Publications by authors named "Victor Guryev"

107 Publications

The 'un-shrunk' partial correlation in Gaussian graphical models.

BMC Bioinformatics 2021 Sep 7;22(1):424. Epub 2021 Sep 7.

Bernoulli Institute, University of Groningen, Groningen, 9747 AG, The Netherlands.

Background: In systems biology, it is important to reconstruct regulatory networks from quantitative molecular profiles. Gaussian graphical models (GGMs) are one of the most popular methods to this end. A GGM consists of nodes (representing the transcripts, metabolites or proteins) inter-connected by edges (reflecting their partial correlations). Learning the edges from quantitative molecular profiles is statistically challenging, as there are usually fewer samples than nodes ('high dimensional problem'). Shrinkage methods address this issue by learning a regularized GGM. However, it remains open to study how the shrinkage affects the final result and its interpretation.

Results: We show that the shrinkage biases the partial correlation in a non-linear way. This bias does not only change the magnitudes of the partial correlations but also affects their order. Furthermore, it makes networks obtained from different experiments incomparable and hinders their biological interpretation. We propose a method, referred to as 'un-shrinking' the partial correlation, which corrects for this non-linear bias. Unlike traditional methods, which use a fixed shrinkage value, the new approach provides partial correlations that are closer to the actual (population) values and that are easier to interpret. This is demonstrated on two gene expression datasets from Escherichia coli and Mus musculus.

Conclusions: GGMs are popular undirected graphical models based on partial correlations. The application of GGMs to reconstruct regulatory networks is commonly performed using shrinkage to overcome the 'high-dimensional problem'. Besides it advantages, we have identified that the shrinkage introduces a non-linear bias in the partial correlations. Ignoring this type of effects caused by the shrinkage can obscure the interpretation of the network, and impede the validation of earlier reported results.
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http://dx.doi.org/10.1186/s12859-021-04313-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8424921PMC
September 2021

InvertypeR: Bayesian inversion genotyping with Strand-seq data.

BMC Genomics 2021 Jul 31;22(1):582. Epub 2021 Jul 31.

Terry Fox Laboratory, British Columbia Cancer Agency, Vancouver, British Columbia, V5Z 1L3, Canada.

Background: Single cell Strand-seq is a unique tool for the discovery and phasing of genomic inversions. Conventional methods to discover inversions with Strand-seq data are blind to known inversion locations, limiting their statistical power for the detection of inversions smaller than 10 Kb. Moreover, the methods rely on manual inspection to separate false and true positives.

Results: Here we describe "InvertypeR", a method based on a Bayesian binomial model that genotypes inversions using fixed genomic coordinates. We validated InvertypeR by re-genotyping inversions reported for three trios by the Human Genome Structural Variation Consortium. Although 6.3% of the family inversion genotypes in the original study showed Mendelian discordance, this was reduced to 0.5% using InvertypeR. By applying InvertypeR to published inversion coordinates and predicted inversion hotspots (n = 3701), as well as coordinates from conventional inversion discovery, we furthermore genotyped 66 inversions not previously reported for the three trios.

Conclusions: InvertypeR discovers, genotypes, and phases inversions without relying on manual inspection. For greater accessibility, results are presented as phased chromosome ideograms with inversions linked to Strand-seq data in the genome browser. InvertypeR increases the power of Strand-seq for studies on the role of inversions in phenotypic variation, genome instability, and human disease.
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http://dx.doi.org/10.1186/s12864-021-07892-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8325862PMC
July 2021

Whole genome sequencing of nearly isogenic WMI and WLI inbred rats identifies genes potentially involved in depression and stress reactivity.

Sci Rep 2021 Jul 20;11(1):14774. Epub 2021 Jul 20.

University of Tennessee Health Science Center, Memphis, TN, USA.

The WMI and WLI inbred rats were generated from the stress-prone, and not yet fully inbred, Wistar Kyoto (WKY) strain. These were selected using bi-directional selection for immobility in the forced swim test and were then sib-mated for over 38 generations. Despite the low level of genetic diversity among WKY progenitors, the WMI substrain is significantly more vulnerable to stress relative to the counter-selected WLI strain. Here we quantify numbers and classes of genomic sequence variants distinguishing these substrains with the long term goal of uncovering functional and behavioral polymorphism that modulate sensitivity to stress and depression-like phenotypes. DNA from WLI and WMI was sequenced using Illumina xTen, IonTorrent, and 10X Chromium linked-read platforms to obtain a combined coverage of ~ 100X for each strain. We identified 4,296 high quality homozygous SNPs and indels between the WMI and WLI. We detected high impact variants in genes previously implicated in depression (e.g. Gnat2), depression-like behavior (e.g. Prlr, Nlrp1a), other psychiatric disease (e.g. Pou6f2, Kdm5a, Reep3, Wdfy3), and responses to psychological stressors (e.g. Pigr). High coverage sequencing data confirm that the two substrains are nearly coisogenic. Nonetheless, the small number of sequence variants contributes to numerous well characterized differences including depression-like behavior, stress reactivity, and addiction related phenotypes. These selected substrains are an ideal resource for forward and reverse genetic studies using a reduced complexity cross.
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http://dx.doi.org/10.1038/s41598-021-92993-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8292482PMC
July 2021

The sputum transcriptome better predicts COPD exacerbations after the withdrawal of inhaled corticosteroids than sputum eosinophils.

