Dual-specificity phosphatase (DUSP) genetic variants predict pulmonary hypertension in patients with bronchopulmonary dysplasia.

BACKGROUND: Pulmonary hypertension (PH) in patients with bronchopulmonary dysplasia (BPD) results from vasoconstriction and/or vascular remodeling, which can be regulated by mitogen-activated protein kinases (MAPKs). MAPKs are deactivated by dual-specificity phosphatases (DUSPs). We hypothesized that single-nucleotide polymorphisms (SNPs) in DUSP genes could be used to predict PH in BPD. METHODS: Preterm infants diagnosed with BPD (n = 188) were studied. PH was defined by echocardiographic criteria. Genomic DNA isolated from patient blood samples was analyzed for 31 SNPs in DUSP genes. Clinical characteristics and minor allele frequencies were compared between BPD-PH (cases) and BPD-without PH (control) groups. Biomarker models to predict PH in BPD using clinical and SNP data were tested by calculations of area under the ROC curve. RESULTS: In our BPD cohort, 32% (n = 61) had PH. Of the DUSP SNPs evaluated, DUSP1 SNP rs322351 was less common, and DUSP5 SNPs rs1042606 and rs3793892 were more common in cases than in controls. The best fit biomarker model combines clinical and DUSP genetic data with an area under the ROC curve of 0.76. CONCLUSION: We identified three DUSP SNPs as potential BPD-PH biomarkers. Combining clinical and DUSP genetic data yields the most robust predictor for PH in BPD.

Reclassification of Variants of Uncertain Significance in Children with Inherited Arrhythmia Syndromes is Predicted by Clinical Factors.

Genetic testing is important to augment clinical diagnosis and inform management of inherited arrhythmias syndromes (IAS), but variants of uncertain significance (VUS) are common and remain a challenge in clinical practice. In 2015, American College of Medical Genetics (ACMG) published updated guidelines for interpretation of genetic results. Despite increasing understanding of human genomic variation, there are no guidelines for reinterpretation of prior genetic test results. Patients at a single tertiary children's hospital with genetic testing for an IAS that demonstrated a VUS were re-evaluated using 2015 ACMG guidelines, clinical information, and publically available databases. Search of the electronic medical record identified 116 patients with genetic testing results available, and 24/116 (21%) harbored a VUS for an IAS. 23 unique VUS were evaluated from 12 genes. Over half of the VUS (12/23 (52%)) were reclassified using 2015 criteria, and 8 (35%) changed to pathogenic and 4 (17%) to benign. Relative risk of reclassification of VUS to a pathogenic variant in a patient with confirmed clinical diagnosis was 4.1 (95% CI 1.23-15.4). Reclassification was not associated with initial testing year. These data demonstrate 52% of VUS in children with IAS are reclassified with application of 2015 ACMG guidelines. Strength of phenotyping is associated with eventual pathogenic classification of genetic variants and periodic re-evaluation of VUS identified on genetic testing for IAS is warranted.

EPG5-related Vici syndrome: a paradigm of neurodevelopmental disorders with defective autophagy.

