BACKGROUND: Early detection of colorectal cancer (CRC) significantly improves its management and patients' survival. Circular RNAs (circRNAs) are peculiar covalently closed transcripts involved in gene expression modulation whose dysregulation has been extensively reported in CRC cells. However, little is known about their alterations in the early phases of colorectal carcinogenesis. METHODS: In this study, we performed an integrative analysis of circRNA profiles in RNA-sequencing (RNA-Seq) data of 96 colorectal cancers, 27 adenomas, and matched adjacent mucosa tissues. We also investigated the levels of cognate linear transcripts and those of regulating RNA-binding proteins (RBPs). Levels of circRNA-interacting microRNAs (miRNAs) were explored by integrating data of small RNA-Seq performed on the same samples. RESULTS: Our results revealed a significant dysregulation of 34 circRNAs (paired adj. p < 0.05), almost exclusively downregulated in tumor tissues and, prevalently, in early disease stages. This downregulation was associated with decreased expression of circRNA host genes and those encoding for RBPs involved in circRNA biogenesis, including NOVA1, RBMS3, and MBNL1. Guilt-by-association analysis showed that dysregulated circRNAs correlated with increased predicted activity of cell proliferation, DNA repair, and c-Myc signaling pathways. Functional analysis showed interactions among dysregulated circRNAs, RBPs, and miRNAs, which were supported by significant correlations among their expression levels. Findings were validated in independent cohorts and public datasets, and the downregulation of circLPAR1(2,3) and circLINC00632(5) was validated by ddPCR. CONCLUSIONS: These results support that multiple altered regulatory mechanisms may contribute to the reduction of circRNA levels that characterize early colorectal carcinogenesis.

• Publication type
• Journal Article MeSH

Single-cell RNA-seq methods can be used to delineate cell types and states at unprecedented resolution but do little to explain why certain genes are expressed. Single-cell ATAC-seq and multiome (ATAC + RNA) have emerged to give a complementary view of the cell state. It is however unclear what additional information can be extracted from ATAC-seq data besides transcription factor binding sites. Here, we show that ATAC-seq telomere-like reads counter-inituively cannot be used to infer telomere length, as they mostly originate from the subtelomere, but can be used as a biomarker for chromatin condensation. Using long-read sequencing, we further show that modern hyperactive Tn5 does not duplicate 9 bp of its target sequence, contrary to common belief. We provide a new tool, Telomemore, which can quantify nonaligning subtelomeric reads. By analyzing several public datasets and generating new multiome fibroblast and B-cell atlases, we show how this new readout can aid single-cell data interpretation. We show how drivers of condensation processes can be inferred, and how it complements common RNA-seq-based cell cycle inference, which fails for monocytes. Telomemore-based analysis of the condensation state is thus a valuable complement to the single-cell analysis toolbox.

• MeSH
• Single-Cell Analysis * methods   MeSH
• B-Lymphocytes metabolism   cytology   MeSH
• Cell Cycle * genetics   MeSH
• Chromatin Immunoprecipitation Sequencing methods   MeSH
• Chromatin * metabolism   chemistry   genetics   MeSH
• Fibroblasts metabolism   cytology   MeSH
• Humans MeSH
• RNA-Seq methods   MeSH
• Telomere * genetics   MeSH
• Binding Sites MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH

Single-cell RNA sequencing (scRNA-seq) methods are widely used in life sciences, including immunology. Typical scRNA-seq analysis pipelines quantify the abundance of particular transcripts without accounting for alternative splicing. However, a well-established pan-leukocyte surface marker, CD45, encoded by the PTPRC gene, presents alternatively spliced variants that define different immune cell subsets. Information about some of the splicing patterns in particular cells in the scRNA-seq data can be obtained using isotype-specific DNA oligo-tagged anti-CD45 antibodies. However, this requires generation of an additional sequencing DNA library. Here, we present IDEIS, an easy-to-use software for CD45 isoform quantification that uses single-cell transcriptomic data as the input. We showed that IDEIS accurately identifies canonical human CD45 isoforms in datasets generated by 10× Genomics 5' sequencing assays. Moreover, we used IDEIS to determine the specificity of the Ptprc splicing pattern in mouse leukocyte subsets.

