PharmVar Tutorial on CYP2D6 Structural Variation Testing and Recommendations on Reporting.
Journal
Clinical pharmacology and therapeutics
ISSN: 1532-6535
Titre abrégé: Clin Pharmacol Ther
Pays: United States
ID NLM: 0372741
Informations de publication
Date de publication:
Dec 2023
Dec 2023
Historique:
received:
16
06
2023
accepted:
23
08
2023
pubmed:
5
9
2023
medline:
5
9
2023
entrez:
5
9
2023
Statut:
ppublish
Résumé
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.
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Pagination
1220-1237Subventions
Organisme : NHGRI NIH HHS
ID : U24 HG010135
Pays : United States
Organisme : NHGRI NIH HHS
ID : U24 HG010615
Pays : United States
Organisme : NIH HHS
ID : U24HG010135
Pays : United States
Organisme : NIH HHS
ID : U24 HG010615
Pays : United States
Organisme : NIH HHS
ID : U24HG010135
Pays : United States
Organisme : NIH HHS
ID : U24 HG010615
Pays : United States
Informations de copyright
© 2023 The Authors. Clinical Pharmacology & Therapeutics © 2023 American Society for Clinical Pharmacology and Therapeutics.
Références
Taylor, C., Crosby, I., Yip, V., Maguire, P., Pirmohamed, M. & Turner, R.M. A review of the important role of CYP2D6 in pharmacogenomics. Genes (Basel) 11, 1295 (2020).
Saravanakumar, A., Sadighi, A., Ryu, R. & Akhlaghi, F. Physicochemical properties, biotransformation, and transport pathways of established and newly approved medications: a systematic review of the top 200 most prescribed drugs vs. the FDA-approved drugs between 2005 and 2016. Clin. Pharmacokinet. 58, 1281-1294 (2019).
Caudle, K.E. et al. Standardizing CYP2D6 genotype to phenotype translation: consensus recommendations from the clinical pharmacogenetics implementation consortium and Dutch pharmacogenetics working group. Clin. Transl. Sci. 13, 116-124 (2020).
Whirl-Carrillo, M. et al. An evidence-based framework for evaluating pharmacogenomics knowledge for personalized medicine. Clin. Pharmacol. Ther. 110, 563-572 (2021).
Nofziger, C. et al. PharmVar GeneFocus: CYP2D6. Clin. Pharmacol. Ther. 107, 154-170 (2020).
Jarvis, J.P., Peter, A.P. & Shaman, J.A. An evidence-based framework for evaluating pharmacogenomics knowledge for personalized medicine. Front. Psych. 10, 432 (2019).
Bousman, C.A. et al. Clinical pharmacogenetics implementation consortium (CPIC) guideline for CYP2D6, CYP2C19, CYP2B6, SLC6A4, and HTR2A genotypes and serotonin reuptake inhibitor antidepressants. Clin. Pharmacol. Ther. 114, 51-68 (2023).
Hicks, J.K. et al. Clinical pharmacogenetics implementation consortium guideline (CPIC) for CYP2D6 and CYP2C19 genotypes and dosing of tricyclic antidepressants: 2016 update. Clin. Pharmacol. Ther. 102, 37-44 (2017).
Crews, K.R. et al. Clinical pharmacogenetics implementation consortium guideline for CYP2D6, OPRM1, and COMT genotypes and select opioid therapy. Clin. Pharmacol. Ther. 110, 888-896 (2021).
Goetz, M.P. et al. Clinical pharmacogenetics implementation consortium (CPIC) guideline for CYP2D6 and tamoxifen therapy. Clin. Pharmacol. Ther. 103, 770-777 (2018).
Brown, J.T. et al. Clinical pharmacogenetics implementation consortium guideline for cytochrome P450 (CYP) 2D6 genotype and atomoxetine therapy. Clin. Pharmacol. Ther. 106, 94-102 (2019).
Bell, G.C. et al. Clinical pharmacogenetics implementation consortium (CPIC) guideline for CYP2D6 genotype and use of ondansetron and tropisetron. Clin. Pharmacol. Ther. 102, 213-218 (2017).