Vici syndrome is a progressive neurodevelopmental multisystem disorder due to recessive mutations in the key autophagy gene EPG5. We report genetic, clinical, neuroradiological, and neuropathological features of 50 children from 30 families, as well as the neuronal phenotype of EPG5 knock-down in Drosophila melanogaster. We identified 39 different EPG5 mutations, most of them truncating and predicted to result in reduced EPG5 protein. Most mutations were private, but three recurrent mutations (p.Met2242Cysfs*5, p.Arg417*, and p.Gln336Arg) indicated possible founder effects. Presentation was mainly neonatal, with marked hypotonia and feeding difficulties. In addition to the five principal features (callosal agenesis, cataracts, hypopigmentation, cardiomyopathy, and immune dysfunction), we identified three equally consistent features (profound developmental delay, progressive microcephaly, and failure to thrive). The manifestation of all eight of these features has a specificity of 97%, and a sensitivity of 89% for the presence of an EPG5 mutation and will allow informed decisions about genetic testing. Clinical progression was relentless and many children died in infancy. Survival analysis demonstrated a median survival time of 24 months (95% confidence interval 0-49 months), with only a 10th of patients surviving to 5 years of age. Survival outcomes were significantly better in patients with compound heterozygous mutations (P = 0.046), as well as in patients with the recurrent p.Gln336Arg mutation. Acquired microcephaly and regression of skills in long-term survivors suggests a neurodegenerative component superimposed on the principal neurodevelopmental defect. Two-thirds of patients had a severe seizure disorder, placing EPG5 within the rapidly expanding group of genes associated with early-onset epileptic encephalopathies. Consistent neuroradiological features comprised structural abnormalities, in particular callosal agenesis and pontine hypoplasia, delayed myelination and, less frequently, thalamic signal intensity changes evolving over time. Typical muscle biopsy features included fibre size variability, central/internal nuclei, abnormal glycogen storage, presence of autophagic vacuoles and secondary mitochondrial abnormalities. Nerve biopsy performed in one case revealed subtotal absence of myelinated axons. Post-mortem examinations in three patients confirmed neurodevelopmental and neurodegenerative features and multisystem involvement. Finally, downregulation of epg5 (CG14299) in Drosophila resulted in autophagic abnormalities and progressive neurodegeneration. We conclude that EPG5-related Vici syndrome defines a novel group of neurodevelopmental disorders that should be considered in patients with suggestive features in whom mitochondrial, glycogen, or lysosomal storage disorders have been excluded. Neurological progression over time indicates an intriguing link between neurodevelopment and neurodegeneration, also supported by neurodegenerative features in epg5-deficient Drosophila, and recent implication of other autophagy regulators in late-onset neurodegenerative disease.

A single nucleotide polymorphism in the dimethylarginine dimethylaminohydrolase gene is associated with lower risk of pulmonary hypertension in bronchopulmonary dysplasia.

AIM: Pulmonary hypertension (PH) develops in 25-40% of bronchopulmonary dysplasia (BPD) patients, substantially increasing mortality. We have previously found that asymmetric dimethylarginine (ADMA), an endogenous inhibitor of nitric oxide (NO) production, is elevated in patients with BPD-associated PH. ADMA is metabolised by N(á´³) ,N(á´³) -dimethylarginine dimethylaminohydrolase (DDAH). Presently, we test the hypothesis that there are single nucleotide polymorphisms (SNPs) in DDAH1 and/or DDAH2 associated with the development of PH in BPD patients. METHODS: BPD patients were enrolled (n = 98) at Nationwide Children's Hospital. Clinical characteristics and 36 SNPs in DDAH1 and DDAH2 were compared between BPD-associated PH patients (cases) and BPD-alone patients (controls). RESULTS: In BPD patients, 25 (26%) had echocardiographic evidence of PH (cases). In this cohort, DDAH1 wild-type rs480414 was 92% sensitive and 53% specific for PH in BPD, and the DDAH1 SNP rs480414 decreased the risk of PH in an additive model of inheritance (OR = 0.39; 95% CI [0.18-0.88], p = 0.01). CONCLUSION: The rs480414 SNP in DDAH1 may be protective against the development of PH in patients with BPD. Furthermore, the DDAH1 rs480414 may be a useful biomarker in developing predictive models for PH in patients with BPD.

ßIV-Spectrin and CaMKII facilitate Kir6.2 regulation in pancreatic beta cells.

Identified over a dozen years ago in the brain and pancreatic islet, ßIV-spectrin is critical for the local organization of protein complexes throughout the nervous system. ßIV-Spectrin targets ion channels and adapter proteins to axon initial segments and nodes of Ranvier in neurons, and ßIV-spectrin dysfunction underlies ataxia and early death in mice. Despite advances in ßIV-spectrin research in the nervous system, its role in pancreatic islet biology is unknown. Here, we report that ßIV-spectrin serves as a multifunctional structural and signaling platform in the pancreatic islet. We report that ßIV-spectrin directly associates with and targets the calcium/calmodulin-dependent protein kinase II (CaMKII) in pancreatic islets. In parallel, ßIV-spectrin targets ankyrin-B and the ATP-sensitive potassium channel. Consistent with these findings, ßIV-spectrin mutant mice lacking CaMKII- or ankyrin-binding motifs display selective loss of expression and targeting of key protein components, including CaMKIIδ. ßIV-Spectrin-targeted CaMKII directly phosphorylates the inwardly-rectifying potassium channel, Kir6.2 (alpha subunit of KATP channel complex), and we identify the specific residue, Kir6.2 T224, responsible for CaMKII-dependent regulation of KATP channel function. CaMKII-dependent phosphorylation alters channel regulation resulting in KATP channel inhibition, a cellular phenotype consistent with aberrant insulin regulation. Finally, we demonstrate aberrant KATP channel phosphorylation in ßIV-spectrin mutant mice. In summary, our findings establish a broader role for ßIV-spectrin in regulation of cell membrane excitability in the pancreatic islet, define the pathway for CaMKII local control in pancreatic beta cells, and identify the mechanism for CaMKII-dependent regulation of KATP channels.

