Using Genetic Information in Practice for Tailoring Lipid-lowering Interventions

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Gary Wiggins
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of Human Nutritional Sciences and Food Sciences University of Manitoba, Winnipeg,

1 Using Genetic Information in Practice for Tailoring Lipid-lowering Interventions Peter J Jones, PhD, Director Richardson Centre for Functional Foods and Nutraceuticals Professor of Human Nutritional Sciences and Food Sciences University of Manitoba, Winnipeg, Manitoba, Canada Advances & Controversies in Clinical Nutrition National Harbor, MD December 5th, 2014

2 Conflict of Interest Groupe Danone Kellogg Company DuPont USA Unilever Global Nutritional Fundamentals for Health

3 High variability exists in biomarker response to natural health products Bioactive Average Response Response Range Plant Sterols LDL-C 10% -40 to +20% Long Chain n-3 Fatty Acids LDL-C 4% TAG 20% Rideout et al., to +133% -16 to -45% Harris et al., 2007 Soluble Fibers Total-C 5% LDL-C 10% -3 to -17% -4 to -20% Ganji et al., 2007

4 Consumers possessing confidence in a product s efficacy will be long term consumers You want the right patients using the right products.

5 Overview Explore whether genetic factors play a role in explaining the variability of responsiveness of disease risk related biomarkers to : Plant sterols Omega-3 fatty acids Dietary fiber Can we develop tests to personalize supplement use for patients?

6 Overview Explore whether genetic factors play a role in explaining the variability of responsiveness of disease risk related biomarkers to : Plant sterols Omega-3 fatty acids Dietary fiber Can we develop tests to personalize supplement use for patients?

Plant sterols are natural components of the

adults 150-400 mg/day Major food sources: fruits

nuts Recommended intake of plant sterol-enriched

effect 2 g/day* * International Atherosclerosis

7 Plant sterols are natural components of the human diet Average daily plant sterol intake of adults mg/day Major food sources: fruits and vegetables fat and oils bread and cereals nuts Recommended intake of plant sterol-enriched foods for a significant cholesterol-lowering effect 2 g/day* * International Atherosclerosis Society, 2003; NCEP III Expert Panel, JAMA 2001, EAS 2014

9 Plant sterol action in the intestinal cell Cholesterol Bile salt Phytosterol ABC Transporters G5 G8 Chylomicron Niemann Pick C1L1 Transporter Intestinal Lumen Enterocyte Enterocyte

10 Plant sterol/ stanol meta-analyses results show 84 trials, 141 trial arms high variability between trials 114 trials, 182 trial arms Pooled LDL- C reduction, 2.15 g/d -8.8% CV = (9.4, 8.3) Maximal average LDL- C lowering, PSa vs PSe: 17.3 vs 8.4%* Demonty et al. J Nutr 2009 Musa-Veloso et al. PLEFA 2011

11 Large variability exists in LDL-C response to plant sterols within trials Healthy individuals were given 2g/d plant sterols for 4 wks consumed under supervision Rideout et al Lipids Health Dis 2009

12 Sterol Intake Phase 2 (% Reduction) Sterol Intake Phase 2 (% Reduction) Plant sterol response is a phenotype Percentage Reduction Low-Density Lipoprotein Cholesterol Compared Control 50 R 2 = Is response to plant sterols repeatable? Sterol Intake Phase 1 (% Reduction) Percentage Reduction in Total Cholesterol Compared to Control R 2 = Sterol Intake Phase 1 (% Reduction) Rudkowska I, Abumweis SS, Nicolle C, Jones PJ. Association between nonresponsiveness to plant sterol intervention and polymorphisms in cholesterol metabolism genes: A case-control study. Appl Physiol Nutr Metab 2008;33:

$Change Mean in (±SE) LDL-C cholesterol in subjects fractional stratified synthesis by low (25%) rates medium (% pool/d) (50%), in responder and and$

13 Change Mean in (±SE) LDL-C cholesterol in subjects fractional stratified synthesis by low (25%) rates medium (% pool/d) (50%), in responder and and high non-responder (25%) basal cholesterol subgroups fractional during control synthesis and rate plant sterol supplemented phases. Rideout et al AJCN 2010

Campesterol/cholesterol or 13C-cholesterol uptake Santosa S, Varady K, AbuMweis S, & Jones PJ (2007).

