Characterization of the Novel DefectiveCYP2C9^*24Allele

Abstract

ABSTRACT:CYP2C9 is one of the major drug-metabolizing enzymes, and it is involved in the oxidative metabolism of approximately 10% of clinically important drugs, among which some, such as the anticoagulant warfarin, have a narrow therapeutic index. The human CYP2C9 gene is highly polymorphic. We found a new sequence variation in exon 7 of the CYP2C9 gene (1060G>A) resulting in a substitution of acidic amino acid glutamate to basic lysine (E354K) when translated. The allele, designated CYP2C9*24, was present in heterozygous state in one warfarin-treated patient. To characterize the CYP2C9*24 allele, we expressed the wild-type and CYP2C9.24 protein in a recombinant yeast expression system and a human embryonic kidney (HEK)-293 cell system. Carbon monoxide difference spectra were recorded on dithionite-reduced microsomes, and protein was determined by Western blotting. Transfection with CYP2C9.1 cDNA resulted in detectable CYP2C9 protein in yeast or HEK-293 cells, whereas only small amounts of the protein were detected in yeast transfected with CYP2C9.24 cDNA. A strong differential absorption peak at 450 nm was observed with microsomes of yeast transfected with CYP2C9.1 cDNA, whereas no peak was detected with microsomes of yeast transfected with CYP2C9.24 cDNA or empty pYeDP60 plasmid. These results suggest that CYP2C9.24 may be improperly folded, both in yeast and mammalian cells, resulting in improper heme incorporation and rapid intracellular degradation. The data obtained in the expression systems are consistent with our findings in vivo. In conclusion, we have identified a novel defective CYP2C9 variant allele of potential importance for drug metabolism in vivo.CYP2C9 is involved in the metabolism of more than 100 currently used drugs, corresponding to about 10 to 20% of commonly prescribed drugs (Kirchheiner and Brockmoller, 2005). Some of them (e.g., anticoagulant warfarin) have a narrow therapeutic index (Miners and Birkett, 1998). Clinically available warfarin is a racemic mixture, and CYP2C9 is responsible for the metabolism of more potent Senantiomer (Kaminsky and Zhang, 1997). The human CYP2C9 gene is highly polymorphic (http://www.imm.ki.se/CYPalleles/). The two most common variant alleles in Caucasian populations are CYP2C9*2, with a point mutation in exon 3 (430CϾT), and CYP2C9*3, with a point mutation in exon 7 (1075AϾC). Both are associated with a decrease in the catabolic activity of the enzyme (Rettie et al., 1994;Sullivan-Klose et al., 1996). It was shown in many studies that the patients with polymorphic alleles require significantly lower doses of warfarin and are more susceptible to bleeding complications than the carriers of two wild-type alleles (Kirchheiner and Brockmoller, 2005).We have previously reported a novel 1060GϾA sequence variant in exon 7 of CYP2C9 gene, leading to substitution of acidic amino acid glutamate to basic lysine (E354K). The new allele, designated CYP2C9*24, was present in the heterozygous state in one warfarintreated patient, who was also a heterozygous carrier of t...

In vitro functional characterization of 37 CYP2C9 allelic isoforms found in Chinese Han population

“…In insect microsomes, relatively lower expressed proteins could be detected for variant CYP2C9.36 when compared with which in COS-7 cells. Herman et al reported a similar phenomenon for the CYP2C9.24 variant [16] . As Maekawa et al proposed, this discrepancy might be because of the different degradation rates of improperly folded proteins in insect and mammalian cell expression systems [8] .…”

Substrate-Dependent Functional Alterations of Seven CYP2C9 Variants Found in Japanese Subjects

“…Guo et al (2005a) reported that the protein expression level of CYP2C9.13 was 39% of that of CYP2C9.1 in the COS-1 expression system by Western blotting. The discrepancy in total protein levels between their study and ours might be due to different rates of degradation of improperly folded proteins between the two expression systems, as observed for CYP2C9.24 between yeast and mammalian systems (Herman et al, 2007). The mammalian cell system seems to be more relevant to assess the potential effects of CYP2C9*13 on its protein expression in vivo although accurate estimation of its holoprotein levels might be difficult in this system because of the low expression levels.…”

Toward predicting CYP2D6-mediated variable drug response from CYP2D6 gene sequencing data

“…It has been shown that in vitro results can give a reliable indication of the in vivo effect of a specific variant (35,36). Both the neural network predictions, which are in silico predictions based on in vivo data, and the in vitro results showed the same impact (decreased, normal, or increased activity) of the investigated variants.…”

“…4A). To confirm the contributions predicted by the model, four variants and the *2 allele were expressed in HEK (human embryonic kidney) 293 cells, which are completely devoid of any other metabolic enzymes (35,36), and incubated with bufuralol (table S2). The direction of our predictions (decrease or increase) was in concordance with the in vitro results, with accuracy increasing as the variant frequency increases.…”