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Wastewater contains an extensive reservoir of genetic information, yet largely unexplored. Here, we analyzed by high-throughput sequencing total nucleic acids extracted from wastewater samples collected during a 17 month-period in Berlin, Germany. By integrating global wastewater datasets and applying a novel computational approach to accurately identify viral strains within sewage RNA-sequencing data, we demonstrated the emergence and global dissemination of a specific astrovirus strain. Astrovirus abundance and sequence variation mirrored temporal and spatial patterns of infection, potentially serving as footprints of specific timeframes and geographical locations. Additionally, we revealed more than 100,000 sequence contigs likely originating from novel viral species, exhibiting distinct profiles in total RNA and DNA datasets and including undescribed bunyaviruses and parvoviruses. Finally, we identified thousands of new CRISPR-associated protein sequences, including Transposase B (TnpB), a class of compact, RNA-guided DNA editing enzymes. Collectively, our findings underscore the potential of high-throughput sequencing of total nucleic acids derived from wastewater for a broad range of applications.

Summary: Noonan syndrome patients harboring causative variants in LZTR1 are particularly at risk to develop severe and early-onset hypertrophic cardiomyopathy. In this study, we investigate the mechanistic consequences of a homozygous variant LZTR1L580P by using patient-specific and CRISPR-Cas9-corrected induced pluripotent stem cell (iPSC) cardiomyocytes. Molecular, cellular, and functional phenotyping in combination with in silico prediction identify an LZTR1L580P-specific disease mechanism provoking cardiac hypertrophy. The variant is predicted to alter the binding affinity of the dimerization domains facilitating the formation of linear LZTR1 polymers. LZTR1 complex dysfunction results in the accumulation of RAS GTPases, thereby provoking global pathological changes of the proteomic landscape ultimately leading to cellular hypertrophy. Furthermore, our data show that cardiomyocyte-specific MRAS degradation is mediated by LZTR1 via non-proteasomal pathways, whereas RIT1 degradation is mediated by both LZTR1-dependent and LZTR1-independent pathways. Uni- or biallelic genetic correction of the LZTR1L580P missense variant rescues the molecular and cellular disease phenotype, providing proof of concept for CRISPR-based therapies.

Abstract Background Long-read sequencing is increasingly used to uncover structural variants in the human genome, both functionally neutral and deleterious. Structural variants occur more frequently in regions with a high homology or repetitive segments, and one rearrangement may predispose to additional events. Bartter syndrome type 3 (BS 3) is a monogenic tubulopathy caused by deleterious variants in the chloride channel gene CLCNKB, a high proportion of these being large gene deletions. Multiplex ligation-dependent probe amplification, the current diagnostic gold standard for this type of mutation, will indicate a simple homozygous gene deletion in biallelic deletion carriers. However, since the phenotypic spectrum of BS 3 is broad even among biallelic deletion carriers, we undertook a more detailed analysis of precise breakpoint regions and genomic structure. Methods Structural variants in 32 BS 3 patients from 29 families and one BS4b patient with CLCNKB deletions were investigated using long-read and synthetic long-read sequencing, as well as targeted long-read sequencing approaches. Results We report a ~3 kb duplication of 3′-UTR CLCNKB material transposed to the corresponding locus of the neighbouring CLCNKA gene, also found on ~50 % of alleles in healthy control individuals. This previously unknown common haplotype is significantly enriched in our cohort of patients with CLCNKB deletions (45 of 51 alleles with haplotype information, 2.2 kb and 3.0 kb transposition taken together, p=9.16×10−9). Breakpoint coordinates for the CLCNKB deletion were identifiable in 28 patients, with three being compound heterozygous. In total, eight different alleles were found, one of them a complex rearrangement with three breakpoint regions. Two patients had different CLCNKA/CLCNKB hybrid genes encoding a predicted CLCNKA/CLCNKB hybrid protein with likely residual function. Conclusions The presence of multiple different deletion alleles in our cohort suggests that large CLCNKB gene deletions originated from many independently recurring genomic events clustered in a few hot spots. The uncovered associated sequence transposition haplotype apparently predisposes to these additional events. The spectrum of CLCNKB deletion alleles is broader than expected and likely still incomplete, but represents an obvious candidate for future genotype/phenotype association studies. We suggest a sensitive and cost-efficient approach, consisting of indirect sequence capture and long-read sequencing, to analyse disease-relevant structural variant hotspots in general.

Abstract Craniosynostosis is characterized by the premature fusion and ossification of one or more of the sutures of the calvaria, often resulting in abnormal features of the face and the skull. In cases in which growth of the brain supersedes available space within the skull, developmental delay or cognitive impairment can occur. A complex interplay of different cell types and multiple signaling pathways are required for correct craniofacial development. In this study, we report on two siblings with craniosynostosis and a homozygous missense pathogenic variant within the IL11RA gene (c.919 T > C; p.W307R). The patients present with craniosynostosis, exophthalmos, delayed tooth eruption, mild platybasia, and a basilar invagination. The p.W307R variant is located within the arginine-tryptophan-zipper within the D3 domain of the IL-11R, a structural element known to be important for the stability of the cytokine receptor. Expression of IL-11R-W307R in cells shows impaired maturation of the IL-11R, no transport to the cell surface and intracellular retention. Accordingly, cells stably expressing IL-11R-W307R do not respond when stimulated with IL-11, arguing for a loss-of-function mutation. In summary, the IL-11R-W307R variant, reported here for the first time to our knowledge, is most likely the causative variant underlying craniosynostosis in these patients.

Introduction: Coagulation Factor VIII (FVIII) plays a pivotal role in the coagulation cascade, and deficiencies in its levels, as seen in Hemophilia A, can lead to significant health implications. Liver sinusoidal endothelial cells (LSECs) are the main producers and contributors of FVIII in blood, a fact we have previously elucidated through mRNA expression profiling when comparing these cells to other endothelial cell types.Methods: Our current investigation focuses on small microRNAs, analyzing their distinct expression patterns across various endothelial cells and hepatocytes.Results: The outcome of this exploration underscores the discernible microRNAs expression differences that set LSECs apart from both hepatocytes (193 microRNAs at p < 0.05) and other endothelial cells (72 microRNAs at p < 0.05). Notably, the 134 and 35 overexpressed microRNAs in LSECs compared to hepatocytes and other endothelial cells, respectively, shed light on the unique functions of LSECs in the liver.Discussion: Our investigation identified a panel of 10 microRNAs (miR-429, miR-200b-3p, miR-200a-3p, miR-216b-5p, miR-1185-5p, miR-19b-3p, miR-192-5p, miR-122-5p, miR-30c-2-3p, and miR-30a-5p) that distinctly define LSEC identity. Furthermore, our scrutiny extended to microRNAs implicated in F8 regulation, revealing a subset (miR-122-5p, miR-214-3p, miR-204-3p, and miR-2682-5p) whose expression intricately correlates with F8 expression within LSECs. This microRNA cohort emerges as a crucial modulator of F8, both directly through suppression and indirect effects on established F8-related transcription factors. The above microRNAs emerged as potential targets for innovative therapies in Hemophilia A patients.

