Sebastian Fröhler

An Oncogenic Role for Alternative NF-κB Signaling in DLBCL Revealed upon Deregulated BCL6 Expression

Diffuse large B cell lymphoma (DLBCL) is a complex disease comprising diverse subtypes and genetic profiles. Possibly because of the prevalence of genetic alterations activating canonical NF-κB activity, a role for oncogenic lesions that activate the alternative NF-κB pathway in DLBCL has remained elusive. Here, we show that deletion/mutation of TRAF3, a negative regulator of the alternative NF-κB pathway, occurs in ∼15% of DLBCLs and that it often coexists with BCL6 translocation, which prevents terminal B cell differentiation. Accordingly, in a mouse model constitutive activation of the alternative NF-κB pathway cooperates with BCL6 deregulation in DLBCL development. This work demonstrates a key oncogenic role for the alternative NF-κB pathway in DLBCL development.

Identification of PENDRIN (SLC26A4) Mutations in Patients With Congenital Hypothyroidism and “Apparent” Thyroid Dysgenesis

CONTEXT Congenital hypothyroidism, the most frequent endocrine congenital disease, can occur either based on a thyroid hormone biosynthesis defect or can predominantly be due to thyroid dysgenesis. However, a genetic cause could so far only be identified in less than 10% of patients with a thyroid dysgenesis. OBJECTIVES Exome sequencing was used for the first time to find additional genetic defects in thyroid dysgenesis. PATIENTS AND METHODS In a consanguineous family with thyroid dysgenesis, exome sequencing was applied, and findings were further validated by Sanger sequencing in a cohort of 94 patients with thyroid dysgenesis. RESULTS By exome sequencing we identified a homozygous missense mutation (p.Leu597Ser) in the SLC26A4 gene of a patient with hypoplastic thyroid tissue, who was otherwise healthy. In the cohort of patients with thyroid dysgenesis, we observed a second case with a homozygous missense mutation (p.Gln413Arg) in the SLC26A4 gene, who was additionally affected by severe hearing problems. Both mutations were previously described as loss-of-function mutations in patients with Pendred syndrome and nonsyndromic enlarged vestibular aqueduct. CONCLUSION We unexpectedly identified SLC26A4 mutations that were hitherto diagnosed in thyroid dyshormonogenesis patients, now for the first time in patients with structural thyroid defects. This result resembles the historic description of thyroid atrophy in patients with the so-called myxedematous form of cretinism after severe iodine deficiency. Most likely the thyroid defect of the two homozygous SLC26A4 gene mutation carriers represents a kind of secondary thyroid atrophy, rather than a primary defect of thyroid development in the sense of thyroid agenesis. Our study extends the variable clinical spectrum of patients with SLC26A4 mutations and points out the necessity to analyze the SLC26A4 gene in patients with apparent thyroid dysgenesis in addition to the known candidate genes TSHR, PAX8, NKX2.1, NKX2.5, and FOXE1.

Exome sequencing helped the fine diagnosis of two siblings afflicted with atypical Timothy syndrome (TS2)

BackgroundLong-QT syndrome (LQTS) causes a prolongation of the QT-interval in the ECG leading to life threatening tachyarrhythmia and ventricular fibrillation. One atypical form of LQTS, Timothy syndrome (TS), is associated with syndactyly, immune deficiency, cognitive and neurological abnormalities as well as distinct cranio-facial abnormalities.Case presentationOn a family with both children diagnosed with clinical LQTS, we performed whole exome sequencing to comprehensively screen for causative mutations after a targeted candidate gene panel screen for Long-QT syndrome target genes failed to identify any underlying genetic defect. Using exome sequencing, we identified in both affected children, a p.402G > S mutation in exon 8 of the CACNA1C gene, a voltage-dependent Ca2+ channel. The mutation was inherited from their father, a mosaic mutation carrier. Based on this molecular finding and further more careful clinical examination, we refined the diagnosis to be Timothy syndrome (TS2) and thereby were able to present new therapeutic approaches.ConclusionsOur study highlights the difficulties in accurate diagnosis of patients with rare diseases, especially those with atypical clinical manifestation. Such challenge could be addressed with the help of comprehensive and unbiased mutation screening, such as exome sequencing.

Epigenetic dynamics of monocyte-to-macrophage differentiation

BackgroundMonocyte-to-macrophage differentiation involves major biochemical and structural changes. In order to elucidate the role of gene regulatory changes during this process, we used high-throughput sequencing to analyze the complete transcriptome and epigenome of human monocytes that were differentiated in vitro by addition of colony-stimulating factor 1 in serum-free medium.ResultsNumerous mRNAs and miRNAs were significantly up- or down-regulated. More than 100 discrete DNA regions, most often far away from transcription start sites, were rapidly demethylated by the ten eleven translocation enzymes, became nucleosome-free and gained histone marks indicative of active enhancers. These regions were unique for macrophages and associated with genes involved in the regulation of the actin cytoskeleton, phagocytosis and innate immune response.ConclusionsIn summary, we have discovered a phagocytic gene network that is repressed by DNA methylation in monocytes and rapidly de-repressed after the onset of macrophage differentiation.

