scholarly journals Methodology for clinical genotyping of CYP2D6 and CYP2C19

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Beatriz Carvalho Henriques ◽  
Avery Buchner ◽  
Xiuying Hu ◽  
Yabing Wang ◽  
Vasyl Yavorskyy ◽  
...  
Keyword(s):  
Copy Number Variant ◽  
Cytochrome P450 Enzymes ◽  
Clinical Implementation ◽  
Clinical Workflow ◽  
Single Nucleotide ◽  
Chain Reactions ◽  
Polymerase Chain Reactions ◽  
Therapeutic Drugs ◽  
Automated Translation ◽  
Polymerase Chain

AbstractMany antidepressants, atomoxetine, and several antipsychotics are metabolized by the cytochrome P450 enzymes CYP2D6 and CYP2C19, and guidelines for prescribers based on genetic variants exist. Although some laboratories offer such testing, there is no consensus regarding validated methodology for clinical genotyping of CYP2D6 and CYP2C19. The aim of this paper was to cross-validate multiple technologies for genotyping CYP2D6 and CYP2C19 against each other, and to contribute to feasibility for clinical implementation by providing an enhanced range of assay options, customizable automated translation of data into haplotypes, and a workflow algorithm. AmpliChip CYP450 and some TaqMan single nucleotide variant (SNV) and copy number variant (CNV) data in the Genome-based therapeutic drugs for depression (GENDEP) study were used to select 95 samples (out of 853) to represent as broad a range of CYP2D6 and CYP2C19 genotypes as possible. These 95 included a larger range of CYP2D6 hybrid configurations than have previously been reported using inter-technology data. Genotyping techniques employed were: further TaqMan CNV and SNV assays, xTAGv3 Luminex CYP2D6 and CYP2C19, PharmacoScan, the Ion AmpliSeq Pharmacogenomics Panel, and, for samples with CYP2D6 hybrid configurations, long-range polymerase chain reactions (L-PCRs) with Sanger sequencing and Luminex. Agena MassARRAY was also used for CYP2C19. This study has led to the development of a broader range of TaqMan SNV assays, haplotype phasing methodology with TaqMan adaptable for other technologies, a multiplex genotyping method for efficient identification of some hybrid haplotypes, a customizable automated translation of SNV and CNV data into haplotypes, and a clinical workflow algorithm.

scholarly journals Validation of methodology for efficient genotyping of CYP2D6 and CYP2C19

2021 ◽  
Author(s):  
Beatriz Carvalho Henriques ◽  
Amanda Buchner ◽  
Xiuying Hu ◽  
Yabing Wang ◽  
Vasyl Yavorskyy ◽  
...  
Keyword(s):  
Sanger Sequencing ◽  
Copy Number Variant ◽  
Large Datasets ◽  
Sequencing Data ◽  
Single Nucleotide ◽  
Chain Reactions ◽  
Polymerase Chain Reactions ◽  
Polymerase Chain ◽  
Consensus Genotypes ◽  
Hybrid Genes

Abstract There are some data associating variants in the CYP2D6 and/or CYP2C19 genes with concentration-to-dose ratios, efficacy, and retention in treatments. However, much of the above arises from relatively small studies or large datasets with limited genotyping methodologies. Our aim was to develop and validate comprehensive and accurate genotyping methodology for these two genes to facilitate regenotyping in large datasets and hence the generation of more accurate clinical associations. TaqMan copy number variant (CNV) assays for CYP2D6 were used to identify samples from a relevant large dataset (GENDEP study, N = 853) with particularly challenging genotypes to call. These and those representing as broad a range of CYP2D6 and CYP2C19 genotypes as possible by prior available data (AmpliChip CYP450 and TaqMan CYP2C19*17) were chosen for further analysis (N = 96). Genotyping techniques employed were: Luminex CYP2D6 xTAGv3 and Luminex CYP2C19 xTAGv3, PharmacoScan, the Ion S5 AmpliSeq Pharmacogenomics Panel, TaqMan single nucleotide variant (SNV) assays, and, for the CYP2D6 hybrids, long-range polymerase chain reactions (L-PCRs) with Sanger sequencing. Agena was also used for CYP2C19. The TaqMan SNV assays were able to assist with identifying which gene was duplicated or in tandem for multiple copy variants. A multiplex assay was adaptable for analysis of CYP2D6 hybrid genes, with Sanger sequencing data being consistent with the data arising; we provide these data for efficient genotyping of such CYP2D6 hybrid genes with adaptable multiplex methods. Consensus genotypes generated to date resulted in revision of assigned enzyme activity score for 28/96(29%) and 2/93 samples (2.2%) for CYP2D6 and CYP2C19, respectively.

