Document Detail

MALDI-TOF mass spectrometry for multiplex genotyping of CYP2B6 single-nucleotide polymorphisms.
MedLine Citation:
PMID:  17082249     Owner:  NLM     Status:  MEDLINE    
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-of-flight 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: -82T --> C (*22); 86G --> C (R29T, *17); 136A --> G (M46V, *11); 296G --> A (G99E, *12); 415A --> G (K139E, *8, *13); 419G --> A (R140Q, *14); 516G --> T (Q172H, *6, *7, *9, *13, *19, *20), 547G --> A (V183I); 769G --> A (D257N); 785A --> G (K262R, *4, *6, *7, *13, *16, *19, *20); 983T-->C (I328T, *16, *18); 1006C --> T (R336C, *19); 1172T --> A (I391N, *15); 1282C --> A (P428T, *21); 1459C --> T (R487C, *5, *7). In 9 DNA samples showing discrepant genotypes, correctness of the MALDI-TOF MS result was confirmed by direct sequencing.
CONCLUSIONS: This genotyping method enabled sensitive, specific, accurate, and comprehensive determination of 15 relevant SNPs of CYP2B6. The assay design allows analysis of SNP subsets, incorporation of additional SNPs, and performance of high-throughput genotyping.
Julia K Blievernicht; Elke Schaeffeler; Kathrin Klein; Michel Eichelbaum; Matthias Schwab; Ulrich M Zanger
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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't     Date:  2006-11-02
Journal Detail:
Title:  Clinical chemistry     Volume:  53     ISSN:  0009-9147     ISO Abbreviation:  Clin. Chem.     Publication Date:  2007 Jan 
Date Detail:
Created Date:  2007-01-04     Completed Date:  2007-02-06     Revised Date:  2014-01-07    
Medline Journal Info:
Nlm Unique ID:  9421549     Medline TA:  Clin Chem     Country:  United States    
Other Details:
Languages:  eng     Pagination:  24-33     Citation Subset:  IM    
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MeSH Terms
Aryl Hydrocarbon Hydroxylases / genetics*
European Continental Ancestry Group
Oxidoreductases, N-Demethylating / genetics*
Polymerase Chain Reaction
Polymorphism, Single Nucleotide*
Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
Reg. No./Substance:
EC 1.14.13.-/cytochrome P-450 CYP2B6; EC Hydrocarbon Hydroxylases; EC 1.5.-/Oxidoreductases, N-Demethylating

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