New Resources to Identify Characterized DNA Reference Materials for Pharmacogenetic (PGx) and Human Leukocyte Antigen (HLA) Testing

Abstract

Regulations, accreditation standards, and professional guidance require laboratories to use reference materials for assay development, validation, quality control, and proficiency testing of clinical genetic tests. There are, however, few publicly available reference materials for most genetic tests. To address this issue, the CDC's Genetic Testing Reference Material Program (GeT-RM), the Coriell Institute for Medical Research, and the genetic testing community have conducted 19 studies, including nine for pharmacogenetic (PGx) and human leukocyte antigen (HLA) testing, to generate characterized, renewable, and publicly available DNA samples for use as reference materials. Because new PGx alleles are frequently identified, and allele designations change over time, many samples were reanalyzed for the same gene(s) in subsequent GeT-RM studies. These studies used more comprehensive and sensitive methods and panels that examined additional single-nucleotide variants and/or star alleles to expand and update the consensus genotypes. Up-to-date information is available in two newly established resources: the GeT-RM Consolidated PGx and HLA Table and the GeT-RM PGx Search Tool. These resources contain all available PGx and HLA genotypes for 363 publicly available samples characterized during nine GeT-RM PGx or HLA studies for 34 genes/loci in a consolidated and searchable format.

Reference materials are critical for many activities in clinical laboratories, including quality control, development and validation of tests, proficiency testing, validating variant calling algorithms, and interlaboratory standardization, and are needed to comply with regulations, accreditation standards, and professional guidance1, 2, 3, 4, 5 [American College of Medical Genetics and Genomics, https://www.acmg.net/PDFLibrary/ACMG%20Technical%20Lab%20Standards%20Section%20G.pdf, last accessed July 12, 2024; Washington State Legislature, http://app.leg.wa.gov/WAC/default.aspx?cite=246-338-090, last accessed July 12, 2024; College of American Pathologists (Northfield, IL), https://www.cap.org, last accessed July 12, 2024; New York State Clinical Laboratory Evaluation Program, https://www.wadsworth.org/regulatory/clep, last accessed July 12, 2024; and Morbidity and Mortality Weekly Report (MMWR), https://www.cdc.gov/mmwr/preview/mmwrhtml/rr5806a1.htm, last accessed July 12, 2024].

Clinical laboratories often develop pharmacogenetic (PGx) and other genetic tests as laboratory-developed tests or procedures, and use residual patient samples, genomic DNA from cell lines, or synthetic DNA containing variants of interest as reference materials. However, despite the regulatory and professional guidelines requiring their use, there are few, if any, publicly available reference materials for most clinical genetic tests.

To address this issue, the CDC created the Genetic Testing Reference Materials Program in 2004 (GeT-RM, https://www.cdc.gov/lab-quality/php/get-rm/index.html, last accessed November 4, 2024).⁶ The GeT-RM is a collaborative program whose goal is to improve the availability of genomic DNA reference materials for genetic testing. GeT-RM works with a variety of partners, including clinical and research laboratories, in vitro diagnostics manufacturers, professional organizations, patient advocacy groups, and the Coriell Institute for Medical Research (Camden, NJ), to identify reference material needs and generate publicly available and renewable genomic DNA reference materials.

GeT-RM has generated cell-line–based genomic DNA reference materials for a large number of clinically important genes for hereditary genetic disorders, including cystic fibrosis, fragile X, Rett syndrome, and Duchenne muscular dystrophy,7, 8, 9, 10 11 human leukocyte antigen (HLA) loci,¹¹ and many pharmacogenes and loci.12, 13, 14, 15, 16, 17, 18, 19 Each sample is experimentally characterized in two or more laboratories using a variety of analytical methods, including targeted genotyping and Sanger sequencing. For some genes, experimental testing also includes copy number variants and structural variation analysis or phasing of variants into star alleles.²⁰ In addition, publicly available high-coverage (30×) whole-genome sequencing data from the 1000 Genomes Project²¹ or 10x Genomics Linked-Read data [Illumina (San Diego, CA), https://github.com/Illumina/Polaris/wiki/HiSeqX-PGx-Cohort, last accessed August 19, 2024] were also analyzed. Results were assessed for quality, discordances, and determination of a consensus genotype for each sample. All characterized genomic DNA samples are publicly available from the National Institute of General Medical Sciences Human Genetic Cell Repository or the National Human Genome Research Institute Sample Repository for Human Genetic Research, which are both housed at the Coriell Institute for Medical Research.