Abstract Objective The phenotypic and genotypic spectrum of adult patients with epilepsy and intellectual disability (ID) is less clear than in children. We investigated an adult patient cohort to further elucidate this and inform the genetic testing approach. Methods Fifty‐two adult patients (30 male, 22 female) with epilepsy, at least mild ID and no known genetic or acquired cause were included and phenotyped. Variants identified through exome sequencing were evaluated using ACMG criteria. Identified variants were compared with commercially available gene panels. Cluster analysis of two features, age at seizure onset and age at ascertainment of cognitive deficits, was performed. Results Median age was 27 years (range 20‐57 years) with median seizure onset at 3 years and median ascertainment of cognitive deficits at 1 year. Likely pathogenic/pathogenic variants were identified in 16/52 patients (31%) including 14 (27%) single nucleotide variants and 2 (4%) copy number variants. Simulated yield of commercial gene panels varied between 13% in small (≤144 genes) and 27% in large panels (≥1478 genes). Cluster analysis (optimal number 3 clusters) identified a cluster with early seizure onset and early developmental delay (developmental and epileptic encephalopathy, n = 26), a cluster with early developmental delay but late seizure onset (ID with epilepsy, n = 16) and a third cluster with late ascertainment of cognitive deficits and variable seizure onset (n = 7). The smaller gene panels particularly missed the genes identified in the cluster with early ascertainment of cognitive deficits and later onset of epilepsy (0/4) as opposed to the cluster with developmental and epileptic encephalopathy (7/10). Significance Our data indicates that adult patients with epilepsy and ID represent a heterogeneous cohort that includes grown‐up patients with DEE but also patients with primary ID and later onset of epilepsy. To maximize diagnostic yield in this cohort either large gene panels or exome sequencing should be used.

Abstract Background The term congenital ocular motor apraxia (COMA), coined by Cogan in 1952, designates the incapacity to initiate voluntary eye movements performing rapid gaze shift, so called saccades. While regarded as a nosological entity by some authors, there is growing evidence that COMA designates merely a neurological symptom with etiologic heterogeneity. In 2016, we reported an observational study in a cohort of 21 patients diagnosed as having COMA. Thorough re-evaluation of the neuroimaging features of these 21 subjects revealed a previously not recognized molar tooth sign (MTS) in 11 of them, thus leading to a diagnostic reassignment as Joubert syndrome (JBTS). Specific MRI features in two further individuals indicated a Poretti–Boltshauser syndrome (PTBHS) and a tubulinopathy. In eight patients, a more precise diagnosis was not achieved. We pursued this cohort aiming at clarification of the definite genetic basis of COMA in each patient. Results Using a candidate gene approach, molecular genetic panels or exome sequencing, we detected causative molecular genetic variants in 17 of 21 patients with COMA. In nine of those 11 subjects diagnosed with JBTS due to newly recognized MTS on neuroimaging, we found pathogenic mutations in five different genes known to be associated with JBTS, including KIAA0586, NPHP1, CC2D2A, MKS1, and TMEM67. In two individuals without MTS on MRI, pathogenic variants were detected in NPHP1 and KIAA0586, arriving at a diagnosis of JBTS type 4 and 23, respectively. Three patients carried heterozygous truncating variants in SUFU, representing the first description of a newly identified forme fruste of JBTS. The clinical diagnoses of PTBHS and tubulinopathy were confirmed by detection of causative variants in LAMA1 and TUBA1A, respectively. In one patient with normal MRI, biallelic pathogenic variants in ATM indicated variant ataxia telangiectasia. Exome sequencing failed to reveal causative genetic variants in the remaining four subjects, two of them with clear MTS on MRI. Conclusions Our findings indicate marked etiologic heterogeneity in COMA with detection of causative mutations in 81% (17/21) in our cohort and nine different genes being affected, mostly genes associated with JBTS. We provide a diagnostic algorithm for COMA.

Summary: Collagen XII, belonging to the fibril-associated collagens, is a homotrimeric secreted extracellular matrix (ECM) protein encoded by the COL12A1 gene. Mutations in the human COL12A1 gene cause an Ehlers-Danlos/myopathy overlap syndrome leading to skeletal abnormalities and muscle weakness. Here, we studied the role of collagen XII in joint pathophysiology by analyzing collagen XII deficient mice and human patients. We found that collagen XII is widely expressed across multiple connective tissue of the developing joint. Lack of collagen XII in mice destabilizes tendons and the femoral trochlear groove to induce patellar subluxation in the patellofemoral joint. These changes are associated with an ECM damage response in tendon and secondary quadriceps muscle degeneration. Moreover, patellar subluxation was also identified as a clinical feature of human patients with collagen XII deficiency. The results provide an explanation for joint hyperlaxity in mice and human patients with collagen XII deficiency.

Abstract Several high-throughput antibody-free methods for RNA modification detection from sequencing data have been developed. We present JACUSA2 as a versatile software solution and comprehensive analysis framework for RNA modification detection assays that are based on either the Illumina or Nanopore platform. Importantly, JACUSA2 can integrate information from multiple experiments, such as replicates and different conditions, and different library types, such as first- or second-strand cDNA libraries. We demonstrate its utility, showing analysis workflows for N6-methyladenosine (m6A) and pseudouridine (Ψ) detection on Illumina and Nanopore sequencing data sets. Our software and its R helper package are available as open source solutions.

Summary: CSNK2B encodes for casein kinase II subunit beta (CK2β), the regulatory subunit of casein kinase II (CK2), which is known to mediate diverse cellular pathways. Variants in this gene have been recently identified as a cause of Poirier-Bienvenu neurodevelopmental syndrome (POBINDS), but functional evidence is sparse. Here, we report five unrelated individuals: two of them manifesting POBINDS, while three are identified to segregate a new intellectual disability-craniodigital syndrome (IDCS), distinct from POBINDS. The three IDCS individuals carried two different de novo missense variants affecting the same codon of CSNK2B. Both variants, NP_001311.3; p.Asp32His and NP_001311.3; p.Asp32Asn, lead to an upregulation of CSNK2B expression at transcript and protein level, along with global dysregulation of canonical Wnt signaling. We found impaired interaction of the two key players DVL3 and β-catenin with mutated CK2β. The variants compromise the kinase activity of CK2 as evident by a marked reduction of phosphorylated β-catenin and consequent absence of active β-catenin inside nuclei of the patient-derived lymphoblastoid cell lines (LCLs). In line with these findings, whole-transcriptome profiling of patient-derived LCLs harboring the NP_001311.3; p.Asp32His variant confirmed a marked difference in expression of genes involved in the Wnt signaling pathway. In addition, whole-phosphoproteome analysis of the LCLs of the same subject showed absence of phosphorylation for 313 putative CK2 substrates, enriched in the regulation of nuclear β-catenin and transcription of the target genes. Our findings suggest that discrete variants in CSNK2B cause dominant-negative perturbation of the canonical Wnt signaling pathway, leading to a new craniodigital syndrome distinguishable from POBINDS.

