Johanna Sistonen

Pharmacogenetic variation at CYP2C9, CYP2C19, and CYP2D6 at global and microgeographic scales.

Objectives CYP2C9, CYP2C19, and CYP2D6 belong to a subfamily of cytochrome P450 (CYP) enzymes, associated mainly with the metabolism of exogenous compounds in the human body. The genes coding for these enzymes are highly polymorphic and thus of major pharmacogenetic importance. By systematically retrieving data from the literature and genotyping new population samples, we aimed at describing the worldwide distribution of genetic variation at these loci. We created a comprehensive resource of frequency data for the most important CYP2C9, CYP2C19, and CYP2D6 genetic variants in 129, 146, and 138 different population samples, respectively. Furthermore, we showed how demographic history can affect pharmacogenetic variation at a microgeographic scale by analyzing regional samples from Finland, which represents a well-known genetic isolate. Methods Data were obtained from the literature from 1991 to 2007 as well as by genotyping new population samples at four CYP2C9, two CYP2C19, and 12 CYP2D6 variable sites affecting enzymatic activity. Results and conclusion Our study shows that: (i) altered activity variants of CYP2C9, CYP2C19, and CYP2D6 occur globally in all geographic regions, reaching extremely high frequencies in some populations; (ii) each of the CYP genes studied shows a distinct geographic pattern of variation; (iii) population substructure can strongly affect the variation seen in pharmacogenetic loci; and (iv) several geographic regions of pharmacogenetic interest are still poorly characterized.

A fatal doxepin poisoning associated with a defective CYP2D6 genotype.

It has been suggested that the polymorphism of the CYP2D6 gene can contribute to occurrence of fatal adverse effects. We therefore investigated postmortem toxicology cases of fatal drug poisonings related to CYP2D6 substrates, with the manner of death denoted as accidental or undetermined. CYP2D6 genotypes were determined in 11 consecutive cases with samples available for DNA analysis. A case of fatal doxepin poisoning with an undetermined manner of death was found to coincide with a completely nonfunctional CYP2D6 genotype (*3/*4), indicating a total absence of CYP2D6 enzyme and suggesting a poor metabolizer phenotype. The doxepin concentration was 2.4 mg/L, the concentration of nordoxepin 2.9 mg/L, and the doxepin/nordoxepin ratio 0.83, the lowest found among the 35 nordoxepin-positive postmortem cases analyzed during the same year. No alcohols or other drugs were detected in the case. The CYP2C19 genotype was determined as that of an extensive metabolizer. The high N-desmethylmetabolite concentration is not consistent with acute intoxication. It is therefore probable that the defective genotype has contributed to the death, possibly involving repeated high dosage of doxepin. Our case strongly emphasizes that a pharmacogenetic analysis in postmortem forensic setting may reveal new insight to the cause or manner of death.

Molecular diversity at the CYP2D6 locus in the Mediterranean region

Despite the importance of cytochrome P450 in the metabolism of many drugs, several aspects of molecular variation at one of the main loci coding for it, CYP2D6, have never been analysed so far. Here we show that it is possible to rapidly and efficiently genotype the main European allelic variants at this locus by a SNaPshot method identifying chromosomal rearrangements and nine single-nucleotide polymorphisms. Haplotypes could be reconstructed from data on 494 chromosomes in six populations of the Mediterranean region. High levels of linkage disequilibrium were found within the chromosome region screened, suggesting that CYP2D6 may be part of a genomic recombination block, and hence that, aside from unequal crossingover that led to large chromosomal rearrangements, its haplotype diversity essentially originated through the accumulation of mutations. With the only, albeit statistically insignificant, exception of Syria, haplotype frequencies do not differ among the populations studied, despite the presence among them of three well-known genetic outliers, which could be the result of common selective pressures playing a role in shaping CYP2D6 variation over the area of Europe that we surveyed.

Evolution of detoxifying systems: the role of environment and population history in shaping genetic diversity at human CYP2D6 locus.