ERJ Open Res 2021 Jul 5;7(3). Epub 2021 Jul 5.

Dept of Pulmonary Diseases, University of Groningen, University Medical Center Groningen, Groningen, The Netherlands.

Introduction: Continuing inhaled corticosteroid (ICS) use does not benefit all patients with COPD, yet it is difficult to determine which patients may safely sustain ICS withdrawal. Although eosinophil levels can facilitate this decision, better biomarkers could improve personalised treatment decisions.

Methods: We performed transcriptional profiling of sputum to explore the molecular biology and compared the predictive value of an unbiased gene signature sputum eosinophils for exacerbations after ICS withdrawal in COPD patients. RNA-sequencing data of induced sputum samples from 43 COPD patients were associated with the time to exacerbation after ICS withdrawal. Expression profiles of differentially expressed genes were summarised to create gene signatures. In addition, we built a Bayesian network model to determine coregulatory networks related to the onset of COPD exacerbations after ICS withdrawal.

Results: In multivariate analyses, we identified a gene signature (, , , , , ) associated with the time to first exacerbation after ICS withdrawal. The addition of this gene signature to a multiple Cox regression model explained more variance of time to exacerbations compared to a model using sputum eosinophils. The gene signature correlated with sputum eosinophil as well as macrophage cell counts. The Bayesian network model identified three coregulatory gene networks as well as sex to be related to an early late/nonexacerbation phenotype.

Conclusion: We identified a sputum gene expression signature that exhibited a higher predictive value for predicting COPD exacerbations after ICS withdrawal than sputum eosinophilia. Future studies should investigate the utility of this signature, which might enhance personalised ICS treatment in COPD patients.
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http://dx.doi.org/10.1183/23120541.00097-2021DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8255541PMC
July 2021

Estimates of gene ensemble noise highlight critical pathways and predict disease severity in H1N1, COVID-19 and mortality in sepsis patients.

Sci Rep 2021 05 24;11(1):10793. Epub 2021 May 24.

Federal Research Centre, Institute of Cytology and Genetics, SB RAS, Novosibirsk, Russia.

Finding novel biomarkers for human pathologies and predicting clinical outcomes for patients is challenging. This stems from the heterogeneous response of individuals to disease and is reflected in the inter-individual variability of gene expression responses that obscures differential gene expression analysis. Here, we developed an alternative approach that could be applied to dissect the disease-associated molecular changes. We define gene ensemble noise as a measure that represents a variance for a collection of genes encoding for either members of known biological pathways or subunits of annotated protein complexes and calculated within an individual. The gene ensemble noise allows for the holistic identification and interpretation of gene expression disbalance on the level of gene networks and systems. By comparing gene expression data from COVID-19, H1N1, and sepsis patients we identified common disturbances in a number of pathways and protein complexes relevant to the sepsis pathology. Among others, these include the mitochondrial respiratory chain complex I and peroxisomes. This suggests a Warburg effect and oxidative stress as common hallmarks of the immune host-pathogen response. Finally, we showed that gene ensemble noise could successfully be applied for the prediction of clinical outcome namely, the mortality of patients. Thus, we conclude that gene ensemble noise represents a promising approach for the investigation of molecular mechanisms of pathology through a prism of alterations in the coherent expression of gene circuits.
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http://dx.doi.org/10.1038/s41598-021-90192-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8144599PMC
May 2021

Construction of Whole Genomes from Scaffolds Using Single Cell Strand-Seq Data.

Int J Mol Sci 2021 Mar 31;22(7). Epub 2021 Mar 31.

Terry Fox Laboratory, BC Cancer Agency, Vancouver, BC V5Z 1L3, Canada.

Accurate reference genome sequences provide the foundation for modern molecular biology and genomics as the interpretation of sequence data to study evolution, gene expression, and epigenetics depends heavily on the quality of the genome assembly used for its alignment. Correctly organising sequenced fragments such as contigs and scaffolds in relation to each other is a critical and often challenging step in the construction of robust genome references. We previously identified misoriented regions in the mouse and human reference assemblies using Strand-seq, a single cell sequencing technique that preserves DNA directionality Here we demonstrate the ability of Strand-seq to build and correct full-length chromosomes by identifying which scaffolds belong to the same chromosome and determining their correct order and orientation, without the need for overlapping sequences. We demonstrate that Strand-seq exquisitely maps assembly fragments into large related groups and chromosome-sized clusters without using new assembly data. Using template strand inheritance as a bi-allelic marker, we employ genetic mapping principles to cluster scaffolds that are derived from the same chromosome and order them within the chromosome based solely on directionality of DNA strand inheritance. We prove the utility of our approach by generating improved genome assemblies for several model organisms including the ferret, pig, Xenopus, zebrafish, Tasmanian devil and the Guinea pig.
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http://dx.doi.org/10.3390/ijms22073617DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8037727PMC
March 2021

Sperm DNA damage causes genomic instability in early embryonic development.

