2.2 Genotypic polymorphism
MTHFR genetic variations were detected by PCR following Sanger sequencing. The PCR was performed as follows: 94 °C for 5 min, followed by 94 °C for 30 s, 58 °C for 30 s, and 72 °C for 30 s for 32 cycles, and a final extension at 72°C for 10 min. MTHFR c.677C > T and c.1298 A > C were 246 bp and 256 bp, respectively. The sequences of the primers used were as follows: c.677C>T, forward: 5′-TGCCCAGTCCCTGTGGTCTC-3′, reverse: 5′-GGCAAGTGATGCCCATGTCG-3′; c.1298A>C, forward: 5′-TTTGGGGAGCTGAAGGACTA-3′, reverse: 5′-ACAGGATGGGGAAGTCACAG-3′. The genotypes are presented in Fig. S1 .