Age-related glomerulosclerosis and interstitial fibrosis in Milan normotensive rats: A podocyte disease

Age-related glomerulosclerosis and interstitial fibrosis in Milan normotensive rats: A podocyte disease. In Milan normotensive (MNS) rats glomerulosclerosis and interstitial fibrosis develop spontaneously in the absence of hypertension. Renal changes were sequentially assessed in these rats between 2 and 10 months of age. At 10 months, rats were characterized by heavy proteinuria, increased serum creatinine, focal or global glomerulosclerosis in 51 ± 12% of the glomeruli as well as tubulointerstitial injury involving > 25% of the section area. Cell injury in podocytes (evidenced as increased expression of desmin and by electron microscopy) and interstitial fibroblasts (increased expression of α-smooth muscle actin) and mild glomerular hypertrophy were witnessed as early as three to four months of age and preceded glomerulosclerosis and interstitial fibrosis. Only minor evidence of mesangial cell activation (as assessed by glomerular de novo α-smooth muscle actin or type I collagen expression or increased cell proliferation) was noted throughout the observation period. Later stages of the disease were characterized by glomerular and/or tubulointerstitial macrophage influx and osteopontin expression (a chemoattractant), mild accumulation of lymphocytes, platelets, fibrinogen, as well as by a progressive accumulation of various matrix proteins. Progressive renal disease in MNS rats is thus noteworthy for the relative lack of mesangial cell activation. Rather, early podocyte damage, induced by yet unknown mechanisms, may underlie the development of glomerulosclerosis and subsequent interstitial fibrosis

Expanding the clinical spectrum associated with defects in CNTNAP2 and NRXN1

RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are.Abstract Background Heterozygous copy-number and missense variants in CNTNAP2 and NRXN1 have repeatedly been associated with a wide spectrum of neuropsychiatric disorders such as developmental language and autism spectrum disorders, epilepsy and schizophrenia. Recently, homozygous or compound heterozygous defects in either gene were reported as causative for severe intellectual disability. Methods 99 patients with severe intellectual disability and resemblance to Pitt-Hopkins syndrome and/or suspected recessive inheritance were screened for mutations in CNTNAP2 and NRXN1. Molecular karyotyping was performed in 45 patients. In 8 further patients with variable intellectual disability and heterozygous deletions in either CNTNAP2 or NRXN1, the remaining allele was sequenced. Results By molecular karyotyping and mutational screening of CNTNAP2 and NRXN1 in a group of severely intellectually disabled patients we identified a heterozygous deletion in NRXN1 in one patient and heterozygous splice-site, frameshift and stop mutations in CNTNAP2 in four patients, respectively. Neither in these patients nor in eight further patients with heterozygous deletions within NRXN1 or CNTNAP2 we could identify a defect on the second allele. One deletion in NRXN1 and one deletion in CNTNAP2 occurred de novo, in another family the deletion was also identified in the mother who had learning difficulties, and in all other tested families one parent was shown to be healthy carrier of the respective deletion or mutation. Conclusions We report on patients with heterozygous defects in CNTNAP2 or NRXN1 associated with severe intellectual disability, which has only been reported for recessive defects before. These results expand the spectrum of phenotypic severity in patients with heterozygous defects in either gene. The large variability between severely affected patients and mildly affected or asymptomatic carrier parents might suggest the presence of a second hit, not necessarily located in the same gene.Peer Reviewe

Optimizing Genetic Workup in Pheochromocytoma and Paraganglioma by Integrating Diagnostic and Research Approaches

Pheochromocytomas and paragangliomas (PPGL) are rare neuroendocrine tumors with a strong hereditary background and a large genetic heterogeneity. Identification of the underlying genetic cause is crucial for the management of patients and their families as it aids differentiation between hereditary and sporadic cases. To improve diagnostics and clinical management we tailored an enrichment based comprehensive multi-gene next generation sequencing panel applicable to both analyses of tumor tissue and blood samples. We applied this panel to tumor samples and compared its performance to our current routine diagnostic approach. Routine diagnostic sequencing of 11 PPGL susceptibility genes was applied to blood samples of 65 unselected PPGL patients at a single center in Dresden, Germany. Predisposing germline mutations were identified in 19 (29.2%) patients. Analyses of 28 PPGL tumor tissues using the dedicated PPGL panel revealed pathogenic or likely pathogenic variants in known PPGL susceptibility genes in 21 (75%) cases, including mutations in IDH2, ATRX and HRAS. These mutations suggest sporadic tumor development. Our results imply a diagnostic benefit from extended molecular tumor testing of PPGLs and consequent improvement of patient management. The approach is promising for determination of prognostic biomarkers that support therapeutic decision-making.Acknowledgments: We thank the patients and their families who have made this research possible. We want to thank JacquesW. Lenders for his support. We further thank Alexander Krüger, Lydia Rossow and Franziska Stübner for technical support as well as Katharina Langton and Uwe Siemon for their assistance in patient administration.S