The GeT-RM has conducted nine studies over the past 14 years to generate reference materials for PGx and HLA testing.11, 12, 13, 14, 15, 16, 17, 18, 19 In many cases, samples were reanalyzed for the same gene(s) in subsequent studies using more comprehensive and sensitive methods and panels with additional single-nucleotide variants and/or star alleles that were not included in previous characterizations along with other data that became publicly available. This approach allowed updating and expanding consensus genotypes for a considerable number of samples. Often, additional samples were added in later characterization studies to identify single-nucleotide variants and star alleles that were defined after the original study. In addition, samples characterized during the earliest studies may also have been assigned outdated designations as allele definitions evolve over time and new alleles are identified frequently. These allele designations were updated during subsequent studies.

Two resources were generated, the GeT-RM Consolidated PGx and HLA Table as well as a searchable database tool, GeT-RM PGx Search, to provide the most up-to-date pharmacogenetic genotypes for each characterized sample. Both resources provide information about 363 DNA samples that were characterized during nine GeT-RM PGx or HLA studies for 34 genes/loci, including CYP2C9, CYP2C19, CYP3A4, CYP3A5, CYP2D6, TPMT, NUDT15, DPYD, and 11 HLA loci.11, 12, 13, 14, 15, 16, 17, 18, 19

Materials and Methods

GeT-RM Samples

For each GeT-RM study, genomic DNA from cell lines identified by study team members to contain variants of interest were selected from the National Institute of General Medical Sciences Human Genetic Cell Repository and/or the National Human Genome Research Institute Sample Repository for Human Genetic Research at the Coriell Institute for Medical Research. Each sample was characterized using a variety of methods and test platforms by two or more laboratories.11, 12, 13, 14, 15, 16, 17, 18, 19 Results were assessed for quality, discordances, and determination of consensus genotype for each sample. These data were subsequently used to generate two consolidated information resources.

Generation of the GeT-RM Consolidated PGx and HLA Table

Consensus genotypes for all samples (n = 363) characterized during the nine GeT-RM PGx or HLA reference material studies were consolidated into a single, publicly available spreadsheet (in Excel format), referred to as the GeT-RM Consolidated PGx and HLA Table (https://www.cdc.gov/lab-quality/php/get-rm/reference-materials.html, last accessed November 4, 2024). This consolidated table (Figure 1) includes information regarding the study or studies in which each sample was characterized, the National Institute of General Medical Sciences or National Human Genome Research Institute Repository sample identifier, and each sample's genotype for the interrogated genes and loci (Table 1). The GeT-RM Consolidated PGx and HLA Table also provides information regarding the availability of binary alignment map files, FASTQ files, as well as high-quality 30× coverage whole-genome sequencing data from the 1000 Genomes Project.²¹ Each allele annotation is accompanied by the associated reference(s) that include a detailed description of the methods used to characterize the sample and construct the consensus genotype or star allele annotation. In cases where a given annotation was described in more than one corresponding publication, and the consensus annotation was consistent, all references are included. In cases where a given annotation was described in more than one corresponding publication, and the consensus annotations were inconsistent, the most recent publication and annotation are shown. The GeT-RM consolidated PGx and HLA Table also contains tabs with information about the columns and definitions of terms used, as well as GeT-RM references.

Figure 1.

