Functional characterization of 40 CYP2B6 allelic variants by assessing efavirenz 8-hydroxylation

Watanabe, Takashi; Saito, Takahiro; Rico, Evelyn Marie Gutiérrez; Hishinuma, Eiji; Kumondai, Masaki; Maekawa, Masamitsu; Oda, Akifumi; Saigusa, Daisuke; Saitō, Sakae; Yasuda, Jun; Nagasaki, Masao; Minegishi, Naoko; Yamamoto, Masayuki; Yamaguchi, Hiroaki; Mano, Nariyasu; Hirasawa, Noriyasu; Hiratsuka, Masahiro

doi:10.1016/j.bcp.2018.09.010

Cited by 18 publications

(29 citation statements)

References 28 publications

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“…For example, the IRUD project (Japan's Initiative on Rare and Undiagnosed Diseases 9 ) used the reference panel to reduce the discovery of false positive single nucleotide variants (SNVs) during the exome analyses of undiagnosed patients. In another project, CYP SNVs included in the reference panels were selected and analyzed systematically for their effect on drug metabolism [10][11][12] . As seen from these examples, the previous versions of the reference panels worked well; however, there are some limitations.…”

Section: Background and Summarymentioning

confidence: 99%

3.5KJPNv2, An allele frequency panel of 3,552 Japanese Individuals

Tadaka

Katsuoka

Ueki

et al. 2019

Preprint

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The first step towards realizing personalized healthcare is to catalog the genetic variations in a population. Since the dissemination of individual-level genomic information is strictly controlled, it will be useful to construct population-level allele frequency panels and to provide them through easy-to-use interfaces.In the Tohoku Medical Megabank Project, we have sequenced nearly 4,000 individuals from a Japanese population, and constructed an allele frequency panel of 3,552 individuals after removing related samples. The panel is called the 3.5KJPNv2. It was constructed by using a standard pipeline including the 1KGP and gnomAD algorithms to reduce technical biases and to allow comparisons to other populations. Our database is the first largescale panel providing the frequencies of variants present on the X chromosome and on the mitochondria in the Japanese population. All the data are available on our original database at https://jmorp.megabank.tohoku.ac.jp. new panel construction by using a pipeline similar to the 1KGP and gnomAD pipelines. We also report the variant frequencies of X chromosome and those of mitochondria, which makes this the first such report to do so on a largescale for the Japanese population. Methods Sample InformationParticipants' declared age, and their sex as determined from X-and Y-chromosome sequencing are presented in Table 1 (b). Samples with irregular karyotypes, such as those with Turner syndrome, were excluded. Close relatives of individual subjects, based on mean identityby-descent (IBD; PIHAT in PLINK version 1.07) values indicating relatedness closer than between third cousins degree relatives, were excluded. Whole-genome sequencingLibrary preparation and sequencing were performed as described earlier with minor modifications 5 . Briefly, genomic DNA extracted from the buffy coat was fragmented by sonication to an average target size of 550 bp. After library quantification using the quantitative MiSeq method 15 , sequencing was performed on the HiSeq 2500 system (Illumina). The TruSeq Rapid PE Cluster V1 and SBS Kits (1 sample per flowcell), and the TruSeq Rapid PE Cluster Kit V2 and SBS Kit (2 samples per flowcell) were used for 162-bp and 259-bp paired-end protocols, respectively.

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Section: Background and Summarymentioning

confidence: 99%

3.5KJPNv2, An allele frequency panel of 3,552 Japanese Individuals

Tadaka

Katsuoka

Ueki

et al. 2019

Preprint

Self Cite

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“…With other substrates, CYP2B6.4 was more active than CYP2B6.1 toward methadone (Gadel et al, 2013(Gadel et al, , 2015 and artmether (Honda et al, 2011) but less active toward cyclophosphamide (Ariyoshi et al, 2011), ifosfamide (Calinski et al, 2015), bupropion (Zhang et al, 2011), and ketamine (Wang et al, 2018). CYP2B6.6 (516G.T, 785A.G, Q172H/K262R) had lesser activity toward S-efavirenz (53% of wild-type), consistent with most (20%-50%) (Ariyoshi et al, 2011;Zhang et al, 2011;Xu et al, 2012) but not all (Radloff et al, 2013;Watanabe et al, 2018) reports. CYP2B6.6 was also less active than CYP2B6.1 toward methadone (Gadel et al, 2013(Gadel et al, , 2015, ketamine (Wang et al, 2018), and bupropion (Zhang et al, 2011) but more active toward artmether and cyclophosphamide (Ariyoshi et al, 2011;Honda et al, 2011).…”

Section: Downloaded Frommentioning

confidence: 81%

“…CYP2B6.16 and CYP2B6.18. Kinetic parameters for S-efavirenz 8-hydroxylation by CYP2B6 variants, mainly CYP2B6.1, CYP2B6.4, CYP2B6.6, and CYP2B6.9, have been reported (Table 5) (Bumpus et al, 2006;Ariyoshi et al, 2011;Zhang et al, 2011;Xu et al, 2012;Radloff et al, 2013;Watanabe et al, 2018). CYP2B6.4 (785G.T, K262R) activity was greater than wild-type when expressed in T. ni (144%, this investigation), Sf9 cells (142%) (Ariyoshi et al, 2011), and E. coli (170%) (Bumpus et al, 2006), or similar to wild-type in E. coli (96%) (Zhang et al, 2011).…”

Section: Downloaded Frommentioning

confidence: 99%

Efavirenz Metabolism: Influence of Polymorphic CYP2B6 Variants and Stereochemistry