Mitochondrial DNA (mtDNA) is particularly vulnerable to somatic mutagenesis. Potential mechanisms include DNA polymerase γ (POLG) errors and the effects of mutagens, such as reactive oxygen species. Here, we studied the effects of transient hydrogen peroxide (H2O2 pulse) on mtDNA integrity in cultured HEK 293 cells, applying Southern blotting, ultra-deep short-read and long-read sequencing. In wild-type cells, 30 min after the H2O2 pulse, linear mtDNA fragments appear, representing double-strand breaks (DSB) with ends characterized by short GC stretches. Intact supercoiled mtDNA species reappear within 2–6 h after treatment and are almost completely recovered after 24 h. BrdU incorporation is lower in H2O2-treated cells compared to non-treated cells, suggesting that fast recovery is not associated with mtDNA replication, but is driven by rapid repair of single-strand breaks (SSBs) and degradation of DSB-generated linear fragments. Genetic inactivation of mtDNA degradation in exonuclease deficient POLG p.D274A mutant cells results in the persistence of linear mtDNA fragments with no impact on the repair of SSBs. In conclusion, our data highlight the interplay between the rapid processes of SSB repair and DSB degradation and the much slower mtDNA re-synthesis after oxidative damage, which has important implications for mtDNA quality control and the potential generation of somatic mtDNA deletions.

Abstract Cell-free DNA (cfDNA) analysis has become essential in cancer diagnostics and prenatal testing. We present cfNOMe, a two-in-one method of measuring cfDNA cytosine methylation and nucleosome occupancy in a single assay using non-disruptive enzymatic cytosine conversion and a custom bioinformatic pipeline. We show that enzymatic cytosine conversion better preserves cfDNA fragmentation information than does bisulfite conversion. Whereas previously separate experiments were required to study either epigenetic marking, cfNOMe delivers reliable results for both, enabling more comprehensive and inexpensive epigenetic cfDNA profiling. cfNOMe has the potential to advance biomarker discovery and diagnostic usage in diseases with systemic perturbations of cfDNA composition.

Single‐cell transcriptomics have revolutionized our understanding of the cell composition of tumors and allowed us to identify new subtypes of cells. Despite rapid technological advancements, single‐cell analysis remains resource‐intense hampering the scalability that is required to profile a sufficient number of samples for clinical associations. Therefore, more scalable approaches are needed to understand the contribution of individual cell types to the development and treatment response of solid tumors such as esophageal adenocarcinoma where comprehensive genomic studies have only led to a small number of targeted therapies. Due to the limited treatment options and late diagnosis, esophageal adenocarcinoma has a poor prognosis. Understanding the interaction between and dysfunction of individual cell populations provides an opportunity for the development of new interventions. In an attempt to address the technological and clinical needs, we developed a protocol for the separation of esophageal carcinoma tissue into leukocytes (CD45+), epithelial cells (EpCAM+), and fibroblasts (two out of PDGFRα, CD90, anti‐fibroblast) by fluorescence‐activated cell sorting and subsequent RNA sequencing. We confirm successful separation of the three cell populations by mapping their transcriptomic profiles to reference cell lineage expression data. Gene‐level analysis further supports the isolation of individual cell populations with high expression of CD3, CD4, CD8, CD19, and CD20 for leukocytes, CDH1 and MUC1 for epithelial cells, and FAP, SMA, COL1A1, and COL3A1 for fibroblasts. As a proof of concept, we profiled tumor samples of nine patients and explored expression differences in the three cell populations between tumor and normal tissue. Interestingly, we found that angiogenesis‐related genes were upregulated in fibroblasts isolated from tumors compared with normal tissue. Overall, we suggest our protocol as a complementary and more scalable approach compared with single‐cell RNA sequencing to investigate associations between clinical parameters and transcriptomic alterations of specific cell populations in esophageal adenocarcinoma.

Abstract Background Mismatch repair (MMR)-deficiency increases the risk of colorectal tumorigenesis. To determine whether the tumors develop on a normal or disturbed epigenetic background and how radiation affects this, we quantified genome-wide histone H3 methylation profiles in macroscopic normal intestinal tissue of young radiated and untreated MMR-deficient VCMsh2 LoxP/LoxP (Msh2 −/− ) mice months before tumor onset. Results Histone H3 methylation increases in Msh2 −/− compared to control Msh2 +/+ mice. Activating H3K4me3 and H3K36me3 histone marks frequently accumulate at genes that are H3K27me3 or H3K4me3 modified in Msh2 +/+ mice, respectively. The genes recruiting H3K36me3 enrich in gene sets associated with DNA repair, RNA processing, and ribosome biogenesis that become transcriptionally upregulated in the developing tumors. A similar epigenetic effect is present in Msh2 +/+ mice 4 weeks after a single-radiation hit, whereas radiation of Msh2 −/− mice left their histone methylation profiles almost unchanged. Conclusions MMR deficiency results in genome-wide changes in histone H3 methylation profiles preceding tumor development. Similar changes constitute a persistent epigenetic signature of radiation-induced DNA damage.

T-cell prolymphocytic leukemia (T-PLL) is a poor-prognostic mature T-cell malignancy. It typically presents with exponentially rising lymphocyte counts, splenomegaly, and bone marrow infiltration. Effective treatment options are scarce and a better understanding of TPLL’s pathogenesis is desirable. Activation of the TCL1 proto-oncogene and loss-of-function perturbations of the tumor suppressor ATM are TPLL’s genomic hallmarks. The leukemic cell reveals a phenotype of active T-cell receptor (TCR) signaling and aberrant DNA damage responses. Regulatory networks based on the profile of microRNA (miR) have not been described for T-PLL. In a combined approach of small-RNA and transcriptome sequencing in 46 clinically and moleculary well-characterized T-PLL, we identified a global T-PLL-specific miR expression profile that involves 34 significantly deregulated miR species. This pattern strikingly resembled miR-ome signatures of TCR-activated T cells. By integrating these T-PLL miR profiles with transcriptome data, we uncovered regulatory networks associated with cell survival signaling and DNA damage response pathways. Despite a miR-ome that discerned leukemic from normal T cells, there were also robust subsets of T-PLL defined by a small set of specific miR. Most prominently, miR-141 and the miR- 200c-cluster separated cases into two major subgroups. Furthermore, increased expression of miR-223-3p as well as reduced expression of miR-21 and the miR-29 cluster were associated with more activated Tcell phenotypes and more aggressive disease presentations. Based on the implicated pathobiological role of these miR deregulations, targeting strategies around their effectors appear worth pursuing. We also established a combinatorial miR-based overall survival score for T-PLL (miROS-T-PLL), that might improve current clinical stratifications.

