Anders Rane

A genome-wide association study confirms VKORC1, CYP2C9, and CYP4F2 as principal genetic determinants of warfarin dose.

We report the first genome-wide association study (GWAS) whose sample size (1,053 Swedish subjects) is sufficiently powered to detect genome-wide significance (p<1.5×10−7) for polymorphisms that modestly alter therapeutic warfarin dose. The anticoagulant drug warfarin is widely prescribed for reducing the risk of stroke, thrombosis, pulmonary embolism, and coronary malfunction. However, Caucasians vary widely (20-fold) in the dose needed for therapeutic anticoagulation, and hence prescribed doses may be too low (risking serious illness) or too high (risking severe bleeding). Prior work established that ∼30% of the dose variance is explained by single nucleotide polymorphisms (SNPs) in the warfarin drug target VKORC1 and another ∼12% by two non-synonymous SNPs (*2, *3) in the cytochrome P450 warfarin-metabolizing gene CYP2C9. We initially tested each of 325,997 GWAS SNPs for association with warfarin dose by univariate regression and found the strongest statistical signals (p<10−78) at SNPs clustering near VKORC1 and the second lowest p-values (p<10−31) emanating from CYP2C9. No other SNPs approached genome-wide significance. To enhance detection of weaker effects, we conducted multiple regression adjusting for known influences on warfarin dose (VKORC1, CYP2C9, age, gender) and identified a single SNP (rs2108622) with genome-wide significance (p = 8.3×10−10) that alters protein coding of the CYP4F2 gene. We confirmed this result in 588 additional Swedish patients (p<0.0029) and, during our investigation, a second group provided independent confirmation from a scan of warfarin-metabolizing genes. We also thoroughly investigated copy number variations, haplotypes, and imputed SNPs, but found no additional highly significant warfarin associations. We present power analysis of our GWAS that is generalizable to other studies, and conclude we had 80% power to detect genome-wide significance for common causative variants or markers explaining at least 1.5% of dose variance. These GWAS results provide further impetus for conducting large-scale trials assessing patient benefit from genotype-based forecasting of warfarin dose.

Use of doping agents, particularly anabolic steroids, in sports and society

The use of doping agents, particularly anabolic androgenic steroids (AAS), has changed from being a problem restricted to sports to one of public-health concern. We review the prevalence of misuse, the evidence that some drugs improve performance in sport, their side-effects, and the long-term consequences of AAS misuse for society at large. There is substantial under-reporting of the side-effects of AAS to health authorities. We describe neuropsychiatric side-effects of AAS and their possible neurobiological correlates, with particular emphasis on violent behaviour. Analytical methods and laboratories accredited by the World Anti-Doping Agency can detect the misuse of all doping agents; although the analysis of testosterone requires special techniques, and recently discovered interethnic differences in testosterone excretion should be taken into account. The prevention of misuse of doping agents should include random doping analyses, medical follow-ups, pedagogic interventions, tougher legislation against possession of AAS, and longer disqualifications of athletes who use AAS.

Doping Test Results Dependent on Genotype of Uridine Diphospho-Glucuronosyl Transferase 2B17, the Major Enzyme for Testosterone Glucuronidation

CONTEXT Testosterone abuse is conventionally assessed by the urinary testosterone/epitestosterone (T/E) ratio, levels above 4.0 being considered suspicious. The large variation in testosterone glucuronide (TG) excretion and its strong association with a deletion polymorphism in the uridine diphospho-glucuronosyl transferase (UGT) 2B17 gene challenge the accuracy of the T/E ratio test. OBJECTIVE Our objective was to investigate whether genotype-based cutoff values will improve the sensitivity and specificity of the test. DESIGN This was an open three-armed comparative study. PARTICIPANTS A total of 55 healthy male volunteers with either two, one, or no allele [insertion/insertion, insertion/deletion, or deletion/deletion (del/del)] of the UGT2B17 gene was included in the study. INTERVENTION A single im dose of 500 mg testosterone enanthate was administered. MAIN OUTCOME MEASURES Urinary excretion of TG after dose and the T/E ratio during 15 d were calculated. RESULTS The degree and rate of increase in the TG excretion rate were highly dependent on the UGT2B17 genotype with a 20-fold higher average maximum increase in the insertion/insertion group compared with the del/del group. Of the del/del subjects, 40% never reached the T/E ratio of 4.0 on any of the 15 d after the dose. When differentiated cutoff levels for the del/del (1.0) and the other genotypes (6.0) were applied, the sensitivity increased substantially for the del/del group, and false positives in the other genotypes were eliminated. CONCLUSIONS Consideration of the genetic variation in disposition of androgens will improve the sensitivity and specificity of the testosterone doping test. This is of interest not only for combating androgen doping in sports, but also for detecting and preventing androgen abuse in society.

