The Genetic Testing Reference Materials Coordination Program

The Genetic Testing Reference Materials (GeT-RM) Coordination Program was established in 2004 at CDC, which has consistently operated collaboratively with the genetic testing community to characterize publicly available genomic DNA reference materials.¹ The Program was initially centered on characterizing reference materials for Mendelian genetic disorders commonly tested by clinical laboratories. In 2010, the GeT-RM Program reported the first collaborative effort to characterize five clinically actionable genes implicated in interindividual drug response variability: CYP2D6, CYP2C19, CYP2C9, VKORC1, and UGT1A1.² Notably, this expansion by the GeT-RM Program to pharmacogenomic reference material characterization included both the analytical consensus targeted genotyping results identified across laboratories and genotyping platforms, as well as the inferred star (∗) allele diplotypes for the genes that adopt this unique nomenclature system.

Over 10 years later, the GeT-RM Program continues to support the growing interests around implementing clinical pharmacogenomics through multiple efforts on expanded gene panels and cell lines, rare and complex diplotype characterization, as well as a synergistic collaboration with the Association for Molecular Pathology (AMP) Pharmacogenetics Working Group. This issue of The Journal of Molecular Diagnostics includes the most recent GeT-RM pharmacogenomics work product, which characterizes genomic DNA reference materials for two clinically actionable cytochrome P450 genes, CYP3A4 and CYP3A5,³ as a companion article and supportive effort to the AMP Pharmacogenetics Working Group CYP3A4 and CYP3A5 star (∗) allele genotyping recommendations.⁴

Cell lines with confirmed genotypes are critical accuracy controls for DNA-based genetic testing, which are now routinely leveraged for laboratory-developed test development and validation, commercial assay in vitro diagnostic product regulatory package submissions, quality control protocols and workflows, as well as the quality assurance proficiency testing programs offered by the College for American Pathologists and other entities. Taken together, the GeT-RM Program has characterized >5800 loci in >450 cell line–based genomic DNA reference materials for fragile X, Ashkenazi Jewish carrier screening panel disorders, cystic fibrosis, Huntington disease, MTHFR-related homocysteinemia, α1-antitrypsin deficiency, multiple endocrine neoplasia, BRCA1- and BRCA2-related cancers, Duchenne muscular dystrophy, myotonic dystrophy, Rett syndrome, spinal muscular atrophy, as well as 11 human leukocyte antigen loci (https://www.cdc.gov/labquality/get-rm, last accessed July 5, 2023). The mechanism of the GeT-RM Program includes testing genomic DNA materials collaboratively through clinical genetic laboratories with diverse genotyping and sequencing platforms, which ultimately leads to the reporting of consensus results for interrogated germline genetic variants. Importantly, these materials are publicly available through the Coriell Cell Repositories (Camden, NJ), and they are a synergistic resource with the benchmarking reference materials provided by the Genome in a Bottle/National Institute of Standards and Technology⁵ and the Global Alliance for Genomics and Health consortia.⁶

Approximately 20% of all medications have response phenotypes that are associated with germline pharmacogenomic variants in genes implicated in drug metabolism,⁷ and the vast majority of the general population carries at least one clinically actionable pharmacogenomic variant. As such, clinical pharmacogenomic testing is increasingly being implemented at certain health care systems and medical centers, which is supported by the practice guidelines from the Clinical Pharmacogenetics Implementation Consortium⁸ and the Dutch Pharmacogenetics Working Group⁹ and the evidence tables published by the US Food and Drug Administration.¹⁰ Additional implementation resources include the Pharmacogenomics Knowledgebase (PharmGKB),¹¹ Pharmacogene Variation (PharmVar) Consortium,¹² the AMP Pharmacogenetics Working Group,¹³ and the American College of Medical Genetics and Genomics pharmacogenomic testing and reporting recommendations.¹⁴

The growing enthusiasm for implementing pharmacogenomics and the increasing availability of clinical evidence and practice guidelines prompted the GeT-RM Program to significantly expand its initial reference material study of 5 genes in 2010 to 28 genes in 2016.¹⁵ A total of 137 Coriell cell lines were selected for this expanded study based on ethnic diversity and partial genotype characterization from earlier testing, which were interrogated by targeted genotyping using several commercially available assays and laboratory-developed tests. Consensus genotype results from two or more laboratories were confirmed in >100 pharmacogenomic alleles, with most discrepancies being attributed to differences in assay design and/or variability in allele nomenclature.¹⁵ The characterization of these reference materials is not intended to indicate that testing these genes is advocated by the GeT-RM Program; rather, it is more so a resource for clinical laboratories that have independently determined if these genes and variants have sufficient evidence to include in their clinical pharmacogenomic testing panels.

