Optimized Strategy for Rapid Cytochrome P450 2D6 Genotyping by Real-Time Long PCR

Abstract

Background: Because of genetic polymorphisms, cytochrome P450 2D6 (Cyp2D6) activity in humans varies widely and alters the metabolism of commonly used drugs such as antidepressants, neuroleptics, and cardioactive agents. Severe adverse effects or resistance to therapy may result. Methods: We performed long PCR on the LightCycler TM and used the product as a template for a previously validated multiplex PCR that examines the *3, *4, *6, *7, and *8 alleles of Cyp2D6. We used real-time PCR to identify the *5 null allele and duplication of Cyp2D6 with detection by either hybridization probes or SYBR Green ® . The *2 ؊1584 C/G polymorphism and the *35 allele were identified by PCR with detection by hybridization probes. Products of all PCRs were visualized with gel electrophoresis using a 0.7-1.5% agarose gel and ethidium bromide. Samples containing the *35 allele were analyzed in parallel by digestion with NlaIII, MslI, and BstXI and SmaI. We analyzed samples from volunteers and patients (105 samples for deletion and duplication and 116 samples for preamplification). Of those samples, 59 were from depressive inpatients taking part in a trial not yet published.

Intermediate metabolizer: increased side effects in psychoactive drug therapy. The key to cost-effectiveness of pretreatment CYP2D6 screening?

“…Also, the response onset was similar (28,34,35 and 34 days for 4 PMs, 33 IMs, 48 EMs and 3 UMs, respectively, P40.6). In the patient group receiving CYP2D6 drugs, a pronounced, significant increase of side effects was observed for the PMs.…”

“…Genotyping CYP2D6 genotyping was carried out as described elsewhere [28][29][30] for detection of the gene duplication and deletion (CYP2D6*5), for the functional alleles *1 and *2 (formerly *2G); for the dysfunctional alleles *3, *4, *6, *7 and *8; for the alleles with reduced function *9, *10, *41 and for the differentiation of gene duplication as *1 Â N, *2 Â N and *4 Â N. 31 The *41 (formerly *2C) was characterized with a fluorescence-based LightCycler hybridization probes assay by the novel polymorphism 2988G4A within intron 6, which was in almost complete linkage to the former À1584C4G change. The following CYP2D6 allele frequencies-similar to published data 4,5 -were found: *1 (39.0%), *2 (18.1%), *3 (2.6%), *4 (21%), *5 (2.9%), *6 (1.1%), *9 (2.2%), *10 (2.2%), *41 (8.2%), *1 Â N (1.6%), *2 Â N (1.0%) and *4 Â N (0.1%).…”

Amitriptyline or Not, That Is the Question: Pharmacogenetic Testing of CYP2D6 and CYP2C19 Identifies Patients with Low or High Risk for Side Effects in Amitriptyline Therapy

“…CYP2D6 genotype was determined by real-time and conventional PCR (18,19 ). The most important alleles in a Caucasian population were assessed: fully functional alleles (*1 and *2); completely dysfunctional alleles (*3-*8); alleles with reduced function (*9, *10, and *41); and duplicated alleles with enhanced function.…”

The clinical effectiveness and cost-effectiveness of testing for cytochrome P450 polymorphisms in patients with schizophrenia treated with antipsychotics: a systematic review and economic evaluation

“…This was particularly the case for CYP2D6, although five of the six US studies also investigated polymorphisms of 90 Heller 2006, 91 Hersberger 2000, 92 James 2004, 93 Lee 2007, 94 Melis 2006, 95c Muller 2003, 96 Neville 2002, 97 Nielsen 2007, 98 Roberts 2000, 99 Roche 2004, 100 Schaeffeler 2003, 101 Soderback 2005, 102 Stamer 2007, 103 Stuven 1996, 104 118 Muthiah 2004, 119 Oyama 1995, 120 Rohrbacher 2006, 121 Weise 2004, 122 Weise 2006, 123 Wen 2004, 124 Wu 2002 125 a The other CYP polymorphisms are CYP1A1 (n = 4), CYP1B1 (n = 1), CYP2B6 (n = 1), CYP2C8 (n = 3), CYP2E1 (n = 1), Studies varied in size from 40 subjects being genotyped in the smallest study 112 to 428 in the largest, 118 although the number of samples being compared by the reference method varied from just six samples tested by AS-PCR in Oyama et al 120 to 1400 samples in Lee et al 94 …”