An Unusual Melting Curve on a LightCycler Due to a New Mutation in Methylenetetrahydrofolate Reductase

Abstract

Methylenetetrahydrofolate reductase (MTHFR; E.C. 1.5.1.20) catalyzes the reduction of 5,10-methylenetetrahydofolate to 5-methytetrahydrofolate, and its activity is affected by a number of polymorphisms and mutations, the best known being MTHFR c.677C > T (OMIM 607093.003). The c.677C > T mutation is often analyzed by melting curve analysis after real-time polymerase chain reaction. We observed in one patient upon dual hybridization probe analysis in a Roche LightCycler 1.2 an unusual melting curve peak at 53.4°C instead of one expected at either 51.6°C or 60.4°C. We sequenced the region of MTHFR interrogated by the dual hybridization probes, which revealed a c.685A > G base substitution that would result in an Ile225Val substitution. In conclusion, we identified the cause of an unusual melting curve in a commonly used genotyping technique for MTHFR c.677C > T as due to a novel mutation highlighting the ability of melting analysis to identify new sequence variants.