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