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. Author manuscript; available in PMC: 2020 Nov 1.
Published in final edited form as: Hum Mutat. 2019 Nov;40(11):e37–e51. doi: 10.1002/humu.23855

Structural variation at the CYP2C locus: Characterization of deletion and duplication alleles

Mariana R Botton 1,2,*, Xingwu Lu 1,2, Geping Zhao 2, Elena Repnikova 3,4, Yoshinori Seki 2, Andrea Gaedigk 4,5, Eric E Schadt 1,2, Lisa Edelmann 1,2, Stuart A Scott 1,2,†
PMCID: PMC6810756  NIHMSID: NIHMS1039268  PMID: 31260137

Abstract

The human CYP2C locus harbors the polymorphic CYP2C18, CYP2C19, CYP2C9 and CYP2C8 genes, and of these, CYP2C19 and CYP2C9 are directly involved in the metabolism of ~15% of all medications. All variant CYP2C19 and CYP2C9 star (*) allele haplotypes currently catalogued by the Pharmacogene Variation (PharmVar) Consortium are defined by sequence variants. To determine if structural variation also occurs at the CYP2C locus, the 10q23.33 region was interrogated across deidentified clinical chromosomal microarray (CMA) data from 20,642 patients tested at two academic medical centers. Fourteen copy number variants that affected the coding region of CYP2C genes were detected in the clinical CMA cohorts, which ranged in size from 39.2–1,043.3 kb. Selected deletions and duplications were confirmed by MLPA or ddPCR. Analysis of the clinical CMA and an additional 78,839 cases from the Database of Genomic Variants (DGV) and ClinGen (total n=99,481) indicated that the carrier frequency of a CYP2C structural variant is ~1 in 1000, with ~1 in 2,000 being a CYP2C19 full-gene or partial-gene deletion carrier, designated by PharmVar as CYP2C19*36 and *37, respectively. Although these structural variants are rare in the general population, their detection will likely improve metabolizer phenotype prediction when interrogated for research and/or clinical testing.

Keywords: CYP2C, CYP2C19, CYP2C9, copy number variation, deletion, duplication, pharmacogenomics, chromosomal microarray, database

INTRODUCTION

The human cytochrome P450 (CYP) enzyme superfamily is responsible for the oxidative metabolism of many drugs, xenobiotics, and other endogenous substances. The polymorphic CYP2C locus at chromosome 10q23.33 is comprised of the CYP2C18, CYP2C19, CYP2C9 and CYP2C8 genes, which encode enzymes that together are involved in the hepatic metabolism of ~25% of commonly prescribed drugs. Moreover, CYP2C19 and CYP2C9 metabolize ~15% of drugs currently listed on the U.S. Food and Drug Administration (FDA) Pharmacogenomic Biomarkers in Drug Labeling table (www.fda.gov/Drugs/ScienceResearch/ResearchAreas/Pharmacogenetics/ucm083378.htm, which includes medications commonly used in neurology, rheumatology, psychiatry, cardiology, gastroenterology, gynecology, and infectious disease.

Currently 35 CYP2C19 and 60 CYP2C9 variant star (*) allele haplotypes are catalogued in the Pharmacogene Variation Consortium (PharmVar) database (www.pharmvar.org) (Gaedigk et al., 2018; Gaedigk et al., 2019). Without exception, all are defined by sequence variants. As such, no CYP2C19 or CYP2C9 star (*) alleles currently include structural variation (e.g., copy number variants (CNV)), which is consistent with our previously reported pilot study that employed multiplex ligation-dependent probe amplification (MLPA) screening of this gene region across a multi-ethnic cohort of ~500 individuals (Martis et al., 2013). However, pharmacogenomic CNV alleles can play important roles in enzyme activity and drug response variability (He, Hoskins, & McLeod, 2011), which have been characterized among some cytochrome P450 (CYP2B6, CYP2D6), glutathione S-transferase (GSTT1, GSTM1), and sulfotransferase (SULT1A1, SULT2A1) genes (Gaedigk, Gaedigk, & Leeder, 2010; Gaedigk, Twist, & Leeder, 2012; Gjerde et al., 2008; Martis et al., 2013; Schulze et al., 2013; Vijzelaar et al., 2018). Notably, interrogation of publicly available sequencing data has recently indicated that some populations harbor CNV alleles at the CYP2C gene region (Santos et al., 2018). The potential clinical significance of low frequency structural variation at the CYP2C gene region in the general population prompted our interrogation of chromosomal microarray (CMA) data from multiple database sources for pharmacogenomic CNV discovery.

DATA SPECIFICATIONS

Data type Tables of copy number variant (CNV) coordinates
Data acquisition method Chromosomal microarray (CMA)
Data format Analyzed
Experimental factors Germline DNA tested by CMA for clinical indication or involvement in population genomics research
Experimental features CYP2C gene region [chr10:96100000_97100000 (GRCh37/hg19)] was interrogated across CMA data from 99,481 individuals
Data source location New York, NY, Kansas City, MO, publicly available CNV databases
Data accessibility Data in this study are available at public repositories:
Leiden Open Variation Database (LOVD) (https://databases.lovd.nl; DOI: 10.1002/humu.21438) cases:
https://databases.lovd.nl/shared/individuals/00239123
https://databases.lovd.nl/shared/individuals/00239124
https://databases.lovd.nl/shared/individuals/00239125
https://databases.lovd.nl/shared/individuals/00239126
https://databases.lovd.nl/shared/individuals/00239127
https://databases.lovd.nl/shared/individuals/00239128
https://databases.lovd.nl/shared/individuals/00239129
https://databases.lovd.nl/shared/individuals/00239130
https://databases.lovd.nl/shared/individuals/00239131
https://databases.lovd.nl/shared/individuals/00239132
https://databases.lovd.nl/shared/individuals/00239133
https://databases.lovd.nl/shared/individuals/00239134
https://databases.lovd.nl/shared/individuals/00239135
https://databases.lovd.nl/shared/individuals/00239136
https://databases.lovd.nl/shared/individuals/00239137
Clinical Genome Consortium (http://dbsearch.clinicalgenome.org/search/; DOI: 10.1056/NEJMsr1406261)
Database of Genomic Variants (http://dgv.tcag.ca; DOI: 10.1093/nar/gkt958)
DECIPHER (https://decipher.sanger.ac.uk; DOI: 10.1016/j.ajhg.2009.03.010)
Pharmacogene Variation Consortium (PharmVar) database (www.pharmvar.org; DOI: 10.1002/cpt.1268)
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IMPACT OF DATA

