Předkládáme případ 5,5letého chlapce s těžkým globálním vývojovým opožděním, hypotonií, malým proporcionálním vzrůstem, neprospíváním, kraniofaciální dysmorfií a dalšími potížemi, u kterého se podařilo stanovit diagnózu pomocí celogenomového sekvenování (WGs). diagnostika byla realizována v rámci českého národního projektu BabyFox, který se zaměřuje na zhodnocení diagnostické a klinické využitelnosti rapidního celogenomového sekvenování (rapid trioWGs) u dětí hospitalizovaných na jednotkách intenzivní péče s podezřením na genetickou podstatu jejich potíží. u pacienta byla identifikována patogenní varianta v genu RNU4-2, která je od roku 2024 spojována s prevalentním onemocněním rnu4-2 syndromem neurovývojového opoždění, také známým pod názvem renu syndrom. ačkoli se jedná o nově popsané onemocnění, je renu syndrom jedním z nejrozšířenějších monogenních neurovývojových onemocnění, přičemž se odhaduje, že patogenní varianty v genu RNU4-2 jsou příčinou přibližně 0,4 % všech neurovývojových poruch. tento gen nekóduje protein, ale malou jadernou rna u4, díky čemuž jeho sekvence není stanovována při standardním celoexomovém sekvenování ani v rámci cílených sekvenačních panelů. Jeho význam tak zůstával až do roku 2024 přehlížen. případ renu syndromu zdůrazňuje význam celogenomového sekvenování pokrývajícího i nekódující části genomu v diagnostice vzácných genetických onemocnění a ukazuje, jak implementace moderních genomických metod může přispět k ukončení diagnostické odysey pacientů s dosud neobjasněnými neurovývojovými poruchami.
We present the case of a 5 and half year-old boy with severe global developmental delay, hypotonia, severe growth failure, failure to thrive, craniofacial dysmorphism, and other difficulties, who was diagnosed by whole genome sequencing (WGs). the diagnosis was carried out within the czech national project BabyFox. a pathogenic variant in the RNU4-2 gene was identified in the patient, which has been associated with the prevalent disorder known as rnu4-2 neurodevelopmental delay syndrome, also known as renu syndrome since 2024. although it is a newly described diseases, renu syndrome is one of the most prevalent monogenic neurodevelopmental disorder, with pathogenic variants in the rnu4-2 gene estimated to cause approximately 0.4% of all neurodevelopmental disorders. this gene does not encode a protein but rather a small nuclear rna u4, which means its sequence is not captured by standard whole-exome sequencing or targeted sequencing panels. as a result, its significance remained overlooked until 2024. the case of renu syndrome highlights the importance of whole-genome sequencing, which covers non-coding regions of the genome, in the diagnosis of rare genetic disorders. it also demonstrates how the implementation of modern genomic methods can help end the diagnostic odyssey for patients with previously unexplained neurodevelopmental disorders.
- Klíčová slova
- gen RNU4-2, ReNU syndrom,
- MeSH
- diferenciální diagnóza MeSH
- lidé MeSH
- mikrocefalie diagnóza etiologie genetika MeSH
- neprospívání diagnóza etiologie MeSH
- neurovývojové poruchy * diagnóza genetika klasifikace MeSH
- předškolní dítě MeSH
- RNA malá jaderná genetika MeSH
- sekvenování celého genomu metody MeSH
- Check Tag
- lidé MeSH
- mužské pohlaví MeSH
- předškolní dítě MeSH
- Publikační typ
- kazuistiky MeSH
Colorectal cancer (CRC) ranks as the second most prevalent malignancy globally, highlighting the urgent need for more effective diagnostic and therapeutic strategies, as well as a deeper understanding of its molecular basis. Extensive research has demonstrated that cells actively secrete extracellular vesicles (EVs) to mediate intercellular communication at both proximal and distal sites. In this study, we conducted a comprehensive analysis of the RNA content of small extracellular vesicles (sEVs) secreted into the culture media of five frequently utilised CRC cell lines (RKO, HCT116, HCT15, HT29, and DLD1). RNA sequencing data revealed significant insights into the RNA profiles of these sEVs, identifying nine protein-coding genes and fourteen long non-coding RNA (lncRNA) genes that consistently ranked among the top 30 most abundant across all cell lines. Notably, the genes found in sEVs were highly similar among the cell lines, indicating a conserved molecular signature. Several of these genes have been previously documented in the context of cancer biology, while others represent novel discoveries. These findings provide valuable insights into the molecular cargo of sEVs in CRC, potentially unveiling novel biomarkers and therapeutic targets.
