The CYP2D gene subfamily: analysis of the molecular basis of the debrisoquine 4-hydroxylase deficiency in DA rats

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Abstract
The DA rat has been proposed as an animal model for the human debrisoquine 4-hydroylase/bufuralol 1''-hydroxylase genetic deficiency. To determine the mechanism of this deficiency, we isolated and sequenced five cDNAs in the CYP2D gene subfamily including a new IID1 allele and two cDNAs of a novel P450s, designated IID3 and IID5. IID3 and IID5 cDNA-deduced amino acid sequences contained 500 and 504 residues with calcuated molecular weights of 56683 and 57801, respectively. IID5 displayed 20 amino acid differences with the IID1, yet bore only 72% and 76% similarity to IID2 and IID3. Despite an overall nucleotide similarity of 80-98% between the 4 cDNAs, a region of 134 nucleotides of sequence exists that contains only 1 base differences. This region is probably the result of gene conversion events between the P450 IID genes. Although all IID cDNAs were expressed in immunodetectable proteins using the COS cell SV40-based expression system, only IID1 could effectively catalyse the oxidation of the prototype substrate bufuralol. Expression of a cDNA isolated in an earlier study [Gonzalez, F. J., Matsunaga, T., Nagata, K., Meyer, U. A., Nebert, D. W. Pastewka, J., Kozak, C. A., Gillette, J., Gelboin, H. V., and Hardwick, J. P. (1987) DNA 6, 149-161], previously called db1 and now designated IID1v, produced a protein with a drastically reduced activity as compared to cDNA-expressed IID1 despite only four amino acid differences between the two cDNA-deduced protein sequences. IID1 and IID1v appear to be allelic variants of the same gene. To determine the mechanism of the debrisoquine/bufuralol drug oxidation deficiency in DA rat, specific cDNA and oligonucleotide probes were used to quantitate levels of each mRNA in Sprague-Dawley and DA rat livers. The former rat strain expressed IID1, IID2, IID3, and IID5 mRNAs, whereas the DA rat expressed only IID2, IID3, and IID5 mRNAs; the IID1 gene was not expressed in this rat strain. Moreover, immunoinhibition studies using a strongly inhibitory antibody suggested a major contribution of IID1 to bufuralol metabolism in SD but not in DA rats. These results establish that the DA rat drug oxidation polymorphism is due to the absence of expression of the IID1 gene.