Sci Adv 2020 Apr 15;6(16):eaaz7602. Epub 2020 Apr 15.

Center for Molecular Medicine and Oncode Institute, University Medical Center Utrecht, Utrecht University, Universiteitsweg 100, Utrecht 3584 CG, Netherlands.

Genomic instability is common in human embryos, but the underlying causes are largely unknown. Here, we examined the consequences of sperm DNA damage on the embryonic genome by single-cell whole-genome sequencing of individual blastomeres from bovine embryos produced with sperm damaged by γ-radiation. Sperm DNA damage primarily leads to fragmentation of the paternal chromosomes followed by random distribution of the chromosomal fragments over the two sister cells in the first cell division. An unexpected secondary effect of sperm DNA damage is the induction of direct unequal cleavages, which include the poorly understood heterogoneic cell divisions. As a result, chaotic mosaicism is common in embryos derived from fertilizations with damaged sperm. The mosaic aneuploidies, uniparental disomies, and de novo structural variation induced by sperm DNA damage may compromise fertility and lead to rare congenital disorders when embryos escape developmental arrest.
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http://dx.doi.org/10.1126/sciadv.aaz7602DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7159919PMC
April 2020

A Novel Role for Bronchial MicroRNAs and Long Noncoding RNAs in Asthma Remission.

Am J Respir Crit Care Med 2020 08;202(4):614-618

University Medical Center Groningen, University of GroningenGroningen, the Netherlands.

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http://dx.doi.org/10.1164/rccm.201908-1610LEDOI Listing
August 2020

Smooth-muscle-derived WNT5A augments allergen-induced airway remodelling and Th2 type inflammation.

Sci Rep 2020 04 21;10(1):6754. Epub 2020 Apr 21.

Department of Molecular Pharmacology, University of Groningen, Groningen, The Netherlands.

Asthma is a heterogeneous disease characterized by chronic inflammation and structural changes in the airways. The airway smooth muscle (ASM) is responsible for airway narrowing and an important source of inflammatory mediators. We and others have previously shown that WNT5A mRNA and protein expression is higher in the ASM of asthmatics compared to healthy controls. Here, we aimed to characterize the functional role of (smooth muscle-derived) WNT5A in asthma. We generated a tet-ON smooth-muscle-specific WNT5A transgenic mouse model, enabling in vivo characterization of smooth-muscle-derived WNT5A in response to ovalbumin. Smooth muscle specific WNT5A overexpression showed a clear trend towards enhanced actin (α-SMA) expression in the ASM in ovalbumin challenged animals, but had no effect on collagen content. WNT5A overexpression in ASM also significantly enhanced the production of the Th2-cytokines IL4 and IL5 in lung tissue after ovalbumin exposure. In line with this, WNT5A increased mucus production, and enhanced eosinophilic infiltration and serum IgE production in ovalbumin-treated animals. In addition, CD4 T cells of asthma patients and healthy controls were stimulated with WNT5A and changes in gene transcription assessed by RNA-seq. WNT5A promoted expression of 234 genes in human CD4 T cells, among which the Th2 cytokine IL31 was among the top 5 upregulated genes. IL31 was also upregulated in response to smooth muscle-specific WNT5A overexpression in the mouse. In conclusion, smooth-muscle derived WNT5A augments Th2 type inflammation and remodelling. Our findings imply a pro-inflammatory role for smooth muscle-derived WNT5A in asthma, resulting in increased airway wall inflammation and remodelling.
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http://dx.doi.org/10.1038/s41598-020-63741-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7174298PMC
April 2020

Eleven grand challenges in single-cell data science.

Genome Biol 2020 02 7;21(1):31. Epub 2020 Feb 7.

Genome Biology Unit, European Molecular Biology Laboratory, Heidelberg, Germany.

The recent boom in microfluidics and combinatorial indexing strategies, combined with low sequencing costs, has empowered single-cell sequencing technology. Thousands-or even millions-of cells analyzed in a single experiment amount to a data revolution in single-cell biology and pose unique data science problems. Here, we outline eleven challenges that will be central to bringing this emerging field of single-cell data science forward. For each challenge, we highlight motivating research questions, review prior work, and formulate open problems. This compendium is for established researchers, newcomers, and students alike, highlighting interesting and rewarding problems for the coming years.
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http://dx.doi.org/10.1186/s13059-020-1926-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7007675PMC
February 2020

Integrated proteogenomic approach identifying a protein signature of COPD and a new splice variant of SORBS1.

