Toward robust clinical genome interpretation: Developing a consistent terminology to characterize Mendelian disease-gene relationships-allelic requirement, inheritance modes, and disease mechanisms.

PURPOSE: The terminology used for gene-disease curation and variant annotation to describe inheritance, allelic requirement, and both sequence and functional consequences of a variant is currently not standardized. There is considerable discrepancy in the literature and across clinical variant reporting in the derivation and application of terms. Here, we standardize the terminology for the characterization of disease-gene relationships to facilitate harmonized global curation and to support variant classification within the ACMG/AMP framework. METHODS: Terminology for inheritance, allelic requirement, and both structural and functional consequences of a variant used by Gene Curation Coalition members and partner organizations was collated and reviewed. Harmonized terminology with definitions and use examples was created, reviewed, and validated. RESULTS: We present a standardized terminology to describe gene-disease relationships, and to support variant annotation. We demonstrate application of the terminology for classification of variation in the ACMG SF 2.0 genes recommended for reporting of secondary findings. Consensus terms were agreed and formalized in both Sequence Ontology (SO) and Human Phenotype Ontology (HPO) ontologies. Gene Curation Coalition member groups intend to use or map to these terms in their respective resources. CONCLUSION: The terminology standardization presented here will improve harmonization, facilitate the pooling of curation datasets across international curation efforts and, in turn, improve consistency in variant classification and genetic test interpretation.

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.

Information-seeking preferences in diverse patients receiving a genetic testing result in the Clinical Sequencing Evidence-Generating Research (CSER) study.

PURPOSE: Accurate and understandable information after genetic testing is critical for patients, family members, and professionals alike. METHODS: As part of a cross-site study from the Clinical Sequencing Evidence-Generating Research consortium, we investigated the information-seeking practices among patients and family members at 5 to 7 months after genetic testing results disclosure, assessing the perceived utility of a variety of information sources, such as family and friends, health care providers, support groups, and the internet. RESULTS: We found that individuals placed a high value on information obtained from genetics professionals and health care workers, independent of genetic testing result case classifications as positive, inconclusive, or negative. The internet was also highly utilized and ranked. Study participants rated some information sources as more useful for positive results compared with inconclusive or negative outcomes, emphasizing that it may be difficult to identify helpful information for individuals receiving an uncertain or negative result. There were few data from non-English speakers, highlighting the need to develop strategies to reach this population. CONCLUSION: Our study emphasizes the need for clinicians to provide accurate and comprehensible information to individuals from diverse populations after genetic testing.

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.

Utilizing ClinGen gene-disease validity and dosage sensitivity curations to inform variant classification.

Understanding whether there is enough evidence to implicate a gene's role in a given disease, as well as the mechanisms by which variants in this gene might cause this disease, is essential to determine clinical relevance. The National Institutes of Health-funded Clinical Genome Resource (ClinGen) has developed evaluation frameworks to assess both the strength of evidence supporting a relationship between a gene and disease (gene-disease validity), and whether loss (haploinsufficiency) or gain (triplosensitivity) of individual genes or genomic regions is a mechanism for disease (dosage sensitivity). ClinGen actively applies these frameworks across multiple disease domains, and makes this information publicly available via its website (https://www.clinicalgenome.org/) for use in multiple applications, including clinical variant classification. Here, we describe how the results of these curation processes can be utilized to inform the appropriate application of pathogenicity criteria for both sequence and copy number variants, as well as to guide test development and inform genomic filtering pipelines.

Newborn screening for neurodevelopmental diseases: Are we there yet?

In the US, newborn screening (NBS) is a unique health program that supports health equity and screens virtually every baby after birth, and has brought timely treatments to babies since the 1960's. With the decreasing cost of sequencing and the improving methods to interpret genetic data, there is an opportunity to add DNA sequencing as a screening method to facilitate the identification of babies with treatable conditions that cannot be identified in any other scalable way, including highly penetrant genetic neurodevelopmental disorders (NDD). However, the lack of effective dietary or drug-based treatments has made it nearly impossible to consider NDDs in the current NBS framework, yet it is anticipated that any treatment will be maximally effective if started early. Hence there is a critical need for large scale pilot studies to assess if and how NDDs can be effectively screened at birth, if parents desire that information, and what impact early diagnosis may have. Here we attempt to provide an overview of the recent advances in NDD treatments, explore the possible framework of setting up a pilot study to genetically screen for NDDs, highlight key technical, practical, and ethical considerations and challenges, and examine the policy and health system implications.

