Complement Factor H Haplotypes and Smoking in Age-related Macular Degeneration (CFH&AMD)
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|ClinicalTrials.gov Identifier: NCT01115231|
Recruitment Status : Unknown
Verified January 2016 by VA Office of Research and Development.
Recruitment status was: Active, not recruiting
First Posted : May 4, 2010
Last Update Posted : January 6, 2016
|First Submitted Date||April 15, 2010|
|First Posted Date||May 4, 2010|
|Last Update Posted Date||January 6, 2016|
|Study Start Date||October 2010|
|Actual Primary Completion Date||June 2015 (Final data collection date for primary outcome measure)|
|Current Primary Outcome Measures
||Assessment of Age-related Macular Degeneration [ Time Frame: patients will be examined for AMD at a routine visit; if they present themselves with AMD or qualify as control, they will be recruited to the study. Information will be used in publication at the end of the 4-year study. ]|
|Original Primary Outcome Measures
||Assessment of Age-related Macular Degeration [ Time Frame: patients will be examined for AMD at a routine visit; if they present themselves with AMD or qualify as control, they will be recruited to the study. Information will be used in publication at the end of the 4-year study. ]|
|Change History||Complete list of historical versions of study NCT01115231 on ClinicalTrials.gov Archive Site|
|Current Secondary Outcome Measures
|Original Secondary Outcome Measures||Same as current|
|Current Other Outcome Measures||Not Provided|
|Original Other Outcome Measures||Not Provided|
|Brief Title||Complement Factor H Haplotypes and Smoking in Age-related Macular Degeneration|
|Official Title||Complement Factor H Haplotypes and Smoking in Age-related Macular Degeneration|
|Brief Summary||Risk factors for Age-related Macular Degeneration (AMD) involves genetic variations in the alternative pathway of complement inhibitor factor H. The complement system is part of the innate and adaptive immune system. Smoking is the only environmental factor known to increase the risk of Age-related Macular Degeneration (AMD). Using serum samples of Age-related Macular Degeneration (AMD) patients and controls the investigators will test the hypothesis that smoking increases Age-related Macular Degeneration (AMD) by increasing complement activation; and that this is positively correlated with known disease variations in the complement factor H (CFH) gene.|
ABSTRACT Age-related macular degeneration (AMD) is a slowly progressing multifactorial disease involving genetic abnormalities and environmental insults. AMD is the leading cause of blindness for Americans over the age of sixty. As the population ages, the prevalence of AMD will continue to grow, reaching a maximum risk rate of ~30% by the age of 70 years. Since smoking increases the risk of AMD, and there is a 20% higher incidence of smoking in the Veteran population than in the U.S. adult civilian population, the U.S. Department of Veterans Affairs healthcare system will have to provide care for potentially up to 7 million or more AMD cases. Currently available treatments focus on the late stage of the disease (choroidal neovascularization); however, those treatments come with significant risks and only target subpopulations of AMD patients. No treatment is available for early AMD disease which includes >85% of all cases. Thus, it is of paramount importance that the investigators learn how to detect AMD early and develop treatments that allow for early disease prevention. While mechanistic studies have shown that inflammation and smoking are fundamental components of both the wet and dry forms of AMD, genetic studies have demonstrated that polymorphisms in different complement proteins each increase the risk for developing AMD. One of the most detrimental mutations occurs in factor H, an essential inhibitor in the complement cascade. Overall, it has been hypothesized that inadequate control of complement-driven inflammation may be a major factor in disease pathogenesis in AMD. Here the investigators wish to answer an essential question: do smoking and complement act synergistically in the AMD disease process. For this proposal the investigators will be guided by the investigators' overall hypothesis that pathological activation of the alternative complement pathway has direct effect on the retinal pigment epithelium, generating a permissive cellular environment for AMD pathology. Thus, the investigators will recruit case subjects with AMD and control subjects, selecting both smokers and non-smokers in both groups, to determine whether smoking influences complement activation, or whether smoking acts through a yet undefined pathway to promote AMD development. A complete eye exam to determine pathology and visual impairment will be performed on Day 1 of the study. Serum will be analyzed to measure complement factor H activity. Plasma will be analyzed to measure complement breakdown products such as C3a, C5a and Bb. The cells that remain will be used for genotyping.