Kane, M. & Dean, L. Eliglustat therapy and CYP2D6 genotype. In Medical Genetics Summaries (eds. Pratt, V.M., Scott, S.A., Pirmohamed, M., Esquivel, B., Kattman, B.L. & Malheiro, A.J.) (National Center for Biotechnology Information, Bethesda, MD, 2012).
PharmGKB. Annotation of FDA Label for tetrabenazine and CYP2D6. <https://www.pharmgkb.org/labelAnnotation/PA166104829>.
PharmGKB. Annotation of FDA Label for pimozide and CYP2D6. <https://www.pharmgkb.org/labelAnnotation/PA166104870>.
Lenk, H.C. et al. The polymorphic nuclear factor NFIB regulates hepatic CYP2D6 expression and influences risperidone metabolism in psychiatric patients. Clin. Pharmacol. Ther. 111, 1165-1174 (2022).
Boone, E.C. et al. Long-distance phasing of a tentative "enhancer" single-nucleotide polymorphism with CYP2D6 star allele definitions. Front. Pharmacol. 11, 486 (2020).
Dinh, J.C. et al. The impact of the CYP2D6 "enhancer" single nucleotide polymorphism on CYP2D6 activity. Clin. Pharmacol. Ther. 111, 646-654 (2022).
Khor, C.C. et al. Cross-ancestry genome-wide association study defines the extended CYP2D6 locus as the principal genetic determinant of endoxifen plasma concentrations. Clin. Pharmacol. Ther. 113, 712-723 (2023).
Sanchez-Spitman, A.B., Moes, D.A., Gelderblom, H., Dezentje, V.O., Swen, J.J. & Guchelaar, H.J. The effect of rs5758550 on CYP2D6*2 phenotype and formation of endoxifen in breast cancer patients using tamoxifen. Pharmacogenomics 18, 1125-1132 (2017).
Wang, D., Poi, M.J., Sun, X., Gaedigk, A., Leeder, J.S. & Sadee, W. Common CYP2D6 polymorphisms affecting alternative splicing and transcription: long-range haplotypes with two regulatory variants modulate CYP2D6 activity. Hum. Mol. Genet. 23, 268-278 (2014).
Brunetti-Pierri, N., Patel, A., Brown, C.W., Rauch, R.A. & Heptulla, R.A. De novo terminal 22q12.3q13.3 duplication with pituitary hypoplasia. Am. J. Med. Genet. A 149A, 2554-2556 (2009).
Upadia, J. et al. A previously unrecognized 22q13.2 microdeletion syndrome that encompasses TCF20 and TNFRSF13C. Am. J. Med. Genet. A 176, 2791-2797 (2018).
Dyar, B., Meaddough, E., Sarasua, S.M., Rogers, C., Phelan, K. & Boccuto, L. Enetic findings as the potential basis of personalized pharmacotherapy in Phelan-McDermid syndrome. Genes (Basel) 12, 1192 (2021).
Ricciardello, A., Tomaiuolo, P. & Persico, A.M. Genotype-phenotype correlation in Phelan-McDermid syndrome: a comprehensive review of chromosome 22q13 deleted genes. Am. J. Med. Genet. A 185, 2211-2233 (2021).
PharmVar. CYP2D6 gene page. <https://www.pharmvar.org/gene/CYP2D6>.
Kimura, S., Umeno, M., Skoda, R.C., Meyer, U.A. & Gonzalez, F.J. The human debrisoquine 4-hydroxylase (CYP2D) locus: sequence and identification of the polymorphic CYP2D6 gene, a related gene, and a pseudogene. Am. J. Hum. Genet. 45, 889-904 (1989).
Gaedigk, A., Blum, M., Gaedigk, R., Eichelbaum, M. & Meyer, U.A. Deletion of the entire cytochrome P450 CYP2D6 gene as a cause of impaired drug metabolism in poor metabolizers of the debrisoquine/sparteine polymorphism. Am. J. Hum. Genet. 48, 943-950 (1991).
Gaedigk, A., Bradford, L.D., Alander, S.W. & Leeder, J.S. CYP2D6*36 gene arrangements within the CYP2D6 locus: association of CYP2D6*36 with poor metabolizer status. Drug Metab. Dispos. 34, 563-569 (2006).
