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Pharmacogenetics from a Forensic Perspective: CYP2D6 and CYP2C19 genotype distributions in autopsy cases
Linköpings universitet, Institutionen för medicin och hälsa, Klinisk farmakologi. Linköpings universitet, Hälsouniversitetet.
2009 (engelsk)Doktoravhandling, med artikler (Annet vitenskapelig)
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.

sted, utgiver, år, opplag, sider
Linköping: Linköping University Electronic Press , 2009. , s. 82
Serie
Linköping University Medical Dissertations, ISSN 0345-0082 ; 1124
HSV kategori
Identifikatorer
URN: urn:nbn:se:liu:diva-17936ISBN: 978-91-7393-639-2 (tryckt)OAI: oai:DiVA.org:liu-17936DiVA, id: diva2:213011
Disputas
2009-05-15, Berzeliussalen, Campus US, Linköpings Universitet, Linköping, 09:00 (svensk)
Opponent
Veileder
Tilgjengelig fra: 2009-04-27 Laget: 2009-04-27 Sist oppdatert: 2020-02-26bibliografisk kontrollert
Delarbeid
1. Identification of CYP2D6 alleles by single nucleotide polymorphism analysis using pyrosequencing
Åpne denne publikasjonen i ny fane eller vindu >>Identification of CYP2D6 alleles by single nucleotide polymorphism analysis using pyrosequencing
2003 (engelsk)Inngår i: European Journal of Clinical Pharmacology, ISSN 0031-6970, E-ISSN 1432-1041, Vol. 59, nr 7, s. 521-526Artikkel i tidsskrift (Fagfellevurdert) Published
Abstract [en]

OBJECTIVE: To develop a single nucleotide polymorphism (SNP) analysis for identification of cytochrome P450 (CYP)2D6 alleles by pyrosequencing.

METHODS: Swedish blood donors ( n=282) were typed for a partial CYP2D6 genotype comprising the alleles *1 (wild type), *2 (2850C>T), *3 (2549A>del), *4 (1846G>A) and *6 (1707T>del) using polymerase chain reaction (PCR) and pyrosequencing analysis. CYP2D6*5 (CYP2D6 deleted) was identified using an established long multiplex PCR method. Pyrosequencing is a sequencing-by-synthesis method in which a cascade of enzymatic reactions yields detectable light, which is proportional to the incorporated nucleotides. One feature of typing SNPs by pyrosequencing is that each allelic variant will give a unique sequence. These variants can be readily distinguished by pattern recognition software.

RESULTS: Of 281 individuals analysed, 24 (8.5%) were found to be poor metabolisers with two non-functional alleles. This is in the range of 7-10%, previously reported for Caucasians. A total of 126 individuals (45%) had one functional and one non-functional allele and 131 individuals (47%) had two functional alleles.

CONCLUSION: Pyrosequencing was found to be a fast and efficient tool for genotyping. The method is robust, reliable, rapid and has high throughput.

Emneord
CYP2D6 - Pyrosequencing - Pharmacogenetics
HSV kategori
Identifikatorer
urn:nbn:se:liu:diva-17932 (URN)10.1007/s00228-003-0654-7 (DOI)13680033 (PubMedID)
Tilgjengelig fra: 2009-04-27 Laget: 2009-04-27 Sist oppdatert: 2017-12-13bibliografisk kontrollert
2. Fatal intoxication cases: cytochrome P450 2D6 and 2C19 genotype distributions
Åpne denne publikasjonen i ny fane eller vindu >>Fatal intoxication cases: cytochrome P450 2D6 and 2C19 genotype distributions
Vise andre…
2004 (engelsk)Inngår i: European Journal of Clinical Pharmacology, ISSN 0031-6970, E-ISSN 1432-1041, Vol. 60, nr 8, s. 547-552Artikkel i tidsskrift (Fagfellevurdert) Published
Abstract [en]

OBJECTIVE: Many commonly used pharmaceuticals, such as antidepressants and neuroleptics as well as some illegal drugs, are metabolised by the cytochrome P450 enzyme debrisoquine 4-hydroxylase (CYP2D6). Of Caucasians, 7-10% lack this enzyme, which can, upon administration of drugs in normal therapeutic doses, lead to adverse reactions and unexpected intoxication, leading in turn even to a fatal outcome in some cases.

