Universitą degli studi di Pavia
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Grignani research activity
“Pathology and Medical Genetics” PhD program (XXVI), University of Pavia.
As part of my PhD program in "Pathology and Medical Genetics", I am working on the research
“SNPs analysis in drugs metabolism”.
Supervisors: Dott. C. Previderč and the Prof. C. Danesino.
Pharmacogenetics, the study of the role of inheritance in interindividual variability in drug response, has the potential to contribute to the development of more rational pharmacological therapies.
Pharmacogenomic studies aim to elucidate the genetic bases for interindividual differences and to use such genetic information to predict the safety, toxicity, and/or efficacy of drugs.
Biotransformation of drugs is one of the major functions of liver. The CYP450 enzyme family plays a determinant role in the biotransformation of a vast number of structurally diverse drugs.
The cytochromes P450 are a superfamily of hemoproteins that catalyze the metabolism of a large number of xenobiotics and endobiotics. The cytochrome P450 gene family contains around 60 different genes and more than 59 pseudogenes divided among 18 families of cytochrome P450 genes and 43 subfamilies.
The drug biotransformation Phase I reactions may occur by oxidation, reduction, hydrolysis carried out by mixed function oxidases, often in the liver. These oxidative reactions typically involve a cytochrome P450 monooxygenase (often abbreviated CYP). Phase II reactions — usually known as conjugation reactions (e.g., with glucuronic acid, sulfonates, glutathione) — are usually detoxicating in nature, and involve the interactions of the polar functional groups of phase I metabolites. Products of conjugation reactions have increased molecular weight and are usually inactive unlike Phase I reactions which often produce active metabolites.
The variability associated with the CYP450 enzymes in each individual result in a marked difference in response when the same drug and the dose are administered to different individuals. Genetic polymorphism of CYP450 enzymes characterizes the general population into three groups: extensive metabolizers (EM), poor metabolizers (PM) and ultra extensive metabolizers (UM).
The 2C subfamily consists of isoenzymes 2C9, 2C19 and others. CYP2C9 has a polymorphic distribution in the population. The isoenzyme CYP2C19 also exhibits genetic polymorphism.
CYP2C9 is a major liver enzyme responsible of the metabolism of many clinically important drugs as oral anticoagulants (warfarin, acenocoumarol), oral antidiabetic drugs and some non-steroidal anti-inflammatory drugs. The presence of CYP2C9 genetic polymorphisms has been associated with marked interindividual variability in its catalytic activity that could result in drug toxicity. Two coding variants, CYP2C9*2 (C430T, rs1799853) and CYP2C9*3 (A1075C, rs1057910), with functional consequences are common. CYP2C9*2 codes for a R144C substitution and CYP2C9*3 reflects an I359L change in the amino-acid sequence.
CYP2C19 is a monooxygenase that metabolizes a wide variety of drugs, including anticonvulsants, antidepressants, antimalarial drugs. The most common variants alleles are named as CYP2C19*2 contain a variant that is a splice defective mutation in exon 5( rs4244285), with a G-A nucleotide change at 681 of cDNA sequence. This change alters the reading frame of the mRNA starting with amino-acid 215 and produces a premature stop codon 20 amino acids downstream, which results in atruncated, nonfunctional protein. Another common allele CYP2C19*3 has a G-A mutation at position 636 of exon 4 (rs28399504), which also creates a premature stop codon. Recently, a novel variant allele CYP2C19*17, −806C in the 5′- flanking region, has been found to be associated with increased CYP2C19 activity, resulting in ultra-rapid metabolism of CYP2C19 substrates.
I’m develop and test a new PCR multiplex amplification reaction. At the same time the fragments with allelic variants CYP2C9*1, CYP2C9*2, CYP2C9*3 and CYP2C19*1 CYP2C19*2 CYP2C19*3, CYP2C19*17 are amplified in a single tube reaction . Allelic discrimination are obtain by capillary electrophoresis on an ABI PRISM 310 Genetic Analyzer after a multiplex SBE (single base extension) reaction performed by SNaPshot kit (Applied Biosystems).
