Systematic evaluation of genome sequencing for the diagnostic assessment of autism spectrum disorder and fetal structural anomalies.

Short-read genome sequencing (GS) holds the promise of becoming the primary diagnostic approach for the assessment of autism spectrum disorder (ASD) and fetal structural anomalies (FSAs). However, few studies have comprehensively evaluated its performance against current standard-of-care diagnostic tests: karyotype, chromosomal microarray (CMA), and exome sequencing (ES). To assess the clinical utility of GS, we compared its diagnostic yield against these three tests in 1,612 quartet families including an individual with ASD and in 295 prenatal families. Our GS analytic framework identified a diagnostic variant in 7.8% of ASD probands, almost 2-fold more than CMA (4.3%) and 3-fold more than ES (2.7%). However, when we systematically captured copy-number variants (CNVs) from the exome data, the diagnostic yield of ES (7.4%) was brought much closer to, but did not surpass, GS. Similarly, we estimated that GS could achieve an overall diagnostic yield of 46.1% in unselected FSAs, representing a 17.2% increased yield over karyotype, 14.1% over CMA, and 4.1% over ES with CNV calling or 36.1% increase without CNV discovery. Overall, GS provided an added diagnostic yield of 0.4% and 0.8% beyond the combination of all three standard-of-care tests in ASD and FSAs, respectively. This corresponded to nine GS unique diagnostic variants, including sequence variants in exons not captured by ES, structural variants (SVs) inaccessible to existing standard-of-care tests, and SVs where the resolution of GS changed variant classification. Overall, this large-scale evaluation demonstrated that GS significantly outperforms each individual standard-of-care test while also outperforming the combination of all three tests, thus warranting consideration as the first-tier diagnostic approach for the assessment of ASD and FSAs.

Establishing the Medical Actionability of Genomic Variants.

Actionability is an important concept in medicine that does not have a well-accepted standard definition, nor is there a general consensus on how to establish it. Medical actionability is often conflated with clinical utility, a related but distinct concept. This lack of clarity contributes to practice variation and inconsistent coverage decisions in genomic medicine, leading to the potential for systematic bias in the use of evidence-based interventions. We clarify how medical actionability and clinical utility are distinct and then discuss the spectrum of actionability, including benefits for the person, the family, and society. We also describe applications across the life course, including prediction, diagnosis, and treatment. Current challenges in assessing the medical actionability of identified genomic variants include gaps in the evidence, limited contexts with practice guidelines, and subjective aspects of medical actionability. A standardized and authoritative assessment of medical actionability is critical to implementing genomic medicine in a fashion that improves population health outcomes and reduces health disparities.

Implementing evidence-based assertions of clinical actionability in the context of secondary findings: Updates from the ClinGen Actionability Working Group.

PURPOSE: The ClinGen Actionability Working Group (AWG) developed an evidence-based framework to generate actionability reports and scores of gene-condition pairs in the context of secondary findings from genome sequencing. Here we describe the expansion of the framework to include actionability assertions. METHODS: Initial development of the actionability rubric was based on previously scored adult gene-condition pairs and individual expert evaluation. Rubric refinement was iterative and based on evaluation, feedback, and discussion. The final rubric was pragmatically evaluated via integration into actionability assessments for 27 gene-condition pairs. RESULTS: The resulting rubric has a 4-point scale (limited, moderate, strong, and definitive) and uses the highest-scoring outcome-intervention pair of each gene-condition pair to generate a preliminary assertion. During AWG discussions, predefined criteria and factors guide discussion to produce a consensus assertion for a gene-condition pair, which may differ from the preliminary assertion. The AWG has retrospectively generated assertions for all previously scored gene-condition pairs and are prospectively asserting on gene-condition pairs under assessment, having completed over 170 adult and 188 pediatric gene-condition pairs. CONCLUSION: The AWG expanded its framework to provide actionability assertions to enhance the clinical value of their resources and increase their utility as decision aids regarding return of secondary findings.

Genomic Sequencing for Newborn Screening: Results of the NC NEXUS Project.

