Preeclampsia-Associated Alteration of DNA Methylation in Fetal Endothelial Progenitor Cells

ObjectiveThe pregnancy complication preeclampsia represents an independent risk factor for cardiovascular disease. Our previous research shows a diminished function of fetal endothelial colony-forming cells (ECFC), a proliferative subgroup of endothelial progenitor cells (EPC) in preeclampsia. The aim of this study was to further investigate whether DNA methylation of fetal EPC is affected in preeclampsia.MethodsThe genomic methylation pattern of fetal ECFC from uncomplicated and preeclamptic pregnancies was compared for 865918 CpG sites, and genes were classified into gene networks. Low and advanced cell culture passages were compared to explore whether expansion of fetal ECFC in cell culture leads to changes in global methylation status and if methylation characteristics in preeclampsia are maintained with increasing passage.ResultsA differential methylation pattern of fetal ECFC from preeclampsia compared to uncomplicated pregnancy was detected for a total of 1266 CpG sites in passage 3, and for 2362 sites in passage 5. Key features of primary networks implicated by methylation differences included cell metabolism, cell cycle and transcription and, more specifically, genes involved in cell-cell interaction and Wnt signaling. We identified an overlap between differentially regulated pathways in preeclampsia and cardiovascular system development and function. Cell culture passages 3 and 5 showed similar gene network profiles, and 1260 out of 1266 preeclampsia-associated methylation changes detected in passage 3 were confirmed in passage 5.ConclusionMethylation modification caused by preeclampsia is stable and detectable even in higher cell culture passages. An epigenetically modified endothelial precursor may influence both normal morphogenesis and postnatal vascular repair capacity. Further studies on epigenetic modifications in complicated pregnancies are needed to facilitate development of EPC based therapies for cardiovascular alterations

Systematic genetic analysis of pediatric patients with autoinflammatory diseases

Monogenic autoinflammatory diseases (AID) encompass a growing group of inborn errors of the innate immune system causing unprovoked or exaggerated systemic inflammation. Diagnosis of monogenic AID requires an accurate description of the patients’ phenotype, and the identification of highly penetrant genetic variants in single genes is pivotal. We performed whole exome sequencing (WES) of 125 pediatric patients with suspected monogenic AID in a routine genetic diagnostic setting. Datasets were analyzed in a step-wise approach to identify the most feasible diagnostic strategy. First, we analyzed a virtual gene panel including 13 genes associated with known AID and, if no genetic diagnosis was established, we then analyzed a virtual panel including 542 genes published by the International Union of Immunological Societies associated including all known inborn error of immunity (IEI). Subsequently, WES data was analyzed without pre-filtering for known AID/IEI genes. Analyzing 13 genes yielded a definite diagnosis in 16.0% (n = 20). The diagnostic yield was increased by analyzing 542 genes to 20.8% (n = 26). Importantly, expanding the analysis to WES data did not increase the diagnostic yield in our cohort, neither in single WES analysis, nor in trio-WES analysis. The study highlights that the cost- and time-saving analysis of virtual gene panels is sufficient to rapidly confirm the differential diagnosis in pediatric patients with AID. WES data or trio-WES data analysis as a first-tier diagnostic analysis in patients with suspected monogenic AID is of limited benefit

Biallelic loss-of-function variants in <i>CACHD1 </i>cause a novel neurodevelopmental syndrome with facial dysmorphism and multisystem congenital abnormalities

Purpose We established the genetic etiology of a syndromic neurodevelopmental condition characterized by variable cognitive impairment, recognizable facial dysmorphism, and a constellation of extra-neurological manifestations. Methods We performed phenotypic characterization of 6 participants from 4 unrelated families presenting with a neurodevelopmental syndrome and used exome sequencing to investigate the underlying genetic cause. To probe relevance to the neurodevelopmental phenotype and craniofacial dysmorphism, we established two- and three-dimensional human stem cell-derived neural models and generated a stable cachd1 zebrafish mutant on a transgenic cartilage reporter line. Results Affected individuals showed mild cognitive impairment, dysmorphism featuring oculo-auriculo abnormalities, and developmental defects involving genitourinary and digestive tracts. Exome sequencing revealed biallelic putative loss-of-function variants in CACHD1 segregating with disease in all pedigrees. RNA sequencing in CACHD1-depleted neural progenitors revealed abnormal expression of genes with key roles in Wnt signaling, neurodevelopment, and organ morphogenesis. CACHD1 depletion in neural progenitors resulted in reduced percentages of post-mitotic neurons and enlargement of 3D neurospheres. Homozygous cachd1 mutant larvae showed mandibular patterning defects mimicking human facial dysmorphism. Conclusion Our findings support the role of loss-of-function variants in CACHD1 as the cause of a rare neurodevelopmental syndrome with facial dysmorphism and multisystem abnormalities

