Identification of CYP2D6 alleles by single nucleotide polymorphism analysis using pyrosequencing.

OBJECTIVE

To develop a single nucleotide polymorphism (SNP) analysis for identification of cytochrome P450 (CYP)2D6 alleles by pyrosequencing.

METHODS

Swedish blood donors ( n=282) were typed for a partial CYP2D6 genotype comprising the alleles *1 (wild type), *2 (2850C>T), *3 (2549A>del), *4 (1846G>A) and 6 (1707T>del) using polymerase chain reaction (PCR) and pyrosequencing analysis. CYP2D65 (CYP2D6 deleted) was identified using an established long multiplex PCR method. Pyrosequencing is a sequencing-by-synthesis method in which a cascade of enzymatic reactions yields detectable light, which is proportional to the incorporated nucleotides. One feature of typing SNPs by pyrosequencing is that each allelic variant will give a unique sequence. These variants can be readily distinguished by pattern recognition software.

RESULTS

Of 281 individuals analysed, 24 (8.5%) were found to be poor metabolisers with two non-functional alleles. This is in the range of 7-10%, previously reported for Caucasians. A total of 126 individuals (45%) had one functional and one non-functional allele and 131 individuals (47%) had two functional alleles.

CONCLUSION

Pyrosequencing was found to be a fast and efficient tool for genotyping. The method is robust, reliable, rapid and has high throughput.