For all 3 genes, polymerase chain reaction (PCR) was used to amplify genomic DNA fragments from 20 ng of leukocyte DNA in a solution of 10× PCR buffer containing 25mM magnesium chloride, 0.2mM cytidine 5-triphosphate, 0.2mM deoxyadenosine triphosphate, 0.2mM deoxyguanosine triphosphate, 0.2mM deoxythrombotic thrombocytopenic purpura, and 0.5 U of Taq DNA polymerase (USB Corporation, Cleveland, Ohio). For APOE and ELOVL4, 5 M of betaine (Sigma-Aldrich Inc, St Louis, Mo) was added to each PCR. The temperatures used during PCR were as follows: for APOE, 95°C for 5 minutes, followed by 35 cycles at 66°C for 30 seconds, 72°C for 30 seconds, and 95°C for 30 seconds, with a final annealing at 66°C for 1.5 minutes and extension at 72°C for 5 minutes; and for CFH and ELOVL4, 95°C for 5 minutes, followed by 35 cycles at 56°C for 30 seconds, 72°C for 30 seconds, and 95°C for 30 seconds, with a final annealing at 56°C for 1.5 minutes and extension at 72°C for 5 minutes. For sequencing reactions, PCR products were digested according to the manufacturer's protocol using ExoSAP-IT (USB Corporation) and were then subjected to a cycle sequencing reaction using the Big Dye Terminator version 3.1 cycle sequencing kit (Applied Biosystems, Foster City, Calif) according to the manufacturer's protocol. Products were purified using 96-well plates (Performa DTR Ultra; Edge Biosystems, Gaithersburg, Md) to remove excess dye terminators. Samples were sequenced on a DNA sequencer (ABI Prism 3100; Applied Biosystems). Electropherograms generated from the DNA sequencer were analyzed using Lasergene DNA and protein analysis software (DNASTAR, Inc, Madison, Wis). All patients were sequenced in the forward direction (5′-3′), unless variants, mutations, or polymorphisms were identified, in which case confirmation was obtained in some instances by sequencing in the reverse direction.