Newborn screening (NBS) was established as a public health program in the 1960s and is crucial for facilitating detection of certain medical conditions in which early intervention can prevent serious, life-threatening health problems. Genomic sequencing can potentially expand the screening for rare hereditary disorders, but many questions surround its possible use for this purpose. We examined the use of exome sequencing (ES) for NBS in the North Carolina Newborn Exome Sequencing for Universal Screening (NC NEXUS) project, comparing the yield from ES used in a screening versus a diagnostic context. We enrolled healthy newborns and children with metabolic diseases or hearing loss (106 participants total). ES confirmed the participant's underlying diagnosis in 15 out of 17 (88%) children with metabolic disorders and in 5 out of 28 (∼18%) children with hearing loss. We discovered actionable findings in four participants that would not have been detected by standard NBS. A subset of parents was eligible to receive additional information for their child about childhood-onset conditions with low or no clinical actionability, clinically actionable adult-onset conditions, and carrier status for autosomal-recessive conditions. We found pathogenic variants associated with hereditary breast and/or ovarian cancer in two children, a likely pathogenic variant in the gene associated with Lowe syndrome in one child, and an average of 1.8 reportable variants per child for carrier results. These results highlight the benefits and limitations of using genomic sequencing for NBS and the challenges of using such technology in future precision medicine approaches.

Variant Classification Concordance using the ACMG-AMP Variant Interpretation Guidelines across Nine Genomic Implementation Research Studies.

Harmonization of variant pathogenicity classification across laboratories is important for advancing clinical genomics. The two CLIA-accredited Electronic Medical Record and Genomics Network sequencing centers and the six CLIA-accredited laboratories and one research laboratory performing genome or exome sequencing in the Clinical Sequencing Evidence-Generating Research Consortium collaborated to explore current sources of discordance in classification. Eight laboratories each submitted 20 classified variants in the ACMG secondary finding v.2.0 genes. After removing duplicates, each of the 158 variants was annotated and independently classified by two additional laboratories using the ACMG-AMP guidelines. Overall concordance across three laboratories was assessed and discordant variants were reviewed via teleconference and email. The submitted variant set included 28 P/LP variants, 96 VUS, and 34 LB/B variants, mostly in cancer (40%) and cardiac (27%) risk genes. Eighty-six (54%) variants reached complete five-category (i.e., P, LP, VUS, LB, B) concordance, and 17 (11%) had a discordance that could affect clinical recommendations (P/LP versus VUS/LB/B). 21% and 63% of variants submitted as P and LP, respectively, were discordant with VUS. Of the 54 originally discordant variants that underwent further review, 32 reached agreement, for a post-review concordance rate of 84% (118/140 variants). This project provides an updated estimate of variant concordance, identifies considerations for LP classified variants, and highlights ongoing sources of discordance. Continued and increased sharing of variant classifications and evidence across laboratories, and the ongoing work of ClinGen to provide general as well as gene- and disease-specific guidance, will lead to continued increases in concordance.

Association of deep phenotyping with diagnostic yield of prenatal exome sequencing for fetal brain abnormalities.

PURPOSE: To evaluate whether deep prenatal phenotyping of fetal brain abnormalities (FBAs) increases diagnostic yield of trio-exome sequencing (ES) compared with standard phenotyping. METHODS: Retrospective exploratory analysis of a multicenter prenatal ES study. Participants were eligible if an FBA was diagnosed and subsequently found to have a normal microarray. Deep phenotyping was defined as phenotype based on targeted ultrasound plus prenatal/postnatal magnetic resonance imaging, autopsy, and/or known phenotypes of other affected family members. Standard phenotyping was based on targeted ultrasound alone. FBAs were categorized by major brain findings on prenatal ultrasound. Cases with positive ES results were compared with those that have negative results by available phenotyping, as well as diagnosed FBAs. RESULTS: A total of 76 trios with FBAs were identified, of which 25 (33%) cases had positive ES results and 51 (67%) had negative results. Individual modalities of deep phenotyping were not associated with diagnostic ES results. The most common FBAs identified were posterior fossa anomalies and midline defects. Neural tube defects were significantly associated with receipt of a negative ES result (0% vs 22%, P = .01). CONCLUSION: Deep phenotyping was not associated with increased diagnostic yield of ES for FBA in this small cohort. Neural tube defects were associated with negative ES results.

