Genotyping

The genomic DNA was extracted from 2 ml venous blood of all subjects using the QIAamp® DNA Blood Mini Kit (Qiagen, Hilden, Germany) and stored at −80°C. The primer sequence of rs1353058818 locus was: 5′-GCC TAT TGG GGT TGA GAG GG-3′ (forward); 5′-GCT GCG ACC TTT CTT TCA TCC-3′ (reverse). The primer sequence of rs1053004 locus was: 5′-GCT GAG GCA AGG TGG TTT TG-3′ (forward); 5′-CTG TGC GTA TGG GAA CAC CT-3′ (reverse). The PCR reaction was carried out with 100 ng genomic DNA, 2.5 μl 10× buffer, 1.5 μl Mg²⁺ (25 mM), 0.5 μl dNTP (10 mM), 0.25 μl Taq (5 U/μl), 1 ml forward primer and 1 ml reverse primer, with water added to a total volume of 25 μl. The PCR reaction was carried out as follows: pre-denaturation at 95°C for 5 min, then 30 reaction cycles (denaturation at 94°C for 45 s, annealing at 58°C for 45 s, extension at 72°C for 30 s), and extension at 72°C for 7 min. The PCR products were determined by Sanger sequencing.