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.

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)

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.

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 )

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.

Diagonostic MarkersRecently, 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.

Peripheral Monocytes gene expression (High Lp(a) vs. Normal)

Christa Buechler et al Blood 2001;97(4):981-986

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.

Hypothesis1. 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.

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.

Design– Patient group

1-HA w/ RF2-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

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.

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;

Design--Baseline information Questionnaire will be filled out for

acquiring baseline information

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.

Experiment Procedure Monocytes mRNA Rest of the inflammatory cells store w/ RNA later (Ambion) @-80 C10ml bloodSample Biochemical

profile Serum Freeze -80 0C

5ml blood

with anticoagulant

s5ml blood without anticoagulants

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

Schematic of probe preparation, hybridization, scanning

The ProcessCells

Poly-ARNA

AAAA

cDNA

L L L

L

10% Biotin-labeled cRNA

L

Fragment (heat, Mg2+)

Labeledfragments

Hybridize Wash/stain Scan

L

(Transcript labeling Kit: ENZo Diag)

Streptavidin Phycoerythrin

Biotin-Labeled Antistreptavidin phycoerythrin

Superscript cDNASynthesis kit

Hybridization and staining

L

L

GeneChipBiotin

Labeled cRNA

+ L

L

L

L

L

L

L

L

L

L+

SAPEStreptavidin-phycoerythrin

Hybridized Array

Cost for arrays assays

U133A $12000/30 ArraysU133B $12000/30 Arrays

$800 /Patient

SuperScript Choice System for CDNA Synthesis$688 /kit 25 reaction

Enzo Labeling kit

$720 /kit10 reaction

$56 /patient $144 /patient

mRNA extraction and Misc. Buffer

$200/patient

Total Cost ~ $1300/patient

Real-Time PCR The expressed genes that have

more than 2 fold changes detected by Microarray will be further verified by Real-Time PCR

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.

Process of Proteomics Analysis

Blood serum protein preparation

Comparison with public databanks

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.

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

What they did:

53 individuals

53 healthyDuke'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

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.