Thorax 2020 02 14;75(2):180-183. Epub 2020 Jan 14.

Department of Analytical Biochemistry, University of Groningen, Groningen Research Institute of Pharmacy, Groningen, the Netherlands.

Translation of genomic alterations to protein changes in chronic obstructive pulmonary disease (COPD) is largely unexplored. Using integrated proteomic and RNA sequencing analysis of COPD and control lung tissues, we identified a protein signature in COPD characterised by extracellular matrix changes and a potential regulatory role for SUMO2. Furthermore, we identified 61 differentially expressed novel, non-reference, peptides in COPD compared with control lungs. This included two peptides encoding for a new splice variant of SORBS1, of which the transcript usage was higher in COPD compared with control lungs. These explorative findings and integrative proteogenomic approach open new avenues to further unravel the pathology of COPD.
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http://dx.doi.org/10.1136/thoraxjnl-2019-213200DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7029225PMC
February 2020

Gene network approach reveals co-expression patterns in nasal and bronchial epithelium.

Sci Rep 2019 11 1;9(1):15835. Epub 2019 Nov 1.

University of Groningen, University Medical Center Groningen, Department of Pulmonology, Groningen, The Netherlands.

Nasal gene expression profiling is a new approach to investigate the airway epithelium as a biomarker to study the activity and treatment responses of obstructive pulmonary diseases. We investigated to what extent gene expression profiling of nasal brushings is similar to that of bronchial brushings. We performed genome wide gene expression profiling on matched nasal and bronchial epithelial brushes from 77 respiratory healthy individuals. To investigate differences and similarities among regulatory modules, network analysis was performed on correlated, differentially expressed and smoking-related genes using Gaussian Graphical Models. Between nasal and bronchial brushes, 619 genes were correlated and 1692 genes were differentially expressed (false discovery rate <0.05, |Fold-change|>2). Network analysis of correlated genes showed pro-inflammatory pathways to be similar between the two locations. Focusing on smoking-related genes, cytochrome-P450 pathway related genes were found to be similar, supporting the concept of a detoxifying response to tobacco exposure throughout the airways. In contrast, cilia-related pathways were decreased in nasal compared to bronchial brushes when focusing on differentially expressed genes. Collectively, while there are substantial differences in gene expression between nasal and bronchial brushes, we also found similarities, especially in the response to the external factors such as smoking.
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http://dx.doi.org/10.1038/s41598-019-50963-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6825243PMC
November 2019

breakpointR: an R/Bioconductor package to localize strand state changes in Strand-seq data.

Bioinformatics 2020 02;36(4):1260-1261

European Research Institute for the Biology of Ageing, University of Groningen, University Medical Center Groningen, Groningen, The Netherlands.

Motivation: Strand-seq is a specialized single-cell DNA sequencing technique centered around the directionality of single-stranded DNA. Computational tools for Strand-seq analyses must capture the strand-specific information embedded in these data.

Results: Here we introduce breakpointR, an R/Bioconductor package specifically tailored to process and interpret single-cell strand-specific sequencing data obtained from Strand-seq. We developed breakpointR to detect local changes in strand directionality of aligned Strand-seq data, to enable fine-mapping of sister chromatid exchanges, germline inversion and to support global haplotype assembly. Given the broad spectrum of Strand-seq applications we expect breakpointR to be an important addition to currently available tools and extend the accessibility of this novel sequencing technique.

Availability And Implementation: R/Bioconductor package https://bioconductor.org/packages/breakpointR.

Supplementary Information: Supplementary data are available at Bioinformatics online.
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http://dx.doi.org/10.1093/bioinformatics/btz681DOI Listing
February 2020

Proteogenomics: From next-generation sequencing (NGS) and mass spectrometry-based proteomics to precision medicine.

Clin Chim Acta 2019 Nov 14;498:38-46. Epub 2019 Aug 14.

UKM Medical Molecular Biology Institute (UMBI), Universiti Kebangsaan Malaysia, 56000 Kuala Lumpur, Malaysia.

One of the best-established area within multi-omics is proteogenomics, whereby the underpinning technologies are next-generation sequencing (NGS) and mass spectrometry (MS). Proteogenomics has contributed significantly to genome (re)-annotation, whereby novel coding sequences (CDS) are identified and confirmed. By incorporating in-silico translated genome variants in protein database, single amino acid variants (SAAV) and splice proteoforms can be identified and quantified at peptide level. The application of proteogenomics in cancer research potentially enables the identification of patient-specific proteoforms, as well as the association of the efficacy or resistance of cancer therapy to different mutations. Here, we discuss how NGS/TGS data are analyzed and incorporated into the proteogenomic framework. These sequence data mainly originate from whole genome sequencing (WGS), whole exome sequencing (WES) and RNA-Seq. We explain two major strategies for sequence analysis i.e., de novo assembly and reads mapping, followed by construction of customized protein databases using such data. Besides, we also elaborate on the procedures of spectrum to peptide sequence matching in proteogenomics, and the relationship between database size on the false discovery rate (FDR). Finally, we discuss the latest development in proteogenomics-assisted precision oncology and also challenges and opportunities in proteogenomics research.
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http://dx.doi.org/10.1016/j.cca.2019.08.010DOI Listing
November 2019

Peptide microarray of pediatric acute myeloid leukemia is related to relapse and reveals involvement of DNA damage response and repair.