14 Reciprocal relationship between cholesterol synthesis and absorption? Cholesterol synthesis Lathosterol/cholesterol or D incorporation Cholesterol absorption Campesterol/cholesterol or 13C-cholesterol uptake Santosa S, Varady K, AbuMweis S, & Jones PJ (2007). Physiological and therapeutic factors affecting cholesterol metabolism: Does a reciprocal relationship between cholesterol absorption and synthesis really exist? Life Sci 80:

15 Interaction between plant sterol concentrations and T400K (rs ) polymorphism in ABCG8 with response to plant sterol intervention Zhao et al. Lipids 2008

16 Objective: Are genetics associated with effectiveness of lathosterol/cholesterol ratio (synthesis indicator) as a predictor of cholesterol lowering in response to plant sterol consumption?

17 Baseline characteristics of subjects with high and low lathosterol/cholesterol (L/C) ratios All participants (n=63) High Synthesis (HS, n=24) Low Synthesis (LS, n=39) P-values (HS vs. LS) L/C ratio a 1.39 ± ± ± 0.28 p< Gender 24/39 12/12 12/27 p= b (male/female) Age (years) 55.2 ± ± ± 8.5 p= Body weight (kg) 81.6 ± ± ± 14.4 p= BMI (kg/m 2 ) 28.8 ± ± ± 4.2 p= TC (mmol/l) 6.01 ± ± ± 0.89 p= NonHDL (mmol/l) 4.53 ± ± ± 0.85 p= LDL (mmol/l) 3.79 ± ± ± 0.76 p= HDL (mmol/l) 1.48 ± ± ± 0.43 p< Trig.(mmol/L) 1.64 ± ± ± 0.60 p= Glucose (mmol/l) 4.89 ± ± ± 0.52 p= Campesterol a 2.23 ± ± ± 0.91 p= Β-sitosterol a 1.16 ± ± ± 0.51 p= αHC c (nmol/l) ± ± ± p= p-values for HS vs LS by unpaired t-test a= non-cholesterol sterols reported as x10 2 µmol/mmol cholesterol b=calculated by two-tailed fisher s exact test c= values from end of placebo phase, not baseline of trial

18 Study design Dual center (Winnipeg, Beltsville), randomized, crossover, single blinded, placebo controlled design

19 Lathosterol/cholesterol ratio and LDL-C response to plant sterol consumption High L/C ratio ± 0.07 mmol/l, p= LDL-c (mmol/l) High L/C ratio= Low L/C ratio= Low L/C ratio ± 0.05 mmol/l, p< Mackay et al submitted

