CYP3A4 and CYP3A5 Genotyping Recommendations: A Joint Consensus Recommendation of the Association for Molecular Pathology, Clinical Pharmacogenetics Implementation Consortium, College of American Pathologists, Dutch Pharmacogenetics Working Group of the Royal Dutch Pharmacists Association, European Society for Pharmacogenomics and Personalized Therapy, and Pharmacogenomics Knowledgebase.

The goals of the Association for Molecular Pathology Clinical Practice Committee's Pharmacogenomics (PGx) Working Group are to define the key attributes of pharmacogenetic alleles recommended for clinical testing and a minimum set of variants that should be included in clinical PGx genotyping assays. This document series provides recommendations for a minimum panel of variant alleles (tier 1) and an extended panel of variant alleles (tier 2) that will aid clinical laboratories when designing assays for PGx testing. The Association for Molecular Pathology PGx Working Group considered functional impact of the variant alleles, allele frequencies in multiethnic populations, the availability of reference materials, and other technical considerations for PGx testing when developing these recommendations. The goal of this Working Group is to promote standardization of PGx gene/allele testing across clinical laboratories. This document will focus on clinical CYP3A4 and CYP3A5 PGx testing that may be applied to all CYP3A4- and CYP3A5-related medications. These recommendations are not to be interpreted as prescriptive but to provide a reference guide.

Characterization of Reference Materials for CYP3A4 and CYP3A5: A (GeT-RM) Collaborative Project.

Pharmacogenetic testing for CYP3A4 is increasingly provided by clinical and research laboratories; however, only a limited number of quality control and reference materials are currently available for many of the CYP3A4 variants included in clinical tests. To address this need, the Division of Laboratory Systems, CDC-based Genetic Testing Reference Material Coordination Program (GeT-RM), in collaboration with members of the pharmacogenetic testing and research communities and the Coriell Institute for Medical Research, has characterized 30 DNA samples derived from Coriell cell lines for CYP3A4. Samples were distributed to five volunteer laboratories for genotyping using a variety of commercially available and laboratory-developed tests. Sanger and next-generation sequencing were also utilized by some of the laboratories. Whole-genome sequencing data from the 1000 Genomes Projects were utilized to inform genotype. Twenty CYP3A4 alleles were identified in the 30 samples characterized for CYP3A4: CYP3A4∗4, ∗5, ∗6, ∗7, ∗8, ∗9, ∗10, ∗11, ∗12, ∗15, ∗16, ∗18, ∗19, ∗20, ∗21, ∗22, ∗23, ∗24, ∗35, and a novel allele, CYP3A4∗38. Nineteen additional samples with preexisting data for CYP3A4 or CYP3A5 were re-analyzed to generate comprehensive reference material panels for these genes. These publicly available and well-characterized materials can be used to support the quality assurance and quality control programs of clinical laboratories performing clinical pharmacogenetic testing.

TPMT and NUDT15 Genotyping Recommendations: A Joint Consensus Recommendation of the Association for Molecular Pathology, Clinical Pharmacogenetics Implementation Consortium, College of American Pathologists, Dutch Pharmacogenetics Working Group of the Royal Dutch Pharmacists Association, European Society for Pharmacogenomics and Personalized Therapy, and Pharmacogenomics Knowledgebase.

The goals of the Association for Molecular Pathology Clinical Practice Committee's Pharmacogenomics (PGx) Working Group are to define the key attributes of pharmacogenetic alleles recommended for clinical testing and a minimum set of variants that should be included in clinical PGx genotyping assays. This article provides recommendations for a minimum panel of variant alleles (Tier 1) and an extended panel of variant alleles (Tier 2) that will aid clinical laboratories when designing assays for PGx testing. The Association for Molecular Pathology PGx Working Group considered the functional impact of the variant alleles, allele frequencies in multiethnic populations, the availability of reference materials, as well as other technical considerations for PGx testing when developing these recommendations. The ultimate goal of this Working Group is to promote standardization of PGx gene/allele testing across clinical laboratories. This article focuses on clinical TPMT and NUDT15 PGx testing, which may be applied to all thiopurine S-methyltransferase (TPMT) and nudix hydrolase 15 (NUDT15)-related medications. These recommendations are not to be interpreted as prescriptive, but to provide a reference guide.

