A novel somatic mutation in GNB2 provides new insights to the pathogenesis of Sturge-Weber syndrome.

Sturge-Weber syndrome (SWS) is a neurocutaneous disorder characterized by vascular malformations affecting skin, eyes and leptomeninges of the brain, which can lead to glaucoma, seizures and intellectual disability. The discovery of a disease-causing somatic missense mutation in the GNAQ gene, encoding an alpha chain of heterotrimeric G-proteins, has initiated efforts to understand how G-proteins contribute to SWS pathogenesis. The mutation is predominantly detected in endothelial cells and is currently believed to affect downstream MAPK signalling. In this study of six Norwegian patients with classical SWS, we aimed to identify somatic mutations through deep sequencing of DNA from skin biopsies. Surprisingly, one patient was negative for the GNAQ mutation, but instead harbored a somatic mutation in GNB2 (NM_005273.3:c.232A>G, p.Lys78Glu), which encodes a beta chain of the same G-protein complex. The positions of the mutant amino acids in the G-protein are essential for complex reassembly. Therefore, failure of reassembly and continuous signalling is a likely consequence of both mutations. Ectopic expression of mutant proteins in endothelial cells revealed that expression of either mutant reduced cellular proliferation, yet regulated MAPK signalling differently, suggesting that dysregulated MAPK signalling cannot fully explain the SWS phenotype. Instead, both mutants reduced synthesis of Yes-associated protein (YAP), a transcriptional co-activator of the Hippo signalling pathway, suggesting a key role for this pathway in the vascular pathogenesis of SWS. The discovery of the GNB2 mutation sheds novel light on the pathogenesis of SWS and suggests that future research on targets of treatment should be directed towards the YAP, rather than the MAPK, signalling pathway.

Human thymic putative CD8αα precursors exhibit a biased TCR repertoire in single cell AIRR-seq.

Thymic T cell development comprises T cell receptor (TCR) recombination and assessment of TCR avidity towards self-peptide-MHC complexes presented by antigen-presenting cells. Self-reactivity may lead to negative selection, or to agonist selection and differentiation into unconventional lineages such as regulatory T cells and CD8[Formula: see text] T cells. To explore the effect of the adaptive immune receptor repertoire on thymocyte developmental decisions, we performed single cell adaptive immune receptor repertoire sequencing (scAIRR-seq) of thymocytes from human young paediatric thymi and blood. Thymic PDCD1+ cells, a putative CD8[Formula: see text] T cell precursor population, exhibited several TCR features previously associated with thymic and peripheral ZNF683+ CD8[Formula: see text] T cells, including enrichment of large and positively charged complementarity-determining region 3 (CDR3) amino acids. Thus, the TCR repertoire may partially explain the decision between conventional vs. agonist selected thymocyte differentiation, an aspect of importance for the development of therapies for patients with immune-mediated diseases.

Whole-exome sequencing in moyamoya patients of Northern-European origin identifies gene variants involved in Nitric Oxide metabolism: A pilot study.

Introduction: Moyamoya disease (MMD) is a chronic cerebrovascular steno-occlusive disease of largely unknown etiology. Variants in the RNF213 gene are strongly associated with MMD in East-Asia. In MMD patients of Northern-European origin, no predominant susceptibility variants have been identified so far. Research question: Are there specific candidate genes associated with MMD of Northern-European origin, including the known RNF213 gene? Can we establish a hypothesis for MMD phenotype and associated genetic variants identified for further research? Material and methods: Adult patients of Northern-European origin, treated surgically for MMD at Oslo University Hospital between October 2018 to January 2019 were asked to participate. WES was performed, with subsequent bioinformatic analysis and variant filtering. The selected candidate genes were either previously reported in MMD or known to be involved in angiogenesis. The variant filtering was based on variant type, location, population frequency, and predicted impact on protein function. Results: Analysis of WES data revealed nine variants of interest in eight genes. Five of those encode proteins involved in nitric oxide (NO) metabolism: NOS3, NR4A3, ITGAV, GRB7 and AGXT2. In the AGXT2 gene, a de novo variant was detected, not previously described in MMD. None harboured the p.R4810K missense variant in the RNF213 gene known to be associated with MMD in East-Asian patients. Discussion and conclusion: Our findings suggest a role for NO regulation pathways in Northern-European MMD and introduce AGXT2 as a new susceptibility gene. This pilot study warrants replication in larger patient cohorts and further functional investigations.

