Methadone pharmacokinetics in opioid agonist treatment: Influencing factors and clinical implications.

BACKGROUND: A considerable inter-individual variability has been reported in the relationship between methadone doses applied and serum concentrations achieved in methadone maintenance treatment. However, the underlying causes for this variability are not fully understood. OBJECTIVES: We investigated the influence of genetic, pathophysiological and pharmacological factors on serum methadone concentration-to-dose ratio (CDR) and discussed the clinical implications of the findings. METHODS: We used data from two retrospective laboratory databases and a prospective cohort study to investigate the impact on methadone CDR of hepatic cytochrome P450 enzyme system (CYP) genetic polymorphisms, age, sex, concomitant medication, liver fibrosis and body mass index through linear mixed model analyses. FINDINGS: A positive association was found between CDR and the homozygous CYP2B6*6 genotype, concurrent treatment with CYP3A4 inhibitors and body mass index. CDR was lower among women and during concomitant use of CYP inducers. CDR was not associated with age or the degree of liver fibrosis in our investigations. CONCLUSIONS: This research work supports the need for individually tailored dosage considering the various factors that influence methadone CDR. The gained knowledge can contribute to reducing the risks associated with the treatment and optimizing the desired outcomes.

Impact of liver fibrosis and clinical characteristics on dose-adjusted serum methadone concentrations.

BACKGROUND: There is limited knowledge on the causes of large variations in serum methadone concentrations and dose requirements. OBJECTIVES: We investigated the impact of the degree of liver fibrosis on dose-adjusted steady-state serum methadone concentrations. METHODS: We assessed the clinical and laboratory data of 155 Norwegian patients with opioid use disorder undergoing methadone maintenance treatment in outpatient clinics in the period 2016-2020. A possible association between the degree of liver fibrosis and dose-adjusted serum methadone concentration was explored using a linear mixed-model analysis. RESULTS: When adjusted for age, gender, body mass index, and genotypes of CYP2B6 and CYP3A5, the concentration-to-dose ratio of methadone did not increase among the participants with liver fibrosis (Coefficient: 0.70; 95% CI: -2.16, 3.57; P: 0.631), even among those with advanced cirrhosis (-0.50; -4.59, 3.59; 0.810). CONCLUSIONS: Although no correlation was found between the degree of liver stiffness and dose-adjusted serum methadone concentration, close clinical monitoring should be considered, especially among patients with advanced cirrhosis. Still, serum methadone measurements can be considered a supplement to clinical assessments, taking into account intra-individual variations.

Nonadherence by Serum Drug Analyses in Resistant Hypertension: 7-Year Follow-Up of Patients Considered Adherent by Directly Observed Therapy.

Background Measurement of serum concentrations of drugs is a novelty found useful in detecting poor drug adherence in patients taking ≥2 antihypertensive agents. Regarding patients with treatment-resistant hypertension, we previously based our assessment on directly observed therapy. The present study aimed to investigate whether serum drug measurements in patients with resistant hypertension offer additional information regarding drug adherence, beyond that of initial assessment with directly observed therapy. Methods and Results Nineteen patients assumed to have true treatment-resistant hypertension and adherence to antihypertensive drugs based on directly observed therapy were investigated repeatedly through 7 years. Serum concentrations of antihypertensive drugs were measured by ultra-high-performance liquid chromatography-tandem mass spectrometry from blood samples taken at baseline, 6-month, 3-year, and 7-year visits. Cytochrome P450 polymorphisms, self-reported adherence and beliefs about medicine were performed as supplement investigations. Seven patients (37%) were redefined as nonadherent based on their serum concentrations during follow-up. All patients reported high adherence to medications. Nonadherent patients expressed lower necessity and higher concerns regarding intake of antihypertensive medication (P=0.003). Cytochrome P450 polymorphisms affecting metabolism of antihypertensive drugs were found in 16 patients (84%), 21% were poor metabolizers, and none were ultra-rapid metabolizers. Six of 7 patients redefined as nonadherent had cytochrome P450 polymorphisms, however, not explaining the low serum drug concentrations measured in these patients. Conclusions Our data suggest that repeated measurements of serum concentrations of antihypertensive drugs revealed nonadherence in one-third of patients previously evaluated as adherent and treatment resistant by directly observed therapy, thereby improving the accuracy of adherence evaluation. Registration URL: https://www.clinicaltrials.gov; unique identifier: NCT01673516.

