Complement Factor H Haplotypes and Smoking in Age-related Macular Degeneration

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.

Inclusion Criteria

1. Case subjects will have a clear diagnosis of AMD and at least a 20/40 view of the
fundus.

2. Control subjects will have <5 small (<63 um each) hard drusen and at least a 20/40 view
of the fundus.

3. All subjects will have the ability to provide a blood sample.

4. All subjects will demonstrate the absence of exclusion criteria listed below.

5. All subjects must be able to provide their own consent, or have a legal representative
available to provide consent for them.

6. All subjects must be able to complete all aspects of testing, or have a legal
representative available to complete all aspects of testing for them.

7. All subjects must be in generally good medical health in the opinion of the study
physician.

Exclusion Criteria

1. Individuals who are unable to provide consent and who lack a legal representative.

2. Individuals whose best corrected visual acuity for both eyes is worse than 20/40.

3. Individuals who are taking a medication known to cause retinopathy.

4. Individuals unable to cooperate to complete testing.

5. Individuals who present themselves with media opacity (cataract) that will prevent
visualization of the macula; or ocular disease that might simulate AMD or preclude its
diagnosis (e.g., prior laser photocoagulation, cryopexy, media opacity, or inflammatory
diseases).

6. Individuals who exhibit diseases that phenotypically overlap with AMD such as drusen or
pigmentary disturbance of the retinal pigment epithelium; or provide insufficient
evidence to diagnose AMD.

7. Individuals who present themselves with macular dystrophies, toxoplasmosis,
histoplasmosis, degenerative myopia, central serous chorioretinopathy, or any disease
or treatment that would diminish the ability to recognize drusen such as laser
photocoagulation, prior retinal detachment surgery, posterior uveitis, and trauma.

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 [1]). 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 [2], and another that used 274 samples
[3] and was conducted by the investigators' collaborator, Dr. Edwards.

Recruitment

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

1. Chief complaint of vision changes.

2. History taken of vision changes. If AMD-related symptoms (blurry vision,
metamorphopsia, scotoma, etc.), the approximate time it started in the affected eye,
particularly noting whether the patient is taking vitamins (specific type and dosing),
and whether previous treatments for AMD have been provided (type, dosing, frequency).

3. If any other eye problems or disease, stroke or heart attack history, surgery planned
for the future, rheumatologic disease, and kidney disease (MPGN-II).

4. Any family history of AMD.

5. Medications currently taking.

6. Social history, including smoking behavior (years smoked and packs/ week and packs/
year.

Eye examination, including Snellen visual acuity with pinhole testing

1. Pupillary exam, followed by confrontational visual fields or visual field test to
determine the amount of vision loss.

2. Intraocular pressure assessed by Tonopen tonometry.

3. Pharmacologically-dilated pupils using phenylephrine 2.5% and 1% tropicamide.

4. Slit-lamp examination including non-contact-lens exam of the macula and peripheral
retina to determine anatomical alteration of the fundus.

5. If recent significant vision changes have occurred, or blood, exudate or subretinal
fluid were noted on the fundus exam, ancillary testing including macular SD-OCT (both
eyes) and fluorescein angiography (both eyes) will be performed with arterial phase of
the eye of most concern.

Treatment options

1. If active choroidal neovascular membranes are found on examination and ancillary
testing, treatment options (anti-VEGF-based therapies such as Lucentis, Macugen,
Avastin) will be discussed.

2. If intermediate AMD is determined in both eyes, or advanced AMD in one eye, treatment
options (the AREDS vitamin supplements, and the need to self-monitor central vision
changes) will be discussed.

Tests

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.

Tonometry

Intraocular pressure will be measured with a Tonopen in the central cornea in both
primary-gaze and up-gaze.

Eye exam

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 imaging

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.

Fluorescein angiography

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.

Biospecimens

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 [3]; however, the protocol is described here in brief.