Characterization of Reference Materials for TPMT and NUDT15: A GeT-RM Collaborative Project.

Pharmacogenetic testing is increasingly provided by clinical and research laboratories; however, only a limited number of quality control and reference materials are currently available for many of the TPMT and NUDT15 variants included in clinical tests. To address this need, the Division of Laboratory Systems, Centers for Disease Control and Prevention-based Genetic Testing Reference Material (GeT-RM) coordination program, in collaboration with members of the pharmacogenetic testing and research communities and the Coriell Institute for Medical Research, has characterized 19 DNA samples derived from Coriell cell lines. DNA samples were distributed to four volunteer testing laboratories for genotyping using a variety of commercially available and laboratory developed tests and/or Sanger sequencing. Of the 12 samples characterized for TPMT, newly identified variants include TPMT∗2, ∗6, ∗12, ∗16, ∗21, ∗24, ∗32, ∗33, and ∗40; for the 7 NUDT15 reference material samples, newly identified variants are NUDT15∗2, ∗3, ∗4, ∗5, ∗6, and ∗9. In addition, a novel haplotype, TPMT∗46, was identified in this study. Preexisting data on an additional 11 Coriell samples, as well as some supplemental testing, were used to create comprehensive reference material panels for TPMT and NUDT15. These publicly available and well-characterized materials can be used to support the quality assurance and quality control programs of clinical laboratories performing clinical pharmacogenetic testing.

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

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.

Creation of an Expert Curated Variant List for Clinical Genomic Test Development and Validation: A ClinGen and GeT-RM Collaborative Project.

Modern genomic sequencing tests often interrogate large numbers of genes. Identification of appropriate reference materials for development, validation studies, and quality assurance of these tests poses a significant challenge for laboratories. It is difficult to develop and maintain expert knowledge to identify all variants that must be validated to ensure analytic and clinical validity. Additionally, it is usually not possible to procure appropriate and characterized genomic DNA reference materials containing the number and scope of variants required. To address these challenges, the Centers for Disease Control and Prevention's Genetic Testing Reference Material Program (GeT-RM) has partnered with the Clinical Genome Resource (ClinGen) to develop a publicly available list of expert curated, clinically important variants. ClinGen Variant Curation Expert Panels nominated 546 variants found in 84 disease-associated genes, including common pathogenic and difficult-to-detect variants. Variant types nominated included 346 single nucleotide variants, 104 deletions, 37 copy number variants, 25 duplications, 18 deletion-insertions, 5 inversions, 4 insertions, 2 complex rearrangements, 3 difficult-to-sequence regions, and 2 fusions. This expert-curated variant list is a resource that provides a foundation for designing comprehensive validation studies and for creating in silico reference materials for clinical genomic test development and validation.

Recommendations for Clinical CYP2D6 Genotyping Allele Selection: A Joint Consensus Recommendation of the Association for Molecular Pathology, College of American Pathologists, Dutch Pharmacogenetics Working Group of the Royal Dutch Pharmacists Association, and the European Society for Pharmacogenomics and Personalized Therapy.

The goals of the Association for Molecular Pathology Clinical Practice Committee's Pharmacogenomics (PGx) Working Group are to define the key attributes of pharmacogenetic alleles recommended for clinical testing, and to determine a minimal set of variants that should be included in clinical PGx genotyping assays. This document series provides recommendations on a minimal panel of variant alleles (Tier 1) and an extended panel of variant alleles (Tier 2) that will aid clinical laboratories in designing assays for PGx testing. When developing these recommendations, the Association for Molecular Pathology PGx Working Group considered the functional impact of the variant alleles, allele frequencies in multiethnic populations, the availability of reference materials, as well as other technical considerations with regard to PGx testing. The ultimate goal of this Working Group is to promote standardization of PGx gene/allele testing across clinical laboratories. This document is focused on clinical CYP2D6 PGx testing that may be applied to all cytochrome P450 2D6-metabolized medications. These recommendations are not meant to be interpreted as prescriptive but to provide a reference guide for clinical laboratories that may be either implementing PGx testing or reviewing and updating their existing platform.