scholarly journals Cross-validation of technologies for genotyping CYP2D6 and CYP2C19

2019 ◽  
Author(s):  
Beatriz Carvalho Henriques ◽  
Amanda Buchner ◽  
Xiuying Hu ◽  
Vasyl Yavorskyy ◽  
Yabing Wang ◽  
...  
Keyword(s):  
Copy Number Variant ◽  
Data Interpretation ◽  
Gene Duplications ◽  
Cytochrome P450 Enzymes ◽  
Extra Copy ◽  
Single Nucleotide ◽  
Therapeutic Drugs ◽  
Two Samples ◽  
Genes Encoding ◽  
Polymerase Chain

AbstractBackgroundCYP2D6 and CYP2C19 are cytochrome P450 enzymes involved in the metabolism of many medications from multiple therapeutic classes. Associations between patterns of variants (known as haplotypes) in the genes encoding them (CYP2D6 and CYP2C19) and enzyme activities are well described. The genes in fact comprise 21% of biomarkers in drug labels. Despite this, genotyping is not common, partly attributable to its challenging nature (CYP2D6 having >100 haplotypes, including those with sequence from an adjacent pseudogene, and gene duplications). We cross-validated different methodologies for identifying haplotypes in these genes against each other.MethodsNinety-two samples with a variety of CYP2D6 and CYP2C19 genotypes according to prior AmpliChip CYP450 and TaqMan CYP2C19*17 data were selected from the Genome-based therapeutic drugs for depression (GENDEP) study. Genotyping was performed with TaqMan copy number variant (CNV) and single nucleotide variant (SNV) analysis, the next generation sequencing-based Ion S5 AmpliSeq Pharmacogenomics Panel, PharmacoScan, long-range polymerase chain reaction (L-PCR) followed by amplicon analysis, and Agena for CYP2C19. Variant pattern to haplotype translation was automated.ResultsThe inter-platform concordance for CYP2C19 was high (up to 100% for available data). For CYP2D6, the IonS5-PharmacoScan concordance was 94% for a range of variants tested apart from those with at least one extra copy of a CYP2D6 gene (occurring at a frequency of 3.8%, 33/853), or those with substantial sequence derived from pseudogene, known as hybrids (3%, 26/853).ConclusionsInter-platform concordance for CYP2C19 was high, and, moreover, the Ion S5 and PharmacoScan data were 100% concordant with that from a TaqMan CYP2C19*2 assay. We have also demonstrated feasibility of using an NGS platform for genotyping CYP2D6 and CYP2C19, with automated data interpretation methodology. This points the way to a method of making CYP2D6 and CYP2C19 genotyping more readily accessible.

