ABSTRACT
Title
Design and development of pharmacogenetic molecular tests
Authors
E. Bugin
Department of Pharmacology and Anaesthesiology "E. Meneghetti", section of Molecular and Cellular Pharmacology - University of Padua, Italy
AB ANALITICA srl - Padua, Italy
Department of Pharmacology and Anaesthesiology "E. Meneghetti", section of Molecular and Cellular Pharmacology - University of Padua, Italy
AB ANALITICA srl - Padua, Italy
Abstract
At the present time, interest on pharmacogenetic aspects has been rapidly growing by increasing access to awarenessresearch based on Molecular Biology using methods that allow the identification and even prevention of adverse drug reactions. The relationship between tolerance and/or effectiveness of certain drugs and the individual genetic characteristics has been already demonstrated.
In the scientific community the development of methods able to detect mutations and/or SNPs responsible of resistance to cancer drugs or adverse reactions due to pharmacological therapy has received large attention. Previous experiments have allowed the development of a diagnostic kit able to detect the 12-13 K_RAS oncogene mutations by DNA amplification and RFLP analysis. In fact, the K-RAS mutational status represents a predictive marker before starting the anticancer therapy. Accordingly, our main goal was to evaluate the possibility of developing a new system to determine codon 12 -13 K-RAS oncogene mutations, for example by Reverse Dot Blot (RDB). We also focused our attention on the design of a method for the analysis of genetic polymorphisms on VKORK and CYP-450 genes, that can influence warfarin dose requirements.
The experimental model consisted of formalin-fixedparaffin-embedded tissue derived from colorectal tumor and blood samples obtained from patients undergoing warfarin therapy. DNA was extracted using commercial kits and amplified using the PCR technique. Formalin-fixedparaffin-embedded tissue required a proper processing to guarantee the DNA integrity.
One of the biggest problems we had to overcome, especially for the polymorphism genotyping, was the samples recruitment; to ensure an adequate number of samples with certain polymorphisms, we built synthetic genes for the required variations using recombinant DNA technology. Built constructs were also used to assess method sensitivity and specificity.
Another question was on the Cytochromes P-450 enzymes. Cytochromes P450 (CYPs) belong to the superfamily of proteins containing a hemecofactorand their sequences are very similar; moreover, each gene contains repeated sequence blocks that make every isoform’s discrimination a challenge. Therefore to detect single SNPs we first amplified the gene of interest using the PCR technique and making big efforts in choosing specific PCR primers; then we detected amplicons exploiting RDB hybridization assay. RDB offers the advantage of a simultaneous detection of a panel of point mutations with only one hybridization per sample and it can be automated to study many samples. We chose this assay after doing benchmarking that supported our choice.
The results confirmed that RDB is a very accurate and reliable method, that offers cost-effective molecular diagnosis results. The next step would be to develop other tests able to predict the occurrence of resistance to tamoxifen and chemotherapic agents, such as 6-mercaptopurine. Moreover we will pay special attention to genotype the CYP2C19 gene, whose polymorphisms are associated with different abilities to metabolize many drugs commonly used in therapy.
In the scientific community the development of methods able to detect mutations and/or SNPs responsible of resistance to cancer drugs or adverse reactions due to pharmacological therapy has received large attention. Previous experiments have allowed the development of a diagnostic kit able to detect the 12-13 K_RAS oncogene mutations by DNA amplification and RFLP analysis. In fact, the K-RAS mutational status represents a predictive marker before starting the anticancer therapy. Accordingly, our main goal was to evaluate the possibility of developing a new system to determine codon 12 -13 K-RAS oncogene mutations, for example by Reverse Dot Blot (RDB). We also focused our attention on the design of a method for the analysis of genetic polymorphisms on VKORK and CYP-450 genes, that can influence warfarin dose requirements.
The experimental model consisted of formalin-fixedparaffin-embedded tissue derived from colorectal tumor and blood samples obtained from patients undergoing warfarin therapy. DNA was extracted using commercial kits and amplified using the PCR technique. Formalin-fixedparaffin-embedded tissue required a proper processing to guarantee the DNA integrity.
One of the biggest problems we had to overcome, especially for the polymorphism genotyping, was the samples recruitment; to ensure an adequate number of samples with certain polymorphisms, we built synthetic genes for the required variations using recombinant DNA technology. Built constructs were also used to assess method sensitivity and specificity.
Another question was on the Cytochromes P-450 enzymes. Cytochromes P450 (CYPs) belong to the superfamily of proteins containing a hemecofactorand their sequences are very similar; moreover, each gene contains repeated sequence blocks that make every isoform’s discrimination a challenge. Therefore to detect single SNPs we first amplified the gene of interest using the PCR technique and making big efforts in choosing specific PCR primers; then we detected amplicons exploiting RDB hybridization assay. RDB offers the advantage of a simultaneous detection of a panel of point mutations with only one hybridization per sample and it can be automated to study many samples. We chose this assay after doing benchmarking that supported our choice.
The results confirmed that RDB is a very accurate and reliable method, that offers cost-effective molecular diagnosis results. The next step would be to develop other tests able to predict the occurrence of resistance to tamoxifen and chemotherapic agents, such as 6-mercaptopurine. Moreover we will pay special attention to genotype the CYP2C19 gene, whose polymorphisms are associated with different abilities to metabolize many drugs commonly used in therapy.