Limitations and possibilities of low cell number ChIP-seq.

BACKGROUND: Chromatin immunoprecipitation coupled with high-throughput DNA sequencing (ChIP-seq) offers high resolution, genome-wide analysis of DNA-protein interactions. However, current standard methods require abundant starting material in the range of 1-20 million cells per immunoprecipitation, and remain a bottleneck to the acquisition of biologically relevant epigenetic data. Using a ChIP-seq protocol optimised for low cell numbers (down to 100,000 cells/IP), we examined the performance of the ChIP-seq technique on a series of decreasing cell numbers. RESULTS: We present an enhanced native ChIP-seq method tailored to low cell numbers that represents a 200-fold reduction in input requirements over existing protocols. The protocol was tested over a range of starting cell numbers covering three orders of magnitude, enabling determination of the lower limit of the technique. At low input cell numbers, increased levels of unmapped and duplicate reads reduce the number of unique reads generated, and can drive up sequencing costs and affect sensitivity if ChIP is attempted from too few cells. CONCLUSIONS: The optimised method presented here considerably reduces the input requirements for performing native ChIP-seq. It extends the applicability of the technique to isolated primary cells and rare cell populations (e.g. biobank samples, stem cells), and in many cases will alleviate the need for cell culture and any associated alteration of epigenetic marks. However, this study highlights a challenge inherent to ChIP-seq from low cell numbers: as cell input numbers fall, levels of unmapped sequence reads and PCR-generated duplicate reads rise. We discuss a number of solutions to overcome the effects of reducing cell number that may aid further improvements to ChIP performance.

Pet keeping and tobacco exposure influence CD14 methylation in childhood.

BACKGROUND: Several CD14 gene-environment interactions in relation to the development of allergic diseases have been reported, but the underlying biological mechanisms are unclear. We recently showed that CD14 methylation increased during childhood, parallelling a decreased impact of CD14 polymorphisms on soluble CD14 levels. Here, we aim to explore whether environmental stimuli during childhood affects CD14 methylation, thereby providing a biological mechanism through which environment may modulate genetic effect. METHODS: CD14 methylation levels were quantified in 157 children from the prospective Environment and Childhood Asthma birth cohort at ages 2 and 10. Associations between CD14 methylation levels and house dust levels of endotoxin, ß(1,3)-glucans (at 2 yr only), allergens (dog, cat, and house dust mite), pet keeping and tobacco smoke exposure (TSE; questionnaire data) at 2 and 10 yr were explored. RESULTS: Children in homes without pets had larger increases in CD14 methylation through childhood (2-10 yr) compared with children with pets (2.1% increase (p = 0.003) vs. 0.4% decrease (n.s.), global p = 0.04). At 10 yr of age, lower CD14 methylation values were found in children with pets compared with children without pets at both 2 and 10 yr (5.4% vs. 7.5% [p = 0.02]). A similar trend was detected for TSE; children not exposed show larger increases in CD14 methylation, most pronounced in school-age girls exposed vs. not exposed to tobacco (5.5% vs. 7.5% methylation, p = 0.037). CONCLUSION: Pet keeping and TSE appears to limit increase in CD14 methylation from 2 to 10 yr of age. This may partly explain the diverging CD14 allele associations with allergic diseases detected in different environments.

Multimodal human thymic profiling reveals trajectories and cellular milieu for T agonist selection.