In contrast to the well-described pharmacogenomic alleles that are relatively common in the general population (e.g., CYP2C9*2, CYP2C19*2), it is increasingly appreciated that the majority of human genetic variation is actually rare [minor allele frequency (MAF) <1%] (Genomes Project et al., 2010; Tennessen et al., 2012), making association studies between these variants and drug response phenotypes challenging (Verma et al., 2018). To facilitate the discovery of low frequency variants that potentially influence drug response, recent studies have interrogated high-throughput sequencing data across drug target genes (Nelson et al., 2012), CYP450 genes (Gordon et al., 2014), and selected drug absorption, distribution, metabolism and excretion (ADME) and other candidate pharmacogenes (Bush et al., 2016; Li et al., 2014; Santos et al., 2018), all indicating that rare and potentially functional pharmacogenomic variants are prevalent in diverse populations. These studies highlight the importance of interrogating large datasets for rare pharmacogenomic variation discovery, which prompted our interrogation of deidentified CMA data from large databases to further study low frequency pharmacogenomic structural variation at the clinically relevant CYP2C gene cluster region.

MATERIALS AND METHODS

Experimental Design

The design of this study included interrogation of multiple databases of CMA data, including both clinical cytogenomic laboratory CMA data (discovery and replication cohorts), as well as publicly available CMA data from the general population (research and clinical cohorts). Selected CYP2C gene deletion and duplication samples from the discovery and replication cohorts were analytically confirmed by orthogonal copy number methods.

Clinical Cytogenomic Testing Cohort – Discovery

Individuals in the discovery cohort were referred to the Cytogenetics and Cytogenomics Laboratory at Mount Sinai Genomics Inc. (DBA Sema4), New York, from 2010 to 2017 for pre- or postnatal clinical CMA testing. All patients were tested with informed consent and deidentified CMA data were stored in an internal database that enabled interrogation and CNV frequency analyses. A total of 11,096 unique samples were analyzed, which included 6,083 prenatal (amniotic fluid, chorionic villus specimens, fetal blood) and 5,013 postnatal (peripheral blood, products of conception) samples. Although race and ethnicity were not commonly available, the prenatal cohort self-reported as white (82.5%), Asian (8.0%), black (4.7%), Hispanic/Latino (4.5%) and American Indian (0.36%), and the postnatal cohort self-reported as white (59.1%), Hispanic/Latino (19.1%), black (11.8%), Asian (6.3%), and American Indian (3.8%).

Clinical Cytogenomic Testing Cohort – Replication

Individuals in the replication cohort were referred to the Clinical Genetics and Genomics Laboratory at Children’s Mercy, Kansas City, from 2009 to 2018 for pre- or postnatal clinical CMA testing. All patients were tested with informed consent and deidentified CMA data were stored in an internal database that enabled interrogation and CNV frequency analyses. A total of 9,760 unique peripheral blood samples were analyzed as a replication cohort for structural variation at the CYP2C locus. Race and ethnicity demographics of the patient cohort were not available.

Chromosomal Microarray (CMA) Analysis – Discovery

CMA was performed on the discovery cohort using the Agilent Technologies platform (Santa Clara, CA, USA) according to the manufacturer’s instructions and as previously reported (Reiner et al., 2017; Scott et al., 2010). Throughout the period of clinical CMA testing and data analysis (2010–2017), three commercial microarrays were used that had increasing probe density and resolution [44K (design 015141), 105K (design 031750), and 180K (design 029830); Agilent Technologies]; however, all three microarray designs had adequate probe coverage across the CYP2C gene cluster region to detect multi-exon CNVs within CYP2C18 (NG_008373.1), CYP2C19 (NG_008384.3), CYP2C9 (NG_008385.1), and/or CYP2C8 (NG_007972.1) (Figure 1). All genomic coordinates are reported using NCBI human genome reference Build 37 (GRCh37/hg19).

Figure 1.

Figure 1

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Illustration of the chromosome 10q23.33 region with the identified CYP2C copy number variants (CNVs) noted in relation to the location of known human genes and transcripts from the UCSC Genes track, segmental duplications, microarray (purple), MLPA (light green), and ddPCR (dark green) probe locations, structural variants from the Database of Genomic Variants (DGV), ClinGen, and DECIPHER. Blue bars represent copy number gains (duplications), red bars represent copy number losses (deletions). The minimum and maximum CNV sizes in the clinical CMA cohorts are denoted by thick and thin horizontal bars, respectively.

Chromosomal Microarray (CMA) Analysis – Replication

CMA was performed on the replication cohort using the Affymetrix Cytoscan® HD CNV+SNP array platform (Santa Clara, CA, USA) or the Agilent 244K whole genome oligonucleotide microarray (design 014693; Santa Clara, CA, USA) according to the manufacturer’s instructions. The CytoScan® HD microarray contains 1,953,246 non-polymorphic and 743,304 single nucleotide polymorphism (SNP) markers, which are enriched in disease gene areas, and the Agilent 244K microarray contains ~244,000 oligonucleotide probes spaced at a median distance of 6.4 kb across the human genome (Figure 1). Data were analyzed by ChAS 3.2 (Affymetrix) or Genomic Workbench (Agilent Technologies) software as appropriate. As above, all genomic coordinates are reported using NCBI human genome reference Build 37 (GRCh37/hg19).

