In Vitro Functional Assessment of 22 Newly Identified CYP2D6 Allelic Variants in the Chinese Population

Abstract

Cytochrome P450 2D6 (CYP2D6) is one of the most widely investigated CYPs related to genetic polymorphisms and is responsible for one-quarter of the currently used clinical drugs. We previously detected 22 novel, non-synonymous, mutated sites in the Chinese population, but nothing is known about the functional effects of these mutations in terms of specific CYP2D6 substrates. In this study, wild-type CYP2D6, two common allelic variants and 22 newly reported CYP2D6 isoforms were transiently expressed in 293FT cells, and the enzymatic activities of these variants were systematically assessed using dextromethorphan and bufuralol as the probing substrates. Consequently, 19 and 21 allelic variants were found to exhibit significantly decreased enzymatic activities for dextromethorphan and bufuralol, respectively. Of 22 novel CYP2D6 variants, six allelic isoforms (CYP2D6.89, CYP2D6.92, CYP2D6.93, CYP2D6.96, E215K and R440C) exhibited absent or extremely reduced metabolic activities compared with those observed for the wild-type enzyme. Our in vitro functional data can be useful for CYP2D6 phenotype prediction and provide valuable information for the study of clinical impact of these newly found CYP2D6 variants in China.As one of the most important drug-metabolizing enzymes, cytochrome P450 2D6 (CYP2D6) is responsible for approximately 25% of clinically used drugs including analgesics, antiarrhythmics, antiemetics, antipsychotics, antidepressants, beta-adrenergic blockers and chemotherapeutic/hormone replacement agents [1][2][3]. Similar to other P450 subfamily members, the CYP2D6 gene locus is also highly polymorphic across different ethnic populations and individuals [4]. To date, a total of 105 allelic variants are defined and described in the Human CYP Allele Nomenclature database (http://www.cypalleles.ki.se/cyp2d6.htm). These genetic variants can be associated with increased, decreased or abolished enzymatic functions, which results in differences of up to 30-to 40-fold changes in substrate drug clearance values. Consequently, these differences in CYP2D6 activity have been demonstrated to be prominent contributors to interindividual drug response variability [2,5,6].It has been reported that significant ethnic and geographic differences exist in CYP2D6 alleles [7,8]. For Eastern Asians, only 1-2% of individuals have been found to be poor metabolizers (PMs), and approximately 57% of the Chinese population are intermediate metabolizers (IMs) [9,10]. Polymorphic studies have revealed that the IM allele CYP2D6*10 is predominant in Asian populations with frequencies ranging from 30% to 50% [7,11]. In a recent large-scale, genetic screening study, we sequenced all nine exons of the CYP2D6 gene in 2129 unrelated Chinese individuals. As a result, 22 new nonsynonymous, mutated sites were found, and 12 of them were designated as novel alleles (*87-*93, *94A, *94B and *95-*98) by the Human CYP Allele Nomenclature Committee [12]. Considering the fact that there are more than 1.4 billion people living in the mainland ...

Effect of 24 Cytochrome P450 2D6 Variants Found in the Chinese Population on Atomoxetine Metabolism in vitro

“…The results indicated that CYP2D6.10 exhibits the lowest catalytic activities on the metabolism of atomoxetine in vitro. These data were in accordance with most of the previous reported studies [15,16,17,18] and thus indicated that our CYP expression and metabolic activity analysis system was suitable for analyzing the catalytic activities of the other CYP2D6 allelic variants. For CYP2D6.10 allele, which occurs as the most common allele in the Chinese population, our investigation may provide guidance for the proper atomoxetine administration in the clinic.…”

Effect of 24 cytochrome P450 2D6 variants found in the Chinese population on theN-demethylation of amitriptylinein vitro

“…Moreover, some allelic variants showed a different trend. In our study, CYP2D6*90 exhibited the moderate clearance intrinsic value (43.15%), while the corresponding value was more than 60% in previous studies (Dai et al 2015;Cai et al 2016). And, CYP2D6*2, CYP2D6*87, CYP2D6*88, CYP2D6*95 showed the relative clearance intrinsic values 20.34, 9.53, 25.46, 25.06% classified into the seriously defective group, respectively, whereas in previous research ranged from 30% to 60% (Cai et al 2016) and even were above 60% (Dai et al 2015).…”

“…In our study, CYP2D6*90 exhibited the moderate clearance intrinsic value (43.15%), while the corresponding value was more than 60% in previous studies (Dai et al 2015;Cai et al 2016). And, CYP2D6*2, CYP2D6*87, CYP2D6*88, CYP2D6*95 showed the relative clearance intrinsic values 20.34, 9.53, 25.46, 25.06% classified into the seriously defective group, respectively, whereas in previous research ranged from 30% to 60% (Cai et al 2016) and even were above 60% (Dai et al 2015). We speculated that the substrate-dependent intrinsic characteristics and metabolic path (hydroxylation and demethylation) of the CYP2D6 protein might be responsible for these results, as several groups have reported that a given CYP2D6 allelic variant can exhibit different catalytic activities towards different substrates in vitro and/or in vivo.…”

“…Among these novel mutation sites, 22 were nonsynonymous and 12 were named as novel alleles by the Human CYP Allele Nomenclature Committee. Most of these novel allelic CYP2D6 variants exhibited significantly decreased catalytic abilities compared with CYP2D6*1 (Dai et al 2015). When expressed in mammalian cells with probing substrates dextromethorphan and bufuralol.…”

“…So, it is very urgent to further systematical research. Furthermore, the previous study revealed that 22 new identified variants significantly affected the catalytic activity of CYP2D6 towards dextromethorphan and bufuralol in transfected COS-7 cell (Dai et al 2015), but considering the fact that the low expression of CYP2D6 in a mammalian cell expression system, resulting in the difficulty of the analysis of catalytic activity. In the present study, we highly expressed the wild-type CYP2D6*1 and newly found variants in Sf21 insect cell.…”