The Burden of COVID-19 on Caregivers of Children with Suspected Genetic Conditions: A Therapeutic Odyssey.

AIMS: Children with disabilities and rare or undiagnosed conditions and their families have faced numerous hardships of living during the COVID-19 pandemic. For those with undiagnosed conditions, the diagnostic odyssey can be long, expensive, and marked by uncertainty. We, therefore, sought to understand whether and how COVID-19 impacted the trajectory of children's care. METHODS: We conducted semi-structured qualitative interviews with 25 caregivers who, prior to the pandemic, were on a diagnostic odyssey for their children. RESULTS: Most caregivers did not report any interruptions to their child's diagnostic odyssey. The greatest impact was access to therapy services, including the suspension or loss of their child's in-person therapeutic care and difficulties with virtual therapies. This therapy gap caused caregivers to fear that their children were not making progress. CONCLUSION: Although much has been written about the challenges of diagnostic odysseys for children and their families, this study illustrates the importance of expanding the focus of these studies to include therapeutic odysseys. Because therapeutic odysseys continue regardless of whether diagnoses are made, future research should investigate how to support caregivers through children's therapies within and outside of the COVID-19 context.

Testing and extending strategies for identifying genetic disease-related encounters in pediatric patients.

PURPOSE: To better understand health care utilization and develop decision support tools, methods for identifying patients with suspected genetic diseases (GDs) are needed. Previous studies had identified inpatient-relevant International Classification of Diseases (ICD) codes that were possibly, probably, or definitely indicative of GDs. We assessed whether these codes identified GD-related inpatient, outpatient, and emergency department encounters among pediatric patients with suspected GDs from a previous study (the North Carolina Clinical Genomic Evaluation by Next-Generation Exome Sequencing [NCGENES] study). METHODS: Using the electronic medical records of 140 pediatric patients from the NCGENES study, we characterized the presence of ICD codes representing possible, probable, or definite GD-related diagnoses across encounter types. In addition, we examined codes from encounters for which initially no GD-related codes had been found and determined whether these codes were indicative of a GD. RESULTS: Among NCGENES patients with visits between 2014 and 2017, 92% of inpatient, 75% of emergency department, and 63% of outpatient encounters included ≥1 GD-related code. Encounters with highly specific (ie, definite) GD codes had fewer low-specificity GD codes than encounters with only low-specificity GD codes. We identified an additional 32 ICD-9 and 56 ICD-10 codes possibly indicative of a GD. CONCLUSION: Code-based strategies can be refined to assess health care utilization among pediatric patients and may contribute to a systematic approach to identify patients with suspected GDs.

ClinGen's Pediatric Actionability Working Group: Clinical actionability of secondary findings from genome-scale sequencing in children and adolescents.

PURPOSE: Synthesis and curation of evidence regarding the clinical actionability of secondary findings (SFs) from genome-scale sequencing are needed to support decision-making on reporting of these findings. To assess actionability of SFs in children and adolescents, the Clinical Genome Resource established the Pediatric Actionability Working Group (AWG). METHODS: The Pediatric AWG modified the framework of the existing Adult AWG, which included production of summary reports of actionability for genes and associated conditions and consensus actionability scores for specific outcome-intervention pairs. Modification of the adult framework for the pediatric setting included accounting for special considerations for reporting presymptomatic or predictive genetic findings in the pediatric context, such as maintaining future autonomy by not disclosing conditions not actionable until adulthood. The Pediatric AWG then applied this new framework to genes and associated conditions with putative actionability. RESULTS: As of September 2021, the Pediatric AWG applied the new framework to 70 actionability topics representing 143 genes. Reports and scores are publicly available at www.clinicalgenome.org. CONCLUSION: The Pediatric AWG continues to curate gene-condition topics and build an evidence-based resource, supporting clinical communities and decision-makers with policy development on the return of SFs in pediatric populations.

Noninvasive prenatal exome sequencing diagnostic utility limited by sequencing depth and fetal fraction.

