Aberrant Splicing Caused by Single Nucleotide Polymorphism c.516G>T [Q172H], a Marker of CYP2B6*6, Is Responsible for Decreased Expression and Activity of CYP2B6 in Liver

Abstract

CYP2B6 is a polymorphic human drug metabolizing cytochrome P450 with clinical relevance for several drug substrates including cyclophosphamide, bupropion, and efavirenz. The common allele CYP2B6*6 [c. 516GϾT, Q172H, and c.785AϾG, K262R] has previously been associated with lower expression in human liver and with increased plasma levels of efavirenz in human immunodeficiency virus patients, but the molecular mechanism has remained unclear. We present novel data showing that hepatic CYP2B6 mRNA levels are reduced in *6 carriers, suggesting a pretranslational mechanism resulting in decreased expression. As one possibility, we first analyzed the common promoter variant, Ϫ750TϾC, but the results did not suggest a prominent role in phenotype determination. In contrast, analysis of liver mRNA splicing variants demonstrated that the most common form lacking exons 4 to 6 (SV1) was tightly associated with the *6 allele and apparently also with the rare variant c.777CϾA (CYP2B6*3). Further investigation using minigene constructs transfected into eukaryotic cell lines COS-1 and Huh7 demonstrated that the single nucleotide polymorphism c.516GϾT in allele CYP2B6*6 was alone responsible for aberrant splicing resulting in high-splice variant (SV) 1 and low-CYP2B6 expression phenotype. Minigenes carrying the single c.785AϾG polymorphism or the rare c.777CϾA variant resulted in normal and intermediate expression phenotypes, respectively. In conclusion, the mechanism of the common *6 allele involves predominantly aberrant splicing, thus leading to reduced functional mRNA, protein, and activity. These results establish the single nucleotide polymorphism 516GϾT as the causal sequence variation for severely decreased expression and function associated with CYP2B6*6.

Differences in Methadone Metabolism by CYP2B6 Variants

“…Data regarding the functional activity of CYP2B6.9 are said to be rare . In a COS-1 system, the activities of CYP2B6.6 and CYP2B6.9, in which 516G.T is the common polymorphism, were both extremely low, but this was attributed to minimal expression (Hofmann et al, 2008). CYP2B6.9 activity toward artemether was somewhat lower than CYP2B6.1 in COS-7 cells (Honda et al, 2011), and toward 7-ethoxy-4-trifluoromethylcoumarin, bupropion, and efavirenz in a reconstituted bacterial expression system (Zhang et al, 2011).…”

“…Clinical consequences of CYP2B6 allelic variants will depend on both the intrinsic activity of the mutant protein and its level of expression. The CYP2B6*6 allele causes low human hepatic CYP2B expression (Lang et al, 2001;Desta et al, 2007;Hofmann et al, 2008), thus both deficient catalytic efficiency and decreased P450 content combine to cause a diminished CYP2B6 metabolizer phenotype in CYP2B6*6 carriers. Liver microsomes from CYP2B6*6 carriers had diminished methadone Ndemethylation (Gadel et al, 2013).…”

Developmental Expression of CYP2B6: A Comprehensive Analysis of mRNA Expression, Protein Content and Bupropion Hydroxylase Activity and the Impact of Genetic Variation

“…It should be noted that none of the aforementioned studies characterized CYP2B6 activity in their samples. Several other studies have found no difference in CYP2B6 expression or activity in relation to age; however, each of these studies included only a few pediatric microsomal samples between the ages of 2 and 18 in their sample set (Hesse et al, 2004;Parkinson et al, 2004;Desta et al, 2007;Hofmann et al, 2008). …”

“…Previous studies noted marked interindividual variability in CYP2B6 expression and activity in human livers; in vitro CYP2B6 mRNA expression (Lamba et al, 2003;Hofmann et al, 2008), protein levels (Hesse et al, 2004) and activity (Croom et al, 2010) have all been reported to exceed 250-fold. This remarkable variation in CYP2B6 expression and activity has the potential to result in therapeutic and toxic responses to medications metabolized by this enzyme, particularly for those drugs with narrow therapeutic indices.…”

Pharmacogenetics of antidepressant response

“…42 Moreover, the common allele with a G-to-T substitution at nucleotide 516 (516G>T; rs3745274) is associated with lower expression via aberrant splicing events, 43 and the amino acid substitution of leucine to phehenylalanine at position 264 (Leu264Phe) has been reported to have functional consequences on CYP2B6 activity. 44 This is quite interesting because CYP2B6 and CYP3A4 are the main isoforms involved in metabolizing methadone and sibutramine (CYP2B6 only), and it is thought that part of the individual response to these drugs depends on genetic variants of these cytochromes. [45][46][47] The recent results of genetic association tests for CYP2D6, CYP2B6 and CYP2C19 variations are presented in Table 1.…”