~550 founders

Transcription

1 Lipid GWAS in the Amish: New Insights into Old Genes Coleen M. Damcott, PhD Assistant Professor of Medicine Division of Endocrinology, Diabetes and Nutrition Program in Genetics and Genomic Medicine University of Maryland School of Medicine September 29, 2010

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3 Old Order Amish migration to the U.S ~550 founders Lee, Pollin, et al, BMC Medical Genetics 11:68, 2010

4 The Heredity and Phenotype Intervention (HAPI) Heart Study 868 Lancaster County Old Order Amish individuals recruited Designed to study the genetic and environmental predictors of response to four short term cardiovascular interventions Cold pressor stress test High and low salt diets Daily aspirin therapy High fat meal

5 HAPI High Fat Meal Intervention Participants 809 Old Order Amish Individuals with data for all time points Time 0: Participant fed high fat milkshake 77.6% of calories from fat (~60% saturated fat) 782 calories/m 2 body surface area Blood draws 0, 1, 2, 3, 4 and 6 hours after intervention Triglycerides Total serum cholesterol (TSC) High density lipoprotein cholesterol (HDL-C) Particle subfractions measured at 0 and 4 hours using ultracentrifugation t ti

6 HAPI Heart Participant Characteristics (Mean for 809 Completing Fat Load) Trait Men Women N Age (y) BMI (kg/m 2 ) Waist (cm) Total Cholesterol (mg/dl) HDL-C (mg/dl) LDL-C (mg/dl) Triglycerides gy (mg/dl) Median Glucose (mg/dl) Insulin (μu/ml) Median SBP (mm Hg) DBP (mm Hg) 78 76

7 Median Lipid Levels After High Fat Meal mg/d dl CHOL TRIG HDL Time (Hours)

8 Examples: Triglyceride Response in 8 HAPI Heart Participants i t Time (hours) Each line represents a different individual

9 Genome-Wide Association Analysis Genotyping of 500, SNPs performed in all HAPI Heart participants 381,934 autosomal SNPs remained after QC Association evaluated across all SNPs using a measured genotype model adjusting for sex and sex-specific specific age and age 2 and BMI as well as residual covariance among related individuals

10 GWAS of Fasting & Postprandial Triglycerides id

11 rs rs Pollin et al, Science 322:1702, 2008

12 Mean triglyceride (TG) excursion by rs genotype 180 GG 160 AG (mg/dl) TG Time (hours)

13 Individual TG excursion curves in family members by rs genotype Trigly ycerides (mg g/dl) Father Mother Son Time (hours) Pollin et al, Science 322:1702, 2008

14 Chromosome 11q23 positional candidate genes: Apolipoprotein gene cluster k SNPs APOA5 APOA4 APOC3 APOA1 rs MAF = log p rs MAF = 0.06 ~800 kb D = 0.85 r 2 = Position, MB

15 Post fat load triglyceride excursion in the Apoc3 knockout k mouse (POST FAT LOAD) Maeda N, Li H, Lee D, Oliver P, Quarfordt SH, Osada J. Targeted disruption of the apolipoprotein C-III gene in mice results in hypotriglyceridemia and protection ti from postprandial hypertriglyceridemia. id i J Biol Chem Sep 23; 269(38):