To prevent autoimmunity, thymocytes expressing self-reactive T cell receptors (TCRs) are negatively selected, however, divergence into tolerogenic, agonist selected lineages represent an alternative fate. As thymocyte development, selection, and lineage choices are dependent on spatial context and cell-to-cell interactions, we have performed Cellular Indexing of Transcriptomes and Epitopes by sequencing (CITE-seq) and spatial transcriptomics on paediatric human thymu​​s. Thymocytes expressing markers of strong TCR signalling diverged from the conventional developmental trajectory prior to CD4+ or CD8+ lineage commitment, while markers of different agonist selected T cell populations (CD8αα(I), CD8αα(II), T(agonist), Treg(diff), and Treg) exhibited variable timing of induction. Expression profiles of chemokines and co-stimulatory molecules, together with spatial localisation, supported that dendritic cells, B cells, and stromal cells contribute to agonist selection, with different subsets influencing thymocytes at specific developmental stages within distinct spatial niches. Understanding factors influencing agonist T cells is needed to benefit from their immunoregulatory effects in clinical use.

CD14 polymorphisms and serum CD14 levels through childhood: a role for gene methylation?

BACKGROUND: CD14 is a pattern-recognition receptor for environmental LPS, and engagement of the CD14-LPS complex activates innate host defense mechanisms. Single nucleotide polymorphisms (SNPs) in the CD14 gene have been associated with soluble CD14 (sCD14) levels, but inconsistencies between studies suggest the presence of regulatory mechanisms hitherto not well understood. OBJECTIVE: We sought to investigate possible associations between CD14 SNPs and sCD14 levels at different time points in childhood (at birth [cord blood] and 2 and 10 years) and to explore whether these associations were related to CD14 gene methylation. METHODS: Four SNPs, rs2569191 (-1145GA), rs5744455 (-550CT or -651CT), rs2569190 (-159CT or -260CT), and rs4914 in CD14 were genotyped in 762 children from the Environmental and Childhood Asthma study. Genotype frequencies were analyzed for association with sCD14 levels in 660 babies, 346 children at age 2 years, and 360 children at age 10 years. In a subgroup of 157 children with DNA available at both 2 and 10 years of age, CD14 methylation patterns were determined and analyzed against detected CD14 gene-sCD14 associations. RESULTS: rs2569191, rs5744455, and rs2569190 were associated with sCD14 levels at birth and 2 years, but only rs5744455 was associated with sCD14 levels at 10 years. CD14 methylation increased significantly from age 2 to 10 years, and the level of methylation was inversely correlated with sCD14 levels at 10 years. CONCLUSION: The reduced effect of CD14 polymorphisms on sCD14 levels from early to late childhood paralleled a small but significant increase in CD14 methylation during the same period.

Differential Glial Activation in Early Epileptogenesis-Insights From Cell-Specific Analysis of DNA Methylation and Gene Expression in the Contralateral Hippocampus.

Background and Aims: Morphological changes in mesial temporal lobe epilepsy with hippocampal sclerosis (mTLE-HS) are well-characterized. Yet, it remains elusive whether these are a consequence of seizures or originate from a hitherto unknown underlying pathology. We recently published data on changes in gene expression and DNA methylation in the ipsilateral hippocampus (ILH) using the intracortical kainate mouse model of mTLE-HS. In order to explore the effects of epileptic activity alone and also to further disentangle what triggers morphological alterations, we investigated glial and neuronal changes in gene expression and DNA methylation in the contralateral hippocampus (CLH). Methods: The intracortical kainic acid mouse model of mTLE-HS was used to elicit status epilepticus. Hippocampi contralateral to the injection site from eight kainate-injected and eight sham mice were extracted and shock frozen at 24 h post-injection. Glial and neuronal nuclei were sorted by flow cytometry. Alterations in gene expression and DNA methylation were assessed using reduced representation bisulfite sequencing and RNA sequencing. The R package edgeR was used for statistical analysis. Results: The CLH featured substantial, mostly cell-specific changes in both gene expression and DNA methylation in glia and neurons. While changes in gene expression overlapped to a great degree between CLH and ILH, alterations in DNA methylation did not. In the CLH, we found a significantly lower number of glial genes up- and downregulated compared to previous results from the ILH. Furthermore, several genes and pathways potentially involved in anti-epileptogenic effects were upregulated in the CLH. By comparing gene expression data from the CLH to previous results from the ILH (featuring hippocampal sclerosis), we derive potential upstream targets for epileptogenesis, including glial Cox2 and Cxcl10. Conclusion: Despite the absence of morphological changes, the CLH displays substantial changes in gene expression and DNA methylation. We find that gene expression changes related to potential anti-epileptogenic effects seem to dominate compared to the pro-epileptogenic effects in the CLH and speculate whether this imbalance contributes to prevent morphological alterations like neuronal death and reactive gliosis.

