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During the sexual phase of Neurospora crassa, unpaired genes are subject to a silencing mechanism known as meiotic silencing by unpaired DNA (MSUD). MSUD targets the transcripts of an unpaired gene and utilizes typical RNA interference (RNAi) factors for its process. Using a reverse genetic screen, we have identified a meiotic silencing gene called sad-9, which encodes a DEAD-box RNA helicase. While not essential for vegetative growth, SAD-9 plays a crucial role in both sexual development and MSUD. Our results suggest that SAD-9, with the help of the SAD-2 scaffold protein, recruits the SMS-2 Argonaute to the perinuclear region, the center of MSUD activity.

The Pharmacogene Variation Consortium (PharmVar) catalogs star (*) allele nomenclature for the polymorphic human CYP3A5 gene. Genetic variation within the CYP3A5 gene locus impacts the metabolism of several clinically important drugs, including the immunosuppressants tacrolimus, sirolimus, cyclosporine, and the benzodiazepine midazolam. Variable CYP3A5 activity is of clinical importance regarding tacrolimus metabolism. This GeneFocus provides a CYP3A5 gene summary with a focus on aspects regarding standardized nomenclature. In addition, this review also summarizes recent changes and updates, including the retirement of several allelic variants and provides an overview of how PharmVar CYP3A5 star allele nomenclature is utilized by the Pharmacogenomics Knowledgebase (PharmGKB) and the Clinical Pharmacogenetics Implementation Consortium (CPIC).

rs5758550 has been associated with enhanced transcription and suggested to be a useful marker of CYP2D6 activity. As there are limited and inconsistent data regarding the utility of this distant "enhancer" single nucleotide polymorphism (SNP), our goal was to further assess the impact of rs5758550 on CYP2D6 activity toward two probe substrates, atomoxetine (ATX) and dextromethorphan (DM), using in vivo urinary metabolite (DM; n = 188) and pharmacokinetic (ATX; n = 70) and in vitro metabolite formation (ATX and DM; n = 166) data. All subjects and tissues were extensively genotyped, the "enhancer" SNP phased with established CYP2D6 haplotypes either computationally or experimentally, and the impact on CYP2D6 activity investigated using several linear models of varying complexity to determine the proportion of variability in CYP2D6 activity captured by each model. For all datasets and models, the "enhancer" SNP had no or only a modest impact on CYP2D6 activity prediction. An increased effect, when present, was more pronounced for ATX than DM suggesting potential substate-dependency. In addition, CYP2D6*2 alleles with the "enhancer" SNP were associated with modestly higher metabolite formation rates in vitro, but not in vivo; no effect was detected for CYP2D6*1 alleles with "enhancer" SNP. In summary, it remains inconclusive whether the small effects detected in this investigation are indeed caused by the "enhancer" SNP or are rather due to its incomplete linkage with other variants within the gene. Taken together, there does not appear to be sufficient evidence to warrant the "enhancer" SNP be included in clinical CYP2D6 pharmacogenetic testing.

Cytochrome P450 2D6 (CYP2D6) copy number (CN) variation affects the metabolism of numerous prescribed drugs. Sequence variation within primer or probe target regions of hydrolysis probe CN assays can generate false-positive calls for CN loss. Furthermore, CYP2D6–CYP2D7 hybrids and gene conversions can cause difficult to interpret discordant CN calls. The identification of haplotypes with CN variations and structural arrangements is important to predict phenotype accurately. During clinical testing with hydrolysis probe assays targeting three CYP2D6 regions (intron 2, intron 6, and exon 9), samples with haplotypes causing inconsistent CN calls were identified. To resolve these cases, next-generation sequencing and allele-specific Sanger sequencing was performed. Sequence analysis of 16 samples, all but one from subjects of African descent, identified six novel suballeles containing single-nucleotide polymorphisms, which cause false-positive calls for CN loss in introns 2 and 6. Five samples with an exon 9 CN loss contained CYP2D6/CYP2D7 hybrids (∗13 or ∗36) and one sample was found to have a novel haplotype, CYP2D6∗141. Interestingly, CYP2D6∗141 contains a CYP2D7-derived exon 9 conversion and core single-nucleotide polymorphisms that are otherwise found in CYP2D6∗17 and ∗27. Although these variants are rare, they can cause inconsistent CN calls that typically are reported as no calls or indeterminant, and thus may deprive patients, particularly those of African descent, from taking full benefit of pharmacogenetic testing.

