Biology subjects

Besselink, N.

Publications and source records attributed to Besselink, N..

11 recordsLinked to original sources

bioRxiv2024

Bacterial cell-free DNA profiling reveals co-elevation of multiple bacteria in newborn foals with suspected sepsis

BackgroundSepsis is the leading cause of death in newborn foals. This study investigates whether cell-free DNA (cfDNA) sequencing can enhance bacterial pathogen detection in foals with suspected sepsis and addresses existing knowledge gaps and diagnostic challenges. MethodsWe developed a foal cfDNA sequencing for bacteria identification (cfFBI) workflow, integrating wetlab and computational protocols to detect increased bacterial cfDNA abundance in blood. Specifically, cfFBI focusses on enriching bacterial cfDNA molecules and preventing false positive bacterial identifications. cfFBI was applied to blood samples of 25 hospitalized foals categorized according to the neonatal Systemic Inflammatory Response Syndrome (nSIRS) criteria and 7 healthy foals. ResultscfDNA levels of potential sepsis-causing bacterial genera were elevated in all 11 nSIRS-positive foals compared to healthy foals (n=7), and in 8/11 (72.7%) when compared to both nSIRS-negative (n=4; nSIRS=0) and healthy foals, with multiple genera elevated in 5/11 (45.5%). The total cfDNA concentration, bacterial cfDNA fraction and bacterial diversity were not different between the foal groups. However, nSIRS-positive foals showed significantly different end-motifs in host chromosomal cfDNA, and a decrease in host mitochondrial cfDNA fraction. ConclusionsThis study is the first to demonstrate that cfDNA sequencing in blood samples from newborn foals enables detection of pathogenic bacteria and can help identify novel host-related sepsis biomarkers. The elevated presence of multiple sepsis-causing genera in nSIRS-positive foals and the difference in end-motif, suggests that multibacterial elevation may be more common than previously thought. These findings indicate that cfDNA sequencing holds promise as a future diagnostic tool for identifying sepsis in newborn foals.

pathology↗

bioRxiv2024

Nanopore-based consensus sequencing enables accurate multimodal tumor cell-free DNA profiling

Shallow genome-wide cell-free DNA (cfDNA) sequencing holds great promise for non-invasive cancer monitoring by providing reliable copy number alteration (CNA) and fragmentomic profiles. Single nucleotide variations (SNVs) are, however, much harder to identify with low sequencing depth due to sequencing errors. Here we present Nanopore Rolling Circle Amplification (RCA)-enhanced Consensus Sequencing (NanoRCS), which leverages RCA and consensus calling based on genome-wide long-read nanopore sequencing to enable simultaneous multimodal tumor fraction estimation through SNVs, CNAs, and fragmentomics. Efficacy of NanoRCS is tested on 18 cancer patient samples and 3 healthy controls, demonstrating its ability to reliably detect tumor fractions as low as 0.24%. In vitro experiments confirm that SNV measurements are essential for detecting tumor fractions below 2%. NanoRCS provides the opportunity for cost-effective and rapid processing, which aligns well with clinical needs, particularly in settings where quick and accurate cancer monitoring is essential for personalized treatment strategies.

genomics↗

bioRxiv2022

The genome-wide mutational consequences of DNA hypomethylation

DNA methylation is important for establishing and maintaining cell identity and for genomic stability. This is achieved by regulating the accessibility of regulatory and transcriptional elements and the compaction of subtelomeric, centromeric, and other inactive genomic regions. Carcinogenesis is accompanied by a global loss in DNA methylation, which facilitates the transformation of cells. Cancer hypomethylation may also cause genomic instability, for example through interference with the protective function of telomeres and centromeres. However, understanding the role(s) of hypomethylation in tumor evolution is incomplete because the precise mutational consequences of global hypomethylation have thus far not been systematically assessed. Here we made genome-wide inventories of all possible genetic variation that accumulates in single cells upon the long-term global hypomethylation by CRISPR/CAS9-mediated conditional knockdown of DNMT1. Depletion of DNMT1 results in a genomewide reduction in DNA methylation levels. Hypomethylated cells show reduced proliferation rates, reactivation of the inactive X-chromosome and abnormal nuclear morphologies. Prolonged hypomethylation is accompanied by increased chromosomal instability. However, there is no increase in mutational burden, enrichment for certain mutational signatures or structural changes to the genome. We conclude that the primary consequence of global hypomethylation is chromosomal instability and does not necessarily lead to other small-scale or structural mutational effects.