Oncotarget 2019 Jul 23;10(45):4679-4690. Epub 2019 Jul 23.

Department of Pediatric Oncology/Hematology, Beatrix Children's Hospital, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands.

The majority of acute myeloid leukemia (AML) patients suffer from relapse and the exact etiology of AML remains unclear. The aim of this study was to gain comprehensive insights into the activity of signaling pathways in AML. In this study, using a high-throughput PepChip™ Kinomics microarray system, pediatric AML samples were analyzed to gain insights of active signal transduction pathway. Unsupervised hierarchical cluster analysis separated the AML blast profiles into two clusters. These two clusters were independent of patient characteristics, whereas the cumulative incidence of relapse (CIR) was significantly higher in the patients belonging to cluster-2. In addition, cluster-2 samples showed to be significantly less sensitive to various chemotherapeutic drugs. The activated peptides in cluster-1 and cluster-2 reflected the activity of cell cycle regulation, cell proliferation, cell differentiation, apoptosis, PI3K/AKT, MAPK, metabolism regulation, transcription factors and GPCRs signaling pathways. The difference between two clusters might be explained by the higher cell cycle arrest response in cluster-1 patients and higher DNA repair mechanism in cluster-2 patients. In conclusion, our study identifies different signaling profiles in pediatric AML in relation with CIR involving DNA damage response and repair.
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http://dx.doi.org/10.18632/oncotarget.27086DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6659796PMC
July 2019

Exact hypothesis testing for shrinkage-based Gaussian graphical models.

Bioinformatics 2019 12;35(23):5011-5017

Department of Analytical Biochemistry, Groningen Research Institute of Pharmacy.

Motivation: One of the main goals in systems biology is to learn molecular regulatory networks from quantitative profile data. In particular, Gaussian graphical models (GGMs) are widely used network models in bioinformatics where variables (e.g. transcripts, metabolites or proteins) are represented by nodes, and pairs of nodes are connected with an edge according to their partial correlation. Reconstructing a GGM from data is a challenging task when the sample size is smaller than the number of variables. The main problem consists in finding the inverse of the covariance estimator which is ill-conditioned in this case. Shrinkage-based covariance estimators are a popular approach, producing an invertible 'shrunk' covariance. However, a proper significance test for the 'shrunk' partial correlation (i.e. the GGM edges) is an open challenge as a probability density including the shrinkage is unknown. In this article, we present (i) a geometric reformulation of the shrinkage-based GGM, and (ii) a probability density that naturally includes the shrinkage parameter.

Results: Our results show that the inference using this new 'shrunk' probability density is as accurate as Monte Carlo estimation (an unbiased non-parametric method) for any shrinkage value, while being computationally more efficient. We show on synthetic data how the novel test for significance allows an accurate control of the Type I error and outperforms the network reconstruction obtained by the widely used R package GeneNet. This is further highlighted in two gene expression datasets from stress response in Eschericha coli, and the effect of influenza infection in Mus musculus.

Availability And Implementation: https://github.com/V-Bernal/GGM-Shrinkage.

Supplementary Information: Supplementary data are available at Bioinformatics online.
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http://dx.doi.org/10.1093/bioinformatics/btz357DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6901079PMC
December 2019

Multi-platform discovery of haplotype-resolved structural variation in human genomes.

Nat Commun 2019 04 16;10(1):1784. Epub 2019 Apr 16.

Pacific Biosciences, Menlo Park, CA, 94025, USA.

The incomplete identification of structural variants (SVs) from whole-genome sequencing data limits studies of human genetic diversity and disease association. Here, we apply a suite of long-read, short-read, strand-specific sequencing technologies, optical mapping, and variant discovery algorithms to comprehensively analyze three trios to define the full spectrum of human genetic variation in a haplotype-resolved manner. We identify 818,054 indel variants (<50 bp) and 27,622 SVs (≥50 bp) per genome. We also discover 156 inversions per genome and 58 of the inversions intersect with the critical regions of recurrent microdeletion and microduplication syndromes. Taken together, our SV callsets represent a three to sevenfold increase in SV detection compared to most standard high-throughput sequencing studies, including those from the 1000 Genomes Project. The methods and the dataset presented serve as a gold standard for the scientific community allowing us to make recommendations for maximizing structural variation sensitivity for future genome sequencing studies.
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http://dx.doi.org/10.1038/s41467-018-08148-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6467913PMC
April 2019

AGER expression and alternative splicing in bronchial biopsies of smokers and never smokers.