Charakterisation of Leupaxin and its interaction partners in carcinoma cells

Leupaxin (LPXN) wird im Rahmen dieser Arbeit als ein Protein identifiziert, welches das Wachstum von bestehenden TRAMP-Tumoren sowie von Fernmetastasen beschleunigt. Verschiedene Signalwege, die sich als LPXN-abhängig herausgestellt haben, geben Erklärungen für diese verstärkte Progression. Durch die Überexpression von LPXN in Prostatakarzinomzellen (PCa) ist sowohl die Migrations- als auch die Invasionsfähigkeit der Zellen erhöht. Untersuchungen des Zelladhäsionsmoleküls p120CTN in den PCa-Zelllinien PC-3 und DU 145 zeigen, dass die Überexpression von LPXN in einer reduzierten Expression von p120CTN resultiert. Außerdem weisen diese Zelllinien nach Herunterregulierung der p120CTN-Expression eine erhöhte Migrations- und Invasionsfähigkeit im Vergleich zu Kontrollzellen auf. Eine Herunterregulierung der LPXN-Expression resultiert hingegen in einer verminderten Migrations- und Invasionsfähigkeit der Zellen. Die simultane Herunterregulierung der LPXN- und p120CTN-Expression schließlich resultiert in einer Aufhebung der Einzel-Effekte. Untersuchungen zur subzellulären Lokalisation von β-Catenin und zur Expression des β-Catenin-Zielgens MMP-7 in PC-3-Zellen zeigen, dass der knockdown der LPXN-Expression in einer Lokalisation von β-Catenin an der Zellmembran resultiert sowie in einer Reduktion der MMP-7-Expression, während der knockdown der p120CTN-Expression eine Lokalisation von β-Catenin im Kern und eine erhöhte MMP-7-Expression zur Folge hat. Der simultane knockdown der LPXN- und p120CTN-Expression ergibt wiederum eine Aufhebung der Einzel-Effekte. Mit Hilfe eines Antikörper-Arrays sind die Kinasen TAK1 und Yes als herunterregulierte Gene nach LPXN-knockdown identifiziert worden. Abhängig von der Stärke der LPXN-Expression nimmt in PC-3-Zellen auch der Phosphorylierungsstatus der downstream von TAK1 stehenden Kinase JNK zu. Die TAK1-JNK-ATF-2-Zielgene Maspin und Presenilin1 zeigen in PCa-Zellen mit herunterregulierter LPXN-Expression eine signifikant herunterregulierte Expression. LPXN interagiert mit dem Aktin bindenden Protein Caldesmon. In PC-3-Zellen lokalisiert Caldesmon an den F-Aktin-Fasern sowie kolokalisiert mit LPXN an den focal adhesion sites. Die Herunterregulierung der Caldesmon-Expression in PC-3- und DU 145-Zellen resultiert in einer erhöhten Migrations- und Invasionsfähigkeit. Die LPXN-Expression hat keinen Effekt auf die Expression von Caldesmon, beeinflusst aber dessen Phosphorylierungsstatus. Bei einer verminderten LPXN-Expression zeigt sich eine verringerte Phosphorylierung von Caldesmon, was zu einer Reduktion der Migrationsfähigkeit der PCa-Zellen führt. Weiterhin führt eine verminderte LPXN-Expression in PC-3- und DU 145-Zellen sowie den murinen LPXN/TRAMP-Primärzellen im Vergleich zu Kontrollzellen zu einer veränderten Adhäsionsfähigkeit sowie Ausbreitung der Zellen. Da bei der für die Adhärenz und Ausbreitung der Zellen notwendigen Umstrukturierung des Zytoskeletts vor allem die Familie der kleinen Rho-GTPasen eine Rolle spielen, wurden die Aktivitäten von RhoA und Rac1 in PC-3-Zellen mit verminderter LPXN-Expression untersucht: Eine verminderte LPXN-Expression führt zu verringerter RhoA- und verstärkter Rac1-Aktivität

ApoE is a major determinant of hepatic bile acid homeostasis in mice

Apolipoprotein E (ApoE) plays a central role in lipid transport and cholesterol metabolism, with surplus cholesterol being removed from the liver through bile acid (BA) synthesis. Furthermore, BAs are of critical importance in fat absorption by forming intestinal lipid-bile salt mixed micelles. To define ApoE's role in BA homeostasis, the metabolism of cholesterol and BA was investigated in liver tissue and gallbladder bile of ApoE-deficient mice given a chow or high-cholesterol/high-fat diet (HCHF) diet for 6 months. When compared to wild-type mice, muricholic acid (MCA) and chenodeoxycholic acid (CDCA) increased approximately 15-, 82-, 22- and 38-fold, respectively, in hepatic tissue of ApoE-deficient mice given a chow or HCHF diet. Moreover, ApoE-deficient mice on an HCHF diet increased the amounts of hepatic free cholesterol, MCA and CDCA by 61%, 61% and 50% (P<.05). Conversely, total cholesterol and cholesterol esters were unchanged, and the bile acids taurohyodeoxycholic acid, taurodeoxycholic acid and hyodeoxycholic acid decreased to one third as compared to the chow diet (P<.05). Additionally, quantitative reverse-transcription polymerase chain reaction assays revealed induced expression of the bile acid receptor (Fxr) and associated transcription factors, i.e. Fxr, Lrh, Lxra and Srebplc. Transcript expression of Cyp2a12, Cyp1b1, Cyp2e1, Cyp3a16 and Cyp4a10 was also induced. Note that Cyp4a10 catalyzes (omega-hydroxylation of arachidonic acid to epoxy- and hydroxyeicosatrienoic acids to control vascular tone. Altogether, MCA and CDCA synthesis is selectively induced in ApoE-deficient mice. These hydrophilic BAs alter micellar size and structure to lower intestinal cholesterol solubilization. Furthermore, CDCA and MCA are potent FXR agonist and antagonist, respectively, and function in a regulatory loop to mitigate impaired ApoE function. (C) 2017 Elsevier Inc. All rights reserved

