CYP2C8, CYP2C9, and CYP2C19 Characterization Using Next-Generation Sequencing and Haplotype Analysis: A GeT-RM Collaborative Project

Abstract

Pharmacogenetic tests typically target selected sequence variants to identify haplotypes that are often defined by star (∗) allele nomenclature. Due to their design, these targeted genotyping assays are unable to detect novel variants that may change the function of the gene product and thereby affect phenotype prediction and patient care. In the current study, 137 DNA samples that were previously characterized by the Genetic Testing Reference Material (GeT-RM) program using a variety of targeted genotyping methods were recharacterized using targeted and whole genome sequencing analysis. Sequence data were analyzed using three genotype calling tools to identify star allele diplotypes for CYP2C8, CYP2C9, and CYP2C19. The genotype calls from next-generation sequencing (NGS) correlated well to those previously reported, except when novel alleles were present in a sample. Six novel alleles and 38 novel suballeles were identified in the three genes due to identification of variants not covered by targeted genotyping assays. In addition, several ambiguous genotype calls from a previous study were resolved using the NGS and/or long-read NGS data. Diplotype calls were mostly consistent between the calling algorithms, although several discrepancies were noted. This study highlights the utility of NGS for pharmacogenetic testing and demonstrates that there are many novel alleles that are yet to be discovered, even in highly characterized genes such as CYP2C9 and CYP2C19.

Figure 1

CYP2C8 haplotype not recognized by the calling tools. Next-generation sequencing revealed NM_000770.3:c.992T>A (rs146806199) in NA19917 (bold outline in pedigree). This missense variant causes a p.Ile331Thr change in exon 7. The haplotype has two additional variants in the 5′ untranslated region (NM_000770.3:c.-6G>A and NM_000770.3:c.-86A>G). The function of this allele is unknown. As shown in the pedigree, the novel allele was inherited by the offspring (NA19918). The phase of the CYP2C8∗16 allele in NA19917 was further corroborated by 10x Linked-Read technology. Because this allele is not part of any of the allele calling tools, it was called as CYP2C8∗1/∗1. The CYP2C8∗2.002 suballele in NA19916 was also only recently designated by PharmVar. Variants inherited together from mother to the child are shown in red, whereas those present on the father (shown in blue) were not passed to the child. Transcript and genomic reference sequences (RefSeqs) are available from the National Center for Biotechnology information (NCBI) Reference Sequence Database (https://www.ncbi.nlm.nih.gov/refseq, last accessed September 20, 2021).

Figure 2

Novel CYP2C9 and CYP2C19 alleles. 10x Genomics Linked-Read data were utilized to phase observed sequence variants across respective genes. A: A Loupe screenshot showing that the core variants are in cis and thus form a novel CYP2C9 haplotype (CYP2C9∗71) in NA15245. B: A Loupe screenshot showing two haplotypes, one corresponding to the CYP2C19∗2.011 suballele, whereas the second allele represents the novel CYP2C19∗35.002 suballele in NA19122. C and D: All variants found on respective CYP2C9 and CYP2C19 haplotypes of samples NA15245 and NA19122, respectively, are shown.

Figure 3

Discovery of novel CYP2C9∗8 suballeles. The top three lines represent the CYP2C9∗8.001, CYP2C9∗8.002, and CYP2C9∗8.003 suballeles that were defined by PharmVar before the start of the investigation. Of those, only CYP2C9∗8.003 was found among the study samples (the presence of CYP2C8∗8.003 was inferred; no 10x Genomics data were available to confirm this allele call). Two novel CYP2C9∗8 suballeles, designated CYP2C9∗8.004 and CYP2C9∗8.005, were identified. The latter was discovered in NA12815 and the phase of the two variants informed by inheritance in a trio for which data were obtained from the 1000 Genomes Project; the subject in question is a member of a large pedigree. Although this novel allele has NM_000771.4:c.449G>A, p.Arg150His, it lacked NM_000771.4:c.-1766T>C (rs9332094). The core variant of the CYP2C9∗8 allele is highlighted in red. Transcript and genomic reference sequences (RefSeqs) are available from the National Center for Biotechnology information (NCBI) Reference Sequence Database (https://www.ncbi.nlm.nih.gov/refseq, last accessed September 20, 2021).

Figure 4

CYP2C9 missense variant NM_000771.4:c.1147A>T. A missense variant was discovered in NA18966 at NM_000771.4:c.1147A>T, which introduces a stop codon (p.Lys383Ter). A: A forward Sanger sequence trace for NA18966 with the reference c.1147A being the dominant peak. The trace for a CYP2C9∗1/∗1 control sample, NA18564, is shown for comparison. B: Selected WGS-1 and PGRNseq read alignments with most reads having the reference c.1147A. Read distributions for the variant T were 4.8% (PGRNseq v1, shown), 11% (WGS-2), 17.1% (WGS-1, shown), and 18% (ADMEseq) reads. The variant is visualized by red horizontal bars and % reads shown in red font. Transcript and genomic reference sequences (RefSeqs) are available from the National Center for Biotechnology Information (NCBI) Reference Sequence Database (https://www.ncbi.nlm.nih.gov/refseq, last accessed September 20, 2021).