ABSTRACTIntroductionScreening and treatment of seizures (Sz) in neonates suffering from hypoxic-ischemic encephalopathy (HIE) is routine. Understanding if Sz worsen brain injury and outcomes would optimize treatment decisions, recognizing risks and benefits. We hypothesized that serum central nervous system (CNS)-specific biomarkers would discriminate neonates with Sz and relate to outcomes.MethodsThis retrospective cohort study was conducted between April 28, 2009 and November 15, 2019, including neonates diagnosed with seizures (Sz) and/or HIE treated with therapeutic hypothermia (TH), who had sufficient remnant serum for biomarker analysis. Neonates were grouped in i) only Sz without HIE (Sz-no HIE), ii) HIE with Sz (Sz-HIE) and iii) HIE without Sz (no Sz-HIE). Levels of glial fibrillary acidic protein (GFAP, astrocytic reactivity), Tau (neuronal injury) and neurofilament light chain (NF-L, axonal degeneration) were studied at admission, <72h and 72-144h of life against time to full oral feeds and brain injury in MRI.ResultsAfter exclusions 145 neonates were included (61% male; 33% Black). Admission GFAP levels were higher in Sz-HIE than in no Sz-HIE neonates. During the first 72h of life, levels of all 3 biomarkers were similar between Sz groups (Sz-no HIE & Sz-HIE), but higher than in no Sz-HIE. After 72h, NF-L and Tau remained higher in both Sz groups vs. no Sz-HIE. Stroke diagnosed in 31% (Sz-no HIE), 8% (Sz-HIE) and 11% (no Sz-HIE) of neonates had no effect in biomarkers levels. In adjusted regression models higher Tau and NF-L percentiles related to longer time to reach full oral feeding and higher odds of significant brain injury in MRI in both Sz groups.ConclusionsIn this study, Tau and NF-L levels are higher in those neonates developing Sz, irrespective of their HIE diagnosis. These results provide additional support for the notion that Sz may worsen brain injury and outcomes in neonates with HIE even with TH.

Aim: We sought to determine if higher plasma levels of brain injury biomarkers neurofilament light (NfL), phosphorylated tau 181 (pT181), tau, and ubiquitin C-terminal hydrolase L1 (UCHL1) were associated with unfavorable outcomes in children supported on extracorporeal membrane oxygenation (ECMO) with and without preceding cardiac arrest. Methods: We conducted a secondary analysis of a two-center prospective observational study of ECMO patients 0-<18 years. Plasma concentrations of NfL, pT181, tau, and UCHL1 were measured on ECMO days 1, 2 and 3. Unfavorable outcome was defined as in-hospital mortality or discharge Pediatric Cerebral Performance Category (PCPC) >2 with decline from baseline PCPC among survivors. Results: Among 88 children on ECMO, mean tau levels were significantly higher on each of the first three ECMO days in children who underwent extracorporeal cardiopulmonary resuscitation (ECPR) compared to those with non-ECPR cardiac arrest or with no cardiac arrest preceding ECMO. Higher ECMO day 1 tau levels were significantly associated with increased hazard of unfavorable outcome in unadjusted (HR, 1.35, 95% CI 1.09-1.66) and adjusted (HR, 1.42; 95% CI 1.13-1.79) models. Higher levels of NfL or pT181 were not associated with increased hazard for unfavorable outcome in multivariable models. UCHL1 values were outside of detectable limits and thus deferred from analysis. Conclusions: Levels of tau were significantly associated with increased hazard of death or unfavorable neurologic outcome in unadjusted and adjusted models. Biomarkers of brain injury, particularly tau, may aid in detection of neurologic injury and neuroprognostication in patients on ECMO with and without preceding cardiac arrest.

