Ronald M. Krauss - US grants

The major goal of this project is to determine the extent to which changes in plasma levels of specific lipoproteins, lipoprotein subspecies, and apolipoproteins are associated with quantitative changes in coronary atherosclerosis during the course of a controlled three year coronary risk factor intervention program. A second aim is to identify those lipoprotein variables that are related at baseline to extent of coronary atherosclerosis before initiation of treatment. Lipoprotein analyses will be carried out in 300 patients admitted to Stanford University Medical Center for management of coronary artery disease (coronary artery bypass surgery, transluminal percutaneous coronary angioplasty, or medical treatment). Initial lipoprotein analyses will be performed three weeks after surgery and after medical stabilization (1-3 weeks) in other patients. Subfractions of plasma very low density, low density, and high density lipoproteins (VLDL, LDL, HDL) will be measured by analytic ultracentrifugation with a computer-based quantitation procedure. LDL and HDL subspecies will also be measured by high-resolution polyacrylamide gradient gel electrophoresis. Apolipoproteins AI, AII and B will be measured by enzyme-linked immunosorption assay. Coronary angiography will be performed and computerized quantitation of non-bypassed or non-dilated coronary artery segments will be carried out as part of an existing protocol at Stanford University before initial treatment and at three years following medical management (all subjects) and intensive multiple risk factor intervention (one-half of the subjects, randomly assigned). The special intervention program at Stanford includes improved nutrition, weight reduction, increased aerobic exercise, stress management, and individualized treatment including elimination of cigarette use, reduction of hypertension, and LDL reduction. Lipoprotein studies will be repeated after one, two and three years of follow-up in all subjects. Analyses of lipid and lipoprotein measurements in relation to changes in coronary atherosclerosis will make it possible to identify those lipoprotein parameters most closely involved in this disease process. The results will also indicate whether detailed measurements of specific lipoprotein subclasses and apolipoproteins are more informative than conventional lipid measurements in predicting the extent of coronary artery disease, and in assessing the effects of strategies directed at its prevention and treatment.

This research program continues to focus on serum lipoproteins considered as biochemical entities of significance in the etiology of atherosclerosis, lipid disorders and other diseases. Understanding such relationships requires the elucidation of the chemical, physical, and functional characteristics of the lipoproteins. Our research therefore emphasizes detailed characterization, as well as quantification, of serum lipoproteins in terms of classes and subclasses, distributions, interrelationships and origins. Our investigations of the lipoproteins will address a variety of problems and employ a broad scope of experimental techniques and skills. Among the principal tools that we expect to develop and apply to fundamental biophysical and biomedical problems are: analytic ultracentrifugation, electrophoresis, electron microscopy, chromatography, enzymatic methods and computer technology. A new apolipoprotein core unit will be directed toward determination of specific apolipoproteins. Lipoprotein structure and function will be examined in native and partially degraded particles and in physical or enzymatically reassembled model structures. Transformation in lipoprotein distributions will be studied in vitro as well as in vivo; the latter as a function of diet, drugs and disease. Human cord blood will be analyzed for information and insights into the genesis of lipoproteins. A major methodologic effort will be concerned with detection and minimization of potential artifacts and degradation arising from lipoprotein isolation and analysis. The unique methodologic resources of this Program Project will be available for outside collaborations on specialized or unique biomedical problems. The interactive design of developing methodology with application to basic and clinical research problems remains a major feature of our Program Project.

The principal aim of this program project is to delineate the processes that give rise to the multiple subclasses of human plasma lipoproteins, thereby to better understand the influence of genetic and environmental factors on plasma lipid transport and the development of atherosclerosis. A wide range of scientific disciplines and experimental approaches will be used, including human and molecular genetics, metabolism, cell biology, biochemistry, biophysics, mathematics and statistics. The program is organized into five highly interactive Projects and three Core units which provide support services used by each of the projects. Project 1 is aimed at determining the influence of genetic and metabolic factors on the distribution and properties of human low density lipoprotein (LDL) subclasses and to refine genetic models for the heritability of lipoprotein subclass phenotypes. The structural, metabolic, and genetic implications of recently demonstrated differences in glycosylation of apolipoprotein (apo) B across the LDL particle spectrum will be investigated in detail. In Project 2, model lipoprotein systems will be used to define the physical-chemical and metabolic bases for speciation, remodeling and function of nascent and plasma apo-specific high density lipoprotein (HDL) subpopulations. A major focus will be on the influence of triglyceride- rich lipoprotein metabolism on the formation and properties of HDL subclasses, and on their function in intravascular and extravascular cholesterol transport. Project 3 further examines the critical link between triglyceride-rich lipoproteins and the formation and metabolism of LDL and HDL subclasses by investigating the role of lipolysis products in mediating physical interactions and transfers among lipoprotein particles. Specific processes to be studied include the direct removal of surface lipids from remnants by HDL proteins, the binding of LDL to remnants, and the roles of these processes in forming HDL and LDL subclasses and in influencing remnant metabolism. In Project 4, the in vivo origins of HDL subclasses, their structural and regulatory determinants, and their influence on lipoprotein metabolism and atherosclerosis will be investigated in transgenic mice carrying the human genes for apoAI and apoAII, the major HDL protein constituents. Cell culture systems will be employed in Project 5 to delineate the cellular origins of apo-specific HDL subpopulations and to study the influence of apoAI and apoAII gene expression on this process. Hepatic cell cultures will also be used to further investigate the relationship of varying apoB glycosylation with LDL subclass formation and metabolism. The Lipoprotein Analysis Core will provide standardized measurements of lipids, lipoproteins, and apolipoproteins for all Projects, and will also serve as a resource for specialized analytic and preparative procedures. The Computation and Statistics Core will support data acquisition, analysis and storage, while the Administrative Core will oversee and coordinate all scientific and support activities, including the clinic. In summary, this Program Project provides an integrated, multidisciplinary approach to the understanding of mechanisms influencing the properties, functions, interrelationships, and clinical significance of lipoprotein subclasses.