As part of my PhD program in "Pathology and Medical Genetics", I am working on the research
“SNPs analysis in drugs metabolism”.
Supervisors: Dott. C. Previderč and the Prof. C. Danesino.
Pharmacogenetics, the study of the role of inheritance in interindividual variability in drug response, has the potential to contribute to the development of more rational pharmacological therapies.
Pharmacogenomic studies aim to elucidate the genetic bases for interindividual differences and to use such genetic information to predict the safety, toxicity, and/or efficacy of drugs.
Biotransformation of drugs is one of the major functions of liver. The CYP450 enzyme family plays a determinant role in the biotransformation of a vast number of structurally diverse drugs.
The cytochromes P450 are a superfamily of hemoproteins that catalyze the metabolism of a large number of xenobiotics and endobiotics. The cytochrome P450 gene family contains around 60 different genes and more than 59 pseudogenes divided among 18 families of cytochrome P450 genes and 43 subfamilies.
The drug biotransformation Phase I reactions may occur by oxidation, reduction, hydrolysis carried out by mixed function oxidases, often in the liver. These oxidative reactions typically involve a cytochrome P450 monooxygenase (often abbreviated CYP). Phase II reactions — usually known as conjugation reactions (e.g., with glucuronic acid, sulfonates, glutathione) — are usually detoxicating in nature, and involve the interactions of the polar functional groups of phase I metabolites. Products of conjugation reactions have increased molecular weight and are usually inactive unlike Phase I reactions which often produce active metabolites.
The variability associated with the CYP450 enzymes in each individual result in a marked difference in response when the same drug and the dose are administered to different individuals. Genetic polymorphism of CYP450 enzymes characterizes the general population into three groups: extensive metabolizers (EM), poor metabolizers (PM) and ultra extensive metabolizers (UM).
The 2C subfamily consists of isoenzymes 2C9, 2C19 and others. CYP2C9 has a polymorphic distribution in the population. The isoenzyme CYP2C19 also exhibits genetic polymorphism.
CYP2C9 is a major liver enzyme responsible of the metabolism of many clinically important drugs as oral anticoagulants (warfarin, acenocoumarol), oral antidiabetic drugs and some non-steroidal anti-inflammatory drugs. The presence of CYP2C9 genetic polymorphisms has been associated with marked interindividual variability in its catalytic activity that could result in drug toxicity. Two coding variants, CYP2C9*2 (C430T, rs1799853) and CYP2C9*3 (A1075C, rs1057910), with functional consequences are common. CYP2C9*2 codes for a R144C substitution and CYP2C9*3 reflects an I359L change in the amino-acid sequence.
CYP2C19 is a monooxygenase that metabolizes a wide variety of drugs, including anticonvulsants, antidepressants, antimalarial drugs. The most common variants alleles are named as CYP2C19*2 contain a variant that is a splice defective mutation in exon 5( rs4244285), with a G-A nucleotide change at 681 of cDNA sequence. This change alters the reading frame of the mRNA starting with amino-acid 215 and produces a premature stop codon 20 amino acids downstream, which results in atruncated, nonfunctional protein. Another common allele CYP2C19*3 has a G-A mutation at position 636 of exon 4 (rs28399504), which also creates a premature stop codon. Recently, a novel variant allele CYP2C19*17, −806C in the 5′- flanking region, has been found to be associated with increased CYP2C19 activity, resulting in ultra-rapid metabolism of CYP2C19 substrates.
I’m develop and test a new PCR multiplex amplification reaction. At the same time the fragments with allelic variants CYP2C9*1, CYP2C9*2, CYP2C9*3 and CYP2C19*1 CYP2C19*2 CYP2C19*3, CYP2C19*17 are amplified in a single tube reaction . Allelic discrimination are obtain by capillary electrophoresis on an ABI PRISM 310 Genetic Analyzer after a multiplex SBE (single base extension) reaction performed by SNaPshot kit (Applied Biosystems).