16 ApoC-III and Lipoprotein Metabolism - PPAR-α + Fibrates Adapted from Ooi et al, Clinical Science 114: (2008)

17 C>T TGCTCAGTTCATCCCTAGAGGCAGCTGCTCCAGGAACAGAGGTGCCATGC AGCCCCGGGTACTCCTTGTTGTTGCCCTCCTGGCGCTCCTGGCCTCTGCC TGA CGAGCTTCAGAGGCCGAGGATGCCTCCCTTCTCAGCTTCATGCAGGGTTA CATGAAGCACGCCACCAAGACCGCCAAGGATGCACTGAGCAGCGTGCAGG AGTCCCAGGTGGCCCAGCAGGCCAGGGGCTGGGTGACCGATGGCTTCAGT TCCCTGAAAGACTACTGGAGCACCGTTAAGGACAAGTTCTCTGAGTTCTG GGATTTGGACCCTGAGGTCAGACCAACTTCAGCCGTGGCTGCCTGAGACC TCAATACCCCAAGTCCACCTGCCTATCCATCCTGCGAGCTCCTTGGGTCC TGCAATCTCCAGGGCTGCCCCTGTAGGTTGCTTAAAAGGGACAGTATTCT CAGTGCTCTCCTACCCCACCTCATGCCTGGCCCCCCTCCAGGCATGCTGG CCTCCCAATAAAGCTGGACAAGAAGCTGCTATG MQPRVLLVVALLALLASARASEAEDASLLSFMQGYMKHATKTAKDAL SSVQESQVAQQARGWVTDGFSSLKDYWSTVKDKFSEFWDLDPEVR PTSAVAA

18 APOC3 R19X, ApoC-III and TG RR (CC) RX (CT) p = Fasti ing TG (mg g/dl) TG/APOC-III: r = 0.71 p = APOC-III (relative) Pollin et al, Science 322:1702, 2008

19 Mean TG excursion by APOC3 R19X genotype RR (CC) RX (CT) (mg/dl) Trig glycerides Time (hours) Pollin et al, Science 322:1702, 2008

20 APOC3 Deficiency Confers Favorable Plasma Lipid id Profile % 90% 80% RR (CC) RX (CT) p < p = % 60% 50% 40% 30% p = % 10% 0% Optimal LDL-C (<100 mg/dl) Pollin et al, Science 322:1702, 2008 High HDL-C (>=60 mg/dl) Normal TG (<150 mg/dl)

21 ... And therefore lower Framingham Risk Score for Coronary Heart Disease p < Pollin et al, Science 322:1702, 2008

22 Median and IQR of cholesterol remnants: Lower in RX carriers p < p < p < mg/dl RR Remnants RX Remnants RR IDL RX IDL RR VLDL3 RX VLDL3 Pollin et al, Science 322:1702, 2008

23 Less coronary artery calcification (CAC) in RX carriers RR RX Calcification Presence: p = Calcification Score: p = CAC Pollin et al, Science 322:1702, 2008 Age (years)

24 What are the long term effects of lowering apoc-iii production?

25 Origin of APOC3 R19X *

26 Higher Prevalence of Nonagenarians Among APOC3 R19X Carriers All descendants of MRCA (38/409 died > 90 years old) Inferred R19X descendants of MRCA (10/44 died > 90 years old)

27 Broader Implications Example of a rare variant with large effect influencing a common phenotype First case reported of APOC3 null mutation and one of only a few reported coding mutations ti in APOC3 Provides opportunities to further elucidate apoc-iii function Isolates direct reduction of APOC3 expression as a potential modality for treatment/prevention of coronary heart disease

28 GWAS of LDL-Cholesterol

29 Peak association for LDL-cholesterol detected t d on Chromosome 2p

30 SNP (rs ) in intron of C2orf43 showed strongest association with LDL-cholesterol rs log(p-v value) Position (Mb) OSR1 C2of43 APOB LOC FLJ14126

31 Chr 2p24.1 Mutations in APOB cause Familial Defective apob-100 (FDB) Exon26 APOB R3500Q R3531C R3500W FDB mutations are located in the LDL receptor binding domain of apob-100 Altered 3-D structure of the binding domain reduces affinity for the LDL receptor Associated with hypercholesterolemia and premature coronary heart disease

32 APOB R3500Q identified by sequencing and in near perfect LD with rs Chr 2p24.1 C2of43 rs MAF= kb APOB Sequenced APOB exons in 7 Amish subjects heterozygous for rs All 7 subjects had the R3500Q mutation Genotyped R3500Q mutation in 1,531 subjects: rs and R3500Q are in near perfect LD, D =1 and r 2 =0.96