RESEARCH DESIGN AND METHODS
A) Study design
This study is designed to determine whether smoking increases complement activation and whether there are specific AMD genotypes that are particularly sensitive to this elevated level of serum complement components.
Thus, the investigators will recruit patients with AMD and age-matched (within 5 years) control subjects, selecting both smokers and non-smokers, to determine whether smoking influences complement activation, or whether smoking acts through a yet undefined pathway to promote AMD development. A complete eye exam to determine pathology and visual impairment will be performed on Day 1 of the study. Serum will be analyzed to measure complement factor H activity. Plasma will be analyzed to measure complement breakdown products such as C3a, C5a and Bb. The cells that remain will be used for genotyping.
Data collection and analysis will be performed by trained professionals who will be masked to the patient evaluation data.
B) Selection of subjects and controls
Case subjects and age-matched (within 5 years) control subjects will be recruited under a protocol approved by the Johnson and DeBakey VA Medical Centers, and the Medical University of South Carolina (MUSC) Human Investigation Review Board. All patients will be provided a written informed-consent form for their signature of acceptance before participation in the study. The case subjects and the control subjects will be derived from military service Veteran populations. The following criteria will be used for inclusion and exclusion, sample size, and recruitment of subjects in the study based on a previous, successful study conducted by the investigators' consultant, Dr. A O Edwards.
Sample Size and Power Estimation
A total of 150 case subjects and 150 control subjects (age-matched within 5 years) will be recruited for this study. This sample size was determined by statistically simulating the study findings 1,000-times using the following assumptions: an alpha level of 0.05; 2-sided hypothesis testing; and an expected distribution across the CC, CT, and TT factor H genotypes of 8.1%, 52%, and 39.9%, respectively, (combining observed data for Caucasians and African Americans ). The investigators also assumed approximately 35% of the case and control subjects would be current smokers (see http://www.mit.edu/people/jeffrey/HarrisVARept97.pdf; a report commissioned by the Department of Veterans Affairs Assistant Secretary for Policy and Planning). Finally, the investigators assumed that smoking status in combination with haplotype status had a multiplicative interactive effect on the measurement parameter of interest (i.e., complement protein Ba, D and C3d levels). That is, smoking was allowed to have a negligible impact on the measurement parameter among those in the low-risk genotype, but those in the heterozygous and homozygous risk genotypes had a 1.5-fold and 2-fold greater measurement, respectively. The simulation also included a direct effect of AMD status (case/control), with AMD cases having higher values than controls. Through this simulation process, the investigators were able to show that the sample size of 150 cases and 150 controls would provide 85% power to detect a significant smoking by genotype interaction, the main focus of this study. Note that the investigators' sample size is comparable to similar prior studies, one of which used 112 AMD patients and 67 controls , and another that used 274 samples  and was conducted by the investigators' collaborator, Dr. Edwards.
Case and age-matched (within 5 years) control subjects will be recruited. Recruitment will take place in two ways: 1) If patients that have been seen at the VA within the last two years, have been diagnosed with AMD or qualify as a control subject, they will be called to see whether they wish to participate in the study. Those subjects wishing to participate will be given a consent form to read and sign at their next VA visit. They will then be asked to provide information about their smoking status, and a blood sample (two 3 mL tubes) will be collected. 2) Patients will also be recruited after the diagnosis in the doctor's office (i.e., they have been diagnosed with AMD or qualify as a control subject). Upon signed consent, these subjects will also be asked to provide information about their smoking status, and a blood sample (two 3 mL tubes) will be collected. Flyers will be posted at the VA to alert patients of this clinical trila and flyers will be made available to ophthalmologists in the area to provide to their patients.