- Factor B will be quantified using a monoclonal capture antibody P21/15 which recognizes
an epitope present on factor B and Ba and a biotinylated rabbit anti-Bb as a detection
antibody. For measurement of Ba, plasma will be depleted of factor B using Biomag
magnetic beads coupled to an anti-B/Bb antibody (mAb M13/12), followed by Ba
quantification using an anti-B/Ba antibody (mAb P21/15) as a capture antibody and a
biotinylated antibody specific for an epitope on B/Ba (mAb M20/6) as a detection
antibody.

- Factor D will be measured by paired capture and detection antibodies (mAb D10/4 and
biotinylated mAb I8/1) specific to factor D. The capture antibody used in the C3d assay
(mAb I3/15) reacts with a neoepitope present on C3b, iC3b and C3dg and will be in
combination with a biotinylated rabbit anti-C3d detection antibody for quantification
of C3d. For all assays, incubation with the detection antibody will be followed by
application of streptavidin-horseradish peroxidase conjugate. The substrate, 2 mM ABTS
(2,2'-azino-di-(3-ethyl benzthiazolinesulfonate)) in buffer containing 2.5 mM peroxide,
will be added and absorbance at 410 nm (490 nm reference) will be read using a
microplate photometer. All samples will be run in triplicate.

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

Incidence

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 [4].

Fundus photographs

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.

AMD definitions

- AMD will be defined as the presence of large ( 63 um), soft, distinct drusen with
pigmentary irregularities, or indistinct (125 um) or reticular drusen, or atrophic or
neovascular AMD. Atrophic AMD will be defined as any sharply demarcated round or oval
areas of apparent absence of the retinal pigment epithelium larger than 175 um,
irrespective of the distance from the fovea, but within the grid, with visible
choroidal vessels and no neovascular AMD.

- Neovascular AMD will be defined as the presence of a serous or hemorrhagic
neuro-retinal or retinal pigment epithelium detachment, and/or a subretinal neovascular
membrane, and/or a subretinal hemorrhage, and/or a peri-retinal fibrous scar. Lesions
that are considered to be the result of generalized disease (see exclusion criteria)
will be excluded from AMD diagnosis.

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

- Data assembled as normalized serum levels of complement factors Ba, C3d and fD levels
will be initially evaluated with univariate statistics to assure that the quality of
the data is adequate for further analyses. The association between each measured
parameter (i.e., serum levels of complement factors), AMD diagnosis, and smoking will
be assessed in a stratified bivariate fashion using Student t tests or Wilcoxon rank
sum tests, as appropriate, and standard measure assessments will be used to check for
normality, skewing, etc.

- Multivariate analyses will be conducted through the use of general linear mixed models
[5]. The models will include random subject effects to account for dependence among
repeated measurements of subjects. This type of model is ideal when there are multiple
measurements on subjects, such as when laboratory measurements are performed in
triplicate. The dependent variables of interest will be the complement level
measurements (log transformed, if necessary), while independent variables will include
AMD status (case/control), factor H genotype (CC, CT, TT), smoking (current, former,
never), and an interaction-term involving factor H genotype and smoking status. The
interaction-term will help us to determine whether the impact of factor H genotype and
smoking on serum complement levels is linear (additive), or non-linear (e.g.,
multiplicative). The model will also include adjustments for age, gender, and race,
which may all affect complement factors. Thus, any differences among haplotypes will be
adjusted (corrected) for effects that may be attributed to age, gender, or race.
Different correlation structures will be examined for the random subject effects, and
the investigators will use Akaike's Information Criterion to select the most
appropriate model. Secondary analyses will involve excluding never smokers, to assess
the nature of the association (if any) between pack/year histories. factor H genotypes.
and their complement levels. An additional analysis (using conditional logistic
regression) will be conducted to determine whether smoking interacts with a subject's
factor H genotype with respect to the risk of AMD. Again, this model will be adjusted
for age, gender, and race, and the results will be expressed as odds ratios associated
with the risk of AMD.

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 [6], 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.

- Visual field testing. The risks of visual field testing include fatigue of the neck;
pain in the neck; pressure under the chin; sweating; photophobia; or tearing. None of
these are permanent or substantial losses. Change in position or rest will resolve the
complication.

- Tonometry. The risks of tonometry include fatigue of the neck; pain in the neck;
pressure under the chin; or feeling of temporary pressure against the eye. None of
these are permanent or substantial losses. Change in position or rest will resolve the
complication.

