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Department of Pharmacology and Center for Pharmacogenetics, University of Pennsylvania School of Medicine, Philadelphia, PA 19104.
a Address correspondence to this author at: Department of Pharmacology and Center for Pharmacogenetics, University of Pennsylvania School of Medicine, 153 Johnson Pavilion, 3620 Hamilton Walk, Philadelphia, PA 19104-6084. Fax 215-573-9135; e-mail aswhitehead{at}pharm.med.upenn.edu
Background: Hyperhomocysteinemia, which is often associated with low folate status, is an independent risk factor for cardiovascular diseases and several other pathologies. The four most common functional polymorphisms in genes involved in folate/homocysteine metabolism are methylenetetrahydrofolate reductase (MTHFR) C677T and A1298C, methionine synthase (MS) A2756G, and cystathionine ß-synthase (CBS) 844ins68. The pathogenic impact of these variants is under active investigation in many laboratories. However, conventional genotyping methods, mostly using PCR followed by restriction enzyme digestion, often are compromised by partial fragment digestion. There is, therefore, a need to develop more reliable approaches to genotyping the above polymorphisms that may be applied in large-scale studies.
Methods: Sequence-specific heteroduplex generators for each of the MTHFR and MS single nucleotide polymorphisms were generated by site-directed mutagenesis. These were subcloned into a single construct, pHcyHG-1, which could be multiplexed with a simple PCR amplification across the CBS 844ins68 polymorphic 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, MS, 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 common functional variants of enzymes involved in folate/homocysteine metabolism. It is rapid, reproducible, and inexpensive, and requires no special preparative or analytic facilities; consequently, it will facilitate large-scale studies of the genetic basis of hyperhomocysteinemia and the many pathologies that have been associated with this phenotype.
The following articles in journals at HighWire Press have cited this article:
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  K. Robien and C. M. Ulrich 5,10-Methylenetetrahydrofolate Reductase Polymorphisms and Leukemia Risk: A HuGE Minireview Am. J. Epidemiol., April 1, 2003; 157(7): 571 - 582. [Abstract] [Full Text] [PDF]
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 L. A. J. Kluijtmans, I. S. Young, C. A. Boreham, L. Murray, D. McMaster, H. McNulty, J. J. Strain, J. McPartlin, J. M. Scott, and A. S. Whitehead Genetic and nutritional factors contributing to hyperhomocysteinemia in young adults Blood, April 1, 2003; 101(7): 2483 - 2488. [Abstract] [Full Text] [PDF]
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 M. L. Smit, B. A.J. Giesendorf, J. A.M. Vet, F. J.M. Trijbels, and H. J. Blom Semiautomated DNA Mutation Analysis Using a Robotic Workstation and Molecular Beacons Clin. Chem., April 1, 2001; 47(4): 739 - 744. [Abstract] [Full Text] [PDF]
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