Short- and long-term effects of body weight, calorie restriction and gastric bypass on CYP1A2, CYP2C19 and CYP2C9 activity.

AIM: Roux-en-Y gastric bypass (RYGB) may influence drug disposition due to surgery-induced gastrointestinal alterations and/or subsequent weight loss. The objective was to compare short- and long-term effects of RYGB and diet on the metabolic ratios of paraxanthine/caffeine (cytochrome P450 [CYP] 1A2 activity), 5-hydroxyomeprazole/omeprazole (CYP2C19 activity) and losartan/losartan carboxylic acid (CYP2C9 activity), and cross-sectionally compare these CYP-activities with normal-to-overweight controls. METHODS: This trial included patients with severe obesity preparing for RYGB (n = 40) or diet-induced (n = 41) weight loss, and controls (n = 18). Both weight loss groups underwent a 3-week low-energy diet (<1200 kcal/day, weeks 0-3) followed by a 6-week very-low-energy diet or RYGB (both <800 kcal/day, weeks 3-9). Follow-up time was 2 years, with four pharmacokinetic investigations. RESULTS: Mean ± SD weight loss from baseline was similar in the RYGB-group (13 ± 2.4%) and the diet group (10.5 ± 3.9%) at week 9, but differed at year 2 (RYGB -30 ± 6.9%, diet -3.1 ± 6.3%). From weeks 0 to 3, mean (95% confidence interval [CI]) CYP2C19 activity similarly increased in both groups (RYGB 43% [16, 55], diet 48% [22, 60]). Mean CYP2C19 activity increased by 30% (2.6, 43) after RYGB (weeks 3-9), but not in the diet-group (between-group difference -0.30 [-0.63, 0.03]). CYP2C19 activity remained elevated in the RYGB group at year 2. Baseline CYP2C19 activity was 2.7-fold higher in controls compared with patients with obesity, whereas no difference was observed in CYP1A2 and CYP2C9 activities. CONCLUSION: Our findings suggest that CYP2C19 activity is lower in patients with obesity and increases following weight loss. This may be clinically relevant for drug dosing. No clinically significant effect on CYP1A2 and CYP2C9 activities was observed.

Mechanical-Stress-Related Epigenetic Regulation of ZIC1 Transcription Factor in the Etiology of Postmenopausal Osteoporosis.

Mechanical loading exerts a profound influence on bone density and architecture, but the exact mechanism is unknown. Our study shows that expression of the neurological transcriptional factor zinc finger of the cerebellum 1 (ZIC1) is markedly increased in trabecular bone biopsies in the lumbar spine compared with the iliac crest, skeletal sites of high and low mechanical stress, respectively. Human trabecular bone transcriptome analyses revealed a strong association between ZIC1 mRNA levels and gene transcripts characteristically associated with osteoblasts, osteocytes and osteoclasts. This supposition is supported by higher ZIC1 expression in iliac bone biopsies from postmenopausal women with osteoporosis compared with age-matched control subjects, as well as strongly significant inverse correlation between ZIC1 mRNA levels and BMI-adjusted bone mineral density (BMD) (Z-score). ZIC1 promoter methylation was decreased in mechanically loaded vertebral bone compared to unloaded normal iliac bone, and its mRNA levels correlated inversely with ZIC1 promoter methylation, thus linking mechanical stress to epigenetic control of gene expression. The findings were corroborated in cultures of rat osteoblast progenitors and osteoblast-like cells. This study demonstrates for the first time how skeletal epigenetic changes that are affected by mechanical forces give rise to marked alteration in bone cell transcriptional activity and translate to human bone pathophysiology.