De novo loss-of-function variants in NSD2 (WHSC1) associate with a subset of Wolf-Hirschhorn syndrome.

Wolf-Hirschhorn syndrome (WHS) is a rare but recurrent microdeletion syndrome associated with hemizygosity of an interstitial segment of Chromosome 4 (4p16.3). Consistent with historical reports in which overlapping deletions defined a minimal critical region in WHS patients, recent reports from exome sequence analysis have provided further evidence that haploinsufficiency of a specific gene within this critical region, NSD2 (WHSC1), is causal for many features of the syndrome. In this report, we describe three unrelated patients with loss-of-function alterations in NSD2 who presented clinically with WHS features including intrauterine growth retardation and global developmental delay. Two of the three patients also had overlapping features of failure to thrive, short stature, constipation, and hypotonia. This series adds additional cases to expand the phenotypic spectrum of WHS and reports novel NSD2 variants.

Islet Brain 1 Protects Insulin Producing Cells against Lipotoxicity.

Chronic intake of saturated free fatty acids is associated with diabetes and may contribute to the impairment of functional beta cell mass. Mitogen activated protein kinase 8 interacting protein 1 also called islet brain 1 (IB1) is a candidate gene for diabetes that is required for beta cell survival and glucose-induced insulin secretion (GSIS). In this study we investigated whether IB1 expression is required for preserving beta cell survival and function in response to palmitate. Chronic exposure of MIN6 and isolated rat islets cells to palmitate led to reduction of the IB1 mRNA and protein content. Diminution of IB1 mRNA and protein level relied on the inducible cAMP early repressor activity and proteasome-mediated degradation, respectively. Suppression of IB1 level mimicked the harmful effects of palmitate on the beta cell survival and GSIS. Conversely, ectopic expression of IB1 counteracted the deleterious effects of palmitate on the beta cell survival and insulin secretion. These findings highlight the importance in preserving the IB1 content for protecting beta cell against lipotoxicity in diabetes.

Transcription factor ATF3 links host adaptive response to breast cancer metastasis.

Host response to cancer signals has emerged as a key factor in cancer development; however, the underlying molecular mechanism is not well understood. In this report, we demonstrate that activating transcription factor 3 (ATF3), a hub of the cellular adaptive response network, plays an important role in host cells to enhance breast cancer metastasis. Immunohistochemical analysis of patient tumor samples revealed that expression of ATF3 in stromal mononuclear cells, but not cancer epithelial cells, is correlated with worse clinical outcomes and is an independent predictor for breast cancer death. This finding was corroborated by data from mouse models showing less efficient breast cancer metastasis in Atf3-deficient mice than in WT mice. Further, mice with myeloid cell-selective KO of Atf3 showed fewer lung metastases, indicating that host ATF3 facilitates metastasis, at least in part, by its function in macrophage/myeloid cells. Gene profiling analyses of macrophages from mouse tumors identified an ATF3-regulated gene signature that could distinguish human tumor stroma from distant stroma and could predict clinical outcomes, lending credence to our mouse models. In conclusion, we identified ATF3 as a regulator in myeloid cells that enhances breast cancer metastasis and has predictive value for clinical outcomes.

Physiological role of ghrelin as revealed by the ghrelin and GOAT knockout mice.