SFINX—a drug-drug interaction database designed for clinical decision support systems

ObjectiveThe aim was to develop a drug-drug interaction database (SFINX) to be integrated into decision support systems or to be used in website solutions for clinical evaluation of interactions.MethodsKey elements such as substance properties and names, drug formulations, text structures and references were defined before development of the database. Standard operating procedures for literature searches, text writing rules and a classification system for clinical relevance and documentation level were determined. ATC codes, CAS numbers and country-specific codes for substances were identified and quality assured to ensure safe integration of SFINX into other data systems. Much effort was put into giving short and practical advice regarding clinically relevant drug-drug interactions.ResultsSFINX includes over 8,000 interaction pairs and is integrated into Swedish and Finnish computerised decision support systems. Over 31,000 physicians and pharmacists are receiving interaction alerts through SFINX. User feedback is collected for continuous improvement of the content.ConclusionSFINX is a potentially valuable tool delivering instant information on drug interactions during prescribing and dispensing.

Expression of CYP3A isoforms and P-glycoprotein in human stomach, jejunum and ileum.

1 CYP3A isoforms metabolise a diverse array of clinically important drugs and P‐glycoprotein (P‐gp), a transmembrane efflux pump, can extrude a wide variety of drugs from the cell. It has been suggested that the function of CYP3A4 is complementary to that of P‐gp along the gastrointestinal (GI) tract, together forming a coordinated intestinal barrier against xenobiotics. Therefore, the expression of CYP3A4, CYP3A5, CYP3A7 and ABCB1 (P‐gp) genes were quantified in five normal samples from the human stomach, seven from the jejunum and eight from the ileum by real‐time reverse transcription–polymerase chain reaction and western blot analysis. 2 In the tissues examined, measurable mRNA expression of CYP3A was found in almost all samples from the stomach, jejunum and ileum. The rank order for CYP3A mRNA expression was CYP3A4 > CYP3A5 > CYP3A7 in the GI tract studied, whereas median mRNA CYP3A4 expression was highest in the small intestine and lowest in the stomach. Expression of ABCB1 mRNA was found in almost all samples and the median mRNA expression level was comparable in the jejunum and ileum, but lower in the stomach. Our data also show a significant correlation between all mRNA transcripts studied and a wide interindividual variation. 3 At the protein level, CYP3A4 was detected in all stomach and small intestine samples, the levels being substantially higher in the small intestine than in the stomach. P‐Glycoprotein was detected in all GI samples, but no statistically significant difference was found along the GI tract considered. 4 Collectively, these results demonstrate that CYP3A4 is the main CYP3A expressed in the GI tract investigated, an extensive interindividual variability in the expression of the different CYP3A isoforms in all tissues examined and P‐gp apoprotein levels similar in the stomach, jejunum and ileum.

A Pharmacometric Model Describing the Relationship Between Warfarin Dose and INR Response With Respect to Variations in CYP2C9, VKORC1, and Age

The objective of the study was to update a previous NONMEM model to describe the relationship between warfarin dose and international normalized ratio (INR) response, to decrease the dependence of the model on pharmacokinetic (PK) data, and to improve the characterization of rare genotype combinations. The effects of age and CYP2C9 genotype on S‐warfarin clearance were estimated from high‐quality PK data. Thereafter, a temporal dose‐response (K‐PD) model was developed from information on dose, INR, age, and CYP2C9 and VKORC1 genotype, with drug clearance as a covariate. Two transit compartment chains accounted for the delay between exposure and response. CYP2C9 genotype was identified as the single most important predictor of required dose, causing a difference of up to 4.2‐fold in the maintenance dose. VKORC1 accounted for a difference of up to 2.1‐fold in dose, and age reduced the dose requirement by _6% per decade. This reformulated K‐PD model decreases dependence on PK data and enables robust assessment of INR response and dose predictions, even in individuals with rare genotype combinations.

