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ND
SD
AZ
OK
Strong Heart
Family Study
Finding genes that influence risk of
cardiovascular disease
Measuring Heart Disease Risk
is Complicated
•
Heart disease is not an all-or-none trait
- we are all at some risk
•
Risk increases as we age
•
Heart disease risk is influenced by many
factors, e.g. cholesterol levels, obesity,
diabetes
•
Heart disease can cluster in families
Why is it Important to Identify
Heart Disease Risk Genes?
•
To learn more about the disease process
•
To find out who should modify lifestyle
•
To develop personalized therapies
Why Look for Heart Disease Risk Genes
In American Indians?
•
Heart disease is a serious and increasing
health problem in American Indians
•
Are the same genes important in every
tribe?
•
What therapies will be most effective in
American Indians?
Drawing Family Trees: Conventions
Female
Male
Married couple
Parents and daughter
Parents and 3 children
Man, 2 wives, 7 children
The Strong Heart Family Study
We identified extended families using name matching
Identifying extended families using name matching
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Identifying extended families using name matching
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Identifying extended families using name matching
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Identifying extended families using name matching
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2
3
Identifying extended families using name matching
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2
3
Identifying extended families using name matching
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2
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Identifying extended families using name matching
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≥ 3 sibs who are members of original cohort
≥ 2 additional sibs
≥ 12 offspring
> 1,200 family members from each field center
The Strong Heart Family Study
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2
3
Arizona: 30 families, sizes 35-288
Dakotas: 27 families, sizes 37-476
Oklahoma: 36 families, sizes 42-243
Who Are the Participants?
•
> 3600 members of American Indian
families in Arizona, Oklahoma, and South
Dakota
•
People who
- wish to participate
- are at least 15 years old
- have a relative in the Strong Heart
Study cohort
- are in large families
•
Most participants are relatively healthy
At Each Field Center:
•
> 1200 people recruited from Strong Heart
Study families
•
For each family member:
- explained the study and get consent
- completed a Family Information Form
- administered questionnaires
- conducted a physical exam
- took a blood sample for DNA and to
measure risk factors
•
Sent Family Information Forms and blood
samples (buffy coats) for DNA to San Antonio
Strong Heart Family Study
• To what extent do genes influence
risk of heart disease beginning when
we are young and healthy?
• What are the genes?
• What traits (risk factors) should we
study?
What Traits Are We Studying?
• Total cholesterol, LDL-C, HDL-C, triglycerides
• LDL size, ApoAI, ApoB, ApoE phenotype
• Insulin, glucose, glycated hemoglobin
• Urine microalbumin, plasma creatinine
• Body fat, BMI, waist circumference
• Systolic and diastolic blood pressure
• Carotid ultrasound measures
• ECG measures
• Etc.
Heritability
The proportion of variance in a trait that
is due to the additive effects of genes
Strong Heart Study:
Heritabilities for Selected Risk
Factors
Total cholesterol
LDL-C
HDL-C
Triglycerides
Fibrinogen
Creatinine
LV mass
0.39
0.32
0.41
0.42
0.42
0.36
0.42
Fasting insulin
Fasting glucose
HbA1C
Height
Weight
BMI
Waist
0.41
0.37
0.59
0.75
0.56
0.51
0.54
If we want to identify the genes that make us
susceptible to CVD, we first have to find them.
Human
Chromosomes
(n = 46)
Karyotype
APOA2
ATH-1
APOB
HMGCS
LFABP
HMGCR
CR39-1
1
LPA
IFABP
APOD
CRBP
5
CRBP-II 4
2
6
Candidate Loci
for
Atherosclerosis
3
LPL
7
8
10
9
CR39-15
LIPC
13
19
14
15
LDLR
APOC1
APOC2
APOE
20
21
11
APOA1
APOC3
APOA4
12
CETP
LCAT
16
22
17
18
CR39-3
Y
X
Adapted from Lusis, 1989
4
6
F8VWFP
D22S57
D22S75
11
6
2
3
5
BCR1
D22S257
D22S156
TOP1P2
D22S41
10
2
3
5
2
2
1
4
5
7
2
7
D22S85
D22S29
MB
D22S102
IL2RB
G22P1
D22S90
D22S92
PDGFB
CYP2DP8
D22S97
D22S94
D22S21
10
D22S55
Human Chromosome 22
Genetic Marker
a polymorphic gene whose exact chromosomal
location is known, but whose function usually is
not known
Mapping Disease
Genes on
Chromosomes
Create a “road map”
of 400 genetic
markers whose
locations are known
Genotype
genetic constitution; the kinds of
genes (= alleles) that a person has
received from her/his parents
12
34
13
12
34
14
12
34
23
12
34
24
ABI 377 Image of 9 Genetic Markers
(Microsatellite Polymorphisms)
Segregating Genotypes: Electrophoretograms
father
mother
child 1
child 2
child 3
Strong Heart Family Study
High-Throughput Genotyping
• >3600 family members
have been genotyped for
about 400 anonymous
markers
• Linkage analyses to detect
gene locations for risk
factor genes are in
progress
“Data Cleaning”: Resolving
Genotyping Discrepancies
AB
AC
DD
AD CD AD
BC
AD
BD AB
BB
BD
BB
CD
AC
AB
BD
AD
CC
BC AC CD AC
BE
AA
AB
Recognizing Genotype Discrepancies
•
•
•
Child doesn’t have
either allele of
one of the parents
AD
Child has an allele
that neither
parent has
AD CC
BB
BB
BC
More than four
alleles in sibship
BC AD AC BD
BE
Causes of Genotype Discrepancies
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Sample mixup
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Genotyping error
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Data entry error
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Incorrect information
concerning family relationships
Resolution of Genotype Discrepancies
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Check for data entry errors
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Re-examine gel
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Re-genotype
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(Re-draw blood)
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For one or more family members,
delete genotype information
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- for this marker
- for all markers
Break link between parent and child
How do genetic markers help us find genes
that influence disease phenotypes?