Missense Variants in the Histone Acetyltransferase Complex Component Gene TRRAP Cause Autism and Syndromic Intellectual Disability.

Acetylation of the lysine residues in histones and other DNA-binding proteins plays a major role in regulation of eukaryotic gene expression. This process is controlled by histone acetyltransferases (HATs/KATs) found in multiprotein complexes that are recruited to chromatin by the scaffolding subunit transformation/transcription domain-associated protein (TRRAP). TRRAP is evolutionarily conserved and is among the top five genes intolerant to missense variation. Through an international collaboration, 17 distinct de novo or apparently de novo variants were identified in TRRAP in 24 individuals. A strong genotype-phenotype correlation was observed with two distinct clinical spectra. The first is a complex, multi-systemic syndrome associated with various malformations of the brain, heart, kidneys, and genitourinary system and characterized by a wide range of intellectual functioning; a number of affected individuals have intellectual disability (ID) and markedly impaired basic life functions. Individuals with this phenotype had missense variants clustering around the c.3127G>A p.(Ala1043Thr) variant identified in five individuals. The second spectrum manifested with autism spectrum disorder (ASD) and/or ID and epilepsy. Facial dysmorphism was seen in both groups and included upslanted palpebral fissures, epicanthus, telecanthus, a wide nasal bridge and ridge, a broad and smooth philtrum, and a thin upper lip. RNA sequencing analysis of skin fibroblasts derived from affected individuals skin fibroblasts showed significant changes in the expression of several genes implicated in neuronal function and ion transport. Thus, we describe here the clinical spectrum associated with TRRAP pathogenic missense variants, and we suggest a genotype-phenotype correlation useful for clinical evaluation of the pathogenicity of the variants.

A systematic review of the methodological quality of economic evaluations in genetic screening and testing for monogenic disorders.

PURPOSE: Understanding the value of genetic screening and testing for monogenic disorders requires high-quality, methodologically robust economic evaluations. This systematic review sought to assess the methodological quality among such studies and examined opportunities for improvement. METHODS: We searched PubMed, Cochrane, Embase, and Web of Science for economic evaluations of genetic screening/testing (2013-2019). Methodological rigor and adherence to best practices were systematically assessed using the British Medical Journal checklist. RESULTS: Across the 47 identified studies, there were substantial variations in modeling approaches, reporting detail, and sophistication. Models ranged from simple decision trees to individual-level microsimulations that compared between 2 and >20 alternative interventions. Many studies failed to report sufficient detail to enable replication or did not justify modeling assumptions, especially for costing methods and utility values. Meta-analyses, systematic reviews, or calibration were rarely used to derive parameter estimates. Nearly all studies conducted some sensitivity analysis, and more sophisticated studies implemented probabilistic sensitivity/uncertainty analysis, threshold analysis, and value of information analysis. CONCLUSION: We describe a heterogeneous body of work and present recommendations and exemplar studies across the methodological domains of (1) perspective, scope, and parameter selection; (2) use of uncertainty/sensitivity analyses; and (3) reporting transparency for improvement in the economic evaluation of genetic screening/testing.

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.

US private payers' perspectives on insurance coverage for genome sequencing versus exome sequencing: A study by the Clinical Sequencing Evidence-Generating Research Consortium (CSER).

PURPOSE: There is limited payer coverage for genome sequencing (GS) relative to exome sequencing (ES) in the U.S. Our objective was to assess payers' considerations for coverage of GS versus coverage of ES and requirements payers have for coverage of GS. The study was conducted by the NIH-funded Clinical Sequencing Evidence-Generating Research Consortium (CSER). METHODS: We conducted semi-structured interviews with representatives of private payer organizations (payers, N = 12) on considerations and evidentiary and other needs for coverage of GS and ES. Data were analyzed using thematic analysis. RESULTS: We described four categories of findings and solutions: demonstrated merits of GS versus ES, enhanced methods for evidence generation, consistent laboratory processes/sequencing methods, and enhanced implementation/care delivery. Payers see advantages to GS vs. ES and are open to broader GS coverage but need more proof of these advantages to consider them in coverage decision-making. Next steps include establishing evidence of benefits in specific clinical scenarios, developing quality standards, ensuring transparency of laboratory methods, developing clinical centers of excellence, and incorporating the role of genetic professionals. CONCLUSION: By comparing coverage considerations for GS and ES, we identified a path forward for coverage of GS. Future research should explicitly address payers' conditions for coverage.