The overall aim of this project is to use transgenic and gene- inactivated mouse models to determine metabolic pathways leading to the formation of the multiple forms of LDL and IDL found in human plasma. A particular focus is the delineation of the steps involved in the production of small, dense LDL particles. In humans, a predominance of small, dense LDL is associated with changes in triglyceride-rich lipoprotein metabolism, and with increased risk of atherosclerosis. Preliminary evidence from stable isotope kinetic studies in subjects with the small, dense LDL phenotype indicates an increase in rate of production and a reduction in rate of catabolism of large VLDL subspecies (Sf 60-400). These VLDL enter a metabolic cascade that gives rise to smaller VLDL remnant particles and intermediate density lipoproteins (IDL). We hypothesize that a subset of particles in this cascade can be further processed and remodeled to give rise to small, dense LDL. We further hypothesize a critical role for hepatic lipase in this process based on studies in humans, and preliminary evidence from hepatic lipase transgenic rabbits and hepatic lipase knockout mice. These studies will utilize a human apoB transgenic mouse strain that we have shown, in collaboration with Project 0008, to express high levels of multiple human-like LDL subclasses of high fat diets. Lipoproteins isolated from plasma an desolated perfused livers from these mice, as well s from mice with targeted inactivation of the LDL receptor, will be characterized and tested in vivo as potential metabolic precursors of individual ILD and LDL subspecies. Further genetic manipulations will allow testing the role of hepatic lipase and other regulatory proteins, including apoCIII and cholesteryl ester transfer protein, in modulating the distribution of LDL subclasses in vivo, and in particular, in mediating the formation of increased levels of IDL and small, dense LDL that may be of particular importance with regard to atherosclerosis risk. Once the key metabolic determinants of these species have been identified, future studies can be directed at testing their role in generating the atherogenic small, dense LDL phenotype in humans, and at evaluating the influence of other genetic and and environmental influences on its pathologic manifestations. This information, in turn, can be used to develop improved means of diagnosis and management of individuals who are at risk for atherosclerosis as a result of predisposition to this common trait.

The overall goal of this Program Project is to investigate determinants of atherogenic and antiatheorgenic properties of specific subpopulations of plasma lipoproteins by manipulating genes affecting lipoprotein structure, metabolism, and function in animal models. The Program Project comprises a highly interactive, multidisciplinary group of investigators and collaborators with strengths in lipoprotein biochemistry and metabolism, cell biology, lipoprotein oxidation, molecular genetics, transgenic and gene knockout techniques, and statistics. The specific aims of this Program Project proposal are organized into three complementary projects relating to the 0006 central theme. A continuing goal of Project 0006 has been to determine the origins and metabolic behavior of apoB-containing lipoprotein subclasses, particularly those associated with a human atherogenic phenotype characterized by a predominance of small, dense LDL. This goal will be addressed by testing the effects of specific genetically-induced metabolic alterations in strains of mice expressing high levels of a spectrum of LDL subclasses with properties similar to those found in humans. The studies in Project 0008 extend initial observations regarding atherogenic properties of apo(a) in transgenic mouse models by using genetic techniques to investigate specific structural determinants of this athergenicity, and related effects on targets in the artery wall. In Project 0004, the focus has been on factors responsible for HDL assembly and for the role of HDL proteins in cellular lipid removal. In the present proposal, emphasis will be shifted to the use of cellular systems and transgenic mouse models to assess the biogenesis and antiatherogenic function of HDL subpopulations transporting antioxidant enzymes. The Lipoprotein Analysis Core will provide a range of lipid, lipoprotein, and apolipoprotein measurements and contribute immunochemical expertise to each of the Projects. A second Core unit will create transgenic mice and carry out quantitative measurements of atherosclerosis. In summary, the completion of the proposed aims will advance our understanding of metabolic and structural features of specific lipoprotein subpopulations that are involved in promoting and retarding atherogenesis. This information, in turn, can have considerable impact on the ability to identify atherosclerosis susceptibility in humans, and to devise approaches that can augment the benefits of cholesterol-lowering therapy in the prevention and management of coronary artery disease.