OBJECTIVE: Sequencing cell-free DNA now allows detection of large chromosomal abnormalities and dominant Mendelian disorders in the prenatal period. Improving upon these methods would allow newborn screening programs to begin with prenatal genetics, ultimately improving the management of rare genetic disorders. METHODS: As a pilot study, we performed exome sequencing on the cell-free DNA from three mothers with singleton pregnancies to assess the viability of broad sequencing modalities in a noninvasive prenatal setting. RESULTS: We found poor resolution of maternal and fetal genotypes due to both sampling and technical issues. CONCLUSION: We find broad sequencing modalities inefficient for noninvasive prenatal applications. Alternatively, we suggest a more targeted path forward for noninvasive prenatal genotyping.

Pre-capture multiplexing provides additional power to detect copy number variation in exome sequencing.

BACKGROUND: As exome sequencing (ES) integrates into clinical practice, we should make every effort to utilize all information generated. Copy-number variation can lead to Mendelian disorders, but small copy-number variants (CNVs) often get overlooked or obscured by under-powered data collection. Many groups have developed methodology for detecting CNVs from ES, but existing methods often perform poorly for small CNVs and rely on large numbers of samples not always available to clinical laboratories. Furthermore, methods often rely on Bayesian approaches requiring user-defined priors in the setting of insufficient prior knowledge. This report first demonstrates the benefit of multiplexed exome capture (pooling samples prior to capture), then presents a novel detection algorithm, mcCNV ("multiplexed capture CNV"), built around multiplexed capture. RESULTS: We demonstrate: (1) multiplexed capture reduces inter-sample variance; (2) our mcCNV method, a novel depth-based algorithm for detecting CNVs from multiplexed capture ES data, improves the detection of small CNVs. We contrast our novel approach, agnostic to prior information, with the the commonly-used ExomeDepth. In a simulation study mcCNV demonstrated a favorable false discovery rate (FDR). When compared to calls made from matched genome sequencing, we find the mcCNV algorithm performs comparably to ExomeDepth. CONCLUSION: Implementing multiplexed capture increases power to detect single-exon CNVs. The novel mcCNV algorithm may provide a more favorable FDR than ExomeDepth. The greatest benefits of our approach derive from (1) not requiring a database of reference samples and (2) not requiring prior information about the prevalance or size of variants.

The Clinical Sequencing Evidence-Generating Research Consortium: Integrating Genomic Sequencing in Diverse and Medically Underserved Populations.

The Clinical Sequencing Evidence-Generating Research (CSER) consortium, now in its second funding cycle, is investigating the effectiveness of integrating genomic (exome or genome) sequencing into the clinical care of diverse and medically underserved individuals in a variety of healthcare settings and disease states. The consortium comprises a coordinating center, six funded extramural clinical projects, and an ongoing National Human Genome Research Institute (NHGRI) intramural project. Collectively, these projects aim to enroll and sequence over 6,100 participants in four years. At least 60% of participants will be of non-European ancestry or from underserved settings, with the goal of diversifying the populations that are providing an evidence base for genomic medicine. Five of the six clinical projects are enrolling pediatric patients with various phenotypes. One of these five projects is also enrolling couples whose fetus has a structural anomaly, and the sixth project is enrolling adults at risk for hereditary cancer. The ongoing NHGRI intramural project has enrolled primarily healthy adults. Goals of the consortium include assessing the clinical utility of genomic sequencing, exploring medical follow up and cascade testing of relatives, and evaluating patient-provider-laboratory level interactions that influence the use of this technology. The findings from the CSER consortium will offer patients, healthcare systems, and policymakers a clearer understanding of the opportunities and challenges of providing genomic medicine in diverse populations and settings, and contribute evidence toward developing best practices for the delivery of clinically useful and cost-effective genomic sequencing in diverse healthcare settings.

Rosai-Dorfman Disease and Exocrine Pancreatic Insufficiency in a Patient With a Germline SLC29A3 Mutation.

