Massively parallel reporter assays discover de novo exonic splicing mutants in paralogs of Autism genes.

To determine the contribution of defective splicing in Autism Spectrum Disorders (ASD), the most common neurodevelopmental disorder, a high throughput Massively Parallel Splicing Assay (MaPSY) was employed and identified 42 exonic splicing mutants out of 725 coding de novo variants discovered in the sequencing of ASD families. A redesign of the minigene constructs in MaPSY revealed that upstream exons with strong 5' splice sites increase the magnitude of skipping phenotypes observed in downstream exons. Select hits were validated by RT-PCR and amplicon sequencing in patient cell lines. Exonic splicing mutants were enriched in probands relative to unaffected siblings -especially synonymous variants (7.5% vs 3.5%, respectively). Of the 26 genes disrupted by exonic splicing mutations, 6 were in known ASD genes and 3 were in paralogs of known ASD genes. Of particular interest was a synonymous variant in TNRC6C - an ASD gene paralog with interactions with other ASD genes. Clinical records of 3 ASD patients with TNRC6C variant revealed respiratory issues consistent with phenotypes observed in TNRC6 depleted mice. Overall, this study highlights the need for splicing analysis in determining variant pathogenicity, especially as it relates to ASD.

Individuals heterozygous for ALG8 protein-truncating variants are at increased risk of a mild cystic kidney disease.

ALG8 protein-truncating variants (PTVs) have previously been described in patients with polycystic liver disease and in some cases cystic kidney disease. Given a lack of well-controlled studies, we determined whether individuals heterozygous for ALG8 PTVs are at increased risk of cystic kidney disease in a large, unselected health system-based observational cohort linked to electronic health records in Pennsylvania (Geisinger-Regeneron DiscovEHR MyCode study). Out of 174,172 patients, 236 were identified with ALG8 PTVs. Using ICD-based outcomes, patients with these variants were significantly at increased risk of having any kidney/liver cyst diagnosis (Odds Ratio 2.42, 95% confidence interval: 1.53-3.85), cystic kidney disease (3.03, 1.26-7.31), and nephrolithiasis (1.89, 1.96-2.97). To confirm this finding, blinded radiology review of computed tomography and magnetic resonance imaging studies was completed in a matched cohort of 52 thirty-plus year old ALG8 PTV heterozygotes and related non-heterozygotes. ALG8 PTV heterozygotes were significantly more likely to have cystic kidney disease, defined as four or more kidney cysts (57.7% vs. 7.7%), or bilateral kidney cysts (69.2% vs. 15.4%), but not one or more liver cyst (11.5% vs. 7.7%). In publicly available UK Biobank data, ALG8 PTV heterozygotes were at significantly increased risk of ICD code N28 (other disorders of kidney/ureter) (3.85% vs. 1.33%). ALG8 PTVs were not associated with chronic kidney disease or kidney failure in the MyCode study or the UK Biobank data. Thus, PTVs in ALG8 result in increased risk of a mild cystic kidney disease phenotype.

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.

Estimated Prevalence and Clinical Manifestations of UBA1 Variants Associated With VEXAS Syndrome in a Clinical Population.

