Prof. Fornasari Flashcards
SNPs classification and their effects on CYP450 system
SNP means single polymorphism nucleotide and it means that we have the same gene different for one nucleotide in different individuals. If the nucleotide variation occurs in the right position, it can drastically modify the function of the protein. These types of differences are not point mutation (frequency ≤ 1,1% and it causes a disease) because their frequency is >1% and they cause a variability compatible with life. SNPs are classified in base of their position in the gene that causes different effects on the encoding protein:
- cSNP are present in exons, in the coding part of the genome. It is a substitution of just one nucleotide that will probably (because of the degeneration of the code) cause an amino acid substitution. The substitution of one nucleotide can also introduce a new stop-codon that will result in a truncated protein that doesn’t work in the right way or it can produce a protein with a different function of partially functioned.
These polymorphisms are inherited from our parents and we will transmit them to our progeny, but they are also spontaneous and so new polymorphisms can always occur. We have 2 alleles, one from the mother and the other from the father, if you received the polymorphism from one parent you have 50% of right expression of the gene (some time it isn’t enough), if you have 2 modified alleles you do not have an active gene (healthy until you take the drug).
- pSNP are polymorphisms that occur in peri-genic regions around the gene, it means in regulatory regions, splicing regions, 3’ and 5’ end.
Cis-Acting-Elements are non-coding regulatory regions, able to bind one or more specific transcriptional factors that modulate transcription of nearby genes (whereas trans-acting-elements are sequences coding for transcriptional factor acting in distant genes). SNPs in these regions may implicate different effects: transcriptional factor may no longer bind to the region, bind with less affinity or even bind with more affinity. A peri-genic SNP may also give rise to a new cis-acting element in a place where it wasn’t there. All of these SNPs are involved in gene transcription
regulation, so they have quantitative effects on protein production (not qualitative ones). p-SNPs in splicing junctions have different outcomes if they occur in the 5’ end or in the 3’ end: for 5’ end splicing junction’s SNPs, the phenomenon generated is ‘intron retention’, because the sequence that marks the end of an exon is no longer recognized by splicing factors; introns often contain stop-codons (lacking of evolutionary pressure), so the translation will lead to truncated transcripts and proteins. So, in intron retention, an intron is not removed because the machinery does not recognize the region and does not eliminate it. The transcript will be truncated, with come different aminoacids and the protein will not be able to work in the right way. On the other hand, SNPs in the 3’ end generate ‘exon skipping’, because the sequence that marks the beginning of a new exon is unrecognizable by splicing machinery. In most of these cases the protein, lacking a set of aminoacids, cannot fold correctly. In both cases, synthetized proteins lose their function and are readily degraded.
- rSNP are polymorphisms that occur in intergenic reason (junk DNA). It means that are random SNPs because they stay in casual region of the genome. Only a little part of junk DNA encodes for protein such as miRNA and so SNP in these regions are not so relevant.
In general, there are approximately 6-8 SNPs in exons (for each gene), few hundred thousand of p-SNP and million in junk DNA.
If we consider CYP450 system (cytochrome with different functions form the classical ones), it can be affected from all these polymorphisms and so have some alleles variants. These phase I enzymes, if their genes are polymorphic, can be truncated, can change their functions (functional deletion), can not work anymore. Once you have a polymorphism, you have to understand if and how it could change the drug metabolism and response.
For example, we can have some polymorphisms in CYP2C9, that is able to metabolize Walfarin (anti-coagulant that inhibits the action of VKORC, a factor able to reduce vitamin K and so allow the function of gamma-GCX enzyme that, normally, does a carboxylation before release the coagulation enzyme in the blood circulation). Polymorphisms in CYP2C9 or in VKORC can produce adverse drug reaction. For example, CYP2C93 causes the formation of altered metabolites due to an altered substrate specificity.
Other examples are SNPs in CYP2C19 (it is able to activate Clopidogrel, an anti-platelet drug), for this CYP has been described 19 allelic variants. For example, CYP2C192 and 3 variants lead to a truncated protein due to a stop codon insertion that causes the adverse severe bleeding. Another variant is CYP2C1917 that increases the translation of the CYP itself and so the therapeutic failure should be avoid.
We can have polymorphism also in the CYP3A5, that are able to metabolize Tacrolimus (an immunosuppressor which works by inhibiting the calcineurin system). The most popular polymorphism (*3) for this CYP shows a stop codon and so we obtain a truncated protein that does not function. 90% of Caucasian people has this polymorphism and so, for these people, the dosage of tacrolimus will be halved compared with normal doses.
differences between germinal and somatic pharmacogenetics
Pharmacogenetics is the study of variability in drug response due to heredity. It is the science that study the individual genetic basis responsible to ADR (by studying the individual variation). In this field, we consider the cancer pharmacogenetics, means that we consider the ADR in a patient affected by cancer, that has led to individual mutations. Cancer patient contain two different kind of genome: one is the host genome, and the other is cancer genome. Cancer genome is the ones
that has acquired the mutations and is expanding. Drug toxicity is largely dependent to the host genome, which encodes for the enzymes able to metabolize anti-cancer drugs. If we consider the drug response/sensitivity, we should pay attention to the cancer genome: in fact, cancer cells, in their chaotic accumulation of mutations, are likely to change their sensitivity to drugs (for example over-expressing MDRs, Multi Drug Resistance transporters that mediate drugs’ efflux from the cell). Drug availability, instead, is an issue shared by both genomes.
Cancer stem cells are the ones where tumor derives from and the ones we need to eradicate in order to successfully cure the patients. We treat the tumor but we’re pretty unable to treat cancer stem cells, because unluckily they’re also the most resistant cells: they’re the cancer’s sanctuary and they express lots of ABC (ATP-Binding-Cassette) transporters (MDRs are included in this family), which mediate active efflux of drugs and keep clean the cell’s inside.
