A Rapid Detection System For C677T Polymorphism Of MTHFR Gene

Homocysteine (Hcy) is an amino acid which is a byproduct of the transmethylation and transsulfuration pathway. High concentration of plasma Hcy increases the risks of diseases, such as vascular disease, coronary artery disease (CAD), thromboembolism, stroke, neural tube defects, diabetic nephropathy (DN) and so on.Methylenetetrahydrofolate reductase (MTHFR) is one of the central enzymes for Hcy metabolism. It catalyzes the biologically irreversible reduction of 5,10-methylenetetrahydrofolate (5,10-methylene-THF) to 5-methyltetrahydrofolate (5-methyl-THF). The most common polymorphism of MTHFR is a C→T substitution at 677 bp that cause a substitution of valine for alanine in the catalytic domain. The T allele of the MTHFR polymorphism cause a thermolability of the enzyme, and inhibits the formation of 5-methyltetrahydrofolate which serves as a methyl donator during the remethylation of homocysteine to methionine. The TT homozygotes exhibit higher plasma homocysteine concentrations than CT heterozygotes and CC homozygotes in a majority of studies. This single substitution can increase Hcy by approximately 25% in subjects with the TT genotype.The C677T MTHFR polymorphism is common, and its prevalence varies among populations, the lowest frequency is found in sub-Saharan Africa and northern Canada, while the highest is in peoples of Southern Europe and South America. C677T MTHFR polymorphism was reported to be a genetic risk factor for Alzherimer's disease (AD), cardiovascular disease, dysplasia, cancer, vascular thrombosis and embolism and has also been related to the occurrence of neural tube and other birth defects and pregnancy complications and the possible consequences of drug therapy. The TT genotype is also associated with lower DNA methylation in peripheral leukocytes compared with the CC genotype. A possible reason for this is the reduced availability of 5-methyltetrahydrofolate required for S-adenosylmethionine biosynthesis. In addition, DNA methylation has been considered to be an important factor in carcinogenesis.A low concentration of blood folate and vitamin supplementation enhances the risk associated with the T allele. The combination of the TT genotype and low concentration of folate increases the concentration of Hcy. Individuals with T allele appear to have an increased dietary folate requirement, and may benefit from supplementation with folic acid, Vitamin B12 and Vitamin B6. However, some authors found a supplementation of folate and vitamins can not be generally recommended for all patients to improve clinical outcome. The determination of MTHFR genotypes is helpful for prediction the risk of diseases and medication with nutrition.So far, among most reports, the C677T MTHFR genotype and allele frequencies have been determined by PCR-SSCP and PCR-RFLP methods, which are time consuming and can not give to unequivocal results. It is necessary to establish a rapid and simple genotyping method for clinical and large scare screening. Template-directed dye-terminator incorporation with fluorescence polarization (TDI-FP) is a novel genotype technique. It performs an assay to determine the base at a polymorphic site with several...