Importance: VEXAS (vacuoles, E1-ubiquitin-activating enzyme, X-linked, autoinflammatory, somatic) syndrome is a disease with rheumatologic and hematologic features caused by somatic variants in UBA1. Pathogenic variants are associated with a broad spectrum of clinical manifestations. Knowledge of prevalence, penetrance, and clinical characteristics of this disease have been limited by ascertainment biases based on known phenotypes. Objective: To determine the prevalence of pathogenic variants in UBA1 and associated clinical manifestations in an unselected population using a genomic ascertainment approach. Design, Setting, and Participants: This retrospective observational study evaluated UBA1 variants in exome data from 163 096 participants within the Geisinger MyCode Community Health Initiative. Clinical phenotypes were determined from Geisinger electronic health record data from January 1, 1996, to January 1, 2022. Exposures: Exome sequencing was performed. Main Outcomes and Measures: Outcome measures included prevalence of somatic UBA1 variation; presence of rheumatologic, hematologic, pulmonary, dermatologic, and other findings in individuals with somatic UBA1 variation on review of the electronic health record; review of laboratory data; bone marrow biopsy pathology analysis; and in vitro enzymatic assays. Results: In 163 096 participants (mean age, 52.8 years; 94% White; 61% women), 11 individuals harbored likely somatic variants at known pathogenic UBA1 positions, with 11 of 11 (100%) having clinical manifestations consistent with VEXAS syndrome (9 male, 2 female). A total of 5 of 11 individuals (45%) did not meet criteria for rheumatologic and/or hematologic diagnoses previously associated with VEXAS syndrome; however, all individuals had anemia (hemoglobin: mean, 7.8 g/dL; median, 7.5 g/dL), which was mostly macrocytic (10/11 [91%]) with concomitant thrombocytopenia (10/11 [91%]). Among the 11 patients identified, there was a pathogenic variant in 1 male participant prior to onset of VEXAS-related signs or symptoms and 2 female participants had disease with heterozygous variants. A previously unreported UBA1 variant (c.1861A>T; p.Ser621Cys) was found in a symptomatic patient, with in vitro data supporting a catalytic defect and pathogenicity. Together, disease-causing UBA1 variants were found in 1 in 13 591 unrelated individuals (95% CI, 1:7775-1:23 758), 1 in 4269 men older than 50 years (95% CI, 1:2319-1:7859), and 1 in 26 238 women older than 50 years (95% CI, 1:7196-1:147 669). Conclusions and Relevance: This study provides an estimate of the prevalence and a description of the clinical manifestations of UBA1 variants associated with VEXAS syndrome within a single regional health system in the US. Additional studies are needed in unselected and genetically diverse populations to better define general population prevalence and phenotypic spectrum.

Frequency of truncating FLCN variants and Birt-Hogg-Dubé-associated phenotypes in a health care system population.

PURPOSE: Penetrance estimates of Birt-Hogg-Dubé syndrome (BHD)-associated cutaneous, pulmonary, and kidney manifestations are based on clinically ascertained families. In a health care system population, we used a genetics-first approach to estimate the prevalence of pathogenic/likely pathogenic (P/LP) truncating variants in FLCN, which cause BHD, and the penetrance of BHD-related phenotypes. METHODS: Exomes from 135,990 patient-participants in Geisinger's MyCode cohort were assessed for P/LP truncating FLCN variants. BHD-related phenotypes were evaluated from electronic health records. Association between P/LP FLCN variants and BHD-related phenotypes was assessed using Firth's logistic regression. RESULTS: P/LP truncating FLCN variants were identified in 35 individuals (1 in 3234 unrelated individuals), 68.6% of whom had BHD-related phenotype(s), including cystic lung disease (65.7%), pneumothoraces (17.1%), cutaneous manifestations (8.6%), and kidney cancer (2.9%). A total of 4 (11.4%) individuals had prior clinical BHD diagnoses. CONCLUSION: In this health care population, the frequency of P/LP truncating FLCN variants is 60 times higher than the previously reported prevalence. Although most variant-positive individuals had BHD-related phenotypes, a minority were previously clinically diagnosed, likely because cutaneous manifestations, pneumothoraces, and kidney cancer were observed at lower frequencies than in clinical cohorts. Improved clinical recognition of cystic lung disease and education concerning its association with FLCN variants could prompt evaluation for BHD.

Leveraging population-based exome screening to impact clinical care: The evolution of variant assessment in the Geisinger MyCode research project.