Segregation of a
Disease Risk Factor in a Family
Segregation of a
Disease Risk Factor and a Marker
12
13
14 34
44
34
14
23
12 13
11
13
34
24
12 12
13
14
33
13 34 34 13
23
11
12
Segregation of a
Disease Risk Factor and a Marker
12
1
13
1
14
34
44
-
34
1
2
14
23
2
12
-
13
11
2
13
12
34
2
1
24
13
2
12
1
2
14
1
13
33
34
34
23
11
1
2
13
12
Cosegregation of a
Disease Risk Factor and a Marker
AB
A
AC
A
AD
DD
B
A
BC
AD
A
CD
AD
AB
A
B
A
AC
BD
AD
BB
A
A
BD
CD
BD
BD
AB
B
CC
BC
A
CD
AC
CD
AA
A
B
AC
AB
Cosegregation of a
Disease Risk Factor and a Marker
AB
A
AC
A
AD
DD
B
A
BC
AD
A
CD
AD
AB
A
B
AC
BD
BB
*
B
BD
BD
AB
B
CC
BD
A
A
BD
CD
BC
B
CD
BC
CD
AA
B
B
BC
AB
Linkage Analysis
Does a phenotype cosegregate with
a specific genetic marker?
How do we measure the
strength of the evidence for
cosegregation (= linkage)?
•
•
LOD score - a measure of the odds in
favor of linkage to a specific marker
LOD score > 3 corresponds to
significant evidence (1000 to 1 odds) in
favor of linkage
Strong Heart Family Study:
Tools for Data Cleaning and Analysis
SOLAR
Sequential
Oligogenic
Linkage
Analysis
Routines
Ln Fasting
InsulinAZ,DK
Chromosome
2
Diego VP, Göring HHH, Cole SA, Almasy L, Dyer TD, Blangero J, Duggirala R, Laston S, Wenger C,
Cantu T, Dyke B, North KE, Schurr T, Best LG, Devereux RB, Fabsitz RR, Howard BV, MacCluer JW.
Fasting insulin and obesity-related phenotypes are linked to chromosome 2p: The Strong Heart
Family Study. Diabetes, in press
Some Preliminary Linkage Results
Strong Heart Family Study
Trait
Chromosome
LV mass
Weight
BMI
Fasting insulin (DK)
Ejection fraction
Lean body mass
Weight
Systolic BP (females)
Fasting glucose
LDL-C
Plasma creatinine
12
4
4
2
18
2
4
17
16
17
5
Location (cM)
37
210
210
51
136
68
219
136
91
89
53
LOD Score
5.4 *
5.2 *
5.2 *
3.4 *
3.4 *
3.3 *
3.3 *
3.3 *
2.6
2.6
2.5
* Significant evidence
After a Disease Risk Factor Gene
Has Been Located: Identify it!
•
Further refine the location
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Identify candidate genes in regions with
high LOD scores
•
Do high-density SNP typing & resequencing
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Do extensive statistical analysis
• Examine expression profiles to understand
gene function
After a Disease Risk Factor Gene
Has Been Identified:
•
Find out what it does
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Develop a therapy
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Determine who has the gene
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Provide therapy and/or counseling
The Eventual Payoff:
•
Better understanding of the disease
process
•
More accurate identification of people
who are at risk
•
Development of personalized therapies
•
Better health for future generations
smoking!
Courtesy of Wen-Chi Hsueh
Heart
Disease