Characterization of Reference Materials with an Association for Molecular Pathology Pharmacogenetics Working Group Tier 2 Status: CYP2C9, CYP2C19, VKORC1, CYP2C Cluster Variant, and GGCX: A GeT-RM Collaborative Project.

Pharmacogenetic testing is increasingly available from clinical and research laboratories. However, only a limited number of quality control and other reference materials are currently available for many of the variants that are tested. The Association for Molecular Pathology Pharmacogenetic Work Group has published a series of papers recommending alleles for inclusion in clinical testing. Several of the alleles were not considered for tier 1 because of a lack of reference materials. To address this need, the Division of Laboratory Systems, Centers for Disease Control and Prevention-based Genetic Testing Reference Material (GeT-RM) program, in collaboration with members of the pharmacogenetic testing and research communities and the Coriell Institute for Medical Research, has characterized 18 DNA samples derived from Coriell cell lines. DNA samples were distributed to five volunteer testing laboratories for genotyping using three commercially available and laboratory developed tests. Several tier 2 variants, including CYP2C9∗13, CYP2C19∗35, the CYP2C cluster variant (rs12777823), two variants in VKORC1 (rs61742245 and rs72547529) related to warfarin resistance, and two variants in GGCX (rs12714145 and rs11676382) related to clotting factor activation, were identified among these samples. These publicly available materials complement the pharmacogenetic reference materials previously characterized by the GeT-RM program and will support the quality assurance and quality control programs of clinical laboratories that perform pharmacogenetic testing.

Recommendations for Clinical Warfarin Genotyping Allele Selection: A Report of the Association for Molecular Pathology and the College of American Pathologists.

The goal of the Association for Molecular Pathology (AMP) Clinical Practice Committee's AMP Pharmacogenomics (PGx) Working Group is to define the key attributes of PGx alleles recommended for clinical testing and a minimum set of variants that should be included in clinical PGx genotyping assays. This document series provides recommendations for a minimum panel of variant alleles (tier 1) and an extended panel of variant alleles (tier 2) that will aid clinical laboratories when designing assays for PGx testing. The AMP PGx Working Group considered functional impact of the variants, allele frequencies in multiethnic populations, the availability of reference materials, as well as other technical considerations for PGx testing when developing these recommendations. The ultimate goal is to promote standardization of PGx gene/allele testing across clinical laboratories. These recommendations are not to be interpreted as prescriptive but to provide a reference guide. Of note, a separate article with recommendations for CYP2C9 allele selection was previously developed by the PGx Working Group that can be applied broadly to CYP2C9-related medications. The warfarin allele recommendations in this report incorporate the previous CYP2C9 allele recommendations and additional genes and alleles that are specific to warfarin testing.

Characterization of Reference Materials for Genetic Testing of CYP2D6 Alleles: A GeT-RM Collaborative Project.

Pharmacogenetic testing increasingly is available from clinical and research laboratories. However, only a limited number of quality control and other reference materials currently are available for the complex rearrangements and rare variants that occur in the CYP2D6 gene. To address this need, the Division of Laboratory Systems, CDC-based Genetic Testing Reference Material Coordination Program, in collaboration with members of the pharmacogenetic testing and research communities and the Coriell Cell Repositories (Camden, NJ), has characterized 179 DNA samples derived from Coriell cell lines. Testing included the recharacterization of 137 genomic DNAs that were genotyped in previous Genetic Testing Reference Material Coordination Program studies and 42 additional samples that had not been characterized previously. DNA samples were distributed to volunteer testing laboratories for genotyping using a variety of commercially available and laboratory-developed tests. These publicly available samples will support the quality-assurance and quality-control programs of clinical laboratories performing CYP2D6 testing.