- Pupil dilation. The risks of pupil dilation include fatigue of the neck; pain in the
neck; pressure under the chin; sweating; photophobia; acute angle-closure lasting for
less than 30 minutes, due to the dilating drops; conjunctival injection (red eye), due
to the dilating drops; impaired near vision for less than 24 hours, due to dilating
drops; burning for less than 5 minutes, due to the dilating drops; or anesthesia of the
eye for less than 2 hours, due to the dilating drops. None of these are permanent or
substantial losses. Rest and time will resolve the complication.

- Stratus-OCT. The risks of Stratus-OCT imaging include fatigue of the neck; pain in the
neck; pressure under the chin; sweating; photophobia; or tearing. None of these are
permanent or substantial losses. Change in position or rest will resolve the
complication.

- Fluorescein angiography. The risks of fluorescein angiography include pain from the
needle stick; inflammation at the injection site; allergy to the fluorescein dye;
feeling of warmth or cold, due to the dye; infection at the site of the needle stick;
anaphylactic reaction to the dye; nausea (20% of cases); vomiting (0-7% of cases);
headache; hypotension; convulsions; basilar artery ischemia; cardiac arrest; severe
shock; or thrombophlebitis at the injection site. Clinical staff are trained to deal
with these complications.

- Venipuncture for whole blood. The risks of venipuncture are pain or bruising at the
site of venipuncture; fainting or dizziness; or infection at the site of the needle
stick. None of these are permanent or substantial losses. Clinical staff are trained to
deal with these complications.

- Personal information. Sharing of personal information (blood specimens, personal
history, genetic information, etc.) is not without risk. Research to identify genes
that cause or contribute to a disease or trait is an increasingly important way to try
to understand the role of genes in human disease. The participants were given a consent
form because the Johnson and DeBakey VA Medical Centers investigators want to include
the participants' blood sample in a research project, or because they want to save such
biological specimens for future research.

There are several things participants should know before allowing the blood to be studied or
to be saved.

1. Blood samples will be stored under an alphanumeric identifier which could eventually be
linked to the participants. Sometimes these samples are shared for research purposes
with other investigators at other research sites. If this is done, the other
investigators would not know the participants' name.

2. In addition to name, other information about participants might be connected to blood
samples. For instance, information about race, ethnicity, sex, medical history, and so
forth might be available to investigators studying the blood. Such information is
important for scientific reasons and sometimes for public health. It is possible that
genetic information might come to be associated with the participants' racial or ethnic
group.

3. Genetic information about the participants' will often apply (to one degree or another)
to family members. It is not generally the Johnson or DeBakey VA Medical Center's
policy to provide information about the participants' to family members. However,
certain studies called "pedigree studies", share such information among family members.
For this and related research, the participants will be asked if they are willing to
share genetic information with family members.

4. The participants have the right to refuse to allow their blood to be studied or saved
for future research studies. The participants may withdraw from this study at any time
and remove any samples that contain identifiers from research use after the date of
withdrawal. This means that while the Johnson or DeBakey VA Medical Centers or Medical
University of South Carolina might retain the identified blood samples - the law often
requires this - the samples would not be used for research.

5. South Carolina and Texas law mandates that genetic information, obtained from any tests
or from this research, be kept confidential. South Carolina and Texas law prohibits any
insurer using this information in a discriminatory manner against the participants or
any member of their family in issuing or renewing insurance coverage for the
participants or family. South Carolina and Texas law further prohibits the
investigators from sharing genetic information with anyone except in a few narrow
circumstances, one of these being a research project of this type, approved by the
Institutional Review Board, and then the investigators must take all steps to protect
the participants' identity. The participants will still be responsible for paying for
health care, however. The Charleston or DeBakey VA Medical Centers will not be
responsible for such costs, even if care is provided for a condition revealed during
research or clinical testing.

