Considerations for reporting variants in novel candidate genes identified during clinical genomic testing.

Since the first novel gene discovery for a Mendelian condition was made via exome sequencing, the rapid increase in the number of genes known to underlie Mendelian conditions coupled with the adoption of exome (and more recently, genome) sequencing by diagnostic testing labs has changed the landscape of genomic testing for rare diseases. Specifically, many individuals suspected to have a Mendelian condition are now routinely offered clinical ES. This commonly results in a precise genetic diagnosis but frequently overlooks the identification of novel candidate genes. Such candidates are also less likely to be identified in the absence of large-scale gene discovery research programs. Accordingly, clinical laboratories have both the opportunity, and some might argue a responsibility, to contribute to novel gene discovery, which should, in turn, increase the diagnostic yield for many conditions. However, clinical diagnostic laboratories must necessarily balance priorities for throughput, turnaround time, cost efficiency, clinician preferences, and regulatory constraints and often do not have the infrastructure or resources to effectively participate in either clinical translational or basic genome science research efforts. For these and other reasons, many laboratories have historically refrained from broadly sharing potentially pathogenic variants in novel genes via networks such as Matchmaker Exchange, much less reporting such results to ordering providers. Efforts to report such results are further complicated by a lack of guidelines for clinical reporting and interpretation of variants in novel candidate genes. Nevertheless, there are myriad benefits for many stakeholders, including patients/families, clinicians, and researchers, if clinical laboratories systematically and routinely identify, share, and report novel candidate genes. To facilitate this change in practice, we developed criteria for triaging, sharing, and reporting novel candidate genes that are most likely to be promptly validated as underlying a Mendelian condition and translated to use in clinical settings.

Developmental Changes of the Coronary Sinus Between the First and Second Trimesters: A Pilot Study.

OBJECTIVE: To assess the coronary sinus (CS) presence, size, and CS to atrial ratio (CS/A) in the first trimester (FT) compared with the second trimester (ST). METHODS: In this IRB-approved retrospective study, fetuses with adequate FT cardiac sweeps and normal ST hearts were included. Maternal and fetal characteristics were obtained. CS and atrial diameters were measured by a single sonologist. The CS/A ratio was compared between FT and ST. Linear regression assessed the relationship between biparietal diameter (BPD) and CS and atrial diameters. Statistical significance was set at P < .05. RESULTS: Among 99 fetuses, the CS was seen in 42/53 (79.2%) in the FT and 14/32 (43.8%) in the ST. No significant associations were found between CS visualization and the factors analyzed. The CS/A ratio was significantly higher in the FT versus ST (0.43 vs 0.25; P < .0001). Combined FT and ST data revealed positive correlations between BPD and both CS (slope = 0.018, P < .0001) and atrial diameters (slope = 0.135, P < .0001), suggesting differential growth rates, with the atrium exhibiting a faster growth rate as BPD increased. CONCLUSIONS: The CS appears prominent in the FT compared with the ST, likely due to differential growth rates between the CS and atrium. Remnants of embryonic structures, differences in myocardial drainage, and hemodynamics may also be contributing factors. Larger prospective studies are needed to confirm these findings and assess the value of the FT CS/A ratio.

Ambulatory Care Coordination Data Gathering and Use.

Care coordination is a crucial component of healthcare systems. However, little is known about data needs and uses in ambulatory care coordination practice. Therefore, the purpose of this study was to identify information gathered and used to support care coordination in ambulatory settings. Survey respondents (33) provided their demographics and practice patterns, including use of electronic health records, as well as data gathered and used. Most of the respondents were nurses, and they described varying practice settings and patterns. Although most described at least partial use of electronic health records, two respondents described paper documentation systems. More than 25% of respondents gathered and used most of the 72 data elements, with collection and use often occurring in multiple locations and contexts. This early study demonstrates significant heterogeneity in ambulatory care coordination data usage. Additional research is necessary to identify common data elements to support knowledge development in the context of a learning health system.

Increased diagnostic yield from negative whole genome-slice panels using automated reanalysis.

