Design and Evaluation of a Method for Testing Polymorphisms of Folate-Related Genes Using the Luminex Liquichip...

The methylene tetrahydrofolate reductase (MTHFR) C677T, MTHFR A1298C, and the methionine synthase reductase (MTRR) A66G polymorphisms are the three most common folate metabolism-related loci in the Chinese population. They are associated with numerous birth defects or congenital diseases. To facilitate screening and genetic counseling, we established a method for the simultaneous detection of these three polymorphisms using the Luminex liquid suspension chip and multiple asymmetric polymerase chain reactions (PCRs). The three polymorphisms were amplified by multiplex PCR with biotinylated primers, followed by hybridization with six probe-linked magnetic microspheres. The mean fluorescent intensity value in each microsphere was detected by Luminex Magpix for polymorphism detection in 150 samples and confirmed by sequencing. The consistency between the Luminex liquid suspension chip method and sequencing was 100%. Among the 150 randomized samples, the minor allele frequency (MAF) of MTHFR C677T was 0.41, which was the most common variant allele, followed by MTRR A66G (MAF = 0.24), and finally MTHFR A1298C (MAF = 0.19). The Luminex liquid suspension chip method can replace sequencing to analyze the MTHFR C677T, MTRR A1298C, and MTRR A66G loci simultaneously as a rapid, convenient, accurate, and stable method for large-scale testing.