Pharmacogenetic testing 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 TPMT and NUDT15 variants included in clinical tests. To address this need, the Division of Laboratory Systems, Centers for Disease Control and Prevention-based Genetic Testing Reference Material (GeT-RM) coordination program, in collaboration with members of the pharmacogenetic testing and research communities and the Coriell Institute for Medical Research, has characterized 19 DNA samples derived from Coriell cell lines. DNA samples were distributed to four volunteer testing laboratories for genotyping using a variety of commercially available and laboratory developed tests and/or Sanger sequencing. Of the 12 samples characterized for TPMT, newly identified variants include TPMT∗2, ∗6, ∗12, ∗16, ∗21, ∗24, ∗32, ∗33, and ∗40; for the 7 NUDT15 reference material samples, newly identified variants are NUDT15∗2, ∗3, ∗4, ∗5, ∗6, and ∗9. In addition, a novel haplotype, TPMT∗46, was identified in this study. Preexisting data on an additional 11 Coriell samples, as well as some supplemental testing, were used to create comprehensive reference material panels for TPMT and NUDT15. 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.

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.

Objective: Hmong individuals represent a unique East Asian subpopulation in whom limited information concerning pharmacogenetic variation exists. The objectives of this study were to comprehensively characterize the highly polymorphic CYP2D6 gene in Hmong, estimate allele and phenotype frequencies and to compare results between two testing platforms.Methods: DNA from 48 self-identified Hmong participants were sequenced using a targeted next-generation sequencing (NGS) panel. Star allele calls were made using Astrolabe, manual inspection of NGS variant calls and confirmatory Sanger sequencing. Structural variation was determined by long-range (XL)-PCR and digital droplet PCR (ddPCR). The consensus diplotypes were subsequently translated into phenotype utilizing the activity score system. Clinical grade pharmacogenetic testing was obtained for 12 of the 48 samples enabling an assessment of concordance between the consensus calls and those determined by clinical testing platforms.Results: A total of 13 CYP2D6 alleles were identified. The most common alleles were CYP2D6*10 and its structural arrangements (37.5%, 36/96) and the *5 gene deletion (13.5%, 13/96). Three novel suballeles (*10.007, *36.004, and *75.002) were also identified. Phenotype frequencies were as follows: ultrarapid metabolizers (4.2%, 2/48), normal metabolizers (41.7%, 20/48) and intermediate metabolizers (52.1%, 25/48); none of the 48 participants were predicted to be poor metabolizers. Concordance of diplotype and phenotype calls between the consensus and clinical testing were 66.7 and 50%, respectively.Conclusion: Our study to explore CYP2D6 genotypes in the Hmong population suggests that this subpopulation is unique regarding CYP2D6 allelic variants; also, a higher portion of Hmong participants (50%) are predicted to have an intermediate metabolizer phenotype for CYP2D6 compared to other East Asians which range between 27 and 44%. Results from different testing methods varied considerably. These preliminary findings underscore the importance of thoroughly interrogating unique subpopulations to accurately predict a patient’s CYP2D6 metabolizer status.

Background: The CYP2D6 gene locus has been extensively studied over decades, yet a portion of variability in CYP2D6 activity cannot be explained by known sequence variations within the gene, copy number variation or structural rearrangements. It was proposed that rs5758550 located 116 kb downstream of the CYP2D6 gene locus increases gene expression and thus contributes to variability in CYP2D6 activity. This finding has, however, not been validated. The purpose of the study was to address a major technological barrier, i.e. experimentally linking rs5758550, also referred to as the ‘enhancer’ SNP, to CYP2D6 haplotypes >100 kb away. To overcome this challenge is essential to ultimately determine the contribution of the ‘enhancer’ SNP to interindividual variability in CYP2D6 activity. Methods: A large ethnically mixed population sample (n=3162) was computationally phased to determine linkage between the ‘enhancer’ SNP and CYP2D6 haplotypes (or star alleles). To experimentally validate predicted linkages, DropPhase2D6, a digital droplet PCR (ddPCR)-based method was developed. 10X Genomics Linked-Reads were utilized as a proof of concept. Results: Phasing predicted that the ‘enhancer’ SNP can occur on numerous CYP2D6 haplotypes including CYP2D6 *1, *2, *5 and *41 and suggested that linkage is incomplete, i.e. a portion of these alleles do not have the ‘enhancer’ SNP. Phasing also revealed differences amongst the European and African ancestry data sets regarding the proportion of alleles with and without the ‘enhancer’ SNP. DropPhase2D6 was utilized to confirm or refute the predicted ‘enhancer’ SNP location for individual samples, e.g. of n=3 samples genotyped as *1/*41, rs5758550 was on the *41 allele of two samples and on *1 allele of one sample. Our findings highlights that the location of the ‘enhancer’ SNP must not be assigned by ‘default’. Furthermore, linkage between the ‘enhancer’ SNP and CYP2D6 star allele haplotypes was confirmed with 10X Genomics technology. We were unable, however, to verify selected haplotypes predicted by Ray et al (PMID 30520769). Conclusions: Since the ‘enhancer’ SNP can be present on a portion of normal, decreased, or no function alleles, the phase of the ‘enhancer’ SNP must be considered when investigating the impact of the ‘enhancer’ SNP on CYP2D6 activity.

