Identification and Functional Assessment of a New CYP2C9 Allelic Variant CYP2C9*59

Abstract

CYP2C9, one of the most important drug-metabolizing enzymes, is responsible for metabolizing approximately 15% of clinically important drugs, including warfarin, diclofenac, and losartan. Similar to other CYP members, human CYP2C9 exhibits marked genetic polymorphisms among individuals of different ethnicities. In this study, a novel missense mutation (1300A>T) was identified in a warfarin-sensitive patient after a genetic screen of three candidate genes related to high variability in response to warfarin doses. This base transversion leads to an Ile-toPhe amino acid substitution at codon 434 within the CYP2C9 protein, and this new variant has been named a novel allele, CYP2C9*59, by the Human CYP Allele Nomenclature Committee (http://www.cypalleles.ki. se/cyp2c9.htm). The exogenous expression of CYP2C9.59 in insect cell microsomes revealed that, despite a similar protein expression level as wild-type CYP2C9, variant CYP2C9.59 exhibited significantly reduced maximal velocity, V max , and/or increased Michaelis constant, K m , values toward three CYP2C9-specific substrates. Our data suggest that the 1300A>T mutation can greatly decrease the enzymatic activity of the CYP2C9 protein both in vitro and in vivo. IntroductionCYP2C9 is the major CYP2C isoform in humans and is responsible for the metabolism of approximately 15% of all clinically important drugs (Samer et al., 2013;Chen et al., 2014). Similar to other CYP members, CYP2C9 is highly polymorphic across various racial and ethnic populations. These genetic polymorphisms have been shown to have clinical importance, leading to great inter-individual variability and serious adverse effects in the efficacies of many therapeutic drugs (Chaudhry et al., 2014). Alleles CYP2C9*2 (containing a R144C substitution) and CYP2C9*3 (containing an I359L substitution) have been well studied among different ethnic populations. They have been identified as the most common defective alleles and have exhibited reduced metabolic activities, both in vitro and in vivo, for the CYP2C9 substrates, such as warfarin. In addition to these two common alleles, other allelic variants, such as CYP2C9*4 ( Lee et al., 2007) Nahar et al., 2013), and CYP2C9*58 (Luo et al., 2014), were reported to affect warfarin sensitivity. In this study, we present the case of a Chinese warfarin-sensitive patient who requires only one-half of the normal oral warfarin dose. Genetic analysis in that patient revealed an absence of the common mutations in CYP2C9 (*2, *3), VKORC1 (rs9923231 and rs7294), and CYP4F2 (rs2108622) that are usually related to warfarin sensitivity. However, a novel missense mutation (1300A.T) was identified in exon 9 of the CYP2C9 gene. The in vitro functional assessment of this newly found CYP2C9 variant revealed that the substitution of an Ile to a Phe at codon 434 significantly reduced the enzymatic activity of this variant toward all of the tested substrates. Materials and MethodsStudy Subject. The study subject was a 47-year-old male Han Chinese patient who has taken warfarin...

Genetic Polymorphisms and <i>in Vitro</i> Functional Characterization of CYP2C8, CYP2C9, and CYP2C19 Allelic Variants

“…Continued allele to *60 variants in insect cells and comprehensively analyzed the kinetic parameters for each of the variant enzymes in the metabolism of losartan, glimepiride, tolbutamide, and diclofenac. [48][49][50][51][52][53] They observed that it was difficult to analyze warfarin metabolism using insect cells. It was therefore indicated that mammalian cells have to be used in expressing CYP2C9*1 to *60 variants to analyze the metabolism of warfarin.…”

<p>In Vitro and In Vivo Rat Model Assessments of the Effects of Vonoprazan on the Pharmacokinetics of Venlafaxine</p>

“…In our previous studies, results of in vitro and in vivo experiments showed that vonoprazan inhibited CYP3A4, CYP2C9, CYP2D6, and CYP2B6 in vitro and in vivo. 16 It has been previously established that metabolism of VEN is dominated by CYP2D6 and it is transformed into the active metabolite ODV in the liver, [17][18][19] In addition, CYP3A4/5 also participate partially in the metabolism of VEN. 20 Therefore, VEN is susceptible to CYP450mediated DDI, especially through the CYP2D6 metabolic pathway.…”

<p>Functional Measurement of CYP2C9 and CYP3A4 Allelic Polymorphism on Sildenafil Metabolism</p>

“…Some studies on tolbutamide and carvedilol metabolism have shown that compared with the wild-type CYP2C9*1, the CYP2C9*11 (R335W) allele is linked to a decrease in enzyme activity. 28,29 However, in some other studies, it has been noted that CYP2C9*11 may have little to do with the dosage requirements of warfarin, and that it shows a significant increase in the enzyme activity of bosentan metabolism. 43,44 In this study, the catalytic activity of the CYP2C9*11 allele on sildenafil was not significantly different from that of the wild type.…”

“…9,24,25 In a recent study, Dai et al sequenced all nine exons of CYP2C9 in 2127 26 In subsequent studies, Luo et al further analyzed the CYP2C9 nine exons in previous 2127 healthy Chinese people and found three novel mutation positions that could result in amino acid variation, which have been named CYP2C9*58 (P337T), CYP2C9*59 (I434F) and CYP2C9*60 (L467P). [27][28][29] CYP3A4 is another important and abundantly expressed CYP450 drug-metabolizing enzyme, which belongs to the human CYP3A subfamily and constitutes ~30% of the total CYPs in human liver. 30 It is in charge of clearing approximately half of clinical drugs that experience phase I metabolic reaction, such as anticancer drugs (tamoxifen), Hypnotics (zolpidem), antibiotics (erythrocin), Hormone Drugs (prednisolone), anticonvulsants (carbamazepine), opioids (fentanyl), calcium channel blockers (nifedipine), antihistamines (loratadine) and so forth.…”