In this two-center prospective cohort study of children on ECMO, we assessed a panel of plasma brain injury biomarkers using exploratory factor analysis (EFA) to evaluate their interplay and association with outcomes. Biomarker concentrations were measured daily for the first 3 days of ECMO support in 95 participants. Unfavorable composite outcome was defined as in-hospital mortality or discharge Pediatric Cerebral Performance Category > 2 with decline ≥ 1 point from baseline. EFA grouped 11 biomarkers into three factors. Factor 1 comprised markers of cellular brain injury (NSE, BDNF, GFAP, S100β, MCP1, VILIP-1, neurogranin); Factor 2 comprised markers related to vascular processes (vWF, PDGFRβ, NPTX1); and Factor 3 comprised the BDNF/MMP-9 cellular pathway. Multivariable logistic models demonstrated that higher Factor 1 and 2 scores were associated with higher odds of unfavorable outcome (adjusted OR 2.88 [1.61, 5.66] and 1.89 [1.12, 3.43], respectively). Conversely, higher Factor 3 scores were associated with lower odds of unfavorable outcome (adjusted OR 0.54 [0.31, 0.88]), which is biologically plausible given the role of BDNF in neuroplasticity. Application of EFA on plasma brain injury biomarkers in children on ECMO yielded grouping of biomarkers into three factors that were significantly associated with unfavorable outcome, suggesting future potential as prognostic instruments.

Traumatic brain injury (TBI) is characterized by heterogeneity in terms of injury severity, mechanism, outcome, and pathophysiology. A single biomarker alone is unlikely to capture the heterogeneity of even one injury subtype, necessitating the use of panels of biomarkers. Herein, we focus on traumatic cerebrovascular injury and investigate associations of a panel of 16 vascular injury-related biomarkers with indices of TBI severity and outcomes using data from 159 participants in the Transforming Research and Clinical Knowledge in TBI (TRACK-TBI) Pilot Study. Associations of individual biomarkers and clusters of biomarkers identified using non-linear principal components analysis with TBI severity and outcomes were assessed using logistic regression models and Spearman's correlations. As individual biomarkers, higher levels of thrombomodulin, angiopoietin (Ang)-2, von Willebrand factor, and P-selectin were associated with more severe injury; higher levels of Ang-1, Tie2, vascular endothelial growth factor (VEGF)-C, and basic fibroblast growth factor (bFGF) were associated with less severe injury (all p < 0.05 in age-adjusted models). After false discovery rate correction for multiple comparisons, higher levels of Ang-2 remained associated with more severe injury and higher levels of Ang-1, Tie2, and bFGF remained associated with less severe injury at a p < 0.05 level. In principal components analysis, principal component (PC)1, comprised of Ang1, bFGF, P-selectin, VEGF-C, VEGF-A, and Tie2, was associated with less severe injury (age-adjusted odds ratio [OR]: 0.63, 95% confidence interval [CI]: 0.44-0.88 for head computer tomography [CT] positive vs. negative) and PC2 (Ang-2, E-selectin, Flt-1, placental growth factor, thrombomodulin, and vascular cell adhesion protein 1) was associated with greater injury severity (age-adjusted OR: 2.29, 95% CI: 1.49-3.69 for Glasgow Coma Scale [GCS] 3-12 vs. 13-15 and age-adjusted OR 1.59, 95% CI: 1.11-2.32 for head CT positive vs. negative). Neither individual biomarkers nor PCs were associated with outcomes in adjusted models (all p > 0.05). In conclusion, in this trauma-center based population of acute TBI patients, biomarkers of microvascular injury were associated with TBI severity.

Each year in the United States, ?2.7 million persons seek medical attention for traumatic brain injury (TBI), of which ?85% are characterized as being mild brain injuries. Many different cell types in the brain are affected in these heterogeneous injuries, including neurons, glia, and the brain vasculature. Efforts to identify biomarkers that reflect the injury of these different cell types have been a focus of ongoing investigation. We hypothesized that von Willebrand factor (vWF) is a sensitive biomarker for acute traumatic vascular injury and correlates with symptom severity post-TBI. To address this, blood was collected from professional boxing athletes (n?=?17) before and within 30?min after competition. Plasma levels of vWF and neuron-specific enolase were measured using the Meso Scale Discovery, LLC. (MSD) electrochemiluminescence array-based multi-plex format (MSD, Gaithersburg, MD). Additional symptom and outcome data from boxers and patients, such as the Rivermead symptom scores (Rivermead Post Concussion Symptoms Questionnaire [RPQ-3]), were collected. We found that, subsequent to boxing bouts, there was a 1.8-fold increase in vWF levels within 30?min of injury (p?<?0.0009). Moreover, fold-change in vWF correlates moderately (r?=?0.51; p?=?0.03) with the number of head blows. We also found a positive correlation (r?=?0.69; p?=?0.002) between fold-change in vWF and self-reported post-concussive symptoms, measured by the RPQ-3. The receiver operating curve analysis of vWF plasma levels and RPQ-3 scoring yielded a sensitivity of 94.12% and a specificity of 76.5% with an area under the curve of 83% for boxers after a fight compared to the pre-bout baseline. This study suggests that vWF is a potential blood biomarker measurable in the hyperacute period after blunt mild TBI. This biomarker may prove to be useful in diagnosing and monitoring traumatic vascular injury.

