Director, Nutrition and Genomics Laboratory Gender, Nutrigenomics and CVD Jose M Ordovas, PHD Director, Nutrition and Genomics Laboratory Jean Mayer USDA Human Nutrition Research Center on Aging at Tufts University jose.ordovas@tufts.edu

Do we Really Need to Take this Uncertain Walk Into the Future?

Yes, Considering that this has been the Path of Nutrition Recommendations

Traditional Epidemiology

Take Home Message The Population Mean does not properly describes/represents the individual within the population. One size does not fit all.

Plasma Lipoprotein Metabolism Intestinal epithelial cell Biliary cholesterol Dietary cholesterol Arterial lumen Atherosclerotic plaque/foam cells Ovary Intestine Muscle Skin Adrenal Bile acid uptake Luminal cholesterol Micellar cholesterol MTP CE ACAT excretion (esterification) ABC G5 ABC G8 Cholesterol Transporter HDL CM Free cholesterol HDL Synthesis – Peripheral Tissues Absorption – Intestine SR-B1 Increased liver LDL receptor activity decreases circulating LDL-C Decreased liver LDL receptor activity increases circulating LDL-C LDL Liver LDL LDL/apo B–E Receptor Two sources of cholesterol Conceptually, it might be argued that circulating cholesterol originates from predominantly two sources: synthesis (from liver and peripheral tissues) and absorption (from the intestine). Irrespective of the origins of cholesterol, it is the liver that normally serves as the main regulatory organ that determines LDL-C blood levels. (Cholesterol from endothelial macrophages associated with arterial cholesterol plaques are clinically important, but only a very minor contributor to total circulating cholesterol.) Animal data suggest that most cholesterol synthesis in the body is from peripheral tissues such as intestine, muscle, and skin. The greatest amount of cholesterol produced per gram of tissue is from endocrine organs such as the ovary, adrenal glands, and gastrointestinal tract. This is because cholesterol is the "backbone" precursor for many hormones. The relative contribution of cholesterol from any of these sources is dependent upon genetic predisposition, diet, drug therapies, interplay of enzymatic up- and down-regulations, and other potential factors. Decreased cholesterol contribution to the liver may increase hepatic LDL receptor activity and thus reduce circulating LDL-C blood levels, which in turn is associated with reduced risk for CHD. Thus, different lipid-altering drugs whose mechanism of action reduces different sources of cholesterol may have complementary actions in lowering LDL-C. Additional abbreviation on slide: SR-B1 = scavenger receptor class B, type 1. References: Bays H, Dujovne C. Colesevelam HCl: a non-systemic lipid-altering drug. Expert Opin Pharmacother 2003;4:779-790. Dietschy JM, Turley SD, Spady DK. Role of liver in the maintenance of cholesterol and low density lipoprotein homeostasis in different animal species, including humans. J Lipid Res 1993;34:1637-1659. Spady DK, Dietschy JM. Sterol synthesis in vivo in 18 tissues of the squirrel monkey, guinea pig, rabbit, hamster, and rat. J Lipid Res 1983;24:303-315. Synthesis - Liver Healthier artery with decreased plaque Atherosclerotic plaque Artery with increased plaque Bays H et al. Expert Opin Pharmacother 2003;4:779-790.

Lipoprotein Metabolism Exogenous - Pathway - Endogenous APOE Intestine Dietary Fat & Cholesterol LPL Chylomicron Remnant FFA Liver VLDL IDL Bile Acids + Cholesterol LDL Peripheral Tissues HDL

Since our beginning in 1948, the Framingham Heart Study, under the direction of the National Heart, Lung and Blood Institute; NHLBI (formerly known as the National Heart Institute) has been committed to identifying the common factors or characteristics that contribute to cardiovascular disease (CVD). We follow CVD development over a long period of time in a large group of participants who had not yet developed overt symptoms of CVD or suffered a heart attack or stroke. Our Study began by recruiting an Original Cohort of 5,209 men and women between the ages of 30 and 62 from the town of Framingham, Massachusetts and since has added an Offspring Cohort (1971) and a Third Generation Cohort, which began in 2002. Over the years, careful monitoring of the Framingham Study population has led to the identification of several major CVD risk factors, as well as a collection of valuable information on the effects of these factors such as blood pressure, blood triglyceride and HDL cholesterol levels, age, gender, and psychosocial issues. Risk factors for other physiological conditions such as dementia have been and continue to be investigated. In addition, the relationships between physical traits and genetic patterns are being studied.

CVD rates, plasma Cholesterol and APOE alleles The Framingham Study b c d This slide represents CVD rates in Framingham according to apoE alleles. In women things are according to what it will be expected: E4>E3>E2; however, in men the risk is E4~E3>E2. This is paradoxical because E2 have lower cholesterol levels than E3 and E4. The explanation of this paradox comes from the fact that male carriers of the APOE2 allele have higher levels of another atherogenic particle (remnants) that are not measured in the clinical practice nowadays. These data will be published in the next 2-3 months in Atherosclerosis. HOWEVER, it is IMPORTANT, as I will explain more detail in one of the coming slides, that each of these genes contribute only 1-5% to the variability in lipids and/or CVD and that in addition, there is a strong interaction with the environment that may increase or decrease this contribution. Lahoz C et al. Atherosclerosis. 2001 15;154:529-37.