Copy Number Variation (CNV) Confirmation

Multiplex Ligation-dependent Probe Amplification (MLPA)

Copy number results from CMA testing were validated by multiplex ligation-dependent probe amplification (MLPA) testing on samples from the discovery cohort with available DNA. MLPA was performed using the Cytochrome P-450 MLPA kit (P128-B1; MRC-Holland, Amsterdam, The Netherlands) according to the manufacturer’s instructions and as previously reported (Martis et al., 2013; Vijzelaar et al., 2018). This commercial MLPA probe mix includes three CYP2C19 probes (exons 2, 6, and 9) and five CYP2C9 probes (exons 2, 7, 8 [2 probes], and 9) (Figure 1), plus an additional 34 probes that interrogate 12 other pharmacogenetic genes (CYP2D6, CYP1B1, CYP3A4, CYP3A5, CYP2E1, CYP1A1, CYP1A2, CYP2A6, CYP2B6, GSTP1, GSTT1 and GSTM1) (Martis et al., 2013). Amplified products were separated by capillary gel electrophoresis and analyzed using GeneMarker v1.90 software (SoftGenetics, State College, PA). After quality control and data normalization, copy number was determined according to the following peak ratio ranges: one copy >0.25 and <0.75; two copies >0.75 and <1.25; three copies >1.25 and <1.7; four copies >1.7 and <2.0.

Droplet Digital PCR (ddPCR)

Copy number results from CMA testing were also validated by droplet digital PCR (ddPCR) testing on samples from the replication cohort with available DNA. TaqMan™ copy number assays targeting CYP2C19 exon 2 (Hs05148033_cn) and intron 6 (Hs02932336_cn) were employed and signals normalized against the TERT gene (Cat# 4403316; Thermo Fisher, Waltham, MA) (Figure 1), and analysis was performed using the Bio-Rad QX-200 Droplet Digital PCR System (Bio-Rad, Hercules, CA). Genomic DNA were digested with EcoRI-HF (New England BioLabs, Ipswich, MA) and inactivated at 65ºC. Digested DNA were subsequently combined with 1X ddPCR Supermix for Probes (Bio–Rad, Hercules, CA), TaqMan™ and TERT reference assays. Droplets were generated with the Auto Droplet Generator and cycled in a C1000 Touch Thermocycler using recommended parameters. Droplets were analyzed with the QX200 Droplet Reader instrument and data analysis performed with the Quantasoft™ Software (Bio-Rad, Hercules, CA).

Cytogenomic Copy Number Variation (CNV) Population Cohorts

Structural variation databases were also interrogated to identify CYP2C region CNVs in healthy and clinical cohorts. Three independent sources were utilized: (1) the Database of Genomic Variants (DGV) (http://dgv.tcag.ca), which catalogues structural variation (>50 bp) in healthy population samples; (2) Clinical Genome Consortium (ClinGen) (http://dbsearch.clinicalgenome.org/search/), which catalogues genomic variation in patient population samples; and (3) DECIPHER (https://decipher.sanger.ac.uk), which also catalogues genomic variation in patient population samples. A 1 Mb genomic region was queried across all public databases [chr10:96100000_97100000 (GRCh37/hg19)], and only CNVs that included coding regions of any CYP2C gene were included in the study. Larger overlapping chromosome 10q23.33-q24.1 deletions and duplications (>2 Mb) in the clinical databases were excluded from the CYP2C CNV allele and carrier frequency analyses, as these likely pathogenic aberrations would be more consistent with a syndromic Mendelian phenotype.

DATA:

Chromosomal Microarray (CMA) Detection of CYP2C Deletions and Duplications

The CMA probe coverage across the CYP2C region for all clinical microarrays used in the study are illustrated in Figure 1. In the discovery cohort (ISMMS/Sema4; n=11,096), CYP2C gene region CNVs were detected in nine unrelated patients, including seven deletions and two duplications (Table 1 and Table 2). The identified deletions ranged in size from 52.0 kb (exons 8 to 9 of CYP2C18 and exons 1 to 4 of CYP2C19) to 421.0 kb (including TBC1D12, HELLS, CYP2C18, and CYP2C19) (Table 1 and Figure 1). The identified duplications were larger, 663.8 kb and 1.0 Mb, and included all CYP2C (CYP2C18, CYP2C19, CYP2C9, CYP2C8) and the neighboring ACMS6, PDLIM1 and SORBS1 genes (Table 1 and Figure 1). In the replication cohort (CMH; n=9,760), CYP2C region CNVs were detected in six unrelated patients, including three CYP2C deletions and three CYP2C duplications (Table 1 and Table 2). The deletions ranged in size from 39.2 kb to 61.5 kb (exons 1 to 5 of CYP2C19) (Table 1 and Figure 1), whereas the duplications (130.8 and 131.9 kb) included the 3’ region of CYP2C19 (exon 9 with or without exon 8) and exons 1 to 7 of CYP2C9 (Table 1 and Figure 1). All CYP2C CNV alleles identified in the discovery and replication CMA cohorts were submitted to the Leiden Open Variation Database (LOVD) (https://databases.lovd.nl) (Fokkema et al., 2011), and their unique variant IDs are listed in Table 1.

Table 1.

CYP2C gene region copy number variants identified by clinical chromosomal microarray (CMA) testing.