2019

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Efavirenz (more specifically the S-enantiomer) is a cornerstone antiretroviral therapy for treatment of HIV infection. The major primary metabolite is S-8-hydroxyefavirenz, which does not have antiretroviral activity but is neurotoxic. Cytochrome P450 2B6 (CYP2B6) is the major enzyme catalyzing S-8-hydroxyefavirenz formation. CYP2B6 genetics and drug interactions are major determinants of clinical efavirenz disposition and dose adjustment. In addition, as a prototypic CYP2B6 substrate, S-efavirenz and analogs can inform on the structure, activity, catalytic mechanisms, and stereoselectivity of CYP2B6. Metabolism of R-efavirenz by CYP2B6 remains unexplored. This investigation assessed Sefavirenz metabolism by clinically relevant CYP2B6 genetic variants. This investigation also evaluated R-efavirenz hydroxylation by wildtype CYP2B6.1 and CYP2B6 variants. S-Efavirenz 8-hydroxylation by wild-type CYP2B6.1 and variants exhibited positive cooperativity and apparent cooperative substrate inhibition. On the basis of Cl max values, relative activities for S-efavirenz 8-hydroxylation were in the order CYP2B6.4 > CYP2B6.1 CYP2B6.5 CYP2B6.17 > CYP2B6.6 CYP2B6.7 CYP2B6.9 CYP2B6.19 CYP2B6.26; CYP2B6.16 and CYP2B6.18 showed minimal activity. Rates of R-efavirenz metabolism were approximately 1/10 those of S-efavirenz for wildtype CYP2B6.1 and variants. On the basis of Cl max values, there was 14-fold enantioselectivity (S > R-efavirenz) for wild-type CYP2B6.1, and 5-to 22-fold differences for other CYP2B6 variants. These results show that both CYP2B6 516G > T (CYP2B6*6 and CYP2B6*9) and 983T > C (CYP2B6*16 and CYP2B6*18) polymorphisms cause canonical diminishment or loss-of-function variants for S-efavirenz 8-hydroxylation, provide a mechanistic basis for known clinical pharmacogenetic differences in efavirenz disposition, and may predict additional clinically important variant alleles. Efavirenz is the most stereoselective CYP2B6 drug substrate yet identified and may be a useful probe for the CYP2B6 active site and catalytic mechanisms. SIGNIFICANCE STATEMENT Clinical disposition of the antiretroviral S-efavirenz is affected by CYP2B6 polymorphisms. Expressed CYP2B6 with 516G>T (CYP2B6*6 and CYP2B6*9), and 983T>C (CYP2B6*16 and CYP2B6*18) polymorphisms had a diminishment or loss of function for efavirenz 8-hydroxylation. This provides a mechanistic basis for efavirenz clinical pharmacogenetics and may predict additional clinically important variant alleles. Efavirenz metabolism showed both cooperativity and cooperative substrate inhibition. With greater than 10-fold enantioselectivity (S-vs. Rmetabolism), efavirenz is the most stereoselective CYP2B6 drug substrate yet identified. These findings may provide mechanistic insights.

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“…We have now shown that in 1.5% of the observations ( N = 6 of 415), where an individual is heterozygous for both the *4 and the *9 variant, the variants are located in trans‐conformation ( CYP2B6 *4/*9). Animal and tissue studies have shown conflicting results in regard to the impact of these individual variants on enzyme function, resulting in uncertainty as to what the effect is on enzymatic function and, therefore, what phenotype should be assigned . In the DPWG guidelines, CYP2B6 *6 is designated as nonfunctional.…”

Section: Discussionmentioning

confidence: 99%

“…Animal and tissue studies have shown conflicting results in regard to the impact of these individual variants on enzyme function, resulting in uncertainty as to what the effect is on enzymatic function and, therefore, what phenotype should be assigned. [20][21][22][23][24] In the DPWG guidelines, CYP2B6*6 is designated as nonfunctional. However, this assignment is only for the combination of the two SNVs (CYP2B6*6) and not for the individual variants (CYP2B6*4 and CYP2B6*9).…”

Section: Discussionmentioning

confidence: 99%

Repurposing of Diagnostic Whole Exome Sequencing Data of 1,583 Individuals for Clinical Pharmacogenetics

Lee

Allard

Bollen

et al. 2019

Clin Pharma and Therapeutics

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For ~ 80 drugs, widely recognized pharmacogenetics dosing guidelines are available. However, the use of these guidelines in clinical practice remains limited as only a fraction of patients is subjected to pharmacogenetic screening. We investigated the feasibility of repurposing whole exome sequencing (WES) data for a panel of 42 variants in 11 pharmacogenes to provide a pharmacogenomic profile. Existing diagnostic WES‐data from child‐parent trios totaling 1,583 individuals were used. Results were successfully extracted for 39 variants. No information could be extracted for three variants, located in CYP2C19, UGT1A1, and CYP3A5, and for CYP2D6 copy number. At least one actionable phenotype was present in 86% of the individuals. Haplotype phasing proved relevant for CYP2B6 assignments as 1.5% of the phenotypes were corrected after phasing. In conclusion, repurposing WES‐data can yield meaningful pharmacogenetic profiles for 7 of 11 important pharmacogenes, which can be used to guide drug treatment.

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Functional characterization of 40 CYP2B6 allelic variants by assessing efavirenz 8-hydroxylation

Cited by 18 publications

References 28 publications

3.5KJPNv2, An allele frequency panel of 3,552 Japanese Individuals

3.5KJPNv2, An allele frequency panel of 3,552 Japanese Individuals

Efavirenz Metabolism: Influence of Polymorphic CYP2B6 Variants and Stereochemistry

Repurposing of Diagnostic Whole Exome Sequencing Data of 1,583 Individuals for Clinical Pharmacogenetics

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