Influence of novel CYP2C-haplotype on proton pump inhibitor pharmacokinetics in children.

In this brief report, we provide an analysis of the influence of a novel CYP2C haplotype (CYP2C:TG) on proton pump inhibitor (PPI) pharmacokinetics (PK) in children. The CYP2C:TG haplotype has been proposed to be associated with increased CYP2C19 activity. We sought to determine if this CYP2C:TG haplotype resulted in similar alterations in metabolism for proton pump inhibitors, which are primarily metabolized by CYP2C19. In a cohort of 41 children aged 6-21 participating in a PPI pharmacokinetic study, effects of the CYP2C:TG allele were assessed by fitting two linear regression models for each of the six PK outcomes assessed, the second of which accounted for the presence of the CYP2C:TG allele. The difference in R2 values between the two models was computed to quantify the variability in the outcome that could be accounted for by the CYP2C:TG allele after adjustment for the CYP2C19 genotype. We found the CYP2C:TG haplotype to have no measurable additive impact on CYP2C19-mediated metabolism of PPIs in vivo in older children and adolescents. The findings of this study do not support the clinical utility of routine testing for the CYP2C:TG haplotype to guide PPI dose adjustments in children.

SLCO1B1 Genetic Variation Influence on Atorvastatin Systemic Exposure in Pediatric Hypercholesterolemia.

This clinical study examined the influence of SLCO1B1 c.521T>C (rs4149056) on plasma atorvastatin concentrations in pediatric hypercholesterolemia. The participants (8-21 years), including heterozygous (c.521T/C, n = 13), homozygous (c.521C/C, n = 2) and controls (c.521T/T, n = 13), completed a single-oral-dose pharmacokinetic study. Similar to in adults, the atorvastatin (AVA) area-under-concentration-time curve from 0 to 24 h (AUC0-24) was 1.7-fold and 2.8-fold higher in participants with c.521T/C and c.521C/C compared to the c.521T/T participants, respectively. The inter-individual variability in AVA exposure within these genotype groups ranged from 2.3 to 4.8-fold, indicating that additional factors contribute to the inter-individual variability in the AVA dose-exposure relationship. A multivariate model reinforced the SLCO1B1 c.521T>C variant as the central factor contributing to AVA systemic exposure in this pediatric cohort, accounting for ~65% of the variability in AVA AUC0-24. Furthermore, lower AVA lactone concentrations in participants with increased body mass index contributed to higher exposure within the c.521T/T and c.521T/C genotype groups. Collectively, these factors contributing to higher systemic exposure could increase the risk of toxicity and should be accounted for when individualizing the dosing of atorvastatin in eligible pediatric patients.

Updated DPYD HapB3 haplotype structure and implications for pharmacogenomic testing.

The DPYD gene encodes dihydropyrimidine dehydrogenase, the rate-limiting enzyme for the metabolism of fluoropyrimidines 5-fluorouracil and capecitabine. Genetic variants in DPYD have been associated with altered enzyme activity, therefore accurate detection and interpretation is critical to predict metabolizer status for individualized fluoropyrimidine therapy. The most commonly observed deleterious variation is the causal variant linked to the previously described HapB3 haplotype, c.1129-5923C>G (rs75017182) in intron 10, which introduces a cryptic splice site. A benign synonymous variant in exon 11, c.1236G>A (rs56038477) is also linked to HapB3 and is commonly used for testing. Previously, these single-nucleotide polymorphisms (SNPs) have been reported to be in perfect linkage disequilibrium (LD); therefore, c.1236G>A is often utilized as a proxy for the function-altering intronic variant. Clinical genotyping of DPYD identified a patient who had c.1236G>A, but not c.1129-5923C>G, suggesting that these two SNPs may not be in perfect LD, as previously assumed. Additional individuals with c.1236G>A, but not c.1129-5923C>G, were identified in the Children's Mercy Data Warehouse and the All of Us Research Program version 7 cohort substantiating incomplete SNP linkage. Consequently, testing only c.1236G>A can generate false-positive results in some cases and lead to suboptimal dosing that may negatively impact patient therapy and prospect of survival. Our data show that DPYD genotyping should include the functional variant c.1129-5923C>G, and not the c.1236G>A proxy, to accurately predict DPD activity.