Respir Res 2019 Apr 10;20(1):70. Epub 2019 Apr 10.

Department of Pathology & Medical Biology, University Medical Center Groningen, University of Groningen, Hanzeplein 1, 9713 GZ, Groningen, The Netherlands.

Cigarette smoking is one of the major risk factors for the development of chronic obstructive pulmonary disease (COPD). Evidence is accumulating that Receptor for Advanced Glycation-End products (RAGE)-signaling is a key pathway in the pathophysiology of COPD. To date, it is unknown how smoking affects RAGE expression. In the current study, we investigated the effect of smoking on AGER, the gene encoding RAGE, expression and on alternative splicing of AGER. To this end, we conducted RNA-Seq on bronchial biopsies for asymptomatic smokers (n = 36) and never smokers (n = 40). Total AGER gene expression was accessed using DESeq2, while alternative splicing was investigated by measuring the number of specific split reads spanning exon-exon junctions and the total split reads. One of the major isoforms of RAGE is endogenous soluble (es) RAGE, an anti-inflammatory decoy receptor, making up for approximately 10% of the total amount of soluble (s)RAGE. We found that smokers show decreased total gene expression of AGER in bronchial biopsies, while the relative abundance of the esRAGE isoform is increased. Furthermore, no difference in the serum levels of total sRAGE were observed between smokers and non-smokers. Our data indicates that smoking initiates a protective anti-inflammatory mechanism with decreased expression of the pro-inflammatory gene AGER and increased relative abundance of the anti-inflammatory isoform esRAGE.
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http://dx.doi.org/10.1186/s12931-019-1038-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6456980PMC
April 2019

TGF-β activation impairs fibroblast ability to support adult lung epithelial progenitor cell organoid formation.

Am J Physiol Lung Cell Mol Physiol 2019 07 10;317(1):L14-L28. Epub 2019 Apr 10.

Department of Molecular Pharmacology, Groningen Research Institute for Asthma and COPD, University of Groningen , Groningen , The Netherlands.

Transforming growth factor-β (TGF-β)-induced fibroblast-to-myofibroblast differentiation contributes to remodeling in chronic obstructive pulmonary disease and idiopathic pulmonary fibrosis, but whether this impacts the ability of fibroblasts to support lung epithelial repair remains little explored. We pretreated human lung fibroblasts [primary (phFB) or MRC5 cells] with recombinant human TGF-β to induce myofibroblast differentiation, then cocultured them with adult mouse lung epithelial cell adhesion molecule-positive cells (EpCAM) to investigate their capacity to support epithelial organoid formation in vitro. While control phFB and MRC5 lung fibroblasts supported organoid formation of mouse EpCAM cells, TGF-β pretreatment of both phFB and MRC5 impaired organoid-supporting ability. We performed RNA sequencing of TGF-β-treated phFB, which revealed altered expression of key Wnt signaling pathway components and Wnt/β-catenin target genes, and modulated expression of secreted factors involved in mesenchymal-epithelial signaling. TGF-β profoundly skewed the transcriptional program induced by the Wnt/β-catenin activator CHIR99021. Supplementing organoid culture media recombinant hepatocyte growth factor or fibroblast growth factor 7 promoted organoid formation when using TGF-β pretreated fibroblasts. In conclusion, TGF-β-induced myofibroblast differentiation results in Wnt/β-catenin pathway skewing and impairs fibroblast ability to support epithelial repair likely through multiple mechanisms, including modulation of secreted growth factors.
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http://dx.doi.org/10.1152/ajplung.00400.2018DOI Listing
July 2019

Gene expression variability: the other dimension in transcriptome analysis.

Physiol Genomics 2019 05 15;51(5):145-158. Epub 2019 Mar 15.

European Research Institute for the Biology of Ageing, University of Groningen, University Medical Centre Groningen , Groningen , The Netherlands.

Transcriptome sequencing is a powerful technique to study molecular changes that underlie the differences in physiological conditions and disease progression. A typical question that is posed in such studies is finding genes with significant changes between sample groups. In this respect expression variability is regarded as a nuisance factor that is primarily of technical origin and complicates the data analysis. However, it is becoming apparent that the biological variation in gene expression might be an important molecular phenotype that can affect physiological parameters. In this review we explore the recent literature on technical and biological variability in gene expression, sources of expression variability, (epi-)genetic hallmarks, and evolutionary constraints in genes with robust and variable gene expression. We provide an overview of recent findings on effects of external cues, such as diet and aging, on expression variability and on other biological phenomena that can be linked to it. We discuss metrics and tools that were developed for quantification of expression variability and highlight the importance of future studies in this direction. To assist the adoption of expression variability analysis, we also provide a detailed description and computer code, which can easily be utilized by other researchers. We also provide a reanalysis of recently published data to highlight the value of the analysis method.
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http://dx.doi.org/10.1152/physiolgenomics.00128.2018DOI Listing
May 2019

Quantification of Aneuploidy in Mammalian Systems.