In the field of cancer therapy, we consider two types of pharmacogenetics:
• Germline Pharmacogenetics→ studies polymorphisms of the host genome (all of our cells, gonads included) that have consequences on drug response (most known of them are located in xenobiotics’ metabolism genes). We get these polymorphisms from our parents and we will transmit them to our progeny, because they are SNPs present in the germinal cells that are inherited from parents. Child will have these polymorphisms in all their cells, both germinal and somatic. Considering Irinotecan, that is a prodrug activated by Ce enzyme in SN-38 and then metabolized by UGT1A1. Germline genome acts properly on UGT1A1, it can be polymorphic in the promoter region due to the fact that the individual has inherited from his parents this SNPs. If this enzyme of phase II is mutated, we will have a low expression of that and so a low glucuronidation of the substrate that can cause a sever adverse drug reaction. This fact demonstrates that the toxicity depends on the host genome.
• Somatic Pharmacogenetics→ the genomes of somatic cells that have acquired mutations (not polymorphisms) are studied in order to get information about the variation in the response to drugs. This kind of cells acquired SNPs during the time. This kind of pharmacogenetics studies the tumors. In these situations, we found two genomes: the host genome and the cancer genome.
Here are some examples of somatic pharmacogenetics:
1. Gefitinib is a small molecule drug, used in the therapy for a kind of lung tumor called ‘Non-Small-Cell-Lung-Cancer’. Its action consists in the blocking of EGF-R (Epithelial Growth Factor Receptor). The problem is that, in this type of cancer, EGF-R could be mutated or not, and Gefitinib works only if the receptor is actually mutated. This is a case in which the drug therapeutic effect depends on the cancer genome;
2. In colon-rectal-cancer, EGF-R is over-expressed, and we use a monoclonal antibody, Cetuximab, to block these receptors. In this case, knowing about the cancer’s genome is important because the pathogenesis could involve a mutation in a downstream component of the signaling pathway under EGF-R, named “K-RAS”: this mutation implicates the constitutional activation of K-RAS and giving Cetuximab for the therapy will end up to be useless, since the signaling pathway will stay activated despite EGF-R is blocked.
CNVs and their effects on drug metabolism
CNV means copy number variation, it is a phenomenon in which sections of the genome are repeated and the number of repeats in the genome varies between individuals in the human population. We consider CNV not only as a SNP, but also as a big part of the genome that can be mist or duplicated. It means that you could have duplicated region of DNA from 50 bp (below this number it is consider only as an insertion). Above 50 bp and up to 1 million of bp, we could have piece of DNA which are deleted or repeated. Specific gene involved in structure variation could be repeated and you could have several copies of that gene or also no copy of the same gene. The enzyme could be absent, and, in this case, there are the deletion of the allele. When you have a stop codon in the allele you have a deletion, but we consider it as a functional deletion because the allele does not work. It encodes but the product is quickly degraded. In the opposite situation, you could have the same gene which is repeated several times. For example, in the case of CYP2D6 some people could have 13 copies of the gene. Each single copy is working, so it means that you will have 13 times more CYP2D6 in the patient having CNV. The number of functional CYP2D6 can be identified thanks to genotyping. Depending on the number of copies of the gene encoding CYP2D6, the metabolism of nortriptyline, for example, shows different behaviors. People with 0 functional copy of CYP2D6 gene, accumulate the drug and after 72 hours, patients are full of the drug in their blood because they have problem to eliminate it because they have not the enzyme that metabolize it. In this case it can be toxicity. In the opposite case, in people with 13 functional copies of CYP2D6 gene, after 24 hours the total amount of drug that they had taken is eliminated. If we talk about drugs, a higher number of copies of a gene encoded for a particular enzyme, can help you to eliminate it in less time and they can have therapeutic failure because the drug has been metabolize in a too rapid way. A lower amount of gene that encodes for that enzyme, mean that the drugs will accumulate and can give toxicity. In this case the patient has to take a lower dose of the drug. If we talk about pro-drug, the situation is different, because if the enzyme which you are interested in is a lot, a big amount of pro-drug (ex. Codeine) is activated in drug (ex. Morphine). If the enzyme is not much, less pro-drug is activated in active drug and this can be not enough and can give therapeutic failure. In this case the patient has to take a higher dose of this molecule.
CYP2D6 is also able to activate the pro-drug tamoxifen in endoxifen (active drug), that is useful against breast cancer sensitive to estrogen. It is a selective antagonist of estrogen receptor (SERM). It is given after surgery for 5 years in order to block some possible proliferation of cells remaining in the body. Women that due to CNV, have not CYP2D6 can not do this therapy because it is not useful. If a patient hasn’t CY2D6 is a poor metabolizer and so his tumor becomes aggressive because there is no metabolization of tamoxifen. It means that before treating women with tamoxifen, we should perform their genotyping in order to understand their right allele. We have to use another drug, for example anastrasol which block the conversion of estrogen. Ultra-metabolize: the patient converts tamoxifen to endoxifen too much → the anti-estogenic effect will be out of the breast tissue because now the specificity is something related to dosage and these women will have anti-estrogenic inducing by the therapies. If they have too many copies, we have to reduce the normal dose of tamoxifene.
In the case of use of some tricyclic anti-depressant, if the individual has CNV in CYP2D6, he can undergoes adverse drug reaction such as tachycardia and cardiotoxicity.
glucocorticoids receptors
definition of intrinsic activity
Beta-adrenegin receptors in the target organs of the ANS (functions and drugs acting on them)
Definition of inverse agonist
GPCR