Screenshot of GeT-RM consolidated PGx and HLA table. Consensus genotypes for 34 gene/loci determined during nine GeT-RM studies are available. References for each genotype are provided in the adjacent column. Binary alignment map and FASTQ files are available for some samples, and many have sequence data from the 1000 Genomes Project. The Excel file contains several tabs, shown at the bottom of the figure. The references in the above image do not reflect the citations in this article. ID, identifier; PMID, PubMed Identifier.

Table 1.

Genes and Loci in the GeT-RM Consolidated PGx and HLA Table and GeT-RM PGx Search Tools

Gene or loci	GeT-RM Study(s) PMID
CYP1A1	2662110117
CYP1A2	2662110117
CYP2A6	2662110117
CYP2B6	2662110117
CYP2C8	26621101,17 3513454214
CYP2C9	20889555,18 26621101,17 35134542,14 3402004115
CYP2C19	20889555,18 26621101,17 35134542,14 3402004115
CYP2D6	20889555,18 26621101,17 3140112416
CYP2E1	2662110117
CYP3A4	26621101,17 3735499312
CYP3A5	26621101,17 3735499312
CYP4F2	2662110117
DPYD	26621101,17 3903282219
GSTM1	2662110117
GSTP1	2662110117
GSTT1	2662110117
NAT1	2662110117
NAT2	2662110117
NUDT15	3593134213
SLC15A2	2662110117
SLCO2B1	2662110117
TPMT	26621101,17 3593134213
UGT1A1	20889555,18 2662110117
UGT2B7	2662110117
UGT2B15	2662110117
UGT2B17	2662110117
HLA-A	2995902511
HLA-B	2995902511
HLA-C	2995902511
HLA-DRB1	2995902511
HLA-DRB3	2995902511
HLA-DRB4	2995902511
HLA-DRB5	2995902511
HLA-DQA1	2995902511
HLA-DQB1	2995902511
HLA-DPA1	2995902511
HLA-DPB1	2995902511
CYP2C cluster NC_000010.10∗: g.96405502G>A, rs12777823†	3402004115
GGCX NM_000821.6:c.214 + 597G>A, rs12714145	3402004115
GGCX NM_000821.6:c.2084 + 45G>C rs11676382	3402004115
VKORC1 NM_024006.5:c.-1639G>A, rs9923231	20889555,18 2662110117
VKORC1 NM_024006.6:c.106G>A, rs61742245	3402004115
VKORC1 NM_024006.6:c.196G>A, rs72547529	3402004115

PMID, PubMed Identifier.

^∗NCBI Reference Sequence Database (RefSeq; https://www.ncbi.nlm.nih.gov/refseq, last accessed May 17, 2024).

^†Single Nucleotide Polymorphism Database (dbSNP; https://www.ncbi.nlm.nih.gov/snp, last accessed February 2, 2024).

Generation of a Searchable Database: GeT-RM PGx Search Tool

The GeT-RM Consolidated PGx and HLA Table was used to generate a searchable, web-based tool, GeT-RM PGx Search. For each DNA sample included in the GeT-RM Consolidated PGx and HLA Table, the annotation for each characterized pharmacogene was formatted into a single genotype, apart from DPYD, for which all variants identified are listed. Most included pharmacogenes are annotated with star (∗) alleles. In these cases, the two star alleles are separated by a slash delimiter (/); however, a subset of pharmacogenes and regions (eg, CYP2C cluster, DPYD, GGCX, SLCO2B1, and VKORC1) includes variant-specific annotations. Finally, HLA gene annotations are included, as previously described.¹¹ Reference(s) for each allele annotation are provided as described in Generation of the GeT-RM Consolidated PGx and HLA Table.