Methods Mol Biol 2019 ;1896:159-190

European Research Institute for the Biology of Ageing (ERIBA), University of Groningen, University Medical Center Groningen, Groningen, The Netherlands.

High-throughput next generation sequencing karyotyping has emerged as a powerful tool for the detection of genomic heterogeneity in normal tissues and cancers. Here we describe a single-cell whole genome sequencing (scWGS) platform to assess whole-chromosome aneuploidy, structural aneuploidies involving only chromosome fragments and more local small copy number alterations in individual cells. We provide a detailed protocol for the isolation, library preparation, low coverage sequencing and data analysis of single cells. Since our approach does not involve a whole-genome preamplification step, our method allows for acquisition of reliable high-resolution single-cell copy number profiles. Moreover, the protocol allows multiplexing of 384 single-cell libraries in one sequencing run, thereby significantly reducing sequencing costs and can be completed in 3-4 days starting from single cell isolation to analysis of sequencing data.
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http://dx.doi.org/10.1007/978-1-4939-8931-7_15DOI Listing
August 2019

VEGFC Antibody Therapy Drives Differentiation of AML.

Cancer Res 2018 10 5;78(20):5940-5948. Epub 2018 Sep 5.

Division of Pediatric Oncology/Hematology, Department of Pediatrics, Beatrix Children's Hospital, University Medical Center Groningen, University of Groningen, Groningen, the Netherlands.

High expression of VEGFC predicts adverse prognosis in acute myeloid leukemia (AML). We therefore explored VEGFC-targeting efficacy as an AML therapy using a VEGFC mAb. VEGFC antibody therapy enforced myelocytic differentiation of clonal CD34 AML blasts. Treatment of CD34 AML blasts with the antibody reduced expansion potential by 30% to 50% and enhanced differentiation via FOXO3A suppression and inhibition of MAPK/ERK proliferative signals. VEGFC antibody therapy also accelerated leukemia cell differentiation in a systemic humanized AML mouse model. Collectively, these results define a regulatory function of VEGFC in CD34 AML cell fate decisions via FOXO3A and serve as a new potential differentiation therapy for patients with AML. These findings reveal VEGFC targeting as a promising new differentiation therapy in AML. .
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http://dx.doi.org/10.1158/0008-5472.CAN-18-0250DOI Listing
October 2018

Cigarette smoke exposure decreases CFLAR expression in the bronchial epithelium, augmenting susceptibility for lung epithelial cell death and DAMP release.

Sci Rep 2018 08 20;8(1):12426. Epub 2018 Aug 20.

Department of Pathology and Medical Biology, University Medical Center Groningen, Groningen, The Netherlands.

Cigarette smoking is a major risk factor for the inflammatory disease, chronic obstructive pulmonary disease (COPD). The mechanism by which cigarette smoke (CS) induces chronic lung inflammation is still largely unknown. We hypothesize that immunogenic airway epithelial cell death is involved in the initiation of the inflammatory response. We previously identified CFLAR, the gene encoding the cell death regulator protein c-FLIP, to be associated with CS-induced release of damage-associated molecular patterns (DAMPs). Here, we investigated the effect of CS on expression levels of CFLAR in bronchial biopsies from smokers and non-smokers and CFLAR transcript isoform-expression in a dataset of air-liquid interface-differentiated bronchial epithelial cells. Furthermore, CFLAR was down-regulated by siRNA in lung epithelial A549 cells, followed by investigation of the effects on apoptosis, necrosis and DAMP release. CS exposure significantly decreased CFLAR expression in bronchial epithelial cells. Moreover, we observed a shift in relative abundance of the isoforms c-FLIP and c-FLIP transcripts in bronchial biopsies of current smokers compared to non-smokers, consistent with a shift towards necroptosis. In vitro, down-regulation of CFLAR increased apoptosis at baseline as well as CS extract-induced necrosis and DAMP release. In conclusion, CS exposure decreases CFLAR expression, which might increase susceptibility to immunogenic cell death.
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http://dx.doi.org/10.1038/s41598-018-30602-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6102306PMC
August 2018

Proteomic alterations in early stage cervical cancer.

Oncotarget 2018 Apr 6;9(26):18128-18147. Epub 2018 Apr 6.

Laboratory of Neuro-Oncology, Clinical and Cancer Proteomics, Department of Neurology, Erasmus University Medical Center Rotterdam, Rotterdam 3015 CN, The Netherlands.

