Long-Distance Phasing of a Tentative Enhancer Single-Nucleotide Polymorphism With CYP2D6 Star Allele Definitions

Background The 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. Methods A 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. Results Phasing 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. Conclusions Since 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.