20 Gene SNP Type of SNP Variation Minor allelic frequency Function of gene ABCA1 ATP-binding cassette, sub-family A (ABC1), member 1 Cholesterol efflux pump in the cellular lipid removal pathway. rs NS - missense C to T T=0.410 rs NS - missense T to C C=0.366 ABCG5 ATP-binding cassette, sub-family G (WHITE), member 5 Half-transporter (with ABCG8) that promotes intestinal and biliary rs NS - missense G to C C=0.211 excretion of sterols. rs NS - missense G to A A=0.074 rs UTR A to C C=0.403 rs Near gene-5 G to C C=0.065 ABCG8 ATP-binding cassette, sub-family G (WHITE), member 8 Half-transporter (with ABCG5) that promotes intestinal and biliary rs NS - missense A to G G=0.441 excretion of sterols. rs NS - missense C to A A=0.218 rs NS - missense C to T T=0.106 CETP Cholesteryl ester transfer protein Facilitates the transport of cholesteryl esters and triglycerides between the rs5882 NS - missense A to G G=0.448 lipoproteins CYP7A1 Cholesterol 7 alpha-hydroxylase The rate-limiting enzyme in the synthesis of bile acid in the classic rs Near gene-5 T to G G=0.450 pathway. DHCR7 7-dehydrocholesterol reductase Enzyme which catalyzes the conversion of 7-dehydrocholesterol to rs NS - missense G to A A=0.179 cholesterol. LDLR low density lipoprotein receptor A cell surface protein involved in receptor-mediated endocytosis. rs688 Synonymous A to G G=0.280 LSS Lanosterol synthase The protein encoded by this gene catalyzes the conversion of (S)-2,3 rs NS - missense C to T T=0.137 oxidosqualene to lanosterol. rs NS - missense T to C C=0.136 PCSK9 Proprotein convertase subtilisin/kexin type 9 A convertase belonging to the proteinase K subfamily which induces rs NS - missense A to G G=0.148 LDLR degradation SCAP SREBF chaperone a protein with a sterol sensing domain which is involved in SREBFs rs NS - missense C to T T=0.476 regulation SREBF2 Sterol regulatory element binding transcription factor 2 Transcription factor that controls cholesterol homeostasis by stimulating rs NS - missense G to C C=0.366 transcription of sterol-regulated genes rs NS - missense G to C C=0.066 ApoE variant Typical frequency* Ε2 7.9% Ε3 78.6% Ε4 13.5% Apolipoprotein E is a glycoprotein present in human plasma; ApoE is associated with triglyceride-rich lipoproteins (chylomicrons and VLDLs) and HDL.

21 rs in CYP7A1 is linked to the cholesterol synthesis phenotype High synthesizers (HS) Low synthesizers (LS) All Χ 2 test for distribution between HS vs LS CYP7A1 Cholesterol 7 alpha-hydroxylase rs T/T T/G G/G Pearson Chi- Square=8.996 DF=2 P-value=0.011 The T/T carriers are more likely in the HS group, while G/T and G/G carriers make up more of the LS group. MacKay et al submitted

22 Impact of SNP rs in CYP7A1 on LDL-C response to plant sterols LDL (mmol/l) T/T G/T G/G ± 0.07 mmol/l p=0.5124, n= ± 0.06 mmol/l p= n= ± 0.12 mmol/l p=0.0002, n=8 MacKay et al submitted

24 APOE isoform and rs in CYP7A1 on LDL-C response to plant sterols 1.0 ε2 ε3 ε4 LDL-c (mmol/l) MacKay et al submitted T/T= G/T= G/G=

25 Triglyceride lowering effects of plant sterols Animal studies - Ntanios et al., Volger et al., Vanstone et al., Ntanios et al., Plosch et al., 2006 Clinical studies - Lees et al., Jones et al., Plana et al., Clifton et al., Zhao et al., Sanchez-Muniz et al., Plat et al., 2009

26 rs5882 in CETP associates with TAG lowering in response to plant sterol consumption Gene SNP CETP rs5882 a Treatment p-value Treatment x genotype p-value Simple effects by genotype* TAG (mmol/l), p-value, n p <.0005 p= A/A=+0.01, p =0.6634(n=25) A/G=--0.04, p= (n=28) G/G=-0.47, p= (n=10) Mackay et al in preparation

27 Overview Explore whether genetic factors play a role in explaining the variability of responsiveness of disease risk related biomarkers to : Plant sterols Omega-3 fatty acids Dietary fiber`

Blood levels of EPA+DHA and risk of sudden death Prospective case-control study of healthy men followed for 17 years in the Physician Health Study The Omega-3 Index

28 Blood levels of EPA+DHA and risk of sudden death Prospective case-control study of healthy men followed for 17 years in the Physician Health Study The Omega-3 Index (proportion of EPA and DHA in RBC membranes) is correlated with cardiac membrane EPA + DHA and reflects the intake of omega-3 Albert et al., N Engl J Med 2002;346:

