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Empirical data on conditions that increase risk of coronavirus disease 2019 (COVID-19) progression are needed to identify high risk individuals. We performed a comprehensive quantitative assessment of pre-existing clinical phenotypes associated with COVID-19-related hospitalization. Phenome-wide association study (PheWAS) of SARS-CoV-2-positive patients from an integrated health system (Geisinger) with system-level outpatient/inpatient COVID-19 testing capacity and retrospective electronic health record (EHR) data to assess pre-COVID-19 pandemic clinical phenotypes associated with hospital admission (hospitalization). Of 12,971 individuals tested for SARS-CoV-2 with sufficient pre-COVID-19 pandemic EHR data at Geisinger, 1604 were SARS-CoV-2 positive and 354 required hospitalization. We identified 21 clinical phenotypes in 5 disease categories meeting phenome-wide significance (P<1.60x10-4), including: six kidney phenotypes, e.g. end stage renal disease or stage 5 CKD (OR = 11.07, p = 1.96x10-8), six cardiovascular phenotypes, e.g. congestive heart failure (OR = 3.8, p = 3.24x10-5), five respiratory phenotypes, e.g. chronic airway obstruction (OR = 2.54, p = 3.71x10-5), and three metabolic phenotypes, e.g. type 2 diabetes (OR = 1.80, p = 7.51x10-5). Additional analyses defining CKD based on estimated glomerular filtration rate, confirmed high risk of hospitalization associated with pre-existing stage 4 CKD (OR 2.90, 95% CI: 1.47, 5.74), stage 5 CKD/dialysis (OR 8.83, 95% CI: 2.76, 28.27), and kidney transplant (OR 14.98, 95% CI: 2.77, 80.8) but not stage 3 CKD (OR 1.03, 95% CI: 0.71, 1.48). This study provides quantitative estimates of the contribution of pre-existing clinical phenotypes to COVID-19 hospitalization and highlights kidney disorders as the strongest factors associated with hospitalization in an integrated US healthcare system.

To determine the contribution of defective splicing in Autism Spectrum Disorders (ASD), the most common neurodevelopmental disorder, a high throughput Ma ssively P arallel S plicing Assa y (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.

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.

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.

As whole exome sequencing (WES) becomes more widely used in the clinical realm, a wealth of unanalyzed information will be routinely generated. Using WES read depth data to predict copy number variation (CNV) could extend the diagnostic utility of this previously underutilized data by providing clinically important information such as previously unsuspected deletions or duplications. We evaluated ExomeDepth, a free R package, in addition to an aneuploidy prediction method, to detect CNVs in WES data. First, in a blinded pilot study, five out of five genomic alterations were correctly identified from clinical samples with previously defined chromosomal gains or losses, including submicroscopic deletions, duplications, and chromosomal trisomy. We then examined CNV calls among 53 patients participating in the NCGENES research study and undergoing WES, who had existing clinical chromosomal microarray (CMA) data that could be used for validation. For unique CNVs that overlap well with WES coverage regions, sensitivity was 89% for deletions and 65% for duplications. While specificity of the algorithm calls remains a concern, this is less of an issue at high threshold filtering levels. When applied to all 672 patients from the exome sequencing study, ExomeDepth identified eleven diagnostically relevant CNVs ranging in size from a two exon deletion to whole chromosome duplications, as well as numerous other CNVs with varying clinical significance. This opportunistic analysis of WES data yields an additional 1.6% of patients in this study with pathogenic or likely pathogenic CNVs that are clinically relevant to their phenotype as well as clinically relevant secondary findings. Finally, we demonstrate the potential value of copy number analysis in cases where a single heterozygous likely or known pathogenic single nucleotide alteration is identified in a gene associated with an autosomal recessive condition.

