Solanidine is a sensitive and specific dietary biomarker for CYP2D6 activity.

BACKGROUND: Individual assessment of CYP enzyme activities can be challenging. Recently, the potato alkaloid solanidine was suggested as a biomarker for CYP2D6 activity. Here, we aimed to characterize the sensitivity and specificity of solanidine as a CYP2D6 biomarker among Finnish volunteers with known CYP2D6 genotypes. RESULTS: Using non-targeted metabolomics analysis, we identified 9152 metabolite features in the fasting plasma samples of 356 healthy volunteers. Machine learning models suggested strong association between CYP2D6 genotype-based phenotype classes with a metabolite feature identified as solanidine. Plasma solanidine concentration was 1887% higher in genetically poor CYP2D6 metabolizers (gPM) (n = 9; 95% confidence interval 755%, 4515%; P = 1.88 × 10-11), 74% higher in intermediate CYP2D6 metabolizers (gIM) (n = 89; 27%, 138%; P = 6.40 × 10-4), and 35% lower in ultrarapid CYP2D6 metabolizers (gUM) (n = 20; 64%, - 17%; P = 0.151) than in genetically normal CYP2D6 metabolizers (gNM; n = 196). The solanidine metabolites m/z 444 and 430 to solanidine concentration ratios showed even stronger associations with CYP2D6 phenotypes. Furthermore, the areas under the receiver operating characteristic and precision-recall curves for these metabolic ratios showed equal or better performances for identifying the gPM, gIM, and gUM phenotype groups than the other metabolites, their ratios to solanidine, or solanidine alone. In vitro studies with human recombinant CYP enzymes showed that solanidine was metabolized mainly by CYP2D6, with a minor contribution from CYP3A4/5. In human liver microsomes, the CYP2D6 inhibitor paroxetine nearly completely (95%) inhibited the metabolism of solanidine. In a genome-wide association study, several variants near the CYP2D6 gene associated with plasma solanidine metabolite ratios. CONCLUSIONS: These results are in line with earlier studies and further indicate that solanidine and its metabolites are sensitive and specific biomarkers for measuring CYP2D6 activity. Since potato consumption is common worldwide, this biomarker could be useful for evaluating CYP2D6-mediated drug-drug interactions and to improve prediction of CYP2D6 activity in addition to genotyping.

Diagnostic yield of genetic testing in a heterogeneous cohort of 1376 HCM patients.

BACKGROUND: Genetic testing in hypertrophic cardiomyopathy (HCM) is a published guideline-based recommendation. The diagnostic yield of genetic testing and corresponding HCM-associated genes have been largely documented by single center studies and carefully selected patient cohorts. Our goal was to evaluate the diagnostic yield of genetic testing in a heterogeneous cohort of patients with a clinical suspicion of HCM, referred for genetic testing from multiple centers around the world. METHODS: A retrospective review of patients with a suspected clinical diagnosis of HCM referred for genetic testing at Blueprint Genetics was undertaken. The analysis included syndromic, myopathic and metabolic etiologies. Genetic test results and variant classifications were extracted from the database. Variants classified as pathogenic (P) or likely pathogenic (LP) were considered diagnostic. RESULTS: A total of 1376 samples were analyzed. Three hundred and sixty-nine tests were diagnostic (26.8%); 373 P or LP variants were identified. Only one copy number variant was identified. The majority of diagnostic variants involved genes encoding the sarcomere (85.0%) followed by 4.3% of diagnostic variants identified in the RASopathy genes. Two percent of diagnostic variants were in genes associated with a cardiomyopathy other than HCM or an inherited arrhythmia. Clinical variables that increased the likelihood of identifying a diagnostic variant included: an earlier age at diagnosis (p < 0.0001), a higher maximum wall thickness (MWT) (p < 0.0001), a positive family history (p < 0.0001), the absence of hypertension (p = 0.0002), and the presence of an implantable cardioverter-defibrillator (ICD) (p = 0.0004). CONCLUSION: The diagnostic yield of genetic testing in this heterogeneous cohort of patients with a clinical suspicion of HCM is lower than what has been reported in well-characterized patient cohorts. We report the highest yield of diagnostic variants in the RASopathy genes identified in a laboratory cohort of HCM patients to date. The spectrum of genes implicated in this unselected cohort highlights the importance of pre-and post-test counseling when offering genetic testing to the broad HCM population.

