Browsing by Author "Hofmann, Marco Hans"

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Open Access
Microarray and molecular genetic analysis of aberrant splicing in human drug metabolizing cytochromes P450 CYP2D6 and CYP2B6
(2008) Hofmann, Marco Hans; Schmid, Rolf (Prof. Dr.)
This study was devoted to the detection of alternative splicing within the Cytochrome P450 enzymes 2D6 and 2B6, mapping of the most common splice variants and to draw connections to certain single nucleotide polymorphisms (SNPs) and alleles. For both enzymes a splicing sensitive microarray was developed. The microarray was produced and optimized in all steps including the oligonucleotide probe design, microarray processing and target preparation, optimization of hybridization conditions and the development of a new data quantification method for the used probe design. For the developed splicing platform a design was chosen based on 5 different probes. Within the CYP2D6 gene it was known that the SNP 2988G>A (allele *41) in intron 6 shifts splicing towards a variant lacking exon 6, what explains the intermediate phenotype within allele *41. The splicing platform verified this splicing aberration in allele *41. Using the microarry specific splicing patterns were monitored in human liver tissue within the most common alleles of CYP2D6 *1, *2, *4 and *41. It could be observed that within mRNA from allele *41 carriers additionally to the known transcript variant, which is lacking exon 6, total or partial retention of intron 5 and 6 was enhanced. Transcript patterns of CYP2D6*1 and *2 were similar with 5 times higher amount of the full functional transcript (NP), including all nine exons, compared to allele *41. The splicing array showed to be a valuable tool not only for detection of splicing variants in human liver tissue but additionally for detection for allele specific splicing patterns. The existence of highly homologous Cytochrome P450 pseudogenes, which in some cases, as in CYP2D7 also express alternative splicing variants, results in a major problem of interpreting the data from splicing arrays. The developed splicing platform is the first existing array with which gene and pseudogene specific transcript patterns can be monitored individually. The microarray platform can be easily transferred to other genes as shown for the second gene CYP2B6. Alternative splicing in this gene was so far only reported descriptive. 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 HIV patients, but the molecular mechanism has remained unclear. With the developed splicing array for CYP2B6 allele specific splicing patterns were observed comparing CYP2B6*6 and CYP2B6*1. This lead to the idea that alternative splicing might play an important role in allele *6. This was investigated in more detail using RNA originating from well-documented human liver tissue. Analysis of mRNA in this tissue demonstrated that additional unknown splicing variants exist (SV8, SV7, SV9). Investigations in human liver tissue using RT-PCR and sequencing showed that the most common transcript in CYP2B6*6 was not the normal transcript (NP) but an alternative splicing transcript lacking exons 4 to 6 (SV1). SV1 was tightly associated with the allele*6 and apparently also with the rare variant c.777C>A (CYP2B6*3). The observations lead to the assumptions that alternative splicing might explain the decreased function observed in allele CYP2B6*6. Further investigations in this direction were performed by cloning CYP2B6 minigene constructs including all nine exons and additional intronic regions. 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 allele*6 involves predominantly a pretranslational mechanism resulting in decreased enzyme expression. Aberrant splicing is leading to reduce functional mRNA, protein and activity. These results establish the SNP c.516G>T, a nonsynonymous exonic mutation, as the causal sequence variation for severely decreased expression and function associated with CYP2B6*6. This work emphasizes the role of SNPs in non-consensus splicing elements such as exonic and intronic splicing enhancers as well as the clinical relevance of alternative splicing in context of adverse drug reactions. In both investigated genes CYP2D6 as well as in CYP2B6 there exists a common allele (CYP2D6*41 and CYP2B6*6, respectively) in which aberrant splicing results in reduced amounts of functional transcript, reduced amount of protein and enzyme activity. The findings establishes the SNP c.516G>T as the causal sequence variation that can now be reliably used in pharmacogenetic studies in various clinical settings including prediction of drug plasma concentration, toxicity, drug effectiveness and dose adjustment.