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.

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. 516G>T, Q172H, and c.785A>G, 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, -750T>C, 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.777C>A(CYP2B6*3). Further investigation using minigene constructs transfected into eukaryotic cell lines COS-1 and Huh7 demonstrated that the single nucleotide polymorphism c.516G>T 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.785A>G polymorphism or the rare c.777C>A 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 516G>Tasthe causal sequence variation for severely decreased expression and function associated with CYP2B6*6.

Cytochrome P450 2B6 activity as measured by bupropion hydroxylation: effect of induction by rifampin and ethnicity.

AIM: The aim of this study was to investigate the activity of the drug-metabolizing enzyme cytochrome P450 (CYP) 2B6 before and after in vivo induction by rifampin (INN, rifampicin) in white subjects and Chinese subjects by use of the probe drug bupropion (INN, amfebutamone). METHODS: Healthy male white subjects (n = 9) and Chinese subjects (n = 9) (age range, 19-34 years) of known CYP2B6 genotype received orally administered bupropion (Zyban SR, 150 mg) alone and during daily treatment with rifampin (600 mg). Blood samples were taken for up to 72 hours after each bupropion dose, and plasma concentrations of bupropion and its active metabolites, hydroxybupropion, threohydrobupropion, and erythrohydrobupropion, were measured by HPLC. The subjects' CYP2B6 genotype was determined by use of a matrix-assisted laser desorption /ionization-time of flight (MALDI-TOF) mass spectrometry assay. RESULTS: Rifampin treatment increased the apparent clearance of bupropion in Chinese subjects and white subjects combined (n = 16) from 2.6 L x h(-1) x kg(-1) (95% confidence interval [CI], 2.3-3.0 L x h(-1) x kg(-1)) after bupropion alone to 7.9 L x h(-1) x kg(-1) (95% CI, 6.8-10.1 L x h(-1) x kg(-1)) during rifampin treatment. Rifampin treatment decreased the half-life of bupropion from 15.9 hours (95% CI, 13.5-20.4 hours) to 8.2 hours (95% CI, 6.7-12.4 hours). Rifampin treatment increased the hydroxybupropion maximum concentration from 395 ng/mL (95% CI, 341-497 ng/mL) to 548 ng/mL (95% CI, 490-638 ng/mL), decreased the area under the concentration-time curve extrapolated to infinity of hydroxybupropion from 14.7 microg x h/mL (95% CI, 12.7-18.4 microg x h/mL) to 8.4 microg x h/mL (95% CI, 7.4-10.2 microg x h/mL), and reduced the elimination half-life of hydroxybupropion from 21.9 hours (95% CI, 20.3-24.0 hours) to 10.7 hours (95% CI, 8.6-14.5 hours). There was no significant difference in the pharmacokinetics of bupropion or hydroxybupropion between white subjects and Chinese subjects before and after treatment with rifampin, once corrected for body weight. CONCLUSIONS: Rifampin significantly induces CYP2B6 activity in vivo, and the clinical consequences of potential interactions between rifampin and CYP2B6 substrates deserve further investigation. Rifampin appears to induce the elimination of hydroxybupropion. Differences in bupropion pharmacokinetics that were observed between white subjects and Chinese subjects can be attributed to differences in body weight, suggesting that, for a given subject weight, CYP2B6 activity is similar in white subjects and Chinese subjects.

MALDI-TOF mass spectrometry for multiplex genotyping of CYP2B6 single-nucleotide polymorphisms.

BACKGROUND: CYP2B6 is a highly variable and polymorphic cytochrome P450 (CYP) enzyme involved in the biotransformation of an increasing number of drugs, including cyclophosphamide, bupropion, and the nonnucleosidic reverse transcriptase inhibitor efavirenz. Several nonsynonymous and promoter single-nucleotide polymorphisms (SNPs) in the CYP2B6 gene are associated with altered hepatic expression and function, which affect drug plasma concentrations. METHODS: We used multiplex PCR to amplify relevant gene fragments while avoiding amplification of the CYP2B7P1 pseudogene. Polymorphic sites were analyzed by allele-specific primer extension followed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Method evaluation was performed on a panel of 287 genomic DNA samples previously genotyped by other methods. RESULTS: Five multiplex assays were developed, comprising the following 15 SNPs: -82T --> C (*22); 86G --> C (R29T, *17); 136A --> G (M46V, *11); 296G --> A (G99E, *12); 415A --> G (K139E, *8, *13); 419G --> A (R140Q, *14); 516G --> T (Q172H, *6, *7, *9, *13, *19, *20), 547G --> A (V183I); 769G --> A (D257N); 785A --> G (K262R, *4, *6, *7, *13, *16, *19, *20); 983T-->C (I328T, *16, *18); 1006C --> T (R336C, *19); 1172T --> A (I391N, *15); 1282C --> A (P428T, *21); 1459C --> T (R487C, *5, *7). In 9 DNA samples showing discrepant genotypes, correctness of the MALDI-TOF MS result was confirmed by direct sequencing. CONCLUSIONS: This genotyping method enabled sensitive, specific, accurate, and comprehensive determination of 15 relevant SNPs of CYP2B6. The assay design allows analysis of SNP subsets, incorporation of additional SNPs, and performance of high-throughput genotyping.