A DUAL-LABEL OLIGONUCLEOTIDE LIGATION ASSAY FOR DETECTION OF THE CYP2C19-ASTERISK-1, CYP2C19-ASTERISK-2, AND CYP2C19-ASTERISK-3 ALLELES INVOLVING TIME-RESOLVED FLUOROMETRY

CYP2C19 (S-mephenytoin hydroxylase) is a polymorphically expressed enz yme. Currently, two defective alleles are known-CYP2C192 and CYP2C19* 3. The authors have developed an oligonucleotide ligation assay to det ect these two alleles. This assay combines the hybridization of one co mmon, biotinylated capture probe and two allele-specific probes to the target DNA, with the ability of a DNA ligase to distinguish mismatche d nucleotides. The probes are only ligated if they are base paired cor rectly to the target strand. The biotin is bound to streptavidin, and all DNA not covalently bound to the biotin-labeled capture probe, is r emoved in a washing procedure. The allele-specific probes are labeled with either europium or samarium, and their emission can be measured s imultaneously. The ratio between the emission separates the genotypes. This method was applied on DNA from 19 whites and 21 Vietnamese livin g in Denmark. All genotypes determined by the assay were consistent wi th the results from restriction enzyme cleavage. There were 12 poor me tabolizers; 10 homozygous CYP2C192/CYP2C19*2, one heterozygous CYP2C1 92/CYP2C19*3, and one heterozygous CYP2C19*1/CYP2C19*2. The authors c onclude that this assay is well-suited for a high throughput of sample s in a routine laboratory. The finding of an apparently heterozygous C YP2C191/CYP2C19*2 poor metabolizer, confirms that there are still unk nown mutations in CYP2C19.