Gaedigk, A. & Coetsee, C. The CYP2D6 gene locus in south African Coloureds: unique allele distributions, novel alleles and gene arrangements. Eur. J. Clin. Pharmacol. 64, 465-475 (2008).
Gaedigk, A., Fuhr, U., Johnson, C., Berard, L.A., Bradford, D. & Leeder, J.S. CYP2D7-2D6 hybrid tandems: identification of novel CYP2D6 duplication arrangements and implications for phenotype prediction. Pharmacogenomics 11, 43-53 (2010).
Gaedigk, A. et al. Identification of novel CYP2D7-2D6 hybrids: non-functional and functional variants. Front. Pharmacol. 1, 121 (2010).
Gaedigk, A. et al. Cytochrome P4502D6 (CYP2D6) gene locus heterogeneity: characterization of gene duplication events. Clin. Pharmacol. Ther. 81, 242-251 (2007).
Kramer, W.E. et al. CYP2D6: novel genomic structures and alleles. Pharmacogenet. Genomics 19, 813-822 (2009).
Steen, V.M., Molven, A., Aarskog, N.K. & Gulbrandsen, A.K. Homologous unequal cross-over involving a 2.8 kb direct repeat as a mechanism for the generation of allelic variants of human cytochrome P450 CYP2D6 gene. Hum. Mol. Genet. 4, 2251-2257 (1995).
Lovlie, R., Daly, A.K., Molven, A., Idle, J.R. & Steen, V.M. Ultrarapid metabolizers of debrisoquine: characterization and PCR-based detection of alleles with duplication of the CYP2D6 gene. FEBS Lett. 392, 30-34 (1996).
PharmVar. CYP2D6 structural variation document. <https://www.pharmvar.org/gene/CYP2D6>.
Chen, X. et al. Cyrius: accurate CYP2D6 genotyping using whole-genome sequencing data. Pharmacogenomics J. 21, 251-261 (2021).
Turner, A.J. et al. Characterization of complex structural variation in the CYP2D6-CYP2D7-CYP2D8 gene loci using single-molecule long-read sequencing. Front. Pharmacol. 14, 1195778 (2023).
Riffel, A.K. et al. CYP2D7 sequence variation interferes with TaqMan CYP2D6*15 and *35 genotyping. Front. Pharmacol. 6, 312 (2016).
Del Tredici, A.L. et al. Frequency of CYP2D6 alleles including structural variants in the United States. Front. Pharmacol. 9, 305 (2018).
Black, J.L., Walker, D.L., O'Kane, D.J. & Harmandayan, M. Frequency of undetected CYP2D6 hybrid genes in clinical samples: impact on phenotype prediction. Drug Metab. Dispos. 40, 111-119 (2012).
Gaedigk, A., Sangkuhl, K., Whirl-Carrillo, M., Klein, T. & Leeder, J.S. Prediction of CYP2D6 phenotype from genotype across world populations. Genet. Med. 19, 69-76 (2017).
Hosono, N. et al. CYP2D6 genotyping for functional-gene dosage analysis by allele copy number detection. Clin. Chem. 55, 1546-1554 (2009).
Chan, W., Li, M.S., Sundaram, S.K., Tomlinson, B., Cheung, P.Y. & Tzang, C.H. CYP2D6 allele frequencies, copy number variants, and tandems in the population of Hong Kong. J. Clin. Lab. Anal. 33, e22634 (2019).
Wen, Y.F., Gaedigk, A., Boone, E.C., Wang, W.Y. & Straka, R.J. The identification of novel CYP2D6 variants in US Hmong: results from genome sequencing and clinical genotyping. Front. Pharmacol. 13, 867331 (2022).
PharmGKB. Gene-specific information tables for CYP2D6. <https://www.pharmgkb.org/page/cyp2d6RefMaterials>.
Collins, R.L. et al. A structural variation reference for medical and population genetics. Nature 581, 444-451 (2020).
National Library of Medicine, National Center for Biotechnology Information (NCBI). Overview of structural variation. <https://www.ncbi.nlm.nih.gov/dbvar/content/overview/>.