Characterization of 108 Genomic DNA Reference Materials for 11 Human Leukocyte Antigen Loci: A GeT-RM Collaborative Project.

The highly polymorphic human leukocyte antigen (HLA) genes, located in the human major histocompatibility complex, encode the class I and II antigen-presenting molecules, which are centrally involved in the immune response. HLA typing is used for several clinical applications, such as transplantation, pharmacogenetics, and diagnosis of autoimmune disease. HLA typing is highly complex because of the homology of HLA genes and pseudogenes and the extensive polymorphism in the population. The Centers for Disease Control and Prevention established the Genetic Testing Reference Materials Coordination Program (GeT-RM) in partnership with the genetics community to improve the availability of genomic DNA reference materials necessary for quality assurance of genetic laboratory testing. The GeT-RM together with three clinical laboratories and the Coriell Cell Repositories have characterized genomic DNA obtained from a panel of 108 cell lines for all HLA classic polymorphic loci: HLA-A, B, C, DRB1, DRB3, DRB4, DRB5, DQA1, DQB1, DPA1, and DPB1. The goal was to develop a publicly available and renewable source of well-characterized genomic DNA reference materials to support molecular HLA typing assay development, validation, and verification, quality control, and proficiency testing. These genomic DNA samples are publicly available from the National Institutes of General Medical Science Repository at the Coriell Cell Repositories.

Recommendations for Clinical CYP2C19 Genotyping Allele Selection: A Report of the Association for Molecular Pathology.

This document was developed by the Pharmacogenomics (PGx) Working Group of the Association for Molecular Pathology Clinical Practice Committee, whose aim is to recommend variants for inclusion in clinical pharmacogenomic testing panels. The goals of the Association for Molecular Pathology PGx Working Group are to define the key attributes of PGx alleles recommended for clinical testing and to define a minimum set of variants that should be included in clinical PGx genotyping assays. These recommendations include a minimum panel of variant alleles (tier 1) and an extended panel of variant alleles (tier 2) that will aid clinical laboratories when designing PGx assays. The Working Group considered variant allele frequencies in different populations and ethnicities, the availability of reference materials, and other technical considerations for PGx testing when developing these recommendations. These CYP2C19 genotyping recommendations are the first of a series of recommendations for PGx testing. These recommendations are not to be interpreted as restrictive, but they are meant to provide a helpful guide.

Development and Characterization of Reference Materials for Genetic Testing: Focus on Public Partnerships.

Characterized reference materials (RMs) are needed for clinical laboratory test development and validation, quality control procedures, and proficiency testing to assure their quality. In this article, we review the development and characterization of RMs for clinical molecular genetic tests. We describe various types of RMs and how to access and utilize them, especially focusing on the Genetic Testing Reference Materials Coordination Program (Get-RM) and the Genome in a Bottle (GIAB) Consortium. This review also reinforces the need for collaborative efforts in the clinical genetic testing community to develop additional RMs.

Characterization of 137 Genomic DNA Reference Materials for 28 Pharmacogenetic Genes: A GeT-RM Collaborative Project.

Pharmacogenetic testing is increasingly available from clinical laboratories. However, only a limited number of quality control and other reference materials are currently available to support clinical testing. To address this need, the Centers for Disease Control and Prevention-based Genetic Testing Reference Material Coordination Program, in collaboration with members of the pharmacogenetic testing community and the Coriell Cell Repositories, has characterized 137 genomic DNA samples for 28 genes commonly genotyped by pharmacogenetic testing assays (CYP1A1, CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A4, CYP3A5, CYP4F2, DPYD, GSTM1, GSTP1, GSTT1, NAT1, NAT2, SLC15A2, SLC22A2, SLCO1B1, SLCO2B1, TPMT, UGT1A1, UGT2B7, UGT2B15, UGT2B17, and VKORC1). One hundred thirty-seven Coriell cell lines were selected based on ethnic diversity and partial genotype characterization from earlier testing. DNA samples were coded and distributed to volunteer testing laboratories for targeted genotyping using a number of commercially available and laboratory developed tests. Through consensus verification, we confirmed the presence of at least 108 variant pharmacogenetic alleles. These samples are also being characterized by other pharmacogenetic assays, including next-generation sequencing, which will be reported separately. Genotyping results were consistent among laboratories, with most differences in allele assignments attributed to assay design and variability in reported allele nomenclature, particularly for CYP2D6, UGT1A1, and VKORC1. These publicly available samples will help ensure the accuracy of pharmacogenetic testing.