Abstract The prevalence of germ line mutations in non‐BRCA1/2 genes associated with hereditary breast cancer (BC) is low, and the role of some of these genes in BC predisposition and pathogenesis is conflicting. In this study, 5589 consecutive BC index patients negative for pathogenic BRCA1/2 mutations and 2189 female controls were screened for germ line mutations in eight cancer predisposition genes (ATM, CDH1, CHEK2, NBN, PALB2, RAD51C, RAD51D, and TP53). All patients met the inclusion criteria of the German Consortium for Hereditary Breast and Ovarian Cancer for germ line testing. The highest mutation prevalence was observed in the CHEK2 gene (2.5%), followed by ATM (1.5%) and PALB2 (1.2%). The mutation prevalence in each of the remaining genes was 0.3% or lower. Using Exome Aggregation Consortium control data, we confirm significant associations of heterozygous germ line mutations with BC for ATM (OR: 3.63, 95%CI: 2.67–4.94), CDH1 (OR: 17.04, 95%CI: 3.54–82), CHEK2 (OR: 2.93, 95%CI: 2.29–3.75), PALB2 (OR: 9.53, 95%CI: 6.25–14.51), and TP53 (OR: 7.30, 95%CI: 1.22–43.68). NBN germ line mutations were not significantly associated with BC risk (OR:1.39, 95%CI: 0.73–2.64). Due to their low mutation prevalence, the RAD51C and RAD51D genes require further investigation. Compared with control datasets, predicted damaging rare missense variants were significantly more prevalent in CHEK2 and TP53 in BC index patients. Compared with the overall sample, only TP53 mutation carriers show a significantly younger age at first BC diagnosis. We demonstrate a significant association of deleterious variants in the CHEK2, PALB2, and TP53 genes with bilateral BC. Both, ATM and CHEK2, were negatively associated with triple‐negative breast cancer (TNBC) and estrogen receptor (ER)‐negative tumor phenotypes. A particularly high CHEK2 mutation prevalence (5.2%) was observed in patients with human epidermal growth factor receptor 2 (HER2)‐positive tumors.

Abstract Background Serrated or Hyperplastic Polyposis Syndrome (SPS, HPS) is a yet poorly defined colorectal cancer (CRC) predisposition characterised by the occurrence of multiple and/or large serrated polyps throughout the colon. A serrated polyp-CRC sequence (serrated pathway) of CRC formation has been postulated, however, to date only few molecular signatures of serrated neoplasia (BRAF, KRAS, RNF43 mutations, CpG Island Methylation, MSI) have been described in a subset of SPS patients and neither the etiology of the syndrome nor the distinct genetic alterations during tumorigenesis have been identified. Methods To identify somatic point mutations in potential novel candidate genes of SPS-associated lesions and the involved pathways we performed exome sequencing of eleven early serrated polyps obtained from a 41 year-old female patient with clinically confirmed SPS. For data filtering and analysis, standard pipelines were used. Somatic mutations were identified by comparison with leukocyte DNA and were validated by Sanger sequencing. Results The BRAF p.V600E or KRAS p.G12D mutation was identified in six polyps (~50%) and not found in polyps from the distal colon. In addition, we found seven unique rare somatic alterations of seven different genes in four serrated tumours, all of which are missense variants. The variant in ABI3BP and CATSPERB are predicted to be deleterious. No established cancer gene or candidate genes related to serrated tumorigenesis were affected. Conclusions Somatic mutations seem to be rare events in early hyperplastic and serrated lesions of SPS patients. Neither frequently affected genes nor enrichment of specific pathways were observed. Thus, other alterations such as non-coding variants or epigenetic changes might be the major driving force of tumour progression in SPS.

Olfactory receptors (ORs) are known to be expressed in a variety of human tissues and act on different physiological processes, such as cell migration, proliferation, or secretion and have been found to function as biomarkers for carcinoma tissues of prostate, lung, and small intestine. In this study, we analyzed the OR expression profiles of several different carcinoma tissues, with a focus on breast cancer. The expression of OR2B6 was detectable in breast carcinoma tissues; here, transcripts of OR2B6 were detected in 73% of all breast carcinoma cell lines and in over 80% of all of the breast carcinoma tissues analyzed. Interestingly, there was no expression of OR2B6 observed in healthy tissues. Immunohistochemical staining of OR2B6 in breast carcinoma tissues revealed a distinct staining pattern of carcinoma cells. Furthermore, we detected a fusion transcript containing part of the coding exon of OR2B6 as a part of a splice variant of the histone HIST1H2BO transcript. In addition, in cancer tissues and cell lines derived from lung, pancreas, and brain, OR expression patterns were compared to that of corresponding healthy tissues. The number of ORs detected in lung carcinoma tissues was significantly reduced in comparison to the surrounding healthy tissues. In pancreatic carcinoma tissues, OR4C6 was considerably more highly expressed in comparison to the respective healthy tissues. We detected OR2B6 as a potential biomarker for breast carcinoma tissues.

Genetic Generalized Epilepsy (GGE) and benign epilepsy with centro-temporal spikes or Rolandic Epilepsy (RE) are common forms of genetic epilepsies. Rare copy number variants have been recognized as important risk factors in brain disorders. We performed a systematic survey of rare deletions affecting protein-coding genes derived from exome data of patients with common forms of genetic epilepsies. We analysed exomes from 390 European patients (196 GGE and 194 RE) and 572 population controls to identify low-frequency genic deletions. We found that 75 (32 GGE and 43 RE) patients out of 390, i.e. ~19%, carried rare genic deletions. In particular, large deletions (>400 kb) represent a higher burden in both GGE and RE syndromes as compared to controls. The detected low-frequency deletions (1) share genes with brain-expressed exons that are under negative selection, (2) overlap with known autism and epilepsy-associated candidate genes, (3) are enriched for CNV intolerant genes recorded by the Exome Aggregation Consortium (ExAC) and (4) coincide with likely disruptive de novo mutations from the NPdenovo database. Employing several knowledge databases, we discuss the most prominent epilepsy candidate genes and their protein-protein networks for GGE and RE.

In the arcuate nucleus of the hypothalamus (ARH) satiety signaling (anorexigenic) pro-opiomelanocortin (POMC)-expressing and hunger signaling (orexigenic) agouti-related peptide (AgRP)-expressing neurons are key components of the neuronal circuits that control food intake and energy homeostasis. Here, we assessed whether the catecholamine noradrenalin directly modulates the activity of these neurons in mice. Perforated patch clamp recordings showed that noradrenalin changes the activity of these functionally antagonistic neurons in opposite ways, increasing the activity of the orexigenic NPY/AgRP neurons and decreasing the activity of the anorexigenic POMC neurons. Cell type-specific transcriptomics and pharmacological experiments revealed that the opposing effect on these neurons is mediated by the activation of excitatory α1A - and β- adrenergic receptors in NPY/AgRP neurons, while POMC neurons are inhibited via α2A – adrenergic receptors. Thus, the coordinated differential modulation of the key hypothalamic neurons in control of energy homeostasis assigns noradrenalin an important role to promote feeding.

The analysis and functional characterization of ectopically expressed human olfactory receptors (ORs) is becoming increasingly important, as many ORs have been identified in several healthy and cancerous tissues. OR activation has been demonstrated to have influence on cancer cell growth and progression. Here, ORs were identified using RNA-Seq analyses and RT-PCR. We demonstrated the OR protein localization in HCT116 cells using immunocytochemistry (IHC). In order to analyze the physiological role of OR51B4, we deorphanized the receptor by the use of CRE-Luciferase assays, conducted calcium imaging experiments as well as scratch- and proliferation assays. Furthermore, western blot analyses revealed the involvement of different protein kinases in the ligand-dependent signaling pathway. Receptor knockdown via shRNA was used to analyze the involvement of OR51B4. We identified OR51B4, which is highly expressed in the colon cancer cell line HCT116 and in native human colon cancer tissues. We deorphanized the receptor and identified Troenan as an effective ligand. Troenan stimulation of HCT116 cells has anti-proliferative, anti-migratory and pro-apoptotic effects, mediated by changes in the intracellular calcium level upon PLC activation. These effects cause changes in the phosphorylation levels of p38, mTor and Akt kinases. Knockdown of the receptor via shRNA confirmed the involvement of OR51B4. This study emphasizes the importance of ectopically expressed ORs in the therapy for several diseases. The findings provide the basis for alternative treatments of colorectal cancer.