Targeted and global pharmacometabolomics in everolimus-based immunosuppression: association of co-medication and lysophosphatidylcholines with dose requirement.

INTRODUCTION: The immunosuppressive therapy with everolimus (ERL) after heart transplantation is characterized by a narrow therapeutic window and a substantial variability in dose requirement. Factors explaining this variability are largely unknown. OBJECTIVES: Our aim was to evaluate factors affecting ERL metabolism and to identify novel metabolites associated with the individual ERL dose requirement to elucidate mechanisms underlying ERL dose response variability. METHOD: We used liquid chromatography coupled with mass spectrometry for quantification of ERL metabolites in 41 heart transplant patients and evaluated the effect of clinical and genetic factors on ERL pharmacokinetics. Non-targeted plasma metabolic profiling by ultra-performance liquid chromatography and high resolution quadrupole-time-of-flight mass spectrometry was used to identify novel metabolites associated with ERL dose requirement. RESULTS: The determination of ERL metabolites revealed differences in metabolite patterns that were independent from clinical or genetic factors. Whereas higher ERL dose requirement was associated with co-administration of sodium-mycophenolic acid and the CYP3A5 expressor genotype, lower dose was required for patients receiving vitamin K antagonists. Global metabolic profiling revealed several novel metabolites associated with ERL dose requirement. One of them was identified as lysophosphatidylcholine (lysoPC) (16:0/0:0). Subsequent targeted analysis revealed that high levels of several lysoPCs were significantly associated with higher ERL dose requirement. CONCLUSION: For the first time, this study describes distinct ERL metabolite patterns in heart transplant patients and detected potentially new drug-drug interactions. The global metabolic profiling facilitated the discovery of novel metabolites associated with ERL dose requirement that might represent new clinically valuable biomarkers to guide ERL therapy.

Influence of CYP3A5 genetic variation on everolimus maintenance dosing after cardiac transplantation.

BACKGROUND: Everolimus (ERL) has become an alternative to calcineurin inhibitors (CNIs) due to its renal-sparing properties, especially in heart transplant (HTx) recipients with kidney dysfunction. However, ERL dosing is challenging due to its narrow therapeutic window combined with high interindividual pharmacokinetic variability. Our aim was to evaluate the effect of clinical and genetic factors on ERL dosing in a pilot cohort of 37 HTx recipients. METHODS: Variants in CYP3A5, CYP3A4, CYP2C8, POR, NR1I2, and ABCB1 were genotyped, and clinical data were retrieved from patient charts. RESULTS: While ERL trough concentration (C0 ) was within the targeted range for most patients, over 30-fold variability in the dose-adjusted ERL C0 was observed. Regression analysis revealed a significant effect of the non-functional CYP3A5*3 variant on the dose-adjusted ERL C0 (p = 0.031). ERL dose requirement was 0.02 mg/kg/d higher in patients with CYP3A5*1/*3 genotype compared to patients with CYP3A5*3/*3 to reach the targeted C0 (p = 0.041). ERL therapy substantially improved estimated glomerular filtration rate (28.6 ± 6.6 mL/min/1.73 m(2)) in patients with baseline kidney dysfunction. CONCLUSION: Everolimus pharmacokinetics in HTx recipients is highly variable. Our preliminary data on patients on a CNI-free therapy regimen suggest that CYP3A5 genetic variation may contribute to this variability.

CYP3A5*3 and POR*28 genetic variants influence the required dose of tacrolimus in heart transplant recipients.