genomics↗

bioRxiv2021

Recurrent activating mutations in AHR act as drivers of urinary tract cancer

Bladder cancer has a high recurrence rate and low survival of advanced stage patients. Few genetic drivers of bladder cancer have thus far been identified. We performed in-depth structural variant analysis on whole-genome sequencing data of 206 metastasized urinary tract cancers. In ~10% of the patients, we identified recurrent in-frame deletions of exons 8 and 9 in the aryl hydrocarbon receptor gene (AHR{Delta}e8-9), which codes for a ligand-activated transcription factor. Pan-cancer analyses show that AHR{Delta}e8-9 is highly specific to urinary tract cancer and mutually exclusive with other bladder cancer drivers. In AHR{Delta}e8-9 the ligand-binding domain is disrupted and we show that this results in ligand-independent AHR-pathway activation. In bladder organoids, AHR{Delta}e8-9 induces a transformed phenotype that is characterized by upregulation of AHR target genes, downregulation of differentiation markers and upregulation of genes associated with stemness and urothelial cancer. Furthermore, AHR{Delta}e8-9 expression results in anchorage independent growth of bladder organoids, indicating tumorigenic potential. DNA-binding deficient AHR{Delta}e8-9 fails to induce transformation, suggesting a role for AHR target genes in the acquisition of the oncogenic phenotype. In conclusion, we show that AHR{Delta}e8-9 is a novel driver of urinary tract cancer and that the AHR pathway could be an interesting therapeutic target.

cancer biology↗

bioRxiv2019

GRIDSS, PURPLE, LINX: Unscrambling the tumor genome via integrated analysis of structural variation and copy number

We have developed a novel, integrated and comprehensive purity, ploidy, structural variant and copy number somatic analysis toolkit for whole genome sequencing data of paired tumor/normal samples. We show that the combination of using GRIDSS for somatic structural variant calling and PURPLE for somatic copy number alteration calling allows highly sensitive, precise and consistent copy number and structural variant determination, as well as providing novel insights for short structural variants and regions of complex local topology. LINX, an interpretation tool, leverages the integrated structural variant and copy number calling to cluster individual structural variants into higher order events and chains them together to predict local derivative chromosome structure. LINX classifies and extensively annotates genomic rearrangements including simple and reciprocal breaks, LINE, viral and pseudogene insertions, and complex events such as chromothripsis. LINX also comprehensively calls genic fusions including chained fusions. Finally, our toolkit provides novel visualisation methods providing insight into complex genomic rearrangements.

bioinformatics↗

bioRxiv2019

Prioritization of genes driving congenital phenotypes of patients with de novo genomic structural variants

BackgroundGenomic structural variants (SVs) can affect many genes and regulatory elements. Therefore, the molecular mechanisms driving the phenotypes of patients with multiple congenital abnormalities and/or intellectual disability carrying de novo SVs are frequently unknown.\n\nResultsWe applied a combination of systematic experimental and bioinformatic methods to improve the molecular diagnosis of 39 patients with de novo SVs and an inconclusive diagnosis after regular genetic testing. In seven of these cases (18%) whole genome sequencing analysis detected disease-relevant complexities of the SVs missed in routine microarray-based analyses. We developed a computational tool to predict effects on genes directly affected by SVs and on genes indirectly affected due to changes in chromatin organization and impact on regulatory mechanisms. By combining these functional predictions with extensive phenotype information, candidate driver genes were identified in 16/39 (41%) patients. In eight cases evidence was found for involvement of multiple candidate drivers contributing to different parts of the phenotypes. Subsequently, we applied this computational method to a collection of 382 patients with previously detected and classified de novo SVs and identified candidate driver genes in 210 cases (54%), including 32 cases whose SVs were previously not classified as pathogenic. Pathogenic positional effects were predicted in 25% of the cases with balanced SVs and in 8% of the cases with copy number variants.\n\nConclusionsThese results show that driver gene prioritization based on integrative analysis of WGS data with phenotype association and chromatin organization datasets can improve the molecular diagnosis of patients with de novo SVs.

genomics↗

bioRxiv2019

Mutational impact of chronic alcohol use on stem cells in cirrhotic liver

Excessive alcohol consumption increases the risk of developing liver cancer, but the mechanism through which alcohol drives carcinogenesis is as yet unknown. Here, we determined the mutational consequences of chronic alcohol use on the genome of human liver stem cells prior to cancer development. No change in base substitution rate or spectrum could be detected. Analysis of the trunk mutations in an alcohol-related liver tumor by multi-site whole-genome sequencing confirms the absence of specific alcohol-induced mutational signatures driving the development of liver cancer. However, we did identify an enrichment of nonsynonymous base substitutions in cancer genes in stem cells of the cirrhotic livers, such as recurrent nonsense mutations in PTPRK that disturb Epidermal Growth Factor (EGF)-signaling. Our results thus suggest that chronic alcohol use does not contribute to carcinogenesis through altered mutagenicity, but instead induces microenvironment changes which provide a fertile ground for selection of cells with oncogenic mutations.

