title: Impaired expression of CYP2D6 in intermediate metabolizers carrying the *41 allele caused by the intronic SNP 2988G>A: evidence for modulation of splicing events.
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.
authors.affiliation: Dr Margarete Fischer-Bosch Institute of Clinical Pharmacology, Stuttgart, Germany.
authors.forename: Claudia
authors.initials: C
authors.lastname: Toscano
authors.forename: Kathrin
authors.initials: K
authors.lastname: Klein
authors.forename: Julia
authors.initials: J
authors.lastname: Blievernicht
authors.forename: Elke
authors.initials: E
authors.lastname: Schaeffeler
authors.forename: Tanja
authors.initials: T
authors.lastname: Saussele
authors.forename: Sebastian
authors.initials: S
authors.lastname: Raimundo
authors.forename: Michel
authors.initials: M
authors.lastname: Eichelbaum
authors.forename: Matthias
authors.initials: M
authors.lastname: Schwab
authors.forename: Ulrich M
authors.initials: UM
authors.lastname: Zanger
mesh_terms: D000483:Alleles; D001483:Base Sequence; D019389:Cytochrome P-450 CYP2D6 / Q000235:genetics*; D017931:DNA Primers; D006801:Humans; D007438:Introns; D010957:Plasmids; D020641:Polymorphism, Single Nucleotide; D012326:RNA Splicing; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014158:Transcription, Genetic
chemical_list: D017931:DNA Primers; D019389:Cytochrome P-450 CYP2D6