Abstract Background Gyrate atrophy of the choroid and retina is a rare autosomal recessive metabolic disorder caused by biallelic variants in the OAT gene, encoding the enzyme ornithine δ-aminotransferase. Impaired enzymatic activity leads to systemic hyperornithinaemia, which in turn underlies progressive chorioretinal degeneration. In this study, we describe the clinical and molecular findings in a cohort of individuals with gyrate atrophy. Methods Study participants were recruited through a tertiary UK clinical ophthalmic genetic service. All cases had a biochemical and molecular diagnosis of gyrate atrophy. Retrospective phenotypic and biochemical data were collected using electronic healthcare records. Results 18 affected individuals from 12 families (8 male, 10 female) met the study inclusion criteria. The median age at diagnosis was 8 years (range 10 months – 33 years) and all cases had hyperornithinaemia (median: 800 micromoles/L; range: 458–1244 micromoles/L). Common features at presentation included high myopia (10/18) and nyctalopia (5/18). Ophthalmic findings were present in all study participants who were above the age of 6 years. One third of patients had co-existing macular oedema and two thirds developed pre-senile cataracts. Compliance with dietary modifications was suboptimal in most cases. A subset of participants had extraocular features including a trend towards reduced fat-free mass and developmental delay. Conclusions Our findings highlight the importance of multidisciplinary care in families with gyrate atrophy. Secondary ophthalmic complications such as macular oedema and cataract formation are common. Management of affected individuals remains challenging due to the highly restrictive nature of the recommended diet and the limited evidence-base for current strategies.

Abstract Background There are numerous benefits to performing salivary serology measurements for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative pathogen for coronavirus disease 2019 (COVID-19). Here, we used a sensitive multiplex serology assay to quantitate salivary IgG against 4 SARS-CoV-2 antigens: nucleocapsid, receptor-binding domain, spike, and N-terminal domain. Methods We used single samples from 90 individuals with COVID-19 diagnosis collected at 0 to 42 days postsymptom onset (PSO) and from 15 uninfected control subjects. The infected individuals were segmented in 4 groups (0–7 days, 8–14 days, 15–21 days, and >21 days) based on days PSO, and values were compared to controls. Results Compared to controls, infected individuals showed higher levels of antibodies against all antigens starting from 8 days PSO. When applying cut-offs with at least 93.3% specificity at every time interval segment, nucleocapsid protein serology had the best sensitivity at 0 to 7 days PSO (60% sensitivity [35.75% to 80.18%], ROC area under the curve [AUC] = 0.73, P = 0.034). Receptor-binding domain serology had the best sensitivity at 8 to 14 days PSO (83.33% sensitivity [66.44%–92.66%], ROC AUC = 0.90, P < 0.0001), and all assays except for N-terminal domain had 92% sensitivity (75.03%–98.58%) at >14 days PSO. Conclusions This study shows that our multiplexed immunoassay can distinguish infected from uninfected individuals and reliably (93.3% specificity) detect seroconversion (in 60% of infected individuals) as early as the first week PSO, using easy-to-collect saliva samples.