Sample ISCN 2016 Nomenclature Microarray Size (kb) Genes Included LOVD Variant ID
Discovery Cohort
 ISMMS/Sema4_6 arr[GRCh37] 10q23.33(96488443_96540495)x1 Agilent 180K 52.1 CYP2C18 (exons 8–9), CYP2C19 (exons 1–4) 0000484257
 ISMMS/Sema4_7 arr[GRCh37] 10q23.33(96488443_96540495)x1 Agilent 180K 52.1 CYP2C18 (exons 8–9), CYP2C19 (exons 1–4) 0000484258
 ISMMS/Sema4_5 arr[GRCh37] 10q23.33(96407740_96521658)x1 Agilent 180K 113.9 CYP2C18 (exons 1–8), CYP2C19 (exon 1) 0000484256
 ISMMS/Sema4_4† arr[GRCh37] 10q23.33(96470997_96602860)x1 Agilent 180K 131.9 CYP2C18 (exons 5–8), CYP2C19 (exons 1–7) 0000484255
 ISMMS/Sema4_1 arr[GRCh37] 10q23.33(96447479_96606715)x1 Agilent 105K 159.2 CYP2C18 (exons 2–8), CYP2C19 (exons 1–7) 0000484252
 ISMMS/Sema4_2† arr[GRCh37] 10q23.33(96361629_96612764)x1 Agilent 44K 251.5 HELLS, CYP2C18, CYP2C19 0000484253
 ISMMS/Sema4_3 arr[GRCh37] 10q23.33(96192063_96612764)x1 Agilent 44K 421.1 TBC1D12, HELLS, CYP2C18, CYP2C19 0000484254
 ISMMS/Sema4_8† arr[GRCh37] 10q23.33q24.1(96383288_97047098)x3 Agilent 180K 663.8 CYP2C18, CYP2C19, CYP2C9, CYP2C8, ACMS6, PDLIM1 0000484259
 ISMMS/Sema4_9 arr[GRCh37] 10q23.33q24.1(96161314_97204585)x3 Agilent 180K 1043.3 CYP2C18, CYP2C19, CYP2C9, CYP2C8, ACMS6, PDLIM1, SORBS1 (exons 3–32) 0000484260
Replication Cohort
 CMH_3 arr[GRCh37] 10q23.33(96507212_96546422)x1 Agilent 244K 39.2 CYP2C19 (exons 1–5) 0000484261
 CMH_1 arr[GRCh37] 10q23.33(96497260_96558710)x1 Affymetrix 61.5 CYP2C19 (exons 1–5) 0000484262
 CMH_2 arr[GRCh37] 10q23.33(96497260_96558710)x1 Affymetrix 61.5 CYP2C19 (exons 1–5) 0000484263
 CMH_5 arr[GRCh37] 10q23.33(96610653_96741497)x3 Affymetrix 130.8 CYP2C19 (exon 9), CYP2C9 (exons 1–7) 0000484264
 CMH_6 arr[GRCh37] 10q23.33(96610653_96741497)x3 Affymetrix 130.8 CYP2C19 (exon 9), CYP2C9 (exons 1–7) 0000484265
 CMH_4 arr[GRCh37] 10q23.33(96609567_96741497)x3 Affymetrix 131.9 CYP2C19 (exons 8–9), CYP2C9 (exons 1–7) 0000484266
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†

Ethnicity for individuals ISMMS/Sema4_2, ISMMS/Sema4_4, and ISMMS/Sema4_8 is Hispanic/Latino, White, and Asian, respectively. Ethnicity was not available for all other reported subjects.

Affymetrix: refers to the Cytoscan® microarray; CMH: Children’s Mercy Hospital; ISMMS: Icahn School of Medicine at Mount Sinai; LOVD: Leiden Open Variation Database (https://databases.lovd.nl).

Table 2.

CYP2C copy number variant (CNV) confirmation by multiplex ligation-dependent probe amplification (MLPA).

SAMPLE ID CYP2C19 CYP2C9
Exon 2 Exon 6 Exon 9 Exon 1 Exon 7 Exon 8A Exon 8B Exon 9
ISMMS/Sema4_4 0.499 0.458 0.517 0.928 0.962 0.953 0.989 0.961
ISMMS/Sema4_5 * 0.977 1.030 1.004 1.016 0.995 0.982 0.949 1.075
ISMMS/Sema4_6 0.508 0.903 0.968 0.953 0.959 1.005 1.041 1.014
ISMMS/Sema4_7 0.481 0.854 0.992 1.004 1.010 1.027 1.064 0.984
ISMMS/Sema4_9 1.440 1.665 1.297 1.209 1.561 1.368 1.487 1.577
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*

A 113.9 kb deletion was detected by CMA in this sample that included exons 1–8 of CYP2C18 and only exon 1 of CYP2C19.

Light gray shaded cells indicate heterozygous deletion or duplication by CMA testing. MLPA ratios: one copy >0.25 and <0.75; two copies: >0.75 and <1.25; three copies >1.25 and <1.7.

Confirmation of CYP2C Copy Number Variants (CNVs)

Among all subjects with CYP2C CNVs identified by CMA testing, five samples from the discovery cohort had available DNA for confirmation by MLPA testing. The locations of the MLPA probes in relation to the CMA probes are illustrated in Figure 1. All MLPA results were consistent with the CNVs detected by CMA testing (Table 2). Of note, given that the CMA and MLPA platforms have unique probe locations to interrogate copy number across the CYP2C region, deletions that affected only CYP2C18 and/or only exon 1 of CYP2C19 were not detected by MLPA. As noted in the Materials and Methods, the MLPA probe mix only interrogates exons 2, 6, and 9 of CYP2C19 and exons 2, 7, 8, and 9 of CYP2C9 (Figure 1). In addition, all subjects with CYP2C19 CNVs identified by CMA testing in the replication cohort were confirmed by ddPCR. The locations of the ddPCR probes in relation to the CMA probes are illustrated in Figure 1. All ddPCR results at exon 2 and intron 6 of CYP2C19 were consistent with the partial gene deletions and duplications detected by CMA testing.

Database Detection of CYP2C Copy Number Variants (CNVs)

Structural variants within a 1 Mb region at the CYP2C locus (chr10:96100000_97100000) were also identified in the DGV, which is commonly considered to be a CMA database representative of the general population. These CYP2C CNVs were consistent with those detected in our clinical cohorts and are illustrated in Figure 1 and detailed in the Appendix. A total of 36 CNVs (from 9 independent studies) overlapping the CYP2C18, CYP2C19, CYP2C9, and/or CYP2C8 genes were catalogued in the DGV. The majority (n=27; 75%) were deletions, including 17 and 4 deletions that overlapped coding regions of CYP2C19 and/or CYP2C9, respectively (Table 3).

Table 3.

CYP2C copy number variant (CNV) allele and carrier frequencies.

