The Science and Art of Clinical Genetic Variant Classification and Its Impact on Test Accuracy.

Clinical genetic variant classification science is a growing subspecialty of clinical genetics and genomics. The field's continued improvement is essential for the success of precision medicine in both germline (hereditary) and somatic (oncology) contexts. This review focuses on variant classification for DNA next-generation sequencing tests. We first summarize current limitations in variant discovery and definition, and then describe the current five- and four-tier classification systems outlined in dominant standards and guideline publications for germline and somatic tests, respectively. We then discuss measures of variant classification discordance and the field's bias for positive results, as well as considerations for panel size and population screening in the context of estimates of positive predictive value thatincorporate estimated variant classification imperfections. Finally, we share opinions on the current state of variant classification from some of the authors of the most widely used standards and guideline publications and from other domain experts.

Clinical pharmacogenomic testing and reporting: A technical standard of the American College of Medical Genetics and Genomics (ACMG).

Pharmacogenomic testing interrogates germline sequence variants implicated in interindividual drug response variability to infer a drug response phenotype and to guide medication management for certain drugs. Specifically, discrete aspects of pharmacokinetics, such as drug metabolism, and pharmacodynamics, as well as drug sensitivity, can be predicted by genes that code for proteins involved in these pathways. Pharmacogenomics is unique and differs from inherited disease genetics because the drug response phenotype can be drug-dependent and is often unrecognized until an unexpected drug reaction occurs or a patient fails to respond to a medication. Genes and variants with sufficiently high levels of evidence and consensus may be included in a clinical pharmacogenomic test; however, result interpretation and phenotype prediction can be challenging for some genes and medications. This document provides a resource for laboratories to develop and implement clinical pharmacogenomic testing by summarizing publicly available resources and detailing best practices for pharmacogenomic nomenclature, testing, result interpretation, and reporting.

Laboratory testing for fragile X, 2021 revision: a technical standard of the American College of Medical Genetics and Genomics (ACMG).

Molecular genetic testing of the FMR1 gene is commonly performed in clinical laboratories. Pathogenic variants in the FMR1 gene are associated with fragile X syndrome, fragile X-associated tremor ataxia syndrome (FXTAS), and fragile X-associated primary ovarian insufficiency (FXPOI). This document provides updated information regarding FMR1 pathogenic variants, including prevalence, genotype-phenotype correlations, and variant nomenclature. Methodological considerations are provided for Southern blot analysis and polymerase chain reaction (PCR) amplification of FMR1, including triplet repeat-primed and methylation-specific PCR.The American College of Medical Genetics and Genomics (ACMG) Laboratory Quality Assurance Committee has the mission of maintaining high technical standards for the performance and interpretation of genetic tests. In part, this is accomplished by the publication of the document ACMG Technical Standards for Clinical Genetics Laboratories, which is now maintained online ( http://www.acmg.net ). This subcommittee also reviews the outcome of national proficiency testing in the genetics area and may choose to focus on specific diseases or methodologies in response to those results. Accordingly, the subcommittee selected fragile X syndrome to be the first topic in a series of supplemental sections, recognizing that it is one of the most frequently ordered genetic tests and that it has many alternative methods with different strengths and weaknesses. This document is the fourth update to the original standards and guidelines for fragile X testing that were published in 2001, with revisions in 2005 and 2013, respectively.This versionClarifies the clinical features associated with different FMRI variants (Section 2.3)Discusses important reporting considerations (Section 3.3.1.3)Provides updates on technology (Section 4.1).

Reducing Sanger confirmation testing through false positive prediction algorithms.

PURPOSE: Clinical genome sequencing (cGS) followed by orthogonal confirmatory testing is standard practice. While orthogonal testing significantly improves specificity, it also results in increased turnaround time and cost of testing. The purpose of this study is to evaluate machine learning models trained to identify false positive variants in cGS data to reduce the need for orthogonal testing. METHODS: We sequenced five reference human genome samples characterized by the Genome in a Bottle Consortium (GIAB) and compared the results with an established set of variants for each genome referred to as a truth set. We then trained machine learning models to identify variants that were labeled as false positives. RESULTS: After training, the models identified 99.5% of the false positive heterozygous single-nucleotide variants (SNVs) and heterozygous insertions/deletions variants (indels) while reducing confirmatory testing of nonactionable, nonprimary SNVs by 85% and indels by 75%. Employing the algorithm in clinical practice reduced overall orthogonal testing using dideoxynucleotide (Sanger) sequencing by 71%. CONCLUSION: Our results indicate that a low false positive call rate can be maintained while significantly reducing the need for confirmatory testing. The framework that generated our models and results is publicly available at https://github.com/HudsonAlpha/STEVE .