Exome and genome sequencing are increasingly utilized in research studies and clinical care and can provide clinically relevant information beyond the initial intent for sequencing, including medically actionable secondary findings. Despite ongoing debate about sharing this information with patients and participants, a growing number of clinical laboratories and research programs routinely report secondary findings that increase the risk for selected diseases. Recently, there has been a push to maximize the potential benefit of this practice by implementing proactive genomic screening at the population level irrespective of medical history, but the feasibility of deploying population-scale proactive genomic screening requires scaling key elements of the genomic data evaluation process. Herein, we describe the motivation, development, and implementation of a population-scale variant-first screening pipeline combining bioinformatics-based filtering with a manual review process to screen for clinically relevant findings in research exomes generated through the DiscovEHR collaboration within Geisinger's MyCode® research project. Consistent with other studies, this pipeline yields a screen-positive detection rate between 2.1 and 2.6% (depending on inclusion of those with prior indication-based testing) in 130,048 adult MyCode patient-participants screened for clinically relevant findings in 60 genes. Our variant-first pipeline affords cost and time savings by filtering out negative cases, thereby avoiding analysis of each exome one-by-one, as typically employed in the diagnostic setting. While research is still needed to fully appreciate the benefits of population genomic screening, MyCode provides the first demonstration of a program at scale to help shape how population genomic screening is integrated into routine clinical care.

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.

Defining the Clinical Value of a Genomic Diagnosis in the Era of Next-Generation Sequencing.

As with all fields of medicine, the first step toward medical management of genetic disorders is obtaining an accurate diagnosis, which often requires testing at the molecular level. Unfortunately, given the large number of genetic conditions without a specific intervention, only rarely does a genetic diagnosis alter patient management-which raises the question, what is the added value of obtaining a molecular diagnosis? Given the fast-paced advancement of genomic technologies, this is an important question to address in the context of genome-scale testing. Here, we address the value of establishing a diagnosis using genome-scale testing and highlight the benefits and drawbacks of such testing. We also review and compare recent major studies implementing genome-scale sequencing methods to identify a molecular diagnosis in cohorts manifesting a broad range of Mendelian monogenic disorders. Finally, we discuss potential future applications of genomic sequencing, such as screening for rare conditions.

Performance of ACMG-AMP Variant-Interpretation Guidelines among Nine Laboratories in the Clinical Sequencing Exploratory Research Consortium.

Evaluating the pathogenicity of a variant is challenging given the plethora of types of genetic evidence that laboratories consider. Deciding how to weigh each type of evidence is difficult, and standards have been needed. In 2015, the American College of Medical Genetics and Genomics (ACMG) and the Association for Molecular Pathology (AMP) published guidelines for the assessment of variants in genes associated with Mendelian diseases. Nine molecular diagnostic laboratories involved in the Clinical Sequencing Exploratory Research (CSER) consortium piloted these guidelines on 99 variants spanning all categories (pathogenic, likely pathogenic, uncertain significance, likely benign, and benign). Nine variants were distributed to all laboratories, and the remaining 90 were evaluated by three laboratories. The laboratories classified each variant by using both the laboratory's own method and the ACMG-AMP criteria. The agreement between the two methods used within laboratories was high (K-alpha = 0.91) with 79% concordance. However, there was only 34% concordance for either classification system across laboratories. After consensus discussions and detailed review of the ACMG-AMP criteria, concordance increased to 71%. Causes of initial discordance in ACMG-AMP classifications were identified, and recommendations on clarification and increased specification of the ACMG-AMP criteria were made. In summary, although an initial pilot of the ACMG-AMP guidelines did not lead to increased concordance in variant interpretation, comparing variant interpretations to identify differences and having a common framework to facilitate resolution of those differences were beneficial for improving agreement, allowing iterative movement toward increased reporting consistency for variants in genes associated with monogenic disease.

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.

Navigating the nuances of clinical sequence variant interpretation in Mendelian disease.

Understanding clinical genetic test results in the era of next-generation sequencing has become increasingly complex, necessitating clear and thorough guidelines for sequence variant interpretation. To meet this need the American College of Medical Genetics and Genomics (ACMG) and the Association for Molecular Pathology (AMP) published guidelines for a systematic approach for sequence variant interpretation in 2015. This framework is intended to be adaptable to any Mendelian condition, promoting transparency and consistency in variant interpretation, yet its comprehensive nature yields important challenges and caveats that end users must understand. In this review, we address some of these nuances and discuss the evolving efforts to refine and adapt this framework. We also consider the added complexity of distinguishing between variant-level interpretations and case-level conclusions, particularly in the context of the large gene panel approach to clinical diagnostics.