We evaluated the diagnostic yield using genome-slice panel reanalysis in the clinical setting using an automated phenotype/gene ranking system. We analyzed whole genome sequencing (WGS) data produced from clinically ordered panels built as bioinformatic slices for 16 clinically diverse, undiagnosed cases referred to the Pediatric Mendelian Genomics Research Center, an NHGRI-funded GREGoR Consortium site. Genome-wide reanalysis was performed using Moon™, a machine-learning-based tool for variant prioritization. In five out of 16 cases, we discovered a potentially clinically significant variant. In four of these cases, the variant was found in a gene not included in the original panel due to phenotypic expansion of a disorder or incomplete initial phenotyping of the patient. In the fifth case, the gene containing the variant was included in the original panel, but being a complex structural rearrangement with intronic breakpoints outside the clinically analyzed regions, it was not initially identified. Automated genome-wide reanalysis of clinical WGS data generated during targeted panels testing yielded a 25% increase in diagnostic findings and a possibly clinically relevant finding in one additional case, underscoring the added value of analyses versus those routinely performed in the clinical setting.

The effect of abnormal placentation on maternal serum fetal fraction of cell-free DNA.

OBJECTIVES: Abnormal placentation may affect the maternal serum fraction of cell-free fetal DNA (fetal fraction) determined as part of non-invasive prenatal screening (NIPS). This study aimed to assess whether the fetal fraction can predict placenta accreta spectrum (PAS) with or without placenta previa (PP). We also investigated the impact of trophoblastic invasion depth on the fetal fraction. METHODS: This is a retrospective case-control study of pregnant women with and without abnormal placentation carrying a singleton and having undergone NIPS prior to 20 weeks of gestation. The eligible subjects were selected from a cohort managed at our institution for PAS suspected antenatally. We compared women with normal placentation (controls) to PAS, PP, or PAS + PP cases. Data were abstracted from electronic medical records, and PAS was confirmed histologically. RESULTS: Of the 146 patients in our cohort, 8 controls, 10 PP, 6 PAS, and 7 PAS + PP cases were eligible for the study. Among the groups, there were no significant differences in baseline demographic and clinical characteristics except the median number of prior uterine surgeries. Also, the groups did not significantly differ in their median fetal fraction. The fetal fraction did not discriminate any group when stratified according to the depth of placental invasion, i.e., no PAS, abnormally adherent, and abnormally invasive placenta. CONCLUSIONS: The maternal serum fraction of cell-free fetal DNA measured before 20 weeks of gestation is not predictive of PAS with or without concurrent PP or the depth of trophoblastic invasion.

Psychometric Evaluation of the Affect Regulation Checklist: Clinical and Community Samples, Parent-Reports and Youth Self-Reports.

The Affect Regulation Checklist (ARC) was designed to capture affect dysregulation, suppression, and reflection. Importantly, affect dysregulation has been established as a transdiagnostic mechanism underpinning many forms of psychopathology. We tested the ARC psychometric properties across clinical and community samples and through both parent-report and youth self-report information. Clinical sample: Participants included parents (n = 814; Mage = 43.86) and their child (n = 608; Mage = 13.98). Community sample: Participants included independent samples of parents (n = 578; Mage = 45.12) and youth (n = 809; Mage = 15.67). Exploratory structural equation modeling supported a three-factor structure across samples and informants. Dysregulation was positively associated with all forms of psychopathology. In general, suppression was positively associated with many forms of psychopathology, and reflection was negatively associated with externalizing problems and positively associated with internalizing problems.

Polyphosphate is a primordial chaperone.

Composed of up to 1,000 phospho-anhydride bond-linked phosphate monomers, inorganic polyphosphate (polyP) is one of the most ancient, conserved, and enigmatic molecules in biology. Here we demonstrate that polyP functions as a hitherto unrecognized chaperone. We show that polyP stabilizes proteins in vivo, diminishes the need for other chaperone systems to survive proteotoxic stress conditions, and protects a wide variety of proteins against stress-induced unfolding and aggregation. In vitro studies reveal that polyP has protein-like chaperone qualities, binds to unfolding proteins with high affinity in an ATP-independent manner, and supports their productive refolding once nonstress conditions are restored. Our results uncover a universally important function for polyP and suggest that these long chains of inorganic phosphate may have served as one of nature's first chaperones, a role that continues to the present day.