BCL10 mutation does not represent an important pathogenic mechanism in gastric MALT-type lymphoma, and the presence of the API2-MLT fusion is associated with aberrant nuclear BCL10 expression

Blood ◽  
2002 ◽  
Vol 99 (4) ◽  
pp. 1398-1404 ◽  
Author(s):  
Brigitte Maes ◽  
Anouk Demunter ◽  
Benjamin Peeters ◽  
Christiane De Wolf-Peeters
Keyword(s):  
Fusion Protein ◽  
Direct Sequencing ◽  
Nucleotide Substitutions ◽  
Single Nucleotide ◽  
Chain Reactions ◽  
Polymerase Chain Reactions ◽  
Tissue Sections ◽  
Nuclear Expression ◽  
Polymerase Chain ◽  
Malt Type Lymphoma

Two recurrent translocations have been associated with mucosa-associated lymphoid tissue (MALT)–type lymphoma, t(11;18)(q21;q21) and t(1;14)(p22;q32). The first, t(11;18)(q21;q21), results in the fusion protein API2-MLT (API2-MALT1). Through t(1;14)(p22;q32), the BCL10 gene is entirely transferred to the IgH gene, resulting in its overexpression. Wild-type BCL10 is implicated in apoptosis, and it has been suggested that mutated forms gain oncogenic activity. The occurrence of genomicBCL10 mutations in 35 gastric MALT-type lymphomas with or without t(11;18)(q21;q21) (10 and 25 cases, respectively) was investigated. DNA extracted from either whole tissue sections or microdissected clusters of tumor cells was used. Five polymerase chain reactions amplifying the coding exons were performed and were followed by direct sequencing of the products. Twenty differences with the published BCL10 sequence, all single nucleotide substitutions, were detected in 16 cases. Of these, 12 represented known polymorphisms, either at codon 8, 213, or 5. Of the remaining 8 substitutions, 2 were silent and 6 resulted in amino acid substitutions. Mutation analysis results were correlated with the BCL10 expression pattern. Aberrant nuclear BCL10 expression was detected in 14 cases. No association could be demonstrated between the latter and the presence of BCL10 mutations. In contrast, all 10 cases carrying t(11;18)(q21;q21) showed nuclear expression, whereas this staining pattern was absent in 21 of 25 cases without t(11;18)(q21;q21). These results demonstrate that BCL10mutations are rare in gastric MALT-type lymphoma and are not related to the aberrant nuclear expression of BCL10. In contrast, they indicate that the presence of the API2-MLT fusion protein is associated with aberrant nuclear BCL10 expression.

scholarly journals Investigation of Lethal Concurrent Outbreak of Chlamydiosis and Pigeon Circovirus in a Zoo

Animals ◽  
2021 ◽  
Vol 11 (6) ◽  
pp. 1654
Author(s):  
Wei-Tao Chen ◽  
Chin-Ann Teng ◽  
Cheng-Hsin Shih ◽  
Wei-Hsiang Huang ◽  
Yi-Fan Jiang ◽  
...  
Keyword(s):  
Disease Outbreaks ◽  
Chlamydia Psittaci ◽  
Bursa Of Fabricius ◽  
Chain Reactions ◽  
Renal Epithelium ◽  
Polymerase Chain Reactions ◽  
Transmission Electron ◽  
Intracytoplasmic Inclusion Bodies ◽  
Polymerase Chain ◽  
Streptopelia Orientalis

During the spring, an outbreak of sudden death involving 58 birds occurred in a zoo. Histopathological examinations revealed variable numbers of intracytoplasmic basophilic microorganisms in the macrophages, hepatocytes, and renal epithelium of most birds, along with occasional botryoid intracytoplasmic inclusion bodies within histiocytes in the bursa of Fabricius. Based on the results of histopathological examinations, immunohistochemical staining, transmission electron microscopy, and polymerase chain reactions, genotype B Chlamydia psittaci infection concurrent with pigeon circovirus (PiCV) was diagnosed. A retrospective survey, including two years before the outbreak and the outbreak year, of C. psittaci and PiCV infections of dead birds in the aviaries, revealed that the outbreak was an independent episode. The findings of this study indicate that concurrent infection with C. psittaci and PiCV might lead to lethal outbreaks of chlamydiosis, particularly Streptopelia orientalis. In addition, persistently monitoring both pathogens and identifying potential PiCV carriers or transmitters might also help prevent lethal disease outbreaks.

Sign in / Sign up

Export Citation Format

Share Document