Lipid
p 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
29, 2010
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
(HAPI) Heart
H t Study
St d

868 Lancaster County
y Old Order Amish individuals
recruited 2003 – 2007

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
g fat meal
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/m2 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(HDL-C)
Particle subfractions measured at 0 and 4 hours using
ultracentrifugation
lt
t if
ti
HAPI Heart Participant Characteristics
(Mean
(M
for
f 809 C
Completing
l ti F
Fatt L
Load)
d)
Trait
Men
Women
N
443
366
Age (y)
42
45
BMI (kg/m2)
25 6
25.6
27 7
27.7
Waist (cm)
90.0
84.0
Total Cholesterol (mg/dl)
203
214
HDL-C (mg/dl)
53
59
LDL-C (mg/dl)
137
140
Triglycerides
gy
((mg/dl)
g ) Median
53
59
Glucose (mg/dl)
86
86
Insulin (μU/ml) Median
7.8
8.5
SBP (mm Hg)
122
121
DBP (mm Hg)
78
76
Median Lipid Levels After High Fat Meal
250
202
208
207
208
207
147
154
mg/d
dl
200
150
100
50
54
TRIG
74
55
CHOL
130
118
55
209
53
51
51
HDL 50
4
6
0
0
2
Time (Hours)
Examples: Triglyceride Response in 8
HAPI H
Heartt P
Participants
ti i
t
600
500
400
300
200
100
0
0
2
4
Ti
Time
(hours)
(h
)
Each line represents a different individual
6
Genome--Wide Association Analysis
Genome

G
Genotyping
t i off 500,568
500 568 SNP
SNPs performed
f
d in
i all
ll
HAPI Heart participants

