Genetic disorders and congenital anomalies are the leading causes of infant mortality. Diagnosis of most genetic diseases in neonatal and paediatric intensive care units (NICU and PICU) is not ...sufficiently timely to guide acute clinical management. We used rapid whole-genome sequencing (STATseq) in a level 4 NICU and PICU to assess the rate and types of molecular diagnoses, and the prevalence, types, and effect of diagnoses that are likely to change medical management in critically ill infants.
We did a retrospective comparison of STATseq and standard genetic testing in a case series from the NICU and PICU of a large children's hospital between Nov 11, 2011, and Oct 1, 2014. The participants were families with an infant younger than 4 months with an acute illness of suspected genetic cause. The intervention was STATseq of trios (both parents and their affected infant). The main measures were the diagnostic rate, time to diagnosis, and rate of change in management after standard genetic testing and STATseq.
20 (57%) of 35 infants were diagnosed with a genetic disease by use of STATseq and three (9%) of 32 by use of standard genetic testing (p=0·0002). Median time to genome analysis was 5 days (range 3-153) and median time to STATseq report was 23 days (5-912). 13 (65%) of 20 STATseq diagnoses were associated with de-novo mutations. Acute clinical usefulness was noted in 13 (65%) of 20 infants with a STATseq diagnosis, four (20%) had diagnoses with strongly favourable effects on management, and six (30%) were started on palliative care. 120-day mortality was 57% (12 of 21) in infants with a genetic diagnosis.
In selected acutely ill infants, STATseq had a high rate of diagnosis of genetic disorders. Most diagnoses altered the management of infants in the NICU or PICU. The very high infant mortality rate indicates a substantial need for rapid genomic diagnoses to be allied with a novel framework for precision medicine for infants in NICU and PICU who are diagnosed with genetic diseases to improve outcomes.
Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Human Genome Research Institute, and National Center for Advancing Translational Sciences.
By informing timely targeted treatments, rapid whole-genome sequencing can improve the outcomes of seriously ill children with genetic diseases, particularly infants in neonatal and pediatric ...intensive care units (ICUs). The need for highly qualified professionals to decipher results, however, precludes widespread implementation. We describe a platform for population-scale, provisional diagnosis of genetic diseases with automated phenotyping and interpretation. Genome sequencing was expedited by bead-based genome library preparation directly from blood samples and sequencing of paired 100-nt reads in 15.5 hours. Clinical natural language processing (CNLP) automatically extracted children's deep phenomes from electronic health records with 80% precision and 93% recall. In 101 children with 105 genetic diseases, a mean of 4.3 CNLP-extracted phenotypic features matched the expected phenotypic features of those diseases, compared with a match of 0.9 phenotypic features used in manual interpretation. We automated provisional diagnosis by combining the ranking of the similarity of a patient's CNLP phenome with respect to the expected phenotypic features of all genetic diseases, together with the ranking of the pathogenicity of all of the patient's genomic variants. Automated, retrospective diagnoses concurred well with expert manual interpretation (97% recall and 99% precision in 95 children with 97 genetic diseases). Prospectively, our platform correctly diagnosed three of seven seriously ill ICU infants (100% precision and recall) with a mean time saving of 22:19 hours. In each case, the diagnosis affected treatment. Genome sequencing with automated phenotyping and interpretation in a median of 20:10 hours may increase adoption in ICUs and, thereby, timely implementation of precise treatments.
Neurodevelopmental disorders (NDDs) affect more than 3% of children and are attributable to single-gene mutations at more than 1000 loci. Traditional methods yield molecular diagnoses in less than ...one-half of children with NDD. Whole-genome sequencing (WGS) and whole-exome sequencing (WES) can enable diagnosis of NDD, but their clinical and cost-effectiveness are unknown. One hundred families with 119 children affected by NDD received diagnostic WGS and/or WES of parent-child trios, wherein the sequencing approach was guided by acuity of illness. Forty-five percent received molecular diagnoses. An accelerated sequencing modality, rapid WGS, yielded diagnoses in 73% of families with acutely ill children (11 of 15). Forty percent of families with children with nonacute NDD, followed in ambulatory care clinics (34 of 85), received diagnoses: 33 by WES and 1 by staged WES then WGS. The cost of prior negative tests in the nonacute patients was $19,100 per family, suggesting sequencing to be cost-effective at up to $7640 per family. A change in clinical care or impression of the pathophysiology was reported in 49% of newly diagnosed families. If WES or WGS had been performed at symptom onset, genomic diagnoses may have been made 77 months earlier than occurred in this study. It is suggested that initial diagnostic evaluation of children with NDD should include trio WGS or WES, with extension of accelerated sequencing modalities to high-acuity patients.
Thesis (M.A.)