"Possibly positive or certainly uncertain?": participants' responses to uncertain diagnostic results from exome sequencing.

PurposeClinical genome sequencing produces uncertain diagnostic results, raising concerns about how to communicate the method's inherent complexities in ways that reduce potential misunderstandings and harm. This study investigates clinicians' communications and patient/participant responses to uncertain diagnostic results arising from a clinical exome sequencing research study, contributing empirical data to the debate surrounding disclosure of uncertain genomic information.MethodsWe investigated the communication and impact of uncertain diagnostic results using ethnographic observations of result disclosures with 21 adults and 11 parents of child patients, followed by two semistructured interviews with these same participants.ResultsParticipants understood their uncertain results in ways that were congruent with clinical geneticists' communications. They followed recommendations for further consultation, although family testing to resolve uncertainty was not always done. Participants were prepared for learning an uncertain result and grasped the key concept that it should not be used to guide health-care or other decisions. They did not express regret for having learned the uncertain result; most regarded it as potentially valuable in the future.ConclusionThis study suggests that uncertain diagnostic results from genome sequencing can be relayed to patients in ways they can understand and consistent with providers' interpretations, without causing undue harm.

Organization and dynamics of the nonhomologous end-joining machinery during DNA double-strand break repair.

Nonhomologous end-joining (NHEJ) is a major repair pathway for DNA double-strand breaks (DSBs), involving synapsis and ligation of the broken strands. We describe the use of in vivo and in vitro single-molecule methods to define the organization and interaction of NHEJ repair proteins at DSB ends. Super-resolution fluorescence microscopy allowed the precise visualization of XRCC4, XLF, and DNA ligase IV filaments adjacent to DSBs, which bridge the broken chromosome and direct rejoining. We show, by single-molecule FRET analysis of the Ku/XRCC4/XLF/DNA ligase IV NHEJ ligation complex, that end-to-end synapsis involves a dynamic positioning of the two ends relative to one another. Our observations form the basis of a new model for NHEJ that describes the mechanism whereby filament-forming proteins bridge DNA DSBs in vivo. In this scheme, the filaments at either end of the DSB interact dynamically to achieve optimal configuration and end-to-end positioning and ligation.

A semiquantitative metric for evaluating clinical actionability of incidental or secondary findings from genome-scale sequencing.

PURPOSE: As genome-scale sequencing is increasingly applied in clinical scenarios, a wide variety of genomic findings will be discovered as secondary or incidental findings, and there is debate about how they should be handled. The clinical actionability of such findings varies, necessitating standardized frameworks for a priori decision making about their analysis. METHODS: We established a semiquantitative metric to assess five elements of actionability: severity and likelihood of the disease outcome, efficacy and burden of intervention, and knowledge base, with a total score from 0 to 15. RESULTS: The semiquantitative metric was applied to a list of putative actionable conditions, the list of genes recommended by the American College of Medical Genetics and Genomics (ACMG) for return when deleterious variants are discovered as secondary/incidental findings, and a random sample of 1,000 genes. Scores from the list of putative actionable conditions (median = 12) and the ACMG list (median = 11) were both statistically different than the randomly selected genes (median = 7) (P < 0.0001, two-tailed Mann-Whitney test). CONCLUSION: Gene-disease pairs having a score of 11 or higher represent the top quintile of actionability. The semiquantitative metric effectively assesses clinical actionability, promotes transparency, and may facilitate assessments of clinical actionability by various groups and in diverse contexts.Genet Med 18 5, 467-475.

Requirements for 5'dRP/AP lyase activity in Ku.