BACKGROUND: After heart transplantation (HTx), the interindividual pharmacokinetic variability of immunosuppressive drugs represents a major therapeutic challenge due to the narrow therapeutic window between over-immunosuppression causing toxicity and under-immunosuppression leading to graft rejection. Although genetic polymorphisms have been shown to influence pharmacokinetics of immunosuppressants, data in the context of HTx are scarce. We thus assessed the role of genetic variation in CYP3A4, CYP3A5, POR, NR1I2, and ABCB1 acting jointly in immunosuppressive drug pathways in tacrolimus (TAC) and ciclosporin (CSA) dose requirement in HTx recipients. METHODS: Associations between 7 functional genetic variants and blood dose-adjusted trough (C0) concentrations of TAC and CSA at 1, 3, 6, and 12 months after HTx were evaluated in cohorts of 52 and 45 patients, respectively. RESULTS: Compared with CYP3A5 nonexpressors (*3/*3 genotype), CYP3A5 expressors (*1/*3 or *1/*1 genotype) required around 2.2- to 2.6-fold higher daily TAC doses to reach the targeted C0 concentration at all studied time points (P ≤ 0.003). Additionally, the POR*28 variant carriers showed higher dose-adjusted TAC-C0 concentrations at all time points resulting in significant differences at 3 (P = 0.025) and 6 months (P = 0.047) after HTx. No significant associations were observed between the genetic variants and the CSA dose requirement. CONCLUSIONS: The CYP3A5*3 variant has a major influence on the required TAC dose in HTx recipients, whereas the POR*28 may additionally contribute to the observed variability. These results support the importance of genetic markers in TAC dose optimization after HTx.

Comparative evaluation of the My5-FU™ immunoassay and LC-MS/MS in monitoring the 5-fluorouracil plasma levels in cancer patients.

BACKGROUND: Chemotherapies of solid tumors commonly include 5-fluorouracil (5-FU). With standard doses of 5-FU, substantial inter-patient variability has been observed in exposure levels and treatment response. Recently, improved outcomes in colorectal cancer patients due to pharmacokinetically guided 5-FU dosing were reported. We aimed at establishing a rapid and sensitive method for monitoring 5-FU plasma levels in cancer patients in our routine clinical practice. METHODS: Performance of the Saladax My5-FU™ immunoassay was evaluated on the Roche Cobas® Integra 800 analyzer. Subsequently, 5-FU concentrations of 247 clinical plasma samples obtained with this assay were compared to the results obtained by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and other commonly used clinical analyzers (Olympus AU400, Roche Cobas c6000, and Thermo Fisher CDx90). RESULTS: The My-FU assay was successfully validated on the Cobas Integra 800 analyzer in terms of linearity, precision, accuracy, recovery, interference, sample carryover, and dilution integrity. Method comparison between the Cobas Integra 800 and LC-MS/MS revealed a proportional bias of 7% towards higher values measured with the My5-FU assay. However, when the Cobas Integra 800 was compared to three other clinical analyzers in addition to LC-MS/MS including 50 samples representing the typical clinical range of 5-FU plasma concentrations, only a small proportional bias (≤1.6%) and a constant bias below the limit of detection was observed. CONCLUSIONS: The My5-FU assay demonstrated robust and highly comparable performance on different analyzers. Therefore, the assay is suitable for monitoring 5-FU plasma levels in routine clinical practice and may contribute to improved efficacy and safety of commonly used 5-FU-based chemotherapies.

Inhibition of efflux transporters by poly ADP-ribose polymerase inhibitors.

Poly ADP-ribose polymerase (PARP) inhibitors have been approved for the treatment of various cancers. They share a similar mechanism of action but have differences in pharmacokinetic characteristics and potential for drug-drug interactions (DDI). This study evaluated the potential ATP-binding cassette transporter-mediated interactions between PARP inhibitors (niraparib, olaparib and rucaparib) and statins (atorvastatin and rosuvastatin). We studied the inhibitory activity of PARP inhibitors on breast cancer resistance protein (BCRP), multidrug resistance-associated protein 3 (MRP3) and P-glycoprotein (P-gp) using vesicular transport assays and determined the concentrations required for 50% inhibition (IC50 ). Then, we predicted the increase of statin exposure followed by the administration of PARP inhibitors using a mechanistic static model. Rucaparib was the strongest inhibitor of BCRP-mediated rosuvastatin transport (IC50 13.7 µM), followed by niraparib (42.6 µM) and olaparib (216 µM). PARP inhibitors did not affect MRP3. While niraparib appeared to inhibit P-gp, the inhibition showed large variability. The inhibition of intestinal BCRP by rucaparib, niraparib and olaparib was predicted to elevate rosuvastatin exposure by 52%, 37% and 24%, respectively. The interactions between PARP inhibitors and rosuvastatin are probably of minor clinical significance alone, but combined with other predisposing factors, they may increase the risk of rosuvastatin-associated adverse effects.