Gaedigk, A., Simon, S.D., Pearce, R.E., Bradford, L.D., Kennedy, M.J. & Leeder, J.S. The CYP2D6 activity score: translating genotype information into a qualitative measure of phenotype. Clin. Pharmacol. Ther. 83, 234-242 (2008).
Johansson, I., Lundqvist, E., Bertilsson, L., Dahl, M.L., Sjoqvist, F. & Ingelman-Sundberg, M. Inherited amplification of an active gene in the cytochrome P450 CYP2D locus as a cause of ultrarapid metabolism of debrisoquine. Proc. Natl. Acad. Sci. U. S. A. 90, 11825-11829 (1993).
Dahl, M.L., Johansson, I., Palmertz, M.P., Ingelman-Sundberg, M. & Sjoqvist, F. Analysis of the CYP2D6 gene in relation to debrisoquin and desipramine hydroxylation in a Swedish population. Clin. Pharmacol. Ther. 51, 12-17 (1992).
Bertilsson, L. et al. Molecular basis for rational megaprescribing in ultrarapid hydroxylators of debrisoquine. Lancet 341, 63 (1993).
Aklillu, E., Persson, I., Bertilsson, L., Johansson, I., Rodrigues, F. & Ingelman-Sundberg, M. Frequent distribution of ultrarapid metabolizers of debrisoquine in an ethiopian population carrying duplicated and multiduplicated functional CYP2D6 alleles. J. Pharmacol. Exp. Ther. 278, 441-446 (1996).
McLellan, R.A., Oscarson, M., Seidegard, J., Evans, D.A. & Ingelman-Sundberg, M. Frequent occurrence of CYP2D6 gene duplication in Saudi Arabians. Pharmacogenetics 7, 187-191 (1997).
Panserat, S. et al. An unequal cross-over event within the CYP2D gene cluster generates a chimeric CYP2D7/CYP2D6 gene which is associated with the poor metabolizer phenotype. Br. J. Clin. Pharmacol. 40, 361-367 (1995).
Sim, S.C., Daly, A.K. & Gaedigk, A. CYP2D6 update: revised nomenclature for CYP2D7/2D6 hybrid genes. Pharmacogenet. Genomics 22, 692-694 (2012).
Bruford, E.A. et al. HUGO gene nomenclature committee (HGNC) recommendations for the designation of gene fusions. Leukemia 35, 3040-3043 (2021).
Pratt, V.M. et al. Recommendations for clinical CYP2D6 genotyping allele selection: a joint consensus recommendation of the association for molecular pathology, College of American Pathologists, Dutch pharmacogenetics working Group of the Royal Dutch Pharmacists Association, and the European Society for Pharmacogenomics and Personalized Therapy. J. Mol. Diagn. 23, 1047-1064 (2021).
Langaee, T., Hamadeh, I., Chapman, A.B., Gums, J.G. & Johnson, J.A. A novel simple method for determining CYP2D6 gene copy number and identifying allele(s) with duplication/multiplication. PloS One 10, e0113808 (2015).
Hosono, N. et al. Multiplex PCR-based real-time invader assay (mPCR-RETINA): a novel SNP-based method for detecting allelic asymmetries within copy number variation regions. Hum. Mutat. 29, 182-189 (2008).
Gaedigk, A., Twist, G.P. & Leeder, J.S. CYP2D6, SULT1A1 and UGT2B17 copy number variation: quantitative detection by multiplex PCR. Pharmacogenomics 13, 91-111 (2012).
Scantamburlo, G. et al. Allele drop uut conferred by a frequent CYP2D6 genetic variation for commonly used CYP2D6*3 genotyping assays. Cell. Physiol. Biochem. 43, 2297-2309 (2017).
Gaedigk, A. et al. Characterization of reference materials for genetic testing of CYP2D6 alleles: a GeT-RM collaborative project. J. Mol. Diagn. 21, 1034-1052 (2019).
Wang, W.Y. et al. Characterization of novel CYP2D6 alleles across sub-Saharan African populations. J. Pers. Med. 12, 1575 (2022).