Convenient and widespread serology testing may alter the trajectory of the COVID-19 pandemic. This study seeks to leverage high-throughput, multiplexed serologic assays, which have been adopted as benchmarks for vaccine efficacy, to support large-scale surveys of SARS-CoV-2 immunity using finger-stick blood and/or saliva. Specifically, we optimized MSD’s serology assays, which were analytically validated for serum, to test self-collected finger-stick blood and saliva samples to identify prior infection. We show that these assays can be used with FDA-registered specimen collection devices to obtain quantitative measurements for self-collected samples. First, we show that salivary antibodies are stable without refrigeration or preservatives for at least 5 days. We selected classification thresholds for antibodies against SARS-CoV-2 N, RBD and Spike in finger-stick blood and saliva that provided 98% specificity in a set of individuals without known COVID-19 exposure. Using matched samples, we show that testing of saliva and finger-stick blood equivalently identified individuals with humoral responses to CoV-2 antigens. Moreover, we piloted a simple saliva collection kit that can be used to safely send samples through the mail using written instructions only. This work establishes key parameters to robustly assay self-collected finger-stick blood and saliva using quantitative immunoassays that could support large-scale serology testing.

Serology provides tools for epidemiologic studies, and may have a role in vaccine prioritization and selection. Automated serologic testing of saliva, especially specimens that are self-collected at home and sent to a laboratory via the mail without refrigeration, could be a highly-scalable strategy for population-wide testing. In this prospective study, non-vaccinated patients were recruited after PCR testing to self-collect saliva and return their specimens via mail. Longitudinal specimens were analyzed in order to monitor seroconversion in the weeks after a diagnostic PCR test for SARS-CoV-2. Diverse users self-collected saliva and returned specimens via mail in compliance with shipping regulations. At our pre-established threshold (0.963 AU/mL), salivary IgG reactivity to full-length spike protein achieved 95.8% sensitivity and 92.4% specificity at 2–4 weeks after diagnostic testing, which is comparable to the typical sensitivity and specificity achieved for serum testing. Reactivity to N antigen also was detected with 92.6% sensitivity and 90.7% specificity at 4–8 weeks after diagnostic testing. Moreover, serologic testing for endemic coronaviruses performed in multiplex with SARS-CoV-2 antigens has the potential to identify samples that may require retesting due to effects of pre-analytical factors. The easy-to-use saliva collection kit, coupled with thresholds for positivity and methods of flagging samples for retest, provides a framework for large-scale serosurveillance of SARS-CoV-2.

ABSTRACTPurposeCongenital stationary night blindness (CSNB) is a heterogeneous group of Mendelian retinal disorders that present in childhood. Biallelic variants altering the protein‐coding region of the TRPM1 gene are one of the commonest causes of CSNB. Here, we report the clinical and genetic findings in 10 unrelated individuals with TRPM1‐retinopathy.MethodsStudy subjects were recruited through a tertiary clinical ophthalmic genetic service at Manchester, UK. All participants underwent visual electrodiagnostic testing and panel‐based genetic analysis.ResultsStudy subjects had a median age of 8 years (range: 3–20 years). All probands were myopic and had electroretinographic findings in keeping with complete CSNB. Notably, three probands reported no night vision problems. Fourteen different disease‐associated TRPM1 variants were detected. One individual was homozygous for the NM_001252024.2 (TRPM1):c.965 + 29G>A variant and a mini‐gene assay highlighted that this change results in mis‐splicing and premature protein termination. Additionally, two unrelated probands who had CSNB and mild neurodevelopmental abnormalities were found to carry a 15q13.3 microdeletion. This copy number variant encompasses seven genes, including TRPM1, and was encountered in the heterozygous state and in trans with a missense TRPM1 variant in each case.ConclusionOur findings highlight the importance of comprehensive genomic analysis, beyond the exons and protein‐coding regions of genes, for individuals with CSNB. When this characteristic retinal phenotype is accompanied by extraocular findings (including learning and/or behavioural difficulties), a 15q13.3 microdeletion should be suspected. Focused analysis (e.g. microarray testing) is recommended to look for large‐scale deletions encompassing TRPM1 in patients with CSNB and neurodevelopmental abnormalities.