Uninsurance in North Carolina: Progress and Opportunities.

Virtually all of the Healthy North Carolina 2030 goals rely on access to affordable health care. This commentary provides an overview of how North Carolina's coverage gap impacts the state's health status and how expanding Medicaid could improve the odds of reaching Healthy NC 2030 goals.

Multicenter observational study evaluating the impact of platelet transport bags on product wastage.

BACKGROUND: Platelets are the most commonly discarded blood product in Canada, with the most common cause of in-date product loss being improper storage. Transport containers to maintain temperature and extend acceptable return time may represent a method to reduce wastage. The objective of this study was to evaluate the impact of a validated Platelet Transport Bag (PTB) on platelet wastage. STUDY DESIGN AND METHODS: Thirty-six hospitals with the highest platelet discards were invited to participate in a before-after observational study. Hospitals were instructed to utilize a validated 4-h PTB for clinical situations where immediate transfusion was not planned. Five hospitals audited in-date platelet discards from July 2018 to November 2019 to characterize wastage causes. In-date platelet discard data 12 months before and after the start date for each site were analyzed to determine changes in wastage. RESULTS: Of 36 hospital sites, 16 agreed to participate. Pre- and postdiscards were 277 and 301, respectively, for all sites combined. There were no significant before-after change in wastage rate (+0.05%, p = .51). Fifty discards were included in the detailed audit; the most common reasons were return to the blood bank after more than 60 min outside a PTB (n = 17, 34%) and return in a red cell cooler (n = 10, 20%). CONCLUSION: Implementation of PTB did not improve wastage. Common causes of in-date discards were return after 1 h outside of a PTB and placement in a red cell cooler in error. Further research is required to investigate potential strategies to mitigate in-date platelet wastage.

Clinical outcomes after early ambulatory multidrug therapy for high-risk SARS-CoV-2 (COVID-19) infection.

There is an emergency need for early ambulatory treatment of Coronavirus Disease 2019 (COVID-19) in acutely ill patients in an attempt to reduce disease progression and the risks of hospitalization and death. Such management should be applied in high-risk patients age > 50 years or with one or more medical problems including cardiovascular disease. We evaluated a total of 922 outpatients from March to September 2020. All patients underwent contemporary real-time polymerase chain reaction (PCR) assay tests from anterior nasal swab samples. Patients age 50.5 ± 13.7 years (range 12 to 89), 61.6% women, at moderate or high risk for COVID-19 received empiric management via telemedicine. At least two agents with antiviral activity against SARS-CoV-2 (zinc, hydroxychloroquine, ivermectin) and one antibiotic (azithromycin, doxycycline, ceftriaxone) were used along with inhaled budesonide and/or intramuscular dexamethasone consistent with the emergent science on early COVID-19 treatment. For patients with high severity of symptoms, urgent in-clinic administration of albuterol nebulizer, inhaled budesonide, and intravenous volume expansion with supplemental parenteral thiamine 500 mg, magnesium sulfate 4 grams, folic acid 1 gram, vitamin B12 1 mg. A total of 320/922 (34.7%) were treated resulting in 6/320 (1.9%) and 1/320 (0.3%) patients that were hospitalized and died, respectively. We conclude that early ambulatory (not hospitalized, treated at home), multidrug therapy is safe, feasible, and associated with low rates of hospitalization and death. Early treatment should be considered for high-risk patients as an emergency measure while we await randomized trials and guidelines for ambulatory management.

Colonial Neglect and the Right to Health in Puerto Rico After Hurricane Maria.

The humanitarian crisis revealed as a result of Hurricane Maria in Puerto Rico demonstrates a long history of US colonial neglect and human rights violations. This reality has made it especially difficult for the people of Puerto Rico to achieve their right to the highest attainable standard of health.The impacts are pervasive, resulting in disparities in Puerto Rican health, including water access and quality; wealth, including economic loss and disinvestment; and sustainability of the island's resources. As a result of failed governmental protection and support, public health issues related to access to care, a failing infrastructure, and discrimination all contributed to crisis on the island. A human rights framework is necessary to assess the ongoing human rights violations of the quality of life to support millions of American citizens on the island.This essay utilizes a rights-based approach to reveal historical disenfranchisement of Puerto Rico before the storms, identifies the specific human rights violations that resulted from the US government's lack of emergency preparedness and responsiveness, and demands rebuilding the island to reconcile all that has been lost.

