Lipid GWAS in the Amish:Lipid GWAS in the Amish:ppNew Insights into “Old” GenesNew Insights into “Old” Genes

Coleen M. Damcott, PhDColeen M. Damcott, PhD

Assistant Professor of MedicineAssistant Professor of MedicineDivision of Endocrinology, Diabetes and NutritionDivision of Endocrinology, Diabetes and Nutrition

Program in Genetics and Genomic MedicineProgram in Genetics and Genomic MedicineProgram in Genetics and Genomic MedicineProgram in Genetics and Genomic MedicineUniversity of Maryland School of MedicineUniversity of Maryland School of Medicine

September 29 2010September 29 2010September 29, 2010September 29, 2010

Old Order Amish migration to the U.S.Old Order Amish migration to the U.S.Old Order Amish migration to the U.S.Old Order Amish migration to the U.S.

1727-1757

~550 founders

Lee, Pollin, et al, BMC Medical Genetics 11:68, 2010

The Heredity and Phenotype Intervention The Heredity and Phenotype Intervention (HAPI) H t St d(HAPI) H t St d(HAPI) Heart Study(HAPI) Heart Study

868 Lancaster County Old Order Amish individuals 868 Lancaster County Old Order Amish individuals yyrecruited 2003 recruited 2003 –– 20072007

Designed to study the genetic and environmental Designed to study the genetic and environmental predictors of response to four short term predictors of response to four short term cardiovascular interventionscardiovascular interventionscardiovascular interventionscardiovascular interventions

Cold pressor stress testCold pressor stress test High and low salt dietsHigh and low salt diets Daily aspirin therapyDaily aspirin therapy High fat mealHigh fat mealgg

HAPI High Fat Meal InterventionHAPI High Fat Meal InterventionHAPI High Fat Meal InterventionHAPI High Fat Meal Intervention

ParticipantsParticipants 809 Old Order Amish Individuals with data for all time points809 Old Order Amish Individuals with data for all time points

Time 0:Time 0: Participant fed high fat milkshakeParticipant fed high fat milkshake

77.6% of calories from fat (~60% saturated fat)77.6% of calories from fat (~60% saturated fat) 782 calories/m782 calories/m22 body surface areabody surface area

Blood drawsBlood draws 0, 1, 2, 3, 4 and 6 hours after intervention0, 1, 2, 3, 4 and 6 hours after intervention

TriglyceridesTriglycerides TriglyceridesTriglycerides Total serum cholesterol (TSC)Total serum cholesterol (TSC) High density lipoprotein cholesterol (HDLHigh density lipoprotein cholesterol (HDL--C)C) Particle subfractions measured at 0 and 4 hours using Particle subfractions measured at 0 and 4 hours using

lt t if tilt t if tiultracentrifugationultracentrifugation

HAPI Heart Participant CharacteristicsHAPI Heart Participant Characteristics(M f 809 C l ti F t L d)(M f 809 C l ti F t L d)(Mean for 809 Completing Fat Load)(Mean for 809 Completing Fat Load)

Trait Men WomenN 443 366Age (y) 42 45BMI (kg/m2) 25 6 27 7BMI (kg/m2) 25.6 27.7Waist (cm) 90.0 84.0Total Cholesterol (mg/dl) 203 214HDL-C (mg/dl) 53 59LDL-C (mg/dl) 137 140Triglycerides (mg/dl) Median 53 59g y ( g )Glucose (mg/dl) 86 86Insulin (μU/ml) Median 7.8 8.5SBP (mm Hg) 122 121SBP (mm Hg) 122 121DBP (mm Hg) 78 76

Median Lipid Levels After High Fat MealMedian Lipid Levels After High Fat Meal

202 208 207 208 207 209250

Median Lipid Levels After High Fat MealMedian Lipid Levels After High Fat Meal

202 208 207 208 207 209

118147 154

130150

200

dl

CHOL

5574

118 130

100

150

mg/

d

TRIG

54 55 53 51 51 500

50HDL

0 2 4 6

Time (Hours)

Examples: Triglyceride Response in 8 Examples: Triglyceride Response in 8 HAPI H t P ti i tHAPI H t P ti i tHAPI Heart ParticipantsHAPI Heart Participants

500

600

300

400

100

200

00 2 4 6

Ti (h )Time (hours)

Each line represents a different individual

GenomeGenome--Wide Association AnalysisWide Association AnalysisGenomeGenome Wide Association AnalysisWide Association Analysis