Prevalence of RPGR-Mediated Retinal Dystrophy in an Unselected Cohort of Over 5000 Patients.

Purpose: Comprehensive genetic testing for inherited retinal dystrophy (IRD) is challenged by difficult-to-sequence genomic regions, which are often mutational hotspots, such as RPGR ORF15. The purpose of this study was to evaluate the diagnostic contribution of RPGR variants in an unselected IRD patient cohort referred for testing in a clinical diagnostic laboratory. Methods: A total of 5201 consecutive patients were analyzed with a clinically validated next-generation sequencing (NGS)-based assay, including the difficult-to-sequence RPGR ORF15 region. Copy number variant (CNV) detection from NGS data was included. Variant interpretation was performed per the American College of Medical Genetics and Genomics guidelines. Results: A confirmed molecular diagnosis in RPGR was found in 4.5% of patients, 24.0% of whom were females. Variants in ORF15 accounted for 74% of the diagnoses; 29% of the diagnostic variants were in the most difficult-to-sequence central region of ORF15 (c.2470-3230). Truncating variants made up the majority (91%) of the diagnostic variants. CNVs explained 2% of the diagnostic cases, of which 80% were one- or two-exon deletions outside of ORF15. Conclusions: Our findings indicate that high-throughput, clinically validated NGS-based testing covering the difficult-to-sequence region of ORF15, in combination with high-resolution CNV detection, can help to maximize the diagnostic yield for patients with IRD. Translational Relevance: These results demonstrate an accurate and scalable method for the detection of RPGR-related variants, including the difficult-to-sequence ORF15 hotspot, which is relevant given current and emerging therapeutic opportunities.

Pharmacogenomics of cardiovascular drugs and adverse effects in pediatrics.

Individual response to medication is highly variable. For many drugs, a substantial proportion of patients show suboptimal response at standard doses, whereas others experience adverse drug reactions (ADRs). Pharmacogenomics aims to identify genetic factors underlying this variability in drug response, providing solutions to improve drug efficacy and safety. We review recent advances in pharmacogenomics of cardiovascular drugs and cardiovascular ADRs, including warfarin, clopidogrel, ß-blockers, renin-angiotensin-aldosterone system inhibitors, drug-induced long QT syndrome, and anthracycline-induced cardiotoxicity. We particularly focus on the applicability of pharmacogenomic findings to pediatric patients in whom developmental changes in body size and organ function may affect drug pharmacokinetics and pharmacodynamics. Solid evidence supports the importance of gene variants in CYP2C9 and VKORC1 for warfarin dosing and in CYP2C19 for clopidogrel response in adult patients. For the other cardiovascular drugs or cardiovascular ADRs, further studies are needed to replicate or clarify genetic associations before considering uptake of pharmacogenetic testing in clinical practice. With the exception of warfarin and anthracycline-induced cardiotoxicity, there is lack of pharmacogenomic studies on cardiovascular drug response or ADRs aimed specifically at children or adolescents. The first pediatric warfarin pharmacogenomic study indeed indicates differences from adults, pointing out the importance and need for pediatric-focused pharmacogenomic studies.

CYP2D6 polymorphisms and codeine analgesia in postpartum pain management: a pilot study.