Neuronal and glial DNA methylation and gene expression changes in early epileptogenesis.

BACKGROUND AND AIMS: Mesial Temporal Lobe Epilepsy is characterized by progressive changes of both neurons and glia, also referred to as epileptogenesis. No curative treatment options, apart from surgery, are available. DNA methylation (DNAm) is a potential upstream mechanism in epileptogenesis and may serve as a novel therapeutic target. To our knowledge, this is the first study to investigate epilepsy-related DNAm, gene expression (GE) and their relationship, in neurons and glia. METHODS: We used the intracortical kainic acid injection model to elicit status epilepticus. At 24 hours post injection, hippocampi from eight kainic acid- (KA) and eight saline-injected (SH) mice were extracted and shock frozen. Separation into neurons and glial nuclei was performed by flow cytometry. Changes in DNAm and gene expression were measured with reduced representation bisulfite sequencing (RRBS) and mRNA-sequencing (mRNAseq). Statistical analyses were performed in R with the edgeR package. RESULTS: We observed fulminant DNAm- and GE changes in both neurons and glia at 24 hours after initiation of status epilepticus. The vast majority of these changes were specific for either neurons or glia. At several epilepsy-related genes, like HDAC11, SPP1, GAL, DRD1 and SV2C, significant differential methylation and differential gene expression coincided. CONCLUSION: We found neuron- and glia-specific changes in DNAm and gene expression in early epileptogenesis. We detected single genetic loci in several epilepsy-related genes, where DNAm and GE changes coincide, worth further investigation. Further, our results may serve as an information source for neuronal and glial alterations in both DNAm and GE in early epileptogenesis.

Exploring the influence from whole blood DNA extraction methods on Infinium 450K DNA methylation.

Genome-wide DNA methylation studies are becoming increasingly important in unraveling the epigenetic basis of cell biology, aging and human conditions. The aim of the present study was to explore whether different methods for extracting DNA from whole blood can affect DNA methylation outcome, potentially confounding DNA methylation studies. DNA was isolated from healthy blood donors (n = 10) using three different extraction methods (i.e. two automatic extractions methods based on magnetic beads or isopropanol precipitation, and manual organic extraction). DNA methylation was analyzed using the Infinium HumanMethylation450 Bead Chip (Infinium 450K) (n = 30 samples in total), which is a frequently used method in genome-wide DNA methylation analyses. Overall, the different extraction methods did not have a significant impact on the global DNA methylation patterns. However, DNA methylation differences between organic extraction and each of the automated methods were in general larger than differences between the two automated extraction methods. No CpG sites or regions reached genome-wide significance when testing for differential methylation between extraction methods. Although this study is based on a small sample, these results suggest that extraction method is unlikely to confound Infinium 450K methylation analysis in whole blood.

Characterization of a novel Eph receptor tyrosine kinase, EphA10, expressed in testis.

In mammals, 14 members of the Eph receptor tyrosine kinase family have been described so far. Here we present a not yet described member of this family denoted EphA10. We report the identification of three putative EphA10 isoforms: one soluble and two transmembrane isoforms. One of the latter isoforms lacked the sterile alpha motif commonly found in Eph receptors. The gene encoding EphA10 is located on chromosome 1p34 and expression studies show that EphA10 mRNA is mainly expressed in testis. Binding studies to ephrin ligands suggests that this receptor belongs to the EphA subclass of Eph receptors binding mainly to ephrin-A ligands.

