Accurate and rapid "multiplex heteroduplexing" method for genotyping key enzymes involved in folate/homocysteine metabolism

Background: Hyperhomocysteinemia, which is often associated with low folate status, is an independent risk factor for cardiovascular diseases and seve ral other pathologies. The four most common functional polymorphisms in gen es involved in folate/homocysteine metabolism are methylenetetrahydrofolate reductase (MTHFR) C677T and A1298C, methionine synthase (MS) A2756G, and c ystathionine P-synthase (CBS) 844ins68. The pathogenic impact of these vari ants is under active investigation in many laboratories. However, conventio nal genotyping methods, mostly using PCR followed by restriction enzyme dig estion, often are compromised by partial fragment digestion. There is, ther efore, a need to develop more reliable approaches to genotyping the above p olymorphisms that may be applied in large-scale studies. Methods: Sequence-specific heteroduplex generators for each of the MTHFR an d MS single nucleotide polymorphisms were generated by site-directed mutage nesis. These were subcloned into a single construct, pHcyHG-1, which could be multiplexed with a simple PCR amplification across the CBS 844ins68 poly morphic site to generate composite genotype-specific banding patterns from individual genomic DNA samples that could be electrophoretically resolved. Results: The "multiplex heteroduplexing" method yielded unambiguous MTHFR,M S, and CBS genotypes in a single-tube reaction that could be analyzed in a single gel run. Conclusions: This method permits unambiguous genotyping of the four most co mmon functional variants of enzymes involved in folate/homocysteine metabol ism. It is rapid, reproducible, and inexpensive, and requires no special pr eparative or analytic facilities; consequently, it will facilitate large-sc ale studies of the genetic basis of hyperhomocysteinemia and the many patho logies that have been associated with this phenotype. (C) 2000 American Ass ociation for Clinical Chemistry.