CYP2C18 CYP2C19 CYP2C9 CYP2C8 Any CYP2C gene
Cohort Carriers/
Cohort		 Allele
Frequency (%)		 Carrier
Frequency (%)		 Carriers/
Cohort		 Allele
Frequency (%)		 Carrier
Frequency (%)		 Carriers/
Cohort		 Allele
Frequency (%)		 Carrier
Frequency (%)		 Carriers/
Cohort		 Allele
Frequency (%)		 Carrier
Frequency (%)		 Carriers/
Cohort		 Allele
Frequency (%)		 Carrier
Frequency (%)
Deletion
 ISMMS/Sema4 7/11096 0.032 0.063 7/11096 0.032 0.063 0/11096 - - 0/11096 - - 7/11096 0.032 0.063
 CMH 0/9760 - - 3/9760 0.015 0.031 0/9760 - - 0/9760 - - 3/9760 0.015 0.031
 DGV 38/41392 0.046 0.092 31/41392 0.037 0.075 4/41392 0.005 0.010 3/41392 0.004 0.007 50/41392 0.060 0.121
 ClinGen 7/37447 0.009 0.019 5/37447 0.007 0.013 1/37447 0.001 0.003 1/37447 0.001 0.003 8/37447 0.011 0.021
Total: 52/99695 0.026 0.052 46/99695 0.023 0.046 5/99695 0.003 0.005 4/99695 0.002 0.004 68/99695 0.034 0.068
Duplication
 ISMMS/Sema4 2/11096 0.009 0.018 2/11096 0.009 0.018 2/11096 0.009 0.018 2/11096 0.009 0.018 2/11096 0.009 0.018
 CMH 0/9760 - - 3/9760 0.015 0.031 3/9760 0.015 0.031 0/9760 - - 3/9760 0.015 0.031
 DGV 1/41392 0.001 0.002 3/41392 0.004 0.007 4/41392 0.005 0.010 4/41392 0.005 0.010 9/41392 0.016 0.022
 ClinGen 2/37447 0.003 0.005 3/37447 0.004 0.003 4/37447 0.005 0.011 4/37447 0.005 0.011 5/37447 0.007 0.013
Total: 5/99695 0.002 0.005 10/99695 0.005 0.010 12/99695 0.006 0.012 10/99695 0.005 0.010 18/99695 0.009 0.018
Deletion/Duplication
 ISMMS/Sema4 9/11096 0.041 0.081 9/11096 0.041 0.081 2/11096 0.009 0.018 2/11096 0.009 0.018 9/11096 0.041 0.081
 CMH 0/9760 - - 6/9760 0.031 0.061 3/9760 0.015 0.031 0/9760 - - 6/9760 0.031 0.061
 DGV 39/41392 0.047 0.094 34/41392 0.041 0.082 8/41392 0.010 0.019 7/41392 0.008 0.017 59/41392 0.076 0.143
 ClinGen 9/37447 0.012 0.024 8/37447 0.011 0.021 5/37447 0.007 0.013 5/37447 0.007 0.013 13/37447 0.016 0.033
Total: 57/99695 0.029 0.057 57/99695 0.029 0.057 18/99695 0.009 0.018 14/99695 0.007 0.014 87/99695 0.044 0.087
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Structural variants at the CYP2C gene region were also identified in the ClinGen and DECIPHER databases. Given that these databases are comprised of CMA data from patient cohorts with variable phenotypes, larger deletions and duplications of the chromosome 10q23.33-q24.1 region were present; however, aberrations >2 Mb were not included in the CYP2C CNV analyses as detailed in the Materials and Methods (Appendix). Consistent with the DGV CYP2C CNVs, 22 CNVs were present in ClinGen and DECIPHER that overlapped CYP2C18, CYP2C19, CYP2C9 and/or CYP2C8, including 13 deletions (8.6 kb to 969.7 kb), and eight duplications (8.9 kb to 1.1 Mb) (Figure 1, Table 3 and Appendix). Notably, the most common CYP2C CNVs in all three population databases were deletions that overlapped CYP2C19 (n=26; 44.8% of all CYP2C CNVs).

CYP2C Deletion and Duplication Frequencies and Allele Nomenclature

The CYP2C CNV frequency data from our clinical CMA cohorts and the CNV population databases are summarized in Table 3. The CYP2C CNV data from DECIPHER was not incorporated into the population frequencies given the difficulty with determining an accurate size of this dynamic clinical cohort. Taken together, the overall carrier frequency of a CYP2C CNV is ~1 in 1000 [0.085% (95% CI: 0.067–0.104%)] (Table 3). The data for these CNV alleles were reviewed by the PharmVar Consortium (Gaedigk et al., 2018; Gaedigk et al., 2019), which subsequently classified a full gene CYP2C19 deletion as CYP2C19*36 and a partial gene CYP2C19 deletion (that includes at least exon 1) as CYP2C19*37 [combined carrier frequency of ~1 in 2000; 0.046% (95% CI: 0.033–0.059%)]. The full gene and partial gene CYP2C9 deletion alleles identified in the DGV and ClinGen had a carrier frequency of ~1 in 20,000; 0.005% (95% CI: 0.001–0.009%)] (Table 3 and Figure 1). The CYP2C19, CYP2C9, and CYP2C8 duplications did not receive designated star (*) alleles as PharmVar recommends classifying gene duplications based on their haplotype sequence and total detected copy number (e.g., CYP2C9*1/*1x2), consistent with established CYP2D6 duplication nomenclature (Gaedigk et al., 2018) (www.pharmvar.org). Notably, one sample in the clinical CMA cohort with the largest chromosome 10q23.33 duplication (1043.3 kb) that included multiple CYP2C genes was genotyped for a panel of CYP2C star (*) allele variants, which resulted in the following diplotypes: CYP2C19*17/*17x2, CYP2C9*1/*1x2, and CYP2C8*1/*1x2.

DISCUSSION

Recent studies that utilized large high-throughput sequencing datasets for pharmacogenomic variant discovery prompted our interrogation of deidentified CMA data from 99,695 individuals for pharmacogenomic CNV discovery at the clinically relevant CYP2C gene cluster region. Consistent with the sequencing studies that identified novel coding variants in pharmacogenomic genes (Bush et al., 2016; Gordon et al., 2014; Li et al., 2014; Nelson et al., 2012), our large CNV study resulted in the discovery of pharmacogenomic deletion and duplication alleles at the CYP2C region. The full gene and partial gene CYP2C19 deletions have been designated by the PharmVar Consortium as CYP2C19*36 and CYP2C19*37, respectively (www.pharmvar.org). Of note, the CYP2C19*37 partial deletion allele is consistent with the CYP2C19 partial gene deletions recently identified in the Exome Aggregation Consortium (ExAC) database (Ruderfer et al., 2016; Santos et al., 2018). Dissemination of these newly defined CYP2C19 alleles through the widely utilized PharmVar database will likely increase awareness of these low frequency structural variants across the pharmacogenomics community.

