Rapid identification of CYP2C8 polymorphisms by high resolution melting analysis.
BACKGROUND
Cytochrome P450 (CYP) 2C8 is the principal enzyme responsible for the metabolism of arachidonic acid and various drugs, and influences drug-drug interactions and some associated diseases. Large interindividual differences in CYP2C8 enzymatic activity and several nonsynonymous genetic variations have been reported in different races. Therefore, how to identify CYP2C8 polymorphisms efficiently for genotyping in different populations is very important.


METHODS
A high resolution melting (HRM) analysis was used to characterize the CYP2C8 polymorphism. Genomic DNA was extracted from peripheral blood samples from 95 normal individuals in Taiwan. Nine exons of the CYP2C8 gene were screened by HRM analysis. All results were confirmed by direct DNA sequencing.


RESULTS
Five new single nucleotide polymorphisms (SNPs) were found in this study; two SNPs [1189G>A (D397N) and 1230C>T (G410G)] were in exon 8 and the others [1312G>C (E438Q), 1497T>C (A499A) and 1677delT (559delL)] were in exon 9. The 1497T>C (A499A) was the most common variant with an allele frequency of 20.53% but without amino acid substitution.


CONCLUSIONS
HRM analysis is a fast, reliable, accurate and cost-effective screening method for gene mutations, even very similar cDNA sequences with 83% identities, compared with CYP2C8 and CYP2C9.
Institute of Clinical Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan.
Chun-Chi
CC
Chang
Pei-Chin
PC
Lin
Ching-Hsiung
CH
Lin
Kun-Tu
KT
Yeh
Hsiao-Yu
HY
Hung
Jan-Gowth
JG
Chang
D001189:Aryl Hydrocarbon Hydroxylases / Q000235:genetics*; D001483:Base Sequence; D065727:Cytochrome P-450 CYP2C8; D017931:DNA Primers; D005091:Exons; D006801:Humans; D009154:Mutation; D016133:Polymerase Chain Reaction; D020641:Polymorphism, Single Nucleotide; D013624:Taiwan
D017931:DNA Primers; D001189:Aryl Hydrocarbon Hydroxylases; C450259:CYP2C8 protein, human; D065727:Cytochrome P-450 CYP2C8