- MeSH
- extracelulární vezikuly * metabolismus genetika MeSH
- HCT116 buňky MeSH
- kolorektální nádory * genetika patologie metabolismus MeSH
- lidé MeSH
- nádorové biomarkery genetika metabolismus MeSH
- nádorové buněčné linie MeSH
- regulace genové exprese u nádorů MeSH
- RNA dlouhá nekódující genetika MeSH
- sekvenční analýza RNA MeSH
- stanovení celkové genové exprese MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
In the last decade, undiagnosed disease programs have emerged to address the significant number of individuals with suspected but undiagnosed rare genetic diseases. In our single-center study, we have launched a pilot program for pediatric patients with undiagnosed diseases in the second-largest university hospital in the Czech Republic. This study was prospectively conducted at the Department of Pediatrics at University Hospital Brno between 2020 and 2023. A total of 58 Czech patients with undiagnosed diseases were enrolled in the study. All children underwent singleton WES with targeted phenotype-driven analysis. We identified 28 variants, including 11 pathogenic, 13 likely pathogenic, and 4 VUS according to ACMG guidelines, as diagnostic of genetic diseases in 25 patients, resulting in an overall diagnostic yield of 43%. Eleven variants were novel and had not been previously reported in any public database. The overall clinical utility (actionability) enabling at least one type of change in the medical care of the patient was 76%, whereas the average number of clinical implications to individual patient care was two. Singleton WES facilitated the diagnostic process in the Czech undiagnosed pediatric population. We believe it is an effective approach to enable appropriate counseling, surveillance, and personalized clinical management.
- MeSH
- dítě MeSH
- genetické testování metody MeSH
- kojenec MeSH
- lidé MeSH
- mladiství MeSH
- nediagnostikované nemoci * genetika diagnóza epidemiologie MeSH
- novorozenec MeSH
- předškolní dítě MeSH
- prospektivní studie MeSH
- sekvenování exomu * metody MeSH
- vzácné nemoci * genetika diagnóza MeSH
- Check Tag
- dítě MeSH
- kojenec MeSH
- lidé MeSH
- mladiství MeSH
- mužské pohlaví MeSH
- novorozenec MeSH
- předškolní dítě MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- Geografické názvy
- Česká republika MeSH
Despite significant improvement in the survival of pediatric patients with cancer, treatment outcomes for high-risk, relapsed, and refractory cancers remain unsatisfactory. Moreover, prolonged survival is frequently associated with long-term adverse effects due to intensive multimodal treatments. Accelerating the progress of pediatric oncology requires both therapeutic advances and strategies to mitigate the long-term cytotoxic side effects, potentially through targeting specific molecular drivers of pediatric malignancies. In this report, we present the results of integrative genomic and transcriptomic profiling of 230 patients with malignant solid tumors (the "primary cohort") and 18 patients with recurrent or otherwise difficult-to-treat nonmalignant conditions (the "secondary cohort"). The integrative workflow for the primary cohort enabled the identification of clinically significant single nucleotide variants, small insertions/deletions, and fusion genes, which were found in 55% and 28% of patients, respectively. For 38% of patients, molecularly informed treatment recommendations were made. In the secondary cohort, known or potentially driving alteration was detected in 89% of cases, including a suspected novel causal gene for patients with inclusion body infantile digital fibromatosis. Furthermore, 47% of findings also brought therapeutic implications for subsequent management. Across both cohorts, changes or refinements to the original histopathological diagnoses were achieved in 4% of cases. Our study demonstrates the efficacy of integrating advanced genomic and transcriptomic analyses to identify therapeutic targets, refine diagnoses, and optimize treatment strategies for challenging pediatric and young adult malignancies and underscores the need for broad implementation of precision oncology in clinical settings.
- MeSH
- dítě MeSH
- genomika * metody MeSH
- individualizovaná medicína * metody MeSH
- kohortové studie MeSH
- kojenec MeSH
- lékařská onkologie metody MeSH
- lidé MeSH
- mladiství MeSH
- mladý dospělý MeSH
- nádory * genetika terapie MeSH
- předškolní dítě MeSH
- stanovení celkové genové exprese MeSH
- Check Tag
- dítě MeSH
- kojenec MeSH
- lidé MeSH
- mladiství MeSH
- mladý dospělý MeSH
- mužské pohlaví MeSH
- předškolní dítě MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
OBJECTIVES: Small extracellular vesicles (EVs) contain various signaling molecules, thus playing a crucial role in cell-to-cell communication and emerging as a promising source of biomarkers. However, the lack of standardized procedures impedes their translation to clinical practice. Thus, we compared different approaches for high-throughput analysis of small EVs transcriptome. METHODS: Small EVs were isolated from 150 μL of serum. Quality and quantity were assessed by dynamic light scattering, transmission electron microscopy, and Western blot. Comparison of RNA extraction efficiency was performed, and expression of selected genes was analyzed by RT-qPCR. Whole transcriptome analysis was done using microarrays. RESULTS: Obtained data confirmed the suitability of size exclusion chromatography for isolation of small EVs. Analyses of gene expression showed the best results in case of samples isolated by Monarch Total RNA Miniprep Kit. Totally, 7,182 transcripts were identified to be deregulated between colorectal cancer patients and healthy controls. The majority of them were non-coding RNAs with more than 70 % being lncRNAs, while protein-coding genes represented the second most common gene biotype. CONCLUSIONS: We have optimized the protocol for isolation of small EVs and their RNA from low volume of sera and confirmed the suitability of Clariom D Pico Assays for transcriptome profiling.