Variability in LDL-C response following Diet Therapy Men Women

LDL-C Response to a Therapeutic Diet by APOE allele Lopez-Miranda et al. J Lipid Res. 1994;35:1965-75.

Pharmacogenetics of Statins: Response is Gender Specific

PL PL Ch Ch Ch Ch LCAT HL CE CETP FA Ch CE TG Ch HDL3 HDL3 HDL2b HDL2a Pre-beta2 HDL Ch Pre-beta1 HDL Pre-beta3 HDL LCAT HDL3 ApoA-I HDL3 HDL-R HL HDL2b HDL2a CE CETP FA Ch Liver CE TG Ch To apoB containing lipoproteins To periphery

High Density Lipoprotein The APOA1-APOC3-APOA4-APOA5 locus CHD Risk According to HDL-C Levels: The Framingham Study High Density Lipoprotein apoA-I apoA-II Phospholipids and Free Cholesterol Triglyceride and Cholesteryl Esters High density lipoproteins (HDL) have received the familial name of carriers of the “good cholesterol”. This is based on the evidence that increasing concentrations in blood are protective for coronary heart disease (CHD). The major protein in HDL is apoA-I and this is coded by the APOA1 gene. This gene is part of the APOA1-C3-A4 gene complex on chromosome 11 here showing some of the common polymorphism. We have demonstrated that: The apoC-III/SstI polymorphism may be quite relevant for non insulin dependent diabetes mellitus (NIDDM) and interaction with dietary habits and ethnicity. The ApoA-IV 360 polymorphism is a marker of dietary response. In the following slides we show the importance of taking into consideration interactions between genes and dietary factors to understand the genetics of cardiovascular disease (CVD). We will use as an example the APOA1 gene polymorphism located –75 bases to the APOA1 gene (this polymorphism is known as the –75(G/A) MspI). Jose M. Ordovas SstI 360 347 MspI

Ordovas et al. Am. J. Clin. Nutr. (2002) Mean Plasma HDL-C and Apolipoprotein AI by APOA1(-75G/A) Genotypes in the Framingham Study Ordovas et al. Am. J. Clin. Nutr. (2002)

Ordovas et al. Am. J. Clin. Nutr. (2002) Polyunsaturated fatty acids modulate the effects of the APOA1-75(g/a) polymorphism on HDL-C levels in a gender Specific manner: The Framingham Study P<0.001 Unexpected! More PUFA= More HDLC Expected! More PUFA= LESS HDLC If we go back to the Framingham Study population and we take into consideration the factor “diet” then the picture that emerges is very different to that seen in the previously shown association study. If we concentrate on those subjects with a low consumption of Polyunsaturated fatty acids (PUFA, commonly found in vegetable oils, nuts and fish) (PUFA <4%, blue bars), we see how the A allele is associated in an allele dose dependent manner with lower HDL-C levels. Therefore, for these subjects the presence of the A allele will be detrimental. If we now concentrate of those subjects with an average intake of PUFA (4-8%, green bars), then we observe that the effect of the polymorphism is neutral, and that is in fact what most population studies have shown because the average consumption of PUFa is within that range. However, if we now go to those with a HIGH PUFA intake, then the effect is reverse. The A allele is associated with increased HDL-C levels and in this case, the A allele is potentially protective for CHD. Therefore, this example shows how, depending on the allele we can suggest the most optimal diet for the individual. If one is an AA or GA then probably they should consume a high PUFA diet. Conversely, if one is a GG. Probably the recommended diet should not exceed 8% of PUFA. Ordovas et al. Am. J. Clin. Nutr. In press Jose M. Ordovas Ordovas et al. Am. J. Clin. Nutr. (2002)

Perilipin function and Gene Structure Hormone sensitive lipase Perilipin Triacylglycerols Exon1 Exon2 Exon3 Exon4 Exon5 Exon6 Exon7 Exon8 Exon9 Perilipin 6209 10171 11482 13041 14995 (T>C) (A>T) (G>A) (A>G) (A>T)

Qi, L. Clin Genet. 2004 Oct;66(4):299-310. Combined effect of the PLIN polymorphisms on weight and BMI (Valencia,Spain) PLIN1 PLIN4 PLIN5 PLIN6 Qi, L. Clin Genet. 2004 Oct;66(4):299-310. 22

Qi et al. Obes Res. 2004 Nov;12(11):1758-65 Combined effect of the PLIN polymorphisms on Weight and BMI (Santa Monica, CA) PLIN1 PLIN4 PLIN5 PLIN6 Qi et al. Obes Res. 2004 Nov;12(11):1758-65

PLIN SNPs and Weight Loss

Weight reduction, low caloric diet and PLIN (11482G>A ) polymorphism in obese subjects Corella et al. J Clin Endocrinol Metab 90: 5121–5126, 2005

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PLIN, Diet and Metabolic Syndrome Corella D et al. Perilipin gene variation determines higher susceptibility to insulin resistance in Asian women when consuming a high-saturated fat, low-carbohydrate diet. Diabetes Care 2006 Jun;29(6):1313-9.

Limitations of the current approach

Summary Genotype/Phenotype associations may be gender dependent. Gene-environment interactions are also gender dependent. For this type of studies, gender-specific statistical analyses should be part of the “Standard Operating Procedures” and therefore included as part of the experimental design. There is potential for future personalized dietary recommendations to decrease risk of chronic disorders, but gender must be part of the equation.