Genomic DNA was extracted from whole blood anticoagulated with EDTA using the Puregene DNA Extraction Kit (catalog number D-5000; Gentra Systems, Minneapolis, Minnesota) according to the manufacturer's instructions. DNA was quantified spectrophotometrically and stored in aliquots at −20°C until required. For candidate gene analysis, polymerase chain reaction amplification was performed on a thermocycler (DNA Engine Tetrad; MJ Research Inc, Waltham, Massachusetts) in a total volume of 25 μL, containing 10 ng of DNA, 50mM potassium chloride, 10mM TRIS–hydrochloric acid (pH 9.0), 1.5mM magnesium chloride, 0.1% Triton X-100, 0.25mM of each deoxyribonucleotide triphosphate, 0.8μM of each primer, and 0.5 U of Taq polymerase (Qiagen, Hilden, Germany). For polymerase chain reaction, an initial denaturation step at 95°C for 10 minutes was followed by 40 cycles of denaturation at 95°C for 30 seconds, annealing for 30 seconds at 60°C, and extension at 72°C for 30 seconds followed by a final extension step of 72°C for 10 minutes. All exons and their intronic boundaries for the candidate gene were sequenced using an ET Dye Terminator Cycle Sequencing Kit (Amersham Biosciences, Piscataway, New Jersey) following the manufacturer's instructions. Sequence analysis was performed using the SeqManII module of the Lasergene software package (DNASTAR Inc, Madison, Wisconsin) using normal sequence for comparison.