Identification of families at risk for ovarian cancer offers the opportunity to consider prophylactic surgery thus reducing ovarian cancer mortality. So far, identification of potentially affected families in Germany was solely performed via family history and numbers of affected family members with breast or ovarian cancer. However, neither the prevalence of deleterious variants in BRCA1/2 in ovarian cancer in Germany nor the reliability of family history as trigger for genetic counselling has ever been evaluated.Prospective counseling and germline testing of consecutive patients with primary diagnosis or with platinum-sensitive relapse of an invasive epithelial ovarian cancer. Testing included 25 candidate and established risk genes. Among these 25 genes, 16 genes (ATM, BRCA1, BRCA2, CDH1, CHEK2, MLH1, MSH2, MSH6, NBN, PMS2, PTEN, PALB2, RAD51C, RAD51D, STK11, TP53) were defined as established cancer risk genes. A positive family history was defined as at least one relative with breast cancer or ovarian cancer or breast cancer in personal history.In total, we analyzed 523 patients: 281 patients with primary diagnosis of ovarian cancer and 242 patients with relapsed disease. Median age at primary diagnosis was 58 years (range 16-93) and 406 patients (77.6%) had a high-grade serous ovarian cancer. In total, 27.9% of the patients showed at least one deleterious variant in all 25 investigated genes and 26.4% in the defined 16 risk genes. Deleterious variants were most prevalent in the BRCA1 (15.5%), BRCA2 (5.5%), RAD51C (2.5%) and PALB2 (1.1%) genes. The prevalence of deleterious variants did not differ significantly between patients at primary diagnosis and relapse. The prevalence of deleterious variants in BRCA1/2 (and in all 16 risk genes) in patients

The molecular pathways underlying tumor suppression are incompletely understood. Here, we identify cooperative non-cell-autonomous functions of a single gene that together provide a novel mechanism of tumor suppression in basal keratinocytes of zebrafish embryos. A loss-of-function mutation in atp1b1a, encoding the beta subunit of a Na,K-ATPase pump, causes edema and epidermal malignancy. Strikingly, basal cell carcinogenesis only occurs when Atp1b1a function is compromised in both the overlying periderm (resulting in compromised epithelial polarity and adhesiveness) and in kidney and heart (resulting in hypotonic stress). Blockade of the ensuing PI3K-AKT-mTORC1-NFκB-MMP9 pathway activation in basal cells, as well as systemic isotonicity, prevents malignant transformation. Our results identify hypotonic stress as a (previously unrecognized) contributor to tumor development and establish a novel paradigm of tumor suppression.

Massively parallel sequencing of whole genomes and exomes has facilitated a direct assessment of causative genetic variation, now enabling the identification of genetic factors involved in rare diseases (RD) with Mendelian inheritance patterns on an almost routine basis. Here, we describe the illustrative case of a single consanguineous family where this strategy suffered from the difficulty to distinguish between two etiologically distinct disorders, namely the co-occurrence of hereditary hypophosphatemic rickets (HRR) and congenital myopathies (CM), by their phenotypic manifestation alone. We used parametric linkage analysis, homozygosity mapping and whole exome-sequencing to identify mutations underlying HRR and CM. We also present an approximate approach for assessing the probability of co-occurrence of two unlinked recessive RD in a single family as a function of the degree of consanguinity and the frequency of the disease-causing alleles. Linkage analysis and homozygosity mapping yielded elusive results when assuming a single RD, but whole-exome sequencing helped to identify two mutations in two genes, namely SLC34A3 and SEPN1, that segregated independently in this family and that have previously been linked to two etiologically different diseases. We assess the increase in chance co-occurrence of rare diseases due to consanguinity, i.e. under circumstances that generally favor linkage mapping of recessive disease, and show that this probability can increase by several orders of magnitudes. We conclude that such potential co-occurrence represents an underestimated risk when analyzing rare or undefined diseases in consanguineous families and should be given more consideration in the clinical and genetic evaluation.

Here, we present the first complete mitochondrial genome of the Red Tailed Loach Yasuhikotakia modesta (Teleostei: Botiidae) from Thailand, assembled from next-generation transcriptome sequencing data. The assembled transcript corresponds to the full length mitochondrial genome of Y. modesta, which measured 16,865 bp in length, and contained 13 protein-coding genes, 2 ribosomal RNA genes, and 22 transfer RNA genes. A slight A + T bias was observed in the mitogenome of Y. modesta with an overall base composition of 32.2% A, 25.8% T, 26.4% C, and 15.4% G, and a GC content of 41.8%. The gene arrangement was identical to that of previously described loach mitogenomes.

The chemosensory capacity of the somatosensory system relies on the appropriate expression of chemoreceptors, which detect chemical stimuli and transduce sensory information into cellular signals. Knowledge of the complete repertoire of the chemoreceptors expressed in human sensory ganglia is lacking. This study employed the next-generation sequencing technique (RNA-Seq) to conduct the first expression analysis of human trigeminal ganglia (TG) and dorsal root ganglia (DRG). We analyzed the data with a focus on G-protein coupled receptors (GPCRs) and ion channels, which are (potentially) involved in chemosensation by somatosensory neurons in the human TG and DRG. For years, transient receptor potential (TRP) channels have been considered the main group of receptors for chemosensation in the trigeminal system. Interestingly, we could show that sensory ganglia also express a panel of different olfactory receptors (ORs) with putative chemosensory function. To characterize OR expression in more detail, we performed microarray, semi-quantitative RT-PCR experiments, and immunohistochemical staining. Additionally, we analyzed the expression data to identify further known or putative classes of chemoreceptors in the human TG and DRG. Our results give an overview of the major classes of chemoreceptors expressed in the human TG and DRG and provide the basis for a broader understanding of the reception of chemical cues.

The ability of animals to sense and differentiate among thousands of odorants relies on a large set of olfactory receptors (OR) and a multitude of accessory proteins within the olfactory epithelium (OE). ORs and related signaling mechanisms have been the subject of intensive studies over the past years, but our knowledge regarding olfactory processing remains limited. The recent development of next generation sequencing (NGS) techniques encouraged us to assess the transcriptome of the murine OE. We analyzed RNA from OEs of female and male adult mice and from fluorescence-activated cell sorting (FACS)-sorted olfactory receptor neurons (ORNs) obtained from transgenic OMP-GFP mice. The Illumina RNA-Seq protocol was utilized to generate up to 86 million reads per transcriptome. In OE samples, nearly all OR and trace amine-associated receptor (TAAR) genes involved in the perception of volatile amines were detectably expressed. Other genes known to participate in olfactory signaling pathways were among the 200 genes with the highest expression levels in the OE. To identify OE-specific genes, we compared olfactory neuron expression profiles with RNA-Seq transcriptome data from different murine tissues. By analyzing different transcript classes, we detected the expression of non-olfactory GPCRs in ORNs and established an expression ranking for GPCRs detected in the OE. We also identified other previously undescribed membrane proteins as potential new players in olfaction. The quantitative and comprehensive transcriptome data provide a virtually complete catalogue of genes expressed in the OE and present a useful tool to uncover candidate genes involved in, for example, olfactory signaling, OR trafficking and recycling, and proliferation.