Systemic sclerosis (SSc) is a clinically heterogenous chronic fibrotic disease which affects skin and internal organs. While the pathogenesis of SSc remains unknown, the hallmark of ...both localized and diffuse SSc in the skin is the replacement of normal dermal architecture with excessive deposition of collagen and other connective tissue macromolecules. Progressive replacement of tissue architecture by collagen-rich extracellular matrix (ECM) results in functional impairment of affected organs. Fibrotic damage to these affected organs accounts for much of the morbidity and mortality concomitant with SSc, particularly in the lungs.;
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Myofibroblasts are the primary ECM-secreting cells during wound healing and fibrosis. Myocardin-related transcription factor A (MRTF-A), is an important regulator of myofibroblast differentiation, depending on serum response factor (SRF) for smooth muscle actin (SMA) and Sp1 in the regulation of collagen gene expression. MRTF-A continually shuttles between the nucleus and cytoplasm in unstimulated cells. Signals of stress, mechanical force, and migration control MRTF-A movement by a mechanism in which Rho-activated cytoskeletal actin polymerization induces its relocation from the cytoplasm to the nucleus. The major hypothesis in this thesis is that MRTF-A is dysregulated (impairment of a physiological regulatory mechanisms) and/or activated in SSc patients in part through transforming growth factor beta (TGF-β). To test this hypothesis, immunohistochemistry using MRTF-A antibodies was performed on SSc patient skin lesions and healthy control skin. Staining was observed in the epidermis, epidermal structures, vasculature and dermis of SSc and healthy control skin. In the epidermal layer of patients with SSc, there was significantly more nuclear localization of MRTF-A then in normal controls. Prominent staining is also present in endothelial, perivascular and some perivascular inflammatory cells of SSc patients. Perivascular staining was not seen in healthy controls. Interestingly, there was some accumulation of nuclear MRTF-A in areas typical of myofibroblasts in SSc skin, but this staining is not as striking as vascular staining.;
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TGF-β activates MRTF-A in a cell-specific manner. As SSc typically begins within the skin, human dermal fibroblasts (HDF) were grown in culture. HDFs synthesize and secrete collagen to a greater extent when compared to human lung fibroblasts (IMR90 cells). Treatment with TGF-β enhances cytoplasmic localization of MRTF-A at 4-8 hours in HDFs and prolongs nuclear localization.;
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Transgenic mouse lung cells were isolated from an MRTF-A loss-of-function mouse carrying the 3.6 kb proximal promoter of the rat COL1A1 gene driving topaz green fluorescent protein (GFP) (pOB3.6COLGFPtpz). Since angiotensin II (ANG II) may enhance TGF-β response or collagen transcription directly, wild type (WT) and MRTF-A knockout (KO) cells were treated with ANG II and TGF-β. Quantification of collagen transcription by GFP fluorescence and protein synthesis by Western and secretion by Sircol analysis revealed collagen gene expression is consistently lower in KO fibroblasts compared to WT. Total percentage of fluorescent KO cells were consistently lower in comparison to WT cells as well. KO cells do not respond to TGF-β or ANG II treatment, whereas TGF-β increased collagen gene expression by WT cells, but not KO cells. Furthermore, treatment with ANG II did not up-regulate transcription in WT mouse lung fibroblasts. However, TGF-β receptor kinase 1 (TβR-1) inhibitor SB431542 attenuated collagen transcription in both WT and KO fibroblasts regardless of treatment suggesting that the receptor is active with or without MRTF-A possibly with an endogenous ligand produced by these cells. The activation of MRTF-A is an important protein regulating collagen synthesis and may potentially serve as a therapeutic target in future treatments of fibrotic disease such as SSc.
Hypertension affects over a billion adults worldwide and is a major risk factor for cardiovascular disease. Studies have reported that the microbiota and its metabolites regulate hypertension ...pathophysiology. Recently, tryptophan metabolites have been identified to contribute to and inhibit the progression of metabolic disorders and cardiovascular diseases, including hypertension. Indole propionic acid (IPA) is a tryptophan metabolite with reported protective effects in neurodegenerative and cardiovascular diseases; however, its involvement in renal immunomodulation and sodium handling in hypertension is unknown. In the current study, targeted metabolomic analysis revealed decreased serum and fecal IPA levels in mice with L-arginine methyl ester hydrochloride (L-NAME)/high salt diet-induced hypertension (LSHTN) compared to normotensive control mice. Additionally, kidneys from LSHTN mice had increased T helper 17 (Th17) cells and decreased T regulatory (Treg) cells. Dietary IPA supplementation in LSHTN mice for 3 weeks resulted in decreased systolic blood pressure, along with increased total 24 h and fractional sodium excretion. Kidney immunophenotyping demonstrated decreased Th17 cells and a trend toward increased Treg cells in IPA-supplemented LSHTN mice. In vitro, naïve T cells from control mice were skewed into Th17 or Treg cells. The presence of IPA decreased Th17 cells and increased Treg cells after 3 days. These results identify a direct role for IPA in attenuating renal Th17 cells and increasing Treg cells, leading to improved sodium handling and decreased blood pressure. IPA may be a potential metabolite-based therapeutic option for hypertension.