Declines in SARS-CoV-2 Transmission, Hospitalizations, and Mortality After Implementation of Mitigation Measures- Delaware, March-June 2020.

Mitigation measures, including stay-at-home orders and public mask wearing, together with routine public health interventions such as case investigation with contact tracing and immediate self-quarantine after exposure, are recommended to prevent and control the transmission of SARS-CoV-2, the virus that causes coronavirus disease 2019 (COVID-19) (1-3). On March 11, the first COVID-19 case in Delaware was reported to the Delaware Division of Public Health (DPH). The state responded to ongoing community transmission with investigation of all identified cases (commencing March 11), issuance of statewide stay-at-home orders (March 24-June 1), a statewide public mask mandate (from April 28), and contact tracing (starting May 12). The relationship among implementation of mitigation strategies, case investigations, and contact tracing and COVID-19 incidence and associated hospitalization and mortality was examined during March-June 2020. Incidence declined by 82%, hospitalization by 88%, and mortality by 100% from late April to June 2020, as the mask mandate and contact tracing were added to case investigations and the stay-at-home order. Among 9,762 laboratory-confirmed COVID-19 cases reported during March 11-June 25, 2020, two thirds (6,527; 67%) of patients were interviewed, and 5,823 (60%) reported completing isolation. Among 2,834 contacts reported, 882 (31%) were interviewed and among these contacts, 721 (82%) reported completing quarantine. Implementation of mitigation measures, including mandated mask use coupled with public health interventions, was followed by reductions in COVID-19 incidence and associated hospitalizations and mortality. The combination of state-mandated community mitigation efforts and routine public health interventions can reduce the occurrence of new COVID-19 cases, hospitalizations, and deaths.

COVID-19 Among Workers in Meat and Poultry Processing Facilities - 19 States, April 2020.

Congregate work and residential locations are at increased risk for infectious disease transmission including respiratory illness outbreaks. SARS-CoV-2, the virus that causes coronavirus disease 2019 (COVID-19), is primarily spread person to person through respiratory droplets. Nationwide, the meat and poultry processing industry, an essential component of the U.S. food infrastructure, employs approximately 500,000 persons, many of whom work in proximity to other workers (1). Because of reports of initial cases of COVID-19, in some meat processing facilities, states were asked to provide aggregated data concerning the number of meat and poultry processing facilities affected by COVID-19 and the number of workers with COVID-19 in these facilities, including COVID-19-related deaths. Qualitative data gathered by CDC during on-site and remote assessments were analyzed and summarized. During April 9-27, aggregate data on COVID-19 cases among 115 meat or poultry processing facilities in 19 states were reported to CDC. Among these facilities, COVID-19 was diagnosed in 4,913 (approximately 3%) workers, and 20 COVID-19-related deaths were reported. Facility barriers to effective prevention and control of COVID-19 included difficulty distancing workers at least 6 feet (2 meters) from one another (2) and in implementing COVID-19-specific disinfection guidelines.* Among workers, socioeconomic challenges might contribute to working while feeling ill, particularly if there are management practices such as bonuses that incentivize attendance. Methods to decrease transmission within the facility include worker symptom screening programs, policies to discourage working while experiencing symptoms compatible with COVID-19, and social distancing by workers. Source control measures (e.g., the use of cloth face covers) as well as increased disinfection of high-touch surfaces are also important means of preventing SARS-CoV-2 exposure. Mitigation efforts to reduce transmission in the community should also be considered. Many of these measures might also reduce asymptomatic and presymptomatic transmission (3). Implementation of these public health strategies will help protect workers from COVID-19 in this industry and assist in preserving the critical meat and poultry production infrastructure (4).

A retrospective review of multiple findings in diagnostic exome sequencing: half are distinct and half are overlapping diagnoses.

