feasibility of Nutrigenetics. The new accomplishments of this CRIS described in sections 4A and 4B have the potential to advance the field of the genetics of obesity and its prevention and treatment (4A). In addition, the potential discovery of new pathways involved in the metabolism of HDL (4B) may provide new knowledge to increase the protective HDL in blood and to prevent CVD. These new findings are already showing their potential impact in the scientific and commercial worlds. This research is related to ARS National Program 107 - Human Nutrition, program component 6: Prevention of Obesity and Disease: Relationship Between Diet, Genetics and Lifestyle. and to Performance Measure 4.1.2 Improve Human Health by Better Understanding the Nutrient Requirements of Individuals and the Nutritional Values of Foods. 6.What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end-user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products? Nutrition and Genomics Laboratory Our research findings are communicated by publication and/or public presentations to other scientists, to industry and to the general public. Based on our discoveries, scientists are more aware of the need to include dietary habits in the analyses involving associations between genes and disease. Scientific panels involved in dietary recommendations and guidelines are introducing the concept of more individualization in the guidelines and recommendations based on ethnic groups and disease status in the present time and genotypes in the future. Industry is very interested in this progress, as it will provide with ways to screen individuals for disease risk and for the most appropriate therapy. This approach has already been commercialized by some. For this reason, two patents have filed through Tufts University to protect the new technologies developed within this CRIS. However, it is our belief that given the complexity of CVD and the multiple biological interactions, this technology will most likely not have broader public application until 2007-2008. 7.List your most important publications in the popular press and presentations to organizations and articles written about your work. (NOTE: List your peer reviewed publications below). Nutrition and Genomics Laboratory Selected Media Clips - October 2004-July 2005 Diet and Genes It isn't just what you eat that can kill you, and it isn't just your DNA that can save you—it's how they interact By Anne Underwood and Jerry Adler Newsweek January 17, 2005 Dieting for the Genome Generation Nutrigenomics has yet to prove its worth. So why is it selling? By Leslie A Pray The Scientist, Volume 19 (1) 14, January 17, 2005 Dieta e genética By Christiane Segatto Epoca (Brazilian magazine) February 2005 Diet and Genes It isn't just what you eat that can kill you, and it isn't just your DNA that can save you—it's how they interact By Anne Underwood and Jerry Adler Newsweek International Edition, February 7, 2005 Darwin's Revenge Why are we getting fat? Because our genes date from the last Ice Age. By Fred Guterl Newsweek International - February 7, 2005 Tu (Italian magazine) February 7, 2005 Jose Ordovas, direttore del Nutrition and Genomics Laboratory della Tufts University. Looking for clues in the double helix Scientists say DNA could tell us what to eat, how to live By Lisa Marshall The Daily Camera, February 7, 2005 GeneAlert Gene Influences Weight in Women By Alex Cukan UPI, February 11, 2005 Researchers link gene, obesity UPI, February 11, 2005 The Diet That Fits Analyzing metabolism for personalized nutrition By Gunjan Sinha Scientific American, February 28, 2005 Can genetic tests aid in nutrition? The Wall Street Journal March 1, 2005 Labs Turn DNA Into Personal Health Forecasts By Ariana Eunjung Cha Washington Post April 7, 2005 Los Angeles Times April 11, 2005 Are the clues to diet success in your genes? With 'nutrigenomics,' eating plans are based on DNA. Some experts question the advice. By Hilary E. MacGregor Designer Diets By Gunjan Sinha Nature Medicine July 2005 Presentations Dr. Ordovas has been invited as keynote speaker to talk about the work carried by this CRIS to many international and national conferences sponsored by international and national scientific societies, universities and by the food and pharmaceutical industries. A selection of these events are