Despite the comprehensive approach of the expanded 2016 GeT-RM pharmacogenomics study, a subsequent targeted effort was pursued to further characterize reference materials for CYP2D6, strategically prioritizing cell lines with complex CYP2D6 diplotypes, structural variants, and low-frequency alleles.¹⁶ The CYP2D6 gene region on chromosome 22 is highly complex and difficult to interrogate by commonly employed genotyping and sequencing methods, in large part because of its highly homologous neighboring pseudogenes, CYP2D7 and CYP2D8.¹⁷ Given the clinical significance of CYP2D6 and its pharmacogenomic roles across multiple drug classes and clinical specialties, including the importance of comprehensive CYP2D6 characterization with copy number assessment to more precisely predict enzyme activity and metabolizer phenotype, this focused GeT-RM effort will likely have a direct impact on improving the quality control of CYP2D6 testing among clinical laboratories in the United States. Moreover, as the AMP Pharmacogenetics Working Group program was evolving in parallel at this time, this period marked the emerging synergistic partnership between the AMP and the GeT-RM programs, specifically underlining the utility of generating comprehensive reference materials for individual pharmacogenomic genes as the AMP Pharmacogenetics Working Group publishes recommended alleles for clinical pharmacogenomic testing.

The AMP Pharmacogenetics Working Group develops documents that recommend a minimum set of variant alleles to include in clinical pharmacogenomic test panels using a two-tier strategy. Tier 1 alleles are a minimum set of variant alleles recommended for clinical testing, whereas tier 2 alleles are additional alleles that do not meet all criteria for inclusion in tier 1 but that may be considered for clinical testing. More important, clinical pharmacogenomic-based medication management recommendations are not part of the AMP Pharmacogenetics Working Group curation of tier 1 and 2 alleles, as those are specifically addressed by the implementation resources noted above (ie, Clinical Pharmacogenetics Implementation Consortium, Dutch Pharmacogenetics Working Group, and US Food and Drug Administration). One of the requirements for tier 1 status is the availability of reference materials, which is one of the major reasons why some alleles are triaged to tier 2. As these allele recommendations began to be published, the GeT-RM Program identified an opportunity to further support the Working Group by prioritizing the characterization of tier 2 variants that previously did not have publicly available reference materials. This work ultimately led to the extended availability of reference materials for tier 2 variants in CYP2C9, CYP2C19, the CYP2C cluster variant, VKORC1, and GGCX.¹⁸ As such, future versions of the AMP Pharmacogenetics Working Group recommendations may be updated on the basis of the availability of these reference materials for previously curated tier 2 variants.

In addition, as the genetic testing landscape evolves from targeted genotyping to high-throughput sequencing, it is increasingly appreciated that there is far more pharmacogenomic variation in the general populations than previously expected, which complicates haplotype assessment and star (∗) allele prediction. As such, the GeT-RM Program recently reported its first collaborative sequencing work on CYP2C8, CYP2C9, and CYP2C19 with the cell lines previously characterized by targeted genotyping to determine if these specimens harbored undetected variant alleles and to assess the consistency of commonly used star (∗) allele calling tools.¹⁹ As expected, rare and/or novel alleles were discovered in all three genes by sequencing, which subsequently were submitted to PharmVar for star (∗) allele assignment.

In 2022, the GeT-RM Program again synergistically supported the AMP Pharmacogenetics Working Group by reporting a collaborative characterization of star (∗) alleles in TPMT and NUDT15,²⁰ which are clinically actionable genes implicated in thiopurine toxicity. As noted above, this work was critical in facilitating the Working Group to assign TPMT and NUDT15 alleles in a companion article as either tier 1 or tier 2 recommendations based on the availability of reference materials. This supportive model was again replicated in this issue of The Journal of Molecular Diagnostics through companion GeT-RM and AMP Pharmacogenetics Working Group articles for two additional cytochrome P450 genes in the CYP3A subfamily of isoenzymes, CYP3A4 and CYP3A5.⁴ The CYP3A4 gene currently has a Dutch Pharmacogenetics Working Group guideline for pharmacogenomic-guided quetiapine dosing,²¹ and CYP3A5 has a Clinical Pharmacogenetics Implementation Consortium guideline for pharmacogenomic-guided tacrolimus dosing,²² which is also noted in the US Food and Drug Administration Table of Pharmacogenetic Associations.¹⁰

In conclusion, clinical pharmacogenomic testing and implementation programs continue to expand across the United States and internationally, which is driven by a network of professional resources that together make new discoveries, curate available evidence, publish practice guidelines, measure clinical utility, lobby third-party payers and reimbursement policies, provide education and clinical decision support tools, as well as the laboratories and companies that develop and provide clinical pharmacogenomic tests. For >10 years, the GeT-RM Program at the CDC has made a direct impact on the evolution of clinical pharmacogenomic testing by continuing to identify resource gaps related to reference material characterization, with the overarching result of improving the quality and consistency of delivering accurate pharmacogenomic results to both providers and patients. The genetic testing community is grateful for the efforts of the GeT-RM Program and looks forward to continued collaboration in the future, particularly as the field rapidly evolves from targeted genotyping to more comprehensive short- and long-read sequencing assays, as well as the ongoing management of interrogating and characterizing novel and/or complex pharmacogenomic haplotypes.