29 Cardioprotective effects of EPA+DHA TG, triglyceride; RLP, remnant lipoproteins Kromhout et al., Eur Heart J 2012;33:436-43

30 Conversion of ALA to LCPUFA: Consensus of dietary intervention studies (Each data point represents a study) EPA DHA ALA Adapted from Brenna et al., Prostaglandins Leukot Essent Fatty Acids 2009;80:85 91

STUDY DESIGN (Randomized controlled crossover trial with 3 phases [*Randomized

X-over start HO-Canola Oil* end X-over start Flaxseed Oil* end Phase 1 29 d

Experimental Diets: 70 % of total dietary fat substituted with: 1.

Western dietary fat blend (Control) Butter (12%), EV olive oil (35%), lard

31 STUDY DESIGN (Randomized controlled crossover trial with 3 phases [*Randomized treatment order]) start Omega-3 study design 28 d 28 d W/O W/O Control* end X-over start HO-Canola Oil* end X-over start Flaxseed Oil* end Phase 1 29 d Phase 2 29 d Phase 3 29 d Controlled diets consumed under supervision (d1 d28) Experimental Diets: 70 % of total dietary fat substituted with: 1. High-oleic canola oil 2. 1:1 blend flaxseed oil and high-oleic canola oil 3. Western dietary fat blend (Control) Butter (12%), EV olive oil (35%), lard (35%), & sunflower oil (18%) Treatment Vehicles: Fruit smoothies at breakfast Puddings at lunch and dinner

Plasma Total Fatty Acids (%) Plasma Omega-3 Fatty Acid Composition at the End 5 4 3 2 1 of the Three Experimental Diets 5x a a b a 3x a b 1.

32 Plasma Total Fatty Acids (%) Plasma Omega-3 Fatty Acid Composition at the End of the Three Experimental Diets 5x a a b a 3x a b 1.5x a a b Western diet High-oleic canola oil Flaxseed/High-oleic canola oil blend a,b a b 0 ALA EPA DPA DHA All values are means ± SEM; n = 36; Means for a variable without a common letter are significantly different, P < Mixed model ANOVA (Bonferroni post hoc test for multiple comparisons). Method: Agilent 6890N gas chromatography-flame ionisation detector, Supelcowax 10 column (30mX0.25mmX0.25µm) Gillingham et al., Br J Nutr 2011;105:417 27

33 Plasma 13 C-DPA (mg) Plasma 13 C-DHA (mg) Plasma 13 C-ALA (mg) Plasma 13 C-EPA (mg) Absolute Amounts (mg) of Administered U- 13 C-ALA Recovered as Plasma 13 C-Labeled ALA, EPA, DPA, and DHA C-ALA a b b Western diet High-oleic canola oil Flaxseed/High-oleic canola oil a a P=0.133 a a C-DHA 0.1 NS 13 C-DPA b Time (hours) NS b Time (hours) All values are means ± SEM; n= 26; Means for a variable without a common letter are significantly different, P < Mixed model ANOVA (Bonferroni post hoc test for multiple comparisons). Gillingham et al. Am J Clin Nutr, C-EPA a b c a b b

Plasma 13 C-EPA (% dose) Plasma 13 C-EPA (% dose) Total Fatty Acids (%) FADS Genetic Variants and Dietary Oil Effect in Modulation of Plasma 13 C-EPA and Fatty Acid Composition Minor Allele

34 Plasma 13 C-EPA (% dose) Plasma 13 C-EPA (% dose) Total Fatty Acids (%) FADS Genetic Variants and Dietary Oil Effect in Modulation of Plasma 13 C-EPA and Fatty Acid Composition Minor Allele Homozygotes (n=4) 6.0 Heterozygotes (n=19) 13 C-EPA Major Alleles Homozygotes (n=13) * 3.5-fold 5.0 * * 3.5-fold 50% C-EPA EPA * * * rs (FADS2) Genotype 0.0 WD HOCO FXCO WD HOCO FXCO All values are means ± SEM; n = 26; * Indicates mean values for minor allele homozygotes significantly differ from major allele carriers (Mann-Whitney U test (P < 0.05)) * * * WD HOCO FXCO *