Objective The transition from food collection to food production (FP) modified the nature of selective pressures, and several studies illustrate that genetic adaptation to new lifestyle has occurred in humans since the agricultural revolution. Here we test the hypothesis that high levels of genetic variation at CYP2D6, a locus coding for a detoxifying enzyme of the cytochrome P450 complex, reflect this change. Methods We compared DNA sequences and predicted the levels of enzyme activity across 10 African, Asian and European populations, six of which currently rely on hunting and gathering (HG) while four on food production (FP). Results and conclusion HG and FP showed similar levels of CYP2D6 diversity, but displayed different substitution patterns at coding DNA sites possibly related to selective differences. Comparison with variation at presumably neutral independent loci confirmed this finding, despite the confounding effects of population history, resulting in higher overall variation in Africans than in Eurasians. The differences between HG and FP populations suggest that new lifestyle and dietary habits acquired in the transition to agriculture affected the variation pattern at CYP2D6, leading to an increase in FP populations of the frequency of alleles that are associated with a slower rate of metabolism. These alleles reached a balanced co-existence with other important and previously selected variants. We suggest that the pronounced substrate-dependent activity of most of these enzymes expanded the spectrum of the metabolic response.

IMPROVED IDENTIFICATION BY IN SITU UV SPECTRA IN PLANAR CHROMATOGRAPHY

Novel software was introduced for library searching in planar chromatography, based on corrected Rf values (hRf c), in situ ultraviolet (UV) spectral correlation, and spectrum maximum site comparison. Analysis of autopsy urine samples by thin-layer chromatography (TLC) and overpressured layer chromatography (OPLC) revealed that the new method was superior to the standard method using hRf c and correlation only. The advantage was more pronounced in TLC, which possessed poorer separation power and generally lower quality spectra than OPLC. In 11 cases of 47 in TLC, the hit list position of the correct finding was improved by the new method. This result indicates that the spectrum maximum site function gives a different kind of discrimination than would be obtained by only setting the correlation cut-off at a higher value. In OPLC, the hit lists were originally shorter, but an improvement in the list lengths was evident. OPLC proved to be the planar method of choice in the screening analysis of urine samples for drugs due to its high separation power and speed.

Quantification of drugs in plasma without primary reference standards by liquid chromatography-chemiluminescence nitrogen detection: application to tramadol metabolite ratios.

Lack of availability of reference standards for drug metabolites, newly released drugs, and illicit drugs hinders the analysis of these substances in biologic samples. To counter this problem, an approach is presented here for quantitative drug analysis in plasma without primary reference standards by liquid chromatography-chemiluminescence nitrogen detection (LC-CLND). To demonstrate the feasibility of the method, metabolic ratios of the opioid drug tramadol were determined in the setting of a pharmacogenetic study. Four volunteers were given a single 100-mg oral dose of tramadol, and a blood sample was collected from each subject 1 hour later. Tramadol, O-desmethyltramadol, and nortramadol were determined in plasma by LC-CLND without reference standards and by a gas chromatography-mass spectrometry reference method. In contrast to previous CLND studies lacking an extraction step, a liquid-liquid extraction system was created for 5-mL plasma samples using n-butyl chloride-isopropyl alcohol (98 + 2) at pH 10. Extraction recovery estimation was based on model compounds chosen according to their similar physicochemical characteristics (retention time, pKa, logD). Instrument calibration was performed with a single secondary standard (caffeine) using the equimolar response of the detector to nitrogen. The mean differences between the results of the LC-CLND and gas chromatography-mass spectrometry methods for tramadol, O-desmethyltramadol, and nortramadol were 8%, 32%, and 19%, respectively. The sensitivity of LC-CLND was sufficient for therapeutic concentrations of tramadol and metabolites. A good correlation was obtained between genotype, expressed by the number of functional genes, and the plasma metabolite ratios. This experiment suggests that a recovery-corrected LC-CLND analysis produces sufficiently accurate results to be useful in a clinical context, particularly in instances in which reference standards are not readily accessible.