G-quadruplexes (G4s) are functional elements of the human genome, some of which inhibit DNA replication. We investigated replication of G4s within highly abundant microsatellite (GGGA, GGGT) and transposable element (L1 and SVA) sequences. We found that genome-wide, numerous motifs are located preferentially on the replication leading strand and the transcribed strand templates. We directly tested replicative polymerase ε and δ holoenzyme inhibition at these G4s, compared to low abundant motifs. For all G4s, DNA synthesis inhibition was higher on the G-rich than C-rich strand or control sequence. No single G4 was an absolute block for either holoenzyme; however, the inhibitory potential varied over an order of magnitude. Biophysical analyses showed the motifs form varying topologies, but replicative polymerase inhibition did not correlate with a specific G4 structure. Addition of the G4 stabilizer pyridostatin severely inhibited forward polymerase synthesis specifically on the G-rich strand, enhancing G/C strand asynchrony. Our results reveal that replicative polymerase inhibition at every G4 examined is distinct, causing complementary strand synthesis to become asynchronous, which could contribute to slowed fork elongation. Altogether, we provide critical information regarding how replicative eukaryotic holoenzymes navigate synthesis through G4s naturally occurring thousands of times in functional regions of the human genome.

• MeSH
• Aminoquinolines MeSH
• DNA Polymerase II * antagonists & inhibitors   metabolism   MeSH
• DNA Polymerase III * antagonists & inhibitors   metabolism   MeSH
• DNA chemistry   MeSH
• G-Quadruplexes * MeSH
• Genome, Human * MeSH
• Holoenzymes metabolism   MeSH
• Picolinic Acids pharmacology   MeSH
• Humans MeSH
• Microsatellite Repeats MeSH
• Poly-ADP-Ribose Binding Proteins MeSH
• DNA Replication * MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH

Somatic hypermutation (SHM) and class switch recombination (CSR) diversify immunoglobulin (Ig) genes and are initiated by the activation-induced deaminase (AID), a single-stranded DNA cytidine deaminase thought to engage its substrate during RNA polymerase II (RNAPII) transcription. Through a genetic screen, we identified numerous potential factors involved in SHM, including elongation factor 1 homolog (ELOF1), a component of the RNAPII elongation complex that functions in transcription-coupled nucleotide excision repair (TC-NER) and transcription elongation. Loss of ELOF1 compromises SHM, CSR, and AID action in mammalian B cells and alters RNAPII transcription by reducing RNAPII pausing downstream of transcription start sites and levels of serine 5 but not serine 2 phosphorylated RNAPII throughout transcribed genes. ELOF1 must bind to RNAPII to be a proximity partner for AID and to function in SHM and CSR, and TC-NER is not required for SHM. We propose that ELOF1 helps create the appropriate stalled RNAPII substrate on which AID acts.

• MeSH
• AICDA (Activation-Induced Cytidine Deaminase) MeSH
• B-Lymphocytes * immunology   metabolism   MeSH
• Cytidine Deaminase metabolism   genetics   MeSH
• Phosphoproteins * genetics   metabolism   MeSH
• Phosphorylation MeSH
• Transcription, Genetic MeSH
• Humans MeSH
• Mice, Knockout MeSH
• Mice MeSH
• DNA Repair MeSH
• Immunoglobulin Class Switching * MeSH
• RNA Polymerase II metabolism   genetics   MeSH
• Somatic Hypermutation, Immunoglobulin * MeSH
• Transcriptional Elongation Factors * genetics   metabolism   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH

BACKGROUND AND PURPOSE: CNS embryonal tumor with pleomorphic adenoma gene-like 1 (PLAGL1)/pleomorphic adenoma gene-like 2 (PLAGL2) amplification (ET, PLAGL) is a newly identified, highly malignant pediatric tumor. Systematic MRI descriptions of ET, PLAGL are currently lacking. MATERIALS AND METHODS: MRI data from 19 treatment-naïve patients with confirmed ET, PLAGL were analyzed. Evaluation focused on anatomic involvement, tumor localization, MRI signal characteristics, DWI behavior, and the presence of necrosis and hemorrhage. Descriptive statistics (median, interquartile range, percentage) were assessed. RESULTS: Ten patients had PLAGL1 and nine had PLAGL2 amplifications. The solid components of the tumors were often multinodular with heterogeneous enhancement (mild to intermediate in 47% and intermediate to strong in 47% of cases). Nonsolid components included cysts in 47% and necrosis in 84% of the cases. The tumors showed heterogeneous T2WI hyper- and isointensity (74%), relatively little diffusion restriction (ADC values less than contralateral normal-appearing WM in 36% of cases with available DWI), and tendencies toward hemorrhage/calcification (42%). No reliable distinction was found between PLAGL1- and PLAGL2-amplified tumors or compared with other embryonal CNS tumors. CONCLUSIONS: The study contributes to understanding the imaging characteristics of ET, PLAGL. It underscores the need for collaboration in studying rare pediatric tumors and advocates the use of harmonized imaging protocols for better characterization.