381,934 autosomal SNPs remained after QC

Association evaluated across all SNPs using
a measured g
genotype
y model adjusting
j
g for
sex and sexsex-specific age and age2 and BMI
as well as residual covariance among related
individuals
GWAS of Fasting &
P t
Postprandial
di l Triglycerides
Ti l
id
rs10892151
rs10892151
Pollin et al, Science 322:1702, 2008
Mean triglyceride (TG) excursion by
rs10892151 genotype
180
GG
AG
160
140
TG (mg/dl)
120
100
80
60
40
20
0
0
1
2
3
Time (hours)
4
5
6
Individual TG excursion curves in family
members by rs10892151 genotype
300
Trigly
ycerides (mg
g/dl)
250
200
150
Father
100
Mother
50
Son
0
0
1
2
3
4
Time (hours)
Pollin et al, Science 322:1702, 2008
5
6
7
Chromosome 11q23 positional candidate
genes: Apolipoprotein gene cluster
16
14
12
rs10892151
MAF = 0.03
500k SNPs
APOA5
APOA4
APOC3
APOA1
- log
gp
10
8
6
4
rs681524
MAF = 0.06
~800 kb
D’ = 0.85
r2 = 0.31
2
0
116.2
116.4
116.6
116.8
Position, MB
117.0
Post fat load triglyceride excursion in the
A
Apoc3
3 knockout
k
k t 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
t ti from
f
postprandial
t
di l hypertriglyceridemia.
h
ti l
id i J Biol
Bi l Chem.
Ch
1994 Sep
S 23;
23
269(38):23610-6.
ApoC--III and Lipoprotein Metabolism
ApoC
PPAR-α
Adapted from Ooi et al, Clinical Science 114:611-624 (2008)
+
Fibrates
C>T
TGCTCAGTTCATCCCTAGAGGCAGCTGCTCCAGGAACAGAGGTGCCATGC
AGCCCCGGGTACTCCTTGTTGTTGCCCTCCTGGCGCTCCTGGCCTCTGCC
TGA
CGAGCTTCAGAGGCCGAGGATGCCTCCCTTCTCAGCTTCATGCAGGGTTA
CATGAAGCACGCCACCAAGACCGCCAAGGATGCACTGAGCAGCGTGCAGG
AGTCCCAGGTGGCCCAGCAGGCCAGGGGCTGGGTGACCGATGGCTTCAGT
TCCCTGAAAGACTACTGGAGCACCGTTAAGGACAAGTTCTCTGAGTTCTG
GGATTTGGACCCTGAGGTCAGACCAACTTCAGCCGTGGCTGCCTGAGACC
TCAATACCCCAAGTCCACCTGCCTATCCATCCTGCGAGCTCCTTGGGTCC
TGCAATCTCCAGGGCTGCCCCTGTAGGTTGCTTAAAAGGGACAGTATTCT
CAGTGCTCTCCTACCCCACCTCATGCCTGGCCCCCCTCCAGGCATGCTGG
CCTCCCAATAAAGCTGGACAAGAAGCTGCTATG
MQPRVLLVVALLALLASARASEAEDASLLSFMQGYMKHATKTAKDAL
SSVQESQVAQQARGWVTDGFSSLKDYWSTVKDKFSEFWDLDPEVR
PTSAVAA
APOC3 R19X, ApoCApoC-III and TG
90
RR (CC)
p = 0.0001
RX (CT)
80
Fastiing TG (mg
g/dl)
70
TG/APOC-III:
r = 0.71
p = 0.0002
60
50
40
30
20
10
0
0
0.2
0.4
0.6
0.8
APOC-III (relative)
Pollin et al, Science 322:1702, 2008
1
1.2
1.4
Mean TG excursion by APOC3 R19X genotype
180
RR (CC)
RX (CT)
160
Trig
glycerides (mg/dl)
140
120
100
80
60
40
20
0
0
1
2
3
Time (hours)
Pollin et al, Science 322:1702, 2008
4
5
6
APOC3 Deficiency Confers Favorable
Pl
Plasma
Li
Lipid
id Profile.
P fil . .
100%
90%
RR (CC)
RX (CT)
p = 0.0004
p < 0.0001
80%
70%
60%
50%
40%
30%
p = 0.07
20%
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)
. . . And therefore lower Framingham Risk
Score for Coronary
y Heart Disease
p < 0.0001
Pollin et al, Science 322:1702, 2008
Median and IQR of cholesterol remnants:
L
Lower
in
i RX carriers
i
30
p < 0.0001
p < 0.0001
p < 0.0001
25
mg/dl
20
15
10
5
0
RR
RX
Remnants Remnants
Pollin et al, Science 322:1702, 2008
RR IDL
RX IDL
RR VLDL3 RX VLDL3
Less coronary artery calcification (CAC)
in
RX
carriers
8000
RR Calcification Presence: p = 0.002
7000
RX
Calcification Score: p = 0.005
6000
CAC
5000
4000
3000
2000
1000
0
29
39
Pollin et al, Science 322:1702, 2008
49
59
69
Age (years)
79
89
99
What are the long term
effects of lowering
g apoCapoC
p -III
production?
Origin of APOC3 R19X
*
Higher Prevalence of Nonagenarians
A
Among
APOC3 R19X C
Carriers
i
All descendants of MRCA
(38/409 died > 90 years old)
Inferred R19X descendants of
MRCA (10/44 died > 90 years old)
Broader Implications

Example of a rare variant with large effect
influencing a common phenotype

First case reported of APOC3 null mutation and one
off only
l a few
f
reported
t d coding
di mutations
t ti
in
i APOC3