PURPOSE: We evaluated clinical and genetic features enriched in patients with multiple Mendelian conditions to determine which patients are more likely to have multiple potentially relevant genetic findings (MPRF). METHODS: Results of the first 7698 patients who underwent exome sequencing at Ambry Genetics were reviewed. Clinical and genetic features were examined and degree of phenotypic overlap between the genetic diagnoses was evaluated. RESULTS: Among patients referred for exome sequencing, 2% had MPRF. MPRF were more common in patients from consanguineous families and patients with greater clinical complexity. The difference in average number of organ systems affected is small: 4.3 (multiple findings) vs. 3.9 (single finding) and may not be distinguished in clinic. CONCLUSION: Patients with multiple genetic diagnoses had a slightly higher number of organ systems affected than patients with single genetic diagnoses, largely because the comorbid conditions affected overlapping organ systems. Exome testing may be beneficial for all cases with multiple organ systems affected. The identification of multiple relevant genetic findings in 2% of exome patients highlights the utility of a comprehensive molecular workup and updated interpretation of existing genomic data; a single definitive molecular diagnosis from analysis of a limited number of genes may not be the end of a diagnostic odyssey.

TNFα and IL-1ß but not IL-18 Suppresses Hippocampal Long-Term Potentiation Directly at the Synapse.

CNS inflammatory responses are linked to cognitive impairment in humans. Research in animal models supports this connection by showing that inflammatory cytokines suppress long-term potentiation (LTP), the best-known cellular correlate of memory. Cytokine-induced modulation of LTP has been previously studied in vivo or in brain slices, two experimental approaches containing multiple cell populations responsive to cytokines. In their target cells, cytokines commonly increase the expression of multiple cytokines, thus increasing the complexity of brain cytokine networks even after single-cytokine challenges. Whether cytokines suppress LTP by direct effects on neurons or by indirect mechanisms is still an open question. Here, we evaluated the effect of a major set of inflammatory cytokines including tumor necrosis factor-α (TNFα), interleukin-1ß (IL-1ß) and interleukin-18 (IL-18) on chemically-induced LTP (cLTP) in isolated hippocampal synaptosomes of mice, using fluorescence analysis of single-synapse long-term potentiation (FASS-LTP). We found that TNFα and IL-1ß suppress synaptosomal cLTP. In contrast, cLTP was not affected by IL-18, at a concentration previously shown to block LTP in hippocampal slices. We also found that IL-18 does not impair cLTP or brain-derived neurotrophic factor (BDNF) signaling in primary hippocampal neuronal cultures. Thus, using both synaptosomes and neuron cultures, our data suggest that IL-18 impairs LTP by indirect mechanisms, which may depend on non-neuronal cells, such as glia. Notably, our results demonstrate that TNFα and IL-1ß directly suppress hippocampal plasticity via neuron-specific mechanisms. A better understanding of the brain's cytokine networks and their final molecular effectors is crucial to identify specific targets for intervention.

Topologically associated domains enriched for lineage-specific genes reveal expression-dependent nuclear topologies during myogenesis.

The linear distribution of genes across chromosomes and the spatial localization of genes within the nucleus are related to their transcriptional regulation. The mechanistic consequences of linear gene order, and how it may relate to the functional output of genome organization, remain to be fully resolved, however. Here we tested the relationship between linear and 3D organization of gene regulation during myogenesis. Our analysis has identified a subset of topologically associated domains (TADs) that are significantly enriched for muscle-specific genes. These lineage-enriched TADs demonstrate an expression-dependent pattern of nuclear organization that influences the positioning of adjacent nonenriched TADs. Therefore, lineage-enriched TADs inform cell-specific genome organization during myogenesis. The reduction of allelic spatial distance of one of these domains, which contains Myogenin, correlates with reduced transcriptional variability, identifying a potential role for lineage-specific nuclear topology. Using a fusion-based strategy to decouple mitosis and myotube formation, we demonstrate that the cell-specific topology of syncytial nuclei is dependent on cell division. We propose that the effects of linear and spatial organization of gene loci on gene regulation are linked through TAD architecture, and that mitosis is critical for establishing nuclear topologies during cellular differentiation.

Interstitial telomeric loops and implications of the interaction between TRF2 and lamin A/C.