The renal lymphatic vasculature and the lymphatic endothelial cells that make up this network play important immunomodulatory roles during inflammation. How lymphatics respond to AKI may affect AKI ...outcomes. The authors used single-cell RNA sequencing to characterize mouse renal lymphatic endothelial cells in quiescent and cisplatin-injured kidneys. Lymphatic endothelial cell gene expression changes were confirmed in ischemia-reperfusion injury and in cultured lymphatic endothelial cells, validating renal lymphatic endothelial cells single-cell RNA sequencing data. This study is the first to describe renal lymphatic endothelial cell heterogeneity and uncovers molecular pathways demonstrating lymphatic endothelial cells regulate the local immune response to AKI. These findings provide insights into previously unidentified molecular pathways for lymphatic endothelial cells and roles that may serve as potential therapeutic targets in limiting the progression of AKI.
The inflammatory response to AKI likely dictates future kidney health. Lymphatic vessels are responsible for maintaining tissue homeostasis through transport and immunomodulatory roles. Owing to the relative sparsity of lymphatic endothelial cells in the kidney, past sequencing efforts have not characterized these cells and their response to AKI.
Here, we characterized murine renal lymphatic endothelial cell subpopulations by single-cell RNA sequencing and investigated their changes in cisplatin AKI 72 hours postinjury. Data were processed using the Seurat package. We validated our findings by quantitative PCR in lymphatic endothelial cells isolated from both cisplatin-injured and ischemia-reperfusion injury, by immunofluorescence, and confirmation in in vitro human lymphatic endothelial cells.
We have identified renal lymphatic endothelial cells and their lymphatic vascular roles that have yet to be characterized in previous studies. We report unique gene changes mapped across control and cisplatin-injured conditions. After AKI, renal lymphatic endothelial cells alter genes involved in endothelial cell apoptosis and vasculogenic processes as well as immunoregulatory signaling and metabolism. Differences between injury models were also identified with renal lymphatic endothelial cells further demonstrating changed gene expression between cisplatin and ischemia-reperfusion injury models, indicating the renal lymphatic endothelial cell response is both specific to where they lie in the lymphatic vasculature and the kidney injury type.
In this study, we uncover lymphatic vessel structural features of captured populations and injury-induced genetic changes. We further determine that lymphatic endothelial cell gene expression is altered between injury models. How lymphatic endothelial cells respond to AKI may therefore be key in regulating future kidney disease progression.
Introduction: Hypertension (HTN) is a major cardiovascular disease that can cause and be worsened by renal damage and inflammation. We previously reported that renal lymphatic endothelial cells ...(LECs) increase in response to HTN and that augmenting lymphangiogenesis in the kidneys reduces blood pressure and renal pro-inflammatory immune cells in mice with various forms of HTN. Our aim was to evaluate the specific changes that renal LECs undergo in HTN. Methods: We performed single-cell RNA sequencing. Using the angiotensin II-induced and salt-sensitive mouse models of HTN, we isolated renal CD31+ and podoplanin+ cells. Results: Sequencing of these cells revealed three distinct cell types with unique expression profiles, including LECs. The number and transcriptional diversity of LECs increased in samples from mice with HTN, as demonstrated by 597 differentially expressed genes (p<0.01), 274 significantly enriched pathways (p<0.01), and 331 regulons with specific enrichment in HTN LECs. These changes demonstrate a profound inflammatory response in renal LECs in HTN, leading to an increase in genes and pathways associated with inflammation-driven growth and immune checkpoint activity in LECs. Conclusion: These results reinforce and help to further explain the benefits of renal LECs and lymphangiogenesis in HTN.
