MALDI-TOF Mass Spectrometry for Multiplex Genotyping of CYP2B6 Single-Nucleotide Polymorphisms

Abstract

Background: CYP2B6 is a highly variable and polymorphic cytochrome P450 (CYP) enzyme involved in the biotransformation of an increasing number of drugs, including cyclophosphamide, bupropion, and the nonnucleosidic reverse transcriptase inhibitor efavirenz. Several nonsynonymous and promoter single-nucleotide polymorphisms (SNPs) in the CYP2B6 gene are associated with altered hepatic expression and function, which affect drug plasma concentrations. Methods: We used multiplex PCR to amplify relevant gene fragments while avoiding amplification of the CYP2B7P1 pseudogene. Polymorphic sites were analyzed by allele-specific primer extension followed by matrix-assisted laser desorption/ionization time-offlight mass spectrometry (MALDI-TOF MS). Method evaluation was performed on a panel of 287 genomic DNA samples previously genotyped by other methods. Results: Five multiplex assays were developed, comprising the following 15 SNPs: ؊82T3 C (*22); 86G3 C (R29T, *17); 136A3 G (M46V, *11); 296G3 A (G99E, *12);

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

“…The mean failure rate of about 5% was independent of assay format and is comparable to the success rates of other MALDI assays (30,35 ). Because failed liquid handling during sample preparation and MALDI preparation contributed equally to all missing calls, improved liquid-dispensing techniques would increase the efficiency of our method.…”

Highly Multiplexed Genotyping of Thiopurine S-Methyltransferase Variants Using MALDI-TOF Mass Spectrometry: Reliable Genotyping in Different Ethnic Groups

“…Thiopurine S-methyltransferase is, at present, one of the best examples of genetic polymorphisms that alter drug response (4,5,28 (19,20,33 ). Further advantages are the option for highly automated processes and the relative ease of setting up multiplex assays, thus reducing time and cost while increasing sample throughput.…”

Aberrant Splicing Caused by Single Nucleotide Polymorphism c.516G>T [Q172H], a Marker of CYP2B6*6, Is Responsible for Decreased Expression and Activity of CYP2B6 in Liver

“…The CYP2B6 genotyping for the most important SNPs or respective alleles was performed as described previously (Lang et al, 2001(Lang et al, , 2004Klein et al, 2005;Blievernicht et al, 2007). In brief, 50 ng of whole genomic DNA prepared from whole blood using a QIAamp DNA blood kit (QIAGEN GmbH, Hilden, Germany) was used as a template for amplification of the respective CYP2B6 target sequence employing specific oligonucleotides that discriminate between CYP2B6 and the CYP2B7 pseudogene.…”