6. Genetic research raises difficult questions about informing participants and other
subjects of any results, or of future results. Some people feel anxious about the
possibility of having a defective gene that would place them or their children at risk.
Some people want to know what is found out about them, others do not. The risks of
knowing include anxiety and other psychological distress. The risks of not knowing what
is found include not being aware if there is treatment for the problem being studied.
But these risks can change depending on whether there is a treatment or cure for a
particular disease, and on how clear the results are. If there is a medical reason to
seek specific information from the participants, their doctor will tell them of this. A
process called "genetic counseling" is often appropriate in such cases; the
participants should ask their doctor about this if there are any questions.

Investigators in this study may try to recontact the participants in the future to find
out about their health. If the participants are recontacted and want to know what the
investigators have learned about their samples, the participants should understand that
the following are the kind of things the investigators or the participants' health team
might say:

1. The information is too sketchy to give particular details at this time, but the
participants will receive a newsletter informing them about the results of the
project.

2. The participants carry a gene for a particular disease that can be treated.

3. The participants carry a gene for a particular disease for which there is no
current treatment. This news might cause severe anxiety or other psychological
distress, depending on the severity of the disease.

4. The participants may carry a gene for a disease and might consider informing
relatives that they, too, might carry the gene. It can be very difficult to decide
whether to share such information with relatives. Genetic counselors can help sort
out the various options in such a case.

Also for any future research, the participants may be recontacted with a new consent
form giving the participant additional information for a new study.

7. If the participants are concerned about a potential genetic disorder, the participant
and their doctor might choose to test specifically for it. This would require
additional blood or tissue samples and would not be part of this research project.
Participants should discuss this option with their doctor or genetic counselor.

8. The presence of a genetic marker for a disease does not necessarily mean that the
participants will develop that disease. Informing people of all such markers
independent of medical need can cause unnecessary anxiety. On the other hand, the
absence of a marker does not mean that the participants will not get the disease.
Genetic diseases appear as a result of a complex mixture of heredity, environment,
behavioral and other factors.

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

1. Weale R. (2006) Lancet 368, 1235-1236.

2. Bailey TA, Kanuga N, Romero IA, Greenwood J, Luthert PJ, Cheetham ME. (2004) Invest
Ophthalmol Vis Sci 45, 675-684.

3. Thurman JM, Renner B, Kunchithapautham K, Ferreira VP, Pangburn MK, Ablonczy Z,
Tomlinson S, Holers VM, Rohrer B. (2009) J Biol Chem 284, 16939-16947.

4. van Leeuwen R, Chakravarthy U, Vingerling JR, Brussee C, Hooghart AJ, Mulder PG, de
Jong PT, (2003) Ophthalmology 110, 1540-1544.

5. Schmidt-Erfurth U, Michels S, Michels R, Aue A. (2005) Eur J Ophthalmol 15, 482-485.

6. Husain D, Ambati B, Adamis AP, Miller JW. (2002) Ophthalmol Clin North Am 15, 87-91.

Inclusion Criteria:

- Inclusion criteria for subjects will be a clear diagnosis of Age-related Macular
Degeneration (AMD)

- Inclusion criteria for controls will be less than five small (< 63 um) hard drusen

- At least a 20/40 view of the fundus

- The ability to provide a blood sample, and the absence of exclusion criteria listed

Exclusion Criteria:

- The investigators will exclude individuals with ocular diseases that might simulate
Age-related Macular Dengeration (AMD) or preclude its diagnosis.

- Those might include prior laser photocoagulation, cryopexy, media opacity, and
inflammatory diseases.

- It is important for potential control subjects not to exhibit media opacity (e.g.,
cataract), which will prevent visualization of the macula.

- Also, subjects will be excluded if they exhibit diseases that phenotypically overlap
with Age-related Macular Degeneration (AMD) such as drusen or pigmentary disturbance
of the retinal pigment epithelium (RPE), or that provided insufficient evidence to
diagnose Age-related Macular Degeneration (AMD).

- In addition, subjects with pattern dystrophies, toxoplasmosis, histoplasmosis,
degenerative myopia, central serous chorioretinopathy, or any disease or treatment
that would diminish the ability to recognize drusen such as laser photocoagulation,
prior retinal detachment surgery, posterior uveitis, and trauma will be excluded.