ABSTRACT
Title
Denaturing High-Performance Liquid Chromatography Method for the Identification of Variant of Cytochrome P450 2D6
Authors
L. Bertolaso
Doctoral School of Pharmacological Sciences, University of Padova
Laboratory of Pharmacolgy and Molecular Biology, Dept. of Oncology, Azienda Ulss 18-Rovigo, S. Luca Hospital, Viale U. Grisetti, 265 45027 Trecenta (Ro)
e-mail: bertolaso.laura@gmail.com
Abstract
Cytochrome P450 is one of the most important family of enzymes involved in drug metabolism. The CYP 2D6 isoform is liable for about 20% of bio-transformation of drugs, including Tamoxifen. This drug is a selective modulator of estrogen receptor (ER) widely used for the treatment of estrogen receptor positive breast cancer. Tamoxifen is a prodrug that is converted, by CYP2D6, into more potent antiestrogenic metabolites, that have a 30- to 100-fold higher affinity to ER compared to the parent drug. CYP2D6 is highly polymorphic, resulting in enhancement or reduction, partial or complete, of the enzyme activity. For this reasons CYP2D6 is one of the most significant enzyme that modulates the pharmacological effect of Tamoxifen and its genotyping characterization may have a clinical relevance. In Caucasian population the more frequent polymorphic variants are *4 (null activity), *3, *6, *10, *41 (reduced activity) and the normally functioning *1.
The key polymorphisms of the variants of interest are the following: allele *4 (C974A and A984G); allele *3 (A2549del); allele *6 (T1707del); allele *10 (C100T); allele *41 (G2988A).
The aim of this work was to develop denaturing HPLC (DHPLC) methods for rapidly identifying CYP 2D6 sequence variations that affect enzyme activity.
Mutated alleles, needed for the implementation of DHPLC methods, were synthesized by site-directed mutagenesis using the Stratagene Quick Change Site Directed Mutagenesis Kit; the success of mutagenesis was confirmed through direct sequencing by capillary electrophoresis on a CEQ2000 sequencer (Beckman Coulter).
All the variants of CYP2D6 considered were PCR-amplified, checked by agarose gel electrophoresis and analyzed by an DHPLC system (Transgenomic Co.).
The optimal melting temperature for DHPLC was selected experimentally. Wild type and mutated samples were eluted as a single peak and then mixed together to allow the formation of heteroduplex after a denaturing treatment (94°C) for 5 min and a gradual re-annealing at room temperature. The size of the PCR products ranged from 110 to 322 bp. The retention times were identified in 8 min runs, using a gradient of Buffer A (triethylammonium acetate) and Buffer B (triethylammonium acetate and acetonitrile) at a flow rate of 0.9 ml/min.
For some of the variants evaluated it was possible to obtain a reliable protocol of genotyping; the best observed melting temperature were as follows: *3: 63.5°C, *4: 66.2°C, *6: 66.5. Heteroduplex molecules produced a double peak with specific retention times : *3 at 5.7 min and 5.99 min; *4 at 4.82 min and 4.97 min; and *6 at 4.84 min and 5.05 min.
DHPLC method proved to be reliable to genotype *3, *4 and *6 variants of CYP2D6 gene: it is highly specific and sensitive, rapid and relatively inexpensive. It would be useful for genotyping patients who are treated with Tamoxifen, to predict their pro-drug activation capability.
References
Dezentjé VO et al. (2009) Clin Cancer Res 15: 15
Higgins MJ et al. (2010) Curr Onncol Rep 12: 7
Ezzeldin H et al. (2002) Anal Biochem 306: 63
The key polymorphisms of the variants of interest are the following: allele *4 (C974A and A984G); allele *3 (A2549del); allele *6 (T1707del); allele *10 (C100T); allele *41 (G2988A).
The aim of this work was to develop denaturing HPLC (DHPLC) methods for rapidly identifying CYP 2D6 sequence variations that affect enzyme activity.
Mutated alleles, needed for the implementation of DHPLC methods, were synthesized by site-directed mutagenesis using the Stratagene Quick Change Site Directed Mutagenesis Kit; the success of mutagenesis was confirmed through direct sequencing by capillary electrophoresis on a CEQ2000 sequencer (Beckman Coulter).
All the variants of CYP2D6 considered were PCR-amplified, checked by agarose gel electrophoresis and analyzed by an DHPLC system (Transgenomic Co.).
The optimal melting temperature for DHPLC was selected experimentally. Wild type and mutated samples were eluted as a single peak and then mixed together to allow the formation of heteroduplex after a denaturing treatment (94°C) for 5 min and a gradual re-annealing at room temperature. The size of the PCR products ranged from 110 to 322 bp. The retention times were identified in 8 min runs, using a gradient of Buffer A (triethylammonium acetate) and Buffer B (triethylammonium acetate and acetonitrile) at a flow rate of 0.9 ml/min.
For some of the variants evaluated it was possible to obtain a reliable protocol of genotyping; the best observed melting temperature were as follows: *3: 63.5°C, *4: 66.2°C, *6: 66.5. Heteroduplex molecules produced a double peak with specific retention times : *3 at 5.7 min and 5.99 min; *4 at 4.82 min and 4.97 min; and *6 at 4.84 min and 5.05 min.
DHPLC method proved to be reliable to genotype *3, *4 and *6 variants of CYP2D6 gene: it is highly specific and sensitive, rapid and relatively inexpensive. It would be useful for genotyping patients who are treated with Tamoxifen, to predict their pro-drug activation capability.
References
Dezentjé VO et al. (2009) Clin Cancer Res 15: 15
Higgins MJ et al. (2010) Curr Onncol Rep 12: 7
Ezzeldin H et al. (2002) Anal Biochem 306: 63