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

Abstract

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

PharmVar GeneFocus: CYP2C19

“…Figure illustrates the utility of single molecule real time sequencing methods (e.g., Pacific Biosciences, Menlo Park, CA or Oxford Nanopore Technologies, Oxford, UK, and 10X Genomics Long‐Read mapping, Pleasanton, CA) to establish haplotypes over a range of distances. SNVs can also be phased over long distances of up to 200 kb using digital droplet PCR to establish haplotypes 91,92 . In Figure , we offer an example of a PharmVar submission for CYP2C19*35 .…”

“…SNVs can also be phased over long distances of up to 200 kb using digital droplet PCR to establish haplotypes. 91,92 In Figure 4d, we offer an example of a PharmVar submission for CYP2C19*35. The haplotype of the CYP2C19*35 was determined from whole genome sequence data of each member of the family trio (father, mother, and child).…”

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

“…Since 2014, Wang and colleagues, in a series of papers characterized a novel polymorphic region, located ~ 115 kb downstream of CYP2D6 and comprising two SNPs rs133333A>G and rs5758550A>G in tight linkage disequilibrium (LD) 28–31 . The rs5758550A>G SNP apparently turns the region into an enhancer that interacts with the CYP2D6 promoter and increases transcription.…”

PharmVar GeneFocus: CYP2B6

“…If a sample is heterozygous for more than one SNV, additional analyses will be necessary to establish whether SNVs are in cis or in trans using techniques, such as allele-specific PCR or digital droplet PCR. 111 Haplotypes may also be computationally inferred from sequencing data, but only receive a Lim or Mod evidence level depending on the number of subjects used for phasing, the frequency of the allele and/ or whether uncertainty of SNVs being in cis or trans affects core SNVs. We refer to the recently updated criteria for computationally inferred haplotypes (see the allele definition criteria and evidence level document for more details and examples).…”