Abstract only Hypertension affects roughly half of the U.S. population and is characterized by detrimental pro-inflammatory changes that alter renal function and the cardiovascular system over time. ...The kidney is a major regulator of blood pressure, however, the specific contributions of renal cell types, including lymphatic endothelial cells (LECs), to hypertension have not been thoroughly explored. Previous work from our lab has shown an increase in renal lymphangiogenesis in response to hypertension and that augmentation of renal lymphangiogenesis can reduce blood pressure. CCN1/Cyr61 is an extracellular matrix protein secreted by multiple cell types, particularly LECs and fibroblasts, which contributes to integrin signaling pathways. CCN1 is also known to be involved in lymphangiogenesis, and has previously been implicated in GWAS studies of hypertension. Given these connections, we performed single cell RNA sequencing using angiotensin II and salt sensitive mouse models of hypertension, which were analyzed separately as well as aggregated into one hypertensive group. Kidneys were turned to a single cell suspension and separated for CD31+/Podoplanin+ cells meant to better represent LECs, which are a small fraction of renal cells. Sequencing was performed and the gene reads were run through the Cell Ranger, Seurat, FGSEA, and Slingshot pipelines, then CCN1+ cells were isolated digitally and analyzed separately. In the AngII model, a total of 60 genes (28 up, 32 down) were differentially expressed (p<0.01) between the renal CCN1+ cells in the hypertensive mice compared to controls, while the salt sensitive model had a total of 331 differentially expressed genes (305 up, 26 down). When the hypertension groups were merged, there were a total of 600 differentially expressed genes (503 up, 97 down) with GSEA showing 48 pathways with enrichment scores >3 or <-3. Additionally, the cellular profile of CCN1+ cells shifted, branching out from a fibroblast-like population to other cell types such as endothelium and tubular epithelium. Overall, the expression of CCN1+ renal cells demonstrates notable changes in response to hypertension in murine models and encourages further study of the mechanisms behind CCN1’s role in the development and maintenance of hypertension.
Within the southeast Canada and northeast USA region, a peak in sulphate (SO₄ ²⁻) concentration has been reported for some streams following periods of substantial catchment drying during the summer ...months (ON, Canada; VT, NH and NY, USA). However, it is currently unclear if a SO₄ ²⁻ response to seasonal drying is widespread across the broader region, or to what extent the level of response varies among catchments. In our study, SO₄ ²⁻ response to seasonal drying was compared in 20 catchments from 11 locations across southeastern Canada (ON, QC and NS) and northeastern USA (NH, NY, VT, WV and ME). Using long-term monitoring data of stream discharge and chemistry, the number of days for each month of the dry season (# d) when discharge (Q) was below a threshold level (25th percentile; Q₂₅) was calculated for each catchment to give a measure of ‘seasonal dryness’ (# d Q < Q₂₅). A SO₄ ²⁻ response score (rs) was then calculated for each catchment based on linear regression analysis of # d Q < Q₂₅ versus either the annual SO₄ ²⁻ concentration, or the residual of annual SO₄ ²⁻ concentration as a function of time (year). The final rs values for each catchment provided an estimate of the proportion of variation in annual SO₄ ²⁻ concentration which could be explained by seasonal drying (possible rs range = 0–1). Of the 20 catchments, 13 exhibited some level of a SO₄ ²⁻ response to seasonal drying (rs = 0.04–0.72) with an additional two catchments exhibiting a SO₄ ²⁻ response for one or more seasons. SO₄ ²⁻ response scores were positively related to percent wetland area (w) (rs = 1.000 − 0.978e⁻⁰.⁰⁵⁴* ʷ , r ² = 0.44) and percent saturated area (sat) (rs = 0.481 − 0.488e⁻⁰.¹⁰¹* ˢᵃᵗ, r ² = 0.54) indicating that wetlands/saturated areas were an important driver of regional variation in the SO₄ ²⁻ response to seasonal drying. Our results suggest that any shift towards drier summers as a result of climate change could impact SO₄ ²⁻ dynamics in a large number of catchments throughout the region.
Hemicraniectomy and Durotomy Upon Deterioration From Infarction-Related Swelling Trial (HeADDFIRST) was a randomized pilot study to obtain information necessary to design a Phase III trial to ...evaluate the benefit of surgical decompression for brain swelling from large supratentorial cerebral hemispheric infarction.
All patients with stroke were screened for eligibility (age 18-75 years, National Institutes of Health Stroke Scale≥18 with Item 1a<2 responsive to minor stimulation, and CT demonstrating unilateral, complete middle cerebral artery territory infarction by specific imaging criteria). All enrolled patients were treated using a standardized medical treatment protocol. Those with both≥4 mm of pineal shift and deterioration in level of arousal or ≥7.5 mm of anteroseptal shift within 96 hours of stroke onset were randomized to continued medical treatment only or medical treatment plus surgery. Death at 21 days was the primary outcome measure.
Among 4909 screened patients, only 66 (1.3%) patients were eligible for HeADDFIRST. Forty patients were enrolled, and 26 patients developed the requisite brain swelling for randomization. All who failed to meet randomization criteria were alive at 21 days. Mortality at 21 and 180 days was 40% (4/10) in the medical treatment only and 21% (3/14) and 36% (5/14) in the medical treatment plus surgery arms, respectively.
HeADDFIRST randomization criteria effectively distinguished low from high risk of death from large supratentorial cerebral hemispheric infarction. Lower mortality in the medical treatment only group than in other published trials suggests a possible benefit to standardizing medical management. These results can inform the interpretation of recently completed European trials concerning patient selection and medical management.
This trial was not registered because enrollment began before July 1, 2005.