• MeSH
• Gene Amplification MeSH
• Child MeSH
• DNA-Binding Proteins genetics   MeSH
• Neoplasms, Germ Cell and Embryonal diagnostic imaging   pathology   MeSH
• Infant MeSH
• Humans MeSH
• Magnetic Resonance Imaging * methods   MeSH
• Adolescent MeSH
• Tumor Suppressor Proteins MeSH
• Central Nervous System Neoplasms diagnostic imaging   pathology   MeSH
• Brain Neoplasms diagnostic imaging   pathology   MeSH
• Child, Preschool MeSH
• Cell Cycle Proteins MeSH
• RNA-Binding Proteins MeSH
• Transcription Factors genetics   MeSH
• Check Tag
• Child MeSH
• Infant MeSH
• Humans MeSH
• Adolescent MeSH
• Male MeSH
• Child, Preschool MeSH
• Female MeSH
• Publication type
• Journal Article MeSH

Intracellular trafficking involves an intricate machinery of motor complexes, including the dynein complex, to shuttle cargo for autophagolysosomal degradation. Deficiency in dynein axonemal chains, as well as cytoplasmic light and intermediate chains, have been linked with ciliary dyskinesia and skeletal dysplasia. The cytoplasmic dynein 1 heavy chain protein (DYNC1H1) serves as a core complex for retrograde trafficking in neuronal axons. Dominant pathogenic variants in DYNC1H1 have been previously implicated in peripheral neuromuscular disorders (NMD) and neurodevelopmental disorders (NDD). As heavy-chain dynein is ubiquitously expressed, the apparent selectivity of heavy chain dyneinopathy for motor neuronal phenotypes remains currently unaccounted for. Here, we aimed to evaluate the full DYNC1H1-related clinical, molecular and imaging spectrum, including multisystem features and novel phenotypes presenting throughout life. We identified 47 cases from 43 families with pathogenic heterozygous variants in DYNC1H1 (aged 0-59 years) and collected phenotypic data via a comprehensive standardized survey and clinical follow-up appointments. Most patients presented with divergent and previously unrecognized neurological and multisystem features, leading to significant delays in genetic testing and establishing the correct diagnosis. Neurological phenotypes include novel autonomic features, previously rarely described behavioral disorders, movement disorders and periventricular lesions. Sensory neuropathy was identified in nine patients (median age of onset 10.6 years), of which five were only diagnosed after the second decade of life, and three had a progressive age-dependent sensory neuropathy. Novel multisystem features included primary immunodeficiency, bilateral sensorineural hearing loss, organ anomalies and skeletal manifestations, resembling the phenotypic spectrum of other dyneinopathies. We also identified an age-dependent biphasic disease course with developmental regression in the first decade and, following a period of stability, neurodegenerative progression after the second decade of life. Of note, we observed several cases in whom neurodegeneration appeared to be prompted by intercurrent systemic infections with double-stranded DNA viruses (Herpesviridae) or single-stranded RNA viruses (Ross River fever, SARS-CoV-2). Moreover, the disease course appeared to be exacerbated by viral infections regardless of age and/or severity of neurodevelopmental disorder manifestations, indicating a role of dynein in anti-viral immunity and neuronal health. In summary, our findings expand the clinical, imaging and molecular spectrum of pathogenic DYNC1H1 variants beyond motor neuropathy disorders and suggest a life-long continuum and age-related progression due to deficient intracellular trafficking. This study will facilitate early diagnosis and improve counselling and health surveillance of affected patients.