listed below: Central American Atherosclerosis Society (Panama City, Panama) Santorini Biologie Prospective Conference 2004 From Human Genetic Variations to Prediction of Risks and Responses to the Environment Santorini, Greece First International Nutrition Symposium: Building the scientific foundation to personalized nutrition, health and wellness. Lausanne, Switzerland Fifth International Phytochemical Conference. California State Polytechnic University, Pomona, CA. International Conference on the Healthy Effect of Virgin Olive oil. Jaen, Spain Bruce Ames International Symposium on Nutritional Genomics. Davis CA Living Well to 100. Tufts University (Organizer) Wageningen Centre for Food Sciences. Food Summit. The Netherlands International Olive Oil Council International Nutrigenomics Symposium, Yonsei University, Seoul, Korea International Nutrigenetics Conference, Istambul, Korea The 65th Annual Mary Swartz Rose Memorial Lecture in New York - "Obesity: Nurture or Nature" Review Publications Corella, D., Ordovas, J.M. 2005. Single nucleotide polymorphisms that influence lipid metabolism: interaction with dietary factors. Annual Review of Nutrition. Available online 5/2/2005 at http://arjournals.annualreviews.org/doi/abs/10.1146/annurev.nutr.25.050304 Debusk, R.M., Fogarty, C.P., Ordovas, J.M., Kornman, K.S. 2005. Nutritional genomics in practice: Where do we begin? Journal of American Dietetic Association. 105(4):589-598. Qi, L., Tai, E.S., Tan, C.E., Shen, H., Chew, S.K., Greenberg, A.S., Corella, D., Ordovas, J.M. 2005. Intragenic linkage disequilibrium structure of the human perilipin gene (PLIN) and haplotype associated with increased obesity risk in a multi-ethnic Asian population. Journal of Molecular Medicine. Available at http://springerlink.com (E-Pub) DOI: 10.1007/s00109-004-0630-4 Tai, E.S., Corella, D., Demissie, S., Cupples, L.A., Coltell, O., Schaefer, E., Tucker, K., Ordovas, J.M. 2005. Polyunsaturated fatty acids interact with the PPARA-L162V polymorphism to affect plasma triglyceride and apolipoprotein C-III concentrations in the Framingham Heart Study. Journal of Nutrition. 135:397-403. Larrabee, P.B., Johnson, K.L., Lai, C., Ordovas, J.M., Cowan, J.W., Tantravahi, U., Bianchi, D.W. 2005. Global gene expression analysis of the living human fetus using cell-free messenger RNA in amniotic fluid. Journal of the American Medical Association. 16;293(7):836-42. Ordovas, J.M., Corella, D. 2004. Nutritional genomics. Annual Review of Genomics and Human Genetics. 5:71-118. Qi, L., Corella, D., Sorli, J.V., Portoles, O., Shen, H., Coltell, O., Godoy, D., Greenberg, A.S., Ordovas, J.M. 2004. Genetic variation at the perilipin (PLIN) locus is associated with obesity-related phenotypes in White women. Clinical Genetics. 66(4):299-310. Elosua, R., Ordovas, J.M., Cupples, A.L., Fox, C.S., Polak, J.F., Wolf, P.A., D'Agostino, R.A., O'Donnell, C.J. 2004. Association of APOE genotype with carotid atherosclerosis in men and women: the Framingham Heart Study. Journal of Lipid Research. 45:1868-1875 Tsai, M.Y., Georgopoulos, A., Otvos, J.D., Ordovas, J.M., Hanson, N.Q., Peacock, J.M., Arnett, D.K. 2004. Comparison of ultracentrifugation and nuclear magnetic resonace spectroscopy in the quantification of triglyceride-rich lipoproteins after an oral fat load. Clinical Chemistry. 50:1201-1204. Ganon, A., Corella, D., Guillen, M., Ordovas, J.M., Pocovi, M., 2004. Frequencies of apolipoprotein A-IV gene polymorphisms and association with serum lipid concentrations in two healthy Spanish populations. Human Biology. 76(2):253-266. Corella, D., Ordovas, J.M. 2004. The metabolic syndrome: a crossroad for genotype-phenotype associations in atherosclerosis. Current Atherosclerosis Reports. 6(3):186-196. Elosua, R., Demissie, S., Cupples, L.A., Meigs, J.B., Wilson, P.W., Schaefer, E.J., Corella, D., Ordovas, J.M. 2003. Obesity modulates the association between apolipoprotein E genotype and fasting insulinemia and glucose in men. The Framingham Offspring Study. Obesity Research. 11(12):1502-1508. Yang, Q., Lai, C., Parnell, L.D., Cupples, L., Adiconis, X., Zhu, Y., Wilson, P.W., Housman, D.E., Sherman, A.M., D'Agostino, R.B., Ordovas, J.M. 2005. Genome-wide linkage analyses and candidate gene fine mapping for HDL3 cholesterol: the Framingham Study. Journal of Lipid Research. 46(7):1416-1425.