LightSNiP assay is a good strategy for pharmacogenetics test.

Abstract

The individual response to standard doses of drugs has a large variability depending on intrinsic factors (age, sex, and disease states) and/or extrinsic factors (diet, chemical exposures from the environment). The influence of these factors on drug responses has therefore to be taken into consideration when making decisions on treatment regimes (Thummel and Lin, 2014). Nonetheless, the taking into account of these factors limits, but does not eliminate, the high degree of interindividual variability in terms of efficacy or fatal adverse reactions. The individual genetic background, explains part of the different pharmacokinetic and pharmacodynamic drug responses both in term of efficacy and toxicity (Hertz and McLeod, 2013). Evidence indicates that genetic variations account for an estimated 20–40% of inter-individual differences in drug metabolism and response (Karczewski et al., 2012). The commonest genetic variations are single nucleotide polymorphisms (SNPs), representing approximately 90% of all human genetic variations and occurring every 100–300 base pairs (Crews et al., 2012). Some of these polymorphisms have been identified for many proteins including enzymes, drug receptors, transporters, and targets of the commonest drugs. These polymorphisms can cause alterations in the amount, structure, binding, and/or function of these proteins, influencing how drugs interact with them (Ma et al., 2012; Patel et al., 2013). The study of these genetic variants and their role in improving drug efficacy and reducing side effects, termed pharmacogenetics, is now established in the clinical practice for many drugs including abacavir, irinotecan, and 6 mercaptopurines (Ingelman-Sundberg, 2008; Wang et al., 2011). To facilitate an appropriate clinical implementation of pharmacogenetics, guidelines have been published by the Clinical Pharmacogenetics Implementation Consortium (CPIC) and the Dutch Pharmacogenetics Working Group (DPWG). The aim of these guidelines as they clearly state is to facilitate translation of the genetic laboratory test results into prescribing decisions for specific drugs, or class of drugs, such as vitamin K antagonist, tricyclic antidepressants (Johnson et al., 2011; Caudle et al., 2013; Hicks et al., 2013). So far, several techniques have been described to detect specific SNPs, with the Sanger Sequencing, the Denaturing Gradient Gel Electrophoresis (DGGE), the Single Strand Conformational Polymorphism analysis (SSCP), the Pyrosequencing and Sequenom being the most widely employed. Recently also techniques of high-throughput screening and for large-scale characterization of SNPs have been developed; these platforms, however, are expensive, not flexible and not of practical use for small to medium size laboratories. More user-friendly, SNPs detection methods for pharmacogenetic tests are based on PCR amplification, in conjunction with an appropriate probe technology (real-time PCR) as TaqMan, Scorpion and SimpleProbe®. Among these, SimpleProbe® appears of particular interest. SimpleProbe format is composed of one hybridization probe, labeled with a fluorophore; this oligonucleotide is designed spanning the variant of interest, but does not participate into the amplification process. Once hybridized to its target sequence, the SimpleProbe probe releases more fluorescence than when not hybridized. Changes in fluorescence that are exclusively based on the probe hybridization status are detected by melting curve analyses. Any mismatch positioned under the SimpleProbe® will reduce the hydrogen-bonds, hence the melting temperature, thus enabling analysis of polymorphisms. Diverse variations destabilize the hydrogen-bonds differently, usually yielding different and specific melting points, thus making it possible to identify polymorphisms tightly close to the intended variant, giving it potential advantages over competing technologies. Here, we describe the advantages of real-time PCR assays for the detection of three SNPs selected for their involvement in DPD and UGT1A1 enzymes regulations (Falvella et al., 2015). This SNP detection is based on LightCycler Technology with SimpleProbe® probes (LightSNiP assays); it designed by TIB Molbiol (Berlin, Germany) and is a validated Real Time PCR method used in many areas of molecular diagnostics. From amplification and detection with specific probes by melting curve analysis, it is possible to obtain a visual discrimination of normal and variant alleles in the homozygous and heterozygous status. When selecting the most appropriate technique for a analysis of a given genotype, few general considerations should be taken into account such as correct detection, simplicity in the technology and reproducibility.