Context.—: In silico mutagenesis can be performed to introduce variants into next-generation sequencing data. This method holds potential promise for proficiency testing since rare or novel variants can be modeled that are not found in available proficiency testing materials. Objective.—: To determine whether in silico mutagenesis could be used as a viable proficiency testing methodology for undiagnosed disorders by exome sequencing. Design.—: Laboratories performed exome sequencing on reference samples and uploaded raw sequence data files to the College of American Pathologists. These files were then mutagenized in silico to introduce variants, including those deemed to be causative for the clinical scenario provided. The laboratories processed the mutagenized files through their bioinformatic pipelines and performed interpretation to identify pathogenic and likely pathogenic primary and secondary findings. Responses were evaluated for concordance with intended responses. Results.—: A total of 7 educational (nongraded) proficiency testing challenges were performed between 2018 and 2021. An average of 47 laboratories participated in each program, with each containing between 2 to 5 (average = 3.6) intended response variants. Intended response variant types included substitutions and small insertion/deletion variants. Participating laboratories returned 94.3% of intended response variants across all programs on average (per program range, 91.5%-97.3%). The percentage of laboratories that correctly returned all intended response variants for a program ranged from 70.0% to 96.7%. Conclusions.—: In silico mutagenesis represents a suitable approach for graded intended response-based proficiency testing for exome sequencing that allows laboratories to assess both analytical and interpretative components of the test.

BACKGROUND:Genetic testing has traditionally been divided into molecular genetics and cytogenetics, originally driven by the use of different assays and their associated limitations. Cytogenetic technologies such as karyotyping, fluorescent in situ hybridization or chromosomal microarrays are used to detect large "megabase level" copy number variants and other structural variants such as inversions or translocations. In contrast, molecular methodologies are heavily biased toward subgenic "small variants" such as single nucleotide variants, insertions/deletions, and targeted detection of intragenic, exon level deletions or duplications. The boundaries between these approaches are now increasingly blurred as next-generation sequencing technologies and their use for genome-wide analysis are used by both disciplines, therefore eliminating the historic and somewhat artificial separation driven by variant type. CONTENT:This review discusses the history of genomic nomenclature across both fields, summarizes implementation challenges for the clinical genetics community, and identifies key considerations for enabling a seamless connection of the stakeholders that consume variant descriptions. SUMMARY:Standardization is naturally a lengthy and complex process that requires consensus building between different stakeholders. Developing a standard that not only fits the multitude of needs across the entities that consume genetic variant information but also works equally well for all genetic variant types is an ambitious goal that calls for revisiting this vision.

Clinical implementation of whole-genome and whole-exome sequencing by next-generation sequencing (NGS) allows for comprehensive detection of genomic alterations. However, with the growing number of clinically relevant genes and variants, there is an urgent need for reference materials to optimize, validate, and quality control NGS tests. This pilot study documents the paucity of physical reference materials for widely tested genes and demonstrates the utility of in silico mutagenized reference materials to supplement physical samples when developing NGS tests. We examined published, expert curated lists of clinically relevant variants for these widely tested genes and found that publicly available reference materials were available for only 29.4%. We outline the steps for generating in silico resources and used 49 curated variants to conduct a blinded proof-of-concept study with three experienced NGS laboratories. One laboratory detected all added variants, and two detected all but one. This study revealed common scenarios that could lead to false-negative results when common pathogenic variants cannot be tested during analytical validation. This work highlights the need to establish centralized knowledge bases for common, pathogenic variants, demonstrates the utility of in silico reference materials, and provides guidance for generating in silico reference materials in-house. Additional work will be needed to generate turnkey processes for novice laboratories without in-house bioinformatics expertise.

As the number of genes associated with various germline disorders continues to grow, it is becoming more difficult for clinical laboratories to maintain separate assays for interrogating disease-focused gene panels. One solution to this challenge is termed slice testing, where capture backbone is used to analyze data specific to a set of genes, and for this article, we will focus on exome. A key advantage to this strategy is greater flexibility by adding genes as they become associated with disease or the ability to accommodate specific provider requests. Here, we provide expert consensus recommendations and results from an Association for Molecular Pathology-sponsored survey of clinical laboratories performing exome sequencing to compare a slice testing approach with traditional static gene panels and comprehensive exome analysis. We explore specific considerations for slices, including gene selection, analytic performance, coverage, quality, and interpretation. Our goal is to provide comprehensive guidance for clinical laboratories interested in designing and using slice tests as a diagnostic.