Recommended measures for the efficient care of patients with genetic disorders during the COVID-19 pandemic in low and middle income countries.

The coronavirus disease 2019 (COVID-19) emerged in early 2020 and since, has brought about tremendous cost to economies and healthcare systems universally. Reports of pediatric patients with inherited conditions and COVID-19 infections are emerging. Specific risks for morbidity and mortality that this pandemic carries for different categories of genetic disorders are still mostly unknown. Thus, there are no specific recommendations for the diagnosis, management, and treatment of patients with genetic disorders during the COVID-19 or other pandemics. Emerging publications, from Upper-Middle Income countries (UMIC), discuss the recent experiences of genetic centers in the continuity of care for patients with genetic disorders in the context of this pandemic. Many measures to facilitate the plan to continuous genetic care in a well-developed health system, may not be applicable in Low and Middle Income countries (LMIC). With poorly structured health systems and with the lack of established genetic services, the COVID-19 pandemic will easily exacerbate the access to care for patients with genetic disease in these countries. This article focuses on the unique challenges of providing genetic healthcare services during emergency situations in LMIC countries and provides practical preparations for this and other pandemic situations.

Assessing the strength of evidence for genes implicated in fatty acid oxidation disorders using the ClinGen clinical validity framework.

Newborn screening is an incredibly useful tool for the early identification of many metabolic disorders, including fatty acid oxidation (FAO) disorders. In many cases, molecular tests are necessary to reach a final diagnosis, highlighting the need for a thorough evaluation of genes implicated in FAO disorders. Using the ClinGen (Clinical Genome Resource) clinical validity framework, thirty genes were analyzed for the strength of evidence supporting their association with FAO disorders. Evidence was gathered from the literature by biocurators and presented to disease experts for review in order to assign a clinical validity classification of Definitive, Strong, Moderate, Limited, Disputed, Refuted, or No Reported Evidence. Of the gene-disease relationships evaluated, 22/30 were classified as Definitive, three as Moderate, one as Limited, three as No Reported Evidence and one as Disputed. Gene-disease relationships with a Limited, Disputed, and No Reported Evidence were found on two, six, and up to four panels out of 30 FAO disorder-specific panels, respectively, in the National Institute of Health Genetic Testing Registry, while over 70% of the genes on panels are definitively associated with an FAO disorder. These results highlight the need to systematically assess the clinical relevance of genes implicated in fatty acid oxidation disorders in order to improve the interpretation of genetic testing results and diagnosis of patients with these disorders.

Unique aspects of sequence variant interpretation for inborn errors of metabolism (IEM): The ClinGen IEM Working Group and the Phenylalanine Hydroxylase Gene.

The ClinGen Inborn Errors of Metabolism Working Group was tasked with creating a comprehensive, standardized knowledge base of genes and variants for metabolic diseases. Phenylalanine hydroxylase (PAH) deficiency was chosen to pilot development of the Working Group's standards and guidelines. A PAH variant curation expert panel (VCEP) was created to facilitate this process. Following ACMG-AMP variant interpretation guidelines, we present the development of these standards in the context of PAH variant curation and interpretation. Existing ACMG-AMP rules were adjusted based on disease (6) or strength (5) or both (2). Disease adjustments include allele frequency thresholds, functional assay thresholds, and phenotype-specific guidelines. Our validation of PAH-specific variant interpretation guidelines is presented using 85 variants. The PAH VCEP interpretations were concordant with existing interpretations in ClinVar for 69 variants (81%). Development of biocurator tools and standards are also described. Using the PAH-specific ACMG-AMP guidelines, 714 PAH variants have been curated and will be submitted to ClinVar. We also discuss strategies and challenges in applying ACMG-AMP guidelines to autosomal recessive metabolic disease, and the curation of variants in these genes.

A Clinicopathologic Evaluation of Incidental Fundic Gland Polyps With Dysplasia: Implications for Clinical Management.

