Abstract
BackgroundThe CYP2D6 gene locus has been extensively studied over decades, yet a portion of variability in CYP2D6 activity cannot be explained by known sequence variations within the gene, copy number variation, or structural rearrangements. It was proposed that rs5758550, located 116 kb downstream of the CYP2D6 gene locus, increases gene expression and thus contributes to variability in CYP2D6 activity. This finding has, however, not been validated. The purpose of the study was to address a major technological barrier, i.e., experimentally linking rs5758550, also referred to as the “enhancer” single-nucleotide polymorphism (SNP), to CYP2D6 haplotypes >100 kb away. To overcome this challenge is essential to ultimately determine the contribution of the “enhancer” SNP to interindividual variability in CYP2D6 activity.MethodsA large ethnically mixed population sample (n=3,162) was computationally phased to determine linkage between the “enhancer” SNP and CYP2D6 haplotypes (or star alleles). To experimentally validate predicted linkages, DropPhase2D6, a digital droplet PCR (ddPCR)-based method was developed. 10X Genomics Linked-Reads were utilized as a proof of concept.ResultsPhasing predicted that the “enhancer” SNP can occur on numerous CYP2D6 haplotypes including CYP2D6*1, *2, *5, and *41 and suggested that linkage is incomplete, i.e., a portion of these alleles do not have the “enhancer” SNP. Phasing also revealed differences among the European and African ancestry data sets regarding the proportion of alleles with and without the “enhancer” SNP. DropPhase2D6 was utilized to confirm or refute the predicted “enhancer” SNP location for individual samples, e.g., of n=3 samples genotyped as *1/*41, rs5758550 was on the *41 allele of two samples and on the *1 allele of one sample. Our findings highlight that the location of the “enhancer” SNP must not be assigned by “default.” Furthermore, linkage between the “enhancer” SNP and CYP2D6 star allele haplotypes was confirmed with 10X Genomics technology.ConclusionsSince the “enhancer” SNP can be present on a portion of normal, decreased, or no function alleles, the phase of the “enhancer” SNP must be considered when investigating the impact of the “enhancer” SNP on CYP2D6 activity.
Highlights
The highly polymorphic CYP2D6 gene encodes the cytochrome P450 2D6 enzyme, which contributes to the metabolism and bioactivation of numerous clinically used drugs (Zhou, 2009a; Zhou, 2009b; Saravanakumar et al, 2019)
There are, no additional published studies to date corroborating the impact of the “enhancer” single nucleotide polymorphism (SNP) on CYP2D6 activity in-vivo demonstrating that combined genotyping for rs5758550 and rs16947 is superior over the current methods focusing on SNPs that identify star alleles of interest
When phasing was done on the entire cohort, 16 alleles were predicted to have the “enhancer” SNP on a CYP2D6*29 allele
Summary
The highly polymorphic CYP2D6 gene encodes the cytochrome P450 2D6 enzyme, which contributes to the metabolism and bioactivation of numerous clinically used drugs (Zhou, 2009a; Zhou, 2009b; Saravanakumar et al, 2019). Searching for additional loci beyond the immediate CYP2D6 gene region, Wang et al (2015) described that rs5758550, located 116 kb downstream of exon 9 of the CYP2D6 gene' is associated with CYP2D6 activity This single nucleotide polymorphism (SNP) has been proposed to impact CYP2D6 activity by modulating expression levels, and may account for unexplained variability, especially within a given diplotype (Wang et al, 2014; Wang et al, 2015). The purpose of the study was to address a major technological barrier, i.e., experimentally linking rs5758550, referred to as the “enhancer” single-nucleotide polymorphism (SNP), to CYP2D6 haplotypes >100 kb away To overcome this challenge is essential to determine the contribution of the “enhancer” SNP to interindividual variability in CYP2D6 activity
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