Abstract Objectives Infection by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), the causative pathogen of coronavirus disease 2019 (COVID-19) presents occasionally with an aberrant autoinflammatory response, including the presence of elevated circulating autoantibodies in some individuals. Whether the development of autoantibodies against self-antigens affects COVID-19 outcomes remains unclear. To better understand the prognostic role of autoantibodies in COVID-19, we quantified autoantibodies against 23 markers that are used for diagnosis of autoimmune disease. To this end, we used serum samples from patients with severe [intensive care unit (ICU)] and moderate (ward) COVID-19, across two to six consecutive time points, and compared autoantibody levels to uninfected healthy and ICU controls. Methods Acute and post-acute serum (from 1 to 26 ICU days) was collected from 18 ICU COVID-19-positive patients at three to six time points; 18 ICU COVID-19-negative patients (sampled on ICU day 1 and 3); 21 ward COVID-19-positive patients (sampled on hospital day 1 and 3); and from 59 healthy uninfected controls deriving from two cohorts. Levels of IgG autoantibodies against 23 autoantigens, commonly used for autoimmune disease diagnosis, were measured in serum samples using MSD® U-PLEX electrochemiluminescence technology (MSD division Meso Scale Discovery®), and results were compared between groups. Results There were no significant elevations of autoantibodies for any of the markers tested in patients with severe COVID-19. Conclusions Sample collections at longer time points should be considered in future studies, for assessing the possible development of autoantibody responses following infection with SARS-CoV-2.

Both an increased frequency of chromosome missegregation (chromosomal instability) and the presence of an abnormal complement of chromosomes (aneuploidy) are hallmarks of cancer. Paradoxically, both chromosomal instability and aneuploidy are also associated with substantial decreases in cellular fitness. To better understand how cells are able to adapt to high levels of chromosomal instability, we previously examined yeast cells that were deleted of the gene BIR1 , a member of the chromosomal passenger complex (CPC). The CPC is an essential regulator of chromosome segregation fidelity. We found bir1Δ cells quickly adapted by acquiring specific combinations of beneficial aneuploidies. However, targeted mutations of specific genes were notably absent in the short term. In this study, we monitored these yeast strains for longer periods of time to determine how cells adapt to high levels of both CIN and aneuploidy in the long term. We identify suppressor mutations that mitigate the chromosome missegregation phenotype. The mutated proteins fall into four main categories: outer kinetochore subunits, members of the SCF Cdc4 complex, the mitotic kinase Mps1, and a member of the CPC itself. These mutants function in two distinct ways, as mutations in the outer kinetochore suppress Bir1 deletion indirectly by destabilizing connections between the chromosomes and the mitotic spindle, whereas the other three categories of mutations affect the CPC directly. As a consequence of the accumulation of suppressor point mutations, overall levels of aneuploidy decreased. These experiments demonstrate a timeline of adaptation to high rates of CIN wherein cells first acquire specific aneuploidies that suppress the CIN phenotype, next develop point mutations that more specifically target the source of CIN, and finally reduce the level of aneuploidy to relieve the fitness burden placed by aneuploidy on the cell.

Abstract Objectives Widespread SARS-CoV-2 testing is invaluable for identifying asymptomatic/pre-symptomatic individuals. There remains a technological gap for highly reliable, easy, and quick SARS-CoV-2 diagnostic tests suitable for frequent mass testing. Compared to nasopharyngeal (NP) swab-based tests, saliva-based methods are attractive due to easier and safer sampling. Current saliva-based SARS-CoV-2 rapid antigen tests (RATs) are hindered by limited analytical sensitivity. Here, we report one of the first ultrasensitive, saliva-based SARS-CoV-2 antigen assays with an analytical sensitivity of <0.32 pg/mL, corresponding to four viral RNA copies/µL, which is comparable to that of PCR-based tests. Methods Using the novel electrochemiluminescence (ECL)-based immunoassay, we measured the SARS-CoV-2 nucleocapsid (N) antigen concentration in 105 salivas, obtained from non-COVID-19 and COVID-19 patients. We then verified the results with a second, independent cohort of 689 patients (3.8% SARS-CoV-2 positivity rate). We also compared our method with a widely used point-of-care rapid test. Results In the first cohort, at 100% specificity, the sensitivity was 92%. Our assay correctly identified samples with viral loads up to 35 CT cycles by saliva-based PCR. Paired NP swab-based PCR results were obtained for 86 cases. Our assay showed high concordance with saliva-based and NP swab-based PCR in samples with negative (<0.32 pg/mL) and strongly positive (>2 pg/mL) N antigen concentrations. In the second cohort, at 100% specificity, sensitivity was also 92%. Our assay is about 700-fold more sensitive than the Abbott Panbio Rapid Test. Conclusions We demonstrated the ultrasensitivity and specificity assay and its concordance with PCR. This novel assay is especially valuable when compliance to frequent swabbing may be problematic.

