Massarray type software

Five milliliters of EDTA-anticoagulated whole blood was collected from all patients and stored at −80 °C. Genomic DNA was extracted from peripheral blood lymphocytes using the FlexiGene DNA Purification Kit (Qiagen, Hiden, Germany) and diluted to 20 ng/µL. All DNA samples were stored at −20 °C. The rs1801133 SNP of the MTHFR gene was genotyped using a SequenomMassARRAY.
The SequenomMassARRAY Assay Design 3.0 software was used to design the PCR parameters and detection primers. The PCR products were subsequently used as templates for locus-specific, single-base extension reactions. The resulting products were desalted and transferred to a 384-element SpectroCHIP array (Sequenom Inc., San Diego, CA, USA). MALDI-TOF MS (Sequenom Inc.) was used for allele detection. The mass spectrograms were analyzed using MassARRAY Type software (Sequenom Inc.). We performed quality control of SNPs and samples at a call rate of 99.7% and analyzed the distribution at rs1801133 in the HCs with the Hardy–Weinberg equilibrium (p > 0.0001).

Five milliliters of EDTA-anticoagulated whole blood were collected from all patients and stored at –80 °C. Genomic DNA was extracted from peripheral blood lymphocytes using the FlexiGene DNA Purification Kit (Qiagen, Hilden, Germany) and diluted to 20 ng/µL. All DNA samples were stored at −20 °C. The rs11960458 SNP of AnxA6 was genotyped using a SequenomMassARRAY. The SequenomMassARRAY Assay Design 3.0 Software (Sequenom Inc., San Diego, CA, USA). was used to design the PCR parameters and detection primers. The PCR products were subsequently used as templates for locus-specific single-base extension reactions. The resulting products were desalted and transferred to a 384-element SpectroCHIP array (Sequenom Inc.). MALDI-TOF MS (Sequenom Inc.) was used for allele detection. The mass spectrograms were analyzed using MassARRAY Type software (Sequenom Inc., San Diego, CA, USA). We performed quality control of SNPs and samples at a call rate of 99.2% and analyzed the distribution of the SNPs in the HCs with the Hardy–Weinberg equilibrium (p > 0.0001).

EDTA-anticoagulated whole blood (5 mL) samples were collected from all patients and stored at −80°C. Genomic DNA was extracted from peripheral blood lymphocytes using the FlexiGene DNA Purification Kit (Qiagen, Hiden, Germany) according to the manufacturer's instructions and diluted to 20 ng/μL. The rs1801133 and rs1801131 single nucleotide polymorphism (SNP) of MTHFR was genotyped using SequenomMassARRAY with the SequenomMassARRAY Assay Design 3.0 software used to design the polymerase chain reaction (PCR) and detection primers. The PCR products were subsequently used as templates for locus-specific single-base extension reactions. The resulting products were desalted and transferred to a 384-element SpectroCHIP array (Sequenom Inc.). Matrix-assisted laser desorption/ionization-time of flight mass spectrometry (Sequenom Inc.) was used for allele detection. The mass spectrograms were analyzed using the MassARRAY Type software (Sequenom Inc.). We performed quality control of SNPs and samples at a call rate of 99.7%. The genotype distributions and allele frequencies for MTHFR gene SNP rs1801131 and rs1801133 were shown in Supplementary Table 2. The genotype frequencies of all subjects comply with Hardy-Weinberg equilibrium (p > 0.05).