PharmVar Tutorial on CYP2D6 Structural Variation Testing and Recommendations on Reporting.

The Pharmacogene Variation Consortium (PharmVar) provides nomenclature for the highly polymorphic human CYP2D6 gene locus and a comprehensive summary of structural variation. CYP2D6 contributes to the metabolism of numerous drugs and, thus, genetic variation in its gene impacts drug efficacy and safety. To accurately predict a patient's CYP2D6 phenotype, testing must include structural variants including gene deletions, duplications, hybrid genes, and combinations thereof. This tutorial offers a comprehensive overview of CYP2D6 structural variation, terms, and definitions, a review of methods suitable for their detection and characterization, and practical examples to address the lack of standards to describe CYP2D6 structural variants or any other pharmacogene. This PharmVar tutorial offers practical guidance on how to detect the many, often complex, structural variants, as well as recommends terms and definitions for clinical and research reporting. Uniform reporting is not only essential for electronic health record-keeping but also for accurate translation of a patient's genotype into phenotype which is typically utilized to guide drug therapy.

Impact of CYP2C:TG Haplotype on CYP2C19 Substrates Clearance In Vivo, Protein Content, and In Vitro Activity.

A novel haplotype composed of two non-coding variants, CYP2C18 NM_000772.3:c.*31T (rs2860840) and NM_000772.2:c.819+2182G (rs11188059), referred to as "CYP2C:TG," was recently associated with ultrarapid metabolism of various CYP2C19 substrates. As the underlying mechanism and clinical relevance of this effect remain uncertain, we analyzed existing in vivo and in vitro data to determine the magnitude of the CYP2C:TG haplotype effect. We assessed variability in pharmacokinetics of CYP2C19 substrates, including citalopram, sertraline, voriconazole, omeprazole, pantoprazole, and rabeprazole in 222 healthy volunteers receiving one of these six drugs. We also determined its impact on CYP2C8, CYP2C9, CYP2C18, and CYP2C19 protein abundance in 135 human liver tissue samples, and on CYP2C18/CYP2C19 activity in vitro using N-desmethyl atomoxetine formation. No effects were observed according to CYP2C:TG haplotype or to CYP2C19*1+TG alleles (i.e., CYP2C19 alleles containing the CYP2C:TG haplotype). In contrast, CYP2C19 intermediate (e.g., CYP2C19*1/*2) and poor metabolizers (e.g., CYP2C19*2/*2) showed significantly higher exposure in vivo, lower CYP2C19 protein abundance in human liver microsomes, and lower activity in vitro compared with normal, rapid (i.e., CYP2C19*1/*17), and ultrarapid metabolizers (i.e., CYP2C19*17/*17). Moreover, a tendency toward lower exposure was observed in ultrarapid metabolizers compared with rapid metabolizers and normal metabolizers. Furthermore, when the CYP2C19*17 allele was present, CYP2C18 protein abundance was increased suggesting that genetic variation in CYP2C19 may be relevant to the overall metabolism of certain drugs by regulating not only its expression levels, but also those of CYP2C18. Considering all available data, we conclude that there is insufficient evidence supporting clinical CYP2C:TG testing to inform drug therapy.

Pharmacogenomic variation in the Malagasy population: implications for the antimalarial drug primaquine metabolism.

Aim: Antimalarial primaquine (PQ) eliminates liver hypnozoites of Plasmodium vivax. CYP2D6 gene variation contributes to PQ therapeutic failure. Additional gene variation may contribute to PQ efficacy. Information on pharmacogenomic variation in Madagascar, with vivax malaria and a unique population admixture, is scanty. Methods: The authors performed genome-wide genotyping of 55 Malagasy samples and analyzed data with a focus on a set of 28 pharmacogenes most relevant to PQ. Results: Mainly, the study identified 110 coding or splicing variants, including those that, based on previous studies in other populations, may be implicated in PQ response and copy number variation, specifically in chromosomal regions that contain pharmacogenes. Conclusion: With this pilot information, larger genome-wide association analyses with PQ metabolism and response are substantially more feasible.