Ghrelin is a gastric hormone that has been shown to regulate food intake and energy metabolism. One unique feature of ghrelin is that its activity is regulated post transcriptionally by ghrelin O-acyltransferase (GOAT) through the addition of fatty acid to the serine residue in the N terminal region. Despite much biochemical characterization, to date no other proteins have been shown to be specifically octonylated by GOAT, suggesting a unique matching of the acyl transferase for a single ligand, ghrelin. If this is indeed correct, then genetic deletion of ghrelin or GOAT should produce near identical phenotypes and there should be extensive overlap in expression patterns. This review summarizes the similarities and differences in the phenotypes with the genetic deletion of ghrelin and GOAT in the various knockout mouse lines reported to date. While there is considerable overlap in expression pattern between ghrelin and GOAT, the latter does exhibit some unique tissue expression that could suggest that additional peptides may be acylated and await discovery and characterization.

A method for murine islet isolation and subcapsular kidney transplantation.

Since the early pioneering work of Ballinger and Reckard demonstrating that transplantation of islets of Langerhans into diabetic rodents could normalize their blood glucose levels, islet transplantation has been proposed to be a potential treatment for type 1 diabetes. More recently, advances in human islet transplantation have further strengthened this view. However, two major limitations prevent islet transplantation from being a widespread clinical reality: (a) the requirement for large numbers of islets per patient, which severely reduces the number of potential recipients, and (b) the need for heavy immunosuppression, which significantly affects the pediatric population of patients due to their vulnerability to long-term immunosuppression. Strategies that can overcome these limitations have the potential to enhance the therapeutic utility of islet transplantation. Islet transplantation under the mouse kidney capsule is a widely accepted model to investigate various strategies to improve islet transplantation. This experiment requires the isolation of high quality islets and implantation of islets to the diabetic recipients. Both procedures require surgical steps that can be better demonstrated by video than by text. Here, we document the detailed steps for these procedures by both video and written protocol. We also briefly discuss different transplantation models: syngeneic, allogeneic, syngeneic autoimmune, and allogeneic autoimmune.

Glucose and fatty acids synergize to promote B-cell apoptosis through activation of glycogen synthase kinase 3ß independent of JNK activation.

BACKGROUND: The combination of elevated glucose and free-fatty acids (FFA), prevalent in diabetes, has been suggested to be a major contributor to pancreatic ß-cell death. This study examines the synergistic effects of glucose and FFA on ß-cell apoptosis and the molecular mechanisms involved. Mouse insulinoma cells and primary islets were treated with palmitate at increasing glucose and effects on apoptosis, endoplasmic reticulum (ER) stress and insulin receptor substrate (IRS) signaling were examined. PRINCIPAL FINDINGS: Increasing glucose (5-25 mM) with palmitate (400 µM) had synergistic effects on apoptosis. Jun NH2-terminal kinase (JNK) activation peaked at the lowest glucose concentration, in contrast to a progressive reduction in IRS2 protein and impairment of insulin receptor substrate signaling. A synergistic effect was observed on activation of ER stress markers, along with recruitment of SREBP1 to the nucleus. These findings were confirmed in primary islets. The above effects associated with an increase in glycogen synthase kinase 3ß (Gsk3ß) activity and were reversed along with apoptosis by an adenovirus expressing a kinase dead Gsk3ß. CONCLUSIONS/SIGNIFICANCE: Glucose in the presence of FFA results in synergistic effects on ER stress, impaired insulin receptor substrate signaling and Gsk3ß activation. The data support the importance of controlling both hyperglycemia and hyperlipidemia in the management of Type 2 diabetes, and identify pancreatic islet ß-cell Gsk3ß as a potential therapeutic target.

The roles of ATF3, an adaptive-response gene, in high-fat-diet-induced diabetes and pancreatic beta-cell dysfunction.