Highly significant antiviral activity of HIV-1 LTR-specific tre-recombinase in humanized mice

Stable integration of HIV proviral DNA into host cell chromosomes, a hallmark and essential feature of the retroviral life cycle, establishes the infection permanently. Current antiretroviral combination drug therapy cannot cure HIV infection. However, expressing an engineered HIV-1 long terminal repeat (LTR) site-specific recombinase (Tre), shown to excise integrated proviral DNA in vitro, may provide a novel and highly promising antiviral strategy. We report here the conditional expression of Tre-recombinase from an advanced lentiviral self-inactivation (SIN) vector in HIV-infected cells. We demonstrate faithful transgene expression, resulting in accurate provirus excision in the absence of cytopathic effects. Moreover, pronounced Tre-mediated antiviral effects are demonstrated in vivo, particularly in humanized Rag2−/−γc−/− mice engrafted with either Tre-transduced primary CD4+ T cells, or Tre-transduced CD34+ hematopoietic stem and progenitor cells (HSC). Taken together, our data support the use of Tre-recombinase in novel therapy strategies aiming to provide a cure for HIV

ARTEFACTS: How do we want to deal with the future of our one and only planet?

The European Commission’s Science and Knowledge Service, the Joint Research Centre (JRC), decided to try working hand-in-hand with leading European science centres and museums. Behind this decision was the idea that the JRC could better support EU Institutions in engaging with the European public. The fact that European Union policies are firmly based on scientific evidence is a strong message which the JRC is uniquely able to illustrate. Such a collaboration would not only provide a platform to explain the benefits of EU policies to our daily lives but also provide an opportunity for European citizens to engage by taking a more active part in the EU policy making process for the future. A PILOT PROGRAMME To test the idea, the JRC launched an experimental programme to work with science museums: a perfect partner for three compelling reasons. Firstly, they attract a large and growing number of visitors. Leading science museums in Europe have typically 500 000 visitors per year. Furthermore, they are based in large European cities and attract local visitors as well as tourists from across Europe and beyond. The second reason for working with museums is that they have mastered the art of how to communicate key elements of sophisticated arguments across to the public and making complex topics of public interest readily accessible. That is a high-value added skill and a crucial part of the valorisation of public-funded research, never to be underestimated. Finally museums are, at present, undergoing something of a renaissance. Museums today are vibrant environments offering new techniques and technologies to both inform and entertain, and attract visitors of all demographics.JRC.H.2-Knowledge Management Methodologies, Communities and Disseminatio

Large scale multifactorial likelihood quantitative analysis of BRCA1 and BRCA2 variants: An ENIGMA resource to support clinical variant classification

The multifactorial likelihood analysis method has demonstrated utility for quantitative assessment of variant pathogenicity for multiple cancer syndrome genes. Independent data types currently incorporated in the model for assessing BRCA1 and BRCA2 variants include clinically calibrated prior probability of pathogenicity based on variant location and bioinformatic prediction of variant effect, co-segregation, family cancer history profile, co-occurrence with a pathogenic variant in the same gene, breast tumor pathology, and case-control information. Research and clinical data for multifactorial likelihood analysis were collated for 1,395 BRCA1/2 predominantly intronic and missense variants, enabling classification based on posterior probability of pathogenicity for 734 variants: 447 variants were classified as (likely) benign, and 94 as (likely) pathogenic; and 248 classifications were new or considerably altered relative to ClinVar submissions. Classifications were compared with information not yet included in the likelihood model, and evidence strengths aligned to those recommended for ACMG/AMP classification codes. Altered mRNA splicing or function relative to known nonpathogenic variant controls were moderately to strongly predictive of variant pathogenicity. Variant absence in population datasets provided supporting evidence for variant pathogenicity. These findings have direct relevance for BRCA1 and BRCA2 variant evaluation, and justify the need for gene-specific calibration of evidence types used for variant classification

Large scale multifactorial likelihood quantitative analysis of BRCA1 and BRCA2 variants: An ENIGMA resource to support clinical variant classification