G t i f 500 568 SNP f d i llG t i f 500 568 SNP f d i ll Genotyping of 500,568 SNPs performed in all Genotyping of 500,568 SNPs performed in all HAPI Heart participantsHAPI Heart participants

381,934 autosomal SNPs remained after QC381,934 autosomal SNPs remained after QC

Association evaluated across all SNPs using Association evaluated across all SNPs using a measured genotype model adjusting for a measured genotype model adjusting for g y j gg y j gsex and sexsex and sex--specific age and agespecific age and age22 and BMI and BMI as well as residual covariance among related as well as residual covariance among related individualsindividualsindividualsindividuals

GWAS of Fasting & GWAS of Fasting & P t di l T i l idP t di l T i l idPostprandial TriglyceridesPostprandial Triglycerides

rs10892151

rs10892151

Pollin et al, Science 322:1702, 2008

Mean triglyceride (TG) excursion by Mean triglyceride (TG) excursion by rs10892151 genotypers10892151 genotype

160

180GGAG

rs10892151 genotypers10892151 genotype

120

140

AG

80

100

120

(mg/

dl)

40

60

80

TG

20

40

00 1 2 3 4 5 6

Time (hours)

Individual TG excursion curves in family Individual TG excursion curves in family members by rs10892151 genotypemembers by rs10892151 genotypemembers by rs10892151 genotypemembers by rs10892151 genotype

300

200

250

g/dl

)

150

ycer

ides

(mg

Father

50

100

Trig

ly

MotherSon

00 1 2 3 4 5 6 7

Son

Time (hours)

Pollin et al, Science 322:1702, 2008

Chromosome 11q23 positional candidate Chromosome 11q23 positional candidate genes: Apolipoprotein gene clustergenes: Apolipoprotein gene clustergenes: Apolipoprotein gene clustergenes: Apolipoprotein gene cluster

12

14

16500k SNPsAPOA5APOA4APOC3

rs10892151MAF = 0.03

8

10

12

g p

APOC3APOA1

4

6

8

- log

~800 kbD’ = 0.85r2 = 0.31rs681524

0

2

4 MAF = 0.06

0116.2 116.4 116.6 116.8 117.0

Position, MB

Post fat load triglyceride excursion in the Post fat load triglyceride excursion in the A 3A 3 k k tk k tApoc3Apoc3 knockout mouseknockout 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

t ti f t di l h t i l id i J Bi l Ch 1994 S 23

(POST FAT LOAD)

protection from postprandial hypertriglyceridemia. J Biol Chem. 1994 Sep 23;269(38):23610-6.

ApoCApoC--III and Lipoprotein MetabolismIII and Lipoprotein Metabolism

PPAR-α

-

Fibrates

+

Adapted from Ooi et al, Clinical Science 114:611-624 (2008)

C>T

TGCTCAGTTCATCCCTAGAGGCAGCTGCTCCAGGAACAGAGGTGCCATGCAGCCCCGGGTACTCCTTGTTGTTGCCCTCCTGGCGCTCCTGGCCTCTGCCCGAGCTTCAGAGGCCGAGGATGCCTCCCTTCTCAGCTTCATGCAGGGTTATGACATGAAGCACGCCACCAAGACCGCCAAGGATGCACTGAGCAGCGTGCAGGAGTCCCAGGTGGCCCAGCAGGCCAGGGGCTGGGTGACCGATGGCTTCAGTTCCCTGAAAGACTACTGGAGCACCGTTAAGGACAAGTTCTCTGAGTTCTGGGATTTGGACCCTGAGGTCAGACCAACTTCAGCCGTGGCTGCCTGAGACCTCAATACCCCAAGTCCACCTGCCTATCCATCCTGCGAGCTCCTTGGGTCCTGCAATCTCCAGGGCTGCCCCTGTAGGTTGCTTAAAAGGGACAGTATTCTCAGTGCTCTCCTACCCCACCTCATGCCTGGCCCCCCTCCAGGCATGCTGG

MQPRVLLVVALLALLASARASEAEDASLLSFMQGYMKHATKTAKDAL

CAGTGCTCTCCTACCCCACCTCATGCCTGGCCCCCCTCCAGGCATGCTGGCCTCCCAATAAAGCTGGACAAGAAGCTGCTATG

MQPRVLLVVALLALLASARASEAEDASLLSFMQGYMKHATKTAKDALSSVQESQVAQQARGWVTDGFSSLKDYWSTVKDKFSEFWDLDPEVRPTSAVAA

APOC3 APOC3 R19X, ApoCR19X, ApoC--III and TGIII and TG

80

90RR (CC)RX (CT)

p = 0.0001

60

70

g/dl

)

