Genotyping of MTHFR 677 C>T and 1298 A>C polymorphisms by High Resolution Melting of Small Amplicons

Defects in the enzyme Methylenetetrahydrofolate Reductase (MTHFR) has been associated to thrombosis and neural tube defects, among other diseases, being of importance to evaluate genetic susceptibility to these disorders. High Resolution Melting (HRM) for gene scanning is a very simple, rapid and an inexpensive method. HRM has been adapted for SNPs genotyping after a PCR amplification of small amplicons. We introduced this closed-tube genotyping method in our laboratory for the genotyping of the most common mutations in the MTHFR gene: 677 C>T (rs1801133) and 1298 A>C (rs1801131). We analyzed 107 blinded samples for rs1801133 and 104 blinded samples for rs1801131. Assignments were made in 96.3% of the cases for rs1801133 and in 98.1% of the cases for rs1801131 being all these calls correct. In the case of the 4 (3.7%) and 2 (1.9%) unknown samples for rs1801133 and rs1801131 respectively, by repeating the analysis we reached a 100% of assignments with correct calls. All the samples used in this study were previously genotyped by RHA Kit Thrombo and RFLP. High Resolution Melting of Small Amplicons is not only a simple and inexpensive method, it is also accurate for genotyping and it can be used for molecular diagnostic purposes.

This work was supported by VZFNM 00064203(6112) and Eurogentest.