• MeSH
• Alternative Splicing MeSH
• Single-Cell Analysis methods   MeSH
• Leukocyte Common Antigens * genetics   metabolism   MeSH
• Leukocytes metabolism   immunology   MeSH
• Humans MeSH
• Mice MeSH
• Protein Isoforms genetics   MeSH
• Sequence Analysis, RNA methods   MeSH
• Software * MeSH
• Gene Expression Profiling methods   MeSH
• Transcriptome MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH

Alterations in DNA methylation profiles belong to important mechanisms in cancer development, and their assessment can be utilized for rapid and precise diagnostics. Therefore, establishing datasets of methylation profiles can improve and deepen our understanding of the role of epigenetic changes in cancer development as well as improve our diagnostic capabilities. In this dataset, we generated NGS data for 189 samples of pediatric CNS, soft tissue, and bone tumors. The sequencing libraries were prepared using methyl capture bisulfite sequencing, an effective compromise between whole-genome bisulfite sequencing and array-based methods with a more limited scope of target regions. The larger part of the cohort was processed with the Agilent SureSelectXT Human Methyl-Seq kit (149 samples) and the rest with the Illumina TruSeq Methyl Capture EPIC Library Prep Kit (40 samples). The data presented in this article may help other researchers further elucidate the importance of methylation in diagnosing pediatric CNS tumors, soft tissue, and bone tumors.

• Publication type
• Journal Article MeSH

Environmental exposure is associated with increased incidence of respiratory and cardiovascular diseases and reduced fertility. Exposure to air pollution can influence gene expression through epigenetic mechanisms. In this study, we analysed gene-specific CpG methylation in spermatozoa of city policemen occupationally exposed to air pollution in two Czech cities differing by sources and composition of the air pollution. In Prague, the pollution is mainly formed by NO2 from heavy traffic. Ostrava is a hotspot of industrial air pollution with high concentrations of particular matter (PM) and benzo[a]pyrene (B[a]P). We performed genome-wide methylation sequencing using the SureSelectXT Human Methyl-Seq system (Agilent Technologies) and next-generation sequencing to reveal differentially methylated CpG sites and regions. We identified differential methylation in the region chr5:662169 - 663376 annotated to genes CEP72 and TPPP. The region was then analysed in sperm DNA from 117 policemen using targeted methylation sequencing, which proved its hypermethylation in sperm of Ostrava policemen.

• MeSH
• Adult MeSH
• Air Pollutants * analysis   toxicity   MeSH
• Middle Aged MeSH
• Humans MeSH
• DNA Methylation * drug effects   MeSH
• Particulate Matter analysis   toxicity   MeSH
• Police MeSH
• Occupational Exposure MeSH
• Spermatozoa * drug effects   MeSH
• Air Pollution * adverse effects   analysis   MeSH
• Check Tag
• Adult MeSH
• Middle Aged MeSH
• Humans MeSH
• Male MeSH
• Publication type
• Journal Article MeSH
• Geographicals
• Czech Republic MeSH

Pediatric central nervous system (CNS) tumors represent the most common cause of cancer-related death in children aged 0-14 years. They differ from their adult counterparts, showing extensive clinical and molecular heterogeneity as well as a challenging histopathological spectrum that often impairs accurate diagnosis. Here, we use DNA methylation-based CNS tumor classification in combination with copy number, RNA-seq, and ChIP-seq analysis to characterize a newly identified CNS tumor type. In addition, we report histology, patient characteristics, and survival data in this tumor type. We describe a biologically distinct pediatric CNS tumor type (n = 31 cases) that is characterized by focal high-level amplification and resultant overexpression of either PLAGL1 or PLAGL2, and an absence of recurrent genetic alterations characteristic of other pediatric CNS tumor types. Both genes act as transcription factors for a regulatory subset of imprinted genes (IGs), components of the Wnt/β-Catenin pathway, and the potential drug targets RET and CYP2W1, which are also specifically overexpressed in this tumor type. A derived PLAGL-specific gene expression signature indicates dysregulation of imprinting control and differentiation/development. These tumors occurred throughout the neuroaxis including the cerebral hemispheres, cerebellum, and brainstem, and were predominantly composed of primitive embryonal-like cells lacking robust expression of markers of glial or neuronal differentiation (e.g., GFAP, OLIG2, and synaptophysin). Tumors with PLAGL1 amplification were typically diagnosed during adolescence (median age 10.5 years), whereas those with PLAGL2 amplification were diagnosed during early childhood (median age 2 years). The 10-year overall survival was 66% for PLAGL1-amplified tumors, 25% for PLAGL2-amplified tumors, 18% for male patients, and 82% for female patients. In summary, we describe a new type of biologically distinct CNS tumor characterized by PLAGL1/2 amplification that occurs predominantly in infants and toddlers (PLAGL2) or adolescents (PLAGL1) which we consider best classified as a CNS embryonal tumor and which is associated with intermediate survival. The cell of origin and optimal treatment strategies remain to be defined.