35 Plasma fatty acid concentration (%) Genetic Variants on FADS and Dietary Oil Effects of Fatty acid Composition in the COMIT study Minor alleles homozygotes (n=19) Heterozygotes (n=61) Major alleles homozygotes (n=49) fold 1.5-fold 2 a a 1.5 b a a 1 b ALA EPA DPA DHA ALA EPA DPA DHA FlaxSaff (n-3) CornSaff (n-6) rs (FADS2) Genotype (C/T) All values are means ± SEM, n=129. Different letters indicate significant differences between genotypes (P<0.05). Jones et al. Am J Clin Nutr 2014

36 Overview Explore whether genetic factors play a role in explaining the variability of responsiveness of disease risk related biomarkers to : Plant sterols Omega-3 fatty acids Dietary fiber

37 Fiber: β-glucan in barley (1 3)(1 4)- - D -glucan ( -glucan) Endosperm -glucan Husk Barley β-glucan %, dry weight basis Millhouse 3.92 Germ Bran -glucan McGwire 4.45 Candle 6.64 Fibar 9.66

38 Physiochemical properties of β-glucan may affect its efficacy in lowering blood cholesterol Viscosity is determined by molecular weight (MW) and concentration (C) Findings are inconsistent Pros ( Wolever et al. 2010) Physicochemical properties of oat β- glucan influence its ability to reduce serum LDL cholesterol in humans: a randomized clinical trial (2010) Am J Clin Nutr 92(4): Cons ( Frank et al. 2004) "Yeast-leavened oat breads with high or low molecular weight β-glucan do not differ in their effects on blood concentrations of lipids, insulin, or glucose in humans. (2004) J Nutr 134(6): Wood et al. (2002), Wolever et al. (2010), Frank et al. (2004)

39 Objectives of fiber study To determine the cholesterol-lowering efficacy of β- glucan with different doses and physiochemical properties, in terms of MW and viscosity To examine the mechanisms inhibition of dietary cholesterol absorption interruption of of bile acid mechanism To access whether individuals carrying varied genotypes will respond to β-glucan intervention differently in terms of lowering circulating cholesterol

40 Fibre study design Control 3g LMW 5g LMW 3g HMW Randomized and crossover Phase 1 Phase 2 Phase 3 Phase 4 5-wk 4-wk 5-wk 4-wk 5-wk 4-wk 5-wk 13C-CHOL 13C-CHOL 13C-CHOL 13C-CHOL LMW: low molecular weight; HMW: high molecular weight

Characteristics of experimental diets Average MW of β-glucan

s) Treatment Control 3g LMW 5g LMW 3g HMW 68,000 288,000 292,000

3 MW: molecular weight; MW and viscosity were measured for barley

Breakfast with barley-containing β-glucan or rice and wheat

41 Characteristics of experimental diets Average MW of β-glucan (g/mole) Average Viscosity of β-glucan (mpa.s) Treatment Control 3g LMW 5g LMW 3g HMW 68, , ,000 1,349, MW: molecular weight; MW and viscosity were measured for barley or control food LMW: low MW β-glucan ; HMW: high MW β-glucan Breakfast with barley-containing β-glucan or rice and wheat (control) Controlled study with the rest of daily foods supplied to meet energy requirements: Full feeding 55% carbohydrate 30% fat 15% protein

mmol/l mmol/l mmol/l Changes of blood lipids in response to β-glucan intervention Control 3g LMW 5g LMW 3g HMW Control 3g LMW 5g LMW 3g HMW -0.0-0.0-0.2-0.1-0.4-0.

