Chantaren et al Initial studies on the association between these two genes and PCV among Asians have shown contradictory results. Although the A69S SNP has been widely found to exhibit a strong association with PCV in many Asian populations, studies in Japanese, Chinese and Korean populations could not find a significant link between Y402H and AMD.6–8 Interestingly, few studies revealed an association between PCV and the I62V polymorphism of another locus in the same CFH gene. Recent studies in a Japanese population and a cohort of subjects of European-American descent in the United States, however, have shown a strong association between Y402H and PCV.9,10 The majority of these data have been obtained from East Asian populations, whereas data from other Asian countries are scarce. This study was conducted to validate the associa- tions between the three SNPs, Y402H and I62V in CFH and A69S in the ARMS2 genes, and PCV in a Thai population. Since the Asian population comprises various ethnic groups that may differ in physical appearance, lifestyle, culture, religion, socioeconomic status, and genetic background,11 the results were compared with the compiled data of studies from other Asian populations. The results obtained in this study should provide an insight into the genotypic manifes- tations of PCV with additional data from a Southeast Asian population. Methods Participants This study was conducted under approval of the Ethical Committee on Research Involving Human Subjects of Rajavithi Hospital, Bangkok, Thailand. All participants gave written informed consent. A total of 119 patients with PCV and 173 controls were enrolled at the Eye Clinic of the same institute. The inclusion criteria for PCV were: (1) Thai native; (2) having clinical manifestations of PCV, such as the pres- ence of orange-red subretinal nodules, hemorrhagic retinal pigment epithelial detachment, subretinal hemorrhage, or intraretinal or subretinal fluid with exudates; and (3) having a branching vascular network with terminal polypoidal dilation of choroidal vessels identified by indocyanine green angiography (ICGA). The polyps identified in ICGA should correspond with the orange nodules found in clini- cal examination. The inclusion criteria for controls were: (1) Thai native; (2) age $50 years; (3) absence of serious systemic or known genetic-influenced diseases such as can- cer, diabetes mellitus, and glaucoma; and (4) clinical absence of diseases of the vitreous, retina, retinal pigment epithelium 466 submit your manuscript | www.dovepress.com Dovepress Dovepress and choroid, especially drusen, or retinal pigment epithelial abnormality. The exclusion criteria for both groups were: (1) relatives of any enrolled patients with PCV or controls, (2) any ethnicity other than Thai, (3) any participants whose parents were not born in Thailand, and (4) any participants who did not sign the consent form. Each participant underwent a comprehensive ophthalmic evaluation using a slit lamp biomicroscope including dilated fundus examination and color fundus photography. All participants with PCV further underwent optical coherent tomography and simultaneous fluorescein and ICGA using a scanning laser ophthalmoscope (Heidelberg Retinal Angiography II®, Heidelberg, Germany) to confirm the diagnosis. genotyping The genomic DNA was extracted from buffy coat using Compact Nucleic Acid Isolation Kit (MagNA Pure® Compact System, Roche Diagnostics, Mannheim, Germany). DNA concentration was measured and diluted to the final concen- tration of 50 ng/µL using a spectrophotometer. The genotyping of the rs1061170 (Y402H), the rs800292 (I62V), and the rs10490924 (A69S) polymor- phism were performed in a 480 LightCycler® instrument (Roche Diagnostics) using a commercial real-time assay (LightMix®, Roche Diagnostics). A 10 µL reaction mix- ture was prepared in a 96-well polymerase chain reaction (PCR) plate containing 50 ng genomic DNA, 0.5 µL for probe-primers mix (LightMix for the detection of human SNPs, TIB MOLBIOL GmbH, Berlin, Germany), 1 µL 2X LightCycler® FastStart DNA Master HybProbe (Roche Diagnostics), 2.7 µL deoxyribonuclease (DNase)-free water (PCR-grade), and processed according to the manufac- turer’s instructions (Roche Diagnostics). Real-time PCR of each SNP was performed by an initial denaturation at 95°C for 10 minutes, followed by 45 cycles of 95°C for 10 seconds, 60°C for 10 seconds, and 72°C for 15 seconds. In order to determine the melting points after the amplifica- tion phase, a melting curve analysis was performed at 95°C for 30 seconds and at 40°C for 20 minutes, followed by a slow heating at the rate of 0.2°C per second until reach- ing 75°C. The polymorphism was determined to have a specific melting point of 55°C for the risk and 62.5°C for the nonrisk allele. In addition, nine blood samples were randomly selected for DNA sequencing analysis (Macrogen®, Seoul, Korea) to confirm the genotyping results. Clinical Ophthalmology 2012:6