Laser capture microdissection (LCM) allows the capture of cell types or well-defined structures in tissue. We compared in a semi-quantitative way the proteomes from an equivalent of 8,000 tumor cells from patients with squamous cell cervical cancer (SCC, = 22) with healthy epithelial and stromal cells obtained from normal cervical tissue ( = 13). Proteins were enzymatically digested into peptides which were measured by high-resolution mass spectrometry and analyzed by "all-or-nothing" analysis, Bonferroni, and Benjamini-Hochberg correction for multiple testing. By comparing LCM cell type preparations, 31 proteins were exclusively found in early stage cervical cancer ( = 11) when compared with healthy epithelium and stroma, based on criteria that address specificity in a restrictive "all-or-nothing" way. By Bonferroni correction for multiple testing, 30 proteins were significantly up-regulated between early stage cervical cancer and healthy control, including six members of the MCM protein family. MCM proteins are involved in DNA repair and expected to be participating in the early stage of cancer. After a less stringent Benjamini-Hochberg correction for multiple testing, we found that the abundances of 319 proteins were significantly different between early stage cervical cancer and healthy controls. Four proteins were confirmed in digests of whole tissue lysates by Parallel Reaction Monitoring (PRM). Ingenuity Pathway Analysis using correction for multiple testing by permutation resulted in two networks that were differentially regulated in early stage cervical cancer compared with healthy tissue. From these networks, we learned that specific tumor mechanisms become effective during the early stage of cervical cancer.
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http://dx.doi.org/10.18632/oncotarget.24773DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5915062PMC
April 2018

Reduced expression of C/EBPβ-LIP extends health and lifespan in mice.

Elife 2018 06 4;7. Epub 2018 Jun 4.

European Research Institute for the Biology of Ageing, University Medical Centre Groningen, University of Groningen, Groningen, Netherlands.

Ageing is associated with physical decline and the development of age-related diseases such as metabolic disorders and cancer. Few conditions are known that attenuate the adverse effects of ageing, including calorie restriction (CR) and reduced signalling through the mechanistic target of rapamycin complex 1 (mTORC1) pathway. Synthesis of the metabolic transcription factor C/EBPβ-LIP is stimulated by mTORC1, which critically depends on a short upstream open reading frame (uORF) in the -mRNA. Here, we describe that reduced C/EBPβ-LIP expression due to genetic ablation of the uORF delays the development of age-associated phenotypes in mice. Moreover, female C/EBPβ mice display an extended lifespan. Since LIP levels increase upon aging in wild type mice, our data reveal an important role for C/EBPβ in the aging process and suggest that restriction of LIP expression sustains health and fitness. Thus, therapeutic strategies targeting C/EBPβ-LIP may offer new possibilities to treat age-related diseases and to prolong healthspan.
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http://dx.doi.org/10.7554/eLife.34985DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5986274PMC
June 2018

Identification of Two Protein-Signaling States Delineating Transcriptionally Heterogeneous Human Medulloblastoma.

Cell Rep 2018 03;22(12):3206-3216

Departments of Pediatric Oncology and Hematology/Pediatrics, University of Groningen, University Medical Center Groningen, Hanzeplein 1, 9700 RB Groningen, the Netherlands. Electronic address:

The brain cancer medulloblastoma consists of different transcriptional subgroups. To characterize medulloblastoma at the phosphoprotein-signaling level, we performed high-throughput peptide phosphorylation profiling on a large cohort of SHH (Sonic Hedgehog), group 3, and group 4 medulloblastomas. We identified two major protein-signaling profiles. One profile was associated with rapid death post-recurrence and resembled MYC-like signaling for which MYC lesions are sufficient but not necessary. The second profile showed enrichment for DNA damage, as well as apoptotic and neuronal signaling. Integrative analysis demonstrated that heterogeneous transcriptional input converges on these protein-signaling profiles: all SHH and a subset of group 3 patients exhibited the MYC-like protein-signaling profile; the majority of the other group 3 subset and group 4 patients displayed the DNA damage/apoptotic/neuronal signaling profile. Functional analysis of enriched pathways highlighted cell-cycle progression and protein synthesis as therapeutic targets for MYC-like medulloblastoma.
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http://dx.doi.org/10.1016/j.celrep.2018.02.089DOI Listing
March 2018

Nasal epithelium as a proxy for bronchial epithelium for smoking-induced gene expression and expression Quantitative Trait Loci.

J Allergy Clin Immunol 2018 07 6;142(1):314-317.e15. Epub 2018 Mar 6.

Department of Pulmonology, University Medical Center Groningen, Groningen, The Netherlands; GRIAC (Groningen Research Institute for Asthma and COPD), Beatrix Children's Hospital, University Medical Center Groningen, Groningen, The Netherlands; Department of Pathology & Medical Biology, Section of Medical Biology, University Medical Center Groningen, Groningen, The Netherlands.

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http://dx.doi.org/10.1016/j.jaci.2018.01.047DOI Listing
July 2018
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