Characterization of complex structural variation in the CYP2D6-CYP2D7-CYP2D8 gene loci using single-molecule long-read sequencing.

Complex regions in the human genome such as repeat motifs, pseudogenes and structural (SVs) and copy number variations (CNVs) present ongoing challenges to accurate genetic analysis, particularly for short-read Next-Generation-Sequencing (NGS) technologies. One such region is the highly polymorphic CYP2D loci, containing CYP2D6, a clinically relevant pharmacogene contributing to the metabolism of >20% of common drugs, and two highly similar pseudogenes, CYP2D7 and CYP2D8. Multiple complex SVs, including CYP2D6/CYP2D7-derived hybrid genes are known to occur in different configurations and frequencies across populations and are difficult to detect and characterize accurately. This can lead to incorrect enzyme activity assignment and impact drug dosing recommendations, often disproportionally affecting underrepresented populations. To improve CYP2D6 genotyping accuracy, we developed a PCR-free CRISPR-Cas9 based enrichment method for targeted long-read sequencing that fully characterizes the entire CYP2D6-CYP2D7-CYP2D8 loci. Clinically relevant sample types, including blood, saliva, and liver tissue were sequenced, generating high coverage sets of continuous single molecule reads spanning the entire targeted region of up to 52 kb, regardless of SV present (n = 9). This allowed for fully phased dissection of the entire loci structure, including breakpoints, to accurately resolve complex CYP2D6 diplotypes with a single assay. Additionally, we identified three novel CYP2D6 suballeles, and fully characterized 17 CYP2D7 and 18 CYP2D8 unique haplotypes. This method for CYP2D6 genotyping has the potential to significantly improve accurate clinical phenotyping to inform drug therapy and can be adapted to overcome testing limitations of other clinically challenging genomic regions.

CYP3A4 and CYP3A5 Genotyping Recommendations: A Joint Consensus Recommendation of the Association for Molecular Pathology, Clinical Pharmacogenetics Implementation Consortium, College of American Pathologists, Dutch Pharmacogenetics Working Group of the Royal Dutch Pharmacists Association, European Society for Pharmacogenomics and Personalized Therapy, and Pharmacogenomics Knowledgebase.

The goals of the Association for Molecular Pathology Clinical Practice Committee's Pharmacogenomics (PGx) Working Group are to define the key attributes of pharmacogenetic alleles recommended for clinical testing and a minimum set of variants that should be included in clinical PGx genotyping assays. This document series provides recommendations for a minimum panel of variant alleles (tier 1) and an extended panel of variant alleles (tier 2) that will aid clinical laboratories when designing assays for PGx testing. The Association for Molecular Pathology PGx Working Group considered functional impact of the variant alleles, allele frequencies in multiethnic populations, the availability of reference materials, and other technical considerations for PGx testing when developing these recommendations. The goal of this Working Group is to promote standardization of PGx gene/allele testing across clinical laboratories. This document will focus on clinical CYP3A4 and CYP3A5 PGx testing that may be applied to all CYP3A4- and CYP3A5-related medications. These recommendations are not to be interpreted as prescriptive but to provide a reference guide.

Characterization of Reference Materials for CYP3A4 and CYP3A5: A (GeT-RM) Collaborative Project.

Pharmacogenetic testing for CYP3A4 is increasingly provided by clinical and research laboratories; however, only a limited number of quality control and reference materials are currently available for many of the CYP3A4 variants included in clinical tests. To address this need, the Division of Laboratory Systems, CDC-based Genetic Testing Reference Material Coordination Program (GeT-RM), in collaboration with members of the pharmacogenetic testing and research communities and the Coriell Institute for Medical Research, has characterized 30 DNA samples derived from Coriell cell lines for CYP3A4. Samples were distributed to five volunteer laboratories for genotyping using a variety of commercially available and laboratory-developed tests. Sanger and next-generation sequencing were also utilized by some of the laboratories. Whole-genome sequencing data from the 1000 Genomes Projects were utilized to inform genotype. Twenty CYP3A4 alleles were identified in the 30 samples characterized for CYP3A4: CYP3A4∗4, ∗5, ∗6, ∗7, ∗8, ∗9, ∗10, ∗11, ∗12, ∗15, ∗16, ∗18, ∗19, ∗20, ∗21, ∗22, ∗23, ∗24, ∗35, and a novel allele, CYP3A4∗38. Nineteen additional samples with preexisting data for CYP3A4 or CYP3A5 were re-analyzed to generate comprehensive reference material panels for these genes. These publicly available and well-characterized materials can be used to support the quality assurance and quality control programs of clinical laboratories performing clinical pharmacogenetic testing.

