Diagnostic Systems

Cytochrome P450

In a medicinal therapy the metabolic profiles often turn out to be very different. While some patients benefit from the therapy, others show no improvement or, even worse, unexpected side effects are observed.  The metabolism of medications, just as the one of the body’s own substances, is taking place mainly in the cytochrome P450-system. This system, made up by several enzymes, is preferentially localized to the liver; however, it is also found in many other tissues (e.g. small intestine). It is known, that individual genetic variations in the P450-enzymes are responsible for most of the occurring different metabolic profiles.

Following cytochrome P450 assays are offered by us:

Cytochrome P450 1A1 (CYP1A1)

Cytochrome P450 1A2 (CYP1A2)

Cytochrome P450 2C8 (CYP2C8)

Cytochrome P450 2C9 (CYP2C9)

Cytochrome P450 2C19 (CYP2C19)

Cytochrome P450 CYP3A4 (CYP3A4)

Cytochrome P450 CYP3A5 (CYP3A5)

Cytochrome P450 CYP2D6 (CYP2D6)

Thiopurine-S-Methyltransferase (TPMT)

Thiopurine-S-Methyltransferase (TPMT) genotyping based on Real-Time PCR

✔  real time detection of TPMT SNP genotypes

✔  CE-IVD certification

✔  high sensitivity and specificity

✔  highly stable and reproducible results

✔  manufactured according to DIN EN ISO 13485

✔  32 rxn or 96 rxn

 

 

 

PharmGenomics' Real-Time PCR diagnostic kit is developed for the simultaneous detection of the variants *2, *3A, *3B and *3C, using capillary systems (e.g. LightCycler®, Roche).

The amplification products are analyzed afterwards in a melting-point curve analysis.

Background information

The enzyme TPMT is responsible for the degradation of the majoritys of thiopurines. To its range of substrates belong azathioprine, mercaptopurin and tioguanin. These substances are mainly used in immune suppression after organ transplantation or as cytostatic in tumor therapies. Furthermore, they are used in the treatment of autoimmune, rheumatic and inflammatory intestinal diseases.

Allelic variants

The relevant allele variants are *2, *3A, *3B and *3C which lead to different implications:

The polymorphism TPMT*2 causes an exchange of guanine to cytosine at position 238 of the gene sequence, *3B from guanine to adenine at position 460, and *3C from adenine to guanine at position 719. The allele variant TMPT*3A defines a carrier of the polymorphisms G460A and A719G. The variants *2, *3B and *3C result in a reduced enzyme activity while TPMT*3A causes a complete loss of enzyme activity.

Example for the TPMT*2 (G238C) variant

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Glutathione-S-transferases (GSTs)

GSTs are multifunctional enzymes having a key role in cellular detoxification by protecting the cell through conjugation of glutathione from chemical carcinogens and xenobiotics. These glutathione conjugates are normally less toxic and possess a better water-solubility than the original substances, whereby the excretion from the cell is facilitated. For GSTM1, GSTP1 and GSTT1 a polymorphic distribution in the different populations could be shown.

GSTT1 is involved in the detoxification of carcinogens (e.g. from the smoke of cigarettes), as well as in the formation of toxic metabolites (e.g. from dichlormethan). The deletion of the GSTT1-gene (GSTT1-0 allele) is associated with susceptibility to varying cancers and their prognosis, respectively. To these belong carcinomas of the basal cells and squamous epithelium of the larynx. Approximately 38 % of the population show a complete lack of activity of GSTT1. The polymorphisms of isoleucine to valine at position 105 in the GSTP1-gene is associated with a reduced risk for prostate cancer as well as a favourable prognosis of breast cancer therapy (e.g. with cyclophosphamide). GSTM1 detoxifies several electrophile metabolites, but can also be part of the toxification process. Approximately half of the Caucasian population carries a homozygous deletion (GSTM1*0/*0) and therefore expresses no enzyme. The GSTM1*0 deletion is associated with a small increase in risk for lung, bladder, skin and colon cancer.

Multi Drug Resistance Gene 1 (MDR1)

The MDR1-gene codes for the P-glycoprotein (PGP), an integral cell membrane protein, which is localized in the kidney, liver, blood-brain-barrier, as well as in the adrenal gland and in the colon. Via an ATP-dependent transport it exports toxic substances and metabolites out of the cell (Hoffmeyer et al., PNAS, Vol.97, No.7, 2000). In the blood-brain-barrier the MDR1-gene product conveys i.a. the export of neurotoxic substances. Its broad spectrum of substrates ranges from cytostatics, beta blockers, statins, steroids and calcium antagonists to cyclosporines.

The C3435T polymorphism is an exchange of cytosine to thymidine at position 3435 in exon 26 of the MDR1 gene, which correlates with the expression of MDR1 and thus the PGP-level. The T/T-gene variant is carried by about 24 % of the Caucasian population and is compared to the C/C-genotype related to a 2-fold decreased MDR1 expression and to an even 65-fold lower PGP-level, which in turn increases the bioavailability of most drugs. Heterozygous carriers (C/T-variant) show an intermediate phenotype (Hoffmeyer et al., PNAS, Vol.97, No.7, 2000).

Since the PGP in the blood brain barrier is involved in the control of uptake of for example neurotoxic xenobiotics, differences in expression can have an effect hereupon and thus modulate the individual predisposition for neurologic diseases like Parkinson’s. A clinical study with Parkinson’s patients with early and late manifestation, as well as healthy probands showed that the T/T-genotype was 2-times more frequent in the group of early manifestation, than in the group of healthy probands. Overall, the T-allele was found more frequently in Parkinson’s patients (Furuno et al., Pharmacogenetics 12, 2002).

Furthermore, there is evidence, that the C3435T polymorphism influences the expression of CYP3A4, an important enzyme of the cytochrome P450 system. T/T-carriers showed an 8-times lower expression profile of intestinal CYP3A4-mRNA in comparison to C/C-carriers (Goto et al. Pharmacogenetics 12, 2002).

CYP3A5

CYP3A5 belongs to the most important enzymes of the P450-system, which hepatic part makes up about 40% together with CYP3A4. Additionally it is found in the intestine and the liver. CYP3A5 has a high structural similarity with CYP3A4, so that both enzymes degrade a very similar spectrum of substrates. CYP3A5 degrades among others xenobiotics, toxins and steroids (endogenous substrates).

The mutations CYP3A5*2 and CYP3A5*3 and their implication:

The polymorphism CYP3A5*2 causes an exchange of threonine to asparagine at position 398 in the amino acid sequence. In the Caucasian population about 2% are carriers of this mutation. The polymorphism CYP3A5*3 causes at position 6986 in the gene sequence an exchange of adenine to guanine. This results in a defective splicing site and a changed exon 3, which inserts a stop codon into the mRNA during the translation. Consequently, this leads to an early abort of protein synthesis. Persons with homozygous genotype for CYP3A5*3 have only minor enzyme activity, while person with at least one CYP3A5 wild type allele have sufficient CYP3A5 activity.