42 mmol/l mmol/l mmol/l Changes of blood lipids in response to β-glucan intervention Control 3g LMW 5g LMW 3g HMW Control 3g LMW 5g LMW 3g HMW a ab ab b NS TC changes LDL-C changes Value presented as least square mean (LSM) ± SEM; Value with with different superscripts letters are significantly different among treatments, P <0.05, adjusted with changes of body weight and waist circumference and genotype of cyp7a1 (Tukey- Kramer adjustment, ANCOVA); NS : not significant

43 mmol/l Different CYP7A1 SNP rs genotypes is associated with cholesterol changes in response to 3g HMW treatment mmol/l Changes of TC Changes of LDL NS ** * NS ** * P =0.042 Value with * indicates significantly different when compared the endpoint with baseline within treatment: * P < 0.05, ** P< 0.01, *** P < (two-tail paired-student s t test);, P value are from Tukey- Kramer adjustment, ANCOVA P =0.035

44 Cholesterol changes Cholesterol changes Relationship between viscosity and cholesterol changes based on CYP7A1-204 T>G G/T and G/G T/T Log (viscosity) Log (viscosity) P =0.058 for LDL-C r 2 =0.89 P =0.011 for TC r 2 =0.98 P =0.96 for LDL-C r 2 = P =0.59 for TC r 2 =0.17 Value presented as mean ± SEM

Variability in response is explained in part by genetic polymorphism in key regulatory genes Bioactive Average Response Response Range Plant Sterols LDL-C 10% -40 to +20%

45 Variability in response is explained in part by genetic polymorphism in key regulatory genes Bioactive Average Response Response Range Plant Sterols LDL-C 10% -40 to +20% n-3 Fatty Acids LDL-C 4% TAG 20% Rideout et al., to +133% -16 to -45% Harris et al., 2007 Soluble Fibers Total-C 5% LDL-C 10% -3 to -17% -4 to -20% Ganji et al., 2007

46 Genetic testing Predictive response tests Responders Plant sterols N-3 PUFA Adverse Responders Non-Responders Soluble Fibers Pharmaceutical approaches Pharmaceutical and dietary approaches

47 Conclusions Gene-nutrient interactions can be seen in nutritional intervention trials provided: -Nutrient/ compliance is well controlled -Response is accurately phenotyped -Biological impact of SNP is large enough A place exists in clinical practice for development of functional tests to distinguish between responders vs non-responders for dietary bioactives

48 Acknowledgements Mohammad Abdullah Nancy Ames David Baer Isabelle Demonty Scott Harding Dylan MacKay Catherine Nichole Todd Rideout Iwona Rudkowska Sylvia Santosa Elke Trautwein Krista Varady Yanan Wang Hailin Zhao Canadian Institute for Health Research Natural Sciences and Engineering Research Council Forbes Medi-Tech Inc Unilever Inc Danone Inc Nutritional Fundamentals for Health

49 RCFFN.com Richardson Centre for Functional Foods and Nutraceuticals SmartPark, University of Manitoba, Winnipeg

FASTING LIPIDS AND INFLAMMATORY BIOMARKERS Both high-oleic canola oil and a flaxseed/high-oleic canola oil blend are cardioprotective via hypolipidemic effects High-oleic canola oil TC LDL HDL

50 FASTING LIPIDS AND INFLAMMATORY BIOMARKERS Both high-oleic canola oil and a flaxseed/high-oleic canola oil blend are cardioprotective via hypolipidemic effects High-oleic canola oil TC LDL HDL E-selectin Flaxseed/high-oleic canola oil ALA-rich flaxseed/high-oleic canola oil blend may further target inflammation and atherogenic pathways by reducing plasma E- selectin Substitution of dietary fats common to the Western diet with both high-oleic canola and flaxseed oil is a feasible option target dietary recommendations and traditional & emerging risk factors for CVD

Facts on Fats. Ronald P. Mensink

Facts on Fats. Ronald P. Mensink Facts on Fats Ronald P. Mensink Department of Human Biology NUTRIM, School for Nutrition, Toxicology and Metabolism Maastricht University Maastricht The Netherlands Outline of the Presentation Saturated