Correlation of Body Weight and Composition With Hepatic Activities of Cytochrome P450 Enzymes.

Obesity is associated with comorbidities of which pharmacological treatment is needed. Physiological changes associated with obesity may influence the pharmacokinetics of drugs, but the effect of body weight on drug metabolism capacity remains uncertain. The aim of this study was to investigate ex vivo activities of hepatic drug metabolizing CYP enzymes in patients covering a wide range of body weight. Liver biopsies from 36 individuals with a body mass index (BMI) ranging from 18 to 63 kg/m2 were obtained. Individual hepatic microsomes were prepared and activities of CYP3A, CYP2B6, CYP2C8, CYP2D6, CYP2C9, CYP2C19 and CYP1A2 were determined. The unbound intrinsic clearance (CLint,u) values for CYP3A correlated negatively with body weight (r = -0.43, p < 0.01), waist circumference (r = -0.47, p < 0.01), hip circumference (r = -0.51, p < 0.01), fat percent (r = -0.41, p < 0.05), fat mass (r = -0.48, p < 0.01) and BMI (r = -0.46, p < 0.01). Linear regression analysis showed that CLint,u values for CYP3A decreased with 5% with each 10% increase in body weight (r2 = 0.12, ß = -0.558, p < 0.05). There were no correlations between body weight measures and CLint,u values for the other CYP enzymes investigated. These results indicate reduced hepatic metabolizing capacity of CYP3A substrates in patients with increasing body weight.

The Influence of Combined CYP2D6 and CYP2C19 Genotypes on Venlafaxine and O-Desmethylvenlafaxine Concentrations in a Large Patient Cohort.

PURPOSE: The antidepressant venlafaxine is largely O-desmethylated by CYP2D6, whereas CYP2C19 mediates an alternative metabolic route of venlafaxine through N-desmethylation. The aim of this study was to investigate the combined effect of genotype-predicted CYP2D6 and CYP2C19 phenotypes on serum concentrations of venlafaxine and metabolites in a large patient population. METHODS: Patients were retrospectively included from a therapeutic drug monitoring service at Diakonhjemmet Hospital in Oslo (Norway) between January 01, 2007, and December 31, 2017. The study population was divided into different phenotype subgroups according to the combinations of CYP2D6/CYP2C19 phenotypes; intermediate metabolizers (IMs), poor metabolizers (PMs) and ultrarapid metabolizers, and compared using combined normal metabolizers (NMs) as reference. FINDINGS: The dose-adjusted serum concentration of venlafaxine was 4- and 13-fold increased in combined CYP2D6 IM/CYP2C19 PMs and combined PMs, respectively, compared with combined NMs (P < 0.001). The sum concentration of venlafaxine + ODV (pharmacological active moiety) was increased 1.9 and 3.6-fold, respectively, in the same phenotype groups. Furthermore, the dose-adjusted active moiety exposure was similar in combined IMs as combined CYP2D6 PM/CYP2C19 NMs. CYP2D6 and CYP2C19 phenotypes explained 46% of the interindividual variability in dose-adjusted venlafaxine serum concentrations, whereas CYP2D6 alone explained 24%. CONCLUSIONS: The combined CYP2D6/CYP2C19 phenotype has a significant impact on serum concentrations of venlafaxine and also on the active moiety of venlafaxine + ODV, than CYP2D6 alone. In clinical practice, it is therefore important to take into account phenotype variabilities of both enzymes when assessing the risk of dose-dependent adverse effects during venlafaxine treatment.

Impact of CYP2C19 genotype on sertraline exposure in 1200 Scandinavian patients.

