Impact of genetic variation in CYP2C19, CYP2D6, and CYP3A4 on oxycodone and its metabolites in a large database of clinical urine drug tests.

Urine drug testing (UDT) is a tool for monitoring drug use, including oxycodone. While variation in cytochrome P450 (CYP) genes is known to alter oxycodone metabolism, its impact on UDT results of oxycodone and its metabolites has not been well-studied. Here, multivariate analysis was performed on retrospective UDT results of 90,379 specimens collected from 14,684 genotyped patients prescribed oxycodone. Genetic variation in CYP2D6 and CYP2C19 had a significant impact on oxymorphone/oxycodone ratios, with a 6.9-fold difference between CYP2D6 ultrarapid metabolizers (UMs) and poor metabolizers (PMs; p < 10-300) and a 1.6-fold difference between CYP2C19 UMs and PMs (p = 1.50 × 10-4). CYP2D6 variation also significantly impacted noroxycodone/oxycodone ratios (p = 6.95 × 10-38). Oxycodone-positive specimens from CYP2D6 PMs were ~5-fold more likely to be oxymorphone-negative compared to normal metabolizers. These findings indicate that multivariate analysis of UDT data may be used to reveal the real-world impact of genetic and non-genetic factors on drug metabolism.

Genetic Testing for BCHE Variants Identifies Patients at Risk of Prolonged Neuromuscular Blockade in Response to Succinylcholine.

BACKGROUND: Genetic variants in the BCHE (butyrylcholinesterase) gene are associated with reduced BChE enzyme activity and prolonged post-succinylcholine neuromuscular blockade, which can lead to postanesthetic apnea and respiratory depression. Testing for BChE deficiency is usually performed by biochemical methods and is generally only offered to patients who have a personal or family history of prolonged post-succinylcholine neuromuscular blockade. PURPOSE: Using a clinical test, we investigated the frequencies of BCHE genotypes that are associated with increased risk for prolonged post-succinylcholine neuromuscular blockade. MATERIALS AND METHODS: Five BCHE variants, including the A (atypical, rs1799807), K (Kalow, rs1803274), F1 (fluoride-1, rs28933389), F2 (fluoride-2, rs28933390), and S1 (silent-1, rs398124632), were genotyped in a large (n = 13,301), multi-ethnic cohort in the United States. Subjects were recipients of pharmacogenetic testing ordered by their physicians as part of routine care. RESULTS: The minor allele frequencies of A, K, F1, F2, and S1 were 1.60%, 19.93%, 0.08%, 0.47%, and 0.04%, respectively, in this cohort. Based on a review of biochemical and clinical data of these variants, we grouped BCHE genotypes into four phenotypic categories to stratify the risk for prolonged post-succinylcholine neuromuscular blockade. Approximately 0.06% of patients were predicted to have severe BChE deficiency, 8% were predicted to have moderate BChE deficiency, and 29% were predicted to have mild BChE deficiency. Compared to other ethnic groups, Caucasians were predicted to have the highest frequency of BChE deficiency. CONCLUSION: While severe BChE deficiency is rare in the United States, approximately 8% of Americans are at moderate risk of prolonged post-succinylcholine neuromuscular blockade, suggesting that a sizable percentage of patients may benefit from preoperative genetic testing of BCHE.

A Screening Test for HLA-B∗15:02 in a Large United States Patient Cohort Identifies Broader Risk of Carbamazepine-Induced Adverse Events.

Purpose: HLA-B∗15:02 is strongly associated with life-threatening severe skin hypersensitivity reactions in patients treated with carbamazepine (CBZ) and structurally related medications. FDA-approved labeling recommends HLA-B∗15:02 screening before CBZ therapy in patients of Asian ancestry. In this study, we aimed to (a) identify a direct method for screening HLA-B∗15:02, and (b) evaluate prevalence in a large cohort of United States patients. Methods: Candidate genetic markers were identified by mining public data. Association was tested in 28,897 individuals by comparing SNP results with high-resolution HLA typing. Retrospective analysis of de-identified SNP and ethnicity data from 130,460 individuals was performed to evaluate the ethnic distribution of HLA-B∗15:02 in the United States. Results: 28,897 United States individuals showed 100% concordance between HLA-B∗15:02 and the minor allele of rs144012689 (100% sensitivity/99.97% specificity). Retrospective analysis of 160 positive individuals (66 with physician-reported ethnicity) notably included 28 Asians (42%), 15 African Americans (22%), 11 Caucasians (17%), 2 Hispanics (3%), and 10 "Other" (15%). Conclusion: Screening United States patients for HLA-B∗15:02 without ethnicity-based preselection identifies more than twice the number of carriers at risk of CBZ-related adverse events than screening patients of Asian ancestry alone. Risk assessment based on ethnicity assumptions may not identify a large portion of at-risk patients in the ethnically diverse United States population.

Frequency of CYP2D6 Alleles Including Structural Variants in the United States.

