MutationDistiller: user-driven identification of pathogenic DNA variants

MutationDistiller is a freely available online tool for user-driven analyses of Whole Exome Sequencing data. It offers a user-friendly interface aimed at clinicians and researchers, who are not necessarily bioinformaticians. MutationDistiller combines Mutation- Taster’s pathogenicity predictions with a phenotypebased approach. Phenotypic information is not limited to symptoms included in the Human Phenotype Ontology (HPO), but may also comprise clinical diagnoses and the suspected mode of inheritance. The search can be restricted to lists of candidate genes (e.g. virtual gene panels) and by tissue-specific gene expression. The inclusion of GeneOntology (GO) and metabolic pathways facilitates the discovery of hitherto unknown disease genes. In a novel approach, we trained MutationDistiller’s HPO-based prioritization on authentic genotype–phenotype sets obtained from ClinVar and found it to match or outcompete current prioritization tools in terms of accuracy. In the output, the program provides a list of potential disease mutations ordered by the likelihood of the affected genes to cause the phenotype. MutationDistiller provides links to gene-related information from various resources. It has been extensively tested by clinicians and their suggestions have been valued in many iterative cycles of revisions. The tool, a comprehensive documentation and examples are freely available at https://www.mutationdistiller.org

Novel bi-allelic variants expand the SPTBN4-related genetic and phenotypic spectrum

Neurodevelopmental disorder with hypotonia, neuropathy, and deafness (NEDHND, OMIM #617519) is an autosomal recessive disease caused by homozygous or compound heterozygous variants in SPTBN4 coding for type 4 beta IV-spectrin, a non-erythrocytic member of the beta-spectrin family. Variants in SPTBN4 disrupt the cytoskeletal machinery that controls proper localization of ion channels and the function of axonal domains, thereby generating severe neurological dysfunction. We set out to analyze the genetic causes and describe the clinical spectrum of suspected cases of NEDHND. Variant screening was done by whole exome sequencing; clinical phenotypes were described according to the human phenotype ontology, and histochemical analysis was performed with disease-specific antibodies. We report four families with five patients harboring novel homozygous and compound heterozygous SPTBN4 variants, amongst them a multi-exon deletion of SPTBN4. All patients presented with the key features of NEDHND; severe muscular hypotonia, dysphagia, absent speech, gross motor, and mental retardation. Additional symptoms comprised horizontal nystagmus, epileptiform discharges in EEG without manifest seizures, and choreoathetosis. Muscle histology revealed both characteristics of myopathy and of neuropathy. This report expands the SPTBN4 variant spectrum, highlights the spectrum of morphological phenotypes of NEDHND-patients, and reveals clinical similarities between the NEDHND, non-5q SMA, and congenital myopathies

Systematic Comparison of Three Methods for Fragmentation of Long-Range PCR Products for Next Generation Sequencing

Next Generation Sequencing (NGS) technologies are gaining importance in the routine clinical diagnostic setting. It is thus desirable to simplify the workflow for high-throughput diagnostics. Fragmentation of DNA is a crucial step for preparation of template libraries and various methods are currently known. Here we evaluated the performance of nebulization, sonication and random enzymatic digestion of long-range PCR products on the results of NGS. All three methods produced high-quality sequencing libraries for the 454 platform. However, if long-range PCR products of different length were pooled equimolarly, sequence coverage drastically dropped for fragments below 3,000 bp. All three methods performed equally well with regard to overall sequence quality (PHRED) and read length. Enzymatic fragmentation showed highest consistency between three library preparations but performed slightly worse than sonication and nebulization with regard to insertions/deletions in the raw sequence reads. After filtering for homopolymer errors, enzymatic fragmentation performed best if compared to the results of classic Sanger sequencing. As the overall performance of all three methods was equal with only minor differences, a fragmentation method can be chosen solely according to lab facilities, feasibility and experimental design

Factors Affecting European Farmers’Participation in Biodiversity Policies

This article reports the major findings from an interdisciplinary research project that synthesises key insights into farmers’ willingness and ability to co-operate with biodiversity policies. The results of the study are based on an assessment of about 160 publications and research reports from six EU member states and from international comparative research.We developed a conceptual framework to systematically review the existent literature relevant for our purposes. This framework provides a common structure for analysing farmers’ perspectives regarding the introduction into farming practices of measures relevant to biodiversity. The analysis is coupled and contrasted with a survey of experts. The results presented above suggest that it is important to view support for practices oriented towards biodiversity protection not in a static sense – as a situation determined by one or several influencing factors – but rather as a process marked by interaction. Financial compensation and incentives function as a necessary, though clearly not sufficient condition in this process