Schmittgen, T.D. & Livak, K.J. Analyzing real-time PCR data by the comparative C(T) method. Nat. Protoc. 3, 1101-1108 (2008).
Whale, A.S., Huggett, J.F. & Tzonev, S. Fundamentals of multiplexing with digital PCR. Biomol. Detect. Quantif. 10, 15-23 (2016).
Rae, J.M. et al. Concordance between CYP2D6 genotypes obtained from tumor-derived and germline DNA. J. Natl. Cancer Inst. 105, 1332-1334 (2013).
Hindson, C.M. et al. Absolute quantification by droplet digital PCR versus analog real-time PCR. Nat. Methods 10, 1003-1005 (2013).
Dingle, T.C., Sedlak, R.H., Cook, L. & Jerome, K.R. Tolerance of droplet-digital PCR vs real-time quantitative PCR to inhibitory substances. Clin. Chem. 59, 1670-1672 (2013).
Turner, A.J. et al. Identification of CYP2D6 haplotypes that interfere with commonly used assays for copy number variation characterization. J. Mol. Diagn. 23, 577-588 (2021).
Sicko, R.J. et al. Rare variants in RPPH1 real-time quantitative PCR control assay binding sites result in incorrect copy number calls. J. Mol. Diagn. 24, 33-40 (2022).
Atiq, M.A., Peterson, S.E., Langman, L.J., Baudhuin, L.M., Black, J.L. & Moyer, A.M. Determination of the duplicated CYP2D6 allele using real-time PCR signal: an alternative approach. J. Pers. Med. 13, 883 (2023).
Soderback, E., Zackrisson, A.L., Lindblom, B. & Alderborn, A. Determination of CYP2D6 gene copy number by pyrosequencing. Clin. Chem. 51, 522-531 (2005).
Agena Bioscience. VeriDose CYP2D6 CNV panel. <https://www.agenabio.com/products/panel/veridose-cyp2d6-cnv-panel/>.
Everts, R.E., Lois, A., Nakorchevsky, A., Tao, H., Jackson, K. & Rhodes, K. The VeriDose CYP2D6 CNV Panel: A one-well solution for copy number and hybrid allele detection of CYP2D6. 21 1124-5 (2019).
Qiao, W. et al. Integrated CYP2D6 interrogation for multiethnic copy number and tandem allele detection. Pharmacogenomics 20, 9-20 (2019).
Luminex Corporation. Luminex xTAG. <https://www.luminexcorp.com/>.
Thermo Fisher Scientific. PharmacoScan™ Assay Kit. <https://www.thermofisher.com/order/catalog/product/903026>.
Illumina Inc. The Infinium Global Array with Enhanced PGx. <https://www.illumina.com/products/by-type/microarray-kits/infinium-global-diversity-pgx.html>.
Illumina Inc. HiFi Amplicon Sequencing for Pharmacogenetics: CYP2D6. <https://www.pacb.com/wp-content/uploads/Application-note-HiFi-amplicon-sequencing-for-pharmacogenetics-CYP2D6.pdf>.
Buermans, H.P. et al. Flexible and scalable full-length CYP2D6 long amplicon PacBio sequencing. Hum. Mutat. 38, 310-316 (2017).
Charnaud, S. et al. PacBio long-read amplicon sequencing enables scalable high-resolution population allele typing of the complex CYP2D6 locus. Commun. Biol. 5, 168 (2022).
Caspar, S.M., Schneider, T., Stoll, P., Meienberg, J. & Matyas, G. Potential of whole-genome sequencing-based pharmacogenetic profiling. Pharmacogenomics 22, 177-190 (2021).
Liau, Y., Maggo, S., Miller, A.L., Pearson, J.F., Kennedy, M.A. & Cree, S.L. Nanopore sequencing of the pharmacogene CYP2D6 allows simultaneous haplotyping and detection of duplications. Pharmacogenomics 20, 1033-1047 (2019).
Hari, A. et al. An efficient genotyper and star-allele caller for pharmacogenomics. Genome Res. 33, 61-70 (2023).
Twesigomwe, D. et al. StellarPGx: a Nextflow pipeline for calling star alleles in cytochrome P450 genes. Clin. Pharmacol. Ther. 110, 741-749 (2021).