Development of a genomic DNA reference material panel for Rett syndrome (MECP2-related disorders) genetic testing.

Rett syndrome is a dominant X-linked disorder caused by point mutations (approximately 80%) or by deletions or insertions (approximately 15% to 18%) in the MECP2 gene. It is most common in females but lethal in males, with a distinctly different phenotype. Rett syndrome patients have severe neurological and behavioral problems. Clinical genetic testing laboratories commonly use characterized genomic DNA reference materials to assure the quality of the testing process; however, none are commercially available for MECP2 genetic testing. The Centers for Disease Control and Prevention's Genetic Testing Reference Material Coordination Program, in collaboration with the genetic testing community and the Coriell Cell Repositories, established 27 new cell lines and characterized the MECP2 mutations in these and in 8 previously available cell lines. DNA samples from the 35 cell lines were tested by eight clinical genetic testing laboratories using DNA sequence analysis and methods to assess copy number (multiplex ligation-dependent probe amplification, semiquantitative PCR, or array-based comparative genomic hybridization). The eight common point mutations known to cause approximately 60% of Rett syndrome cases were identified, as were other MECP2 variants, including deletions, duplications, and frame shift and splice-site mutations. Two of the 35 samples were from males with MECP2 duplications. These MECP2 and other characterized genomic DNA samples are publicly available from the NIGMS Repository at the Coriell Cell Repositories.

Current landscape and new paradigms of proficiency testing and external quality assessment for molecular genetics.

CONTEXT: Participation in proficiency testing (PT) or external quality assessment (EQA) programs allows the assessment and comparison of test performance among different clinical laboratories and technologies. In addition to the approximately 2300 tests for individual genetic disorders, recent advances in technology have enabled the development of clinical tests that quickly and economically analyze the entire human genome. New PT/EQA approaches are needed to ensure the continued quality of these complex tests. OBJECTIVES: To review the availability and scope of PT/EQA for molecular genetic testing for inherited conditions in Europe, Australasia, and the United States; to evaluate the successes and demonstrated value of available PT/EQA programs; and to examine the challenges to the provision of comprehensive PT/EQA posed by new laboratory practices and methodologies. DATA SOURCES: The available literature on this topic was reviewed and supplemented with personal experiences of several PT/EQA providers. CONCLUSIONS: Proficiency testing/EQA schemes are available for common genetic disorders tested in many clinical laboratories but are not available for most genetic tests offered by only one or a few laboratories. Provision of broad, method-based PT schemes, such as DNA sequencing, would allow assessment of many tests for which formal PT is not currently available. Participation in PT/EQA improves the quality of testing by identifying inaccuracies that laboratories can trace to errors in their testing processes. Areas of research and development to ensure that PT/EQA programs can meet the needs of new and evolving genetic tests and technologies are identified and discussed.

Molecular diagnostics: harmonization through reference materials, documentary standards and proficiency testing.

There is a great need for harmonization in nucleic acid testing for infectious disease and clinical genetics. The proliferation of assay methods, the number of targets for molecular diagnostics and the absence of standard reference materials contribute to variability in test results among laboratories. This article provides a comprehensive overview of reference materials, related documentary standards and proficiency testing programs. The article explores the relationships among these resources and provides necessary information for people practicing in this area that is not taught in formal courses and frequently is obtained on an ad hoc basis. The aim of this article is to provide helpful tools for molecular diagnostic laboratories.

