Tri-Allelic Haplotypes Determine and Differentiate Functionally Normal Allele CYP2D6*2 and Impaired Allele CYP2D6*41.

Abstract

CYP2D6 metabolizes 20-25% of all clinically used drugs and its complex genetic polymorphism is a major determinant of drug safety and efficacy. We investigated the basis for the functional difference between the two common alleles *2 (g.2851C>T+g.4181G>C, normal function) and *41 (additional intronic g.2989G>A, reduced function). A recently reported far-distant enhancer polymorphism rs5758550A/G linked to *2 has been suggested to play a decisive role. Genotyping of two white cohorts confirmed strong linkage of rs5758550G to *2, whereas no influence was found on metabolic ratio of sparteine or hepatic expression. Genomic plasmid constructs carrying individual variants or combinations thereof were expressed in COS1 and Huh7 cells. Both g.2851C>T(R296C) and g.2989G>A reduced enzyme activity and protein levels similarly by ~50-65% compared to reference (*1), whereas the double variant had only ~20% activity. Although the unexpected loss of function caused by g.2851C>T was compensated by g.4181G>C (mimicking the EM-phenotype of *2), the additional loss of function due to intronic g.2989G>A in the triple variant was not compensated (mimicking the IM-phenotype of *41). We also confirmed increased erroneous splicing in carriers of g.2989G>A but not of g.2851C>T as a likely explanation for the impaired function of *41. In conclusion, our data demonstrate g.2989G>A as causal variant of impaired allele CYP2D6*41 whereas triple-haplotypes have to be considered to explain the functional difference between *2 and *41. These data are important for genotyping strategies and clinical implementation of CYP2D6 pharmacogenetics.