Genome-Wide Linkage Analysis of
Carotid Ultrasound Phenotypes in
Afro-Caribbean Families
A l l i s o n K u i p e r s 1, M i l j k o v i c I 1, K a m m e r e r C M 2, W o o d a r d
G 1, B u n k e r C H 1, P a t r i c k A L 3, W h e e l e r V W 3, S u t t o n - T y r r e l l
K 1, Z m u d a J M 1,2
1D
epartment of Epidemiology, Graduate School of Public Health, University of
Pittsburgh, Pittsburgh, PA, USA;
2D e p a r t m e n t o f H u m a n G e n e t i c s , G r a d u a t e S c h o o l o f P u b l i c H e a l t h , U n i v e r s i t y o f
Pittsburgh, Pittsburgh, PA, USA;
3T h e T o b a g o H e a l t h S t u d i e s O f f i c e , S c a r b o r o u g h , T o b a g o , W e s t I n d i e s
Presented at the 2011 NHLBI Trainee Session at the
American Heart Association EPI/NPAM Conference
March 23, 2011
Marquis Marriott Hotel, Atlanta, GA
Vessel Wall Physiology
 Subclinical cardiovascular disease changes:

Intima layer thickens with atherosclerotic deposition

Adventitial diameter (AD) increases as vessel adapts to changes
in hemodynamics

Lumen diameter (LD) increases with AD, but also decreases as
large plaques occlude lumen
 Ultrasound can image carotid artery and measures
intima-media thickness (IMT), AD and LD

Associated with traditional CVD risk factors and predictive of
future events and mortality
Carotid Ultrasound (US) Genetics
 IMT: heritable, 4 genome-wide association or
linkage studies, >300 candidate gene studies
APOE
lipoproteins/lipids
chr 19, q13.2
ACE
renin-angiotensin system
chr 17, q23.3
MTHFR
homocysteine metabolism
chr 1, p36.3
NOS3
nitric oxide metabolitsm
chr 7, q36
ADD1
vessel wall structure
chr 4, p16.3
9p21.3
unknown function
chr 9, p21.3
 AD and LD: less studied, unknown heritability, few
genome-wide or candidate studies
 Few studies in African ancestry populations
PURPOSE
T o p e r fo r m g e no m e - w i de li n k a ge an a l y s i s o n
c o m m o n c a r o ti d ar t e r y t r a i t s ( I M T , A D a nd L D )
f r o m c ar o t i d u lt r a s o u nd i n A f ri ca n anc e s t r y
families from the Tobago Family Study.
Tobago Family Study
Extended families:

471 individuals (187 men,
284 women) in 7 families

Afro-Caribbean ancestry

Aged 18-92
Subclinical CVD visit:

common carotid artery
ultrasound scan

N = 395
Carotid Ultrasound
 B-mode ultrasonography by Acuson Cypress portable
machine
 Near and far walls of CCA (1cm distal to carotid bulb)

right and left sides had 2 images per side (4 images)

read using semi-automated reading software
 Mean, max (IMT, AD) and min (LD) traits were
analyzed
Statistics and Linkage Analysis
 Covariate model building and proportion of variance
due to genetics (heritability) and covariates (SOLAR)
 Genotyped panel of SNPs from genomic DNA

Applied quality control filters resulting in 1,512 SNPS spaced
~1.92 cM across genome
 Calculated multipoint IBD (Loki); ran QTL linkage
analysis (SOLAR) on fully adjusted traits