- MeSH
- extracelulární vezikuly * genetika metabolismus MeSH
- gelová chromatografie MeSH
- lidé MeSH
- RNA MeSH
- stanovení celkové genové exprese * metody MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články 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.
- Publikační typ
- časopisecké články MeSH
Constitutional mismatch repair deficiency (CMMRD) is a rare syndrome characterized by an increased incidence of cancer. It is caused by biallelic germline mutations in one of the four mismatch repair genes (MMR) genes: MLH1, MSH2, MSH6, or PMS2. Accurate diagnosis accompanied by a proper molecular genetic examination plays a crucial role in cancer management and also has implications for other family members. In this report, we share the impact of the diagnosis and challenges during the clinical management of two brothers with CMMRD from a non-consanguineous family harbouring compound heterozygous variants in the PMS2 gene. Both brothers presented with different phenotypic manifestations and cancer spectrum. Treatment involving immune checkpoint inhibitors significantly contributed to prolonged survival in both patients affected by lethal gliomas. The uniform hypermutation also allowed immune-directed treatment using nivolumab for the B-cell lymphoma, thereby limiting the intensive chemotherapy exposure in this young patient who remains at risk for subsequent malignancies.
- Publikační typ
- časopisecké články MeSH
Colorectal cancer (CRC) is the second most prevalent cancer type worldwide, which highlights the urgent need for non-invasive biomarkers for its early detection and improved prognosis. We aimed to investigate the patterns of long non-coding RNAs (lncRNAs) in small extracellular vesicles (sEVs) collected from low-volume blood serum specimens of CRC patients, focusing on their potential as diagnostic biomarkers. Our research comprised two phases: an initial exploratory phase involving RNA sequencing of sEVs from 76 CRC patients and 29 healthy controls, and a subsequent validation phase with a larger cohort of 159 CRC patients and 138 healthy controls. Techniques such as dynamic light scattering, transmission electron microscopy, and Western blotting were utilized for sEV characterization. Optimized protocol for sEV purification, RNA isolation and preamplification was applied to successfully sequence the RNA content of sEVs and validate the results by RT-qPCR. We successfully isolated sEVs from blood serum and prepared sequencing libraries from a low amount of RNA. High-throughput sequencing identified differential levels of 460 transcripts between CRC patients and healthy controls, including mRNAs, lncRNAs, and pseudogenes, with approximately 20% being lncRNAs, highlighting several tumor-specific lncRNAs that have not been associated with CRC development and progression. The validation phase confirmed the upregulation of three lncRNAs (NALT1, AL096828, and LINC01637) in blood serum of CRC patients. This study not only identified lncRNA profiles in a population of sEVs from low-volume blood serum specimens of CRC patients but also highlights the value of innovative techniques in biomolecular research, particularly for the detection and analysis of low-abundance biomolecules in clinical samples. The identification of specific lncRNAs associated with CRC provides a foundation for future research into their functional roles in cancer development and potential clinical applications.
Bioinformatics may seem to be a scientific field processing primarily large string datasets, as nucleotides and amino acids are represented with dedicated characters. On the other hand, many computational tasks that bioinformatics challenges are mathematical problems understandable as operations with digits. In fact, many computational tasks are solved this way in the background. One of the most widely used digital representations is mapping of nucleotides and amino acids with integers 0-3 and 0-20, respectively. The limitation of this mapping occurs when the digital signal of nucleotides has to be translated into a digital signal of amino acids as the genetic code is degenerated. This causes non-monotonies in a mapping function. Although map for reducing this undesirable effect has already been proposed, it is defined theoretically and for standard genetic codes only. In this study, we derived a novel optimal criterion for reducing the influence of degeneration by utilizing a large dataset of real sequences with various genetic codes. As a result, we proposed a new robust global optimal map suitable for any genetic code as well as specialized optimal maps for particular genetic codes.
- Publikační typ
- časopisecké články MeSH
Species delineation based on bacterial genomes is an essential part of the research of prokaryotes. In silico genome-to-genome comparison methods are computationally demanding, but much less tedious and error prone than the wet-lab methods. In this paper, we present a novel method for the delineation of bacterial genomes based on genomic signal processing. The proposed method uses numerical representations of whole bacterial genomes, phase signal and cumulated phase signal, from which four parameters are derived for each genome. The parameters characterize a genome and their calculation is independent of the other genomes comprising a delineation dataset. The delineation itself is processed as a calculation of the parameters' average similarity. The method was statistically verified on 1826 bacterial genomes. A similarity threshold of 96% was set based on the receiver operating characteristic curve that featured sensitivity of 99.78% and specificity of 97.25%. Additionally, comparative analysis on another 33 bacterial genomes was conducted using standard delineation tools as these tools were not able to process the dataset of 1826 genomes using desktop computer. The proposed method achieved comparable or better delineation results in comparison with the standard tools. Besides the excellent delineation results, another great advantage of the method is its small computational demands, which enables the delineation of thousands of genomes on a desktop computer. The calculation of the parameters takes tens of minutes for thousands of genomes. Moreover, they can be calculated in advance by creating a database, meaning the delineation itself is then completed in a matter of seconds.
- Publikační typ
- časopisecké články MeSH
