Multiplex genotyping of cytochrome p450 single-nucleotide polymorphisms by use of MALDI-TOF mass spectrometry.
BACKGROUND
Polymorphisms in cytochrome P450 (CYP450) genes contribute to interindividual differences in the metabolism of xenobiotic chemicals, including the vast majority of drugs, and may lead to toxicity and adverse drug reactions. Studies on these polymorphisms in research and diagnostic settings typically involve large-scale genotyping and hence require high-throughput assays.


METHODS
We used the previously developed solid-phase capture-single-base extension (SPC-SBE) approach for concurrent analysis of 40 single-nucleotide polymorphisms (SNPs) of CYP2C9 and 50 SNPs of CYP2A13, both genes belonging to the CYP450 family. Desired SNP-containing regions for each gene were amplified in a single-step multiplex PCR. We designed a library of primers to anneal immediately upstream of the selected SNPs and extended it with biotinylated terminators using PCR products as templates. Biotinylated extension products were isolated by affinity purification and analyzed with MALDI-TOF mass spectrometry to determine SNP genotypes.


RESULTS
We analyzed 11 samples for CYP2C9 and 14 samples for CYP2A13 with unambiguous detection of SNPs in all samples. Many samples showed a high occurrence of heterozygotes for both genes, with as many as 10 of 50 SNPs appearing as heterozygotes in 1 sample genotyped for CYP2A13.


CONCLUSIONS
The SPC-SBE method provides an efficient means for genotyping SNPs from the CYP450 family. This approach is suitable for automation and can be extended to other genotyping applications.
Misra
Ashish
A
Department of Chemical and Biochemical Engineering, Rutgers, The State University of New Jersey, Piscataway, NJ 08854, USA.
Hong
Jun-Yan
JY
Kim
Sobin
S
D001189:Aryl Hydrocarbon Hydroxylases / Q000235:genetics*; D002460:Cell Line; D065729:Cytochrome P-450 CYP2C9; D019075:Electrophoresis, Capillary; D005838:Genotype; D006579:Heterozygote; D006801:Humans; D016133:Polymerase Chain Reaction; D020641:Polymorphism, Single Nucleotide; D017422:Sequence Analysis, DNA; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
C450260:CYP2C9 protein, human; D065729:Cytochrome P-450 CYP2C9; D001189:Aryl Hydrocarbon Hydroxylases; C414206:CYP2A13 protein, human