never be the subject of a dedicated study because of a perceived lack of value due to the high cost of genetic association studies and the low rate of uncorrectable vision loss due to mild severity or the availability of surgical interventions. A great deal of work goes into the ascertainment of subjects for disease-specific case-control studies. Some of this effort is specific to each disease. However other factors such as fundus photographs of the macula in studies of AMD can also be used to study the retinal vasculature (e.g., occlusions) and optic nerve (e.g., glaucoma). Similarly, activities such as consent, eye examination, phlebotomy, isolation of genomic DNA, and genotyping would apply to all traits initially. Environmental exposures such as tobacco smoking and body mass index alter the risk of multiple eye diseases and the effort to collect this information would be applicable to a number of complex traits affecting the eye [20–24]. Certainly, there are complex traits that would require collection of unique medical history information such as asking about atopic dermatitis in a study of retinal detachment; however, most of the answers would be negative and our impression is that the additional work is probably modest. Nonetheless, the documen- tation of other traits and biomorphic variables would modestly increase the cost of ascertainment, while also greatly increasing the value of the study. One might argue that the use of clinic-based case-control studies should be minimized in favor of population-based case-control cohorts or family studies as more robust designs for avoiding possible confounding and biases [25,26]. The major limitation of using these methods is the expense of identifying large numbers of subjects with and without the full spectrum of the primary trait. While population-based case-control studies may be ideal for gene discovery, such studies are rarely available because the full expense of the recruitment, diagnoses, and documentation must be covered by the research program. Family-based association is an excellent strategy for early-onset diseases, but is difficult to apply to age- related disease where the parents of the proband are often deceased [27]. Thus, clinic-based, case-control studies are a valuable tool for gene discovery because large numbers of subjects can be rapidly and efficiently ascertained and evaluated in a systematic manner. They are likely to continue to be a primary method used in genetic association studies of ocular traits. The purpose of this study was to better understand the other complex ocular traits affecting the aging eye in the types of clinic- based, case-control studies currently being used by us and others to study the genetics of AMD and other traits. To this end, we documented the density of complex traits present in 100 consecutive patients age 60 or older attending a referral-based vitreoretinal clinic. This information was used to explore the additional information that might be gained by considering secondary ocular traits in genetic-epidemiology studies. The study revealed that most patients age 60 older have greater than 3 distinct diseases affecting their eyes, that most of these diseases were common ocular traits, and that they were typically present at a similar or higher frequency as population estimates. Controls without any other common and potentially hereditary eye diseases were rare. Rather than ignoring the potential confounding of secondary traits, we propose an alternative strategy in which subjects selected based on the presence of a disease be characterized for all other common diseases within that organ system. This approach would enable the detection of genetic risks for secondary traits while accounting for any stratification, confounding, or association between traits and other variables using standard statistical methods. The proposed ‘‘mul- tiple-trait case-control study’’ employing several binary traits is analogous to traditional cohort studies in which the incidence of multiple quantitative traits are ascertained. These observations are Density Complex Ocular Traits important for the design of large-scale genome-wide association studies studying ocular traits and possibly for the study of other organ systems. Results Density of Complex Ocular Traits In order to determine the density (number of traits per patient) of traits affecting the aging eye, the systematically documented diagnoses of 100 consecutive patients seen in the clinic for the first time for any reason from one retinal specialist were extracted from the medical record (Table 1). An average of 3.5 distinct diagnoses was observed in the 100 consecutive patients (Table 2). The 10 common complex traits listed in Table 1 accounted for 2.5 of these distinct diagnoses (Table 2). Only one of the 100 patients did not have at least 1 of the 10 common complex traits listed in Table 1. The density of complex traits in the 45 males was 2.5 compared to 2.6 in the 55 females (P = 0.70, Poisson regression). Effect of Age on Density of Complex Ocular Traits A scatter plot of year of age versus the density of the ten complex ocular traits listed in Table 1 for each patient shows a trend toward an increase in density with age (Figure 1A). Poisson regression modeling showed an effect of age (P = 0.03) and estimated a rate of increase of 0.01760.0074 log-number of complex traits per year across the age range represented in this study (ages 60–94). Using logistic regression, AMD (P,0.0001) and glaucoma (P = 0.01) were observed to increase with age (Figure 1B), while the frequency of posterior vitreous detachment (PVD) and vitreomacular traction (VMT) were not influenced by age. An effect of age was not observed for the combined frequency of the 8 complex traits remaining after removing AMD and glaucoma from the analysis (P = 0.66). Gender-Trait, and Trait-Trait Associations Exploratory analyses were performed looking for gender-trait and trait-trait associations. Fisher’s exact test was used to test for association with gender for all traits with at least 5 patients with the trait and 5 patients without the trait. No gender-trait association was observed for Fuchs, AMD, glaucoma, PVD, VMT, retinal vascular occlusion (RVO), or retinal tears. The low frequency of conditions thought to be associated with gender (e.g., VMT), may explain the failure to observe an association. Trait-trait associations were assessed for each of the ten complex traits having at least 10 patients with and 10 patients without the trait (AMD, Glaucoma, PVD, VMT). The only significant association was AMD with VMT (P = 0.0009) in which VMT was protective. The distribution was as follows: AMD/VMT 4, AMD/No VMT 50, No AMD/VMT 16, and No AMD/No VMT 30. Since both eyes were always examined, it is unlikely that the VMT obscured the diagnosis of AMD, which usually affects both eyes. This observation probably reflects a referral bias in which patients with AMD and VMT are not referred for surgery for VMT, thus leading to a decrease in the number of subjects with AMD and VMT. Modeling of power to detect association with secondary traits in a genetic association study We sought to determine the odds-ratio of a genetic effect that could be detected in a case-control study based on this clinic-based study. Using the frequencies of traits observed in the 100-person sample, we extrapolated to a similar hypothesized clinic-based sample of 2000 subjects. The 2000 subjects effectively form a cross-sectional study, where one defines diseased and not-diseased PLoS ONE | www.plosone.org 2 June 2008 | Volume 3 | Issue 6 | e2510