The significance of structural variation in human disease and phenotypic diversity is increasingly being recognized, and several genomic studies have generated catalogs of CNVs to facilitate a better understanding of their clinical relevance (Johansson & Feuk, 2011; Sudmant et al., 2015). It is estimated that up to 60% of the human genome may contain structural variants in the general population, which typically range in size from 100 bp to 50 kb (Escaramis, Docampo, & Rabionet, 2015), and clinical interpretation of these aberrations when identified by CMA testing is facilitated by professional medical genetics practice guidelines (South et al., 2013). Larger gene-dense aberrations are more likely to result in penetrant syndromic phenotypes; however, some smaller CNVs have increasingly been implicated as susceptibility alleles for several phenotypes, including neurodegenerative disorders, cancer, autism, and psychiatric diseases (Cook & Scherer, 2008; Gonzalez et al., 2005; Han et al., 2017; Nishioka et al., 2006; Rovelet-Lecrux et al., 2006; Sebat et al., 2007). CNVs can influence these human traits by altering the copy number of dosage-sensitive genes (Douglas et al., 2005; Roa, Garcia, & Lupski, 1991) and/or modulating local gene expression (Cahan, Li, Izumi, & Graubert, 2009; Henrichsen, Chaignat, & Reymond, 2009).

Pharmacogenomic structural variation has been previously characterized at several clinically relevant regions (Santos et al., 2018), including CYP450 genes (CYP2A6, CYP2B6, CYP2C cluster, CYP2D6), glutathione S-transferases (GSTT1, GSTM1), and sulfotransferases (SULT1A1, SULT2A1) (Gaedigk et al., 2010; Gjerde et al., 2008; Martis et al., 2013; Schulze et al., 2013; Vijzelaar et al., 2018). Notably, individuals with greater than two functional CYP2D6 copies (e.g. *1xN, *2xN, *35xN) have higher enzyme activity, whereas CYP2D6*5 deletion alleles do not encode a functional CYP2D6 protein. Moreover, common full gene GSTT1 and GSTM1 deletions have been associated with increased risk for chemotherapy toxicity among lymphoma patients (Cho et al., 2010) and susceptibility to tacrine hepatotoxicity among Alzheimer’s patients (Simon et al., 2000).

Loss-of-function CYP2C19 and CYP2C9 alleles have extensive evidence for important roles in clopidogrel, voriconazole, antidepressant, warfarin and/or phenytoin response, which prompted recent Clinical Pharmacogenetics Implementation Consortium (CPIC) and Dutch Pharmacogenetics Working Group (DPWG) practice guidelines for CYP2C19 and CYP2C9 pharmacogenetic-guided prescribing (Caudle et al., 2014; Hicks et al., 2015; Hicks et al., 2017; Johnson et al., 2017; Moriyama et al., 2017; Scott et al., 2013; Swen et al., 2011). Given that the newly defined CYP2C19*36 and *37 deletion alleles are presumed to be nonfunctional, it is expected that they would have the same effects on drug response as the well-known alleles defined by sequence variants (e.g., CYP2C19*2, *3). Interestingly, CYP2C19 deletions have previously been reported in a Northern Finnish case-control study, which identified an association between CYP2C19 deletion and triple-negative breast cancer (ER/PR/HER2 negative), implicating CYP2C19 in estrogen catabolism (Tervasmaki, Winqvist, Jukkola-Vuorinen, & Pylkas, 2014). Our combined analysis of clinical CMA data from two academic medical centers and publicly available databases indicate that the carrier frequency of a CYP2C structural variant is ~1 in 1000, with ~1 in 2000 being a CYP2C19 deletion carrier; however, these aberrations may have higher allele frequencies in specific subpopulations (Santos et al., 2018; Tervasmaki et al., 2014).

Recurrent CNVs are often flanked by segmental duplications, which can act as substrates for non-allelic homologous recombination (NAHR) and the meiotic formation of both deletion and duplication alleles (Carvalho & Lupski, 2016). Importantly, two pairs of segmental duplications are nested within the CYP2C gene cluster region, one directly oriented ~10–20 kb element (~92% identical) that flank CYP2C18 and CYP2C19, and a smaller element (~1.6 kb) directly oriented on the negative strand that are located at the 3’ region of CYP2C8 (Figure 1). In addition to these segmental duplications, the CYP2C subfamily genes have a high degree of sequence homology. This homology and the segmental duplications flanking CYP2C18 and CYP2C19 are likely driving the formation of the more common and recurrent deletions at this region, which is consistent with the NAHR mechanism generally favoring deletions over duplications (Liu, Carvalho, Hastings, & Lupski, 2012).

Notably, the resolution of microarrays used for CMA testing was different across platforms and laboratories. As such, our reported CNV sizes are based on the minimum number of probes that detected gains or losses by a specific microarray; however, based on both our data and those from public databases there are recurrent deletions that affect both CYP2C18 and CYP2C19, as well as larger CNVs that can include multiple CYP2C genes. Unfortunately, precise breakpoints were not feasible to determine given the multiple CMA platforms used across studies, as well as the paucity of available DNA for follow up sequencing.

In conclusion, our interrogation of CMA data from almost 100,000 individuals identified low frequency pharmacogenomic CNVs at the clinically relevant CYP2C region in the general population. These results are consistent with previously reported pharmacogenomic sequencing studies, which identified a spectrum of rare pharmacogenomic variants that are likely to be functional (Bush et al., 2016; Gordon et al., 2014; Li et al., 2014; Nelson et al., 2012). Although the identified CYP2C deletion and duplication alleles have low frequencies in the studied populations, their contribution to an individual’s CYP2C19 and CYP2C9 metabolizer phenotype status is most likely clinically relevant. The nonfunctional deletion alleles can lead to either intermediate or poor metabolizer phenotypes, and the larger CYP2C gene region duplications discovered in our study could lead to an ultrarapid metabolizer phenotype across CYP2C19, CYP2C19, CYPC9 and CYP2C8 if the duplication alleles do not harbor sequence variants that obliterate function. These rare individuals would likely be at risk for atypical CYP2C-mediated metabolism across multiple drugs and drug classes (e.g., voriconazole, clopidogrel, phenytoin, fosphenytoin, phenobarbital, amytriptiline, torsemide). Although the technical infrastructure and additional cost of interrogating copy number at the CYP2C gene region may not currently be feasible for clinical laboratories that offer pharmacogenetic testing, the clinical relevance of these low frequency CNV alleles indicates that future iterations of clinical pharmacogenomic sequencing assays that incorporate computational copy number detection pipelines should include this gene family in addition to the pharmacogenes with more common CNV alleles.