In excess of 12% of human cancer incidents have a viral cofactor. Epidemiological studies of idiopathic human cancers indicate that additional tumor viruses remain to be discovered. Recent advances in sequencing technology have enabled systematic screenings of human tumor transcriptomes for viral transcripts. However, technical problems such as low abundances of viral transcripts in large volumes of sequencing data, viral sequence divergence, and homology between viral and human factors significantly confound identification of tumor viruses. We have developed a novel computational approach for detecting viral transcripts in human cancers that takes the aforementioned confounding factors into account and is applicable to a wide variety of viruses and tumors. We apply the approach to conducting the first systematic search for viruses in neuroblastoma, the most common cancer in infancy. The diverse clinical progression of this disease as well as related epidemiological and virological findings are highly suggestive of a pathogenic cofactor. However, a viral etiology of neuroblastoma is currently contested. We mapped 14 transcriptomes of neuroblastoma as well as positive and negative controls to the human and all known viral genomes in order to detect both known and unknown viruses. Analysis of controls, comparisons with related methods, and statistical estimates demonstrate the high sensitivity of our approach. Detailed investigation of putative viral transcripts within neuroblastoma samples did not provide evidence for the existence of any known human viruses. Likewise, de-novo assembly and analysis of chimeric transcripts did not result in expression signatures associated with novel human pathogens. While confounding factors such as sample dilution or viral clearance in progressed tumors may mask viral cofactors in the data, in principle, this is rendered less likely by the high sensitivity of our approach and the number of biological replicates analyzed. Therefore, our results suggest that frequent viral cofactors of metastatic neuroblastoma are unlikely.

The specific functions of sensory systems depend on the tissue-specific expression of genes that code for molecular sensor proteins that are necessary for stimulus detection and membrane signaling. Using the Next Generation Sequencing technique (RNA-Seq), we analyzed the complete transcriptome of the trigeminal ganglia (TG) and dorsal root ganglia (DRG) of adult mice. Focusing on genes with an expression level higher than 1 FPKM (fragments per kilobase of transcript per million mapped reads), we detected the expression of 12984 genes in the TG and 13195 in the DRG. To analyze the specific gene expression patterns of the peripheral neuronal tissues, we compared their gene expression profiles with that of the liver, brain, olfactory epithelium, and skeletal muscle. The transcriptome data of the TG and DRG were scanned for virtually all known G-protein-coupled receptors (GPCRs) as well as for ion channels. The expression profile was ranked with regard to the level and specificity for the TG. In total, we detected 106 non-olfactory GPCRs and 33 ion channels that had not been previously described as expressed in the TG. To validate the RNA-Seq data, in situ hybridization experiments were performed for several of the newly detected transcripts. To identify differences in expression profiles between the sensory ganglia, the RNA-Seq data of the TG and DRG were compared. Among the differentially expressed genes (> 1 FPKM), 65 and 117 were expressed at least 10-fold higher in the TG and DRG, respectively. Our transcriptome analysis allows a comprehensive overview of all ion channels and G protein-coupled receptors that are expressed in trigeminal ganglia and provides additional approaches for the investigation of trigeminal sensing as well as for the physiological and pathophysiological mechanisms of pain.

In addition to the canonical olfactory receptors, TAARs were currently suggested to be a second class of chemosensory receptors in the olfactory epithelium of vertebrates. In contrast to several deorphanized murine TAARs, agonists for the intact human TAAR genes 2, 5, 6, 8 and 9 that are potentially expressed in the human olfactory epithelium have not been determined so far. Moreover, the physiological relevance of TAARs still remains elusive. We present the first successful functional expression of a human TAAR and agonists of human TAAR5. We performed a ligand screening using recombinantly expressed human TAAR5 in HANA3A cells and Xenopus laevis oocytes. In order to measure receptor activity, we used a cAMP-dependent reporter gene assay and two-electrode voltage clamp technique. As a result, human TAAR5 can be activated in a concentration-dependent manner by trimethylamine and with less efficacy by dimethylethylamine. It could neither be activated by any other of the tested single amines with a related chemical structure (42 in total), nor by any of the tested odorant mixtures. The hypothesis that Single Nucleotide Polymorphisms (SNP) within the reading frame of an olfactory receptor gene can cause a specific anosmia, formed the basis for clarifying the question, if anosmia for trimethylamine is caused by a SNP in a TAAR coding sequence. All functional human TAAR gene reading frames of subjects with specific anosmia for trimethylamine were amplified and products analyzed regarding SNP distribution. We demonstrated that the observed specific anosmia for trimethylamine is not correlated with a SNP in the coding sequence of one of the putatively functional human TAAR genes.

Intracellular Cl(-) concentrations ([Cl(-)](i)) of sensory neurons regulate signal transmission and signal amplification. In dorsal root ganglion (DRG) and olfactory sensory neurons (OSNs), Cl(-) is accumulated by the Na(+)-K(+)-2Cl(-) cotransporter 1 (NKCC1), resulting in a [Cl(-)](i) above electrochemical equilibrium and a depolarizing Cl(-) efflux upon Cl(-) channel opening. Here, we investigate the [Cl(-)](i) and function of Cl(-) in primary sensory neurons of trigeminal ganglia (TG) of wild type (WT) and NKCC1(-/-) mice using pharmacological and imaging approaches, patch-clamping, as well as behavioral testing. The [Cl(-)](i) of WT TG neurons indicated active NKCC1-dependent Cl(-) accumulation. Gamma-aminobutyric acid (GABA)(A) receptor activation induced a reduction of [Cl(-)](i) as well as Ca(2+) transients in a corresponding fraction of TG neurons. Ca(2+) transients were sensitive to inhibition of NKCC1 and voltage-gated Ca(2+) channels (VGCCs). Ca(2+) responses induced by capsaicin, a prototypical stimulus of transient receptor potential vanilloid subfamily member-1 (TRPV1) were diminished in NKCC1(-/-) TG neurons, but elevated under conditions of a lowered [Cl(-)](o) suggesting a Cl(-)-dependent amplification of capsaicin-induced responses. Using next generation sequencing (NGS), we found expression of different Ca(2+)-activated Cl(-) channels (CaCCs) in TGs of mice. Pharmacological inhibition of CaCCs reduced the amplitude of capsaicin-induced responses of TG neurons in Ca(2+) imaging and electrophysiological recordings. In a behavioral paradigm, NKCC1(-/-) mice showed less avoidance of the aversive stimulus capsaicin. In summary, our results strongly argue for a Ca(2+)-activated Cl(-)-dependent signal amplification mechanism in TG neurons that requires intracellular Cl(-) accumulation by NKCC1 and the activation of CaCCs.