BACKGROUND: Codeine, a common opiate prescribed for pain postcesarean section (c-section), is biotransformed by the highly polymorphic Cytochrome P450 enzyme 2D6 (CYP2D6). Ultrarapid metabolizers (UMs), individuals with multiple active copies of CYP2D6, can biotranform up to 50% more codeine into morphine than normal individuals can. In contrast, poor metabolizers (PMs), individuals who have no active CYP2D6 genes, convert almost no codeine into morphine and as a result may take multiple doses of codeine without attaining analgesia. OBJECTIVE: The aim was to study the relationship between CYP2D6 genotype and codeine analgesia among women recovering from c-section. METHODS: Forty-five mothers prescribed codeine provided a blood sample for CYP2D6 genotyping and recorded their pain level 4 times a day for 3 days immediately after a c-section. Codeine was used on an as-needed basis; doses and times were recorded. The relationship between CYP2D6 genotype, pain scores, need for codeine, and adverse events was studied. Theoretical morphine dose, based on CYP2D6 genotype, was estimated. RESULTS: Women at the genotypic extremes reported codeine effects consistent with their genotype: the 2 PMs of codeine reported no analgesia as a result of taking codeine, whereas 2 of the 3 UMs reported immediate pain relief from codeine but stopped taking it due to dizziness and constipation. Much larger numbers are needed to study similar correlations among extensive and intermediate metabolizers. CONCLUSIONS: In this pilot study, the extreme CYP2D6 genotypes (PMs and UMs) seemed to predict pain response and adverse events. Larger sample sizes are needed to correlate the range of genotypes with pain response.

Diagnostic utility of next-generation sequencing-based panel testing in 543 patients with suspected skeletal dysplasia.

BACKGROUND: Skeletal dysplasia is typically diagnosed using a combination of radiographic imaging, clinical examinations, and molecular testing. Identifying a molecular diagnosis for an individual with a skeletal dysplasia can lead to improved clinical care, guide future medical management and treatment, and inform assessment of risk for familial recurrence. The molecular diagnostic utility of multi-gene panel testing using next-generation sequencing (NGS) has not yet been characterized for an unselected population of individuals with suspected skeletal dysplasia. In this study, we retrospectively reviewed patient reports to assess the diagnostic yield, reported variant characteristics, impact of copy number variation, and performance in prenatal diagnostics of panel tests for variants in genes associated with skeletal dysplasia and growth disorders. RESULTS: Clinical reports of consecutive patients with a clinical indication of suspected skeletal dysplasia who underwent panel testing were examined. The 543 patients included in the study submitted samples for diagnostic genetic testing with an indication of suspected skeletal dysplasia or growth disorder and received one of three nested panel tests. A molecular diagnosis was established in 42.0% of patients (n = 228/543). Diagnostic variants were identified in 71 genes, nearly half of which (n = 35, 49.3%) contributed uniquely to a molecular diagnosis for a single patient in this cohort. Diagnostic yield was significantly higher among fetal samples (58.0%, n = 51/88) than postnatal samples (38.9%, n = 177/455; z = 3.32, p < 0.0009). Diagnostic variants in fetal cases were identified across 18 genes. Thirteen diagnostic CNVs were reported, representing 5.7% of diagnostic findings and ranging in size from 241-bp to whole chromosome aneuploidy. Additionally, 11.4% (36/315) of non-diagnostic patient reports had suspicious variants of unknown significance (VUS), in which additional family studies that provide segregation data and/or functional characterization may result in reclassification to likely pathogenic. CONCLUSIONS: These findings demonstrate the utility of panel testing for individuals with a suspected skeletal dysplasia or growth disorder, with a particularly high diagnostic yield seen in prenatal cases. Pursuing comprehensive panel testing with high-resolution CNV analysis can provide a diagnostic benefit, given the considerable phenotype overlap amongst skeletal dysplasia conditions.

GRINL1A Complex Transcription Unit Containing GCOM1, MYZAP, and POLR2M Genes Associates with Fully Penetrant Recessive Dilated Cardiomyopathy.