Disorders of the cardiovascular (CV) system are frequently due to temporal or quantitative changes in the expression of a large, but finite set of genes. Noncoding cis regulatory sequences play a central role in controlling gene expression and inter-species (i.e., human/mouse) genomic sequence comparisons serve as a rapid and accurate means for identifying such noncoding regulatory elements. The central goal of this PGA will be to use a comparative genomic approach first to identify, and them to determine the function of elements regulating the expression of genes affecting the CV system. The activities of this PGA are not centered on the discovery of new genes, but rather upon using comparative genomics to understand the role of cis regulating elements in the expression of genes already being studied by CV researchers. In this integrated program to "genomically" explore the regulation of CV genes, 200 human genomic intervals (=~200 BACs), each containing a CV gene(s), will be comparatively characterized. The components of this program will include: (1) The acquisition of orthologous human/mouse and other mammalian genomic sequence for a set of prioritized CV genes. Sequences will either be accessed from publicly funded databases or generated by the sequencing component of this PGA. (2) The creation of a cardiovascular comparative genomic database that will contain extensively annotated human and mouse sequences including the localization of conserved noncoding elements in proximity to well studied CV genes. (3) Genome-wide expression profiling to discover genes co-regulated with CV genes and identify shared noncoding regulatory elements, through intra-species analysis. (4) The identification of SNPs within conserved non- coding sequences, and analysis of their effect on CV gene expression in humans. (5) Analysis in genetically engineered mice of a prioritized set of the conserved noncoding elements for their role in CV gene expression. (6) The establishment of an educational program for cardiovascular researchers in the use of genomic databases and tools.

DESCRIPTION (provided by applicant): The goal of this Pharmacogenetics Research Group is to identify common gene variants that contribute to interindividual differences in response to drugs used to reduce risk for cardiovascular disease (CVD). The Group comprises a multidisciplinary team of investigators with expertise in lipoprotein metabolism and blood pressure regulation; genomics and related computational methodology; clinical trials; human genetics and genetic epidemiology; transgenic mouse models, and database management and biostatistics. For the present study, the drugs chosen are atorvastatin, an HMG CoA reductase inhibitor that lowers plasma lipid levels, and ramipril, an ACE inhibitor that lowers blood pressure. Candidate genes are those with products in metabolic pathways that are potential targets of these drugs. DNA sequence variations in 50 genes will be determined in 24 Caucasians and 24 African-Americans, two ethnic groups with differing degrees of sequence diversity. Based on patterns of single nucleotide polymorphisms (SNPs), haplotypes will be constructed for each gene in both ethnic groups, and groups of SNP genotypes will be identified that are characteristic for the 2-4 most common haplotypes in each gene, present in at least 10 percent of the population. Haplotypes for genes related to atorvastatin effects (27 genes) will be determined in 600 individuals (300 from each ethnic group) who will receive 10 mg/day of this drug for 8 weeks. Haplotypes for genes related to ramipril effects (23 genes) will be determined in 600 individuals (300 from each ethnic group) who will receive 10 mg/day of this drug for 12 weeks. Detailed measurements of phenotypes related to lipoprotein and blood pressure regulation will be performed in the two respective cohorts, and associations will be sought with each of the respective candidate haplotypes. Functional effects of specific sequence variants will be tested in appropriate transgenic mouse models. Future studies will corroborate positive findings using samples from large ongoing clinical endpoint trials of atorvastatin and ACE inhibitor therapy. Data will be transmitted to the Pharmacogenetics Knowledge Base and other genomic databases. The findings will advance our fundamental understanding of the roles of specific genes and their variants in modulating biologic pathways of importance in the pathogenesis and management of CVD.

DESCRIPTION (provided by applicant): Overall Objectives of the Lipids and Chronic Diseases Research Core: High intakes of lipids and calories are associated with increased incidence and severity of chronic diseases including diabetes, obesity, certain cancers, and cardiovascular diseases (CVD). As a consequence of high intakes of fat, lipid metabolism is altered and contributes to the initiation and development of disease processes. The disease process in turn alters metabolism further contributing to additional complications. One of the most prevalent risk factors and targets of therapy for these diseases is dyslipidemias, which are known to be affected by diet. Minority populations have disproportionately high incidences and morbidities of all chronic diseases and specifically prostate cancer (Powell and Meyskens 2001), diabetes (Kamel, Rodriguez-Saldana et al. 1999), asthma (Strunk, Ford et al. 2002), obesity (including childhood obesity (Crawford, Story et al. 2001), and cardiovascular diseases (CVD). Observations from the largest survey of dietary habits and health status in the U.S., the Third National Health and Nutrition Examination Survey (NHANES), have established that older black and Mexican American women and black men were at greatest risk for CVD (Winkleby, Kraemer et al. 1998; Sundquist, Winkleby et al. 2001), paralleling the heightened risk of CVD among younger ethnic minority populations (Winkleby, Robinson et al. 1999). Multiple CVD risk factors, including plasma lipids as well as dietary fat, obesity, hypertension, and diabetes, contribute to these ethnic differences.

