PharmVar GeneFocus: CYP2C9

Abstract

The Pharmacogene Variation Consortium (PharmVar) catalogues star (*) allele nomenclature for the polymorphic human CYP2C9 gene. Genetic variation within the CYP2C9 gene locus impacts the metabolism or bioactivation of many clinically important drugs, including nonsteroidal anti-inflammatory drugs, phenytoin, antidiabetic agents, and angiotensin receptor blockers. Variable CYP2C9 activity is of particular importance regarding efficacy and safety of warfarin and siponimod as indicated in their package inserts. This GeneFocus provides a comprehensive overview and summary of CYP2C9 and describes how haplotype information catalogued by PharmVar is utilized by the Pharmacogenomics Knowledgebase and the Clinical Pharmacogenetics Implementation Consortium.

Figure 1. Overview of the gene locus and allelic variation

Panel (a) provides a graphical overview of the CYP2C gene locus containing CYP2C18, CYP2C19, CYP2C9, and CYP2C8. The latter is encoded on the reverse strand (arrow) while the other three genes are encoded on the forward strand. CYP2C9 is composed of nine exons, covering 51.5 kb (GRCh37, 96,698,415 – 96,749,147; GRCh38, 94,938,658 – 94,990,091). Panel (b) shows the core sequence variations defining CYP2C9*2,*3, *5, *6, *8, *10, *11, *12, *13 and *15 representing Tier 1 and Tier 2 alleles per AMP allele recommendation guidelines (34). p.R144C is also present on CYP2C9*35 and *61 and is thus not unique to CYP2C9*2. Likewise, p.I359L is also part of several haplotypes, i.e., CYP2C9*3, *18, and *68.

Figure 2. Allele assignment by ‘default’, a commonly used strategy

Pharmacogenetic test platforms typically comprise the more commonly observed SNVs and do not test for all star (*) alleles catalogued by PharmVar. Consequently, some alleles may not be identified or receive an assignment by ‘default’. Therefore, it is important to understand which SNVs are tested and how alleles were called and translated into phenotype. Panel (a) shows that the relatively common SNV at c.430C>T (p.R144C) is not only a core SNV of CYP2C9*2, but also *35 and *61. Unless c.374G>T and c.1370A>G are also tested, a CYP2C9*35 or *61 allele will be called as a *2 potentially leading to an incorrect phenotype assignment. Since CYP2C9*2 and *61 are both classified as decreased function by CPIC, defaulting CYP2C9*61 as *2 will not impact the patient’s phenotype call. Panel (b) demonstrates that c.1075A>C (p.I359L) is also present in CYP2C9*18 and the recently discovered *68 alleles. c.1075A>C on its own causes severely decreased function and thus, CYP2C9*3 is classified by CPIC as ‘no function’ allele. CYP2C9*68 has an additional SNV interfering with splicing predicted to be deleterious/probably damaging (93). It remains unknown though whether p.D379A on CYP2C9*18 also impacts function. The yellow ‘zigzag’ arrow and the red X denote decreased and no function, respectively. Comparative Allele ViewEr (CAVE) outputs are shown for both examples to demonstrate the utility of this tool. The graph visualizes which core alleles have the tested SNVs and may be not be identified by limited testing and default allele assignments. Blue boxes indicate the presence of core SNVs. The function () symbol indicates that a core SNV alters function and the PharmVar () symbol highlights that a core SNV is unique to a star allele. SNV positions refer to the transcript (NM_000771.4).

Figure 3. Overview of core allele and suballele categorization

Panel (a) shows the CYP2C9*2 and *8 core allele definitions (gray bar) with NM_000771.4 as the reference sequence. Core SNVs, PharmVar ID (PVID), and evidence level is shown for each allele. All currently defined CYP2C9*2 suballeles and selected CYP2C9*8 suballeles are displayed underneath the core allele bar. Legacy allele designations are cross-referenced (e.g., *2.001 corresponds to *2A and *2B which have been merged). Panel (b) is a graphical representation of the CYP2C9*2 and *8 core alleles. Each is characterized by a single core SNV is highlighted in red (CYP2C9*2 by c.430C>T, p.R144C and CYP2C9*8 by c.449G>A, R150H). While c.449G>A is unique to CYP2C9*8, c.430C>T is not only found in the CYP2C9*2 haplotype (see Figure 2 for more details). Gray boxes represent the nine exons (scale is approximated); 3’UTR denotes the 3’ untranslated region. Panel (c) shows the CYP2C9*2 and *8 suballeles defined to date. As shown, suballeles of CYP2C9*2 only differ in their upstream region (graph only shows this portion of the gene), while those for CYP2C9*8 also vary in their 3’UTR regions (graph showing respective regions). ‘Lim’, ‘Mod’ and ‘Def’ symbols denote ‘Limited’, ‘Moderate’ and ‘Definitive’ haplotype evidence levels.

Figure 4. Characterization of novel allelic variants

Panels (a-d) provide examples of alleles submitted to PharmVar for naming or to confirm existing allele definitions. Variants of submitted alleles are highlighted by red lines. All submissions utilized WGS data which were either confirmed by WES or targeted NGS-based sequencing panels. Panel (a) exemplifies a subject whose haplotype can be unequivocally be deduced and Panel (b) shows a subject whose three heterozygous SNVs were placed on the same chromosome using 10X Genomics Linked-Read (long-distance) phasing technology; CYP2C9*1.009 and *71 both received an evidence level of ‘Definitive’ (‘Def’). Panel (c) depicts two examples for which haplotypes were computationally inferred. Due to uncertainty regarding the phase of the variants, CYP2C9*63 and CYP2C9*67 received evidence levels of ‘Moderate’ (Mod) and ‘Limited’ (Lim), respectively. Panel (d) illustrates how haplotype can be inferred using inheritance in a family trio.