ACKNOWLEDGEMENTS

The study was supported, in part, by Sema4, a Mount Sinai venture, Stamford, CT. This study makes use of data generated by the DECIPHER community. A full list of centers who contributed to the generation of the data is available from http://decipher.sanger.ac.uk and via email from decipher@sanger.ac.uk. Funding for the project was provided by the Wellcome Trust.

SOURCES OF SUPPORT:

This research was supported, in part, by Sema4, a Mount Sinai venture, Stamford, CT, and by the National Institutes of Health through grant R24 GM123930 (A.G.; Pharmacogene Variation (PharmVar) Consortium).

APPENDIX

Appendix Table 1.

CYP2C copy number variants (CNVs) identified in the Database of Genomic Variants (DGV), ClinGen, and DECIPHER.

CNV ID Genes CNV type Region size Frequency Study population Ref.
DGV
esv3624253 CYP2C18 Deletion 118,150 1/2504 Healthy (Genomes Project et al., 2015)
esv2672446 CYP2C18 Deletion 118,136 1/1092 Healthy (Genomes Project et al., 2012)
esv3624254 CYP2C18 Deletion 79,667 3/2504 Healthy (Genomes Project et al., 2015)
dgv955n100 CYP2C18 Deletion 111,509 6/29084 Intellectual disability and/or developmental delay (Coe et al., 2014)
nsv551954 CYP2C18 Deletion 115,921 2/15767 Intellectual disability and/or developmental delay (Cooper et al., 2011)
dgv956n100 CYP2C18 Deletion 69,696 2/29084 Intellectual disability and/or developmental delay (Coe et al., 2014)
esv2657299 CYP2C18 Deletion 8,618 1/1092 Healthy (Genomes Project et al., 2012)
nsv522538 CYP2C18, CYP2C19 Deletion 142,135 1/2026 Healthy (Shaikh et al., 2009)
dgv1355n54 CYP2C18, CYP2C19 Deletion 246,123 3/15767 Intellectual disability and/or developmental delay (Cooper et al., 2011)
esv2674280 CYP2C18, CYP2C19 Deletion 155,809 1/1092 Healthy (Genomes Project et al., 2012)
nsv1046308 CYP2C18, CYP2C19 Deletion 191,713 1/29084 Intellectual disability and/or developmental delay (Coe et al., 2014)
dgv1355n54 CYP2C18, CYP2C19 Deletion 246,123 3/15767 Intellectual disability and/or developmental delay (Cooper et al., 2011)
dgv957n100 CYP2C18, CYP2C19 Deletion 70,440 5/29084 Intellectual disability and/or developmental delay (Coe et al., 2014)
esv3624256 CYP2C18, CYP2C19 Deletion 44,223 2/2504 Healthy (Genomes Project et al., 2015)
esv2761617 CYP2C18, CYP2C19 Deletion 53,597 1/1109 Healthy (Vogler et al., 2010)
esv3624258 CYP2C18, CYP2C19 Duplication 80,151 1/2504 Healthy (Genomes Project et al., 2015)
dgv160e214 CYP2C18, CYP2C19 Deletion 80,151 1/2504 Healthy (Genomes Project et al., 2015)
dgv1356n54 CYP2C18, CYP2C19 Deletion 61,544 6/15767 Intellectual disability and/or developmental delay (Cooper et al., 2011)
esv3891886 CYP2C18, CYP2C19, CYP2C9 Deletion 271,380 1/3017 Infectious diseases, Thyrotoxic Hypokalemic Periodic Paralysis (THPP) and Hb E/b-thalassemia (Suktitipat et al., 2014)
nsv1035409 CYP2C18, CYP2C19, CYP2C9, CYP2C8 Deletion 336,881 1/29084 Intellectual disability and/or developmental delay (Coe et al., 2014)
nsv551962 CYP2C19 Deletion 54,075 1/15767 Intellectual disability and/or developmental delay (Cooper et al., 2011)
esv2659638 CYP2C19 Deletion 61,848 1/1092 Healthy (Genomes Project et al., 2012)
esv3624260 CYP2C19 Deletion 39,678 2/2504 Healthy (Genomes Project et al., 2015)
nsv516555 CYP2C19 Deletion 12,880 2/2026 Healthy (Shaikh et al., 2009)
nsv523259 CYP2C19 Duplication 159,144 1/2026 Healthy (Shaikh et al., 2009)
nsv1052578 CYP2C19 Deletion 103,868 1/29084 Intellectual disability and/or developmental delay (Coe et al., 2014)
nsv1047782 CYP2C19, CYP2C9 Duplication 137,386 1/29084 Intellectual disability and/or developmental delay (Coe et al., 2014)
esv2741937 CYP2C19, CYP2C9 Deletion 138,826 1/96 Healthy (Wong et al., 2013)
esv3624265 CYP2C9 Duplication 72,590 1/2504 Healthy (Genomes Project et al., 2015)
nsv551963 CYP2C9 Deletion 23,682 1/15767 Intellectual disability and/or developmental delay (Cooper et al., 2011)
nsv7497 CYP2C9 Duplication 34,308 1/8 Healthy (Kidd et al., 2008)
esv3624264 CYP2C9, CYP2C8 Duplication 175,419 1/2504 Healthy (Genomes Project et al., 2015)
nsv1050544 CYP2C8 Duplication 47,756 1/29084 Intellectual disability and/or developmental delay (Coe et al., 2014)
nsv1044182 CYP2C8 Duplication 30,694 1/29084 Intellectual disability and/or developmental delay (Coe et al., 2014)
esv2761618 CYP2C8 Duplication 30,682 1/1109 Healthy (Vogler et al., 2010)
nsv551964 CYP2C8 Deletion 56,916 1/15767 Intellectual disability and/or developmental delay (Cooper et al., 2011)
nsv467436 CYP2C8 Deletion 56,916 1/2493 Healthy (Itsara et al., 2009)
ClinGen
Pathogenic
nssv13651550_unk 150 genes Deletion 7,901,553 Behavioral abnormality, global development delay, microcephaly (Miller et al., 2010)
nssv13646178_unk 2,161 genes Duplication 135,285,622 Development delay and/or other significant development or morphological phenotypes (Miller et al., 2010)