The CYP2D6 gene encodes an enzyme important in the metabolism of many commonly used medications. Variation in CYP2D6 is associated with inter-individual differences in medication response, and genetic testing is used to optimize medication therapy. This report describes a retrospective study of CYP2D6 allele frequencies in a large population of 104,509 de-identified patient samples across all regions of the United States (US). Thirty-seven unique CYP2D6 alleles including structural variants were identified. A majority of these alleles had frequencies which matched published frequency data from smaller studies, while eight had no previously published frequencies. Importantly, CYP2D6 structural variants were observed in 13.1% of individuals and accounted for 7% of the total variants observed. The majority of structural variants detected (73%) were decreased-function or no-function alleles. As such, structural variants were found in approximately one-third (30%) of CYP2D6 poor metabolizers in this study. This is the first CYP2D6 study to evaluate, with a consistent methodology, both structural variants and single copy alleles in a large US population, and the results suggest that structural variants have a substantial impact on CYP2D6 function.

Developmental genetics of secretory vesicle acidification during Caenorhabditis elegans spermatogenesis.

Secretory vesicles are used during spermatogenesis to deliver proteins to the cell surface. In Caenorhabditis elegans, secretory membranous organelles (MO) fuse with the plasma membrane to transform spermatids into fertilization-competent spermatozoa. We show that, like the acrosomal vesicle of mammalian sperm, MOs undergo acidification during development. Treatment of spermatids with the V-ATPase inhibitor bafilomycin blocks both MO acidification and formation of functional spermatozoa. There are several spermatogenesis-defective mutants that cause defects in MO morphogenesis, including spe-5. We determined that spe-5, which is on chromosome I, encodes one of two V-ATPase B paralogous subunits. The spe-5 null mutant is viable but sterile because it forms arrested, multi-nucleate spermatocytes. Immunofluorescence with a SPE-5-specific monoclonal antibody shows that SPE-5 expression begins in spermatocytes and is found in all subsequent stages of spermatogenesis. Most SPE-5 is discarded into the residual body during spermatid budding, but a small amount remains in budded spermatids where it localizes to MOs as a discrete dot. The other V-ATPase B subunit is encoded by vha-12, which is located on the X chromosome. Usually, spe-5 mutants are self-sterile in a wild-type vha-12 background. However, an extrachromosomal transgene containing wild-type vha-12 driven by its own promoter allows spe-5 mutant hermaphrodites to produce progeny, indicating that VHA-12 can at least partially substitute for SPE-5. Others have shown that the X chromosome is transcriptionally silent in the male germline, so expression of the autosomally located spe-5 gene ensures that a V-ATPase B subunit is present during spermatogenesis.

SPE-39 family proteins interact with the HOPS complex and function in lysosomal delivery.

Yeast and animal homotypic fusion and vacuole protein sorting (HOPS) complexes contain conserved subunits, but HOPS-mediated traffic in animals might require additional proteins. Here, we demonstrate that SPE-39 homologues, which are found only in animals, are present in RAB5-, RAB7-, and RAB11-positive endosomes where they play a conserved role in lysosomal delivery and probably function via their interaction with the core HOPS complex. Although Caenorhabditis elegans spe-39 mutants were initially identified as having abnormal vesicular biogenesis during spermatogenesis, we show that these mutants also have disrupted processing of endocytosed proteins in oocytes and coelomocytes. C. elegans SPE-39 interacts in vitro with both VPS33A and VPS33B, whereas RNA interference of VPS33B causes spe-39-like spermatogenesis defects. The human SPE-39 orthologue C14orf133 also interacts with VPS33 homologues and both coimmunoprecipitates and cosediments with other HOPS subunits. SPE-39 knockdown in cultured human cells altered the morphology of syntaxin 7-, syntaxin 8-, and syntaxin 13-positive endosomes. These effects occurred concomitantly with delayed mannose 6-phosphate receptor-mediated cathepsin D delivery and degradation of internalized epidermal growth factor receptors. Our findings establish that SPE-39 proteins are a previously unrecognized regulator of lysosomal delivery and that C. elegans spermatogenesis is an experimental system useful for identifying conserved regulators of metazoan lysosomal biogenesis.

The Caenorhabditis elegans spe-39 gene is required for intracellular membrane reorganization during spermatogenesis.

Caenorhabditis elegans spermatid formation involves asymmetric partitioning of cytoplasm during the second meiotic division. This process is mediated by specialized ER/Golgi-derived fibrous body-membranous organelles (FB-MOs), which have a fibrous body (FB) composed of bundled major sperm protein filaments and a vesicular membranous organelle (MO). spe-39 mutant spermatocytes complete meiosis but do not usually form spermatids. Ultrastructural examination of spe-39 spermatocytes reveals that MOs are absent, while FBs are disorganized and not surrounded by the membrane envelope usually observed in wild type. Instead, spe-39 spermatocytes contain many small vesicles with internal membranes, suggesting they are related to MOs. The spe-39 gene was identified and it encodes a novel hydrophilic protein. Immunofluorescence with a specific SPE-39 antiserum reveals that it is distributed through much of the cytoplasm and not specifically associated with FB-MOs in spermatocytes and spermatids. The spe-39 gene has orthologs in Drosophila melanogaster and humans but no homolog was identified in the yeast genome. This suggests that the specialized membrane biogenesis steps that occur during C. elegans spermatogenesis are part of a conserved process that requires SPE-39 homologs in other metazoan cell types.