The Gene Curation Coalition: A global effort to harmonize gene-disease evidence resources.

PURPOSE: Several groups and resources provide information that pertains to the validity of gene-disease relationships used in genomic medicine and research; however, universal standards and terminologies to define the evidence base for the role of a gene in disease and a single harmonized resource were lacking. To tackle this issue, the Gene Curation Coalition (GenCC) was formed. METHODS: The GenCC drafted harmonized definitions for differing levels of gene-disease validity on the basis of existing resources, and performed a modified Delphi survey with 3 rounds to narrow the list of terms. The GenCC also developed a unified database to display curated gene-disease validity assertions from its members. RESULTS: On the basis of 241 survey responses from the genetics community, a consensus term set was chosen for grading gene-disease validity and database submissions. As of December 2021, the database contained 15,241 gene-disease assertions on 4569 unique genes from 12 submitters. When comparing submissions to the database from distinct sources, conflicts in assertions of gene-disease validity ranged from 5.3% to 13.4%. CONCLUSION: Terminology standardization, sharing of gene-disease validity classifications, and resolution of curation conflicts will facilitate collaborations across international curation efforts and in turn, improve consistency in genetic testing and variant interpretation.

Quality of care metrics for patients with inborn errors of metabolism.

Despite recent calls to action and a heavy emphasis on timeliness of care in guidelines for common inborn errors of metabolism, there is a dearth of specific measurable quality metrics for these conditions and little to no electronic decision support for their management. We have developed a novel set of process-oriented metrics based on the aforementioned guidelines that can be calculated from data already contained in most major EHRs, which we believe are responsive to the needs of the metabolism community.

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.

Development and initial testing of a multi-stakeholder intervention for Lynch syndrome cascade screening: an intervention mapping approach.

BACKGROUND: Lynch syndrome is an underdiagnosed hereditary condition carrying an increased lifetime risk for colorectal and endometrial cancer and affecting nearly 1 million people in the United States. Cascade screening, systematic screening through family members of affected patients, could improve identification of Lynch syndrome, but this strategy is underused due to multi-level barriers including low knowledge about Lynch syndrome, low access to genetics services, and challenging family dynamics. METHODS: We used intervention mapping, a 6-step methodology to create stakeholder-driven interventions that meet the needs of a target population, to develop an intervention to improve cascade screening for Lynch syndrome. The intervention development process was guided by input from key stakeholders in Lynch syndrome care and patients. We conducted usability testing on the intervention with Lynch syndrome patients using qualitative semi-structured interviewing and rapid qualitative analysis. RESULTS: We developed a workbook intervention named Let's Talk that addresses gaps in knowledge, skills, self-efficacy, outcome expectancy and other perceived barriers to cascade screening for Lynch syndrome. Let's Talk contained educational content, goal setting activities, communication planning prompts and supplemental resources for patients to plan family communication. Evidence-based methods used in the workbook included information chunking, guided practice, goal setting and gain-framing. We conducted usability testing focused on the complexity and relative advantage of the intervention through 45-min virtual interviews with 10 adult patients with Lynch syndrome recruited from a national advocacy organization in the United States. Usability testing results suggested the intervention was acceptable in terms of complexity and relative advantage to other available resources, but additional information for communication with young or distant family members and a web-based platform could enhance the intervention's usability. CONCLUSIONS: Intervention mapping provided a framework for intervention development that addressed the unique needs of Lynch syndrome patients in overcoming barriers to cascade screening. Future work is needed to transform Let's Talk into a web-based tool and evaluate the effectiveness of the intervention in clinical practice with patients and genetic counselors. Intervention mapping can be useful to researchers as an evidence-based technique to develop stakeholder-centered interventions for addressing the needs of other unique populations.

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.

Selective serotonin reuptake inhibitors ameliorate MEGF10 myopathy.