Combining transcription factor binding affinities with open-chromatin data for accurate gene expression prediction

The binding and contribution of transcription factors (TF) to cell specific gene expression is often deduced from open-chromatin measurements to avoid costly TF ChIP-seq assays. Thus, it is important to develop computational methods for accurate TF binding prediction in open-chromatin regions (OCRs). Here, we report a novel segmentation-based method, TEPIC, to predict TF binding by combining sets of OCRs with position weight matrices. TEPIC can be applied to various open-chromatin data, e.g. DNaseI-seq and NOMe-seq. Additionally, Histone-Marks (HMs) can be used to identify candidate TF binding sites. TEPIC computes TF affinities and uses open-chromatin/HM signal intensity as quantitative measures of TF binding strength. Using machine learning, we find low affinity binding sites to improve our ability to explain gene expression variability compared to the standard presence/absence classification of binding sites. Further, we show that both footprints and peaks capture essential TF binding events and lead to a good prediction performance. In our application, gene-based scores computed by TEPIC with one open-chromatin assay nearly reach the quality of several TF ChIP-seq data sets. Finally, these scores correctly predict known transcriptional regulators as illustrated by the application to novel DNaseI-seq and NOMe-seq data for primary human hepatocytes and CD4+ T-cells, respectively.

Discovering the mechanisms underlying serotonin (5-HT)2A and 5-HT2C receptor regulation following nicotine withdrawal in rats

We have previously demonstrated that nicotine withdrawal produces depression‐like behavior and that serotonin (5‐HT)2A/2C receptor ligands modulate that mood‐like state. In the present study we aimed to identify the mechanisms (changes in radioligand binding, transcription or RNA‐editing) related to such a behavioral outcome. Rats received vehicle or nicotine (0.4 mg/kg, s.c.) for 5 days in home cages. Brain 5‐HT2A/2C receptors were analyzed on day 3 of nicotine withdrawal. Nicotine withdrawal increased [3H]ketanserin binding to 5‐HT2A receptors in the ventral tegmental area and ventral dentate gyrus, yet decreased binding in the nucleus accumbens shell. Reduction in [3H]mesulergine binding to 5‐HT2C receptors was seen in the ventral dentate gyrus. Profound decrease in the 5‐HT2A receptor transcript level was noted in the hippocampus and ventral tegmental area. Out of five 5‐HT2C receptor mRNA editing sites, deep sequencing data showed a reduction in editing at the E site and a trend toward reduction at the C site in the hippocampus. In the ventral tegmental area, a reduction for the frequency of CD 5‐HT2C receptor transcript was seen. These results show that the reduction in the 5‐HT2A receptor transcript level may be an auto‐regulatory response to the increased receptor density in the hippocampus and ventral tegmental area during nicotine withdrawal, while decreased 5‐HT2C receptor mRNA editing may explain the reduction in receptor labeling in the hippocampus. Serotonin (5‐HT)2A/2C receptor ligands alleviate depression‐like state in nicotine‐withdrawn rats. Here, we show that the reduction in 5‐HT2A receptor transcript level may be an auto‐regulatory response to the increased receptor number in the hippocampus and ventral tegmental area during nicotine withdrawal, while attenuated 5‐HT2C receptor mRNA editing in the hippocampus might explain reduced inverse agonist binding to 5‐HT2C receptor and suggest a shift toward a population of more active receptors. 5‐HT, serotonin; 5‐HT2AR, 5‐HT2A receptor; 5‐HT2CR, 5‐HT2C receptor.

3PD: Rapid design of optimal primers for chromosome conformation capture assays

BackgroundHigher eukaryotes control the expression of their genes by mechanisms that we are just beginning to understand. A complex layer of control is the dynamic spatial organization of the nucleus.ResultsWe present a bioinformatics solution (3PD) to support the experimentalist in detecting long-ranging intra or inter chromosomal contacts by Chromosome conformation capture (3C) assays. 3C assays take a snapshot of chromosomal contacts by a fixation step and quantify them by PCR. Our contribution is to rapidly design an optimal primer set for the crucial PCR step. Our primer design reduces the level of experimental error as primers are highly similar in terms of physical properties and amplicon length. All 3C primers are compatible with multiplex PCR reactions. Primer uniqueness is checked genome-wide with a suitable index structure.ConclusionsIn summary, our software 3PD facilitates genome-wide primer design for 3C experiments in a matter of seconds. Our software is available as a web server at: http://www.pristionchus.org/3CPrimerDesign/.

MOESM11 of Epigenetic dynamics of monocyte-to-macrophage differentiation

Additional file 11: Table S5. Overview over the donors and sample nomenclature used in the analyses presented in the manuscript.