Pharmacogenetic testing increasingly is available from clinical and research laboratories. However, only a limited number of quality control and other reference materials currently are available for the complex rearrangements and rare variants that occur in the CYP2D6 gene. To address this need, the Division of Laboratory Systems, CDC-based Genetic Testing Reference Material Coordination Program, in collaboration with members of the pharmacogenetic testing and research communities and the Coriell Cell Repositories (Camden, NJ), has characterized 179 DNA samples derived from Coriell cell lines. Testing included the recharacterization of 137 genomic DNAs that were genotyped in previous Genetic Testing Reference Material Coordination Program studies and 42 additional samples that had not been characterized previously. DNA samples were distributed to volunteer testing laboratories for genotyping using a variety of commercially available and laboratory-developed tests. These publicly available samples will support the quality-assurance and quality-control programs of clinical laboratories performing CYP2D6 testing.

In Neurospora, genes not paired during meiosis are targeted by meiotic silencing by unpaired DNA (MSUD). Here, our bimolecular fluorescence complementation (BiFC) study suggests that RNA-directed RNA polymerase, Dicer, Argonaute, and others form a silencing complex in the perinuclear region, with intimate interactions among the majority of them. We have also shown that SAD-2 is likely the anchor for this assembly.

Meiotic silencing by unpaired DNA (MSUD) is a process that detects unpaired regions between homologous chromosomes and silences them for the duration of sexual development. While the phenomenon of MSUD is well recognized, the process that detects unpaired DNA is poorly understood. In this report, we provide two lines of evidence linking unpaired DNA detection to a physical search for DNA homology. First, we have found that a putative SNF2-family protein (SAD-6) is required for efficient MSUD in Neurospora crassa. SAD-6 is closely related to Rad54, a protein known to facilitate key steps in the repair of double-strand breaks by homologous recombination. Second, we have successfully masked unpaired DNA by placing identical transgenes at slightly different locations on homologous chromosomes. This masking falls apart when the distance between the transgenes is increased. We propose a model where unpaired DNA detection during MSUD is achieved through a spatially constrained search for DNA homology. The identity of SAD-6 as a Rad54 paralog suggests that this process may be similar to the searching mechanism used during homologous recombination.

During meiosis in the filamentous fungus Neurospora crassa, unpaired genes are identified and silenced by a process known as meiotic silencing by unpaired DNA (MSUD). Previous work has uncovered six proteins required for MSUD, all of which are also essential for meiotic progression. Additionally, they all localize in the perinuclear region, suggesting that it is a center of MSUD activity. Nevertheless, at least a subset of MSUD proteins must be present inside the nucleus, as unpaired DNA recognition undoubtedly takes place there. In this study, we identified and characterized two new proteins required for MSUD, namely SAD-4 and SAD-5. Both are previously uncharacterized proteins specific to Ascomycetes, with SAD-4 having a range that spans several fungal classes and SAD-5 seemingly restricted to a single order. Both genes appear to be predominantly expressed in the sexual phase, as molecular study combined with analysis of publicly available mRNA-seq datasets failed to detect significant expression of them in the vegetative tissue. SAD-4, like all known MSUD proteins, localizes in the perinuclear region of the meiotic cell. SAD-5, on the other hand, is found in the nucleus (as the first of its kind). Both proteins are unique compared to previously identified MSUD proteins in that neither is required for sexual sporulation. This homozygous-fertile phenotype uncouples MSUD from sexual development and allows us to demonstrate that both SAD-4 and SAD-5 are important for the production of masiRNAs, which are the small RNA molecules associated with meiotic silencing.