TG/APOC-III:r = 0.71p = 0.0002

40

50

ing

TG (m

g p

20

30Fast

i

0

10

00 0.2 0.4 0.6 0.8 1 1.2 1.4

APOC-III (relative)Pollin et al, Science 322:1702, 2008

Mean TG excursion by Mean TG excursion by APOC3APOC3 R19X genotypeR19X genotype180

140

160

180RR (CC)RX (CT)

100

120

140

(mg/

dl)

80

100

glyc

erid

es

40

60

Trig

0

20

0 1 2 3 4 5 60 1 2 3 4 5 6Time (hours)

Pollin et al, Science 322:1702, 2008

APOC3APOC3 Deficiency Confers Favorable Deficiency Confers Favorable Pl Li id P filPl Li id P filPlasma Lipid Profile. . .Plasma Lipid Profile. . .

100%

80%

90%

100%RR (CC)RX (CT)

p < 0.0001

p = 0.0004

50%

60%

70%

30%

40%

50%

p = 0.07

0%

10%

20%

0%Optimal LDL-C (<100

mg/dl)High HDL-C (>=60

mg/dl)Normal TG (<150

mg/dl)Pollin et al, Science 322:1702, 2008

. . . And therefore lower Framingham Risk . . . And therefore lower Framingham Risk Score for Coronary Heart DiseaseScore for Coronary Heart Diseaseyy

p < 0.0001

Pollin et al, Science 322:1702, 2008

Median and IQR of cholesterol remnants: Median and IQR of cholesterol remnants: L i RX iL i RX iLower in RX carriersLower in RX carriers

25

30p < 0.0001 p < 0.0001p < 0.0001

20

25

15

mg/

dl

10

0

5

RRRemnants

RXRemnants

RR IDL RX IDL RR VLDL3 RX VLDL3

Pollin et al, Science 322:1702, 2008

Less coronary artery calcification (CAC) Less coronary artery calcification (CAC) in RX carriersin RX carriers

7000

8000RR

RX

in RX carriersin RX carriersCalcification Presence: p = 0.002Calcification Score: p = 0.005

6000

4000

5000

CA

C

2000

3000

1000

029 39 49 59 69 79 89 99

Age (years)Pollin et al, Science 322:1702, 2008

What are the long termWhat are the long termWhat are the long term What are the long term effects of lowering apoCeffects of lowering apoC--III III g pg p

production?production?

Origin ofOrigin of APOC3APOC3 R19XR19XOrigin of Origin of APOC3APOC3 R19XR19X

*

Higher Prevalence of Nonagenarians Higher Prevalence of Nonagenarians AA APOC3APOC3 R19X C iR19X C iAmong Among APOC3APOC3 R19X CarriersR19X Carriers

All descendants of MRCA Inferred R19X descendants ofAll descendants of MRCA(38/409 died > 90 years old)

Inferred R19X descendants of MRCA (10/44 died > 90 years old)

Broader ImplicationsBroader ImplicationsBroader ImplicationsBroader Implications Example of a rare variant with large effect Example of a rare variant with large effect

influencing a common phenotypeinfluencing a common phenotypeinfluencing a common phenotypeinfluencing a common phenotype

First case reported of First case reported of APOC3APOC3 null mutation and one null mutation and one f l f t d di t ti if l f t d di t ti i APOC3APOC3of only a few reported coding mutations in of only a few reported coding mutations in APOC3APOC3

Provides opportunities to further elucidate apoCProvides opportunities to further elucidate apoC--IIIIII Provides opportunities to further elucidate apoCProvides opportunities to further elucidate apoC III III functionfunction

I l tI l t di t d ti fdi t d ti f APOC3APOC3 ii IsolatesIsolates direct reduction of direct reduction of APOC3APOC3 expression as a expression as a potential modality for treatment/prevention of potential modality for treatment/prevention of coronary heart diseasecoronary heart disease

GWAS of LDLGWAS of LDL--CholesterolCholesterol

Peak association for LDLPeak association for LDL--cholesterol cholesterol d t t d Ch 2 24 1d t t d Ch 2 24 1detected on Chromosome 2p24.1 detected on Chromosome 2p24.1

SNP (rs4971516) in intron of C2orf43 SNP (rs4971516) in intron of C2orf43 showed strongest association withshowed strongest association withshowed strongest association with showed strongest association with

LDLLDL--cholesterol cholesterol

70

40

50

60

valu

e)

rs4971516

20

30

40

-log(

p-v

0

10

19 20 21 22 23 24Position

(Mb)