Most people with type 2 diabetes (T2D) have reduced beta-cell mass, and apoptosis is a key factor for this reduction. Previously, we showed that ATF3, an adaptive-response gene, is induced by various stress signals relevant to T2D, such as high glucose and high fatty acid. Because ATF3 is proapoptotic in beta-cells, we tested the hypothesis that ATF3 plays a detrimental role and contributes to the development of T2D. We compared wild-type (WT) and ATF3 knockout (KO) mice in an animal model for T2D, high-fat diet-induced diabetes. We also used INS-1 beta-cells and primary islets to analyze the roles of ATF3 in beta-cell function, including insulin gene expression and glucose-induced insulin secretion. Surprisingly, WT mice performed better in glucose tolerance test than KO mice, suggesting a protective, rather than detrimental, role of ATF3. At 12 wk on high-fat diet, no beta-cell apoptosis was observed, and the WT and KO mice had comparable beta-cell areas. However, ATF3 deficiency significantly reduced serum insulin levels in the KO mice without affecting insulin sensitivity, suggesting reduced beta-cell function in the KO mice. Analyses using INS-1 cells and primary islets support the notion that this defect is due, at least partly, to reduced insulin gene transcription in the KO islets without detectable reduction in glucose-induced calcium influx, a critical step for insulin secretion. In conclusion, our results support a model in which, before apoptosis becomes obvious, expression of ATF3 can be beneficial by helping beta-cells to cope with higher metabolic demand.

Adipocyte CREB promotes insulin resistance in obesity.

Increases in adiposity trigger metabolic and inflammatory changes that interfere with insulin action in peripheral tissues, culminating in beta cell failure and overt diabetes. We found that the cAMP Response Element Binding protein (CREB) is activated in adipose cells under obese conditions, where it promotes insulin resistance by triggering expression of the transcriptional repressor ATF3 and thereby downregulating expression of the adipokine hormone adiponectin as well as the insulin-sensitive glucose transporter 4 (GLUT4). Transgenic mice expressing a dominant-negative CREB transgene in adipocytes displayed increased whole-body insulin sensitivity in the contexts of diet-induced and genetic obesity, and they were protected from the development of hepatic steatosis and adipose tissue inflammation. These results indicate that adipocyte CREB provides an early signal in the progression to type 2 diabetes.

The repression of IRS2 gene by ATF3, a stress-inducible gene, contributes to pancreatic beta-cell apoptosis.

OBJECTIVE: beta-Cell failure is an essential component of all types of diabetes, and the insulin receptor substrate 2 (IRS2) branch of signaling plays a key role in beta-cell survival and function. We tested the hypothesis that activating transcription factor 3 (ATF3), a stress-inducible proapoptotic gene, downregulates the expression of IRS2 in beta-cells. RESEARCH DESIGN AND METHODS: We used both the gain- and loss-of-function approaches to test the effects of ATF3 on IRS2 gene expression. We also analyzed the binding of ATF3 to the IRS2 promoter by chromatin immunoprecipitation assay and the transcription of the IRS2 gene by polymerase II occupancy assay. Furthermore, we tested the ability of IRS2 to alleviate the proapoptotic effects of ATF3 in cultured beta-cells and in transgenic mice using the rat insulin promoter to drive the transgenes. RESULTS: Expression of ATF3 is sufficient to reduce IRS2 gene expression; in contrast, knockdown or knockout of ATF3 reduces the ability of stress signals to downregulate IRS2 expression. ATF3 binds to the IRS2 promoter in vivo, and the binding of ATF3 correlates with decreased IRS2 gene transcription. Functionally, expression of IRS2 protects beta-cells from ATF3-induced apoptosis. CONCLUSIONS: IRS2 is a target gene of ATF3, and its repression by ATF3 contributes, at least partly, to the apoptosis induced by ATF3. Because ATF3 is a stress-inducible gene, our work provides a direct link to explain how environmental stress factors can modulate IRS2 gene transcription.

Antimicrobial activities and structures of two linear cationic peptide families with various amphipathic beta-sheet and alpha-helical potentials.