Sertraline is an (SSRI-)antidepressant metabolized by the polymorphic CYP2C19 enzyme. The aim of this study was to investigate the impact of CYP2C19 genotype on the serum concentrations of sertraline in a large patient population. Second, the proportions of patients in the various CYP2C19 genotype-defined subgroups obtaining serum concentrations outside the therapeutic range of sertraline were assessed. A total of 2190 sertraline serum concentration measurements from 1202 patients were included retrospectively from the drug monitoring database at Diakonhjemmet Hospital in Oslo. The patients were divided into CYP2C19 genotype-predicted phenotype subgroups, i.e. normal (NMs), ultra rapid (UMs), intermediate (IMs), and poor metabolisers (PMs). The differences in dose-harmonized serum concentrations of sertraline and N-desmethylsertraline-to-sertraline metabolic ratio were compared between the subgroups, with CYP2C19 NMs set as reference. The patient proportions outside the therapeutic concentration range were also compared between the subgroups with NMs defined as reference. Compared with the CYP2C19 NMs, the sertraline serum concentration was increased 1.38-fold (95% CI 1.26-1.50) and 2.68-fold (95% CI 2.16-3.31) in CYP2C19 IMs and PMs, respectively (p < 0.001), while only a marginally lower serum concentration (-10%) was observed in CYP2C19 UMs (p = 0.012). The odds ratio for having a sertraline concentration above the therapeutic reference range was 1.97 (95% CI 1.21-3.21, p = 0.064) and 8.69 (95% CI 3.88-19.19, p < 0.001) higher for IMs and PMs vs. NMs, respectively. CYP2C19 IMs and PMs obtain significantly higher serum concentrations of sertraline than NMs. Based on the relative differences in serum concentrations compared to NMs, dose reductions of 60% and 25% should be considered in PMs and IMs, respectively, to reduce the risk of sertraline overexposure in these patients.

A Comparative Analysis of Cytochrome P450 Activities in Paired Liver and Small Intestinal Samples from Patients with Obesity.

The liver and small intestine restrict oral bioavailability of drugs and constitute the main sites of pharmacokinetic drug-drug interactions. Hence, detailed data on hepatic and intestinal activities of drug metabolizing enzymes is important for modeling drug disposition and optimizing pharmacotherapy in different patient populations. The aim of this study was to determine the activities of seven cytochrome P450 (P450) enzymes in paired liver and small intestinal samples from patients with obesity. Biopsies were obtained from 20 patients who underwent Roux-en-Y gastric bypass surgery following a 3-week low-energy diet. Individual hepatic and intestinal microsomes were prepared and specific probe substrates in combined incubations were used for determination of CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, and CYP3A activities. The activities of CYP2C8, CYP2C9, CYP2D6, and CYP3A were quantified in both human liver microsomes (HLM) and human intestinal microsomes (HIM), while the activities of CYP1A2, CYP2B6, and CYP2C19 were only quantifiable in HLM. Considerable interindividual variability was present in both HLM (9- to 23-fold) and HIM (5- to 55-fold). The median metabolic HLM/HIM ratios varied from 1.5 for CYP3A to 252 for CYP2C8. The activities of CYP2C9 in paired HLM and HIM were positively correlated (r = 0.74, P < 0.001), while no interorgan correlations were found for activities of CYP2C8, CYP2D6, and CYP3A (P > 0.05). Small intestinal CYP3A activities were higher in females compared with males (P < 0.05). Hepatic CYP2B6 activity correlated negatively with body mass index (r = -0.72, P < 0.001). These data may be useful for further in vitro-in vivo predictions of drug disposition in patients with obesity. SIGNIFICANCE STATEMENT: Hepatic and intestinal drug metabolism is the key determinant of oral drug bioavailability. In this study, paired liver and jejunum samples were obtained from 20 patients with obesity undergoing gastric bypass surgery following a 3-week low-energy diet. We determined the hepatic and small intestinal activities of clinically important P450 enzymes and provide detailed enzyme kinetic data relevant for predicting in vivo disposition of P450 substrates in this patient population.

Effect of CYP4F2, VKORC1, and CYP2C9 in Influencing Coumarin Dose: A Single-Patient Data Meta-Analysis in More Than 15,000 Individuals.

The cytochrome P450 (CYP)4F2 gene is known to influence mean coumarin dose. The aim of the present study was to undertake a meta-analysis at the individual patients level to capture the possible effect of ethnicity, gene-gene interaction, or other drugs on the association and to verify if inclusion of CYP4F2*3 variant into dosing algorithms improves the prediction of mean coumarin dose. We asked the authors of our previous meta-analysis (30 articles) and of 38 new articles retrieved by a systematic review to send us individual patients' data. The final collection consists of 15,754 patients split into a derivation and validation cohort. The CYP4F2*3 polymorphism was consistently associated with an increase in mean coumarin dose (+9% (95% confidence interval (CI) 7-10%), with a higher effect in women, in patients taking acenocoumarol, and in white patients. The inclusion of the CYP4F2*3 in dosing algorithms slightly improved the prediction of stable coumarin dose. New pharmacogenetic equations potentially useful for clinical practice were derived.

Combined Effect of CYP2B6 Genotype and Other Candidate Genes on a Steady-State Serum Concentration of Methadone in Opioid Maintenance Treatment.

BACKGROUND: A considerable interindividual variability in methadone pharmacokinetics is seen in patients on methadone maintenance treatment. The aim of this study was to clarify the impact of the reduced function CYP2B6*6 variant allele together with variants in other candidate genes on a steady-state methadone concentration in a naturalistic setting. METHODS: Information of methadone serum concentration, dose, age, sex, and CYP2C9, CYP2C19, and CYP2D6 genotypes were collected from a routine therapeutic drug monitoring database, whereas variant alleles in CYP2B6 and CYP3A5 were retrospectively genotyped. Linear mixed model analyses were used to study the impact of gene variants on methadone serum concentration/dose (C/D) ratios, including age, sex, and time since the last dose intake as covariates. RESULTS: Overall, 155 serum samples from 62 patients were included in this study. The estimated mean methadone C/D ratios was 17.8 nmol·L·mg for homozygous carriers of CYP2B6*6, which was significantly (P < 0.001) higher than noncarriers (9.2 nmol·L·mg). There was no difference in C/D ratios between heterozygous carriers of CYP2B6*6 (9.1 nmol·L·mg) and noncarriers. An increase in mean methadone C/D ratios was also seen for homozygous carriers of CYP3A5*3 and heterozygous carriers of CYP2C9*2 or *3 and CYP2C19*2 or *3. CONCLUSIONS: Patients homozygous for CYP2B6*6 had a >90% higher methadone C/D ratio. Genotyping of CYP2B6 may therefore be of value when assessing dose requirements in methadone maintenance treatment.

Distinct DNA methylation profiles in bone and blood of osteoporotic and healthy postmenopausal women.

DNA methylation affects expression of associated genes and may contribute to the missing genetic effects from genome-wide association studies of osteoporosis. To improve insight into the mechanisms of postmenopausal osteoporosis, we combined transcript profiling with DNA methylation analyses in bone. RNA and DNA were isolated from 84 bone biopsies of postmenopausal donors varying markedly in bone mineral density (BMD). In all, 2529 CpGs in the top 100 genes most significantly associated with BMD were analyzed. The methylation levels at 63 CpGs differed significantly between healthy and osteoporotic women at 10% false discovery rate (FDR). Five of these CpGs at 5% FDR could explain 14% of BMD variation. To test whether blood DNA methylation reflect the situation in bone (as shown for other tissues), an independent cohort was selected and BMD association was demonstrated in blood for 13 of the 63 CpGs. Four transcripts representing inhibitors of bone metabolism-MEPE, SOST, WIF1, and DKK1-showed correlation to a high number of methylated CpGs, at 5% FDR. Our results link DNA methylation to the genetic influence modifying the skeleton, and the data suggest a complex interaction between CpG methylation and gene regulation. This is the first study in the hitherto largest number of postmenopausal women to demonstrate a strong association among bone CpG methylation, transcript levels, and BMD/fracture. This new insight may have implications for evaluation of osteoporosis stage and susceptibility.

Mortality among head trauma patients taking preinjury antithrombotic agents: a retrospective cohort analysis from a Level 1 trauma centre.

BACKGROUND: Bleeding represents the most well-known and the most feared complications caused by the use of antithrombotic agents. There is, however, limited documentation whether pre-injury use of antithrombotic agents affects outcome after head trauma. The aim of this study was to define the relationship between the use of preinjury antithrombotic agents and mortality among elderly people sustaining blunt head trauma. METHODS: A retrospective cohort analysis was performed on the hospital based trauma registry at Oslo University Hospital. Patients aged 55 years or older sustaining blunt head trauma between 2004 and 2006 were included. Multivariable logistic regression analyses were used to identify independent predictors of 30-day mortality. Separate analyses were performed for warfarin use and platelet inhibitor use. RESULTS: Of the 418 patients admitted with a diagnosis of head trauma, 137 (32.8 %) used pre-injury antithrombotic agents (53 warfarin, 80 platelet inhibitors, and 4 both). Seventy patients died (16.7 %); 15 (28.3 %) of the warfarin users, 12 (15.0 %) of the platelet inhibitor users, and two (50 %) with combined use of warfarin and platelet inhibitors, compared to 41 (14.6 %) of the non-users. There was a significant interaction effect between warfarin use and the Triage Revised Trauma Score collected upon the patients' arrival at the hospital. After adjusting for potential confounders, warfarin use was associated with increased 30-day mortality among patients with normal physiology (adjusted OR 8,3; 95 % CI, 2.0 to 34.8) on admission, but not among patients with physiological derangement on admission. Use of platelet inhibitors was not associated with increased mortality. CONCLUSIONS: The use of warfarin before trauma was associated with increased 30-day mortality among a subset of patients. Use of platelet inhibitors before trauma was not associated with increased mortality. These results indicate that patients on preinjury warfarin may need closer monitoring and follow up after trauma despite normal physiology on admission to the emergency department.

Changes of 5-hydroxymethylcytosine distribution during myeloid and lymphoid differentiation of CD34+ cells.

BACKGROUND: Hematopoietic stem cell renewal and differentiation are regulated through epigenetic processes. The conversion of 5-methylcytosine into 5-hydroxymethylcytosine (5hmC) by ten-eleven-translocation enzymes provides new insights into the epigenetic regulation of gene expression during development. Here, we studied the potential gene regulatory role of 5hmC during human hematopoiesis. RESULTS: We used reduced representation of 5-hydroxymethylcytosine profiling (RRHP) to characterize 5hmC distribution in CD34+ cells, CD4+ T cells, CD19+ B cells, CD14+ monocytes and granulocytes. In all analyzed blood cell types, the presence of 5hmC at gene bodies correlates positively with gene expression, and highest 5hmC levels are found around transcription start sites of highly expressed genes. In CD34+ cells, 5hmC primes for the expression of genes regulating myeloid and lymphoid lineage commitment. Throughout blood cell differentiation, intragenic 5hmC is maintained at genes that are highly expressed and required for acquisition of the mature blood cell phenotype. Moreover, in CD34+ cells, the presence of 5hmC at enhancers associates with increased binding of RUNX1 and FLI1, transcription factors essential for hematopoiesis. CONCLUSIONS: Our study provides a comprehensive genome-wide overview of 5hmC distribution in human hematopoietic cells and new insights into the epigenetic regulation of gene expression during human hematopoiesis.

Copy number variation in the ATP-binding cassette transporter ABCC6 gene and ABCC6 pseudogenes in patients with pseudoxanthoma elasticum.

Single mutations in the ATP-binding cassette transporter (ABCC6) gene (OMIM 603234) are known to cause the rare autosomal recessive disease pseudoxanthoma elasticum (PXE). Recently, we have found that copy number variations (CNVs) in pseudogenes of the ABCC6 gene are quite common. The aim of this study was to investigate the frequency and possible contribution of CNV in ABCC6 and its pseudogenes in PXE. Genomic DNA from 212 PXE individuals were examined for copy number by pyrosequencing and quantitative polymerase chain reaction (PCR) and compared with healthy individuals. The frequency of PXE individuals with any CNV was higher than in healthy individuals. The majority of variation comprised known and possibly new deletions in the ABCC6 gene and duplications of the ABCC6P1 and ABCC6P2 genes. ABCC6 deletions and ABCC6P2 duplications were not observed in 142 healthy individuals. In conclusion, by pyrosequencing and quantitative PCR, we were able to detect known and possibly new deletions in the ABCC6 gene that may have caused the PXE phenotype. Pyrosequencing may be used in PXE patients who have obtained incomplete genotype from conventional techniques. The frequency of ABCC6P2 pseudogene duplication was more common in PXE patients than healthy individuals and may affect the PXE phenotype.

RNA-sequencing analysis of HepG2 cells treated with atorvastatin.

The cholesterol-lowering drug atorvastatin is among the most prescribed drug in the world. Alternative splicing in a number of genes has been reported to be associated with variable statin response. RNA-seq has proven to be a powerful technique for genome-wide splice variant analysis. In the present study, we sought to investigate atorvastatin responsive splice variants in HepG2 cells using RNA-seq analysis to identify novel candidate genes implicated in cholesterol homeostasis and in the statin response. HepG2 cells were treated with 10 µM atorvastatin for 24 hours. RNA-seq and exon array analyses were performed. The validation of selected genes was performed using Taqman gene expression assays. RNA-seq analysis identified 121 genes and 98 specific splice variants, of which four were minor splice variants to be differentially expressed, 11 were genes with potential changes in their splicing patterns (SYCP3, ZNF195, ZNF674, MYD88, WHSC1, KIF16B, ZNF92, AGER, FCHO1, SLC6A12 and AKAP9), and one was a gene (RAP1GAP) with differential promoter usage. The IL21R transcript was detected to be differentially expressed via RNA-seq and RT-qPCR, but not in the exon array. In conclusion, several novel candidate genes that are affected by atorvastatin treatment were identified in this study. Further studies are needed to determine the biological significance of the atorvastatin responsive splice variants that have been uniquely identified using RNA-seq.

Serum bilirubin concentration in healthy adult North-Europeans is strictly controlled by the UGT1A1 TA-repeat variants.

The major enzyme responsible for the glucuronidation of bilirubin is the uridine 5'-diphosphoglucose glucuronosyltransferase A1 (UGT1A1) enzyme, and genetic variation in the UGT1A1 gene is reported to influence the bilirubin concentration in the blood. In this study, we have investigated which gene-/haplotype variants may be useful for genetic testing of Gilbert's syndrome. Two groups of samples based on serum bilirubin concentrations were obtained from the Nordic Reference Interval Project Bio-bank and Database (NOBIDA): the 150 individuals with the highest bilirubin (>17.5 µmol/L) and the 150 individuals with normal bilirubin concentrations (<17.5 µmol/L). The individuals were examined for the TA6>TA7 variant in the UGT1A1 promoter and 7 tag-SNPs in an extended promoter region of UGT1A1 (haplotype analysis) and in selected SNPs in candidate genes (SLCO1B3, ABCC2 and NUP153). We found significant odds ratios for high bilirubin level for all the selected UGT1A1 variants. However, in stepwise multivariate logistic regression analysis of all genetic variants together with age, sex, country of origin and fasting time, the repeat variants of UGT1A1 TA6>TA7 and SLCO1B3 rs2117032 T>C were the only variants significantly associated with higher bilirubin concentrations. Most individuals with high bilirubin levels were homozygous for the TA7-repeat (74%) while only 3% were homozygous for the TA7-repeat in individuals with normal bilirubin levels. Among individuals heterozygous for the TA7-repeat, a low frequent UGT1A1-diplotype harboring the rs7564935 G-variant was associated with higher bilirubin levels. In conclusion, our results demonstrate that in testing for Gilbert's syndrome, analyzing for the homozygous TA7/TA7-genotype would be appropriate.

UGT1A1*28 is associated with decreased systemic exposure of atorvastatin lactone.

BACKGROUND: Atorvastatin is commonly used to reduce cholesterol. Atorvastatin acid is converted to its corresponding lactone form spontaneously or via glucuronidation mediated by uridine diphosphate glucuronosyltransferase (UGT) 1A1 and 1A3. Atorvastatin lactone is pharmacologically inactive, but is suspected to be muscle toxic and cause statin-induced myopathy (SIM). A several fold increase in systemic exposure of atorvastatin lactone has previously been observed in patients with SIM compared with healthy control subjects. In this study we aimed to investigate the association between polymorphisms in the UGT1A gene locus and plasma atorvastatin lactone levels. METHODS: DNA was extracted from whole blood obtained from a previous pharmacokinetic study of patients carefully diagnosed as having true SIM (n = 13) and healthy control subjects (n = 15). The UGT1A1*28(TA) 7 , UGT1A3*2, UGT1A3*3, and UGT1A3*6 polymorphisms were detected by pyrosequencing. RESULTS: Carriers of the low-expression allele UGT1A1*28(TA) 7 tended to have lower levels of atorvastatin lactone (p < 0.05) than carriers with the normal-activity allele (TA) 6 . CONCLUSION: The low-expression UGT1A1*28(TA) 7 allele seems to be associated with decreased systemic exposure of the suspected muscle-toxic metabolite atorvastatin lactone.

A novel 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR) splice variant with an alternative exon 1 potentially encoding an extended N-terminus.

BACKGROUND: The major rate-limiting enzyme for de novo cholesterol synthesis is 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR). HMGCR is sterically inhibited by statins, the most commonly prescribed drugs for the prevention of cardiovascular events. Alternative splicing of HMGCR has been implicated in the control of cholesterol homeostasis. The aim of this study was to identify novel alternatively spliced variants of HMGCR with potential physiological importance. RESULTS: Bioinformatic analyses predicted three novel HMGCR transcripts containing an alternative exon 1 (HMGCR-1b, -1c, -1d) compared with the canonical transcript (HMGCR-1a). The open reading frame of the HMGCR-1b transcript potentially encodes 20 additional amino acids at the N-terminus, compared with HMGCR-1a. Reverse transcription quantitative polymerase chain reaction (RT-qPCR) was used to examine the mRNA levels of HMGCR in different tissues; HMGCR-1a was the most highly expressed variant in most tissues, with the exception of the skin, esophagus, and uterine cervix, in which HMGCR-1b was the most highly expressed transcript. Atorvastatin treatment of HepG2 cells resulted in increased HMGCR-1b mRNA levels, but unaltered proximal promoter activity compared to untreated cells. In contrast, HMGCR-1c showed a more restricted transcription pattern, but was also induced by atorvastatin treatment. CONCLUSIONS: The gene encoding HMGCR uses alternative, mutually exclusive exon 1 sequences. This contributes to an increased complexity of HMGCR transcripts. Further studies are needed to investigate whether HMGCR splice variants identified in this study are physiologically functional.