C2of43 FLJ14126OSR1 LOC645949APOB

Mutations in Mutations in APOBAPOB cause Familial cause Familial D f ti BD f ti B 100 (FDB)100 (FDB)Defective apoBDefective apoB--100 (FDB)100 (FDB)

Exon26Chr 2p24.1 APOB

R3500QR3531CR3531CR3500W

FDB mutations are located in the LDLFDB mutations are located in the LDL FDB mutations are located in the LDL FDB mutations are located in the LDL receptor binding domain of apoBreceptor binding domain of apoB--100100

Altered 3Altered 3--D structure of the binding domainD structure of the binding domainAltered 3Altered 3 D structure of the binding domain D structure of the binding domain reduces affinity for the LDL receptorreduces affinity for the LDL receptor

Associated with hypercholesterolemia and Associated with hypercholesterolemia and ypyppremature coronary heart diseasepremature coronary heart disease

APOB APOB R3500QR3500Q identifiedidentified byby sequencing sequencing and inand in near perfect LD withnear perfect LD with rs4971516rs4971516

Chr 2p24.1C2of43 APOB

300kband inand in near perfect LD withnear perfect LD with rs4971516rs4971516

Sequenced APOB exons in 7 Amish subjects heterozygous for rs4971516

rs4971516MAF=0.056

All 7 subjects had the R3500Q mutation

Genotyped R3500Q mutation in 1,531 subjects:

rs4971516 and R3500Q are in near perfect LD, D’=1 and r2=0.96

Population Frequency ofPopulation Frequency of APOBAPOB R3500QR3500QPopulation Frequency of Population Frequency of APOBAPOB R3500QR3500Q

fCountry/Ethnicity N % heterozygotes Reference

United States/Multi-ethnic 5,160 0.08%, 0.1% Bersot et al, 1993

Denmark/Danish 9,255 0.08% Tybjaerg-Hansen et al, 1998

Denmark/Danish 5,000 0.08% Hansen et al, 1994

United States/Old Order Amish 1,531 12%

Switzerland/Swiss 728 0.41% Miserez et al, 1994

,

*Modified from table by Austin MA Am J Epdiemiol 2004

ElevatedElevated LDLLDL--C in C in APOB APOB R3500QR3500Q carriers carriers constant across age groupsconstant across age groups

250

constant across age groupsconstant across age groups

200

250

mg/

dL)

Carriers

100

150

este

rol (

Non-carriers59mg/dL

50

100

LDL-

chol

00 1 2 3 4 5 6 7

L

20-30 30-40 40-50 50-60 60-70 >70Age group

R3500Q accounted for 26% of the variation in LDL-C level

Age group

APOB APOB R3500Q had modest effect on HDLR3500Q had modest effect on HDL--C,C,but no effect on TG or LDLbut no effect on TG or LDL--C Particle PatternsC Particle Patternsbut no effect on TG or LDLbut no effect on TG or LDL C Particle PatternsC Particle Patterns

70

dL)

p = 0.30

56

58

40

50

60

ycer

ide

9mg/

d

p = 0.038

50

52

54

56

L-C

(mg/

dL)

30

40

RR RQ QQ

Trig

l

46

48

50

RR RQ QQ

HD

L

40

50

60

70

LDL

parti

cle

n A

>B

p = 0.54

RR RQ QQ

0

10

20

30Pe

rcen

tage

of

patte

rn

RR RQ QQ

P

Adjusted for age, age2, sex, lipid medication usage, and family structure

Coronary Artery Calcification (CAC) was more common Coronary Artery Calcification (CAC) was more common and extensive (score ≥ 400) in and extensive (score ≥ 400) in APOBAPOB R3500Q carriers R3500Q carriers

across age groupsacross age groupsacross age groupsacross age groups

100

) ≥400

80

CA

C (%

) ≥400< 400

NC = Non-carrierC = Carrier

40

60

nce

of C C = Carrier

20

40

Prev

alen

040-50 50-60 60-70 >70

P

NC=203 C=27 NC=234 C=25 NC=222 C=25 NC=148 C=22

40-50 50-60 60-70 >70Age Group:

R3500Q accounted for 4.5% of CAC presence and 12.8% of extensive CAC

g p

R3500QR3500Q carriers had additional increased risk carriers had additional increased risk for CAC and extensive CAC independent of for CAC and extensive CAC independent of o C C a d e te s e C C depe de t oo C C a d e te s e C C depe de t o

effects on LDLeffects on LDL--cholesterolcholesterol

OR (95% CI)Model 1

OR (95% CI)Model1 + LDL-C

CAC 4.65 (2.73-7.90)*** 3.26 (1.79-5.94)***

Extensive CAC 8.54 (2.79-26.16)** 4.75 (1.33-16.92)*( ) ( )