The protein-DNA complexes that compose the end of mammalian chromosomes-telomeres-serve to stabilize linear genomic DNA and are involved in cellular and organismal aging. One mechanism that protects telomeres from premature degradation is the formation of structures called t-loops, in which the single-stranded 3' overhang present at the terminal end of telomeres loops back and invades medial double-stranded telomeric DNA. We identified looped structures formed between terminal chromosome ends and interstitial telomeric sequences (ITSs), which are found throughout the human genome, that we have termed interstitial telomeric loops (ITLs). While they form in a TRF2-dependent manner similar to t-loops, ITLs further require the physical interaction of TRF2 with the nuclear intermediate filament protein lamin A/C. Our findings suggest that interactions between telomeres and the nucleoskeleton broadly impact genomic integrity, including telomere stability, chromosome structure, and chromosome fragility. Here, we review the roles of TRF2 and lamin A/C in telomere biology and consider how their interaction may relate telomere homeostasis to cellular and organismal aging.

Correction: Candidate-gene criteria for clinical reporting: diagnostic exome sequencing identifies altered candidate genes among 8% of patients with undiagnosed diseases.

In the published version of this paper, some of the columns in the last three rows of Table 3 were mistakenly transposed. The corrected table appears below. In col. 6 of the row for DNMT3A, "S3" was published in the original article. However, in the revised table for the corrigendum, it has been corrected to "S1". In col. 6 of the row for SON, "S3" was published in the original article. However, in the revised table for the corrigendum, it has been corrected to "S2".

An additional case of Hennekam lymphangiectasia-lymphedema syndrome caused by loss-of-function mutation in ADAMTS3.

Hennekam lymphangiectasia-lymphedema syndrome (HKLLS) is a genetically heterogeneous lymphatic dysplasia with characteristic of facial dysmorphism, neurocognitive impairments, and abnormalities of the pericardium, intestinal tract, and extremities. It is an autosomal recessive condition caused by biallelic mutations in CCBE1 (collagen- and calcium-binding epidermal growth factor domain-containing protein 1) (HKLLS1; OMIM 235510) or FAT4 (HKLLS2; OMIM 616006). CCBE1 acts via ADAMTS3 (a disintegrin and metalloprotease with thrombospondin motifs-3 protease) to enhance vascular endothelial growth factor C signaling. There is report of one family supporting mutations in ADAMTS3 as causative for the phenotype labeled as HKLLS3. Here, we report an additional case of HKLLS that appears to be associated with homozygous nonsense mutation of ADAMTS3.

Synapse-specific IL-1 receptor subunit reconfiguration augments vulnerability to IL-1ß in the aged hippocampus.

In the aged brain, synaptic plasticity and memory show increased vulnerability to impairment by the inflammatory cytokine interleukin 1ß (IL-1ß). In this study, we evaluated the possibility that synapses may directly undergo maladaptive changes with age that augment sensitivity to IL-1ß impairment. In hippocampal neuronal cultures, IL-1ß increased the expression of the IL-1 receptor type 1 and the accessory coreceptor AcP (proinflammatory), but not of the AcPb (prosurvival) subunit, a reconfiguration that potentiates the responsiveness of neurons to IL-1ß. To evaluate whether synapses develop a similar heightened sensitivity to IL-1ß with age, we used an assay to track long-term potentiation (LTP) in synaptosomes. We found that IL-1ß impairs LTP directly at the synapse and that sensitivity to IL-1ß is augmented in aged hippocampal synapses. The increased synaptic sensitivity to IL-1ß was due to IL-1 receptor subunit reconfiguration, characterized by a shift in the AcP/AcPb ratio, paralleling our culture data. We suggest that the age-related increase in brain IL-1ß levels drives a shift in IL-1 receptor configuration, thus heightening the sensitivity to IL-1ß. Accordingly, selective blocking of AcP-dependent signaling with Toll-IL-1 receptor domain peptidomimetics prevented IL-1ß-mediated LTP suppression and blocked the memory impairment induced in aged mice by peripheral immune challenge (bacterial lipopolysaccharide). Overall, this study demonstrates that increased AcP signaling, specifically at the synapse, underlies the augmented vulnerability to cognitive impairment by IL-1ß that occurs with age.