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Determination of CYP2D6 gene copy number by pyrosequencing
Biotage AB, Uppsala, Sweden.
Linköping University, Department of Medicine and Health Sciences, Clinical Pharmacology . Linköping University, Faculty of Health Sciences.
Linköping University, Department of Clinical and Experimental Medicine. Linköping University, Faculty of Health Sciences.
Biotage AB, Uppsala, Sweden.
2005 (English)In: Clinical Chemistry, ISSN 0009-9147, Vol. 51, no 3, 522-531 p.Article in journal (Refereed) Published
Abstract [en]

BACKGROUND: Identification of CYP2D6 alleles *5 (deletion of the whole CYP2D6 gene) and *2xN (gene duplication) is very important because they are associated with decreased or increased metabolism of many drugs. The most commonly used method for analysis of these alleles is, however, considered to be laborious and unreliable.

METHODS: We developed a method to determine the copy number of the CYP2D6*5 and CYP2D6*2xN alleles by use of Pyrosequencing technology. A single set of PCR and sequencing primers was used to coamplify and sequence a region in the CYP2D6 gene and the equivalent region in the CYP2D8P pseudogene, and relative quantification between these fragments was performed. The CYP2D8P-specific Pyrosequencing peak heights were used as references for the CYP2D6-specific peak heights.

RESULTS: Analysis of 200 pregenotyped samples showed that this approach reliably resolved 0-4 genome copies of the CYP2D6 gene. In 15 of these samples, the peak pattern from one analyzed position was unexpected but could be solved by conclusive results from a second position. The method was verified on 270 other samples, of which 267 gave results that corresponded to the expected genotype. One of the samples could not be interpreted. The reproducibility of the method was high.

CONCLUSIONS: CYP2D6 gene copy determination by Pyrosequencing is a reliable and rapid alternative to other methods. The use of an internal CYP2D8P control as well as generation of a sequence context ensures a robust method and hence facilitates method validation.

Place, publisher, year, edition, pages
2005. Vol. 51, no 3, 522-531 p.
National Category
Medical and Health Sciences
URN: urn:nbn:se:liu:diva-17934DOI: 10.1373/clinchem.2004.043182PubMedID: 15650034OAI: diva2:213008
Available from: 2009-04-27 Created: 2009-04-27 Last updated: 2009-04-27Bibliographically approved
In thesis
1. Pharmacogenetics from a Forensic Perspective: CYP2D6 and CYP2C19 genotype distributions in autopsy cases
Open this publication in new window or tab >>Pharmacogenetics from a Forensic Perspective: CYP2D6 and CYP2C19 genotype distributions in autopsy cases
2009 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

In Sweden about 550 individuals die every year due to drug intoxication. A challenge for the forensic toxicologist is to determine whether or not the analytical results can explain intoxication as a cause of death. The most common drugs found among intoxication cases are psychiatric drugs and analgesics. Many of these drugs are metabolised by CYP-enzymes such as CYP2D6 and CYP2C19. Genetic variations, polymorphisms, in the genes coding for these enzymes can lead to an inactive enzyme resulting in poor metabolism, which can lead to adverse drug reactions, even with fatal outcome. The CYP2D6 gene can be multiplied, which can lead to an ultra-rapid metabolism if the alleles are active. Another polymorphism, in the CYP2C19 gene, can also lead to an ultra-rapid metabolism. This increased metabolism can result in insufficient drug plasma concentration and, with that, failed treatment. Alternately, if the drug is a pro-drug and has to be activated by these enzymes, it can lead to a high amount of active metabolites. There is a large inter-individual variation of these polymorphisms and also a large variation between different populations. Additional information about an individual’s pharmacogenetics may possibly facilitate the interpretation of the postmortem result and contribute to solve the “toxicological puzzle”.

The general aim of this thesis was to study if genetic variation in the drug metabolising enzymes, CYP2D6 and CYP2C19 can contribute to fatal intoxication. Reliable and rapid SNP and CNV assays suitable for forensic samples using PCR and pyrosequencing were developed for CYP2D6 and genotype frequencies in a Swedish population were shown to be in concordance with earlier published data. SNP assays were established for polymorphisms in the CYP2C19 gene.

Genotype distributions in fatal intoxication cases were compared with Swedish blood donors and significant difference between the materials were established. The allele CYP2D6*4 was found to be less frequent among the intoxication cases, as compared with the blood donors. No differences in CYP2C19 genotype frequencies were found between the materials. These findings are the opposite of our hypothesis that we expected to find an increased number of individuals carrying genetic variations, leading to poor metabolism among fatal intoxication cases. However, we are convinced that information concerning an individual’s genotype can be of importance in specific intoxication cases. Further studies are required to illuminate this question. Two further autopsy materials were studied; suicide cases (intoxications excluded) and natural death cases. A significant increased number of individuals carrying more than two active CYP2D6 alleles among the suicide cases were found compared to natural death cases. Furthermore, we found some significant differences between the materials when the individuals in each material were grouped according to how many active CYP2D6 alleles they carry in combination with the CYP2C19 genotype, which was divided into six subgroups. We do not currently have any explanation for the differences between the materials.

Place, publisher, year, edition, pages
Linköping: Linköping University Electronic Press, 2009. 82 p.
Linköping University Medical Dissertations, ISSN 0345-0082 ; 1124
National Category
Medical and Health Sciences
urn:nbn:se:liu:diva-17936 (URN)978-91-7393-639-2 (ISBN)
Public defence
2009-05-15, Berzeliussalen, Campus US, Linköpings Universitet, Linköping, 09:00 (Swedish)
Available from: 2009-04-27 Created: 2009-04-27 Last updated: 2009-08-21Bibliographically approved

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Zackrisson, Anna-LenaLindblom, Bertil
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