Clinical Pharmacogenetics Implementation Consortium (CPIC) Guideline for CYP2D6, CYP2C19, CYP2B6, SLC6A4, and HTR2A Genotypes and Serotonin Reuptake Inhibitor Antidepressants.

Serotonin reuptake inhibitor antidepressants, including selective serotonin reuptake inhibitors (SSRIs; i.e., citalopram, escitalopram, fluoxetine, fluvoxamine, paroxetine, and sertraline), serotonin and norepinephrine reuptake inhibitors (i.e., desvenlafaxine, duloxetine, levomilnacipran, milnacipran, and venlafaxine), and serotonin modulators with SSRI-like properties (i.e., vilazodone and vortioxetine) are primary pharmacologic treatments for major depressive and anxiety disorders. Genetic variation in CYP2D6, CYP2C19, and CYP2B6 influences the metabolism of many of these antidepressants, which may potentially affect dosing, efficacy, and tolerability. In addition, the pharmacodynamic genes SLC6A4 (serotonin transporter) and HTR2A (serotonin-2A receptor) have been examined in relation to efficacy and side effect profiles of these drugs. This guideline updates and expands the 2015 Clinical Pharmacogenetics Implementation Consortium (CPIC) guideline for CYP2D6 and CYP2C19 genotypes and SSRI dosing and summarizes the impact of CYP2D6, CYP2C19, CYP2B6, SLC6A4, and HTR2A genotypes on antidepressant dosing, efficacy, and tolerability. We provide recommendations for using CYP2D6, CYP2C19, and CYP2B6 genotype results to help inform prescribing these antidepressants and describe the existing data for SLC6A4 and HTR2A, which do not support their clinical use in antidepressant prescribing.

Advancing CYP2D6 Pharmacogenetics through a Pharmacoequity Lens.

Over 20% of US Food and Drug Administration (FDA)-approved drugs in the United States are metabolized by the hepatic enzyme cytochrome P450 2D6 (CYP2D6). The gene encoding CYP2D6 is highly polymorphic and genetic variation has been shown to impact drug response for many commonly dispensed drugs including opioids and antidepressants. Thus, it is important to understand an individual's CYP2D6 metabolizer status to optimize treatment outcomes for patients taking medications that are metabolized by this enzyme. Consequently, clinical CYP2D6 pharmacogenetic testing is being implemented by a growing number of health centers. Furthermore, clinical guidelines currently recommend adapting therapeutic regimens based on CYP2D6 genotype-informed phenotype. However, CYP2D6 genetic variation varies considerably across global populations and many allelic variants, or star alleles, are predominantly found in certain ancestral populations. Although CYP2D6 genetic variation has been extensively studied, there is still a paucity of information for many non-European populations. As has been shown for other pharmacogenes in randomized controlled trials, results from European populations cannot simply be extrapolated to other groups and, in some cases, even has the potential to cause harm. Therefore, enhanced inclusion in pharmacogenetic studies is urgently needed to increase ancestral representation, determine the extent of global CYP2D6 genetic variation (e.g., ancestry-specific variants), and determine the clinical impact of this variation on clinical treatment outcome. This review highlights knowledge gaps, challenges, and future directions in CYP2D6 pharmacogenomics through a unique pharmacoequity lens to address health inequities that hamper our ability to optimize drug therapy for improved pharmacological outcomes in diverse populations globally.

Evaluating treatment outcomes in pharmacogenomic-guided care for major depression: A rapid review and meta-analysis.

