Complement Factor H Haplotypes and Smoking in Age-related Macular Degeneration
Condition: Macular Degeneration
MeSH terms: Macular Degeneration
Study type: Observational

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.
    

Detailed description: 
      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.

      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.

      Inclusion Criteria

        -  Case subjects with a clear diagnosis of AMD and at least a 20/40 view of the fundus.

        -  Control subjects with <5 small hard drusen and at least a 20/40 view of the fundus.

        -  All subjects will have the ability to provide a blood sample, demonstrate the absence of
           exclusion criteria listed below, provide their own consent, or have a legal
           representative available to provide consent for them, able to complete all aspects of
           testing, and be in generally good medical health in the opinion of the study physician.

      Exclusion Criteria

        -  Individuals who are unable to provide consent and who lack a legal representative, whose
           best corrected visual acuity for both eyes is worse than 20/40, who are taking a
           medication known to cause retinopathy, unable to cooperate to complete testing, and who
           present themselves with media opacity, 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.

        -  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 will
      be recruited. Sample size was determined by statistically simulating the study findings 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, [1 and assuming ~35% of the subjects being current smokers. This sample size
      would provide 85% power to detect a significant smoking by genotype interaction, the main
      focus of this study.

      Recruitment Case and age-matched (within 5 years) control subjects will be recruited.
      Recruitment will take place in two ways: 1) they will be called or recruited after the
      diagnosis in the doctor's office. 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.

      C) Outcome measures Incidence of AMD will have been determined in the prior clinical visit
      based on Fundus photographs and accepted AMD definitions. Additional outcome measurements
      that will help characterize the severity of AMD disease might include the visual field test,
      OCT and fluorescein angiograms.

      D) Data analyses

        -  Data assembled as normalized serum levels of complement factors Ba, C3d and fD and fH
           activity 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, genotype
           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.

      E) Potential risks

        -  Subjects will have received a comprehensive eye examination that was not part of this
           study. The potential risks cannot be described.

        -  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.

        -  Blood samples will be stored under an alphanumeric identifier which could eventually be
           linked to the participants.

        -  In addition to name, other information about participants might be connected to blood
           samples.

        -  Genetic information about the participants' will often apply to family members.

        -  The participants have the right to refuse to allow their blood to be studied or saved
           for future research studies.

        -  South Carolina law mandates that genetic information, obtained from any tests or from
           this research, be kept confidential.

        -  Genetic research raises difficult questions about informing participants and other
           subjects of any results, or of future results.

        -  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.

        -  The presence of a genetic marker for a disease does not necessarily mean that the
           participants will develop that disease.

      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.
    

Observational model: Cohort
Time perspective: Prospective
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.
      
Biospecimen description: 
      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.
    
Keywords: Age-Related Macular Degeneration; Age-Related Maculopathies; Age-Related Maculopathy; Macular Dystrophy; Maculopathies, Age-Related; Maculopathy, Age-Related