Rosai-Dorfman disease (RDD) typically presents as bulky lymphadenopathy. Somatic mutations in RAS/MAP kinase pathway genes are common but germline mutations are rare. A patient with RDD and exocrine pancreatic insufficiency was found to have a homozygous germline mutation in SLC29A3, which has been associated with the Histiocytosis/Lymphadenopathy Plus Syndrome. His RDD also was positive for a somatic mutation in lymphoid enhancer binding factor 1 (LEF1). The concurrence of RDD and pancreatic insufficiency should raise consideration of SLC29A3 mutations. Other cases will be needed to confirm this observation and a possible contribution of LEF1 to the development of RDD.

An approach to integrating exome sequencing for fetal structural anomalies into clinical practice.

PURPOSE: We investigated the diagnostic and clinical performance of trio exome sequencing (ES) in parent-fetus trios where the fetus had sonographic abnormalities but normal karyotype, microarray and, in some cases, normal gene-specific sequencing. METHODS: ES was performed from DNA of 102 anomalous fetuses and from peripheral blood from their parents. Parents provided consent for the return of diagnostic results in the fetus, medically actionable findings in the parents, and identification as carrier couple for significant autosomal recessive conditions. RESULTS: In 21/102 (20.6%) fetuses, ES provided a positive-definitive or positive-probable diagnosis. In 10/102 (9.8%), ES provided an inconclusive-possible result. At least 2/102 (2.0%) had a repeat pregnancy during the study period and used the information from the study for prenatal diagnosis in the next pregnancy. Six of 204 (2.9%) parents received medically actionable results that affected their own health and 3/102 (2.9%) of couples received results that they were carriers for the same autosomal recessive condition. CONCLUSION: ES has diagnostic utility in a select population of fetuses where a genetic diagnosis was highly suspected. Challenges related to genetics literacy, variant interpretation, and various types of diagnostic results affecting both fetal and parental health must be addressed by highly tailored pre- and post-test genetic counseling.

Correction: An approach to integrating exome sequencing for fetal structural anomalies into clinical practice.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Referencing BRCA in hereditary cancer risk discussions: In search of an anchor in a sea of uncertainty.

As panel testing and exome sequencing are increasingly incorporated into clinical care, clinicians must grapple with how to communicate the risks and treatment decisions surrounding breast cancer genes beyond BRCA1 and BRCA2. In this paper, we examine clinicians' practice of employing BRCA1 and BRCA2 to help contextualize less certain genetic information regarding cancer risk and the possible implications of this practice for patients within the context of an exome sequencing study, NCGENES. We audio-recorded return of results appointments for 14 women who participated in NCGENES, previously had breast cancer, and were suspected of having a hereditary cancer predisposition. These patients were also interviewed four weeks later regarding their understanding of their results. We found that BRCA1 and BRCA2 were held as the gold standard, where clinicians compared what is known about BRCA to the limited understanding of other breast cancer-related genes. BRCA1 and BRCA2 were used as anchors to shape patients' understandings of genetic knowledge, risk, and management, illustrating how the information clinicians provide to patients may work as an external anchor. Yet, presenting BRCA1 and BRCA2 as a means of scientific reassurance can run the risk of patients conflating knowledge about certainty of risk with degree of risk after receiving a result for a moderate penetrance gene. This can be further complicated by misperceptions of the precision of cancer predictability attributed to these or other described 'cancer genes' in public media.

Factors influencing NCGENES research participants' requests for non-medically actionable secondary findings.

PURPOSE: Genomic sequencing can reveal variants with limited to no medical actionability. Previous research has assessed individuals' intentions to learn this information, but few report the decisions they made and why. METHODS: The North Carolina Clinical Genomic Evaluation by Next Generation Exome Sequencing (NCGENES) project evaluated adult patients randomized to learn up to six types of non-medically actionable secondary findings (NMASF). We previously found that most participants intended to request NMASF and intentions were strongly predicted by anticipated regret. Here we examine discrepancies between intentions and decisions to request NMASF, hypothesizing that anticipated regret would predict requests but that this association would be mediated by participants' intentions. RESULTS: Of the 76% who expressed intentions to learn results, only 42% made one or more requests. Overall, only 32% of the 155 eligible participants requested NMASF. Analyses support a plausible causal link between anticipated regret, intentions, and requests. CONCLUSIONS: The discordance between participants' expressed intentions and their actions provides insight into factors that influence patients' preferences for genomic information that has little to no actionability. These findings have implications for the timing and methods of eliciting preferences for NMASF and suggest that decisions to learn this information have cognitive and emotional components.