GenOtoScope: Towards automating ACMG classification of variants associated with congenital hearing loss.

Since next-generation sequencing (NGS) has become widely available, large gene panels containing up to several hundred genes can be sequenced cost-efficiently. However, the interpretation of the often large numbers of sequence variants detected when using NGS is laborious, prone to errors and is often difficult to compare across laboratories. To overcome this challenge, the American College of Medical Genetics and Genomics and the Association for Molecular Pathology (ACMG/AMP) have introduced standards and guidelines for the interpretation of sequencing variants. Additionally, disease-specific refinements have been developed that include accurate thresholds for many criteria, enabling highly automated processing. This is of particular interest for common but heterogeneous disorders such as hearing impairment. With more than 200 genes associated with hearing disorders, the manual inspection of possible causative variants is particularly difficult and time-consuming. To this end, we developed the open-source bioinformatics tool GenOtoScope, which automates the analysis of all ACMG/AMP criteria that can be assessed without further individual patient information or human curator investigation, including the refined loss of function criterion ("PVS1"). Two types of interfaces are provided: (i) a command line application to classify sequence variants in batches for a set of patients and (ii) a user-friendly website to classify single variants. We compared the performance of our tool with two other variant classification tools using two hearing loss data sets, which were manually annotated either by the ClinGen Hearing Loss Gene Curation Expert Panel or the diagnostics unit of our human genetics department. GenOtoScope achieved the best average accuracy and precision for both data sets. Compared to the second-best tool, GenOtoScope improved the accuracy metric by 25.75% and 4.57% and precision metric by 52.11% and 12.13% on the two data sets, respectively. The web interface is accessible via: http://genotoscope.mh-hannover.de:5000 and the command line interface via: https://github.com/damianosmel/GenOtoScope

Ciliary Ultrastructure Assessed by Transmission Electron Microscopy in Adults with Bronchiectasis and Suspected Primary Ciliary Dyskinesia but Inconclusive Genotype

Whole-exome sequencing has expedited the diagnostic work-up of primary ciliary dyskinesia (PCD), when used in addition to clinical phenotype and nasal nitric oxide. However, it reveals variants of uncertain significance (VUS) in established PCD genes or (likely) pathogenic variants in genes of uncertain significance in approximately 30% of tested individuals. We aimed to assess genotype–phenotype correlations in adults with bronchiectasis, clinical suspicion of PCD, and inconclusive whole-exome sequencing results using transmission electron microscopy (TEM) and ciliary image averaging by the PCD Detect software. We recruited 16 patients with VUS in CCDC39, CCDC40, CCDC103, DNAH5, DNAH5/CCDC40, DNAH8/HYDIN, DNAH11, and DNAI1 as well as variants in the PCD candidate genes DNAH1, DNAH7, NEK10, and NME5. We found normal ciliary ultrastructure in eight patients with VUS in CCDC39, DNAH1, DNAH7, DNAH8/HYDIN, DNAH11, and DNAI1. In six patients with VUS in CCDC40, CCDC103, DNAH5, and DNAI1, we identified a corresponding ultrastructural hallmark defect. In one patient with homozygous variant in NME5, we detected a central complex defect supporting clinical relevance. Using TEM as a targeted approach, we established important genotype–phenotype correlations and definite PCD in a considerable proportion of patients. Overall, the PCD Detect software proved feasible in support of TEM

Activation frequency ratios for VCEP-HL data set.

Log ratios calculated for each of the three classes classified by the VCEP-HL: (A) “Benign” broader class, (B) “VUS” class and (C) “Pathogenic” broader class.</p

ACMG/AMP classification scheme based on evidence-based criteria.

The table contains 2 columns. The right column contains sufficient conditions of triggered criteria that result to the left column, pathogenicity class. Sufficient combination of criteria specified for HL are marked with (HL). Pathogenicity probability and its relaxed version are shown for the criteria combinations with the lowest strength that can result to “likely benign” or “likely pathogenic” class. (TIF)</p

Precision-recall curves and AUC scores of all classification tools for VCEP-HL data set.

(A) Prediction of “Benign” broader class versus “Pathogenic” broader class and “VUS” class (B) Prediction of “Pathogenic” broader class versus “Benign” broader class and “VUS” class (C) Prediction of “VUS” class versus “Benign” broader class and “Pathogenic” broader class.</p