Background: Familial dilated cardiomyopathy (DCM) is a monogenic disorder typically inherited in an autosomal dominant pattern. We have identified two Finnish families with familial cardiomyopathy that is not explained by a variant in any previously known cardiomyopathy gene. We describe the cardiac phenotype related to homozygous truncating GCOM1 variants. Methods and Results: This study included two probands and their relatives. All the participants are of Finnish ethnicity. Whole-exome sequencing was used to test the probands; bi-directional Sanger sequencing was used to identify the GCOM1 variants in probands' family members. Clinical evaluation was performed, medical records and death certificates were obtained. Immunohistochemical analysis of myocardial samples was conducted. A homozygous GCOM1 variant was identified altogether in six individuals, all considered to be affected. None of the nine heterozygous family members fulfilled any cardiomyopathy criteria. Heart failure was the leading clinical feature, and the patients may have had a tendency for atrial arrhythmias. Conclusions: This study demonstrates the significance of GCOM1 variants as a cause of human cardiomyopathy and highlights the importance of searching for new candidate genes when targeted gene panels do not yield a positive outcome.

Biallelic loss-of-function in NRAP is a cause of recessive dilated cardiomyopathy.

BACKGROUND: Familial dilated cardiomyopathy (DCM) is typically a monogenic disorder with dominant inheritance. Although over 40 genes have been linked to DCM, more than half of the patients undergoing comprehensive genetic testing are left without molecular diagnosis. Recently, biallelic protein-truncating variants (PTVs) in the nebulin-related anchoring protein gene (NRAP) were identified in a few patients with sporadic DCM. METHODS AND RESULTS: We determined the frequency of rare NRAP variants in a cohort of DCM patients and control patients to further evaluate role of this gene in cardiomyopathies. A retrospective analysis of our internal variant database consisting of 31,639 individuals who underwent genetic testing (either panel or direct exome sequencing) was performed. The DCM group included 577 patients with either a confirmed or suspected DCM diagnosis. A control cohort of 31,062 individuals, including 25,912 individuals with non-cardiac (control group) and 5,150 with non-DCM cardiac indications (Non-DCM cardiac group). Biallelic (n = 6) or two (n = 5) NRAP variants (two PTVs or PTV+missense) were identified in 11 unrelated probands with DCM (1.9%) but none of the controls. None of the 11 probands had an alternative molecular diagnosis. Family member testing supports co-segregation. Biallelic or potentially biallelic NRAP variants were enriched in DCM vs. controls (OR 1052, p<0.0001). Based on the frequency of NRAP PTVs in the gnomAD reference population, and predicting full penetrance, biallelic NRAP variants could explain 0.25%-2.46% of all DCM cases. CONCLUSION: Loss-of-function in NRAP is a cause for autosomal recessive dilated cardiomyopathy, supporting its inclusion in comprehensive genetic testing.

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.

CYP2D6 and CYP2C19 genotypes and amitriptyline metabolite ratios in a series of medicolegal autopsies.

In a series of 202 postmortem toxicology cases, the CYP2D6 and CYP2C19 genes were genotyped, and the concentrations of amitriptyline (AT) and six metabolites were analyzed. The polymorphic CYP2D6 and CYP2C19 genes encode enzymes participating in the metabolism of several potentially toxic drugs, and mutations in these genes may lead to adverse drug reactions, possibly even intoxications. AT was chosen as the substrate of interest because it is mainly metabolized by these enzymes, is considered relatively toxic, and ranks among the major causes of fatal drug poisoning in Finland. Our objective was to evaluate genetically determined interindividual variation in conjunction with metabolite ratios of drugs found in toxicological analysis in a series of medicolegal autopsies. Positive correlations were found between the proportion of trans-hydroxylated metabolites and the number of functional copies of CYP2D6 and between the proportion of demethylated metabolites and the number of functional copies of CYP2C19. None of the accidental or undetermined AT poisonings coincided with the CYP2D6 or CYP2C19 genotype which predicts a poor metabolizer phenotype. However, an unusually high femoral blood concentration of AT, 60mg/l, was found in one suicide case with no functional CYP2D6 genes. Our study shows a concordance of AT metabolite patterns with CYP2D6 and CYP2C19 genotypes in the presence of confounding factors typical for postmortem material. This result demonstrates the feasibility of postmortem pharmacogenetic analysis and supports the dominant role of genes in drug metabolism.

Dihydropyrimidinase and ß-ureidopropionase gene variation and severe fluoropyrimidine-related toxicity.