1-Naphthaleneheptanoic acid, 1,2,6,7,8,8a-hexahydro-beta,delta,6-trihydroxy-2-methyl-8-(2-methyl-1-oxobutoxy)-, (1S-(1alpha(betaS*,deltaS*),2alpha,6alpha,8beta(R*),8aalpha))-; 24-methylcholesterol; 3-Hydroxy-3-methylglutaryl CoA Reductase; 5 alpha-cholest-7-en-3 beta-ol; 6-Heptenoic acid, 7-(4-(4-fluorophenyl)-6-(1-methylethyl)-2-(ethyl(methylsulfonyl)amino)-5-pyrimidinyl)-3,5-dihydroxy-, (3R,5S,6E); AMAC-1; AMAC1; Address; African American; Afro American; Afroamerican; Age; Aged 65 and Over; Allele Frequency; American; Analgesic Management; Apo-B; ApoB; Apolipoproteins B; Apoplexy; Apoproteins; Arts; Atheroscleroses; Atherosclerosis; Atherosclerotic Cardiovascular Disease; Biologic Marker; Biological Markers; Black Populations; Black or African American; Blood Plasma; Body Tissues; Bone; Bone and Bones; Bones and Bone Tissue; C-reactive protein; CCL18; CCL18 gene; CKb7; Candidate Disease Gene; Candidate Gene; Cardiovascular; Cardiovascular Body System; Cardiovascular Diseases; Cardiovascular system; 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Research Projects; Research Projects, R-Series; Researchers; Resequencing; Reticuloendothelial System, Serum, Plasma; Risk; Risk Factors; Risk Reduction; SCYA18; SNP; SNPs; Sampling; Science of Medicine; Serum, Plasma; Sham Treatment; Signature Molecule; Simvastatin; Single Nucleotide Polymorphism; Standards; Standards of Weights and Measures; Sterols; Stratification; Stroke; Study Subject; Study, Interdisciplinary; Sum; Susceptibility; Synvinolin; Testing; Therapeutic; Tissues; Toxic effect; Toxicities; Triacylglycerol; Triglycerides; Urinary System, Kidney; Variant; Variation; Variation (Genetics); Vascular Accident, Brain; Vascular, Heart; advanced age; allelic frequency; allelic variant; alpha-Lipoprotein Cholesterol; alpha-Lipoproteins; association test; atheromatosis; atherosclerotic vascular disease; atorvastatin; base; beta-Lipoprotein Cholesterol; beta-Lipoproteins; biomarker; black American; bone; brain attack; campesterol; cardiac pharmacology; cardiology pharmacology; cardiovascular disease risk; 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white race; whole genome association studies; whole genome association study

DESCRIPTION (provided by applicant): This proposal stems from a long-standing NHLBI-funded post-doctoral training program based at the Lawrence Berkeley National Laboratory that has evolved over time to maintain focus on the most cutting edge topics in cardiovascular research, and has trained many scientists with a wide range of backgrounds who have been actively recruited by academia as well as industry. The leadership and management of this multidisciplinary program will move to Children's Hospital Oakland Research Institute, and it will retain some of the original faculty from the Lawrence Berkeley National Laboratory. A major emphasis of the training is the use of genomic technologies to investigate mechanisms involved in atherosclerotic cardiovascular disease. The eight program mentors each run successful and well-funded laboratories, and bring varied and complementary expertise in cellular, molecular, biophysical, computational, genetic, and genomic aspects of lipoprotein metabolism, atherogenesis and cardiovascular biology. We offer training in state-of-the art areas such as manipulation the genomes of mice, next-generation sequencing, computational analysis of sequence data, analysis of genetic association, computational analysis of human sequence variation to predict its potential role in disease, gene expression profiling, and epigenetics, as well as biophysical studies of lipoprotein structure. In addition, there is strong emphasis on genomic approaches to study gene regulation with a focus on the cardiovascular system and on resequencing and genotyping to identify human sequence variation responsible for cardiovascular disorders. Our location within a major research institution and in close proximity to the University of California, Berkeley creates a rich training environment with potential for numerous collaborations and training opportunities beyond the scope of this grant. The cross- disciplinary training that the trainees receive will provide them with the skills to develop independent research programs that exploit the experimental and computational approaches required for modern biomedical research. RELEVANCE (See instructions): This proposal supports the training of the next generation of scientist who will understand the genetic and biochemical basis of cardiovascular disease.