Intra-individual changes in DNA methylation not mediated by cell-type composition are correlated with aging during childhood.

BACKGROUND: Several studies have reported age-associated changes in DNA methylation in the first few years of life and in adult populations, but the extent of such changes during childhood is less well studied. The goals of this study were to investigate to what degree intra-individual changes in DNA methylation are associated with aging during childhood and dissect the methylation changes directly associated with aging from the effect mediated through variation in cell-type composition (CTC). RESULTS: We performed reduced representation bisulfite sequencing (RRBS) in peripheral whole-blood samples collected at 2, 10, and 16 years of age. We identified age-associated longitudinal changes in DNA methylation at 346 CpGs in 178 genes. Analyses separating the effect mediated by CTC variability across age identified 26 CpGs located in 12 genes that associated directly with age. Hence, the CTC changes across age appear to act as a mediator of the observed DNA methylation associated with age. The results were replicated using EpiTYPER in a second sample set selected from the same cohort. Gene ontology analyses revealed enrichment of transcriptional regulation and developmental processes. Further, comparisons of the mean DNA methylation differences between the time points reveal greater differences between 2 to 10 years and 10 to 16 years, suggesting that the identified age-associated DNA methylation patterns manifests in early childhood. CONCLUSIONS: This study reveals insights into the epigenetic dynamics associated with aging early in life. Such information could ultimately provide clues and point towards molecular pathways that are susceptible to aging-related disease-associated epigenetic dysregulation.

A mild form of Mucopolysaccharidosis IIIB diagnosed with targeted next-generation sequencing of linked genomic regions.

Next-generation sequencing (NGS) techniques have already shown their potential in the identification of mutations underlying rare inherited disorders. We report here the application of linkage analysis in combination with targeted DNA capture and NGS to a Norwegian family affected by an undiagnosed mental retardation disorder with an autosomal recessive inheritance pattern. Linkage analysis identified two loci on chromosomes 9 and 17 which were subject to target enrichment by hybridization to a custom microarray. NGS achieved 20-fold or greater sequence coverage of 83% of all protein-coding exons in the target regions. This led to the identification of compound heterozygous mutations in NAGLU, compatible with the diagnosis of Mucopolysaccharidosis IIIB (MPS IIIB or Sanfilippo Syndrome type B). This diagnosis was confirmed by demonstrating elevated levels of heparan sulphate in urine and low activity of α-N-acetyl-glucosaminidase in cultured fibroblasts. Our findings describe a mild form of MPS IIIB and illustrate the diagnostic potential of targeted NGS in Mendelian disease with unknown aetiology.

Ephrin-A1 stimulates migration of CD8+CCR7+ T lymphocytes.

We have previously demonstrated that binding of ephrin-A1 to Eph receptors on human CD4+ T cells stimulates migration. Here, we show that a distinct population of CD8+ T lymphocytes, expressing the chemokine receptor CCR7, also binds ephrin-A1 and is stimulated to migrate after binding. The Eph receptor signaling pathway taking part in the migration event was here investigated. Induced tyrosine phosphorylation of several proteins was seen after ephrin-A1 binding. In particular, induced phosphorylation and kinase activity of the Src kinase family member Lck was observed. An Lck inhibitor inhibited ephrin-A1-induced migration, indicating the involvement of Lck in the migration event. In addition, we observed an induced association of the focal adhesion-like kinase proline-rich tyrosine kinase 2 (Pyk2) and the guanidine exchange factor Vav1 with Lck. PI3K inhibitors also inhibited migration, and studies in transfectants indicate an association of PI3K with EphA1. Further, ephrin-A1-induced migration could be related to the activation of Rho GTPases. This was also observed by using an inhibitor of the Rho-associated kinase ROCK, a downstream effector of Rho. Our results suggest that stimulation of Eph receptors on CD8+CCR7+ T cells leads to migration involving activation of Lck, Pyk2, PI3K, Vav1 and Rho GTPase.