The non-homologous end joining (NHEJ) pathway is used in diverse species to repair chromosome breaks, and is defined in part by a requirement for Ku. We previously demonstrated mammalian Ku has intrinsic 5' deoxyribosephosphate (5'dRP) and apurinic/apyrimidinic (AP) lyase activity, and showed this activity is important for excising abasic site damage from ends. Here we employ systematic mutagenesis to clarify the protein requirements for this activity. We identify lysine 31 in the 70 kD subunit (Ku70 K31) as the primary candidate nucleophile required for catalysis, but additional mutation of Ku70 K160 and six other lysines within Ku80 were required to eliminate all activity. Ku from Saccharomyces cerevisiae also possesses 5'dRP/AP lyase activity, and robust activity was also reliant on lysines in Ku70 analogous to K31 and K160. By comparison, these lysines are not conserved in Xenopus laevis Ku, and Ku from this species has negligible activity. A role for residues flanking Ku70 K31 in expanding the range of abasic site contexts that can be used as substrate was also identified. Our results suggest an active site well located to provide the substrate specificity required for its biological role.

The phenotype of multiple congenital anomalies-hypotonia-seizures syndrome 1: report and review.

The Multiple Congenital Anomalies-Hypotonia-Seizures Syndrome 1 (MCAHS1) has been described in two families to date. We describe a 2-year-old Mexican American boy with the syndrome and additional manifestations not yet reported as part of the phenotype. The patient presented with severe hypotonia, microphallus and left cryptorchidism, and was later diagnosed with epilepsy and severe cortical visual impairment. He also had supernumerary nipples, pectus excavatum, a short upturned nose, fleshy ear lobes, and a right auricular pit. Massively parallel exome sequencing and analysis revealed two novel compound heterozygous missense (Trp136Gly and Ser859Thr) variants in the PIGN gene. This report extends and further defines the phenotype of this syndrome.

Genome-first approach of the prevalence and cancer phenotypes of pathogenic or likely pathogenic germline TP53 variants.

Pathogenic or likely pathogenic (P/LP) germline TP53 variants are the primary cause of Li-Fraumeni syndrome (LFS), a hereditary cancer predisposition disorder characterized by early-onset cancers. The population prevalence of P/LP germline TP53 variants is estimated to be approximately one in every 3,500 to 20,000 individuals. However, these estimates are likely impacted by ascertainment biases and lack of clinical and genetic data to account for potential confounding factors, such as clonal hematopoiesis. Genome-first approaches of cohorts linked to phenotype data can further refine these estimates by identifying individuals with variants of interest and then assessing their phenotypes. This study evaluated P/LP germline (variant allele fraction ≥30%) TP53 variants in three cohorts: UK Biobank (UKB, n = 200,590), Geisinger (n = 170,503), and Penn Medicine Biobank (PMBB, n = 43,731). A total of 109 individuals were identified with P/LP germline TP53 variants across the three databases. The TP53 p.R181H variant was the most frequently identified (9 of 109 individuals, 8%). A total of 110 cancers, including 47 hematologic cancers (47 of 110, 43%), were reported in 71 individuals. The prevalence of P/LP germline TP53 variants was conservatively estimated as 1:10,439 in UKB, 1:3,790 in Geisinger, and 1:2,983 in PMBB. These estimates were calculated after excluding related individuals and accounting for the potential impact of clonal hematopoiesis by excluding heterozygotes who ever developed a hematologic cancer. These varying estimates likely reflect intrinsic selection biases of each database, such as healthcare or population-based contexts. Prospective studies of diverse, young cohorts are required to better understand the population prevalence of germline TP53 variants and their associated cancer penetrance.

Generating Clinical-Grade Gene-Disease Validity Classifications Through the ClinGen Data Platforms.

Clinical genetic laboratories must have access to clinically validated biomedical data for precision medicine. A lack of accessibility, normalized structure, and consistency in evaluation complicates interpretation of disease causality, resulting in confusion in assessing the clinical validity of genes and genetic variants for diagnosis. A key goal of the Clinical Genome Resource (ClinGen) is to fill the knowledge gap concerning the strength of evidence supporting the role of a gene in a monogenic disease, which is achieved through a process known as Gene-Disease Validity curation. Here we review the work of ClinGen in developing a curation infrastructure that supports the standardization, harmonization, and dissemination of Gene-Disease Validity data through the creation of frameworks and the utilization of common data standards. This infrastructure is based on several applications, including the ClinGen GeneTracker, Gene Curation Interface, Data Exchange, GeneGraph, and website.