New CDC Partnerships to Advance the Development and Validation of Next Generation Sequencing Tests: A Publicly Available List of Expert Curated Variants

Over the last decade, genetic testing has evolved from examining a few well-defined variants in one or a few genes to the capability to examine much of the human genome using next generation sequencing (NGS). These analyses are particularly useful for disorders with locus and allelic heterogeneity, and are now the norm in several clinical areas, such as hereditary cancers and cardiomyopathy.

Clinical tests in the United States are regulated under the Clinical Laboratory Improvement Amendments (CLIA), which require laboratories to establish performance characteristics such as accuracy, precision,  analytical sensitivity and specificity, reportable range of test results for the test system, reference intervals/normal values, and any other performance characteristic required for test performance. Analytic validation studies are designed to demonstrate that a test can reliably analyze clinically relevant sequences and identify disease-associated variants.  Such studies demonstrate that the test platform provides reliable sequence across the genomic regions targeted by the test, that the test system can correctly identify disease-associated variants in genomic regions targeted by the test, and that the informatics pipeline can reliably analyze the data to produce accurate sequence.  Characterized reference materials, such as genomic DNA known to contain disease-associated variants, are used in test development and validation as a vital tool to establish that a genetic test performs as intended. Use of genomic DNA reference materials has generally worked well for tests targeting one or a few genes but presents challenges for assuring the quality of sequencing tests with more than a few genes.

Development and validation of large NGS assays can be difficult.  Laboratories often cannot obtain reference materials that encompass the necessary scope of variants and variant types because the supply of available characterized genomic DNA samples from cell lines or patient samples is limited and does not cover many clinically relevant variants and/or variant types. Even if available, the cost to test the large number of samples needed would be prohibitive.

These challenges may be solved, at least in part, by supplementing genomic DNA samples with in silico (electronic) reference materials to develop and validate the informatics pipeline used to analyze the data generated during the physical sequencing of the patient sample.  In silico reference materials are created by electronically adding variants to NGS sequencing files. The “mutagenized” files can then be analyzed using the NGS informatics pipeline to see if the added variants are detected. In silico reference materials are truly scalable and can be tailored to meet the needs of any NGS assay regardless of size, including whole exome/whole genome.  The Food and Drug Administration and professional societies including the Association for Molecular Pathology and the College of American Pathologists    support the development and use of in silico reference materials for validation studies.

It can be challenging for individual laboratories to recognize the clinically relevant variant spectrum and have knowledge of difficult to sequence genomic regions, such as those with high GC content, highly homologous genes, or repetitive sequences for all genes included in the test.  Laboratories may face challenges in selecting clinically important and/or difficult to detect variants that should be included, either as DNA samples or as part of in silico reference materials, in validation studies.

To address these issues, 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 variants. As part of this study, the ClinGen Variant Curation Expert Panels nominated 546 variants found in 84 disease associated genes (link to table of genes ), including common pathogenic and difficult to detect variants. Variant types nominated included 346 SNVs, 104 deletions, 37 CNVs, 25 duplications, 18 deletion-insertions, 5 inversions, 4 insertions, 2 complex rearrangements, 3 in difficult to sequence regions, and 2 fusions. The nominated variants are associated with a wide range of diseases that include heritable cancers, inborn errors of metabolism, cardiomyopathy, diabetes, and immune disorders.

This expert-curated resource can help laboratories to identify a uniform set of important variants and variant types for each gene included in their NGS panel.  The list can inform selection of genomic DNA samples and aid design of in silico reference materials.  This facilitates development and standardization of well-designed and validated clinical assays that provide accurate and reliable results, enabling accurate diagnosis and appropriate treatment decisions.  In addition, the capability to create and manipulate electronic reference materials will support evolving precision medicine and public health applications.