• MeSH
• Child MeSH
• DNA-Binding Proteins genetics   metabolism   MeSH
• Infant MeSH
• Humans MeSH
• DNA Methylation MeSH
• Tumor Suppressor Proteins genetics   MeSH
• Central Nervous System Neoplasms * genetics   MeSH
• Child, Preschool MeSH
• Neuroectodermal Tumors, Primitive * genetics   MeSH
• Cell Cycle Proteins genetics   MeSH
• RNA-Binding Proteins genetics   MeSH
• Wnt Signaling Pathway genetics   MeSH
• Transcription Factors genetics   metabolism   MeSH
• Check Tag
• Child MeSH
• Infant MeSH
• Humans MeSH
• Male MeSH
• Child, Preschool MeSH
• Female MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Hereditární angioedém (HAE) je charakterizován rekurentími atakami otoků a je spojen se sníženou hladinou a/nebo funkcí C1 inhibitoru (SERPING1), což vede k lokální nadprodukci bradykininu a následnému rozvoji angioedému. I v rodinách se stejnou mutací v SERPING1 se HAE projevuje s různou závažností. Genetická determinace této různorodosti je dosud neobjasněna. Projekt by měl vysvětlit, které genetické faktory přispívají k této variabilitě. S použitím cílené DNA-seq bude charakterizováno celkem 70 genů souvisejících s funkčností bradykininu u pacientů s různou závažností HAE. Další faktory, které mohou způsobovat rozdílnost fenotypu se týkají regulace genové exprese, což nemusí souviset přímo se změnami sekvencí těchto genů, ale spíše se změnami regulačních faktorů, které ovlivňují expresi jednotlivých genů. Proto budeme pomocí cíleného RNA-seq sledovat expresi vybraných genů (včetně SERPING1) v monocytech/makrofázích u vybrané skupiny pacientů. Unikátní kombinace genomických i transkriptomických dat by měla přispět k porozumění mechanismu angioedémů a cílenější medikaci.; Hereditary angioedema (HAE) is one of bradykinin-associated angioedema characterized by recurrent attacks of swelling. It is characterized by decreased level and/or function of C1 inhibitor (SERPING1), which results in local bradykinin overproduction and subsequent angioedema development. The severity of HAE in terms of frequency and extent of attacks varies from very mild to very serious even in families with the same SERPING1 mutation, but the genetic determination of this heterogeneity remains to be clarified. We would like to elucidate which genetic factors largely contribute to this variability. We will determine genetic background of patients with diverse severity of HAE in 70 genes associated with bradykinin function using targeted DNA-seq. Another explanation labours with disrupted regulation of gene expression. Thus expression of selected genes (incl. SERPING1) in patients' macrophages will be determined using RNA-seq in a defined set of patients. Unique combination of transcriptomic and genomic approaches may improve our knowledge in all bradykinin-mediated angioedema.

• Keywords
• Next generation sequencing, genová exprese, gene expression, hereditary angioedema, hereditární angioedém, bradykinin, C1 inhibitor, SERPING1, asociační studie, sekvenování příští generace, bradykinin, c1 inhibitor, SERPING1, association study,

• NML Publication type
• závěrečné zprávy o řešení grantu AZV MZ ČR

BACKGROUND: Molecular aberrations occurring in primary ovarian clear cell carcinoma (OCCC) can be of diagnostic, predictive, and prognostic significance. However, a complex molecular study including genomic and transcriptomic analysis of large number of OCCC has been lacking. METHODS: 113 pathologically confirmed primary OCCCs were analyzed using capture DNA NGS (100 cases; 727 solid cancer related genes) and RNA-Seq (105 cases; 147 genes) in order to describe spectra and frequency of genomic and transcriptomic alterations, as well as their prognostic and predictive significance. RESULTS: The most frequent mutations were detected in genes ARID1A, PIK3CA, TERTp, KRAS, TP53, ATM, PPP2R1A, NF1, PTEN, and POLE (51,47,27,18,13,10,7,6,6, and 4%, respectively). TMB-High cases were detected in 9% of cases. Cases with POLEmut and/or MSI-High had better relapse-free survival. RNA-Seq revealed gene fusions in 14/105 (13%) cases, and heterogeneous expression pattern. The majority of gene fusions affected tyrosine kinase receptors (6/14; four of those were MET fusions) or DNA repair genes (2/14). Based on the mRNA expression pattern, a cluster of 12 OCCCs characterized by overexpression of tyrosine kinase receptors (TKRs) AKT3, CTNNB1, DDR2, JAK2, KIT, or PDGFRA (p < 0.00001) was identified. CONCLUSIONS: The current work has elucidated the complex genomic and transcriptomic molecular hallmarks of primary OCCCs. Our results confirmed the favorable outcomes of POLEmut and MSI-High OCCC. Moreover, the molecular landscape of OCCC revealed several potential therapeutical targets. Molecular testing can provide the potential for targeted therapy in patients with recurrent or metastatic tumors.