Treatment of EGFR-mutant non-small cell lung cancer patients with the tyrosine kinase inhibitors erlotinib or gefitinib results in high response rates and prolonged progression-free survival. Despite the development of sensitive mutation detection approaches, a thorough validation of these in a clinical setting has so far been lacking. We performed, in a clinical setting, a systematic validation of dideoxy 'Sanger' sequencing and pyrosequencing against massively parallel sequencing as one of the most sensitive mutation detection technologies available. Mutational annotation of clinical lung tumor samples revealed that of all patients with a confirmed response to EGFR inhibition, only massively parallel sequencing detected all relevant mutations. By contrast, dideoxy sequencing missed four responders and pyrosequencing missed two responders, indicating a dramatic lack of sensitivity of dideoxy sequencing, which is widely applied for this purpose. Furthermore, precise quantification of mutant alleles revealed a low correlation (r(2) = 0.27) of histopathological estimates of tumor content and frequency of mutant alleles, thereby questioning the use of histopathology for stratification of specimens for individual analytical procedures. Our results suggest that enhanced analytical sensitivity is critically required to correctly identify patients responding to EGFR inhibition. More broadly, our results emphasize the need for thorough evaluation of all mutation detection approaches against massively parallel sequencing as a prerequisite for any clinical implementation.

Abstract Background Telomere maintenance mechanisms (TMM) are a hallmark of high-risk neuroblastoma, and are conferred by activation of telomerase or alternative lengthening of telomeres (ALT). However, detection of TMM is not yet part of the clinical routine, and consensus on TMM detection, especially on ALT assessment, remains to be achieved. Methods Whole genome sequencing (WGS) data of 68 primary neuroblastoma samples were analyzed. Telomere length was calculated from WGS data or by telomere restriction fragment analysis (n = 39). ALT was assessed by C-circle assay (CCA, n = 67) and detection of ALT-associated PML nuclear bodies (APB) by combined fluorescence in situ hybridization and immunofluorescence staining (n = 68). RNA sequencing was performed (n = 64) to determine expression of TERT and telomeric long non-coding RNA (TERRA). Telomerase activity was examined by telomerase repeat amplification protocol (TRAP, n = 15). Results Tumors were considered as telomerase-positive if they harbored a TERT rearrangement, MYCN amplification or high TERT expression (45.6%, 31/68), and ALT-positive if they were positive for APB and CCA (19.1%, 13/68). If all these markers were absent, tumors were considered TMM-negative (25.0%, 17/68). According to these criteria, the majority of samples were classified unambiguously (89.7%, 61/68). Assessment of additional ALT-associated parameters clarified the TMM status of the remaining seven cases with high likelihood: ALT-positive tumors had higher TERRA expression, longer telomeres, more telomere insertions, a characteristic pattern of telomere variant repeats, and were associated with ATRX mutations. Conclusions We here propose a workflow to reliably detect TMM in neuroblastoma. We show that unambiguous classification is feasible following a stepwise approach that determines both, activation of telomerase and ALT. The workflow proposed in this study can be used in clinical routine and provides a framework to systematically and reliably determine telomere maintenance mechanisms for risk stratification and treatment allocation of neuroblastoma patients.

Abstract Activation of MAPK signaling via BRAF mutations may limit the activity of EGFR inhibitors in EGFR-mutant lung cancer patients. However, the impact of BRAF mutations on the selection and fitness of emerging resistant clones during anti-EGFR therapy remains elusive. We tracked the evolution of subclonal mutations by whole-exome sequencing and performed clonal analyses of individual metastases during therapy. Complementary functional analyses of polyclonal EGFR-mutant cell pools showed a dose-dependent enrichment of BRAF V600E and a loss of EGFR inhibitor susceptibility. The clones remain stable and become vulnerable to combined EGFR, RAF, and MEK inhibition. Moreover, only osimertinib/trametinib combination treatment, but not monotherapy with either of these drugs, leads to robust tumor shrinkage in EGFR-driven xenograft models harboring BRAF V600E mutations. These data provide insights into the dynamics of clonal evolution of EGFR-mutant tumors and the therapeutic implications of BRAF co-mutations that may facilitate the development of treatment strategies to improve the prognosis of these patients.

Objective: Nonalcoholic fatty liver disease (NAFLD) ranges from steatosis to nonalcoholic steatohepatitis (NASH), which often progresses to hepatocellular carcinoma (HCC) through a largely undefined mechanism. NASH and HCC depend on inflammatory signaling, whose master regulator is the NFκB transcription factor family, activated by canonical and non-canonical pathways. Methods: Here, we investigated non-canonical NFκB-inducing kinase (NIK/MAP3K14) in metabolic NASH, NASH to HCC transition, and DEN-induced HCC. To this end, we performed dietary and chemical interventions in mice that were analyzed via single nucleus sequencing, gene expression and histochemical methods. Ultimately, we verified our mouse results in human patient samples. Results: We revealed that hepatocyte-specific NIK deficiency (NIKLKO) ameliorated metabolic NASH complications and reduced hepatocarcinogenesis, independent of its role in the NFκB pathway. Instead, hepatic NIK attenuated hepatoprotective JAK2/STAT5 signaling that is a prerequisite for NASH and NASH to HCC progression in mice and humans. Conclusions: Our data suggest NIK-mediated inhibitory JAK2 phosphorylation at serine 633 that might be amenable for future therapeutic interventions in patients.

STAG2 is a component of the large, evolutionarily highly conserved cohesin complex, which has been linked to various cellular processes like genome organization, DNA replication, gene expression, heterochromatin formation, sister chromatid cohesion, and DNA repair. A wide spectrum of germline variants in genes encoding subunits or regulators of the cohesin complex have previously been identified to cause distinct but phenotypically overlapping multisystem developmental disorders belonging to the group of cohesinopathies. Pathogenic variants in STAG2 have rarely been implicated in an X-linked cohesinopathy associated with undergrowth, developmental delay, and dysmorphic features. Here, we describe for the first time a mosaic STAG2 variant in an individual with developmental delay, microcephaly, and hemihypotrophy of the right side. We characterized the grade of mosaicism by deep sequencing analysis on DNA extracted from EDTA blood, urine and buccal swabs. Furthermore, we report an additional female with a novel de novo splice variant in STAG2. Interestingly, both individuals show supernumerary nipples, a feature that has not been reported associated to STAG2 before. Remarkably, additional analysis of STAG2 transcripts in both individuals showed only wildtype transcripts, even after blockage of nonsense-mediated decay using puromycin in blood lymphocytes. As the phenotype of STAG2-associated cohesinopathies is dominated by global developmental delay, severe microcephaly, and brain abnormalities, we investigated the expression of STAG2 and other related components of the cohesin complex during Bioengineered Neuronal Organoids (BENOs) generation by RNA sequencing. Interestingly, we observed a prominent expression of STAG2, especially between culture days 0 and 15, indicating an essential function of STAG2 in early brain development. In summary, we expand the genotypic and phenotypic spectrum of STAG2-associated cohesinopathies and show that BENOs represent a promising model to gain further insights into the critical role of STAG2 in the complex process of nervous system development.

We introduce Single-cell Nanopore Spatial Transcriptomics (scNaST), a software suite to facilitate the analysis of spatial gene expression from second- and third-generation sequencing, allowing to generate a full-length near-single-cell transcriptional landscape of the tissue microenvironment. Taking advantage of the Visium Spatial platform, we adapted a strategy recently developed to assign barcodes to long-read single-cell sequencing data for spatial capture technology. Here, we demonstrate our workflow using four short axis sections of the mouse heart following myocardial infarction. We constructed a de novo transcriptome using long-read data, and successfully assigned 19,794 transcript isoforms in total, including clinically-relevant, but yet uncharacterized modes of transcription, such as intron retention or antisense overlapping transcription. We showed a higher transcriptome complexity in the healthy regions, and identified intron retention as a mode of transcription associated with the infarct area. Our data revealed a clear regional isoform switching among differentially used transcripts for genes involved in cardiac muscle contraction and tissue morphogenesis. Molecular signatures involved in cardiac remodeling integrated with morphological context may support the development of new therapeutics towards the treatment of heart failure and the reduction of cardiac complications.