nssv13638976_unk, nssv13640749_unk 2,161 genes Duplication 135,327,117 Abnormal facial shape, Intrauterine growth retardation, Micrognathia, Syndactyly, Ventricular septal defect (Miller et al., 2010)
nssv13655969_unk 122 genes Deletion 6,302,504 Developmental delay and/or other significant developmental or morphological phenotypes (Miller et al., 2010)
nssv13655409_unk 928 genes Deletion 53,839,386 Global developmental delay (Miller et al., 2010)
nssv13653237_unk 717 genes Duplication 42,143,651 Patent ductus arteriosus (Miller et al., 2010)
nssv577306_dnovo 109 genes Deletion 5,128,423 Dilatation, Hydronephrosis (Kaminsky et al., 2011)
nssv577307_dnovo 176 genes Deletion 8,175,579 Abnormal facial shape (Kaminsky et al., 2011)
nssv1494941_unk 54 genes Deletion 2,827,219 Autism, Failure to thrive (Miller et al., 2010)
Benign
nssv581618_unk CYP2C18, CYP2C19 Deletion 158,935 Intellectual disability (Miller et al., 2010)
nssv1608783_unk CYP2C19 Deletion 20,211 Developmental delay and/or other significant developmental or morphological phenotypes (Miller et al., 2010)
Likely Benign
nssv13650654_unk CYP2C18, CYP2C19 Deletion 121,822 Developmental delay and/or other significant developmental or morphological phenotypes (Miller et al., 2010)
nssv13655256_unk CYP2C18 Deletion 114,674 Developmental delay and/or other significant developmental or morphological phenotypes (Miller et al., 2010)
nssv13647268_unk CYP2C19, CYP2C9 Duplication 180,257 Developmental delay and/or other significant developmental or morphological phenotypes (Miller et al., 2010)
nssv13650288_unk CYP2C18 Deletion 48,162 Developmental delay and/or other significant developmental or morphological phenotypes (Miller et al., 2010)
nssv585041_unk CYP2C18 Deletion 112,487 Developmental delay and/or other significant developmental or morphological phenotypes (Miller et al., 2010)
nssv13655998_unk CYP2C8 Duplication 8,867 Developmental delay and/or other significant developmental or morphological phenotypes (Miller et al., 2010)
Uncertain
nssv13650363_unk CYP2C18, CYP2C19 Deletion 240,911 Seizures (Miller et al., 2010)
nssv583937_pat PLCE1, NOC3L, TBC1D12, HELLS, CYP2C18, CYP2C19, CYP2C9, CYP2C8, PDLM1, ACSM6 Deletion 969,724 Low-set ears (Miller et al., 2010)
nssv580748_unk CYP2C9, CYP2C8, PDLIM1, SORBS1 Duplication 473,822 Global developmental delay (Kaminsky et al., 2011)
nssv1495397_unk TBC1D12, HELLS, CYP2C18, CYP2C19, CYP2C9, CYP2C8, PDLIM1, SORBS1, ACSM6 Duplication 1,043,272 Autistic behavior, Global developmental delay (Miller et al., 2010)
nssv3395040_unk TBC1D12, HELLS, CYP2C18, CYP2C19, CYP2C9, CYP2C8, PDLIM1, SORBS1, ACSM6 Duplication 1,089,626 Seizures (Miller et al., 2010)
DECIPHER
184 175 genes Deletion 12,434,019 Anterior creases of earlobe, capillary hemangiomas, hypoglycemia, intellectual disability, microcephaly (Firth et al., 2009)
341717 122 genes Duplication 8,517,656 Emotional lability, growth delay, intellectual disability (Firth et al., 2009)
2578 232 genes Duplication 17,187,727 Behavioral abnormality, constipation, deeply set eye, delayed speech and language development, intellectual disability, macrocephaly, pectus excavatum, plagiocephaly, short stature, ventricular septal defect (Firth et al., 2009)
337109 PLCE1, NOC3L, TBC1D12, HELLS, CYP2C18, CYP2C19, CYP2C9, CYP2C8, PDLIM1 Duplication 1,080,102 Intellectual disability - moderate, oromotor apraxia, severe expressive language delay (Firth et al., 2009)
292398 PLCE1, NOC3L, TBC1D12, HELLS, CYP2C18, CYP2C19, CYP2C9, CYP2C8, PDLIM1 Duplication 1,080,102 Anxiety, cognitive impairment (Firth et al., 2009)
265438 PLCE1, NOC3L, TBC1D12, HELLS, CYP2C18, CYP2C19, CYP2C9, CYP2C8, PDLIM1 Duplication 1,080,102 - (Firth et al., 2009)
260874 PLCE1, NOC3L, TBC1D12, HELLS, CYP2C18, CYP2C19, CYP2C9, CYP2C8, PDLIM1 Duplication 1,080,102 Delayed speech and language development, incomprehensible speech, intellectual disability - moderate, oromotor apraxia (Firth et al., 2009)
318690 CYP2C18, CYP2C19 Deletion 120,242 Delayed speech and language development, EEG abnormality, moderate global developmental delay (Firth et al., 2009)
300098 CYP2C18, CYP2C19 Deletion 158,935 Cognitive impairment (Firth et al., 2009)
283724 CYP2C18, CYP2C19 Deletion 140,797 Abnormal facial shape, intellectual disability (Firth et al., 2009)
278463 CYP2C19 Deletion 61,682 Behavioral abnormality (Firth et al., 2009)
270359 CYP2C19, CYP2C8, CYP2C9 Duplication 288,534 Hypospadias, rudimentary fibula (Firth et al., 2009)
256495 34 genes (CYP2C18 is not included) Deletion 2,093,635 - (Firth et al., 2009)
305972 CYP2C19, CYP2C8, CYP2C9 Duplication 344,191 - (Firth et al., 2009)
272275 CYP2C8, CYP2C9, PDLIM1, SORBS1 Deletion 694,656 - (Firth et al., 2009)
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Footnotes

CONFLICT OF INTEREST

X.L., G.Z., Y.S., E.E.S., L.E., and S.A.S. are paid employees of Sema4, a Mount Sinai venture, Stamford, CT.

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