METHODS: Individuals (n=242) who had died due to intoxication by pharmaceuticals were genotyped for CYP2D6 and CYP2C19 and compared with a reference group of 281 blood donors. A single nucleotide polymorphism (SNP) method was used to identify five CYP2D6 alleles: *1 (wt), *2, *3, *4 and *6. The allele *5, a complete gene deletion, was identified by a multiplex amplification of long DNA fragments. Four CYP2C19 alleles *1 (wt), *2, *3 and *4 were also identified by SNP analysis.

RESULTS: The prevalence of the CYP2D6 poor metaboliser (PM) genotypes in individuals with fatal intoxication was lower (4.7%) than expected from the frequencies of these genotypes in the blood donors (8.5%). A significantly lower frequency P<0.005 (0.03 with correction according to Bonferroni) was found for the CYP2D6*4 allele among the fatal intoxication cases. The CYP2C19 genotype analyses showed the same results for the fatal intoxication cases and for the blood donors.

CONCLUSIONS: The findings in this study confirm our earlier observations of a lower frequency of CYP2D6 PM genotypes in cases of fatal intoxication. To our knowledge, it has not been shown previously that intoxication victims might have a lower frequency of PMs than the general population.

Emneord
CYP2D6, CYP2C19, Post-mortem
HSV kategori
Identifikatorer
urn:nbn:se:liu:diva-17933 (URN)10.1007/s00228-004-0800-x (DOI)15349706 (PubMedID)
Tilgjengelig fra: 2009-04-27 Laget: 2009-04-27 Sist oppdatert: 2018-09-01bibliografisk kontrollert
3. Determination of CYP2D6 gene copy number by pyrosequencing
Åpne denne publikasjonen i ny fane eller vindu >>Determination of CYP2D6 gene copy number by pyrosequencing
2005 (engelsk)Inngår i: Clinical Chemistry, ISSN 0009-9147, E-ISSN 1530-8561, Vol. 51, nr 3, s. 522-531Artikkel i tidsskrift (Fagfellevurdert) 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.

HSV kategori
Identifikatorer
urn:nbn:se:liu:diva-17934 (URN)10.1373/clinchem.2004.043182 (DOI)15650034 (PubMedID)
Tilgjengelig fra: 2009-04-27 Laget: 2009-04-27 Sist oppdatert: 2017-12-13bibliografisk kontrollert
4. High Frequency of Occurrence of CYP2D6 Gene Duplication/Multiduplication Indicating Ultrarapid Metabolism Among Suicide Cases: High frequency of CYP2D6 ultra-rapid metabolizers among suicide cases
Åpne denne publikasjonen i ny fane eller vindu >>High Frequency of Occurrence of CYP2D6 Gene Duplication/Multiduplication Indicating Ultrarapid Metabolism Among Suicide Cases: High frequency of CYP2D6 ultra-rapid metabolizers among suicide cases
2009 (engelsk)Inngår i: Clinical Pharmacology and Therapeutics, ISSN 0009-9236, E-ISSN 1532-6535Artikkel i tidsskrift (Annet vitenskapelig) Published
Abstract [en]

In Sweden about 550 individuals die every year due to intoxication with drugs. Many of these drugs are metabolized by CYP-enzymes, such as CYP2D6 and CYP2C19. Lack of these enzymes, resulting in a poor metabolism, can lead to adverse reactions, even leading to a fatal outcome. On the other hand, socalled ultra-rapid metabolism can lead to insufficient drug plasma concentration and with that failed treatment or it can lead to a high amount of active/toxic metabolites. The aim of this project was to study the genetic distributions of CYP2D6 and CYP2C19 in fatal intoxication cases (242), suicide cases (intoxications excluded) (262) and natural death cases (212). PCR followed by pyrosequencing was used for all the analyses. Surprisingly, we found an increased number of individuals carrying more than two active CYP2D6 alleles, corresponding to the phenotype of an ultra-rapid metabolizer, among the suicide cases as compared to the natural death cases (p=0.007).

HSV kategori
Identifikatorer
urn:nbn:se:liu:diva-17935 (URN)10.1038/clpt.2009.216 (DOI)
Tilgjengelig fra: 2009-04-27 Laget: 2009-04-27 Sist oppdatert: 2017-12-13bibliografisk kontrollert

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