Many naturally occurring antimicrobial peptides comprise cationic linear sequences with the potential to adopt an amphipathic alpha-helical conformation. We designed a linear 18-residue peptide that adopted an amphipathic beta-sheet structure when it was bound to lipids. In comparison to a 21-residue amphipathic alpha-helical peptide of equal charge and hydrophobicity, this peptide possessed more similar antimicrobial activity and greater selectivity in binding to and inducing leakage in vesicles composed of bacterial membrane lipids than vesicles composed of mammalian membrane lipids (J. Blazyk, R. Weigand, J. Klein, J. Hammer, R. M. Epand, R. F. Epand, W. L. Maloy, and U. P. Kari, J. Biol. Chem. 276:27899-27906, 2001). Here, we compare two systematically designed families of linear cationic peptides to evaluate the importance of amphipathicity for determination of antimicrobial activity. Each peptide contains six lysine residues and is amidated at the carboxyl terminus. The first family consists of five peptides with various capacities to form amphipathic beta-sheet structures. The second family consists of six peptides with various potentials to form amphipathic alpha helices. Only those peptides that can form a highly amphipathic structure (either a beta sheet or an alpha helix) possessed significant antimicrobial activities. Striking differences in the abilities to bind to and induce leakage in membranes and lipid vesicles were observed for the two families. Overall, the amphipathic beta-sheet peptides are less lytic than their amphipathic alpha-helical counterparts, particularly toward membranes containing phosphatidylcholine, a lipid commonly found in mammalian plasma membranes. Thus, it appears that antimicrobial peptides that can form an amphipathic beta-sheet conformation may offer a selective advantage in targeting bacterial cells.

Influence of tryptophan on lipid binding of linear amphipathic cationic antimicrobial peptides.

We recently demonstrated that a linear 18-residue peptide, (KIGAKI)(3)-NH(2), designed to form amphipathic beta-sheet structure when bound to lipid bilayers, possessed potent antimicrobial activity and low hemolytic activity. The ability of (KIGAKI)(3)-NH(2) to induce leakage from lipid vesicles was compared to that of the amphipathic alpha-helical peptide, (KIAGKIA)(3)-NH(2), which had equivalent antimicrobial activity. Significantly, the lytic properties of (KIGAKI)(3)-NH(2) were enhanced for mixed acidic-neutral lipid vesicles containing phosphatidylethanolamine instead of phosphatidylcholine as the neutral component, while the potency of (KIAGKIA)(3)-NH(2) was significantly reduced [Blazyk, J., et al. (2001) J. Biol. Chem. 276, 27899-27906]. In this paper, we measured the lytic properties of these peptides, as well as several fluorescent analogues containing a single tryptophan residue, by monitoring permeability changes in large unilamellar vesicles with varying lipid compositions and in Escherichia coli cells. The binding of these peptides to lipid bilayers with defined compositions was compared using surface plasmon resonance, circular dichroism, and fluorescence spectroscopy. Surprisingly large differences were observed in membrane binding properties, particularly in the case of KIGAKIKWGAKIKIGAKI-NH(2). Since all of these peptides possess the same charge and very similar mean hydrophobicities, the binding data cannot be explained merely in terms of electrostatic and/or hydrophobic interactions. In light of their equivalent antimicrobial and hemolytic potencies, some of these peptides may employ mechanisms beyond simply increasing plasma membrane permeability to exert their lethal effects.

Mismatch uracil glycosylase from Escherichia coli: a general mismatch or a specific DNA glycosylase?

The gene for the mismatch-specific uracil glycosylase (MUG) was identified in the Escherichia coli genome as a sequence homolog of the mammalian thymine DNA glycosylase, with activity against uracil in U.G mismatches. Subsequently, 3,N4-ethenocytosine (epsilonC), thymine, 5-hydroxymethyluracil, and 8-(hydroxymethyl)-3,N4-ethenocytosine have been proposed as possible substrates for this enzyme. The evaluation of various DNA adducts as substrates is complicated by the biphasic nature of the kinetics of this enzyme. Our results demonstrate that product release by the enzyme is very slow and hence comparing the "steady-state" parameters of the enzyme for different substrates is of limited use. Consequently, the ability of the enzyme to excise a variety of damage products of purines and pyrimidines was studied under single turnover conditions. Although the enzyme excised both epsilonC and U from DNA, the former adduct was significantly better as a substrate in terms of binding and hydrolysis. Some products of oxidative and alkylation damage are also moderately good substrates for the enzyme, but thymine is a poor substrate. This comparison of different substrates under single turnover conditions provides a rational basis for comparing substrates of MUG and we relate these conclusions to the known crystal structures of the enzyme and its catalytic mechanism.