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.

The Impact of the CYP2D6 “Enhancer” Single Nucleotide Polymorphism on CYP2D6 Activity

“…Consistent with other in vivo reports 17,18 our data did not find convincing evidence that the “enhancer” SNP is associated with a change in activity that would support revising values for AS calculation or CYP2D6 phenotype classification.…”

“…Although our cumulative data could be interpreted as being supportive of the “enhancer” SNP having a small impact on CYP2D6 activity, this may also be explained by recently published new findings 14 regarding the combinatorial effect of 2851C>T (p.R296C) and 4181G>C (p.S486T) 18 showing that reduced activity caused by 2851C>T can be compensated by 4181G>C and explain why CYP2D6*2 has activity that is similar to that of CYP2D6*1 . For CYP2D6*41 , Zanger et al .…”

“…to conclude that the “enhancer” SNP may account for unexplained variability in CYP2D6 activity 14 and propose a modified CYP2D6 activity score (AS) system including the “enhancer” SNP 16 . However, the impact of the “enhancer” SNP could not be confirmed in vitro 12 or in vivo 17,18 or remains inconclusive 17,19 . In fact, Zanger et al .…”

“…In fact, Zanger et al . elegantly demonstrated that variability in activity can rather be explained by the presence and combination of three SNPs (rs16947, rs28371725, and rs1135840) that define and differentiate the CYP2D6*2 and *41 haplotypes rather than the “enhancer” SNP 18 …”

Functional Characterization of 40 CYP3A4 Variants by Assessing Midazolam 1′-Hydroxylation and Testosterone 6β-Hydroxylation

“…Gutierrez Rico et al, 2020;Kumondai et al, 2018;Lee et al, 2005;Maekawa et al, 2010;Saito et al, 2018;Watanabe et al, 2018 DMD-AR-2020-000261 10 intronic CYP2D6 mutation was evaluated using a mini-gene expression system with transfected whole CYP2D6 sequences (<5 kb) (Zanger et al, 2020). Mini-genes may aid the assessment of CYP3A4 levels.…”