2.2. Genotyping

Genomic DNA was extracted from EDTA anticoagulated peripheral blood leukocytes using a commercial DNA isolation kit (Sigma‐Aldrich, Taufkirchen, Germany). The C677T and A1298C variants in MTHFR gene were determined by polymerase chain reaction (PCR) followed by restriction fragment length polymorphisms (RFLP).7 The MTHFR C677T variant was analyzed by using forward (F) 5′‐TGA AGG AGA AGG TGT CTG CGG GA‐3′ and reverse (R) 5′‐AGG ACG GTG CGG TGA GAG TG‐3′ primers. The PCR protocol was consisted of an initial melting step of 5 minutes at 94°C; followed by 35 cycles of 30 seconds at 94°C, 30 seconds at 61°C, and 30 seconds at 72°C; and a final elongation step of 5 minutes at 72°C. After amplification, the 198 bp of PCR product was digested with Hinf I in a 15 μL reaction solution containing 10 μL of PCR product, 1.5 μL of 10× buffer, and two units of Hinf I at 37°C overnight. The digestion products were separated on 3% agarose gels, and fragments stained with the ethidium bromide were photographed on an ultraviolet transilluminator. Wild type (CC) individuals were identified by only a 198 bp fragment, heterozygotes (CT) by both the 175/23 bp and 198 bp, and homozygote variants (TT) by the 175/23 bp.

MTHFR A1298C variant amplification was performed using primers forward (F) 5′‐CTT TGG GGA GGT GAA GGA CTA CTA C‐3′ and reverse (R) 5′‐CAC TTT GTG AGC ATT CCG GTT TG‐3′ and the protocol described previously by El‐Baz et al.13 Restriction digestion of PCR product with MboII endonuclease was performed. Wild type (AA) was produced 4 fragments (176, 30, 28, and 22 bp), whereas heterozygous (AC) yielded 5 fragments (204, 176, 30, 28, and 22 bp) and homozygous mutant (CC) variant produced 3 fragments (204, 30, and 22 bp). Second PCR was performed to confirm samples whose results were not clear.