CONTEXT.—:Next-generation sequencing (NGS)-based assays are used for diagnosis of diverse inherited disorders. Limited data are available pertaining to interlaboratory analytical performance of these assays. OBJECTIVE.—:To report on the College of American Pathologists (CAP) NGS Germline Program, which is methods based, and explore the evolution in laboratory testing practices. DESIGN.—:Results from the NGS Germline Program from 2016-2020 were analyzed for interlaboratory analytical performance. Self-reported laboratory testing practices were also evaluated. RESULTS.—:From 2016-2020, a total of 297 laboratories participated in at least 1 program mailing. Of the 289 laboratories that provided information on tests offered, 138 (47.8%) offered only panel testing throughout their enrollment, while 35 (12.1%) offered panels and exome testing, 30 (10.4%) offered only exomes, 9 (3.1%) offered only genomes, and 15 (5.2%) offered panels, exomes, and genomes. The remainder (62 laboratories, 21.4%) changed their test offerings during the 2016-2020 timeframe. Considering each genomic position/interval, the median detection percentage at variant positions across the 2016-2020 mailings ranged from 94.3% to 100%, while at reference positions (no variant detected), the median correct response percentage was 100% across all mailings. When considering performance of individual laboratories, 89.5% (136 of 152) to 98.0% (149 of 152) of laboratories successfully met the detection threshold (≥90% of the variants present), while 94.6% (87 of 92) to 100% (163 of 163) of laboratories met the 95% specificity threshold across mailings. CONCLUSIONS.—:Since the inception of this program, laboratories have consistently performed well. The median sensitivity and specificity of detection of sequence variants included in this program (eg, single nucleotide variants, insertions, and deletions) were 100.0%.

As the number of genes associated with various germline disorders continues to grow, it is becoming more difficult for clinical laboratories to maintain separate assays for interrogating disease -focused gene panels. One solution to this challenge is termed slice testing, where capture backbone is used to analyze data specific to a set of genes, and for this article, we will focus on exome. A key advantage to this strategy is greater flexibility by adding genes as they become associated with disease or the ability to accommodate specific provider requests. Here, we provide expert consensus recommendations and results from an Association for Molecular Pathologyesponsored survey of clinical laboratories performing exome sequencing to compare a slice testing approach with traditional static gene panels and comprehensive exome analysis. We explore specific considerations for slices, including gene selection, analytic performance, coverage, quality, and interpretation. Our goal is to provide comprehensive guidance for clinical laboratories interested in designing and using slice tests as a diagnostic. (J Mol Diagn 2024, 26: 159e167; https://doi.org/10.1016/j.jmoldx.2023.11.008)

In 2017, the ClinGen Cardiomyopathy Variant Curation Expert Panel published an adaptation of the American College of Medical Genetics and Genomics (ACMG) and the Association of Molecular Pathology (AMP) Standards for Variant Interpretation for the MYH7 gene, one of the most common causes of hypertrophic cardiomyopathy (HCM). HCM is a genetically heterogeneous condition, characterized by age-related incomplete penetrance.

PurposeThe specialty of Laboratory Genetics and Genomics (LGG) was created in 2017 in an effort to reflect the increasing convergence in technologies and approaches between clinical molecular genetics and clinical cytogenetics. However, there has not yet been any formal evaluation of the merging of these disciplines and the challenges faced by Program Directors (PDs) tasked with ensuring the successful training of laboratory geneticists under the new model.MethodsAn electronic multi-question Qualtrics survey was created and was sent to the PD for each of the ACGME-accredited LGG fellowship programs at the time. The data was collected and the responses were aggregated for each question.ResultsAll of the responding PDs had started training at least one LGG fellow. PDs noted challenges with funding, staff shortages, molecular/cytogenetics content integration, limited total training time, increased remote work, increased sendout testing, and a lack of prior cytogenetics knowledge among incoming fellows.ConclusionThis survey attempted to assess the challenges that LGG PDs have been facing in offering and integrating clinical molecular genetics and clinical cytogenetics fellowship training. Common challenges between programs were noted, and a set of six concluding comments are provided to facilitate future discussion.

Complex health related disorders, including some forms of cardiovascular disease, diabetes, asthma, autism, and cancer, arise through the relative contributions of genetic, environmental, and lifestyle factors over long periods of time. Unlike monogenic disorders, complex disorders develop via a cumulative effect across many genomic loci, each conferring small individual risks. Polygenic risk scores (PRSs)1 combine these small individual variant effects to predict risk for developing complex disorders (Box 1) and may be combined with monogenic disease risk and nongenetic risk factors in an integrated risk model to predict disease risk more accurately.

Genetic counseling for patients who are pursuing genetic testing in the absence of a medical indication, referred to as elective genomic testing (EGT), is becoming more common. This type of testing has the potential to detect genetic conditions before there is a significant health impact permitting earlier management and/or treatment. Pre‐ and post‐test counseling for EGT is similar to indication‐based genetic testing. Both require a complete family and medical history when ordering a test or interpreting a result. However, EGT counseling has some special considerations including greater uncertainties around penetrance and clinical utility and a lack of published guidelines. While certain considerations in the selection of a high‐quality genetic testing laboratory are universal, there are some considerations that are unique to the selection of a laboratory performing EGT. This practice resource intends to provide guidance for genetic counselors and other healthcare providers caring for adults seeking pre‐ or post‐test counseling for EGT. Genetic counselors and other genetics trained healthcare providers are the ideal medical professionals to supply accurate information to individuals seeking counseling about EGT enabling them to make informed decisions about testing and follow‐up.