Pharmacogenomic (PGx) testing may increase the probability of remission and response in patients with major depressive disorder (MDD) undergoing pharmacotherapy. Given the potential implications of these outcomes and recent proliferation of PGx studies, we conducted a systematic review to evaluate the effectiveness of PGx testing on clinical outcomes in patients with MDD as compared to treatment as usual (TAU). MEDLINE, Embase, PsycInfo, and CENTRAL were searched for English-language articles from 2000 to 2021 for randomized controlled trials (RCTs) comparing PGx-guided treatment vs. TAU in patients with MDD. Meta-analyses were conducted in R. Ten RCTs were included: eight reported remission and seven reported response. The best available evidence suggests that PGx-guided care for moderate-to-severe adult depression is more likely to result in remission and response than TAU (both risk ratios significant). However, there are limitations in the evidence base, including high risk of bias and inconsistency between trials. Despite the consequent very low certainty in the magnitude of effect, there is confidence in the direction. Though modest, the beneficial effects of PGx for adults with moderate-severe MDD could - as a result of the scope and scale of the condition and its impacts - have important ramifications for patients and the health system.

PharmVar GeneFocus: SLCO1B1.

The Pharmacogene Variation Consortium (PharmVar) is now providing star (*) allele nomenclature for the highly polymorphic human SLCO1B1 gene encoding the organic anion transporting polypeptide 1B1 (OATP1B1) drug transporter. Genetic variation within the SLCO1B1 gene locus impacts drug transport, which can lead to altered pharmacokinetic profiles of several commonly prescribed drugs. Variable OATP1B1 function is of particular importance regarding hepatic uptake of statins and the risk of statin-associated musculoskeletal symptoms. To introduce this important drug transporter gene into the PharmVar database and serve as a unified reference of haplotype variation moving forward, an international group of gene experts has performed an extensive review of all published SLCO1B1 star alleles. Previously published star alleles were self-assigned by authors and only loosely followed the star nomenclature system that was first developed for cytochrome P450 genes. This nomenclature system has been standardized by PharmVar and is now applied to other important pharmacogenes such as SLCO1B1. In addition, data from the 1000 Genomes Project and investigator-submitted data were utilized to confirm existing haplotypes, fill knowledge gaps, and/or define novel star alleles. The PharmVar-developed SLCO1B1 nomenclature has been incorporated by the Clinical Pharmacogenetics Implementation Consortium (CPIC) 2022 guideline on statin-associated musculoskeletal symptoms.

Developmental pharmacokinetics of indomethacin in preterm neonates: Severely decreased drug clearance in the first week of life.

Indomethacin is used commonly in preterm neonates for the prevention of intracranial hemorrhage and closure of an abnormally open cardiac vessel. Due to biomedical advances, the infants who receive this drug in the neonatal intensive care unit setting have become younger, smaller, and less mature (more preterm) at the time of treatment. To develop a pharmacokinetics (PK) model to aid future dosing, we designed a prospective cohort study to characterize indomethacin PK in a dynamically changing patient population. A population PK base model was created using NONMEM, and a covariate model was developed in a primary development cohort and subsequently was tested for accuracy in a validation cohort. Postnatal age was a significant covariate for hepatic clearance (CLH ) and renal clearance (CLR ). The typical value of the total clearance (CL, the sum of CLR and CLH ) was 3.09 ml/h and expressed as CL/WTmedian  = 3.96 ml/h/kg, where WTmedian is the median body weight. The intersubject variability of CLR and CLH were 61% and 207%, respectively. The typical value of the volume of distribution Vp  = 366 ml (Vp /WTmedian  = 470 ml/kg), and its intersubject variability was 38.8%. Half-life was 82.1 h. Compared with more mature and older preterm populations studied previously, indomethacin CL is considerably lower in this contemporary population. Model-informed precision dosing incorporating important covariates other than weight alone offers an opportunity to individualize dosing in a susceptible patient undergoing rapid change.

Pediatric CYP2D6 metabolizer status and post-tonsillectomy nausea and vomiting after ondansetron.

