SHAPE Society

1. Search for Genomic and
Proteomic Risk Factors and
Protective Factors
Associated with Coronary
Heart Disease
Xiaohong Wu MD, Mehran Haidari MD, Silvio Litovsky
MD, Ward Casscells MD, James T Willerson MD, and
Morteza Naghavi MD.

2. Background
 Coronary heart disease (CHD), the most important clinical
manifestation of atherosclerosis, is still the main cause of death in
developed societies and has been predicted to remain so for years
to come (Murray CJ et al Lancet 1997; 349:1498-505)
 It has become increasingly apparent that atherosclerosis is an
inflammatory disease (Shan PK. Et al Cardiology clinical 1999;17:271-281).
 Leukocyte infiltration has been documented in virtually every stage
of atherosclerotic progress, from the fatty streak to the complex
atheromatous plaque ( Shan PK. Et al Circulation 200;101:1758-1759, Ross R. New
Engl J Med 1999;340:115-126)

3. Background
 The role of genetic background for relative resistance to atherosclerosis is
highlighted by the study with familial hypercholesterolemia in whom high plasma
cholesterol levels has not curtailed their expected life span (Goldstein JL et al The
Metabolic &Molecular Basees of Inherited Disease, Vol.11, 2001:2863-913 Chapter
120, Part 12)
 Several gene products has been identified to affect cholesterol absorption:
apolipoprotein-E, scavenger receptor-B1 (Friedman et al Arterioscler Thromb Vasc
Bio 2000;20:2459-64)
 An international team led by Duke University Medical Center researchers has
discovered that a genetic variant of an immune system receptor appears to
simultaneously dampen the body’s immune response to bacteria and other microbial
toxins and to provide some protection against atherosclerosis, or clogging of the
arteries. Furthermore, all of these results were virtually unchanged when we
statistically adjusted for other common cardio-vascular risk factors.” The scientists
believe their discovery suggests a possible new approach to anti-atherosclerosis
drugs. David Schwartz, M.D et al ( July 18, 2002) in the New England Journal of
Medicine.

4. Peripheral Leukocyte gene
expression (different renal disease)
 It is anticipated that more precise delineation of the patterns of gene
expression will help to identify molecular targets for the prevention and
treatment of atherosclerotic disease. Microarray technology application has
made gene profile in a mRNA sample feasible.
 David Alcorta et al (Experiment of Naphrology 2002;10:139-149 )

5. Costomized “Lymphochip”
 Alizadeh et al (Cold Spring Harb Symp Quant
Biol. 1999;64:71-8 )
Based on the gene profile from analying data, they
dentified the subset of genes important in various stages of
lymphocyte development and in leukemia and custom
designed the ‘LymphoChip’ with specific gene profile in
normal and malignant lymphocytes, which tailored the gene
arrays to contain only the most relevant genes, perhaps
only several thousand, instead of 60,000 gene fragments,
made the use of the array less expensive.

6. Diagonostic Markers
Recently, Matthias et al Invented
markers that is differentially
expressed polynucleotides in
ruptured and stable atherosclerotic
plaques which may be useful in the
diagnosis, prevention and treatment
of atherosclerotic disorders.

7. Peripheral Monocytes gene expression
(High Lp(a) vs. Normal)
Christa Buechler et al Blood 2001;97(4):981-986

8. Objective
 Search for genetic and proteomic
risk factors and protective factors
associated with coronary heart
disease in order for developing new
diagnostic techniques and therapies
for coronary heart disease.

9. Hypothesis
1. We hypothesize that the gene expression pattern of
inflammatory cells in the peripheral blood are distinct
among groups of patients who have heart attack with or
without risk factors and groups of patients who have no
heart attack but with risk factors.
2. We hypothesize that systemic proteomics study of blood
serum from different group of patients will identify
additional candidate markers, hence result in a much
greater capacity to determine individual risk profiles.

10. Design--Study population
 This study will be an analytic case-
control study and either sex, 40 to 80
years old patients will be recruited in
the study. Based on the criteria of the
heart attack and risk factor patients
will be classified into five groups.

11. Design– Patient group
1-HA w/ RF 2-HA w/o
RF
3-Young w/
RF w/o HA
4-Elderly w/
RF w/o HA
5-Normal
(elderly
without
RF)
Gene
profile

12. Design--Criteria for Heart Attack
 Patients who admitted to Hermann and St. Luke’s
Episcopal Hospital with first time myocardial infarction
or acute coronary syndrome.