• MeSH
• Cytoplasmic Dyneins * genetics   MeSH
• Child MeSH
• Adult MeSH
• Phenotype MeSH
• Infant MeSH
• Middle Aged MeSH
• Humans MeSH
• Adolescent MeSH
• Young Adult MeSH
• Neurodevelopmental Disorders genetics   MeSH
• Infant, Newborn MeSH
• Child, Preschool MeSH
• Check Tag
• Child MeSH
• Adult MeSH
• Infant MeSH
• Middle Aged MeSH
• Humans MeSH
• Adolescent MeSH
• Young Adult MeSH
• Male MeSH
• Infant, Newborn MeSH
• Child, Preschool MeSH
• Female MeSH
• Publication type
• Journal Article MeSH

AIMS: Spindle-cell/sclerosing rhabdomyosarcomas (SS-RMS) are clinically and genetically heterogeneous. They include three well-defined molecular subtypes, of which those with EWSR1/FUS::TFCP2 rearrangements were described only recently. This study aimed to evaluate five new cases of SS-RMS and to perform a clinicopathological and statistical analysis of all TFCP2-rearranged SS-RMS described in the English literature to more comprehensively characterize this rare tumour type. METHODS AND RESULTS: Cases were retrospectively selected and studied by immunohistochemistry, fluorescence in situ hybridization with EWSR1/FUS and TFCP2 break-apart probes, next-generation sequencing (Archer FusionPlex Sarcoma kit and TruSight RNA Pan-Cancer Panel). The PubMed database was searched for relevant peer-reviewed English reports. Five cases of SS-RMS were found. Three cases were TFCP2 rearranged SS-RMS, having FUSex6::TFCP2ex2 gene fusion in two cases and triple gene fusion EWSR1ex5::TFCP2ex2, VAX2ex2::ALKex2 and VAX2intron2::ALKex2 in one case. Two cases showed rhabdomyoblastic differentiation and spindle-round cell/sclerosing morphology, but were characterized by novel genetic fusions including EWSR1ex8::ZBTB41ex7 and PLOD2ex8::RBM6ex7, respectively. In the statistical analysis of all published cases, CDKN2A or ALK alterations, the use of standard chemotherapy and age at presentation in the range of 18-24 years were negatively correlated to overall survival. CONCLUSION: EWSR1/FUS::TFCP2-rearranged SS-RMS is a rare rhabdomyosarcoma subtype, affecting predominantly young adults with average age at presentation 34 years (median 29.5 years; age range 7-86 years), with a predilection for craniofacial bones, rapid clinical course with frequent bone and lung metastases, and poor prognosis (3-year overall survival rate 28%).

• MeSH
• Child MeSH
• DNA-Binding Proteins genetics   MeSH
• Adult MeSH
• Gene Fusion MeSH
• In Situ Hybridization, Fluorescence MeSH
• Middle Aged MeSH
• Humans MeSH
• Procollagen-Lysine, 2-Oxoglutarate 5-Dioxygenase genetics   MeSH
• Adolescent MeSH
• Young Adult MeSH
• Biomarkers, Tumor genetics   MeSH
• RNA-Binding Protein EWS genetics   MeSH
• RNA-Binding Proteins genetics   MeSH
• Retrospective Studies MeSH
• Rhabdomyosarcoma * genetics   pathology   MeSH
• Aged, 80 and over MeSH
• Aged MeSH
• Transcription Factors * genetics   MeSH
• Check Tag
• Child MeSH
• Adult MeSH
• Middle Aged MeSH
• Humans MeSH
• Adolescent MeSH
• Young Adult MeSH
• Aged, 80 and over MeSH
• Aged MeSH
• Publication type
• Journal Article MeSH
• Review MeSH

SMARCB1-deficient sinonasal adenocarcinoma is a rare variant of SWI/SNF-deficient malignancies with SMARCB1 loss and adenocarcinoma features. More than 200 high-grade epithelial sinonasal malignancies were retrieved. A total of 14 cases exhibited complete SMARCB1 (INI1) loss and glandular differentiation. SMARCA2 and SMARCA4 were normal, except for one case with a loss of SMARCA2. Next-generation sequencing (NGS) and/or fluorescence in situ hybridization (FISH) revealed an alteration in the SMARCB1 gene in 9/13 cases, while 2/13 were negative. Two tumors harbored SMARCB1 mutations in c.157C > T p.(Arg53Ter) and c.842G > A p.(Trp281Ter). One harbored ARID1B mutations in c.1469G > A p.(Trp490Ter) and MGA c.3724C > T p.(Arg1242Ter). Seven tumors had a SMARCB1 deletion. One carried an ESR1 mutation in c.644-2A > T, and another carried a POLE mutation in c.352_374del p.(Ser118GlyfsTer78). One case had a PAX3 mutation in c.44del p.(Gly15AlafsTer95). Histomorphology of SMARCB1-deficient adenocarcinoma was oncocytoid/rhabdoid and glandular, solid, or trabecular in 9/14 cases. Two had basaloid/blue cytoplasm and one showed focal signet ring cells. Yolk sac tumor-like differentiation with Schiller-Duval-like bodies was seen in 6/14 cases, with 2 cases showing exclusively reticular-microcystic yolk sac pattern. Follow-up of a maximum of 26 months (median 10 months) was available for 8/14 patients. Distant metastasis to the lung, liver, mediastinum, bone, and/or retroperitoneum was seen in 4/8 cases. Locoregional failure was seen in 75% of patients, with 6/8 local recurrences and 3 cervical lymph node metastases. At the last follow-up, 5 of 8 (62%) patients had died of their disease 2 to 20 months after diagnosis (median 8.2 months), and 3 were alive with the disease. The original diagnosis was usually high-grade non-intestinal-type adenocarcinoma or high-grade myoepithelial carcinoma. A correct diagnosis of these aggressive tumors could lead to improved targeted therapies with potentially better overall disease-specific survival.

