High-level multiplex genotyping of polymorphisms involved in folate or homocysteine metabolism by matrix-assisted laser desorption/ionization mass spectrometry.
BACKGROUND
Increased plasma total homocysteine (tHcy), a risk factor for cardiovascular disease, is related to genetic, environmental, and nutritional factors, in particular folate status. Future large epidemiologic studies of the genetic basis of hyperhomocysteinemia will require high-throughput assays for polymorphisms of genes related to folate and Hcy metabolism.


METHODS
We developed a high-level multiplex genotyping method based on matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) for the detection of 12 polymorphisms in 8 genes involved in folate or Hcy metabolism. The assay includes methylenetetrahydrofolate reductase (MTHFR) 677C>T and 1298A>C, methionine synthase (MTR) 2756A>G, methionine synthase reductase (MTRR) 66A>G, cystathionine beta-synthase (CBS) 844ins68 and 699C>T, transcobalamin II (TCII) 776C>G and 67A>G, reduced folate carrier-1 (RFC1) 80G>A, paraoxonase-1 (PON1) 575A>G and 163T>A, and betaine homocysteine methyltransferase (BHMT) 742G>A.


RESULTS
The failure rate of the assay was < or = 1.7% and was attributable to unsuccessful DNA purification, nanoliter dispensing, and spectrum calibration. Most errors were related to identification of heterozygotes as homozygotes. The mean error rate was 0.26%, and error rates differed for the various single-nucleotide polymorphisms. Identification of CBS 844ins68 was carried out by a semiquantitative approach. The throughput of the MALDI-TOF MS assay was 1152 genotypes within 20 min.


CONCLUSIONS
This high-level multiplex method is able to genotype 12 polymorphisms involved in folate or Hcy metabolism. The method is rapid and reproducible and could facilitate large-scale studies of the genetic basis of hyperhomocysteinemia and associated pathologies.
Meyer
Klaus
K
LOCUS for Homocysteine and Related Vitamins, University of Bergen, Bergen, Norway. klaus.meyer@farm.uib.no
Fredriksen
Ase
A
Ueland
Per Magne
PM
D005492:Folic Acid / Q000378:metabolism*; D005838:Genotype; D006710:Homocysteine / Q000378:metabolism*; D006801:Humans; D016133:Polymerase Chain Reaction; D011110:Polymorphism, Genetic; D020641:Polymorphism, Single Nucleotide; D015203:Reproducibility of Results; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D006710:Homocysteine; D005492:Folic Acid