OBJECTIVES: Fundic gland polyps (FGPs) can rarely exhibit dysplasia of the surface epithelium. Based on retrospective data, FGPs with dysplasia (FGPDs) are thought to be a strong marker for familial adenomatous polyposis (FAP), although sporadic, non-syndromic FGPDs also occur. Owing to the significant syndromic association, diagnosis of an apparently sporadic FGPD may prompt clinical evaluation for FAP, especially its attenuated variant. We sought to evaluate the positive predictive value of incidental FGPDs for FAP. We also characterized the clinicopathologic features of incidental FGPDs to advance clinical management. METHODS: Incidental FGPDs were identified from 2004 to 2015 in patients without FAP at biopsy. All clinical follow-up data were reviewed, and germline analysis for APC and MUTYH mutations was performed in consenting patients. RESULTS: We identified 25 incidental FGPDs in patients not known to have FAP (11.6% of FGPDs, 1.0% of all FGPs). Four patients had a family history of gastric polyps or gastrointestinal cancers. Clinical management included completion polypectomy and gastric endoscopic surveillance (44%), endoscopic surveillance alone (32%), no follow-up (24%), colonoscopy referral (12%), and genetic counseling (4%). Colonoscopies on record revealed 0-7 cumulative adenomas. Follow-up averaged 4.4 years (range 0.3-10.6). No clinical evidence of FAP, gastric cancer, death, or surgery occurred. None of the 11 patients consenting to germline APC and MUTYH testing had genomic alterations. CONCLUSIONS: Incidental FGPDs in this series were all found to be sporadic (25/25) by endoscopic, clinical, and molecular findings, and thus FGPDs were not harbingers of FAP. As isolated findings, FGPDs do not appear to warrant follow-up genetic counseling or testing.

Molecular genetic testing for cystic fibrosis: laboratory performance on the College of American Pathologists external proficiency surveys.

BACKGROUND: Molecular testing for cystic fibrosis mutations is widespread and routine in reproductive decision making and diagnosis. Our objective was to assess the level of performance of laboratories for this test. METHODS: The College of American Pathologists administers external proficiency testing with multiple DNA samples distributed biannually. RESULTS are analyzed, reviewed, and graded by the joint College of American Pathologists/American College of Medical Genetics and Genomics Biochemical and Molecular Genetics Committee. Assessment is based on genotype and associated clinical interpretation. RESULTS: Overall, 357 clinical laboratories participated in the proficiency testing survey between 2003 and 2013 (322 in the United States and 35 international). In 2013, US participants reported performing nearly 120,000 tests monthly. Analytical sensitivity and specificity of US laboratories were 98.8% (95% confidence interval: 98.4-99.1%) and 99.6% (95% confidence interval: 99.4-99.7%), respectively. Analytical sensitivity improved between 2003 and 2008 (from 97.9 to 99.3%; P = 0.007) and remained steady thereafter. Clinical interpretation matched the intended response for 98.8, 86.0, and 91.0% of challenges with no, one, or two mutations, respectively. International laboratories performed similarly. DISCUSSION: Laboratory testing for cystic fibrosis in the United States has improved since 2003, and these data demonstrate a high level of quality. Neither the number of samples tested nor test methodology affected performance.

Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology.

The American College of Medical Genetics and Genomics (ACMG) previously developed guidance for the interpretation of sequence variants.(1) In the past decade, sequencing technology has evolved rapidly with the advent of high-throughput next-generation sequencing. By adopting and leveraging next-generation sequencing, clinical laboratories are now performing an ever-increasing catalogue of genetic testing spanning genotyping, single genes, gene panels, exomes, genomes, transcriptomes, and epigenetic assays for genetic disorders. By virtue of increased complexity, this shift in genetic testing has been accompanied by new challenges in sequence interpretation. In this context the ACMG convened a workgroup in 2013 comprising representatives from the ACMG, the Association for Molecular Pathology (AMP), and the College of American Pathologists to revisit and revise the standards and guidelines for the interpretation of sequence variants. The group consisted of clinical laboratory directors and clinicians. This report represents expert opinion of the workgroup with input from ACMG, AMP, and College of American Pathologists stakeholders. These recommendations primarily apply to the breadth of genetic tests used in clinical laboratories, including genotyping, single genes, panels, exomes, and genomes. This report recommends the use of specific standard terminology-"pathogenic," "likely pathogenic," "uncertain significance," "likely benign," and "benign"-to describe variants identified in genes that cause Mendelian disorders. Moreover, this recommendation describes a process for classifying variants into these five categories based on criteria using typical types of variant evidence (e.g., population data, computational data, functional data, segregation data). Because of the increased complexity of analysis and interpretation of clinical genetic testing described in this report, the ACMG strongly recommends that clinical molecular genetic testing should be performed in a Clinical Laboratory Improvement Amendments-approved laboratory, with results interpreted by a board-certified clinical molecular geneticist or molecular genetic pathologist or the equivalent.