Elucidation of genetic disorders in neuropediatrics with novel sequencing technique

Die hier vorgestellten Arbeiten dienen der Aufklärung krankheitsverursachender genetischer Defekte aus dem Fachgebiet der Neuropädiatrie. Angewendet wurden verschiedene moderne molekulargenetische Analyseverfahren. Es handelt sich bei den Projekten um forschungsträchtige Themen aus dem klinischen Alltag, die ich in die Grundlagenforschung eingebracht habe. Nach Aufdeckung des jeweiligen Gendefektes wurde die funktionelle Relevanz in zellbiologischen Experimentalsystemen untersucht. Die Ergebnisse der vorgestellten Arbeiten verbessern unsere Fähigkeiten, Familien mit seltenen Erkrankungen zu beraten und bereichern unser Wissen auf dem Gebiet der Entwicklungsstörungen des Nervensystems.The aim of this work was to find disease causing mutations in children with hereditary neurologic disorders using novel sequencing techniques and link the genetic findings to their clinical phenotype. Once a novel gene defect was found, its functional relevance was investigated by different cell biological methods. This work will improve our ability to counsel families with orphan disorders and forward our understanding of the pathogenesis of developmental disorders of the nervous system

The neurotoxicity of phenobarbital in the immature rat brain: Analysis of the expression of neurotrophins and downstream proteins

Titelblatt und Inhaltsverzeichnis Einleitung Material und Methoden Ergebnisse Diskussion Zusammenfassung Literaturverzeichnis AbkürzungsverzeichnisEpilepsie ist eine weit verbreitete neurologische Erkrankung vor allem junger Menschen und betrifft 1-2 % der Population weltweit. Die höchste Inzidenz liegt im ersten Lebensjahr. Phenobarbital ist eines der Medikamente, das zur Therapie von Säuglingen, Kindern und Schwangeren mit Epilepsien eingesetzt wird. Es hat sich gezeigt, dass sowohl die pränatale Exposition, als auch die Behandlung mit Phenobarbital im Säuglings- und Kleinkindesalter bleibende negative Auswirkungen auf die kognitive Entwicklung von Kindern haben kann, die bis ins Erwachsenenalter reichen. Im Tierversuch konnte gezeigt werden, dass Phenobarbital, welches die inhibitorische Wirkung des Neurotransmitters GABA verstärkt, dosisabhängig eine apoptotische Neurodegeneration im sich entwickelnden Gehirn der Ratte bewirkt. Über zugrundeliegende potentielle Pathomechanismen, über die Phenobarbital Apoptose im unreifen Gehirn auslöst, war bisher nichts bekannt. Ziel dieser Arbeit war es, Änderungen der Konzentrationen neurotropher Faktoren und ihrer Effektorsignalproteine im infantilen Rattengehirn nach Phenobarbitalexposition zu untersuchen. Die Neurotrophine BDNF (Brain Derived Neurotrophic Factor) und NT-3 (Neurotophin 3) aktivieren intrazelluläre Überlebenssignale und ihr Entzug führt im unreifen Gehirn zu neuronalem Zelltod. Es konnte anhand von RT-PCR-Analysen gezeigt werden, dass Phenobarbital die mRNA-Expression von BDNF und NT-3 im Thalamus sieben Tage alter Ratten vermindert. Mittels Western-Blot-Analysen wurden die abhängigen Neurotrophin-vermittelten Signaltransduktionskaskaden untersucht. Es konnte gezeigt werden, dass Phenobarbital die Menge der phosphorylierten aktiven Isoformen ERK1/2 und p-Raf sowie die Menge der aktiven Isoform der Serin-Threonin-Kinase AKT (p-Akt) vermindert. Solche Veränderungen sind Ausdruck eines Ungleichgewichts zwischen neuroprotektiven und neurodestruktiven Mechanismen, was in einer bestimmten Entwicklungsphase des Gehirns apoptotische Neurodegeneration fördern könnte. Die Ergebnisse legen nahe, dass eine verminderte Synthese von Wachstumsfaktoren und die daraus resultierende Beeinträchtigung abhängiger Signalwege einen möglicher Mechanismus der Phenobarbital-vermittelten Neurotoxizität darstellen. Die größte Vulnerabilität des Gehirns gegenüber dem proapoptotischen Effekt von Phenobarbital stimmt mit dem Zeitraum des maximalen Hirnwachstums überein, einer Phase, die beim Menschen vom letzten Trimester der Schwangerschaft bis zum dritten Lebensjahr andauert. Eine durch Phenobarbital hervorgerufene apoptotische Neurodegeneration in dieser kritischen Entwicklungsphase vermag möglicherweise die Beeinträchtigung der intellektuellen Fähigkeiten prä- und/oder postnatal exponierter Kinder zu erklären. Diese Ergebnisse verdeutlichen, dass mit Phenobarbital für die Therapie der Epilepsien bei Säuglingen und Kleinkindern kein optimales Antiepileptikum zur Verfügung steht. Um die Prognose für eine normale intellektuelle Entwicklung bei frühkindlichen Epilepsien zu verbessern, müssen neue Substanzen ohne oder mit geringeren neurotoxischen Nebenwirkungen sowie adjuvante neuroprotektive Strategien entwickelt werden.Epilepsy ist he most common neurological disorder of young humans. Phenobarbital, used to treat seizures in children, infants, and pregnant women, cause cognitive impairment, microcephaly, and birth defects. Phenobarbital causes apoptosis in the developing brain. Neuronal death is associated with reduced expression of neurotrophins NT-3 und BDNF and decreased concentrations of survival-promoting proteins (phosphorylated forms of c-RAF, ERK ½, and AKT). Such changes reflect impairment of survival- promoting signals and an imbalance between neuroprotective and neurodestructive mechanisms in the brain. The vulnerability period to the proapoptotic effect of phenobarbital coincides with the brain growth spurt period, which in the rat spans the first 2 weeks of life In humans, the comparable period beginns in the third trimester of gestation and extends to several years after birth. Apoptotic neurodegeneration triggered by phenobarbital can at least partly account for reduced head circumference and impaired intellectual skills observed in prenatally and postnatally exposed humans