Abstract Spatial organization and gene expression of mammalian chromosomes are maintained and regulated in conjunction with cell cycle progression. This is perturbed once cells enter senescence and the highly abundant HMGB1 protein is depleted from nuclei to act as an extracellular proinflammatory stimulus. Despite its physiological importance, we know little about the positioning of HMGB1 on chromatin and its nuclear roles. To address this, we mapped HMGB1 binding genome‐wide in two primary cell lines. We integrated ChIP‐seq and Hi‐C with graph theory to uncover clustering of HMGB1‐marked topological domains that harbor genes involved in paracrine senescence. Using simplified Cross‐Linking and Immuno‐Precipitation and functional tests, we show that HMGB1 is also a bona fide RNA‐binding protein (RBP) binding hundreds of mRNAs. It presents an interactome rich in RBPs implicated in senescence regulation. The mRNAs of many of these RBPs are directly bound by HMGB1 and regulate availability of SASP‐relevant transcripts. Our findings reveal a broader than hitherto assumed role for HMGB1 in coordinating chromatin folding and RNA homeostasis as part of a regulatory loop controlling cell‐autonomous and paracrine senescence.

Abstract Innate immunity triggers responsible for viral control or hyperinflammation in COVID‐19 are largely unknown. Here we show that the SARS‐CoV‐2 spike protein (S‐protein) primes inflammasome formation and release of mature interleukin‐1β (IL‐1β) in macrophages derived from COVID‐19 patients but not in macrophages from healthy SARS‐CoV‐2 naïve individuals. Furthermore, longitudinal analyses reveal robust S‐protein‐driven inflammasome activation in macrophages isolated from convalescent COVID‐19 patients, which correlates with distinct epigenetic and gene expression signatures suggesting innate immune memory after recovery from COVID‐19. Importantly, we show that S‐protein‐driven IL‐1β secretion from patient‐derived macrophages requires non‐specific monocyte pre‐activation in vivo to trigger NLRP3‐inflammasome signaling. Our findings reveal that SARS‐CoV‐2 infection causes profound and long‐lived reprogramming of macrophages resulting in augmented immunogenicity of the SARS‐CoV‐2 S‐protein, a major vaccine antigen and potent driver of adaptive and innate immune signaling.

Degradation of nonsense mediated mRNA decay (NMD) substrates is carried out by two seemingly independent pathways, SMG6-mediated endonucleolytic cleavage and/or SMG5-SMG7-induced accelerated deadenylation. Here the authors show that SMG5-SMG7 maintain NMD activity by permitting SMG6 activation.

XPA-binding protein (XAB)-2 is the human homologue of the yeast pre-mRNA splicing factor Syf1. Here the authors use an in vivo biotinylation tagging approach to show XAB2’s role in DNA repair, RNA splicing and transcription during mammalian development.

Members of the CHD chromatin remodeler family are implicated in human pathologies, however CHD6 remained poorly studied. Here, the authors show that CHD6 binds to and regulates autophagy and stress response genes across cell types. They identify a clinical mutation that affects its ability to recruit cofactors, leading to impaired autophagy induction and DNA repair.

Abstract Faithful genome duplication requires regulation of origin firing to determine loci, timing and efficiency of replisome generation. Established kinase targets for eukaryotic origin firing regulation are the Mcm2-7 helicase, Sld3/Treslin/TICRR and Sld2/RecQL4. We report that metazoan Sld7, MTBP (Mdm2 binding protein), is targeted by at least three kinase pathways. MTBP was phosphorylated at CDK consensus sites by cell cycle cyclin-dependent kinases (CDK) and Cdk8/19-cyclin C. Phospho-mimetic MTBP CDK site mutants, but not non-phosphorylatable mutants, promoted origin firing in human cells. MTBP was also phosphorylated at DNA damage checkpoint kinase consensus sites. Phospho-mimetic mutations at these sites inhibited MTBP’s origin firing capability. Whilst expressing a non-phospho MTBP mutant was insufficient to relieve the suppression of origin firing upon DNA damage, the mutant induced a genome-wide increase of origin firing in unperturbed cells. Our work establishes MTBP as a regulation platform of metazoan origin firing.

Abstract SCNN1B encodes the beta subunit of the epithelial sodium channel ENaC. Previously, we reported an association between SNP markers of SCNN1B gene and disease severity in cystic fibrosis-affected sibling pairs. We hypothesized that factors interacting with the SCNN1B genomic sequence are responsible for intrapair discordance. Concordant and discordant pairs differed at six SCNN1B markers (Praw = 0.0075, Pcorr = 0.0397 corrected for multiple testing). To identify the factors binding to these six SCNN1B SNPs, we performed an electrophoretic mobility shift assay and captured the DNA–protein complexes. Based on protein mass spectrometry data, the epithelial splicing regulatory protein ESRP2 was identified when using SCNN1B-derived probes and the ESRP2-SCNN1B interaction was independently confirmed by coimmunoprecipitation assays. We observed an alternative SCNN1B transcript and demonstrated in 16HBE14o− cells that levels of this transcript are decreased upon ESRP2 silencing by siRNA. Furthermore, we confirmed that mildly and severely affected siblings have different ESPR2 genetic backgrounds and that ESRP2 markers are linked to the response of CF patients’ nasal epithelium to amiloride, indicating ENaC involvement (Pbest = 0.0131, Pcorr = 0.068 for multiple testing). Our findings demonstrate that sibling pairs clinically discordant for CF can be used to identify meaningful DNA regulatory elements and interacting factors.

Abstract Functional studies giving insight into the biology of circulating tumor cells (CTCs) remain scarce due to the low frequency of CTCs and lack of appropriate models. Here, we describe the characterization of a novel CTC‐derived breast cancer cell line, designated CTC‐ITB‐01, established from a patient with metastatic estrogen receptor‐positive (ER+) breast cancer, resistant to endocrine therapy. CTC‐ITB‐01 remained ER+ in culture, and copy number alteration (CNA) profiling showed high concordance between CTC‐ITB‐01 and CTCs originally present in the patient with cancer at the time point of blood draw. RNA‐sequencing data indicate that CTC‐ITB‐01 has a predominantly epithelial expression signature. Primary tumor and metastasis formation in an intraductal PDX mouse model mirrored the clinical progression of ER+ breast cancer. Downstream ER signaling was constitutively active in CTC‐ITB‐01 independent of ligand availability, and the CDK4/6 inhibitor Palbociclib strongly inhibited CTC‐ITB‐01 growth. Thus, we established a functional model that opens a new avenue to study CTC biology.

DNA damage is associated with metabolic disorders, but the mechanism in unclear. Here, the authors show that persistent DNA damage induced by lack of the endonuclease XPF-ERCC1 triggers extracellular vesicle biogenesis in tissue infiltrating macrophages, and that vesicle uptake stimulates glucose uptake in recipient cells, leading to increased inflammation.