Development and characterization of reference materials for MTHFR, SERPINA1, RET, BRCA1, and BRCA2 genetic testing.

Well-characterized reference materials (RMs) are integral in maintaining clinical laboratory quality assurance for genetic testing. These RMs can be used for quality control, monitoring of test performance, test validation, and proficiency testing of DNA-based genetic tests. To address the need for such materials, the Centers for Disease Control and Prevention established the Genetic Testing Reference Material Coordination Program (GeT-RM), which works with the genetics community to improve public availability of characterized RMs for genetic testing. To date, the GeT-RM program has coordinated the characterization of publicly available genomic DNA RMs for a number of disorders, including cystic fibrosis, Huntington disease, fragile X, and several genetic conditions with relatively high prevalence in the Ashkenazi Jewish population. Genotypic information about a number of other cell lines has been collected and is also available. The present study includes the development and commutability/genotype characterization of 10 DNA samples for clinically relevant mutations or sequence variants in the following genes: MTHFR; SERPINA1; RET; BRCA1; and BRCA2. DNA samples were analyzed by 19 clinical genetic laboratories using a variety of assays and technology platforms. Concordance was 100% for all samples, with no differences observed between laboratories using different methods. All DNA samples are available from Coriell Cell Repositories and characterization information can be found on the GeT-RM website.

Development of genomic reference materials for cystic fibrosis genetic testing.

The number of different laboratories that perform genetic testing for cystic fibrosis is increasing. However, there are a limited number of quality control and other reference materials available, none of which cover all of the alleles included in commercially available reagents or platforms. The alleles in many publicly available cell lines that could serve as reference materials have neither been confirmed nor characterized. The Centers for Disease Control and Prevention-based Genetic Testing Reference Material Coordination Program, in collaboration with members of the genetic testing community as well as Coriell Cell Repositories, have characterized an extended panel of publicly available genomic DNA samples that could serve as reference materials for cystic fibrosis testing. Six cell lines [containing the following mutations: E60X (c.178G>T), 444delA (c.312delA), G178R (c.532G>C), 1812-1G>A (c.1680-1G>A), P574H (c.1721C>A), Y1092X (c.3277C>A), and M1101K (c.3302T>A)] were selected from those existing at Coriell, and seven [containing the following mutations: R75X (c.223C>T), R347H (c.1040G>A), 3876delA (c.3744delA), S549R (c.1646A>C), S549N (c.1647G>A), 3905insT (c.3773_3774insT), and I507V (c.1519A>G)] were created. The alleles in these materials were confirmed by testing in six different volunteer laboratories. These genomic DNA reference materials will be useful for quality assurance, proficiency testing, test development, and research and should help to assure the accuracy of cystic fibrosis genetic testing in the future. The reference materials described in this study are all currently available from Coriell Cell Repositories.

Consensus characterization of 16 FMR1 reference materials: a consortium study.

Fragile X syndrome, which is caused by expansion of a (CGG)(n) repeat in the FMR1 gene, occurs in approximately 1:3500 males and causes mental retardation/behavioral problems. Smaller (CGG)(n) repeat expansions in FMR1, premutations, are associated with premature ovarian failure and fragile X-associated tremor/ataxia syndrome. An FMR1-sizing assay is technically challenging because of high GC content of the (CGG)(n) repeat, the size limitations of conventional PCR, and a lack of reference materials available for test development/validation and routine quality control. The Centers for Disease Control and Prevention and the Association for Molecular Pathology, together with the genetic testing community, have addressed the need for characterized fragile X mutation reference materials by developing characterized DNA samples from 16 cell lines with repeat lengths representing important phenotypic classes and diagnostic cutoffs. The alleles in these materials were characterized by consensus analysis in nine clinical laboratories. The information generated from this study is available on the Centers for Disease Control and Prevention and Coriell Cell Repositories websites. DNA purified from these cell lines is available to the genetics community through the Coriell Cell Repositories. The public availability of these reference materials should help support accurate clinical fragile X syndrome testing.