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Objective: Hmong individuals represent a unique East Asian subpopulation in whom limited information concerning pharmacogenetic variation exists. The objectives of this study were to comprehensively characterize the highly polymorphic CYP2D6 gene in Hmong, estimate allele and phenotype frequencies and to compare results between two testing platforms.Methods: DNA from 48 self-identified Hmong participants were sequenced using a targeted next-generation sequencing (NGS) panel. Star allele calls were made using Astrolabe, manual inspection of NGS variant calls and confirmatory Sanger sequencing. Structural variation was determined by long-range (XL)-PCR and digital droplet PCR (ddPCR). The consensus diplotypes were subsequently translated into phenotype utilizing the activity score system. Clinical grade pharmacogenetic testing was obtained for 12 of the 48 samples enabling an assessment of concordance between the consensus calls and those determined by clinical testing platforms.Results: A total of 13 CYP2D6 alleles were identified. The most common alleles were CYP2D6*10 and its structural arrangements (37.5%, 36/96) and the *5 gene deletion (13.5%, 13/96). Three novel suballeles (*10.007, *36.004, and *75.002) were also identified. Phenotype frequencies were as follows: ultrarapid metabolizers (4.2%, 2/48), normal metabolizers (41.7%, 20/48) and intermediate metabolizers (52.1%, 25/48); none of the 48 participants were predicted to be poor metabolizers. Concordance of diplotype and phenotype calls between the consensus and clinical testing were 66.7 and 50%, respectively.Conclusion: Our study to explore CYP2D6 genotypes in the Hmong population suggests that this subpopulation is unique regarding CYP2D6 allelic variants; also, a higher portion of Hmong participants (50%) are predicted to have an intermediate metabolizer phenotype for CYP2D6 compared to other East Asians which range between 27 and 44%. Results from different testing methods varied considerably. These preliminary findings underscore the importance of thoroughly interrogating unique subpopulations to accurately predict a patient’s CYP2D6 metabolizer status.

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’ 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=3162) 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 amongst 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 *1 allele of one sample. Our findings highlights 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. We were unable, however, to verify selected haplotypes predicted by Ray et al (PMID 30520769). 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.