The goal of this study was to determine whether CYP2D6 metabolizer status within the ondansetron-treated pediatric tonsillectomy population is associated with risk of postoperative nausea and vomiting (PONV) in the post-anesthesia care unit. We conducted a retrospective cohort study of pediatric patients (<18 years) who underwent tonsillectomy and received ondansetron on the day of the procedure. Data were obtained from BioVU, an institutional biobank that links DNA to de-identified electronic health record data. Subjects were tested for 10 CYP2D6 allelic variants and copy number variation, and genotype data translated into CYP2D6 metabolizer status. The cohort included 652 individuals, 105 (16.1%) of whom had PONV. Rates of PONV were similar across groups: ultrarapid metabolizers (UMs), 1 of 9 (11.1%); normal metabolizers (NMs), 64 of 354 (18.1%); intermediate metabolizers (IMs), 33 of 234 (14.1%); poor metabolizers (PMs), 6 of 39 (15.4%); and ambiguous phenotypes, 1 of 16 (6.3%). In multivariable analysis adjusted for age, sex, and time under anesthesia, CYP2D6 metabolizer status was not associated with PONV, with an odds ratio of 1.37 (95% confidence interval 0.9, 2.1) when comparing PM/IM versus NM/UM. In this large pediatric population, no significant differences were detected for PONV based on CYP2D6 metabolizer status. Further investigation is needed to determine mechanisms for ondansetron inefficacy in children.

Characterization of Novel CYP2D6 Alleles across Sub-Saharan African Populations.

The CYP2D6 gene has been widely studied to characterize variants and/or star alleles, which account for a significant portion of variability in drug responses observed within and between populations. However, African populations remain under-represented in these studies. The increasing availability of high coverage genomes from African populations has provided the opportunity to fill this knowledge gap. In this study, we characterized computationally predicted novel CYP2D6 star alleles in 30 African subjects for whom DNA samples were available from the Coriell Institute. CYP2D6 genotyping and resequencing was performed using a variety of commercially available and laboratory-developed tests in a collaborative effort involving three laboratories. Fourteen novel CYP2D6 alleles and multiple novel suballeles were identified. This work adds to the growing catalogue of validated African ancestry CYP2D6 allelic variation in pharmacogenomic databases, thus laying the foundation for future functional studies and improving the accuracy of CYP2D6 genotyping, phenotype prediction, and the refinement of clinical pharmacogenomic implementation guidelines in African and global settings.

A longitudinal study of cytochrome P450 2D6 (CYP2D6) activity during adolescence.

CYP2D6 substrates are among the most highly prescribed medications in teenagers and also commonly associated with serious adverse events. To investigate the relative contributions of genetic variation, growth, and development on CYP2D6 activity during puberty, healthy children and adolescents 7-15 years of age at enrollment participated in a longitudinal phenotyping study involving administration of 0.3 mg/kg dextromethorphan (DM) and 4-h urine collection every 6 months for 3 years (7 total visits). At each visit, height, weight, and sexual maturity were recorded, and CYP2D6 activity was determined as the urinary molar ratio of DM to its metabolite dextrorphan (DX). A total of 188 participants completed at least one visit, and 102 completed all seven study visits. Following univariate analysis, only CYP2D6 activity score (p < 0.001), urinary pH (p < 0.001), weight (p = 0.018), and attention-deficit/hyperactivity disorder (ADHD) diagnosis (p < 0.001) were significantly correlated with log(DM/DX). Results of linear mixed model analysis with random intercept, random slope covariance structure revealed that CYP2D6 activity score had the strongest effect on log(DM/DX), with model-estimated average log(DM/DX) being 3.8 SDs higher for poor metabolizers than for patients with activity score 3. A moderate effect on log(DM/DX) was observed for sex, and smaller effects were observed for ADHD diagnosis and urinary pH. The log(DM/DX) did not change meaningfully with age or pubertal development. CYP2D6 genotype remains the single, largest determinant of variability in CYP2D6 activity during puberty. Incorporation of genotype-based dosing guidelines should be considered for CYP2D6 substrates given the prevalent use of these agents in this pediatric age group.