C) Clinical tests in the diagnosis of AMD
Flowchart of patient medical history workup
Eye examination, including Snellen visual acuity with pinhole testing
Confrontational visual field
The examiner will ask the patient to cover one eye and stare at the examiner. The examiner will then move his/her hand out of the patient's visual field and then bring it back in. The patient will signal when the hand comes back into view. This will be frequently done by the examiner as a simple and preliminary test.
Intraocular pressure will be measured with a Tonopen in the central cornea in both primary-gaze and up-gaze.
Eyes will be dilated (mydriasis with one drop of each (2.5% phenylepinephrin and 1% tropicamide) to achieve at least 6 mm pupil diameter, and the back of the eye examined by slit-lamp.
OCT images will be acquired with a Stratus-OCT imaging system (Carl Zeiss Meditec). Images will be obtained using the high-resolution "Radial Lines" protocol of six high-resolution B-scans (transverse resolution of 512 A-scans per B-scan). Total retinal volume values will be provided by the "Retinal Map Analysis" program of the Stratus software. Data will be analyzed qualitatively or based on the automated measurements provided by the Stratus-OCT software. Choroidal neovascularization (CNV) size will be quantitatively measured, including the maximum CNV diameter as well as the maximum CNV thickness. Retinal structure will be qualitatively judged as (1) wet, revealing unchanged fluid conditions; (2) dry, revealing no fluid; or (3) less edema, revealing incomplete fluid regression.
The fluorescein angiogram will contain stereoscopic views of 2 fields at specified times after injection. These fields include the macula (Field 2) and the disc field (Field 1). Stereoscopic red-free photographs will be taken of the macula prior to the injection of the fluorescein dye. Fluorescein is injected rapidly (less than 5 seconds) into either the anticubital or other convenient vein according to usual clinic protocol. Photographs will be taken at time zero and at the moment the injection is complete as control photographs, to document the integrity of the interference filters and to document the rate of injection. Stereo pairs of Field 2 and then of Field 1M of the study eyes will be taken between minutes 1 and 3, followed by two additional pairs at 5 minutes and 10 minutes.
Two tubes (3 mL each) of whole blood will be collected in the Johnson or DeBakey VA Medical Center by the laboratory services personnel and processed to the freezer (-80 C) within 3 hours from collection. One tube will be collected and allowed to clot. Serum will be separated from this clot by centrifugation (10 minutes at 3,000 rpm) and frozen at -80 degrees centigrade until analyzed. Serum will be used to measure complement factor H activity. The other tube will contain dipotassium ethylenediaminetetraacetic acid (EDTA), resulting in a final EDTA concentration of 4.5 mM when the blood is added. Plasma will be separated from this whole blood by centrifugation (10 minutes at 3,000 rpm) and frozen at -80 degrees centigrade until analyzed. Plasma will be used to measure complement breakdown products. The cells that remain will be used for genotyping assays.
ELISA protein analyses
ELISA examinations have been described in detail by the investigators' collaborator, Dr. Edwards ; however, the protocol is described here in brief.
Assessment of smoking
Regular cigarette smoking will be assessed by a brief questionnaire administered by clinic staff, to determine whether the subject has ever smoked on a regular basis and whether he/she is a current smoker; and if so, their pack/month and pack/year history.
D) Outcome measures
The incidence of AMD will be defined based on the published definition of the Rotterdam Study, a population-based prospective cohort study in Rotterdam, The Netherlands, in which 6,780 out of a total population of 10,275 participants over the age of 55 years, participated in ophthalmic examination .
The fundus photographs will be graded at 12.5x magnification, according to the International Classification and Grading System for ARM and AMD (The International ARM Epidemiological Study Group, 1995). In this system, all ARM fundus signs within a standard circular area (diameter 6,000 m) around the fovea will be recorded. Graders, trained according to the Wisconsin ARM grading system will grade the photographs blinded to the patient information.