cancer biology↗

bioRxiv2019

5-Fluorouracil treatment induces characteristic T>G mutations in human cancer

5-Fluorouracil (5-FU) is a chemotherapeutic drug component that is commonly used for the treatment of solid cancers. It is proposed that 5-FU possesses anticancer properties via the interference with nucleotide synthesis and incorporation into DNA. As both mechanisms may have a mutational impact on both surviving tumor and healthy cells, we treated intestinal organoids with 5-FU followed by whole genome sequencing analysis and uncovered a highly characteristic mutational pattern that is dominated by T>G substitutions in a CTT context. Analysis of tumor whole genome sequencing data confirmed that this signature can also be identified in vivo in colorectal and breast cancer patients that have undergone treatment with 5-FU. We also found that more 5-FU mutations are induced in TP53 null backgrounds which may be of clinical relevance. Taken together, our results demonstrate that 5-FU is mutagenic and may drive tumor evolution and increase the risk of secondary malignancies. Furthermore, the identified signature shows a strong resemblance to COSMIC signature 17, the hallmark signature of treatment-naive esophageal and gastric tumors, which indicates that distinct endogenous and exogenous triggers can converge onto highly similar mutational signatures.

cancer biology↗

bioRxiv2018

Early divergence of mutational mechanisms drives genetic heterogeneity of fetal tissues

A developing human fetus needs to balance rapid cellular expansion with maintaining genomic stability. Here, we accurately quantified and characterized somatic mutation accumulation in fetal tissues by analyzing individual stem cells from human fetal liver and intestine. Fetal mutation rates were ~5-fold higher than in tissue-matched adult stem cells. The mutational landscape of fetal intestinal stem cells resembled that of adult intestinal stem cells, while the mutation spectrum of fetal liver stem cells is distinct from stem cells of the fetal intestine and the adult liver. Our analyses indicate that variation in mutational mechanisms, including oxidative stress and spontaneous deamination of methylated cytosines, contribute to the observed divergence in mutation accumulation patterns and drive genetic mosaicism in humans.\n\nOne Sentence SummaryLiver and intestinal cells accumulate elevated amounts and diverged types of somatic DNA mutations during early human fetal development

genomics↗

bioRxiv2018

Mutational impact of culturing human pluripotent and adult stem cells

Genetic changes acquired during in vitro culture pose a potential risk for the successful application of stem cells in regenerative medicine. To assess mutation accumulation risks induced by culturing, we determined genetic aberrations in individual human induced pluripotent stem cells (iPS cells) and adult stem cells (ASCs) by whole genome sequencing analyses. Individual iPS cells, intestinal ASCs and liver ASCs accumulated 3.5{+/-}0.5, 7.2{+/-}1.0 and 8.4{+/-}3.6 base substitutions per population doubling, respectively. The annual in vitro mutation accumulation rate of ASCs adds up to [~]1600 base pair substitutions, which is [~]40-fold higher than the in vivo rate of [~]40 base pair substitutions per year. Mutational analysis revealed a distinct in vitro induced mutational signature that is irrespective of stem cell type and distinct from the in vivo mutational signature. This in vitro signature is characterized by C to A changes that have previously been linked to oxidative stress conditions. Additionally, we observed stem cell-specific mutational signatures and differences in transcriptional strand bias, indicating differential activity of DNA repair mechanisms between stem cell types in culture. We demonstrate that the empirically defined mutation rates, spectra, and genomic distribution enable risk assessment by modelling the accumulation of specific oncogenic mutations during typical in vitro expansion, manipulation or screening experiments using human stem cells. Taken together, we have here for the first time accurately quantified and characterized in vitro mutation accumulation in human iPS cells and ASCs in a direct comparison. These results provide insights for further optimization of culture conditions for safe in vivo utilization of these cell types for regenerative purposes.

genomics↗

bioRxiv2017

Deficiency of global genome nucleotide excision repair explains mutational signature observed in cancer

Nucleotide excision repair (NER) is one of the main DNA repair pathways that protect cells against genomic damage. Disruption of this pathway can contribute to the development of cancer and accelerate aging. Tumors deficient in NER are more sensitive to cisplatin treatment. Characterization of the mutational consequences of NER-deficiency may therefore provide important diagnostic opportunities. Here, we analyzed the somatic mutational profiles of adult stem cells (ASCs) from NER-deficient Ercc1-/{Delta} mice, using whole-genome sequencing analysis of clonally derived organoid cultures. Our results indicate that NER-deficiency increases the base substitution load in liver, but not in small intestinal ASCs, which coincides with a tissue-specific aging-pathology observed in these mice. The mutational landscape changes as a result of NER-deficiency in ASCs of both tissues and shows an increased contribution of Signature 8 mutations, which is a pattern with unknown etiology that is recurrently observed in various cancer types. The scattered genomic distribution of the acquired base substitutions indicates that deficiency of global-genome NER (GG-NER) is responsible for the altered mutational landscape. In line with this, we observed increased Signature 8 mutations in a GG-NER-deficient human organoid culture in which XPC was deleted using CRISPR-Cas9 gene-editing. Furthermore, genomes of NER-deficient breast tumors show an increased contribution of Signature 8 mutations compared with NER-proficient tumors. Elevated levels of Signature 8 mutations may therefore serve as a biomarker for NER-deficiency and could improve personalized cancer treatment strategies.

molecular biology↗