• MeSH
• Adenocarcinoma, Clear Cell * genetics   MeSH
• Gene Fusion MeSH
• Genomics MeSH
• Humans MeSH
• Neoplasm Recurrence, Local * MeSH
• Gene Expression Profiling MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH

BACKGROUND: The international, multicenter registry LOGGIC Core BioClinical Data Bank aims to enhance the understanding of tumor biology in pediatric low-grade glioma (pLGG) and provide clinical and molecular data to support treatment decisions and interventional trial participation. Hence, the question arises whether implementation of RNA sequencing (RNA-Seq) using fresh frozen (FrFr) tumor tissue in addition to gene panel and DNA methylation analysis improves diagnostic accuracy and provides additional clinical benefit. METHODS: Analysis of patients aged 0 to 21 years, enrolled in Germany between April 2019 and February 2021, and for whom FrFr tissue was available. Central reference histopathology, immunohistochemistry, 850k DNA methylation analysis, gene panel sequencing, and RNA-Seq were performed. RESULTS: FrFr tissue was available in 178/379 enrolled cases. RNA-Seq was performed on 125 of these samples. We confirmed KIAA1549::BRAF-fusion (n = 71), BRAF V600E-mutation (n = 12), and alterations in FGFR1 (n = 14) as the most frequent alterations, among other common molecular drivers (n = 12). N = 16 cases (13%) presented rare gene fusions (eg, TPM3::NTRK1, EWSR1::VGLL1, SH3PXD2A::HTRA1, PDGFB::LRP1, GOPC::ROS1). In n = 27 cases (22%), RNA-Seq detected a driver alteration not otherwise identified (22/27 actionable). The rate of driver alteration detection was hereby increased from 75% to 97%. Furthermore, FGFR1 internal tandem duplications (n = 6) were only detected by RNA-Seq using current bioinformatics pipelines, leading to a change in analysis protocols. CONCLUSIONS: The addition of RNA-Seq to current diagnostic methods improves diagnostic accuracy, making precision oncology treatments (MEKi/RAFi/ERKi/NTRKi/FGFRi/ROSi) more accessible. We propose to include RNA-Seq as part of routine diagnostics for all pLGG patients, especially when no common pLGG alteration was identified.

• MeSH
• Child MeSH
• DNA-Binding Proteins genetics   MeSH
• Glioma * pathology   MeSH
• Precision Medicine MeSH
• Humans MeSH
• Pathology, Molecular MeSH
• Proto-Oncogene Proteins B-raf * genetics   MeSH
• Proto-Oncogene Proteins genetics   MeSH
• RNA-Seq MeSH
• Transcription Factors genetics   MeSH
• Protein-Tyrosine Kinases MeSH
• Check Tag
• Child MeSH
• Humans MeSH
• Publication type
• Journal Article MeSH
• Multicenter Study MeSH

Transfer RNAs acquire a large plethora of chemical modifications. Among those, modifications of the anticodon loop play important roles in translational fidelity and tRNA stability. Four human wobble U-containing tRNAs obtain 5-methoxycarbonylmethyluridine (mcm5U34) or 5-methoxycarbonylmethyl-2-thiouridine (mcm5s2U34), which play a role in decoding. This mark involves a cascade of enzymatic activities. The last step is mediated by alkylation repair homolog 8 (ALKBH8). In this study, we performed a transcriptome-wide analysis of the repertoire of ALKBH8 RNA targets. Using a combination of HITS-CLIP and RIP-seq analyses, we uncover ALKBH8-bound RNAs. We show that ALKBH8 targets fully processed and CCA modified tRNAs. Our analyses uncovered the previously known set of wobble U-containing tRNAs. In addition, both our approaches revealed ALKBH8 binding to several other types of noncoding RNAs, in particular C/D box snoRNAs.

• MeSH
• AlkB Homolog 8, tRNA Methyltransferase genetics   MeSH
• Anticodon MeSH
• Chromatin Immunoprecipitation Sequencing * MeSH
• Humans MeSH
• RNA, Untranslated genetics   MeSH
• RNA, Transfer * genetics   metabolism   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH