Abstract
rs1801133 is a single nucleotide polymorphism (SNP) located in the sequence of MTHFR on human chromosome 1. The alleles of this SNP affect the activity of the MTHFR enzyme. People bearing C/T genotype have 66% activity of MTHFR while people with T/T genotype have only 25% activity. These reduced activities of MTHFR cause homocysteinemia. There are several publications on the relationship between homocysteinemia and human diseases such as cardiovascular disease, neurological diseases, abnormal fetus, infertility and cancer. In this study, we built a molecular protocol for genotyping rs1801133 using real-time PCR HRM technique. This protocol could be used for diagnosis of molecular mechanism of homocysteinemia causing the mentoned above diseases as well as for the study of the relationship between rs1801133 and other human diseases. We successfully designed the primer pairs for genotyping and nucleotide sequencing rs1801133 by real-time PCR HRM and Sanger sequencing method. We also examined the optimal MgCl2 concentration for clear differentiation of three rs1801133 genotypes. Performance characteristics of the real-time PCR HRM protocol included of specificity, repeatability, reproducibility was evaluated and it showed good results. Comparison of genotyping results of rs1801133 between the realtime PCR HRM method and the Sanger nucleotide sequencing method showed good concordances. Finally, this real-time PCR HRM protocol for rs1801133 genotyping was applied on 100 human DNA samples to evaluate the clinical utility of the protocol.
Highlights
MEthylenetetrahydrofolate reductase (MTHFR) is an enzyme that is involved in the methylation cycle in the human body
C is at nucleotide 677 coded for alanine whereas T is at the same nucleotide coded for valine resulting in the decrease in 5,10methylenetetrahydrofolate reductase activity of the MTHFR enzyme [3]
The CN5-CN6 primer pair occupies most of the nucleotide sequence of the PCR product except for the position of rs1801133 resulting that different human DNAs containing rs1801133 will be distinguished merely at rs1801133 (Fig. 1)
Summary
MEthylenetetrahydrofolate reductase (MTHFR) is an enzyme that is involved in the methylation cycle in the human body. This enzyme is located on chromosome 1 at the position 1p63.3 [1]. The MTHFR gene has many variants in the nucleotide sequence due to different SNPs located in the nucleotide sequence of the gene These different variants of the gene affect the methylenetetrahydrofolate reduction of MTHFR in which the C677T variant (rs1801133) was most studied. This variant has two alleles, cytosine (C) and thymine (T). MTHFR requires the lavin adenine dinucleotide (FAD) cofactor for its activity and studies have shown that the human recombinant MTHFR protein with the 677T variant lost its FAD cofactor three times much more than the MTHFR with the 677C variant [4, 5]
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