Incidence and predictors of severe bleeding during warfarin treatment

BackgroundOptimal warfarin prescription requires correct, individualized assessment of the warfarin-related bleeding risk, which randomised controlled trials may underestimate . Observational studies have reported a range of bleeding risks that differ 40-fold. This variation may be caused by time trends, variation in bleeding definition and study subject selection. We investigated the incidence of, and risk factors for severe bleeding in un-selected warfarin-treated patients from Sweden.MethodsBetween 2001 and 2005, 40 centres recruited warfarin-naïve patients commencing warfarin therapy and followed them prospectively with continuous registration of clinical data. The primary outcome was severe bleeding, according to the WHO universal definition of severe adverse drug reactions. The influence of potential risk factors was investigated by means of a Cox proportional-hazards model.ResultA total of 1523 patients contributed 1276 warfarin-exposed patient-years. The incidence of first-time severe bleeding was 2.3 per 100 patient-years (95% confidence interval 1.4 to 3.1). Male sex and use of drugs potentially interacting with warfarin were the only independent risk factors of severe bleeding, with hazard ratios of 2.8 and 2.3, respectively. Age, target International Normalized Ratio (INR), time spent outside target INR range, and warfarin dose requirement were not significantly associated with bleeding risk.ConclusionsThe risk of severe bleeding in a large naturalistic, prospective cohort of first-time warfarin users was lower than reported in some previous reports. Male gender was an independent predictor of severe bleeding as was the receipt of warfarin-interacting medications at the onset of anticoagulation therapy. Further studies are required to evaluate the effect these findings may have on the quality of current risk-benefit analysis involved in warfarin prescription.

Ontogeny, distribution and potential roles of 5-hydroxymethylcytosine in human liver function.

BackgroundInterindividual differences in liver functions such as protein synthesis, lipid and carbohydrate metabolism and drug metabolism are influenced by epigenetic factors. The role of the epigenetic machinery in such processes has, however, been barely investigated. 5-hydroxymethylcytosine (5hmC) is a recently re-discovered epigenetic DNA modification that plays an important role in the control of gene expression.ResultsIn this study, we investigate 5hmC occurrence and genomic distribution in 8 fetal and 7 adult human liver samples in relation to ontogeny and function. LC-MS analysis shows that in the adult liver samples 5hmC comprises up to 1% of the total cytosine content, whereas in all fetal livers it is below 0.125%. Immunohistostaining of liver sections with a polyclonal anti-5hmC antibody shows that 5hmC is detected in most of the hepatocytes. Genome-wide mapping of the distribution of 5hmC in human liver samples by next-generation sequencing shows significant differences between fetal and adult livers. In adult livers, 5hmC occupancy is overrepresented in genes involved in active catabolic and metabolic processes, whereas 5hmC elements which are found in genes exclusively in fetal livers and disappear in the adult state, are more specific to pathways for differentiation and development.ConclusionsOur findings suggest that 5-hydroxymethylcytosine plays an important role in the development and function of the human liver and might be an important determinant for development of liver diseases as well as of the interindividual differences in drug metabolism and toxicity.

Radical prostatectomy: Influence on serum and urinary androgen levels

The role of the prostate as an active endocrine organ and the hormonal changes after radical prostatectomy (RP) has not been well studied. The objective of this study was to investigate the serum and urine hormonal changes after RP.

The UGT2B17 Gene Deletion is Not Associated With Prostate Cancer Risk

Deletion polymorphism of the UDP‐glucuronosyltransferase 2B17 (UGT2B17) gene has been associated with an increased prostate cancer risk in two previous independent studies. Here we determine the risk in a large‐scale population‐based case–control study.