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Clinical Chemistry 55: 139-149, 2009. First published November 6, 2008; 10.1373/clinchem.2008.115378
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(Clinical Chemistry. 2009;55:139-149.)
© 2009 American Association for Clinical Chemistry, Inc.

Molecular Diagnostics and Genetics

MALDI-TOF MS Genotyping of Polymorphisms Related to 1-Carbon Metabolism Using Common and Mass-Modified Terminators

Klaus Meyer1,2,a, Åse Fredriksen2 and Per Magne Ueland2,3

1 Bevital A/S, Armauer Hansens Hus, Bergen, Norway; 2 Section for Pharmacology, Institute of Medicine, Armauer Hansens Hus, University of Bergen, Bergen, Norway; 3 Laboratory of Clinical Biochemistry, Haukeland University Hospital, Bergen, Norway.

aAddress correspondence to this author at: LOCUS for Homocysteine and Related Vitamins, University of Bergen, N-5021 Bergen, Norway. Fax +47 55974605; e-mail klaus.meyer{at}farm.uib.no.

Background: Large cohort studies may provide sufficient power to disentangle the role of polymorphisms related to 1-carbon metabolism and chronic diseases, but they require fast, accurate, high-throughput genotyping techniques. MALDI-TOF mass spectrometry has been adapted to rapid fine mapping using various approaches for allele discrimination. We developed a genotyping method based on MALDI-TOF MS and compared assay performance for formats based on standard and mass-modified terminators.

Methods: The assay includes 20 polymorphisms of 14 genes involved in 1-carbon metabolism (BHMT 742G>A, CBS 844ins68 and 699C>T, CTH 1364G>T, DHFR del19, NOS3 –786T>C and 894G>T, FOLR1 1314G>A, MTHFD1 –105T>C and 1958G>A, MTHFR 677C>T and 1298A>C, MTR 2756A>G, MTRR 66A>G and 524C>T, SLC19A1 80G>A, SHMT1 1420C>T, TCN2 67A>G and 776C>G, and TYMS 1494del6).

Results: Missing calls were observed for 4.7% of the DNA samples, attributed to failed liquid sample handling. Highly accurate genotyping was obtained by mass-modified as well as standard ddNTPs, with an average error rate of ≤0.1% by analysis of sample duplicates. A semiquantitative approach enabled unambiguous identification of the CBS 844ins68. Cluster plots of the relative allele intensities showed allele-specific bias according to type of minisequencing terminator and revealed a potential structural variation in the BHMT gene.

Conclusions: MALDI-TOF MS–based genotyping using either standard or mass-modified terminators allows the accurate determination of single nucleotides as well as structural genetic variants. This was demonstrated with 20 polymorphisms involved in 1-carbon metabolism.






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