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A novel genotyping algorithm for the CYP2D6*10 allele in Asians using real-time, rapid-cycle PCR and multiplex PCR

University of British Columbia

Purpose: The CYP2D6*10 allele (C188T) is common among Asians and is associated with decreased metabolism of various CYP2D6 substrates. Based on reported allele frequencies among Asians, to accurately genotype patients for CYP2D6*10, it is necessary to rule out CYP2D6H (C188T, G1934A) and CYP2D6*5 (gene deletion) before inferring the presence of CYP2D6*wt (CYP2D6*1 or CYP2D6*2; C at position 188). The objectives of this project were to develop and validate genotyping methods for detecting the C188T and G1934A single nucleotide polymorphisms (SNPs) and CYP2D6*5, to use these methods to genotype Asian subjects from a pilot clinical study, and to devise a genotyping algorithm for CYP2D6*10 in Asians. Methods: Long PCR was used to amplify the CYP2D6 gene. Nested real-time, rapidcycle polymerase chain reaction (PCR) methods for detecting the C188T and G1934A SNPs were developed and validated by restriction fragment length polymorphism (RFLP) and sequence analyses of reference samples with genotypes CYP2D6*1/*1, CYP2D6*l/*4, and CYP2D6*4/*4. A multiplex PCR method for detecting CYP2D6*5 using published primer sequences was optimized and validated by analyzing reference samples with genotypes CYP2D6*1/*1, CYP2D6*l/*5, CYP2D6*5/*5. These methods were used to genotype Asian subjects from a pilot clinical study. Results: C188T and G1934A genotyping results using nested real-time, rapid-cycle PCR were consistent with results from RFLP and sequence analyses, as well as the genotypes of the reference samples. CYP2D6*5 genotyping results were also in agreement with the genotypes of the reference samples. CYP2D6 genotype frequencies were determined in 36 Asian subjects: CYP2D6*10/*10 (50.0%; n=18), CYP2D6*wt/*10 (33.3%; n=12), CYP2D6*wt/*wt (11.1%; n=4), and CYP2D6*wt/*5 (5.6%; n=2). Conclusions: The novel genotyping algorithm for identifying Asian patients with the CYP2D6*wt/*wt, CYP2D6*wt/*10, and CYP2D6*10/*10 genotypes involves the use of: 1) long PCR to amplify the CYP2D6 gene; 2) nested real-time, rapid-cycle PCR to detect the C188T SNP (CYP2D6*10 or CYP2D6*4); 3) nested real-time, rapid-cycle PCR to detect the G1934A SNP (CYP2D6*4) in those carrying a C188T SNP; and 4) multiplex PCR to detect CYP2D6*5 in those who appear homozygous for C at 188 (CYP2D6*wt/wt or CYP2D6*wt/*5) or T at 188 (CYP2D6*10/*10 or CYP2D6*10/*5). This algorithm can be used in future clinical studies that require genotyping Asians with respect to CYP2D6*10.

Thesis/Dissertation

application/pdf

2009-11-24

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http://hdl.handle.net/2429/15602

Pharmaceutical Sciences

University of British Columbia

UBCV

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