Characterizing genetic variants for clinical action.

Genome-wide association studies, DNA sequencing studies, and other genomic studies are finding an increasing number of genetic variants associated with clinical phenotypes that may be useful in developing diagnostic, preventive, and treatment strategies for individual patients. However, few variants have been integrated into routine clinical practice. The reasons for this are several, but two of the most significant are limited evidence about the clinical implications of the variants and a lack of a comprehensive knowledge base that captures genetic variants, their phenotypic associations, and other pertinent phenotypic information that is openly accessible to clinical groups attempting to interpret sequencing data. As the field of medicine begins to incorporate genome-scale analysis into clinical care, approaches need to be developed for collecting and characterizing data on the clinical implications of variants, developing consensus on their actionability, and making this information available for clinical use. The National Human Genome Research Institute (NHGRI) and the Wellcome Trust thus convened a workshop to consider the processes and resources needed to: (1) identify clinically valid genetic variants; (2) decide whether they are actionable and what the action should be; and (3) provide this information for clinical use. This commentary outlines the key discussion points and recommendations from the workshop.

Results of the College of American Pathology/American College of Medical Genetics and Genomics external proficiency testing from 2006 to 2013 for three conditions prevalent in the Ashkenazi Jewish population.

PURPOSE: The purpose of this study was to determine analytic performance of laboratories offering molecular testing for conditions such as Tay-Sachs disease, Canavan disease, and familial dysautonomia, which are prevalent in the Ashkenazi Jewish population. METHODS: The College of American Pathologists and the American College of Medical Genetics and Genomics cosponsor molecular proficiency testing for these disorders. Responses from 2006 to 2013 were analyzed for accuracy (genotyping and interpretations). RESULTS: Between 11 and 36 laboratories participated in each Tay-Sachs disease distribution. Samples tested per month were constant (2,900) from 2006 to 2011 but recently increased. Participants reporting <10 samples tested per month had longer turnaround times (42 vs. 7%, longer than 14 days; P = 0.03). Analytic sensitivity and specificity for US participants were 97.2% (95% confidence interval: 94.7-98.7%) and 99.8% (95% confidence interval: 99.1-99.9%), respectively. Of 11 genotyping errors, 2 were due to sample mix-up. Analytic interpretations were correct in 99.3% of challenges (956/963; 95% confidence interval: 98.5-99.7%). Better performance was found for Canavan disease and familial dysautonomia. International laboratories performed equally well. CONCLUSION: These results demonstrated high analytic sensitivity and specificity along with excellent analytic interpretation performance, confirming the genetics community impression that laboratories provide accurate test results in both diagnostic and screening settings. Proficiency testing can identify potential laboratory issues and helps document overall laboratory performance.

Three-year experience of a CAP/ACMG methods-based external proficiency testing program for laboratories offering DNA sequencing for rare inherited disorders.

PURPOSE: Thousands of genetic tests are now offered clinically, but many are for rare disorders that are offered by only a few laboratories. The classic approach to disease-specific external proficiency testing programs is not feasible for such testing, yet calls have been made to provide external oversight. METHODS: A methods-based Sequencing Educational Challenge Survey was launched in 2010, under joint administration of the College of American Pathologists and the American College of Medical Genetics and Genomics. Three sets of Sanger ABI sequence data were distributed twice per year. Participants were asked to identify, formally name, and interpret the sequence variant(s). RESULTS: Between 2010 and 2012, 117 laboratories participated. Using a proposed assessment scheme (e.g., at least 10 of 12 components correct), 98.3% of the 67 US participants had acceptable performance (235 of 239 challenges; 95% confidence interval: 95.8-99.5%) as compared with 88.9% (136 of 153; 95% confidence interval: 82.8-93.4%) for the 50 international participants. CONCLUSION: These data provide a high level of confidence that most US laboratories offering rare disease testing are providing consistent and reliable clinical interpretations. Methods-based proficiency testing programs may be one part of the solution to assessing genetic testing based on next-generation sequencing technology.