An Age-Based Framework for Evaluating Genome-Scale Sequencing Results in Newborn Screening.

OBJECTIVE: To assess the performance of a standardized, age-based metric for scoring clinical actionability to evaluate conditions for inclusion in newborn screening and compare it with the results from other contemporary methods. STUDY DESIGN: The North Carolina Newborn Exome Sequencing for Universal Screening study developed an age-based, semiquantitative metric to assess the clinical actionability of gene-disease pairs and classify them with respect to age of onset or timing of interventions. This categorization was compared with the gold standard Recommended Uniform Screening Panel and other methods to evaluate gene-disease pairs for newborn genomic sequencing. RESULTS: We assessed 822 gene-disease pairs, enriched for pediatric onset of disease and suspected actionability. Of these, 466 were classified as having childhood onset and high actionability, analogous to conditions selected for the Recommended Uniform Screening Panel core panel. Another 245 were classified as having childhood onset and low to no actionability, 25 were classified as having adult onset and high actionability, 19 were classified as having adult onset and low to no actionability, and 67 were excluded due to controversial evidence and/or prenatal onset. CONCLUSIONS: This study describes a novel method to facilitate decisions about the potential use of genomic sequencing for newborn screening. These categories may assist parents and physicians in making informed decisions about the disclosure of results from voluntary genomic sequencing in children.

ClinGen Allele Registry links information about genetic variants.

Effective exchange of information about genetic variants is currently hampered by the lack of readily available globally unique variant identifiers that would enable aggregation of information from different sources. The ClinGen Allele Registry addresses this problem by providing (1) globally unique "canonical" variant identifiers (CAids) on demand, either individually or in large batches; (2) access to variant-identifying information in a searchable Registry; (3) links to allele-related records in many commonly used databases; and (4) services for adding links to information about registered variants in external sources. A core element of the Registry is a canonicalization service, implemented using in-memory sequence alignment-based index, which groups variant identifiers denoting the same nucleotide variant and assigns unique and dereferenceable CAids. More than 650 million distinct variants are currently registered, including those from gnomAD, ExAC, dbSNP, and ClinVar, including a small number of variants registered by Registry users. The Registry is accessible both via a web interface and programmatically via well-documented Hypertext Transfer Protocol (HTTP) Representational State Transfer Application Programming Interface (REST-APIs). For programmatic interoperability, the Registry content is accessible in the JavaScript Object Notation for Linked Data (JSON-LD) format. We present several use cases and demonstrate how the linked information may provide raw material for reasoning about variant's pathogenicity.

Anticipated responses of early adopter genetic specialists and nongenetic specialists to unsolicited genomic secondary findings.

PURPOSE: Secondary findings from genomic sequencing are becoming more common. We compared how health-care providers with and without specialized genetics training anticipated responding to different types of secondary findings. METHODS: Providers with genomic sequencing experience reviewed five secondary-findings reports and reported attitudes and potential clinical follow-up. Analyses compared genetic specialists and physicians without specialized genetics training, and examined how responses varied by secondary finding. RESULTS: Genetic specialists scored higher than other providers on four-point scales assessing understandings of reports (3.89 vs. 3.42, p = 0.0002), and lower on scales assessing reporting obligations (2.60 vs. 3.51, p < 0.0001) and burdens of responding (1.73 vs. 2.70, p < 0.0001). Nearly all attitudes differed between findings, although genetic specialists were more likely to assert that laboratories had no obligations when findings had less-established actionability (p < 0.0001 in interaction tests). The importance of reviewing personal and family histories, documenting findings, learning more about the variant, and recommending familial discussions also varied according to finding (all p < 0.0001). CONCLUSION: Genetic specialists felt better prepared to respond to secondary findings than providers without specialized genetics training, but perceived fewer obligations for laboratories to report them, and the two groups anticipated similar clinical responses. Findings may inform development of targeted education and support.