Genotyping of CYP2D6 Polymorphisms by MALDI-TOF Mass Spectrometry in Sardinian People

The CYP2D6 enzyme is involved in the metabolism of many commonly prescribed drugs. The presence of CYP2D6 gene SNPs can alter CYP2D6 enzymatic activity with effects ranging considerably within a population. Objectives. In this study, we have developed a genotyping platform able to determine the alleles related to interindividual variability in the CYP2D6 gene. Design and Methods. We used a long PCR strategy coupled to MALDI-TOF mass spectrometry (Sequenom) to develop a SNPs genotyping method. Furthermore, an amplification allele specific was carried out to infer the correct allelic phase. Results. We tested the multiplex platform in 250 DNA Sardinian samples and found it to be 100% concordant with the sequencing results of our previous work. Conclusions. The MALDI-TOF-based multiplexing system allowed simultaneous and efficient genotyping of a set of CYP2D6 SNPs, evidencing its potential use in diagnostic test development to predict drug responses and clinical outcomes. 1. Introduction The presence of polymorphisms in the cytochrome P450 2D6 (CYP2D6) gene may modulate enzyme levels affecting individual responses to pharmacological treatment in drug level, response, and adverse reactions and includes individuals with ultrarapid (UM), extensive (EM), intermediate (IM), and poor (PM) metabolizer status. Furthermore, the presence of multiple functional gene copies or the deletion of the entire gene results in increased or absent drug metabolism, respectively [1, 2]. Genotypic analysis to identify individual polymorphisms has become increasingly important during drug development and for selection of individualized therapies. For this reason, high-throughput technology for rapid, accurate, and efficient genotyping is needed. Several strategies and methods for SNPs genotyping have been developed. Techniques commonly used, such as PCR-RFLP, real-time PCR, and the TaqMan allele-specific assays from Applied Biosystems (CA, USA), are often laborious, and a restricted number of alleles can be simultaneously detected by these techniques. Conversely, the high-throughput oligonucleotide microarray technology, such as the Affymetrix/Roche (CA, USA) AmpliChip CYP450 GeneChip test, has the disadvantage over other assays in that it cannot be customized by the user, and the benefits of this technology are often not compensable due to unfavourable costs [3]. In our previous work [4], we tried to create a complete genotyping platform for the simultaneous analysis of CYP3A4, CYP3A5, CYP2C9, CYP2C19, and CYP2D6 SNPs. The genotyping of the CYP2D6 gene was difficult due to its