ORIGINAL ARTICLESImpaired expression of CYP2D6 in intermediate metabolizers carrying the *41 allele caused by the intronic SNP 2988G>A: evidence for modulation of splicing eventsToscano, Claudia; Klein, Kathrin; Blievernicht, Julia; Schaeffeler, Elke; Saussele, Tanja; Raimundo, Sebastiana; Eichelbaum, Michel; Schwab, Matthias; Zanger, Ulrich M.Author Information Dr Margarete Fischer-Bosch Institute of Clinical Pharmacology, Stuttgart, Germany Correspondence and requests for reprints to Dr Ulrich M. Zanger, Dr Margarete Fischer-Bosch Institute of Clinical Pharmacology, Auerbachstr. 112, 70376 Stuttgart, Germany Tel: +49 (0)711 81 01 37 04; fax:+49 (0)711 85 92 95; e-mail: [email protected] Sponsorship: This work was supported by the German Federal Ministry of Education and Research (Grant 0313080D) and by the Robert Bosch Foundation (Stuttgart, Germany). aPresent address: Sebastian Raimundo, Institute for Cell Biology, Department of Molecular Biology, University of Tuebingen, Auf der Morgenstelle 15, 72076 Tuebingen, Germany. aPresent address: Sebastian Raimundo, Institute for Cell Biology, Department of Molecular Biology, University of Tuebingen, Auf der Morgenstelle 15, 72076 Tuebingen, Germany. Preliminary data of this study were presented in poster form at the 7th International ISSX Meeting, 29 August–2 September 2004, Vancouver, Canada, and published as abstract: Toscano C, Raimundo S, Klein K, Schaeffeler E, Eichelbaum M, Schwab M, Zanger UM. Molecular analysis of the CYP2D6*41 ‘intermediate metabolizer’ allele. Drug Metab Rev 2004; 36 (Suppl 1):116. Received 21 December 2005 Accepted 19 April 2006 Pharmacogenetics and Genomics: October 2006 - Volume 16 - Issue 10 - p 755-766 doi: 10.1097/01.fpc.0000230112.96086.e0 Buy Metrics Abstract We investigated the molecular basis for low expression and activity of CYP2D6 associated with the CYP2D6*41 allele in about 10–15% of Caucasians with intermediate metabolizer phenotype. With respect to two previously described polymorphisms in the promoter (−1584C>G) and in intron 6 (2988G>A; c.985+39G>A), the three most frequent functional alleles have the distinct haplotypes 2D6*1[CG], 2D6*2[GG] and 2D6*41[CA], respectively. Reporter gene analyses in transiently transfected HepG2 and Huh7 hepatoma cells did not indicate changes in transcription rate by these polymorphisms. By reverse-transcription polymerase chain reaction analysis of liver RNA of genotyped patients, however, we discovered that the 2988G>A change was associated with increased levels of a nonfunctional splice variant lacking exon 6. Quantification by denaturing high-performance liquid chromatography revealed up to 7.3-fold increased levels of the splice variant and up to 2.9-fold less functional transcript in carriers of 2D6*41, in good concordance with concomitant changes in immunoquantified CYP2D6 protein. Recombinant expression of the entire genomic sequence coding for 2D6*41, 2D6*2 and 2D6*1 alleles but lacking the upstream region in COS-1 and Huh7 cell lines resulted in two-fold to five-fold reduced levels of CYP2D6 mRNA containing exon 6, apoprotein and enzyme activity of 2D6*41. These experiments establish the causal relationship between the intron 6 single-nucleotide polymorphism 2988G>A and the low expression phenotype associated with allele 2D6*41. These data improve the CYP2D6 genotype–phenotype relationship and they demonstrate that major phenotype changes occurring in large population subgroups can be caused by intronic polymorphisms outside of splice site consensus sequences. © 2006 Lippincott Williams & Wilkins, Inc.