To demonstrate the utility of vWF as a biomarker of TBI

The Amelanchier-Malacomeles-Peraphyllum (AMP) clade consists of ca. 26 species distributed in North and Central America, Europe, Asia, and northwestern Africa. While molecular and morphological data strongly support this clade, relationships of its genera are uncertain. Support for the monophyly of Amelanchier and for the phylogenetic positions of Malacomeles and Peraphyllum has varied between studies. Our goals were to reconstruct a robust phylogeny of the AMP clade in the framework of Maleae and clarify the phylogenetic placements of Malacomeles and Peraphyllum. This study employs sequences of the whole plastome and nuclear ribosomal DNA (nrDNA) repeats assembled using genome skimming with 131 samples representing 115 species in 31 genera of Rosaceae, especially Maleae. Maximum likelihood (ML) and Bayesian analysis (BI) of whole plastome datasets strongly supported Amelanchier as not monophyletic, with Peraphyllum sister to eastern North American Amelanchier and Malacomeles sister to the western North American-Eurasian Amelanchier. In contrast, nrDNA recovered the monophyly of Amelanchier, with Peraphyllum sister to Amelanchier and Malacomeles sister to the Amelanchier-Peraphyllum clade. The strong topological conflicts between plastome and nrDNA phylogenies of Peraphyllum and of Malacomeles are best explained by ancient chloroplast capture that occurred in SW North America.

Small whorled pogonia, Isotria medeoloides, is a rare terrestrial orchid of temperate forests in eastern North America. It is federally threatened, and 1992 recovery criteria in place for this species need to be evaluated in light of current data. Study of I. medeoloides demography is complicated by the potential for belowground dormancy lasting one to three or more years. From 1986 to 2000, we observed 404 individuals in monitoring plots at four sites in Maine, USA, with up to 241 additional plants followed less intensively outside those plots. We used a life cycle diagram to summarize stage distribution data regarding emergence, recruitment, fruiting, and dormancy. A frequency analysis of the stage dynamics of individuals revealed no consistent patterns. Within plots, 17.9% of individuals bore fruits. Of plants that flowered, mean fruit set overall was 55%. Plant abundance within plots declined over time, possibly due to increasing shade and weather-related effects. In 1993, we removed 33% of the tree basal area over subsections of the populations at two sites while also leaving a portion of both sites untreated. We used mixed-effect regressions to test for increased recruitment and rate of capsule production as a result of the canopy treatment. As of 1993, for 187 plants in the treated zones mean capsule production per flowering plant did not decline, while it did decline in the untreated zones. In a subset of 100 paired plants matched by year and site, 95.8% of those impacted by severe herbivory were dead within 4 years, compared to 58% of counterparts with no herbivory. We used these data to evaluate the recovery criteria and recommend a change from a focus on percent flowering to a minimum of at least two capsules produced per year on average over a ten-year period.

We report on an experiment that investigated how people naturally communicate with computational devices using speech and gaze. Our approach follows from the idea that human-human conversation involves the establishment of common ground, the use of gaze direction to indicate attention and turn-taking, and awareness of other's knowledge and abilities. Our goal is to determine whether it is easier to communicate with several devices, each with its own specialized functions and abilities, or with a single system that can control several devices. If conversations with devices resemble conversations with people, we would expect interaction with several devices to require extra effort—both in building common ground and in specifying turn-taking. To test this, we observed participants in an office mock-up where information was accessed on displays through speech input only. Between groups, we manipulated what participants were told: in one case, that they were speaking to a single controlling system, and in the other, that they were speaking to a set of individually controlled devices. Based on language use and gaze patterns, our results suggest that the office environment was more efficient and easier to use when participants believed they were talking to a single system than when they believed they were talking to a several devices.