Abstract The multifactorial likelihood analysis method has demonstrated utility for quantitative assessment of variant pathogenicity for multiple cancer syndrome genes. Independent data types currently incorporated in the model for assessing BRCA1 and BRCA2 variants include clinically calibrated prior probability of pathogenicity based on variant location and bioinformatic prediction of variant effect, co-segregation, family cancer history profile, co-occurrence with a pathogenic variant in the same gene, breast tumor pathology, and case-control information. Research and clinical data for multifactorial likelihood analysis were collated for 1395 BRCA1/2 predominantly intronic and missense variants, enabling classification based on posterior probability of pathogenicity for 734 variants: 447 variants were classified as (likely) benign, and 94 as (likely) pathogenic; 248 classifications were new or considerably altered relative to ClinVar submissions. Classifications were compared to information not yet included in the likelihood model, and evidence strengths aligned to those recommended for ACMG/AMP classification codes. Altered mRNA splicing or function relative to known non-pathogenic variant controls were moderately to strongly predictive of variant pathogenicity. Variant absence in population datasets provided supporting evidence for variant pathogenicity. These findings have direct relevance for BRCA1 and BRCA2 variant evaluation, and justify the need for gene-specific calibration of evidence types used for variant classification. This article is protected by copyright. All rights reserved.Peer reviewe

Untersuchungen zur Expression der murinen Gene Pkd1 und Pkd2, den orthologen Genen der Autosomal Dominanten Polyzystischen Nierenerkrankung (ADPKD)

Hackmann K. Untersuchungen zur Expression der murinen Gene Pkd1 und Pkd2, den orthologen Genen der Autosomal Dominanten Polyzystischen Nierenerkrankung (ADPKD). Bielefeld (Germany): Bielefeld University; 2005.Die Autosomal Dominante Polyzystische Nierenerkrankung (ADPKD) ist eine der häufigsten monogenen Erbkrankheiten des Menschen. Mindestens 0,1 Prozent der Bevölkerung sind von dieser Krankheit betroffen. Neben völligem Versagen der stark vergrößerten und mit Zysten übersäten Nieren kann es zu weiteren lebensbedrohlichen Symptomen in anderen Organen und Systemen kommen. Zwei Gene sind bei über 99 Prozent der bekannten Fälle in die Entstehung der Krankheit involviert: PKD1 und PKD2. Nach Entdeckung der homologen Gene der Maus wurden murine Modelle erzeugt, die zum Verständnis der Pathogenese der ADPKD beitragen sollen. In dieser Arbeit wurde ein Konstrukt erstellt, das ein Reportergen unter der Promotorkontrolle des murinen Pkd1-Gens trägt. Zur Erzeugung des Konstrukts wurde das so genannte "ET-Cloning" verwendet. Die erzeugte transgene Mauslinie zeigte keinen auffällig veränderten Phänotyp. Parallel wurden zwölf Antiseren gegen verschiedene Epitope des humanen Polycystin-1 auf Sektionen von Mausembryonen getestet. Nur das Antiserum PK6 aus einem Huhn zeigte eine Aktivität, die mit der aus vorherigen Untersuchungen scheinbar übereinstimmte. Da keine Präimmunseren vorhanden waren, sollte das von PK6 erzeugte Muster mit dem der transgenen Reportermäuse verglichen werden. Wie sich herausstellte, wurde das Transgen durch Methylierung inaktiviert. Im zweiten Teil der Arbeit wurden vier unbekannte Spleißformen des Pkd2-Gens identifiziert. Diese konnten auch in cDNAs humaner Zelllinien bzw. fötalen Gewebes nachgewiesen werden. Pkd2[Delta]6 und Pkd2[Delta]9 werden aufgrund ihrer vorzeitigen Termination der Translation wahrscheinlich vom NMD-Stoffwechselweg (nonsense mediated RNA decay) abgebaut. Pkd2[Delta]12-13 konnte nur als Fragment, aber nicht mit dem gesamten codierenden Bereich amplifiziert werden. Pkd2[Delta]7 ist eine der am häufigsten gefundenen Spleißformen und scheint für ein funktionelles Protein zu codieren. Diese Variante wurde in höchster Menge im Gehirn von Mäusen unterschiedlicher Entwicklungsstadien gefunden. Es konnte gezeigt werden, dass sowohl Pkd2 als auch Pkd2[Delta]7 entwicklungsspezifisch transkribiert werden. Das Polycystin-2[Delta]7-Protein konnte transient stabil exprimiert werden. Durch Co-Immunopräzipitation konnte gezeigt werden, dass die Variante nicht mit Polycystin-1 interagiert, im Gegensatz zum normalen Polycystin-2. Versuche mit Fusionsproteinen geben Hinweise, dass die Polycystin-2[Delta]7-Variante trotzdem in ihrer subzellulären Lokalisation durch die Gegenwart von Polycystin-1 beeinflusst werden könnte