Provides opportunities to further elucidate apoCapoC-III
function

I l t direct
Isolates
di t reduction
d ti off APOC3 expression
i
as a
potential modality for treatment/prevention of
coronary heart disease
GWAS of LDLLDL-Cholesterol
Peak association for LDL
LDL--cholesterol
d t t d on Chromosome
detected
Ch
2p24.1
2 24 1
SNP (rs4971516) in intron of C2orf43
showed strongest association with
LDL--cholesterol
LDL
70
rs4971516
-log(p-v
value)
60
50
40
30
20
10
0
19
20
OSR1
21
22
C2of43 APOB LOC645949
23
24
FLJ14126
Position
(Mb)
Mutations in APOB cause Familial
D f ti apoBDefective
apoB
B-100 (FDB)
Chr 2p24.1
APOB
Exon26
R3500Q
R3531C
R3500W
 FDB mutations are located in the LDL
receptor binding domain of apoBapoB-100
 Altered 33-D structure of the binding domain
reduces affinity for the LDL receptor
yp
and
 Associated with hypercholesterolemia
premature coronary heart disease
APOB R3500Q identified by sequencing
and in near perfect LD with rs4971516
300kb
Chr 2p24.1
C2of43
rs4971516
MAF=0.056
APOB
Sequenced APOB exons in 7 Amish
subjects heterozygous for rs4971516
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 of APOB R3500Q
Country/Ethnicity
N
f
% heterozygotes Reference
United States/Multi-ethnic
5,160
0.08%, 0.1%
Denmark/Danish
9,255
0.08%
Tybjaerg-Hansen et al, 1998
Denmark/Danish
5,000
0.08%
Hansen et al, 1994
728
0.41%
Miserez et al, 1994
Switzerland/Swiss
United States/Old Order Amish
1,531
,
12%
*Modified from table by Austin MA Am J Epdiemiol 2004
Bersot et al, 1993
Elevated LDLLDL-C in APOB R3500Q carriers
constant across age groups
L
LDL-chol
esterol (mg/dL)
250
Carriers
200
150
59mg/dL
Non-carriers
100
50
0
0
1
2
20-30 30-40
3
4
40-50 50-60
Age group
5
60-70
6
>70
7
R3500Q accounted for 26% of the variation in LDL-C level
APOB R3500Q had modest effect on HDLHDL-C,
but no effect on TG or LDLLDL-C Particle Patterns
p = 0.30
p = 0.038
56
54
60
50
40
30
RR
52
50
48
46
RR
RQ
QQ
Percentage of LDL particle
P
n A>B
pattern
HDL
L-C (mg/dL)
58
Triglyceride 9mg/d
dL)
70
RQ
QQ
p = 0.54
70
60
50
40
30
20
10
0
RR
Adjusted for age, age2, sex, lipid medication usage, and family structure
RQ
QQ
Coronary Artery Calcification (CAC) was more common
and extensive (score ≥ 400) in APOB R3500Q carriers
across age groups
P
Prevalen
nce of C
CAC (%))
100
80
60
≥400
< 400
NC = Non-carrier
C = Carrier
40
20
0
40-50
NC=203
C=27
Age
g Group:
p 40-50
50-60
NC=234
C=25
50-60
60-70
NC=222
C=25
60-70
>7 0
NC=148
C=22
>70
R3500Q accounted for 4.5% of CAC presence
and 12.8% of extensive CAC
R3500Q carriers had additional increased risk
for
o CAC
C Ca
and
de
extensive
te s e C
CAC
C independent
depe de t o
of
effects on LDLLDL-cholesterol
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,
g , sex,, BMI,, smoking,
g, SBP,, DBP,, HDLC, 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 Implications
p

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,
population due to a founder effect

Provides opportunities to further elucidate the clinical
implications of R3500Q in a large number of carriers (n=185)
(n 185)

Understanding the role of APOB through the impact of genetic
utat o s o
on c
cholesterol
o este o metabolism
etabo s a
and
d de
development
e op e t o
of
mutations
atherosclerosis could have major impacts on
treatment/prevention of coronary heart disease
Lessons to be learned from a founder
population
l ti and
d a pair
i off old
ld genes…..

Amish are ideal for identification of rare
variants with large effects on complex traits:

Linkage detected by association

Many--of
Many
of--few concept provides:

Increased # of alleles in the population, which
boosts power to detect effect

Ease of recruitment by genotype for more detailed hypothesishypothesisdriven phenotyping

Provides opportunity to better understand the
biology of genes/diseases

APOC3 & APOB = proof of concept
Illumina Product Infomercial

Whole Genome Expression
p
Beadchips:
p
HumanWG--6 in lymphocytes of 250 Amish
HumanWG
subjects in the Amish Family Longevity
Study

Custom Genotyping: 384384-plex GoldenGate
assay in 1,636 Amish wellwell-characterized for
CVD--related traits
CVD

Future Directions: Human Omni5 BeadChip
Acknowledgements
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UMB Division of EDN
Alan Shuldiner
Braxton Mitchell
T i Pollin
Toni
P lli
Haiqing Shen
Richard Horenstein
Linda Kao
Patrick McArdle
John McLenithan
Michael Miller
Jeff O’Connell
Sandy Ott
Wendy
y Post
Evadnie Rampersaud
Kathy Ryan
Jack Shelton
Jing Yin
Yiju Zhao
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Amish Research Clinic Staff
Amish Liaisons
Amish Community
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Larry Bielak
Patricia Peyser
Julie Douglas
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PROGENI Consortium
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Funded by NHLBI, NIDDK
and ADA
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