Understanding the penetrance of pathogenic variants identified as secondary findings (SFs) is of paramount importance with the growing availability of genetic testing. We estimated penetrance through large-scale analyses of individuals referred for diagnostic sequencing for hypertrophic cardiomyopathy (HCM; 10,400 affected individuals, 1,332 variants) and dilated cardiomyopathy (DCM; 2,564 affected individuals, 663 variants), using a cross-sectional approach comparing allele frequencies against reference populations (293,226 participants from UK Biobank and gnomAD). We generated updated prevalence estimates for HCM (1:543) and DCM (1:220). In aggregate, the penetrance by late adulthood of rare, pathogenic variants (23% for HCM, 35% for DCM) and likely pathogenic variants (7% for HCM, 10% for DCM) was substantial for dominant cardiomyopathy (CM). Penetrance was significantly higher for variant subgroups annotated as loss of function or ultra-rare and for males compared to females for variants in HCM-associated genes. We estimated variant-specific penetrance for 316 recurrent variants most likely to be identified as SFs (found in 51% of HCM- and 17% of DCM-affected individuals). 49 variants were observed at least ten times (14% of affected individuals) in HCM-associated genes. Median penetrance was 14.6% (±14.4% SD). We explore estimates of penetrance by age, sex, and ancestry and simulate the impact of including future cohorts. This dataset reports penetrance of individual variants at scale and will inform the management of individuals undergoing genetic screening for SFs. While most variants had low penetrance and the costs and harms of screening are unclear, some individuals with highly penetrant variants may benefit from SFs.

Exome reanalysis is useful for providing molecular diagnoses for previously uninformative samples. However, challenges exist in implementing a practical solution for clinicians and laboratories. This study complements the current literature by providing practical considerations for patient-level and cohort-level reanalyses. The Clinical and Laboratory Standards Institute assembled the Document Development Committee and an interpretation working group that developed the framework for reevaluation of exome-based data. We describe two distinct but complementary approaches toward exome reanalyses: clinician-initiated patient-level reanalysis, and laboratory-initiated cohort-level reanalysis. We highlight the advantages and constraints for both approaches, and provide a high-level conceptual guide for ordering clinicians and laboratories through the critical decision pathways. Because clinical exome sequencing continues to be the standard of care in genetics, exome reanalysis would be critical in increasing the overall diagnostic yield. A systematic guide will facilitate the efficient adoption of reevaluation of exome data for laboratories, health care professionals, genetic counselors, and clinicians.

The ryanodine receptors (RyR1) is the main sarcoplasmic reticulum Ca2+ channel in skeletal muscle and acts as a connecting link between electrical stimulation and Ca2+-dependent muscle contraction. RYR1 is expressed predominantly in fast and slow-twitch skeletal muscle and also in the esophagus and in cerebellar Purkinje cells in the brain. RYR2 is the predominant isoform in cardiac muscle and brain. Its expression in the brain, brain stem, and spinal cord is widespread, but it is absent from the pituitary. RYR3 is differentially expressed in the brain. Abnormal RyR1 activity compromises normal muscle function. RYR1-associated myopathies have been described as rare, histopathologically and clinically heterogeneous, and slowly progressive neuromuscular disorders. RYR1 variants can lead to dysfunctional RyR1-mediated calcium release, malignant hyperthermia susceptibility, elevated oxidative stress, deleterious post-translational modifications, and decreased RyR1 expression. RYR1 mutations cause disorders such as malignant hyperthermia, central core disease, congenital fibre type disproportion and centronuclear myopathy. However, RYR1 is one of the largest genes of the human genome and accumulates numerous missense variants of uncertain significance (VUS), precluding an efficient molecular diagnosis for many patients and families. Here we describe several RYR mutations and correlate them to the diseases. We also validated large deletion in ryanodine receptor 1 (RYR1) using droplet digital PCR. The ryanodine receptors (RyR1) is the main sarcoplasmic reticulum Ca2+ channel in skeletal muscle and acts as a connecting link between electrical stimulation and Ca2+-dependent muscle contraction. RYR1 is expressed predominantly in fast and slow-twitch skeletal muscle and also in the esophagus and in cerebellar Purkinje cells in the brain. RYR2 is the predominant isoform in cardiac muscle and brain. Its expression in the brain, brain stem, and spinal cord is widespread, but it is absent from the pituitary. RYR3 is differentially expressed in the brain. Abnormal RyR1 activity compromises normal muscle function. RYR1-associated myopathies have been described as rare, histopathologically and clinically heterogeneous, and slowly progressive neuromuscular disorders. RYR1 variants can lead to dysfunctional RyR1-mediated calcium release, malignant hyperthermia susceptibility, elevated oxidative stress, deleterious post-translational modifications, and decreased RyR1 expression. RYR1 mutations cause disorders such as malignant hyperthermia, central core disease, congenital fibre type disproportion and centronuclear myopathy. However, RYR1 is one of the largest genes of the human genome and accumulates numerous missense variants of uncertain significance (VUS), precluding an efficient molecular diagnosis for many patients and families. Here we describe several RYR mutations and correlate them to the diseases. We also validated large deletion in ryanodine receptor 1 (RYR1) using droplet digital PCR.

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.