33 Population Frequency of APOB R3500Q Country/Ethnicity N % heterozygotes Reference United States/Multi-ethnic 5, %, 0.1% Bersot et al, 1993 Denmark/Danish 9, % Tybjaerg-Hansen et al, 1998 Denmark/Danish 5, % Hansen et al, 1994 Switzerland/Swiss % Miserez et al, 1994 United States/Old Order Amish 1,531 12% *Modified from table by Austin MA Am J Epdiemiol 2004

34 Elevated LDL-C in APOB R3500Q carriers constant across age groups esterol ( mg/dl) LDL-chol Carriers 59mg/dL Non-carriers >70 Age group R3500Q accounted for 26% of the variation in LDL-C level

35 APOB R3500Q had modest effect on HDL-C, but no effect on TG or LDL-C Particle Patterns 70 p = 0.30 HDL L-C (mg/dl) p = RR RQ QQ yceride 9mg/d dl) Trigl Percentage of LDL particle pattern A>B RR RQ QQ RR RQ QQ p = 0.54 Adjusted for age, age 2, sex, lipid medication usage, and family structure

36 Prevalen nce of CAC (%) Coronary Artery Calcification (CAC) was more common and extensive (score 400) in APOB R3500Q carriers across age groups Age Group: 400 < 400 NC = Non-carrier C = Carrier NC= C=27 NC= C=25 NC= C=25 NC=148 >70 C= >70 R3500Q accounted for 4.5% of CAC presence and 12.8% of extensive CAC

37 R3500Q carriers had additional increased risk for CAC C and extensive e CAC C independent of effects on LDL-cholesterol OR (95% CI) Model 1 OR (95% CI) Model1 + LDL-C CAC 4.65 ( )*** 3.26 ( )*** Extensive CAC 8.54 ( )** 4.75 ( )* Model 1: covariates include age, sex, BMI, smoking, SBP, DBP, HDL- C, lipid-lowering medication and sibships CAC: CAC 1 vs CAC<1; Extensive CAC: CAC 400 vs CAC <1 ***P<0.0001, **P=0.0002, *P=0.016

38 Broader Implications Another example of a rare variant with a large effect influencing a common phenotype APOB R3500Q carrier frequency is 12% in OOA, the highest ever found in any population, due to a founder effect Provides opportunities to further elucidate the clinical implications of R3500Q in a large number of carriers (n=185) Understanding the role of APOB through the impact of genetic mutations on cholesterol o metabolism and development e e of atherosclerosis could have major impacts on treatment/prevention of coronary heart disease

39 Lessons to be learned from a founder population and a pair of old genes.. Amish are ideal for identification of rare variants with large effects on complex traits: Linkage detected by association Many-of-few few concept provides: Increased # of alleles in the population, which boosts power to detect effect Ease of recruitment by genotype for more detailed hypothesis- driven phenotyping Provides opportunity to better understand the biology of genes/diseases APOC3 & APOB = proof of concept

40 Illumina Product Infomercial Whole Genome Expression Beadchips: HumanWG-6 in lymphocytes of 250 Amish subjects in the Amish Family Longevity Study Custom Genotyping: 384-plex GoldenGate assay in 1,636 Amish well-characterized for CVD-related traits Future Directions: Human Omni5 BeadChip

41 Acknowledgements UMB Division of EDN Alan Shuldiner Braxton Mitchell Toni Pollin Haiqing Shen Richard Horenstein Linda Kao Patrick McArdle John McLenithan Michael Miller Jeff O Connell Sandy Ott Wendy Post Evadnie Rampersaud Kathy Ryan Jack Shelton Jing Yin Yiju Zhao Amish Research Clinic Staff Amish Liaisons Amish Community Larry Bielak Patricia Peyser Julie Douglas PROGENI Consortium Funded by NHLBI, NIDDK and ADA