Results

The GeT-RM Consolidated PGx and HLA Table (https://www.cdc.gov/lab-quality/php/get-rm/reference-materials.html, last accessed November 4, 2024) shows consensus genotypes determined during the nine GeT-RM PGx studies for all 363 samples in an Excel format. This table was used to develop an interactive, web-based search tool, GeT-RM PGx Search, which is available on the Coriell Institute for Medical Research website (https://www.coriell.org/GetRM/PGxSearch, last accessed August 13, 2024). Each gene is given a unique key in a Structured Query Language Server relational database that is linked to sample-level data. Users can query GeT-RM PGx Search using a web-based interface and select a gene of interest from the drop-down menu (Figure 2). GeT-RM PGx Search returns the following: links to Pharmacogene Variation Consortium (PharmVar) annotations (https://www.pharmvar.org, last accessed July 22, 2024) and the National Center for Biotechnology Information gene entry (if available) (NCBI, https://www.ncbi.nlm.nih.gov/gene/?term=, last accessed August 13, 2024), additional documentation regarding allele information and allele definitions, an option to export annotations for the chosen gene to an Excel spreadsheet, and an interactive data display (Figure 3). The number of samples viewable in the interactive table can be adjusted to the user's preference, and the annotated samples can be sorted by sample identifier, description, gene, genotype, reference, product, source, or genetic sex. Each sample identifier is hyperlinked to a sample-specific page that includes the detailed overview, characterizations, associated data and publications, and related external links. In addition, the viewable samples can be filtered by star allele, variant(s), or genotype.

Figure 2.

Screenshot of user drop-down menu of genes and loci with available GeT-RM PGx and HLA characterizations. HUGO, Human Genome Organization.

Figure 3.

Screenshot of example interactive GeT-RM PGx search data display: search for the CYP2D6∗17 allele. The display includes annotation definition information and external links to Pharmacogene Variation Consortium (PharmVar) and National Center for Biotechnology Information (NCBI). The user can choose the number of viewable samples, sort samples by sample identifier, description, gene, genotype, reference, product, source, or genetic sex, export the information to an Excel spreadsheet, and click on any sample hyperlink to access a sample-specific page that includes the detailed overview, characterizations, associated data and publications, and related external links.

Discussion

Reference material organizations, including the National Institute of Standards and Technology (https://www.nist.gov, last accessed January 3, 2025), the National Institute for Biological Standards and Control (Hertfordshire, UK), and the Joint Research Centre (Geel, Belgium), as well as commercial vendors produce reference materials and standards for product control and forensic testing. Availability of reference materials encompassing a range of human genetic variation, however, has been an ongoing challenge for clinical laboratorians. This limited availability has negatively impacted the ability to develop and validate new tests and provide quality control and ongoing quality assurance for existing tests. To address this gap, the GeT-RM has characterized hundreds of publicly available and renewable genomic DNA reference materials, many of these for pharmacogenetic testing. Unlike other PGx annotation approaches,22, 23, 24 the GeT-RM requires that each sample be experimentally characterized in two or more laboratories using a variety of analytical methods. These reference materials can then be used by laboratories as they implement clinical practice guidelines, such as those developed by the Association for Molecular Pathology PGx Working Group.25, 26, 27, 28, 29, 30

Most pharmacogenes are highly polymorphic,^31,32 with new alleles and haplotypes still being discovered, especially in non-European poulations^33,34; thus, the catalog of haplotype-based (ie, star allele) definitions continues to grow and is anticipated to be updated regularly.^20,35,36 In addition, there has been a rapid evolution of the technologies and methods available to perform pharmacogenetic testing. Over the past 15 years, pharmacogenetic tests, which started as targeted genotyping and Sanger sequencing assays, have come to incorporate more comprehensive analyses,³⁷ including testing for copy number variation, as well as short- and long-read next-generation sequencing.^38,39 This has enhanced the understanding of many pharmacogenetic loci, such as CYP2D6, which often requires extensive analysis to resolve its many structural variants.⁴⁰ Historically, simpler assays were only able to detect full CYP2D6 gene deletions, duplications, and multiplications, whereas recent more advanced assays can also detect hybrid genes and more comprehensively characterize complex genotypes. These higher-resolution analyses have resulted in the identification and naming of many new CYP2D6 star alleles by PharmVar. Although copy number analysis is now commonly performed for CYP2D6, testing for copy number variation is not widely used for other loci. For example, the most recent GeT-RM study used copy number analysis and identified a relatively common intragenic deletion in DPYD.¹⁹ Finally, at present, the phase of most pharmacogene variants is determined empirically. As single-molecule long-read next-generation sequencing becomes more widely available, it is anticipated that previously analyzed samples may be retested to determine phase, which may require further updates to the gold standard genotype data in GeT-RM for some samples. Although the reference material DNA does not change over time, the ability to precisely characterize genotype continues to improve.