• MeSH
• Adenocarcinoma * genetics   pathology   MeSH
• Diagnosis, Differential MeSH
• DNA-Binding Proteins genetics   deficiency   MeSH
• Adult MeSH
• SMARCB1 Protein * deficiency   genetics   MeSH
• In Situ Hybridization, Fluorescence MeSH
• Middle Aged MeSH
• Humans MeSH
• Mutation * MeSH
• Myoepithelioma * genetics   pathology   MeSH
• Biomarkers, Tumor genetics   MeSH
• Paranasal Sinus Neoplasms * genetics   pathology   MeSH
• Aged, 80 and over MeSH
• Aged MeSH
• Neoplasm Grading MeSH
• Transcription Factors * genetics   deficiency   MeSH
• High-Throughput Nucleotide Sequencing MeSH
• Check Tag
• Adult MeSH
• Middle Aged MeSH
• Humans MeSH
• Male MeSH
• Aged, 80 and over MeSH
• Aged MeSH
• Female MeSH
• Publication type
• Journal Article MeSH

DNA double-strand breaks (DSBs) represent a lethal form of DNA damage that can trigger cell death or initiate oncogenesis. The activity of RNA polymerase II (RNAPII) at the break site is required for efficient DSB repair. However, the regulatory mechanisms governing the transcription cycle at DSBs are not well understood. Here, we show that Integrator complex subunit 6 (INTS6) associates with the heterotrimeric sensor of ssDNA (SOSS1) complex (comprising INTS3, INIP and hSSB1) to form the tetrameric SOSS1 complex. INTS6 binds to DNA:RNA hybrids and promotes Protein Phosphatase 2A (PP2A) recruitment to DSBs, facilitating the dephosphorylation of RNAPII. Furthermore, INTS6 prevents the accumulation of damage-associated RNA transcripts (DARTs) and the stabilization of DNA:RNA hybrids at DSB sites. INTS6 interacts with and promotes the recruitment of senataxin (SETX) to DSBs, facilitating the resolution of DNA:RNA hybrids/R-loops. Our results underscore the significance of the tetrameric SOSS1 complex in the autoregulation of DNA:RNA hybrids and efficient DNA repair.

• MeSH
• DNA-Binding Proteins metabolism   MeSH
• DNA Helicases metabolism   genetics   MeSH
• DNA * metabolism   chemistry   MeSH
• DNA Breaks, Double-Stranded * MeSH
• Phosphorylation MeSH
• Homeostasis genetics   MeSH
• Humans MeSH
• DNA Repair * MeSH
• Protein Phosphatase 2 metabolism   genetics   MeSH
• R-Loop Structures MeSH
• RNA Helicases metabolism   genetics   MeSH
• RNA Polymerase II * metabolism   MeSH
• RNA * metabolism   genetics   chemistry   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH

BACKGROUND: Genetic factors are involved in the pathogenesis of familial and sporadic amyotrophic lateral sclerosis (ALS) and constitute a link to its association with frontotemporal dementia (FTD). Gene-targeted therapies for some forms of ALS (C9orf72, SOD1) have recently gained momentum. Genetic architecture in Czech ALS patients has not been comprehensively assessed so far. OBJECTIVE: We aimed to deliver pilot data on the genetic landscape of ALS in our country. METHODS: A cohort of patients with ALS (n = 88), recruited from two Czech Neuromuscular Centers, was assessed for hexanucleotide repeat expansion (HRE) in C9orf72 and also for genetic variations in other 36 ALS-linked genes via next-generation sequencing (NGS). Nine patients (10.1%) had a familial ALS. Further, we analyzed two subgroups of sporadic patients - with concomitant FTD (n = 7) and with young-onset of the disease (n = 22). RESULTS: We detected the pathogenic HRE in C9orf72 in 12 patients (13.5%) and three other pathogenic variants in FUS, TARDBP and TBK1, each in one patient. Additional 7 novel and 9 rare known variants with uncertain causal significance have been detected in 15 patients. Three sporadic patients with FTD (42.9%) were harbouring a pathogenic variant (all HRE in C9orf72). Surprisingly, none of the young-onset sporadic patients harboured a pathogenic variant and we detected no pathogenic SOD1 variant in our cohort. CONCLUSION: Our findings resemble those from other European populations, with the highest prevalence of HRE in the C9orf72 gene. Further, our findings suggest a possibility of a missing genetic variability among young-onset patients.