DESCRIPTION (provided by applicant): Statins are widely used to reduce cardiovascular disease risk by lowering LDL cholesterol (C), but the magnitude of this effect varies widely among individuals. The overall objective of this proposal is to provide evidence in support of a novel pharmacogenetic mechanism contributing to this variation that involves alternative splicing of the mRNA for HMG-CoA reductase (HMGCR), the rate-limiting enzyme in cholesterol biosynthesis that is the target of statin inhibition. We have recently shown using lymphocyte cell lines derived from subjects in the Cholesterol and Pharmacogenetics (CAP) study, that increased magnitude of simvastatin-induced expression of a spliced HMGCR transcript lacking exon 13 in vitro is significantly correlated with smaller reductions in plasma total and LDL-C in response to simvastatin treatment of the same individuals in vivo. Moreover, we have used selective siRNA knockdown of the normal HMGCR transcript (containing exon 13) in cultured cells to provide preliminary evidence that the spliced variant encodes an HMGCR isoform that is relatively resistant to statin inhibition. Finally, we have found that the extent of exon 13 skipping is associated with a common HMGCR single nucleotide polymorphism (SNP) in intron 13 that we have also found to be associated with the magnitude of plasma LDL-C response to simvastatin. These observations lead to the hypotheses that: 1) the HMGCR mRNA without exon 13 encodes a statin resistant enzyme isoform and;2) the level of expression of this isoform modulates inhibition of cellular cholesterol synthesis in response to statin treatment. Overall, confirmation of these hypotheses would lead to the first demonstration that genetically-influenced variation in alternative splicing can contribute to inter-individual differences in statin response. To test these hypotheses, we will: 1) determine the effects of exon 13 deletion on HMGCR catalytic activity and sensitivity to statin inhibition using purified recombinant HMGCR catalytic domains with and without exon 13 both independently (1A) and as part of a heterogenous tetramer comprised of both HMGCR isoforms (1B);and 2) assess statin effects on HMGCR catalytic activity, cholesterol biosynthesis, and cellular cholesterol content in both cells over-expressing varying amounts of the HMGCR exon 13 splice variant (2A) and immortalized lymphocyte lines that express high versus low levels of the exon 13 splice variant in response to statin treatment (2B). Since HMGCR alternative splicing is genetically regulated, elucidating the basis for the relationship of HMGCR alternative splicing to statin response, would have a major impact on the field of pharmacogenetics by establishing a new role for post-transcriptional genetic regulation in modulating drug efficacy. Moreover since nearly 1/3 of statin-treated patients do not meet lipid-lowering goals, the identification of a statin-resistant HMGCR isoform directly related to its expression has potential utility in the emerging field of personalized medicine as a means of helping to predict an individual's response to statin treatment, with the potential of optimizing use of statin drugs in cardiovascular disease prevention. PUBLIC HEALTH RELEVANCE: The goal of this proposal is to test a new genetically-influenced mechanism that can contribute to inter- individual variation in drug response. Specifically, we seek to test whether alternative splicing of HMG-CoA reductase, the target of statin inhibition, produces a statin-resistant isoform whose expression contributes to inter-individual differences in the magnitude of LDL cholesterol reduction observed in statin-treated patients. The results of this research could yield improvement in the ability to identify individuals most likely to achieve cardiovascular benefit from statin treatment, and new pharmacologic approaches for increasing statin efficacy.

DESCRIPTION (provided by applicant): This application addresses the broad Challenge Area (15) Translational Science, and specific Challenge Topic 15-DK-101: Identification of bioactive macronutrients in the diet that impact metabolic state. The alarming increase in the prevalence of obesity is a cause of great concern given its association with many adverse health conditions, including insulin resistance and type 2 diabetes, which are associated with increased cardiovascular disease (CVD) risk. The primary objective of this project is to identify effective dietary strategies, focused on carbohydrate quantity and starch digestibility, to improve outcome variables associated with CVD risk in insulin resistant individuals who express components of the atherogenic lipoprotein phenotype (ALP). Current dietary guidelines emphasize substitution of carbohydrate calories for total and saturated fat calories for prevention and management of chronic disease. Yet, we and others have shown that high-carbohydrate diets increase the expression of the ALP, characterized by increased plasma triglycerides, reduced HDL cholesterol, and increased levels of small, dense LDL particles, and that this phenotype is reversed by moderate carbohydrate restriction. We have also shown that expression of steroyl coenzymeA desaturase (SCD), an enzyme involved in triglyceride synthesis, is reduced with carbohydrate restriction and that this change is correlated with plasma triglyceride response. While carbohydrate restriction is effective for management of ALP, the role of starch quality has not been addressed. Furthermore, there has been no study of the effects of resistant vs. digestible starches incorporated into high- vs. lower carbohydrate diets. Since isolated reports suggest that increased intake of resistant starch lowers plasma triglycerides and postprandial insulinemia, we hypothesize that starch quality is an important determinant of components of ALP, and that this may be mediated in part by reduced adipose tissue SCD expression. Aim 1 of this proposal will address this hypothesis by a controlled dietary intervention in 52 insulin resistant men and women in which changes in plasma lipids, lipoproteins and lipogenic gene expression will be determined after substituting resistant starch for digestible starch in a high- vs. lower-carbohydrate diet. In Aim 2, the fasting and postprandial glucose and insulin responses to a resistant vs. digestible starch meal will be measured to test the hypothesis that starch digestibility improves glycemic and insulinemic control in a way that relates to diet-induced changes in plasma lipids and lipoproteins. Ultimately, understanding the mechanisms by which resistant vs. rapidly digested starches improve insulinemic and lipid control may facilitate the formulation of more specific dietary recommendations aimed at prevention and treatment of insulin resistance, and its associated atherogenic dyslipidemia and CVD risk. PUBLIC HEALTH RELEVANCE: The overall objective of this project is to test the capacity of a specific dietary component, resistant starch, to improve metabolic biomarkers of CVD risk in individuals who are predisposed to type 2 diabetes by virtue of reduced insulin sensitivity. The information gained from this study could facilitate formulation of dietary recommendations for CVD prevention and management that focus on carbohydrate quality as well as quantity

DESCRIPTION (provided by applicant): The overall objective of this project is to test the hypothesis that the effects of saturated fat (SF) on lipoprotein markers of cardiovascular disease (CVD) risk are influenced by food sources of dietary protein. There is growing epidemiological evidence that consumption of red meat is associated with greater incidence of CVD than either white meat or non-meat foods. Pathophysiological support for the validity of this association is provided by preliminary evidence from our group that a high beef diet has a more deleterious effect on lipoprotein measures of CVD risk than we have observed for mixed protein diets. Specifically, we have found that a high protein, high SF diet with a moderate red meat content selectively induces increases in intermediate density lipoproteins (IDL) and larger LDL particles that have been found to be much more weakly associated with CVD risk than smaller LDL. In contrast, a more recent study from our group has found that, with a similar intake of SF, high beef consumption results in a preferential increase in levels of small and medium sized LDL particles, both of which are strongly related to incident CVD. To date however, no studies have directly compared the lipoprotein effects of red meats with other food sources of protein in the context of both high and low saturated fat intake. We specifically hypothesize that increases in plasma levels of LDL cholesterol (C), and apolipoprotein (apo) B, induced by SF are greater when the major food source of protein is red meat rather than either white meat (poultry) or non-meat foods, and that this is due to increased levels of small and medium sized LDL particles. We therefore propose a clinical trial in which 180 healthy men and women will be randomized to high SF (15%) or low SF (7%) diet groups, and within each group, consume diets with equivalent amounts of protein derived from red meat, white meat, and non-meat sources for 4 wk each in random order. Our Specific Aims will test whether: (1) with high SF, the red meat diet, compared to the other food sources of protein, will result in higher levels of LDL-C, apoB, small and medium sized LDL particles, and total/HDL-C; (2) with low SF, dietary protein source will not be related to any of these measurements; (3) with both the white meat and non-meat diets, increased LDL-C with high vs. low SF will be due primarily to increases in IDL and/or large LDL, whereas with red meat the additional increase in small and medium LDL will result in greater increases in apoB. In addition to these aims we will test for possible metabolic determinants of dietary effects on apoB-containing lipoprotein subclasses, including post-heparin plasma hepatic lipase activity, which is critical for production of smaller LDL, and LDL receptor activity as assessed in peripheral blood mononuclear cells, a system demonstrated to reflect physiologically relevant LDL receptor regulation. Finally, we will examine potential dietary influences on other metabolic biomarkers of CVD risk, including HDL subclasses and apoproteins, insulin sensitivity as assessed by HOMA-IR, measures of inflammation including CRP and multiple cytokines, and endothelial function using a non-invasive fingertip method.

DESCRIPTION (provided by applicant): Important progress continues to be made in the treatment of most common cancers, but therapeutic benefit remains difficult to predict and severe or fatal adverse events occur frequently. The Human Genome Project has fueled the notion that genetic information can produce effective and cost-efficient selection of therapies for individual patients, but validated genetic signatures that predict response to most chemotherapy regimens remain to be identified. Numerous genes potentially influence drug response, but current candidate-gene approaches are limited by the requirement of a priori knowledge about the genes involved and the moderate size of most clinical trials often limits the power of in vitro genome wide association studies (GWAS) for cancer pharmacogenomics discovery. In response to these limitations, we have undertaken a thorough, pharmacogenomic assessment of cytotoxic effect of the majority of FDA approved anti-cancer compounds using an ex vivo model system to determine the heritability of drug-induced cell killing to prioritize drugs for pharmacogenomic mapping. These results are an important first step, and while high heritability of a trait does not guarantee successful association mapping results, it represents an important first step and the results will be used to prioritize drugs with high heritabilities for genome-wide association mapping. In the current proposal, GWAS mapping of cytotoxic agents will be performed in a European American population, and then replication GWAS mapping will be performed in an East Asian population. In addition to discovering and validating genetic variants that predict drug response, the wealth of data collected will be used to dissect the underlying etiology of drug response traits, including assessing the relative contribution of genetic, environmental, and interaction components of variation. These results will provide crucial insight to prioritize genetic variants for follow-up in precious clinical population resources, and potentially reveal new insight into the overall etiology of drug responses.

? DESCRIPTION (provided by applicant): The overall objective of the Center Pharmacogenomics of Statin Therapy (POST) is to apply genomic, transcriptomic, and metabolomic analyses, together with studies in cellular and animal models, and innovative informatic tools, to identify and validate biomarkers for efficacy of statin drugs in reducing riskof cardiovascular disease (CVD), and for adverse effects of statins, specifically myopathy and type 2 diabetes. This multidisciplinary approach is enabled by a team of investigators with expertise in genomics (human, mouse, and molecular), statistics and informatics, and clinical medicine and pharmacology. The Center is comprised of three Projects, two Research Cores, and an Administrative Core. A major aim of Project 1 is the identification of cellular transcriptomic and metabolomic markers for clinical efficacy and adverse effects of statins. This will be accomplished by analyses in statin-exposed lymphoblast cell lines derived from patients with major adverse coronary events, or onset of myopathy or type 2 diabetes on statin treatment, compared with unaffected statin- treated controls. In addition, using genome wide genotypes from these patients, DNA variants will be identified that are associated with statin-induced changes in the transcripts and metabolites that most strongly discriminate affected patients and controls. Project 2 will use a unique, well-characterized panel of 100 inbred mouse strains to discover genetic variation associated with statin-induced myopathy and dysglycemia. Mechanisms underlying these effects will be investigated, with emphasis on the role of dysregulation of autophagy by statin treatment. Projects 1 and 2 will also use relevant cellular and mouse models, respectively, to perform functional studies to validate effects of genes identified in all POST projects as strong candidates for modulating statin efficacy or adverse effects. In Project 3, information derived from genome-wide genotypes, electronic health records, and pharmacy data in a very large and diverse population-based patient cohort will be leveraged to identify and replicate genetic associations with statin efficacy (lipid lowering and CVD event reduction) and adverse effects (myopathy and type 2 diabetes), as well as to assess the overall heritability of these responses. The Clinical Core, based in Kaiser Permanente of Northern California, will provide the clinical information and biologic materials for both Projects1 and 3. Investigators in the Informatics Core will optimize data analysis and integration of results across all projects. The Administrative Core will provide scientific leadership and management of the Center, and foster scientific interactions and training opportunities. Overall, the research program of this Center provides an innovative model for a systems approach to pharmacogenomics that incorporates complementary investigative tools to discover and validate genetically influenced determinants of drug response. Moreover, the findings have the potential for guiding more effective use of statins for reducing CVD risk and minimizing adverse effects, and identifying biomarkers of pathways that modulate the multiple actions of this widely used class of drugs.

ADMINISTRATIVE CORE: SUMMARY Under the leadership of PI's Ronald M. Krauss, M.D., and Aldons J. Lusis, Ph.D., the Administrative Core will be responsible for overall scientific leadership and administrative management of the Center Pharmacogenomics of Statin Therapy (POST). Drs. Krauss and Lusis will co-chair the POST Steering Committee, consisting of all key personnel across the projects and cores of the Center, which will meet at least bimonthly by teleconference to foster productive interactions across the Center, assess progress, plan and review publications, and make key decisions affecting research directions. They will also convene the POST Scientific Advisory Board, which will meet face to face with POST members at least once annually to assess scientific progress, and advise Drs. Krauss and Lusis regarding research directions and new scientific opportunities. In addition, Drs. Krauss and Lusis will monitor timelines for each of the POST components, providing guidance in achieving milestones if needed, and will be responsible for preparing annual NIH progress reports. An important activity of this Core will be the provision of opportunities for training of students and postdoctoral fellows in pharmacogenomics research, and in this regard, trainees will be invited to participate and present their work during the open portion of POST Steering Committee meetings, and to receive cross-training in the laboratories of POST investigators. The Core PI's, together with POST scientists and staff, will organize a two day meeting of the Pharmacogenomics Research Network (PGRN), with the proposed topic Systems Pharmacogenomics, and they will represent POST in all PGRN-wide activities. Dr. Krauss, as Contact PI of POST, will be primarily responsible for its administrative management, and will supervise POST's senior administrative coordinator Ms. Myra Gloria. Ms. Gloria will serve as the point of contact for NIH program and budgetary staff as well for administrative staff of all the institutions affiliated with POST. With the Core PI's, she will ensure adherence to all administrative policies and timelines and assist in preparation of required reports. She will also monitor budget expenditures on a regular basis, and work with the Sponsored Projects Offices of the POST affiliates to ensure effective management of subcontracts. In addition, with the Core PI's, she will assess spending plans so as to ensure effective allocation of Center resources. Finally, she will staff the POST Steering Committee and SAB meetings, and prepare draft summaries for the PI's to review and identify action items. Overall, this Core will work to achieve and maintain effective scientific leadership and efficient administrative management of POST in support of achieving its scientific objectives.

PARENT ABSTRACT The overall objective of the Center Pharmacogenomics of Statin Therapy (POST) is to apply genomic, transcriptomic, and metabolomic analyses, together with studies in cellular and animal models, and innovative informatic tools, to identify and validate biomarkers for efficacy of statin drugs in reducing risk of cardiovascular disease (CVD), and for adverse effects of statins, specifically myopathy and type 2 diabetes. This multidisciplinary approach is enabled by a team of investigators with expertise in genomics (human, mouse, and molecular), statistics and informatics, and clinical medicine and pharmacology. The Center is comprised of three Projects, two Research Cores, and an Administrative Core. A major aim of Project 1 is the identification of cellular transcriptomic and metabolomic markers for clinical efficacy and adverse effects of statins. This will be accomplished by analyses in statin-exposed lymphoblast cell lines derived from patients with major adverse coronary events, or onset of myopathy or type 2 diabetes on statin treatment, compared with unaffected statin-treated controls. In addition, using genome wide genotypes from these patients, DNA variants will be identified that are associated with statin-induced changes in the transcripts and metabolites that most strongly discriminate affected patients and controls. Project 2 will use a unique, well- characterized panel of 100 inbred mouse strains to discover genetic variation associated with statin- induced myopathy and dysglycemia. Mechanisms underlying these effects will be investigated, with emphasis on the role of dysregulation of autophagy by statin treatment. Projects 1 and 2 will also use relevant cellular and mouse models, respectively, to perform functional studies to validate effects of genes identified in all POST projects as strong candidates for modulating statin efficacy or adverse effects. In Project 3, information derived from genome-wide genotypes, electronic health records, and pharmacy data in a very large and diverse population-based patient cohort will be leveraged to identify and replicate genetic associations with statin efficacy (lipid lowering and CVD event reduction) and adverse effects (myopathy and type 2 diabetes), as well as to assess the overall heritability of these responses. The Clinical Core, based in Kaiser Permanente of Northern California, will provide the clinical information and biologic materials for both Projects 1 and 3. Investigators in the Informatics Core will optimize data analysis and integration of results across all projects. The Administrative Core will provide scientific leadership and management of the Center, and foster scientific interactions and training opportunities. Overall, the research program of this Center provides an innovative model for a systems approach to pharmacogenomics that incorporates complementary investigative tools to discover and validate genetically influenced determinants of drug response. Moreover, the findings have the potential for guiding more effective use of statins for reducing CVD risk and minimizing adverse effects, and identifying biomarkers of pathways that modulate the multiple actions of this widely used class of drugs. ADMINISTRATIVE SUPPLEMENT ABSTRACT In response to NOT-AG-18-008, our goal is to extend the validation and application of our data integration methodologies into Alzheimer?s disease research. This administrative supplement is designed to extend the work of the existing subaward to the University of Pennsylvania subcontract for the POST Informatics Core. The PGRN POST Informatics Core serves as the central hub for data sharing and coordination across the three POST projects in the PGRN P50 award. One of our jobs is annotating the extensive information that will be collected and providing analysis expertise to the projects as needed. However, to make great strides in scientific progress and ensure that the collective whole of the Center is greater than the sum of the parts, a key function of the Informatics Core is to serve as ?The Integrator? to combine these data and information. We and others have shown that integration of complementary omics-based data can provide emergent insights into biological processes compared to what can be learned through any single approach alone. The methods that we are developing to integrate data for statin pharmacogenomic phenotypes will be equally applicable in the area of Alzheimer?s disease. Additionally, recent emphasis on open data science by Alzheimer?s disease researchers provides ample data for us to interrogate our method. We have developed novel statistical analysis tools such as the Analysis Tool for Heritable and Environmental Network Associations (ATHENA), and data visualization tools, such as PhenoGram, both of which are designed to collect and combine information from diverse data sources. With these tools, we will leverage publicly available Alzheimer?s disease datasets to maximize the knowledge gleaned about disease risk for Alzheimer?s diseases. The methodologies that we have been developing as part of the PGRN POST award for the past 2.5 years are clearly applicable to the study of Alzheimer?s disease risk. An important validation step of the application of our methodologies is to apply them to different types of datasets and in different phenotypic areas. This administrative supplement focused on extended research into having an Alzheimer?s disease focus is a great mechanism to simultaneously allow us to validate our methodologies with different types of data and potentially identify important risk factors and pathways toward a better understanding of the etiology of Alzheimer?s disease. Finally, we may have the opportunity to identify cross biological implications due to the known pleiotropic relationships between Alzheimer?s disease and cardiovascular disease.

Project Summary/ Abstract Millions of middle-aged and older adults are using statins, and despite the attention that has been given to the possibility that statin treatment may reduce risk of Alzheimer?s Disease and Related Dementias (ADRD), there is substantial uncertainty about such a benefit, as well as concern regarding possible adverse effects of statins on cognitive function. Even a small benefit or harm for ADRD risk could be of substantial public health importance, but prior research has not had access to sufficiently large cohorts with detailed covariate data to provide precise estimates with full control for confounding by indication or other threats to internal validity. This project uses the comprehensive electronic health records (EHRs) of Kaiser Permanente Northern California on a diverse, multi-ethnic cohort of over 1 million individuals age 65 and over (34% statin users), with longitudinal data on laboratory measures (including LDL cholesterol) and related clinical conditions, and a survey of 254,000 providing additional socioeconomic and behavioral risk factor data. These data resources combine the advantages of both a large administrative longitudinal data set with the advantages of detailed lab, pharmacy, and covariate control. With these data we will be able to evaluate (AIM 1) the net effect of initiating statins on the risk of ADRD and whether the effects of statins depend on type, timing, duration, or dose. These are all easily modified factors and precise estimates of their potential benefit or harm would have immediate clinical relevance. We will also evaluate (AIM 2) whether effects differ based on social, demographic, clinical, or genetic background. The KPNC membership is exceptionally racially/ethnically diverse, so we will be able to provide rigorous tests of whether effects differ for African Americans, Latinos, Asian Americans, or non-Latino Whites. Genetic information is available for over 67,000 participants, permitting evaluation of whether major ADRD genetic risk factors or genetic variants related to lipid response and cardiovascular disease modify the effects of statins on ADRD. Finally, we will evaluate (AIM 3) whether statins influence ADRD risk via changes in LDL levels, diagnosed cerebrovascular or cardiovascular disease, or via other mechanisms. If so, this opens the path for future research on how to leverage these different mechanisms, either with statins or potentially via other pharmacotherapies or interventions. These aims are independently essential to help explain inconsistent results from prior research on statins and dementia. The research team assembled to accomplish these aims includes expertise on all the data sources, statins, pharmacoepidemiology, statistical genetics, and epidemiologic methods for study of ADRD. The possibility that statins might be judiciously applied to reduce lifetime risk of ADRD would impact current guidelines for use of statins and would be a powerful advance towards a population prevention strategy for ADRD. The main KPNC cohort and linked survey and genetic data provide an unparalleled resource. In the absence of extremely large RCTs, the proposed research will provide the most conclusive evidence for the effects of statins on ADRD.