13. Design--Risk Factor
We classify the key risk factors include:
 1. Gender and Age;
 2. Hyperlipidemia;
 3. Hypertension;
 4. Smoking;
 5. Diabetes mellitus;
 6. Family History;

14. Design--Baseline information
 Questionnaire will be filled out for
acquiring baseline information

15. Design--Sample Size
 In order to obtain necessary information
for evaluation of techniques and
calculation of required sample size, we
will do a pilot study. Twenty five patients
from each group will take part in the
study.

16. Experiment Procedure
Monocytes mRNA
Rest of the
inflammatory
cells store w/
RNA later
(Ambion) @-80 C
10ml blood
Sample
Biochemical profile
Serum
0
5ml blood with
anticoagulants
5ml bloodwithoutanticoagulants

17. Monocytes Isolation and mRNA
Extraction
Monocyte direct mRNA
isolation kit(Dynal
biotech)
mRNA Extraction
RNA measured
at 260nm and 280n
( 260/280 ratio 1.8-2.0
as high quality)
5ug of mRNA for
downward Microarray
Assay

18. Schematic of probe preparation,
hybridization, scanning

19. The Process
Cells
Poly-A
RNA
AAAA
cDNA
L L L
L
10% Biotin-labeled cRNA
L
Fragment (heat, Mg2+
)
Labeled
fragments
Hybridize Wash/stain Scan
L
(Transcript labeling Kit: ENZo Diag)
Streptavidin
Phycoerythrin
Biotin-Labeled
Antistreptavidin
phycoerythrin
Superscript cDNA
Synthesis kit

20. Hybridization and staining
L
L
GeneChip
Biotin
Labeled cRNA
+ L
L
L
L
L
L
L
L
L
L
+
SAPE
Streptavidin-
phycoerythrin
Hybridized Array

21. Cost for arrays assays
U133A $12000/30 Arrays
U133B $12000/30
Arrays
$800 /Patient
SuperScript
Choice System for
CDNA Synthesis
$688 /kit
25 reaction
Enzo
Labeling kit
$720 /kit
10 reaction
$56 /patient $144 /patient
mRNA
extraction
and Misc.
Buffer
$200/
patient
Total Cost ~ $1300/patient

22. Real-Time PCR
 The expressed genes that have more
than 2 fold changes detected by
Microarray will be further verified by
Real-Time PCR

23. Proteomics Analysis
 With the 2-D-gel system proteins are separated by
molecular weight and isoelectric point.
 The gel is silver stained and comparison of 2-D gels
of different materials will indicate protein spots which
differ in identity and/or quantity.
 Interesting proteins ("differentials") can be
characterized further after analyzing protein digests
by mass spectrometry.
 Comparison with public databanks can be done to
reveal the identity and functions of these proteins.

24. Process of Proteomics
Analysis
Blood serum protein
preparation
Comparison with

public databanks

25. Database Develop
 We will cooperate with Dr.Fofanov and Dr.
Christoph F. Eick from UH Department of
Computer Science for developing software
and database for further analyzing the gene
expression profile from different group of
patients in order to gain further genetic
information that would help us to be able to
develop new diagnostic and therapies for
Coronary Heart Disease.

26. An industrial-scale approach to protein analysis
Duke University
Medical Center
GeneProt, Inc
Novartis Pharma
AG
Collaborate to identify how the proteins produced by heart
disease patients differ from those of healthy people
blood
samples
heart disease
patients
normal people
To yield information that leads to new drugs or other
treatments for coronary artery disease.
Chris Granger, a
cardiologist at the Duke
Clinical Research
Institute and lead Duke
investigator for the study.
“Such study are essential for
determining how the code of life
produces the specific proteins that play
a role in heart disease” -- Dr. Granger
“a model for academic/industry relationships
that will benefit our patients in the coming
decade“ --Sandy Williams, M.D., dean of the
Duke University School of Medicine
Switzerland Switzerland

27. What they did:
53 individuals
53 healthy
Duke's Databank for
Cardiovascular Disease
6 liters of blood
each group
(pooled
samples
involved)
Maching charateristic:
Gender; age;
Ethnicity;
“It is necessary to use large
volumes in order to have
sufficient quantities of those
proteins present at very low
concentration -- this involves
pooling, which also serves to
dilute normal differences
with occur between
individuals unrelated to the
disease process," said Keith
Rose, Ph.D., chief scientific
officer for GeneProt.
* GeneProt has completed its analysis of the smaller proteins
and is now analyzing the larger proteins in the samples.
* Some interesting new proteins have already been
synthesized, which validates the vision of an industrial-scale
proteomics approach

28. What next:
 Proteins that are present in one sample but
not the other, or are present in widely differing
amounts, are likely to be associated with the
disease process and would be promising
candidates for further investigation.
 GeneProt will synthesizes the smaller
interesting proteins.
 Novartis will test them in further studies.