ACMG Standards and Guidelines for fragile X testing: a revision to the disease-specific supplements to the Standards and Guidelines for Clinical Genetics Laboratories of the American College of Medical Genetics and Genomics.

Molecular genetic testing of the FMR1 gene is commonly performed in clinical laboratories. Mutations in the FMR1 gene are associated with fragile X syndrome, fragile X tremor ataxia syndrome, and premature ovarian insufficiency. This document provides updated information regarding FMR1 gene mutations, including prevalence, genotype-phenotype correlation, and mutation nomenclature. Methodological considerations are provided for Southern blot analysis and polymerase chain reaction amplification of the FMR1 gene, including triplet repeat-primed and methylation-specific polymerase chain reaction. In addition to report elements, examples of laboratory reports for various genotypes are also included.

ACMG clinical laboratory standards for next-generation sequencing.

Next-generation sequencing technologies have been and continue to be deployed in clinical laboratories, enabling rapid transformations in genomic medicine. These technologies have reduced the cost of large-scale sequencing by several orders of magnitude, and continuous advances are being made. It is now feasible to analyze an individual's near-complete exome or genome to assist in the diagnosis of a wide array of clinical scenarios. Next-generation sequencing technologies are also facilitating further advances in therapeutic decision making and disease prediction for at-risk patients. However, with rapid advances come additional challenges involving the clinical validation and use of these constantly evolving technologies and platforms in clinical laboratories. To assist clinical laboratories with the validation of next-generation sequencing methods and platforms, the ongoing monitoring of next-generation sequencing testing to ensure quality results, and the interpretation and reporting of variants found using these technologies, the American College of Medical Genetics and Genomics has developed the following professional standards and guidelines.

Aneuploidy detection in paraffin embedded tissue from products of conception by mini-STR genotyping.

Autosomal trisomy is the most common genetic abnormality observed in pregnancy loss. We designed a panel of mini-short tandem repeats (mini-STRs) for aneuploidy detection in chromosomes 13, 16, 18 and 21 from fresh and formalin fixed, paraffin embedded (FFPE) samples from products of conception (POC). FFPE POCs with trisomy 13 (n = 6), trisomy 18 (n = 6), trisomy 21 (n = 12), 6 euploid for the chromosomes of interest and two trisomy 16 samples from fresh tissue were tested. Concordance between cytogenetics and genotyping was 100% for non-mosaic samples. Mini-STR genotyping is a viable method for targeted aneuploidy detection in low quality DNA samples.

Laboratory testing of CYP2D6 alleles in relation to tamoxifen therapy.

Tamoxifen, a widely prescribed drug for the treatment and prevention of breast cancer, is metabolized to more potent metabolites by the cytochrome P450 2D6 (CYP2D6) enzyme. Variants in the CYP2D6 gene can cause patients to be either intermediate or poor metabolizers, thereby rendering tamoxifen treatment less effective. Testing for CYP2D6 gene variants is available in Clinical Laboratory Improvement Amendments-certified clinical laboratories; however, the biological complexity of the variants makes result interpretation and phenotype prediction challenging. This article describes the clinical significance of variants as well as important analytical, interpretative, and reporting issues. It is designed to be a guideline for clinical laboratory professionals in performing tests and interpreting results with respect to CYP2D6 genetic variants.

Sickle cell disease resulting from uniparental disomy in a child who inherited sickle cell trait.

Sickle cell disease (SCD) is a classic example of a disorder with recessive Mendelian inheritance, in which each parent contributes one mutant allele to an affected offspring. However, there are exceptions to that rule. We describe here the first reported case of conversion of inherited sickle cell trait to SCD by uniparental disomy (UPD) resulting in mosaicism for SS and AS erythrocytes. A 14-year-old boy presented with splenomegaly and hemolysis. Although his father has sickle cell trait, his mother has no abnormal hemoglobin (Hb). DNA sequencing, performed to rule out Hb S/ß-thalassemia, detected homozygous Hb SS. Further studies revealed mosaic UPD of the ß-globin locus, more SS erythroid progenitors than AS, but a reverse ratio of erythrocytes resulting from the survival advantage of AS erythrocytes. This report exemplifies non-Mendelian genetics wherein a patient who inherited sickle cell trait has mild SCD resulting from postzygotic mitotic recombination leading to UPD.