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METABOLISM, TRANSPORT, AND PHARMACOGENOMICS

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

Dr. Margarete Fischer-Bosch Institute of Clinical Pharmacology, Stuttgart, Germany; and Department of Clinical Pharmacology, University of Tübingen, Stuttgart, Germany

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.

Address correspondence to: Dr. Ulrich M. Zanger, Dr. Margarete Fischer-Bosch Institute of Clinical Pharmacology, Stuttgart, Germany and University of Tübingen, Auerbachstrasse 112, 70376 Stuttgart, Germany. E-mail: uli.zanger{at}ikp-stuttgart.de

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