BackgroundScoliosis is the most common paediatric spinal deformity. More than 80% of scoliosis is idiopathic and appears during the adolescent growth spurt. Spinal fusion surgery is often required for patients with progressive adolescent idiopathic scoliosis (AIS), and the genetic risk factors for severe disease (defined here as curve >35 degrees) are largely unknown.MethodsTo explore the role of rare variants in severe AIS, exome sequence data from 1221 individuals with AIS were compared with both 1397 in-house European ancestry controls and 56885 gnomAD non-Finish European controls. Segregation analysis of variants in prioritised genes was performed in additional family members. A replication study was performed using the Geisinger MyCode cohort. FNDC1 function was investigated in fndc1 null mutant zebrafish.ResultsRare variants were enriched in 84 genes, including RAF1 (Noonan syndrome), FBN1 (Marfan syndrome) and FNDC1, in individuals with severe AIS. FNDC1, which had previously been associated with joint hypermobility, harboured missense variants in 4.0% of individuals with AIS compared with 2.3% of controls (p=0.00764, OR=1.78). FNDC1 variants segregated with AIS in five multiplex families with incomplete penetrance. In addition, FNDC1 rare variants were also associated with scoliosis in the Geisinger MyCode cohort (p=0.0002, OR=3.6). Disruption of the fndc1 locus in zebrafish resulted in increased bone mineral density.ConclusionWe broadened the phenotype associated with RAF1 and FBN1 variants and identified FNDC1 as a novel gene associated with severe AIS. Mechanistic alterations of bone mineral density or joint hypermobility may explain the association of FNDC1 missense variants with AIS.

Purpose Clinical 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. Methods We 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. Results Participants 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. Conclusion This 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.

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. 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. 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). 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.

Nonhomologous end joining (NHEJ) can effectively resolve chromosome breaks despite diverse end structures; however, it is unclear how the steps employed for resolution are determined. We sought to address this question by analysing cellular NHEJ of ends with systematically mispaired and damaged termini. We show NHEJ is uniquely proficient at bypassing subtle terminal mispairs and radiomimetic damage by direct ligation. Nevertheless, bypass ability varies widely, with increases in mispair severity gradually reducing bypass products from 85 to 6%. End-processing by nucleases and polymerases is increased to compensate, although paths with the fewest number of steps to generate a substrate suitable for ligation are favoured. Thus, both the frequency and nature of end processing are tailored to meet the needs of the ligation step. We propose a model where the ligase organizes all steps during NHEJ within the stable paired-end complex to limit end processing and associated errors. DNA double strand breaks result in various types of damaged termini and are resolved by non-homologous end joining, but how cells coordinate the different steps that occur during repair is not clear. Here the authors show that a DNA ligase coordinates processing prior to the ligation step to limit errors.

Abstract Background Lynch syndrome (LS) is a hereditary cancer predisposition that increases risk for colorectal, endometrial, and other cancers. Although population-based genomic screening programs identify individuals with LS, clinical presentation in such genomically ascertained populations is unknown. Methods MyCode is a healthcare system-based biobank that returns clinically actionable genomic screening results to participants, including pathogenic/likely pathogenic (P/LP) variants in LS genes (MLH1, MSH2, MSH6, PMS2). Adult cases (participants with an LS result) and controls (participants without a cancer predisposition variant matched to cases) reported their personal and family cancer histories. Rates of meeting National Comprehensive Cancer Network (NCCN) genetic testing guidelines and rates of colorectal and endometrial cancers in cases, controls, and their family members were calculated and compared. Results A total of 175 cases (10 MLH1, 7 MSH2, 83 MSH6, and 75 PMS2) and 169 controls were included. Of case pedigrees, 62/175 (35.4%) met NCCN criteria for LS evaluation. Case pedigrees were more likely (P < .001) to meet criteria than control pedigrees (4/169, 8.35%). Case probands had significantly higher rates of colorectal and endometrial cancer than controls (7.7% vs 2.4%, P = .03 colorectal; 11.5% vs 0%, P < .001 endometrial), as did their relatives (3.1% vs 0.9%, P < .001 colorectal; 2.2% vs 0.5%, P < .001 endometrial). Conclusions NCCN guidelines missed 65% of cases with P/LP LS variants despite families having higher colorectal and endometrial cancer rates compared with controls. Genomic screening can assist in identifying individuals at risk for LS-related cancers, providing an opportunity to tailor risk management for cancer prevention and early detection.