A Frameshift Mutation and Alternate Splicing in Human Brain Generate a Functional Form of the Pseudogene Cytochrome P4502D7 That Demethylates Codeine to Morphine

Abstract

A frameshift mutation 138delT generates an open reading frame in the pseudogene, cytochrome P4502D7 (CYP2D7), and an alternate spliced functional transcript of CYP2D7 containing partial inclusion of intron 6 was identified in human brain but not in liver or kidney from the same individual. mRNA and protein of the brain variant CYP2D7 were detected in 6 of 12 human autopsy brains. Genotyping revealed the presence of the frameshift mutation 138delT only in those human subjects who expressed the brain variant CYP2D7. Genomic DNA analysis in normal volunteers revealed the presence of functional CYP2D7 in 4 of 8 individuals. In liver, the major organ involved in drug metabolism, a minor metabolic pathway mediated by CYP2D6 metabolizes codeine (pro-drug) to morphine (active drug), whereas norcodeine is the major metabolite. In contrast, when expressed in Neuro2a cells, brain variant CYP2D7 metabolized codeine to morphine with greater efficiency compared with the corresponding activity in cells expressing CYP2D6. Morphine binds to -opioid receptors in certain regions of the central nervous system, such as periaqueductal gray, and produces pain relief. The brain variant CYP2D7 and -opioid receptor colocalize in neurons of the periaqueductal gray area in human brain, indicating that metabolism of codeine to morphine could occur at the site of opioid action. Histiospecific isoforms of P450 generated by alternate splicing, which mediate selective metabolism of pro-drugs within tissues, particularly the brain, to generate active drugs may play an important role in drug action and provide newer insights into the genetics of metabolism.Cytochrome P450 (EC 1.14.14.1; P450) 1 and associated monooxygenases, a family of heme proteins, are the principal class of drug-metabolizing enzymes. A supergene family encodes them, and the member proteins exist as multiple forms having distinct yet overlapping substrate specificities. Multiple forms of P450, which are selectively induced or inhibited by a variety of drugs, are known to exist in liver, the major organ involved in P450-mediated metabolism (1). However, the potential to generate active metabolite(s) at the site of action has generated interest in extrahepatic P450. This has prompted extensive investigations into the xenobiotic metabolizing capability of extrahepatic organs, such as lung, kidney, skin, and nasal epithelium, and the far reaching consequences of such metabolism, in situ, within specific cells in target organs have been recognized in laboratory animals (2) and humans (3). The preferential localization of drug-metabolizing enzymes within specific cell types in these organs renders such cells significantly capable of metabolizing drugs (4). Thus, even minor metabolic pathways of xenobiotic metabolism can produce major effects if they take place at the site of action. These observations have prompted investigation into P450-associated monooxygenases in brain with an effort to determine the capability of the brain to metabolize psychoactive drugs (5, 6). P450-mediated ...

Pharmacokinetics of codeine and its metabolite morphine in ultra-rapid metabolizers due to CYP2D6 duplication

“…Recently, a functional variant of CYP2D7 leading to expression of this pseudogene in brain and to brain metabolism of codeine to morphine has been described in four of eight individuals of whom brain autopsies were obtained. 37 The population frequency of this variant has not yet been studied so far but this mechanism as well might be a possible explanation for opioid effects in PMs after codeine intake.…”

“…The obtained PCR product was used as a template for the single-base primer extension reaction (SNaPshot, Applied Biosystems) designed to genotype the polymorphic position 4180G4C of the upstream gene-copy and positions À1584G4C and 100C4T of the downstream gene-copy. The following primers were used for the extension reactions: À1584G4C, 5 0 -(A) 9 CCT GGA CAA CTT GG AAG AAC C-3 0 ; 100C4T, 5 0 -(A) 31 CAA CGC TGG GCT GCA CGC TAC-3 0 and 4180G4C, 5 0 -(A) 37 CAA AGC TCA TAG GGG GAT GGG-3 0 . The single-base primer extension reactions were performed according to the manufacturer's instructions (SNaPshot, Applied Biosystems, Weiterstadt, Germany).…”

Genetic polymorphisms of cytochrome P450 2D6 (CYP2D6): clinical consequences, evolutionary aspects and functional diversity

“…13,14 The question arises whether such an activity is essential. Interestingly, Pai et al 15 have recently provided evidence that a common variant of the pseudogene CYP2D7P could be expressed in an active …”

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“…It has been observed that sometimes a frame-shifted protein coding gene is still functional [2,14,15,26], but this phenomenon has been taken as special or individual cases, rather than a fundamentally important biological process sharing a common underlying mechanism. In fact, we found that this phenomenon is not rare, but can be observed quite often.…”

“…Therefore, although it was observed that sometimes frame shifted or overlapped genes is functional [12][13][14][15][16][17], a frame-shifted translational product is generally considered to be non-functional, because it is a common sense that it is often possible to inactivate the function of a peptide by changing only one single residue.…”