Background Diagnostic use of gene panel nextgeneration sequencing (NGS) techniques is commonplace for individuals with inherited retinal dystrophies (IRDs), a highly genetically heterogeneous group of disorders. However, these techniques have often failed to capture the complete spectrum of genomic variation causing IRD, including CNVs. This study assessed the applicability of introducing CNV surveillance into first-tier diagnostic gene panel NGS services for IRD. Methods Three read-depth algorithms were applied to gene panel NGS data sets for 550 referred individuals, and informatics strategies used for quality assurance and CNV filtering. CNV events were confirmed and reported to referring clinicians through an accredited diagnostic laboratory. Results We confirmed the presence of 33 deletions and 11 duplications, determining these findings to contribute to the confirmed or provisional molecular diagnosis of IRD for 25 individuals. We show that at least 7% of individuals referred for diagnostic testing for IRD have a CNV within genes relevant to their clinical diagnosis, and determined a positive predictive value of 79% for the employed CNV filtering techniques. Conclusion Incorporation of CNV analysis increases diagnostic yield of gene panel NGS diagnostic tests for IRD, increases clarity in diagnostic reporting and expands the spectrum of known disease-causing mutations.

Conifers are an important living seed plant lineage with an extensive fossil record spanning more than 300 million years. The group therefore provides an excellent opportunity to explore congruence and conflict between dated molecular phylogenies and the fossil record.We surveyed the current state of knowledge in conifer phylogenetics to present a new time-calibrated molecular tree that samples ~90% of extant species diversity. We compared phylogenetic relationships and estimated divergence ages in this new phylogeny with the paleobotanical record, focusing on clades that are species-rich and well known from fossils.Molecular topologies and estimated divergence ages largely agree with the fossil record in Cupressaceae, conflict with it in Araucariaceae, and are ambiguous in Pinaceae and Podocarpaceae. Molecular phylogenies provide insights into some fundamental questions in conifer evolution, such as the origin of their seed cones, but using them to reconstruct the evolutionary history of specific traits can be challenging.Molecular phylogenies are useful for answering deep questions in conifer evolution if they depend on understanding relationships among extant lineages. Because of extinction, however, molecular datasets poorly sample diversity from periods much earlier than the Late Cretaceous. This fundamentally limits their utility for understanding deep patterns of character evolution and resolving the overall pattern of conifer phylogeny.

Background— The 22q11.2 deletion syndrome (22q11.2DS; DiGeorge syndrome/velocardiofacial syndrome) occurs in 1 of 4000 live births, and 60% to 70% of affected individuals have congenital heart disease, ranging from mild to severe. In our cohort of 1472 subjects with 22q11.2DS, a total of 62% (n=906) have congenital heart disease and 36% (n=326) of these have tetralogy of Fallot (TOF), comprising the largest subset of severe congenital heart disease in the cohort. Methods and Results— To identify common genetic variants associated with TOF in individuals with 22q11.2DS, we performed a genome-wide association study using Affymetrix 6.0 array and imputed genotype data. In our cohort, TOF was significantly associated with a genotyped single-nucleotide polymorphism (rs12519770, P =2.98×10 − 8 ) in an intron of the adhesion GPR98 (G-protein–coupled receptor V1) gene on chromosome 5q14.3. There was also suggestive evidence of association between TOF and several additional single-nucleotide polymorphisms in this region. Some genome-wide significant loci in introns or noncoding regions could affect regulation of genes nearby or at a distance. On the basis of this possibility, we examined existing Hi-C chromatin conformation data to identify genes that might be under shared transcriptional regulation within the region on 5q14.3. There are 6 genes in a topologically associated domain of chromatin with GPR98 , including MEF2C (Myocyte-specific enhancer factor 2C). MEF2C is the only gene that is known to affect heart development in mammals and might be of interest with respect to 22q11.2DS. Conclusions— In conclusion, common variants may contribute to TOF in 22q11.2DS and may function in cardiac outflow tract development.