![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
|
|
|
|
|
||
Washington University in St. Louis, St. Louis, Missouri
(Received May 27, 2005; Accepted June 8, 2005)
). The gene lies within the CYP2D locus on chromosome 22q13 downstream of two homologous pseudogenes, 5'-CYP2D8P-CYP2D7P-CYP2D6-3' (Kimura et al., 1989). CYP2D8P is a true pseudogene with many frameshift mutations, whereas CYP2D7P contains a single base insertion in exon 1 (138T) that causes a frameshift and premature termination of translation (Kimura et al., 1989). Gene conversion by deletion of 138T has been hypothesized to produce a functional CYP2D7 protein. This was investigated by Lovlie et al. (2001) in 17 extensive and ultrarapid metabolizers of CYP2D6 substrates of various ancestral backgrounds using nested polymerase chain reaction, but all subjects were homozygous for the insertion.
A recent publication by Pai et al. (2004) reported a CYP2D7 mRNA transcript with a 57-bp insertion in intron 6, found in 6 of 12 cDNA samples prepared from human brain. Using an antibody that recognized the 19-amino acid peptide representing the 57-bp insertion in intron 6 of the CYP2D7 splice variant and not CYP2D6, the authors detected CYP2D7 protein expression in brains carrying the intron 6 insertion but not in the brains of noncarriers. Neuro2 cells were transfected with the cDNA transcript. They produced a CYP2D protein with greater catalytic activity than CYP2D6 toward codeine O-demethylation, a prototypical CYP2D6 reaction. The six brains positive for CYP2D7 protein were also found to carry a 138delT in CYP2D7, which would convert the pseudogene to a functional gene. To confirm the presence of the 138delT polymorphism in germline DNA, the authors amplified and sequenced a 506-bp region of CYP2D7 surrounding the deletion polymorphism in eight individuals of Indian descent. Consistent with the frequency observed in DNA isolated from brain, four of the eight individuals carried 138delT.
We conducted a BLAST search (http://www.ncbi.nlm.nih.gov/BLAST) and found that the forward and reverse primers used by Pai et al. (2004) to amplify genomic DNA (forward: 5'-CTTCCTGCTCCTGGTGGA-3'; reverse: 5'-CACCCCCTTCATCCTCGA-3') were fully complementary to CYP2D7P (GenBank accession number X58467
[GenBank]
; forward primer: 1286..1304; reverse primer: 1792..1775), CYP2D6 (NG_003180
[GenBank]
; forward primer: 7770..7788; reverse primer: 8275..8258, translation start: 7726) and also CYP2D8P2 (NG_000853
[GenBank]
; forward primer: 145... 163; reverse primer: 651..634), suggesting that the primers will produce a mix of products from all of the CYP2D genes with the expected product sizes of 506, 506, and 507 bp, respectively. The sequence NG_002362
[GenBank]
used by Pai et al. (2004) has since been withdrawn from RefSeq.
Pai et al. (2004) presented electropherograms for six of the individuals genotyped. Subjects were homozygous for insertion or deletion, and none were heterozygous. Because the number of heterozygotes was not reported in the paper, it is assumed that none were observed and four individuals carrying the deletion were homozygous, which does not follow Hardy-Weinberg rules and, consequently, is not consistent with a genetic polymorphism. Indeed, these data are more consistent with differential amplification of two or more genes with high sequence homologies varying at this position. Both pseudogenes have 98% sequence homology with CYP2D6, and both pseudogenes vary from the functional gene by the insertion of a T allele. CYP2D6 gene amplification is well characterized, and up to 13 copies of the gene have been reported (Ingelman-Sundberg, 1999). Since Pai et al. (2004) did not test for CYP2D6 amplification in their samples, this cannot be ruled out as a conflicting source of genomic sequence, especially since the primer sequences demonstrated 100% homology with CYP2D6 and CYP2D8P2, and the polymerase chain reaction product sizes differ only by 1 bp and are consequently indistinguishable on a gel or sequencing trace.
Problems with false-positive polymorphism discovery in genes with high homology to other gene family members or pseudogenes have been reported previously (Marsh et al., 2002). In addition, problems with the sequence for CYP2D6 and associated pseudogenes have been discussed (Zanger et al., 2004), and this is further evidenced by the addition and withdrawal of GenBank sequences for this region of chromosome 22. Indeed, the Human Genome Project version of chromosome 22 was completely lacking in CYP2D6 sequence in its original release, implying either problems with sequencing this locus or a deletion of CYP2D6 in the sample used for sequencing (Zanger et al., 2004). Consequently at this juncture, there is not a suitable validated genomic reference sequence for the CYP2D locus.
Given the limitations of the technical approach used by the authors to type genomic DNA, a T deletion at 138 in CYP2D7 may not explain the conversion of this pseudogene to a functional gene. Therefore, a mechanistic explanation for the observed CYP2D7 protein expression in human brain is still needed.
Caution should be exercised when taking this work forward into future studies, and every effort should be made to validate the data using primer sequences and samples well characterized for CYP2D6 polymorphisms and amplification. This case highlights the difficulty and extra care needed when investigating regions of high sequence homology to other genes.
 |
Footnotes |
|---|
ABBREVIATION: bp, base pair(s).
Address correspondence to: Janelle M. Hoskins, School of Medicine, Washington University in St. Louis, 660 S. Euclid Ave, Box 8069, St. Louis, MO, 63110. E-mail: jhoskins{at}im.wustl.edu
|
References |
|---|
 Top References |
|---|
This article has been cited by other articles:
|
|
 |
|
  A. Bhathena, T. Mueller, D. R. Grimm, K. Idler, A. Tsurutani, B. B. Spear, and D. A. Katz Frequency of the Frame-Shifting CYP2D7 138delT Polymorphism in a Large, Ethnically Diverse Sample Population Drug Metab. Dispos., August 1, 2007; 35(8): 1251 - 1253. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Gaedigk and J. S. Leeder COMMENTS ON HOSKINS ET AL. [(2005) DRUG METAB DISPOS 33:1564-1565] Drug Metab. Dispos., March 1, 2006; 34(3): 504 - 505. [Full Text] [PDF]
|
|
|
|
|
|
H. V. Pai and V. Ravindranath RESPONSE TO COMMENTS ON HOSKINS ET AL. [(2005) DRUG METAB DISPOS 33:1564-1565] Drug Metab. Dispos., March 1, 2006; 34(3): 506 - 506. [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
 |
 |
|
 |
 |
 |
