DNA was extracted from the peripheral blood of 231 patients who had kidney transplantation between January 1996 and December 2004 by using the LaboPass Genomic DNA Extraction Kit (COSMO, Seoul, Korea) or QuickGene DNA whole blood kit (Fujifilm, Tokyo, Japan) when HLA genotyping for pre-operation evaluation was performed. For 195 healthy controls, DNA was extracted using LaboPass Genomic DNA Extraction Kit (COSMO, Seoul, Korea) during the period of January 1999 and July 2001. All DNA samples were preserved at –70℃ prior to being used for Foxp3 polymorphisms analyses which were performed during the period of June 2015 and July 2016. Four Foxp3 polymorphisms (rs3761548 A/C, rs2280883 C/T, rs5902434 del/ATT, and rs2232365 A/G) were analyzed by PCR with sequence-specific primers (PCR-SSP) with some modifications [20 (link)] (Table 2). PCR was performed by using a 40-µL reaction mixture containing 40 ng DNA, 0.2mM of each primer, 0.8 µL of 10mM dNTP, 2.0mM MgCl2, 1.0 U Taq DNA polymerase (Roche Applied Science, Basel, Switzerland), and 4 µL of 10× reaction buffer. The PCR protocol consisted of an initial denaturation step at 95℃ for 5 min; 35 cycles of denaturation at 95℃ for 30 sec, annealing (temperatures detailed in Table 2) for 30 sec, and extension at 72℃ for 30 sec; and a final extension step at 72℃ for 5 min.