Lee, S.B. et al. Stargazer: a software tool for calling star alleles from next-generation sequencing data using CYP2D6 as a model. Genet. Med. 21, 361-372 (2019).
Lee, S.B., Shin, J.Y., Kwon, N.J., Kim, C. & Seo, J.S. ClinPharmSeq: a targeted sequencing panel for clinical pharmacogenetics implementation. PloS One 17, e0272129 (2022).
Twesigomwe, D., Wright, G.E.B., Drogemoller, B.I., da Rocha, J., Lombard, Z. & Hazelhurst, S. A systematic comparison of pharmacogene star allele calling bioinformatics algorithms: a focus on CYP2D6 genotyping. NPJ Genom. Med. 5, 30 (2020).
Belkadi, A. et al. Whole-genome sequencing is more powerful than whole-exome sequencing for detecting exome variants. Proc. Natl. Acad. Sci. U. S. A. 112, 5473-5478 (2015).
Ng, D., Hong, C.S., Singh, L.N., Johnston, J.J., Mullikin, J.C. & Biesecker, L.G. Assessing the capability of massively parallel sequencing for opportunistic pharmacogenetic screening. Genet. Med. 19, 357-361 (2017).
Yang, Y., Botton, M.R., Scott, E.R. & Scott, S.A. Sequencing the CYP2D6 gene: from variant allele discovery to clinical pharmacogenetic testing. Pharmacogenomics 18, 673-685 (2017).
van der Lee, M. et al. Repurposing of diagnostic whole exome sequencing data of 1,583 individuals for clinical pharmacogenetics. Clin. Pharmacol. Ther. 107, 617-627 (2020).
Lanillos, J., Carcajona, M., Maietta, P., Alvarez, S. & Rodriguez-Antona, C. Clinical pharmacogenetic analysis in 5,001 individuals with diagnostic exome sequencing data. NPJ Genom. Med. 7, 12 (2022).
Tafazoli, A. et al. Development of an extensive workflow for comprehensive clinical pharmacogenomic profiling: lessons from a pilot study on 100 whole exome sequencing data. Pharmacogenomics J. 22, 276-283 (2022).
Wang, L. et al. Implementation of preemptive DNA sequence-based pharmacogenomics testing across a large academic medical center: the Mayo-Baylor RIGHT 10K study. Genet. Med. 24, 1062-1072 (2022).
Lopes, J.L. et al. Targeted genotyping in clinical pharmacogenomics: what is missing? J. Mol. Diagn. 24, 253-261 (2022).
Rubben, K., Tilleman, L., Deserranno, K., Tytgat, O., Deforce, D. & Van Nieuwerburgh, F. Cas9 targeted nanopore sequencing with enhanced variant calling improves CYP2D6-CYP2D7 hybrid allele genotyping. PLoS Genet. 18, e1010176 (2022).
Gaedigk, A., Riffel, A.K., Berrocal, B.G., Solaesa, V.G., Davila, I. & Isidoro-Garcia, M. Characterization of a complex CYP2D6 genotype that caused an AmpliChip CYP450 test no-call in the clinical setting. Clin. Chem. Lab. Med. 52, 799-807 (2014).
Moyer, A.M. et al. Genotype and phenotype concordance for pharmacogenetic tests through proficiency survey testing. Arch. Pathol. Lab. Med. 144, 1057-1066 (2020).
Bousman, C.A., Jaksa, P. & Pantelis, C. Systematic evaluation of commercial pharmacogenetic testing in psychiatry: a focus on CYP2D6 and CYP2C19 allele coverage and results reporting. Pharmacogenet. Genomics 27, 387-393 (2017).
National Center for Biotechnology Information (NCBI). GTR: genetic testing registry. <https://www.ncbi.nlm.nih.gov/gtr/>.
Centers for Disease Control and Prevention. Genetic testing reference materials (GeT-RM) coordination program Reference Materials for Pharmacogenetics. <https://www.cdc.gov/labquality/get-rm/inherited-genetic-diseases-pharmacogenetics/pharmacogenetics.html>.