A New Mechanism in THRA Resistance: The First Disease-Associated Variant Leading to an Increased Inhibitory Function of THRA2

The nuclear thyroid hormone receptors (THRs) are key mediators of thyroid hormone function on the cellular level via modulation of gene expression. Two different genes encode THRs (THRA and THRB), and are pleiotropically involved in development, metabolism, and growth. The THRA1 and THRA2 isoforms, which result from alternative splicing of THRA, differ in their C-terminal ligand-binding domain (LBD). Most published disease-associated THRA variants are located in the LBD of THRA1 and impede triiodothyronine (T3) binding. This keeps the nuclear receptor in an inactive state and inhibits target gene expression. Here, we investigated a new dominant THRA variant (chr17:g.38,241,010A > G, GRCh37.13 | c.518A > G, NM_199334 | p.(E173G), NP_955366), which is located between the DNA- and ligand-binding domains and affects both splicing isoforms. Patients presented partially with hypothyroid (intellectual disability, motor developmental delay, brain atrophy, and constipation) and partially with hyperthyroid symptoms (tachycardia and behavioral abnormalities) to varying degrees. Functional characterization of THRA1p.(E173G) by reporter gene assays revealed increased transcriptional activity in contrast to THRA1(WT), unexpectedly revealing the first gain-of-function mutation found in THRA1. The THRA2 isoform does not bind T3 and antagonizes THRA1 action. Introduction of p.(E173G) into THRA2 increased its inhibitory effect on THRA1, which helps to explain the hypothyroid symptoms seen in our patients. We used protein structure models to investigate possible underlying pathomechanisms of this variant with a gain-of-antagonistic function and suggest that the p.(E173G) variant may have an influence on the dimerization domain of the nuclear receptor

Comparison of the percentage of missense, deletion and insertion errors in individual sequence reads.

<p>The error frequency was calculated according to Method#1 (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0028240#s3" target="_blank">Materials and Methods</a> section) with respect to the fragmentation method. The error bars depict the standard deviation. In order to classify a position on a sequence read as erroneous, the coverage of the respective position had to be >20 fold and the percentage of the alternative (erroneous) allele to be <20%. *, p<0.05.</p

Workflow for fragmentation and NGS sequencing of long-range PCR fragments.

<p>(<b>A</b>) Graphical illustration of the entire workflow. The red arrows depict a measuring and DNA-quantification step. (<b>B</b>) Analysis of fragment lengths by PAGE before (left panel) and after (right panel) removal of small fragments <500 bp with AMPure™ columns. The red boxes depict the desired size range between 600 and 1,000 bp. Neb, nebulization; Son, sonication; Enz, enzymatic fragmentation.</p