10 research outputs found
Cryptic exon activation in SLC12A3 in Gitelman syndrome
No full textGitelman syndrome (GS) is an autosomal recessive renal tubulopathy characterized by hypokalemic metabolic alkalosis with hypocalciuria and hypomagnesemia. GS clinical symptoms range from mild weakness to muscular cramps, paralysis or even sudden death as a result of cardiac arrhythmia. GS is caused by loss-of-function mutations in the solute carrier family 12 member 3 (SLC12A3) gene, but molecular mechanisms underlying such a wide range of symptoms are poorly understood. Here we report cryptic exon activation in SLC12A3 intron 12 in a clinically asymptomatic GS, resulting from an intronic mutation c.1669+297?T>G that created a new acceptor splice site. The cryptic exon was sandwiched between the L3 transposon upstream and a mammalian interspersed repeat downstream, possibly contributing to inclusion of the cryptic exon in mature transcripts. The mutation was identified by targeted next-generation sequencing of candidate genes in GS patients with missing pathogenic SLC12A3 alleles. Taken together, this work illustrates the power of next-generation sequencing to identify causal mutations in intronic regions in asymptomatic individuals at risk of developing potentially fatal disease complications, improving clinical management of these case
Endothelial VEGFR2-PLCγ signaling regulates vascular permeability and antitumor immunity through eNOS/Src
No full textEndothelial phospholipase C gamma (PLC gamma) is essential for vascular development; however, its role in healthy, mature, or pathological vessels is unexplored. Here, we show that PLC gamma was prominently expressed in vessels of several human cancer forms, notably in renal cell carcinoma (RCC). High PLC gamma expression in clear cell RCC correlated with angiogenic activity and poor prognosis, while low expression correlated with immune cell activation. PLC gamma was induced downstream of vascular endothelial growth factor receptor 2 (VEGFR2) phosphosite Y1173 (pY1173). Heterozygous Vegfr2Y1173F/+ mice or mice lacking endothelial PLC gamma (Plcg1iECKO) exhibited a stabilized endothelial barrier and diminished vascular leakage. Barrier stabilization was accompanied by decreased expression of immunosuppressive cytokines, reduced infiltration of B cells, helper T cells and regulatory T cells, and improved response to chemo-and immunotherapy. Mechanistically, pY1173/PLC gamma signaling induced Ca2+/protein kinase C-dependent activation of endothelial nitric oxide synthase (eNOS), required for tyrosine nitration and activation of Src. Src-induced phosphorylation of VE-cadherin at Y685 was accompanied by disintegration of endothelial junctions. This pY1173/PLC gamma/eNOS/Src pathway was detected in both healthy and tumor vessels in Vegfr2Y1173F/+ mice, which displayed decreased activation of PLC gamma and eNOS and suppressed vascular leakage. Thus, we believe that we have identified a clinically relevant endothelial PLC gamma pathway downstream of VEGFR2 pY1173, which destabilizes the endothelial barrier and results in loss of antitumor immunity
eNOS-induced vascular barrier disruption in retinopathy by c-Src activation and tyrosine phosphorylation of VE-cadherin
No full textBackground: Hypoxia and consequent production of vascular endothelial growth factor A (VEGFA) promote blood vessel leakiness and edema in ocular diseases. Anti-VEGFA therapeutics may aggravate hypoxia; therefore, therapy development is needed. Methods: Oxygen-induced retinopathy was used as a model to test the role of nitric oxide (NO) in pathological neovascularization and vessel permeability. Suppression of NO formation was achieved chemically using L-NMMA, or genetically, in endothelial NO synthase serine to alanine (S1176A) mutant mice. Results: Suppression of NO formation resulted in reduced retinal neoangiogenesis. Remaining vascular tufts exhibited reduced vascular leakage through stabilized endothelial adherens junctions, manifested as reduced phosphorylation of vascular endothelial (VE)-cadherin Y685 in a c-Src-dependent manner. Treatment with a single dose of L-NMMA in established retinopathy restored the vascular barrier and prevented leakage. Conclusions: We conclude that NO destabilizes adheren junctions, resulting in vascular hyperpermeability, by converging with the VEGFA/VEGFR2/c-Src/VE-cadherin pathway.De två första författarna delar förstaförfattarskapet</p
Paladin is a phosphoinositide phosphatase regulating endosomal VEGFR2 signalling and angiogenesis