Suggestive and significant evidence of linkage set at LOD > 2.0
and > 3.3, respectively
RESULTS
Characteristics of the Afro-Caribbean Family Members
Trait
All (n=395)
Men (n=152)
Women (n=243)
Age (years)
42.1 ± 15.7
42.2 ± 15.4
42.1 ± 15.9
BMI (kg/m2)
28.4 ± 6.3
26.8 ± 5.1‡
29.5 ± 6.7‡
Height (cm)
170.5 ± 8.3
177.1 ± 6.8‡
166.4 ± 6.3‡
Waist Circumference (cm)
89.7 ± 15.3
4.3
90.3 ± 12.9
10.7‡
89.4 ± 16.7
0.4‡
>1 Drink per week (%)
12.7
29.1‡
2.5‡
≥25 Min walk per week (%)
48.5
53.0
45.7
Diabetes (%)
8.3
6.4
9.4
Hypertension (%)
26.6
27.8
25.8
Hypertensive Medication (%)
8.4
4.0‡
11.1‡
Systolic Blood Pressure (mmHg)§
118.8 ± 21.8
125.7 ± 19.5‡
114.1 ± 22.1‡
LDL-c (mg/dl)
132.8 ± 40.1
126.9 ± 37.5‡
136.5 ± 41.3‡
HDL-c (mg/dl)
39.8 ± 12.3
40.7 ± 11.6
39.2 ± 12.7
Triglycerides (mg/dl)
88.9 ± 44.9
30.1
96.2 ± 54.1‡
--
84.5 ± 37.6‡
30.1
Parity (%)†
77.0
--
77.0
Oral Contraceptive (%)†
34.4
--
34.4
Current Smoker (%)
Menopausal (%)†
Excluding participants on hypertensive medication
in women only
‡Comparison by sex is statistically significant (p<0.05)
§
†Frequency
Ultrasound Traits in the Afro-Caribbean Family Members
Trait
All
Men
Women
Mean IMT (mm)
Max IMT (mm)
Mean AD (mm)
Max AD (mm)
Mean LD (mm)
Min LD (mm)
0.69 ± 0.15
0.80 ± 0.17
7.23 ± 0.72
7.53 ± 0.76
5.86 ± 0.64
5.65 ± 0.64
0.70 ± 0.15
0.81 ± 0.17
7.49 ± 0.68‡
7.78 ± 0.74‡
6.09 ± 0.63‡
5.89 ± 0.62‡
0.68 ± 0.14
0.79 ± 0.16
7.07 ± 0.70‡
7.36 ± 0.74‡
5.71 ± 0.61‡
5.51 ± 0.61‡
‡Comparison
by sex is statistically significant (p<0.05)
Carotid Ultrasound Traits Variance Components
Trait
Mean IMT
Significant Covariates*
Covariate
Effects
(r2)
Genetic
Effects
(h2r  SE)¥
0.552
0.467±0.110
0.564
0.349±0.101
Max IMT
age, (-) female sex, BMI,
SBP
age, BMI, SBP
Mean AD
age, height, waist, SBP
0.245
0.641±0.120
Max AD
age, height, waist, SBP
0.270
0.622±0.127
Mean LD
(-) age, height, waist, SBP
0.169
0.584±0.123
Min
LD into all
(-)final
age,models,
height,even
waist,
SBP
0.182 alone
* Sex forced
when
not significant predictor
¥ All
residual heritability estimates had a corresponding P-value < 0.0001
0.573±0.120
Genome-wide Linkage LOD Scores for IMT
RED line=mean IMT; BLUE line=max IMT
Genome-wide Linkage LOD Scores for AD
RED line=mean AD; BLUE line=max AD
Genome-wide Linkage LOD Scores for LD
RED line=mean LD; BLUE line=min LD
Conclusions
 All US traits were heritable in this African ancestry
family population

Future study of genetics of vessel diameter warranted
 Evidence of linkage identified for AD/LD on chr. 11

Genes w/ known CVD implications: APOA1/C3/A4/A5 and IL18
 Suggestive linkage on chr. 13 (IMT) and 14 (AD/LD)

No genes w/ strong CVD implications
 Further refinement of linkage peaks needed
Thank You!
ACKNOWLEDGEMENTS:
Zmuda Lab (genotyping)
Cara Nestlerode, MS
Ashley Edwards, BS
Linkage Analysis
Amy Dressen, MS
Ryan Minster, MS
Hu Li, PhD
Ultrasound Study Set-Up
Genevieve Woodard, PhD
Mentors
Joseph Zmuda, PhD
Candace Kammerer, PhD
Kim Sutton-Tyrrell, PhD
Clareann Bunker, PhD
Iva Miljkovic, PhD
Funding
NHLBI: T32-HL083825
NIAMS: R03-AR050107 and
R01-AR049747
No Disclosures