A. APPLICATION FORM
Association of TGF-beta1 and TGF-alpha polymorphisms with disease severity of patients infected with Hepatitis C virus.
2. DESCRIPTION OF THE STUDY
We will receive previously collected DNA samples from patients with HCV and healthy individuals from Dr. Radhika Gade of the Genetic Research Institute of the Desert in Rancho Mirage, CA. This institute has already completed the consent form that has been signed by the patients. The only information that we will receive is if the DNA is from HCV infected patients or healthy individuals. We will examine the frequency of TGF-beta1 and TGF-alpha polymorphisms in these samples to see if there is a correlation between these genes and the progression of HCV disease. (See section B for further information)
3. DURATION OF THE STUDY
The project will be completed in March 2005.
4. MULTICENTER STUDY
This study will be conducted by Weber State Clinical Laboratory Sciences program in conjunction with Dr. Radhika Gade of the Genetic Research Institute of the Desert in Rancho Mirage, CA. Dr. Gade will be sending the DNA samples to us, and all the testing will be performed at Weber State University.
5. NUMBER OF SUBJECTS
We will receive and test 155 patient samples, 35 of these patients have HCV and 120 are normal, healthy individuals. We will not have contact with the patients.
6. HEALTH STATUS OF THE SUBJECTS
The DNA that we will receive is from patients with Hepatitis C and normal, healthy patients. We will not, however, have any contact with these patients.
7. SUBJECT GROUPS EXCLUDED
There will be no subject groups excluded from this study.
8. AGES OF THE SUBJECTS
The study will include from patients of all ages.
9. DESIGN OF THE STUDY
Certain portions of the TGF-beta1 and TGF-alpha genes from DNA samples will be amplified and cut by an enzyme at the polymorphic site. The cut portions (bands) will be differentiated on a gel, and the frequency of the bands will be analyzed by Chi square method of analysis.
10. RISKS TO SUBJECTS
There will be no risk to the subjects.
ANY MODERATE OR HIGH RISK STUDY MUST GO THROUGH A SEPARATE REVIEW PROCEDURE WHICH INCLUDES THE APPROPRIATE ADMINISTRATIVE OFFICER AND THE UNIVERSITY ATTORNEY.
11. BENEFITS TO SUBJECTS AND OTHERS
No benefits will be given to the subjects that participate in the project, or to the research lab that will be providing the samples.
12. COSTS TO BE BORNE BY SUBJECTS
The participant is not financially responsible for any part of the study.
13. IS CONFIDENTIALITY ASSURED
Patient names have already been removed from samples and replaced by an accession number. Students working on this project will not have access to actual identifying patient information.
14. CONTRACT OR GRANT NUMBER
We have not received a grant yet, but we are in the process of applying.
15. NAME OF PRINCIPAL INVESTIGATOR AND DEPARTMENT
Scott Wright from the Clinical Laboratory Sciences Department at Weber State University is the principal investigator.
B. DESCRIPTION OF THE STUDY
1. BACKGROUND INFORMATION
Hepatitis C virus (HCV) is an infectious blood-borne pathogen that usually persists as a chronic liver infection. There are 4 million people in USA that are infected with HCV. The mode of infection is unknown for HCV and there is currently no vaccine available. Studies have shown that 10-15% of the time, patients can fight off the virus, indicating that individual genetic differences could be critical in determining the course of HCV infection. The course of HCV varies among people infected with the virus. Some people respond to treatment, while many do not, some recover completely, while others remain chronically infected. Among those who remain infected, some have mild symptoms while others have severe symptoms.
In recent years, variations in DNA sequences, called polymorphisms, have been identified that may influence the progression of hepatic fibrosis (liver disease) in human and animal studies. There is increasing interest in the influence of genetic factors on hepatic fibrosis, and whether genetic polymorphisms can reliably identify subjects at risk of developing severe disease. Studies indicate that variants of genes such as Transforming Growth Factor-beta1 (TGF-beta1) and Transforming Growth Factor alpha (TGF alpha) are associated with chronic liver disease in HCV. TGF-beta1 is believed to be a potent cytokine (hormone-like protein) that is a determining factor in hepatic fibrosis. TGF-beta1 stimulates the synthesis of liver cell proteins and also prevents their degradation. TGF alpha is a protein that has been shown to stimulate liver cell growth.
In our study we will investigate the correlation in the frequency of TGF-beta1and TGF alpha gene polymorphisms in HCV patients and healthy individuals. If the frequency of these polymorphisms is determined to be significant, this will show that these two genes are associated with HCV and may be used as genetic markers in diagnosis of patients infected with HCV. This study could also show that people who have certain polymorphisms in their genes are genetically predisposed to have a higher chance of getting infected with HCV.
This is a joint project with Dr. Radhika Gade of the Genetic Research Institute of the Desert in Rancho Mirage, CA. The DNA samples from HCV infected patients as well as healthy individuals will be provided by Dr. Gade. The only patient information we will receive is whether or not the patient is infected with HCV.
2. EXPERIMENTAL METHODS
Dr. Gade has agreed to supply us with the DNA samples; including 35 HCV infected patients and 120 healthy individuals. All human DNA samples contain polymorphisms for Transforming Growth Factor-beta1 (TGF-beta1) and Transforming Growth Factor alpha (TGF alpha). We are planning to PCR amplify a specific portion of the gene (about 100-200 base pairs) from HCV individuals and control subjects. The first step in the project will be to design a set of primers (a synthesized small piece of DNA about 20 base pair) using the Primer 3 program at the Whitehead Institute (http://frodo.wi.mit.edu/cgi-bin/primer3_www.cgi). We will then use the primers to PCR amplify a section of the gene (either from TGF-beta1 or TGF alpha). After the PCR amplification we will use an enzyme to cut the synthesized DNA at specific polymorphic sites. The polymorphic sites of interest will be found from the Single Nucleotide Polymorphism database (http://www.ncbi.nlm.nih.gov/SNP). We will then use a gel electrophoresis apparatus to separate the bands (generated small pieces of DNA as the result of enzyme treatment). Statistical Chi square analysis of the frequency of the generated bands between the HCV infected and uninfected individuals will show whether or not these polymorphisms have any role in the progress in HCV disease.
C. INFORMED CONSENT
Informed consent has already been done by Dr. Radhika Gade of the Genetic Research Institute of the Desert in Rancho Mirage, CA.