No full textCell signalling governs cellular behaviour and is therefore subject to tight spatiotemporal regulation. Signalling output is modulated by specialized cell membranes and vesicles which contain unique combinations of lipids and proteins. The phosphatidylinositol 4,5-bisphosphate (PI(4,5)P-2), an important component of the plasma membrane as well as other subcellular membranes, is involved in multiple processes, including signalling. However, which enzymes control the turnover of non-plasma membrane PI(4,5)P-2, and their impact on cell signalling and function at the organismal level are unknown. Here, we identify Paladin as a vascular PI(4,5)P-2 phosphatase regulating VEGFR2 endosomal signalling and angiogenesis. Paladin is localized to endosomal and Golgi compartments and interacts with vascular endothelial growth factor receptor 2 (VEGFR2) in vitro and in vivo. Loss of Paladin results in increased internalization of VEGFR2, over-activation of extracellular regulated kinase 1/2, and hypersprouting of endothelial cells in the developing retina of mice. These findings suggest that inhibition of Paladin, or other endosomal PI(4,5)P-2 phosphatases, could be exploited to modulate VEGFR2 signalling and angiogenesis, when direct and full inhibition of the receptor is undesirable.Shared first authorship: Anja Nitzsche, Riikka Pietilä and Dominic T Love</p
The utility of urinary CD80 as a diagnostic marker in patients with renal diseases
No full textCD80, which regulates T cell activation, may provide a differential diagnostic marker between minimal change disease (MCD) and other renal diseases, including focal segmental glomerular sclerosis (FSGS). However, recent reports show contrasting results. Therefore, we evaluated the utility of urinary CD80 as a diagnostic biomarker. We collected 65 urine samples from 55 patients with MCD (n = 31), FSGS (n = 4), inherited nephrotic syndrome (n = 4), Alport syndrome (n = 5) and other glomerular diseases (n = 11), and control samples (n = 30). We measured urinary CD80 levels by ELISA. Urinary CD80 (ng/gCr) (median, interquartile range) levels were significantly higher in patients with MCD in relapse (91.5, 31.1-356.0), FSGS (376.2, 62.7-1916.0), and inherited nephrotic syndrome (220.1, 62.9-865.3), than in patients with MCD in remission (29.5, 21.7-52.8) (p < 0.05). Elevation of urinary CD80 was observed, even in patients with inherited nephrotic syndrome unrelated to T cell activation. Additionally, urinary CD80 was positively correlated with urinary protein levels. Our results suggest that urinary CD80 is unreliable as a differential diagnostic marker between MCD in relapse and FSGS or inherited kidney diseases. Increased urinary CD80 excretion was present in all patients with active kidney disease
Molecular analysis of the GLA gene inactivation pattern and its contribution to the phenotype in a Colombian women with Fabry disease sample through methylation-based assays
No full textilustraciones, diagramas, tablasEl objetivo del presente estudio fue analizar el patrón de inactivación del gen GLA, a través de ensayos basados en metilación, y su contribución al fenotipo en una muestra de mujeres colombianas con Enfermedad de Fabry EF.
Se reporta una heterogénea severidad de fenotipo en mujeres de casos familiares, pudiendo presentar todos los síntomas y signos documentados en varones con EF; asociado a un marcado deterioro en la percepción subjetiva de la calidad de vida.
Así mismo se reporta una metilación heterogénea de las posiciones CpG en la región analizada del gen GLA. Con base en los resultados del ensayo HUMARA, se observó más del 60% de las muestras con inactivación aleatoria, y no se encontró relación estadística entre el patrón de ICX de las muestras analizadas y la severidad fenotípica.
En conclusión, EF afecta en todos los casos la salud física, psicológica y la calidad de vida de las mujeres heterocigotas para variante patogénica en el gen GLA; y no se encontró asociación estadística entre la severidad del fenotipo sistémico en EF, y el patrón de ICX de las muestras analizadas (Texto tomado de la fuente).The present study aimed to analyze the pattern of GLA gene inactivation, through methylation-based assays, and its contribution to the phenotype in a sample of Colombian women with Fabry disease FD.
A heterogeneous severity of phenotype is reported in women of familial cases, being able to present all the symptoms and signs documented in men with FD; associated with a marked deterioration in the subjective perception of quality of life.
Likewise, a heterogeneous methylation of the CpG positions in the analyzed region of the GLA gene is reported. Based on the results of the HUMARA assay, more than 60% of the samples were observed with random inactivation, and no statistical relationship was found between the ICX pattern of the samples analyzed and phenotypic severity.
In conclusion, in all cases, FD affects the physical and psychological health and quality of life of women heterozygous for the pathogenic variant in the GLA gene; and no statistical association was found between the severity of the systemic phenotype in FD and the ICX pattern of the samples analyzed.MaestríaMagíster en Genética Human