Model 1: covariates include age, sex, BMI, smoking, SBP, DBP, HDL-g , , , g, , ,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

Broader ImplicationsBroader Implicationspp

Another example of a “rare” variant with a large effect Another example of a “rare” variant with a large effect influencing a common phenotypeinfluencing a common phenotypeinfluencing a common phenotypeinfluencing a common phenotype

APOBAPOB R3500Q carrier frequency is 12% in OOA, the highest R3500Q carrier frequency is 12% in OOA, the highest ever found in any population due to a founder effectever found in any population due to a founder effectever found in any population, due to a founder effectever found in any population, due to a founder effect

Provides opportunities to further elucidate the clinical Provides opportunities to further elucidate the clinical implications of R3500Q in a large number of carriers (n=185)implications of R3500Q in a large number of carriers (n=185)implications of R3500Q in a large number of carriers (n 185) implications of R3500Q in a large number of carriers (n 185)

Understanding the role of APOB through the impact of genetic Understanding the role of APOB through the impact of genetic mutations on cholesterol metabolism and development of mutations on cholesterol metabolism and development of utat o s o c o este o etabo s a d de e op e t outat o s o c o este o etabo s a d de e op e t oatherosclerosis could have major impacts on atherosclerosis could have major impacts on treatment/prevention of coronary heart diseasetreatment/prevention of coronary heart disease

Lessons to be learned from a founder Lessons to be learned from a founder l ti d i f ldl ti d i f ldpopulation and a pair of old genes…..population and a pair of old genes…..

Amish are ideal for identification of rare Amish are ideal for identification of rare variants with large effects on complex traits:variants with large effects on complex traits:

Linkage detected by associationLinkage detected by association

ManyMany--ofof--few concept provides:few concept provides:

Increased # of alleles in the population, which Increased # of alleles in the population, which boosts power to detect effectboosts power to detect effect

Ease of recruitment by genotype for more detailed hypothesisEase of recruitment by genotype for more detailed hypothesis--driven phenotypingdriven phenotyping

Provides opportunity to better understand the Provides opportunity to better understand the biology of genes/diseasesbiology of genes/diseases

APOC3APOC3 & & APOBAPOB = proof of concept= proof of concept

Illumina Product InfomercialIllumina Product InfomercialIllumina Product InfomercialIllumina Product Infomercial

Whole Genome Expression Beadchips: Whole Genome Expression Beadchips: p pp pHumanWGHumanWG--6 in lymphocytes of 250 Amish 6 in lymphocytes of 250 Amish subjects in the Amish Family Longevity subjects in the Amish Family Longevity StudyStudyStudyStudy

Custom Genotyping: 384Custom Genotyping: 384 plex GoldenGateplex GoldenGate Custom Genotyping: 384Custom Genotyping: 384--plex GoldenGate plex GoldenGate assay in 1,636 Amish wellassay in 1,636 Amish well--characterized for characterized for CVDCVD--related traitsrelated traits

Future Directions: Human Omni5 BeadChipFuture Directions: Human Omni5 BeadChip

AcknowledgementsAcknowledgements UMB Division of EDNUMB Division of EDN Alan Shuldiner Alan Shuldiner Braxton MitchellBraxton Mitchell

T i P lliT i P lli

Amish Research Clinic StaffAmish Research Clinic Staff Amish LiaisonsAmish Liaisons Amish CommunityAmish Community

Toni Pollin Toni Pollin Haiqing Shen Haiqing Shen Richard Horenstein Richard Horenstein Linda KaoLinda Kao

Larry BielakLarry Bielak Patricia PeyserPatricia Peyser

Linda KaoLinda Kao Patrick McArdle Patrick McArdle John McLenithanJohn McLenithan Michael MillerMichael Miller

Julie DouglasJulie Douglas

PROGENI ConsortiumPROGENI Consortium Michael MillerMichael Miller Jeff O’ConnellJeff O’Connell Sandy Ott Sandy Ott Wendy PostWendy Post

Funded by NHLBI, NIDDK Funded by NHLBI, NIDDK and ADAand ADAyy

Evadnie Rampersaud Evadnie Rampersaud Kathy RyanKathy Ryan Jack SheltonJack Shelton Jing YinJing Yin Yiju ZhaoYiju Zhao