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. CYP2D6*5 (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.


CONCLUSIONS
Pyrosequencing was found to be a fast and efficient tool for genotyping. The method is robust, reliable, rapid and has high throughput.
D000483:Alleles; D019389:Cytochrome P-450 CYP2D6 / Q000235:genetics*; D017931:DNA Primers; D006801:Humans; D016133:Polymerase Chain Reaction; D020641:Polymorphism, Single Nucleotide / Q000235:genetics*; D017422:Sequence Analysis, DNA / Q000379:methods*; D013548:Sweden; D044465:White People / Q000235:genetics*
D017931:DNA Primers; D019389:Cytochrome P-450 CYP2D6