• MeSH
• Amyotrophic Lateral Sclerosis * genetics   MeSH
• DNA-Binding Proteins genetics   MeSH
• Adult MeSH
• DNA Repeat Expansion * MeSH
• Frontotemporal Dementia * genetics   MeSH
• Genetic Predisposition to Disease MeSH
• Cohort Studies MeSH
• Middle Aged MeSH
• Humans MeSH
• C9orf72 Protein * genetics   MeSH
• RNA-Binding Protein FUS genetics   MeSH
• Protein Serine-Threonine Kinases genetics   MeSH
• Aged MeSH
• Age of Onset MeSH
• High-Throughput Nucleotide Sequencing MeSH
• Check Tag
• Adult MeSH
• Middle Aged MeSH
• Humans MeSH
• Male MeSH
• Aged MeSH
• Female MeSH
• Publication type
• Journal Article MeSH
• Geographicals
• Czech Republic MeSH

Replication forks stalled at co-transcriptional R-loops can be restarted by a mechanism involving fork cleavage-religation cycles mediated by MUS81 endonuclease and DNA ligase IV (LIG4), which presumably relieve the topological barrier generated by the transcription-replication conflict (TRC) and facilitate ELL-dependent reactivation of transcription. Here, we report that the restart of R-loop-stalled replication forks via the MUS81-LIG4-ELL pathway requires senataxin (SETX), a helicase that can unwind RNA:DNA hybrids. We found that SETX promotes replication fork progression by preventing R-loop accumulation during S-phase. Interestingly, loss of SETX helicase activity leads to nascent DNA degradation upon induction of R-loop-mediated fork stalling by hydroxyurea. This fork degradation phenotype is independent of replication fork reversal and results from DNA2-mediated resection of MUS81-cleaved replication forks that accumulate due to defective replication restart. Finally, we demonstrate that SETX acts in a common pathway with the DEAD-box helicase DDX17 to suppress R-loop-mediated replication stress in human cells. A possible cooperation between these RNA/DNA helicases in R-loop unwinding at TRC sites is discussed.

• MeSH
• Flap Endonucleases metabolism   genetics   MeSH
• DEAD-box RNA Helicases * metabolism   genetics   MeSH
• DNA-Binding Proteins * metabolism   genetics   MeSH
• DNA Helicases * metabolism   genetics   MeSH
• DNA Ligase ATP metabolism   genetics   MeSH
• DNA metabolism   genetics   MeSH
• Endonucleases * metabolism   genetics   MeSH
• Transcription, Genetic MeSH
• Humans MeSH
• Multifunctional Enzymes * metabolism   genetics   MeSH
• R-Loop Structures * MeSH
• DNA Replication * MeSH
• RNA Helicases * metabolism   genetics   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH

PIWI-interacting RNAs (piRNAs) play a crucial role in safeguarding genome integrity by silencing mobile genetic elements. From flies to humans, piRNAs originate from long single-stranded precursors encoded by genomic piRNA clusters. How piRNA clusters form to adapt to genomic invaders and evolve to maintain protection remain key outstanding questions. Here, we generate a roadmap of piRNA clusters across seven species that highlights both similarities and variations. In mammals, we identify transcriptional readthrough as a mechanism to generate piRNAs from transposon insertions (piCs) downstream of genes (DoG). Together with the well-known stress-dependent DoG transcripts, our findings suggest a molecular mechanism for the formation of piRNA clusters in response to retroviral invasion. Finally, we identify a class of dynamic piRNA clusters in humans, underscoring unique features of human germ cell biology. Our results advance the understanding of conserved principles and species-specific variations in piRNA biology and provide tools for future studies.

• MeSH
• Species Specificity MeSH
• Humans MeSH
• RNA, Small Interfering * metabolism   genetics   MeSH
• Mice MeSH
• Piwi-Interacting RNA MeSH
• Dogs MeSH
• Mammals * genetics   MeSH
• DNA Transposable Elements genetics   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Mice MeSH
• Dogs MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Comparative Study MeSH