AIMS: To assess the association of DPYS and UPB1 genetic variation, encoding the catabolic enzymes downstream of dihydropyrimidine dehydrogenase, with early-onset toxicity from fluoropyrimidine-based chemotherapy. PATIENTS & METHODS: The coding and exon-flanking regions of both genes were sequenced in a discovery subset (164 patients). Candidate variants were genotyped in the full cohort of 514 patients. RESULTS & CONCLUSIONS: Novel rare deleterious variants in DPYS (c.253C > T and c.1217G > A) were detected once each in toxicity cases and may explain the occurrence of severe toxicity in individual patients, and associations of common variants in DPYS (c.1-1T > C: p(adjusted) = 0.003; OR = 2.53; 95% CI: 1.39-4.62, and c.265-58T > C: p(adjusted) = 0.039; OR = 0.61; 95% CI: 0.38-0.97) with 5-fluorouracil toxicity were replicated.

Polymorphisms in MIR27A Associated with Early-Onset Toxicity in Fluoropyrimidine-Based Chemotherapy.

PURPOSE: The microRNA miR-27a was recently shown to directly regulate dihydropyrimidine dehydrogenase (DPD), the key enzyme in fluoropyrimidine catabolism. A common polymorphism (rs895819A>G) in the miR-27a genomic region (MIR27A) was associated with reduced DPD activity in healthy volunteers, but the clinical relevance of this effect is still unknown. Here, we assessed the association of MIR27A germline variants with early-onset fluoropyrimidine toxicity. EXPERIMENTAL DESIGN: MIR27A was sequenced in 514 patients with cancer receiving fluoropyrimidine-based chemotherapy. Associations of MIR27A polymorphisms with early-onset (cycles 1-2) fluoropyrimidine toxicity were assessed in the context of known risk variants in the DPD gene (DPYD) and additional covariates associated with toxicity. RESULTS: The association of rs895819A>G with early-onset fluoropyrimidine toxicity was strongly dependent on DPYD risk variant carrier status (Pinteraction = 0.0025). In patients carrying DPYD risk variants, rs895819G was associated with a strongly increased toxicity risk [OR, 7.6; 95% confidence interval (CI), 1.7-34.7; P = 0.0085]. Overall, 71% (12/17) of patients who carried both rs895819G and a DPYD risk variant experienced severe toxicity. In patients without DPYD risk variants, rs895819G was associated with a modest decrease in toxicity risk (OR, 0.62; 95% CI, 0.43-0.9; P = 0.012). CONCLUSIONS: These results indicate that miR-27a and rs895819A>G may be clinically relevant for further toxicity risk stratification in carriers of DPYD risk variants. Our data suggest that direct suppression of DPD by miR-27a is primarily relevant in the context of fluoropyrimidine toxicity in patients with reduced DPD activity. However, miR-27a regulation of additional targets may outweigh its effect on DPD in patients without DPYD risk variants.

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.

Screening for basic drugs in 2-mL urine samples by dual-plate overpressured layer chromatography and comparison with gas chromatography-mass spectrometry.

A dual-plate overpressured layer chromatography (OPLC) method was evaluated for broad-scale screening of basic drugs in 2-mL autopsy urine samples. Extraction was carried out by mixed-mode solid-phase extraction, and identification was based on automated comparison of corrected Rf values (hRfc) and in situ UV spectra with library values by dedicated software. The day-to-day precision of hRfc values was good in both OPLC1 and OPLC2 systems with median relative standard deviations of 2.4% and 3.4%, respectively. Both Rf and hRfc values were independent of the amount of analyte (0.5-10 microg) applied to the plate. Detection limits were determined for 47 drug substances in 2-mL urine samples, and they varied between 0.05 and 3.5 mg/L with a median of 1.0 mg/L. The performance of OPLC was evaluated by analyzing 30 autopsy urine samples by both OPLC and gas chromatography-mass spectrometry (GC-MS). The majority of findings by OPLC were in agreement with GC-MS. Some substances with low concentrations were not detected by OPLC, whereas GC-MS failed to detect a few polar substances. The OPLC method thus provides an alternative for current planar and column liquid chromatographic drug screening methods with the possibility of lowering detection limits by using a larger sample size.