Additional outcome measurements
Additional outcome measures that will help characterize the severity of AMD disease include the following. In the visual field test, the loss of vision occurring predominantly in the central retina, will be quantified. The choroidal and retinal vasculature will be analyzed using fluorescein angiograms, which should reveal abnormal blood vessels and leakage in subjects with systemic vascular problems.
E) Data analyses
F) Predicted results and interpretation
Published results, as well as data from the investigators' collaborator, Dr. Edwards, have provided ample evidence that AMD is correlated with increased serum levels of Ba, C3d and fD; whereas elevated levels of Ba and C3d were documented in V62I subjects. The investigators expect to confirm and extend the findings in V62I subjects, and to identify a positive correlation between elevated levels of complement activation proteins and Y402H. Since smoke exposure has been shown to result in increased levels of alternative pathway (AP) activation , the investigators predict that some components (i.e., C3d or Ba) might be further elevated in the factor H risk population that smokes.
G) Potential risks
Subjects will receive a comprehensive eye examination as part of this study. This examination will include visual inspection by a trained ophthalmologist, assessment of visual acuity, SD-OCT, visual fields, and fluorescein angiography. The subjects and their physicians will be made aware of any identified abnormalities. The potential risk to subjects is modest. All of the planned tests are identical to those test routinely performed for purposes of diagnosing AMD or other diseases of the eye. Such studies are performed routinely in out-patient settings by ophthalmologists. In addition, information obtained from this study may provide an important link to understanding the pathogenesis of AMD, and its link with complement factor H haplotypes and smoking. Any relationship between the three could potentially lead to future therapies for AMD.
There are several things participants should know before allowing the blood to be studied or to be saved.
These are the best-known risks and challenges of genetic research. There might be other risks the investigators do not know about yet. It is important that the participants talk to their doctor, nurse, or genetic counselor if there are any questions or concerns about this research study.
- Unknown risks. The researchers will let the participants know if they learn of anything that might make a change of mind about participating in this study.
H) Literature cited
|Study Design||Observational Model: Cohort
Time Perspective: Prospective
|Target Follow-Up Duration||Not Provided|
|Biospecimen||Retention: Samples With DNA
Whole blood will be collected into dipotassium ethylenediaminetetraacetic acid (EDTA) tubes resulting in a final EDTA concentration of 4.5 mM. Plasma will be separated from whole blood by centrifugation (10 minutes at 3000 RPM) and frozen at -80 degrees C until further use. Subjects will be genotyped for signal nucleotide polymorphisms for I62V and Y402H using TaqMan single nucleotide polymorphisms (SNP) genotyping assays. Complement pathway protein analysis will be performed by ELISAs.
|Sampling Method||Probability Sample|
|Study Population||The case and control subjects will be derived from a group of Veterans at the Charleston, SC VA Medical Center.|
|Publications *||Not Provided|
* Includes publications given by the data provider as well as publications identified by ClinicalTrials.gov Identifier (NCT Number) in Medline.
|Recruitment Status||Unknown status|
|Original Estimated Enrollment||Same as current|
|Estimated Study Completion Date||December 2016|
|Actual Primary Completion Date||June 2015 (Final data collection date for primary outcome measure)|
|Ages||40 Years to 80 Years (Adult, Older Adult)|
|Accepts Healthy Volunteers||Yes|
|Contacts||Contact information is only displayed when the study is recruiting subjects|
|Listed Location Countries||United States|
|Removed Location Countries|
|Other Study ID Numbers||C7428-R|
|Has Data Monitoring Committee||No|
|U.S. FDA-regulated Product||Not Provided|
|IPD Sharing Statement||Not Provided|
|Responsible Party||VA Office of Research and Development|
|Study Sponsor||VA Office of Research and Development|
|PRS Account||VA Office of Research and Development|
|Verification Date||January 2016|