MEGF10 myopathy is a rare inherited muscle disease that is named after the causative gene, MEGF10. The classic phenotype, early onset myopathy, areflexia, respiratory distress and dysphagia, is severe and immediately life-threatening. There are no disease-modifying therapies. We performed a small molecule screen and follow-up studies to seek a novel therapy. A primary in vitro drug screen assessed cellular proliferation patterns in Megf10-deficient myoblasts. Secondary evaluations were performed on primary screen hits using myoblasts derived from Megf10-/- mice, induced pluripotent stem cell-derived myoblasts from MEGF10 myopathy patients, mutant Drosophila that are deficient in the homologue of MEGF10 (Drpr) and megf10 mutant zebrafish. The screen yielded two promising candidates that are both selective serotonin reuptake inhibitors (SSRIs), sertraline and escitalopram. In depth follow-up analyses demonstrated that sertraline was highly effective in alleviating abnormalities across multiple models of the disease including mouse myoblast, human myoblast, Drosophila and zebrafish models. Sertraline also restored deficiencies of Notch1 in disease models. We conclude that SSRIs show promise as potential therapeutic compounds for MEGF10 myopathy, especially sertraline. The mechanism of action may involve the Notch pathway.

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.

Evaluating the Clinical Validity of Gene-Disease Associations: An Evidence-Based Framework Developed by the Clinical Genome Resource.

With advances in genomic sequencing technology, the number of reported gene-disease relationships has rapidly expanded. However, the evidence supporting these claims varies widely, confounding accurate evaluation of genomic variation in a clinical setting. Despite the critical need to differentiate clinically valid relationships from less well-substantiated relationships, standard guidelines for such evaluation do not currently exist. The NIH-funded Clinical Genome Resource (ClinGen) has developed a framework to define and evaluate the clinical validity of gene-disease pairs across a variety of Mendelian disorders. In this manuscript we describe a proposed framework to evaluate relevant genetic and experimental evidence supporting or contradicting a gene-disease relationship and the subsequent validation of this framework using a set of representative gene-disease pairs. The framework provides a semiquantitative measurement for the strength of evidence of a gene-disease relationship that correlates to a qualitative classification: "Definitive," "Strong," "Moderate," "Limited," "No Reported Evidence," or "Conflicting Evidence." Within the ClinGen structure, classifications derived with this framework are reviewed and confirmed or adjusted based on clinical expertise of appropriate disease experts. Detailed guidance for utilizing this framework and access to the curation interface is available on our website. This evidence-based, systematic method to assess the strength of gene-disease relationships will facilitate more knowledgeable utilization of genomic variants in clinical and research settings.

De Novo Mutations in Protein Kinase Genes CAMK2A and CAMK2B Cause Intellectual Disability.

Calcium/calmodulin-dependent protein kinase II (CAMK2) is one of the first proteins shown to be essential for normal learning and synaptic plasticity in mice, but its requirement for human brain development has not yet been established. Through a multi-center collaborative study based on a whole-exome sequencing approach, we identified 19 exceedingly rare de novo CAMK2A or CAMK2B variants in 24 unrelated individuals with intellectual disability. Variants were assessed for their effect on CAMK2 function and on neuronal migration. For both CAMK2A and CAMK2B, we identified mutations that decreased or increased CAMK2 auto-phosphorylation at Thr286/Thr287. We further found that all mutations affecting auto-phosphorylation also affected neuronal migration, highlighting the importance of tightly regulated CAMK2 auto-phosphorylation in neuronal function and neurodevelopment. Our data establish the importance of CAMK2A and CAMK2B and their auto-phosphorylation in human brain function and expand the phenotypic spectrum of the disorders caused by variants in key players of the glutamatergic signaling pathway.

Parents' perceptions of personal utility of exome sequencing results.

PURPOSE: Clinical genome or exome sequencing (GS/ES) provides a diagnosis for many individuals with suspected genetic disorders, but also yields negative or uncertain results for the majority. This study examines how parents of a child with an undiagnosed condition attribute personal utility to all types of ES results. METHODS: Return of 31 exome sequencing results was observed during clinic sessions, followed by semistructured interviews with parents one month later. Observations and interviews were recorded and transcribed. Data display matrices were used for content analysis and systematic comparisons of parents' perceptions of utility. RESULTS: ES results could not provide all the answers to parents' questions, especially in cases of clinically uninformative results, but parents nonetheless attributed utility to the knowledge gained. Parents across all results categories used the genomic information to rule out possible causes, end or postpone the diagnostic odyssey, and shift focus to treatment and management of symptoms. CONCLUSION: This study suggests that parents value even uninformative ES results while expressing hope for future discoveries. As pediatric genetics moves toward GS/ES as a first-tier test, how parents perceive the personal utility of negative or uncertain results is an important topic for genetic counseling and further research.