Since 2010, GeT-RM has performed eight studies characterizing genomic DNA samples for pharmacogenetic loci12, 13, 14, 15, 16, 17, 18, 19 and one study for 11 HLA loci.¹¹ Because of the changing nature of PGx testing and the identification of important new alleles, reference materials for many of the commonly tested PGx genes, including CYP2C9, CYP2C19, CYP2D6, and DPYD, have been characterized in more than one GeT-RM study.12, 13, 14, 15, 16, 17, 18, 19 Some samples were characterized for the same gene during multiple studies, and their consensus genotype was revised on the basis of the information gleaned from analyses using more comprehensive assays. This has resulted in some sample genotypes seeming to change between GeT-RM studies, which may have been interpreted as being discrepant and caused confusion among users of the reference material samples.

Assignment of PGx genotypes during GeT-RM studies is dependent on the assays used for sample characterization as well as the PharmVar star allele definitions existing at the time of the study. Newly available assay technologies can be used to recharacterize samples to overcome limitations of assays used during earlier studies. For example, genotyping assays, Sanger assays, or short-read next-generation sequencing assays used in previous studies could often not resolve the phase of variants. This limitation may now be overcome by single-molecule long-read next-generation sequencing technologies. In addition, PharmVar may have revised allele definitions and added new star alleles since samples were first characterized. GeT-RM aims to not only update reported genotypes of previously characterized samples by testing with new technologies, but also identify samples with newly discovered star alleles. Genotypes listed in the GeT-RM Consolidated PGx and HLA Table are based on the nomenclature available at the time of analysis and may not be consistent with current nomenclature. This explains, for example, why some genotypes contain letter extensions (eg, for CYP1A2). Furthermore, NAT2 was only transferred to PharmVar in 2024, and its nomenclature underwent substantial changes. Future recharacterization of sample materials for NAT2 will not only include testing for many additional star alleles with the latest technologies but will also report genotypes using PharmVar's current star allele definitions.

There are several bioinformatic tools that can be used to call PGx variants and star alleles for the 1000 Genomes Project samples.22, 23, 24^,41,42 These tools can be useful to identify publicly available samples that may contain PGx variants of interest; however, the variants in these samples should be confirmed using orthogonal methods before use as reference materials.

The GeT-RM Consolidated PGx and HLA Table and the GeT-RM PGx Search tool were generated to provide an easily accessible and searchable resource with up-to-date information about publicly available, well-characterized reference DNA samples that can be used to support quality assurance programs of laboratories performing clinical PGx and HLA testing. The GeT-RM Consolidated PGx and HLA Table and GeT-RM PGx Search tool will be updated and synchronized as additional GeT-RM PGx studies are completed. All reference materials characterized by GeT-RM are publicly available from the National Institute of General Medical Sciences and National Human Genome Research Institute Repositories at the Coriell Institute for Medical Research.

Disclosure Statement

RPRD Diagnostics LLC is a fee-for-service laboratory that offers clinical pharmacogenetic testing. A.J.T.’s efforts were supported in part by RPRD Diagnostics, and A.J.T. holds equity. A.G. is Director of PharmVar. A.J.T, A.M.M., and V.M.P. are members of PharmVar. V.M.P. is an employee of Agena Bioscience.