Wilfred van IJcken
Erasmus MC, Center for Biomics, Faculty Member
- Wilfred van IJcken received his PhD in Molecular Sciences from the Wageningen University and Research Center, The Net... moreWilfred van IJcken received his PhD in Molecular Sciences from the Wageningen University and Research Center, The Netherlands in 2001. During his PhD he sequenced and annotated the SeMNPV virus genome, resulting in the identification of a novel structural virus protein and a novel baculovirus envelope fusion protein. In 2002, he joined as a post-doc the group of Prof. Frank Grosveld at Erasmus MC in Rotterdam, where he initiated and started the genomics core facility. He implemented a wide range of genomics techniques including microarrays, imaging and high throughput automation. In 2007 he was one of the first offering Next Generation Sequencing to the Dutch research community. He was appointed as assistant professor in 2008 to boost innovation on next generation sequencing technology. Implementation of Next generation sequencing in clinical diagnostics was achieved in 2012 and further professionalized by achieving ISO15189 accreditation in 2016. He is co-founder of a startup company focusing on methylation sequencing. Currently, he is responsible for the daily management (both financial and operational) of the genomics core facility with 10 full-time direct reporting technicians and bioinformaticians. He is also actively involved in medical student teaching and trainings. He is participating in several national programs and organizer of many NGS data analysis courses. He performs consultancy for several life science companies in and outside The Netherlands. Dr. ir. van IJcken is a regular speaker at international conferences and co-applicant of numerous successful grants. Teaming up with national and international research groups has yielded over 250 publications in international peer-reviewed journals.edit
Microbial community analysis of aquatic environments showed that an important component of its microbial diversity consists of bacteria with cell sizes of ;0.1 mm. Such small bacteria can show genomic reductions and metabolic dependencies... more
Microbial community analysis of aquatic environments showed that an
important component of its microbial diversity consists of bacteria with cell sizes of
;0.1 mm. Such small bacteria can show genomic reductions and metabolic dependencies
with other bacteria. However, so far, no study has investigated if such bacteria
exist in terrestrial environments like soil. Here, we isolated soil bacteria that
passed through a 0.1-mm filter. The complete genome of one of the isolates was
sequenced and the bacterium was identified as Hylemonella gracilis. A set of coculture
assays with phylogenetically distant soil bacteria with different cell and genome
sizes was performed. The coculture assays revealed that H. gracilis grows better
when interacting with other soil bacteria like Paenibacillus sp. AD87 and Serratia plymuthica.
Transcriptomics and metabolomics showed that H. gracilis was able to
change gene expression, behavior, and biochemistry of the interacting bacteria without
direct cell-cell contact. Our study indicates that in soil there are bacteria that
can pass through a 0.1-mm filter. These bacteria may have been overlooked in previous
research on soil microbial communities. Such small bacteria, exemplified here by
H. gracilis, can induce transcriptional and metabolomic changes in other bacteria
upon their interactions in soil. In vitro, the studied interspecific interactions allowed
utilization of growth substrates that could not be utilized by monocultures, suggesting
that biochemical interactions between substantially different sized soil bacteria
may contribute to the symbiosis of soil bacterial communities.
important component of its microbial diversity consists of bacteria with cell sizes of
;0.1 mm. Such small bacteria can show genomic reductions and metabolic dependencies
with other bacteria. However, so far, no study has investigated if such bacteria
exist in terrestrial environments like soil. Here, we isolated soil bacteria that
passed through a 0.1-mm filter. The complete genome of one of the isolates was
sequenced and the bacterium was identified as Hylemonella gracilis. A set of coculture
assays with phylogenetically distant soil bacteria with different cell and genome
sizes was performed. The coculture assays revealed that H. gracilis grows better
when interacting with other soil bacteria like Paenibacillus sp. AD87 and Serratia plymuthica.
Transcriptomics and metabolomics showed that H. gracilis was able to
change gene expression, behavior, and biochemistry of the interacting bacteria without
direct cell-cell contact. Our study indicates that in soil there are bacteria that
can pass through a 0.1-mm filter. These bacteria may have been overlooked in previous
research on soil microbial communities. Such small bacteria, exemplified here by
H. gracilis, can induce transcriptional and metabolomic changes in other bacteria
upon their interactions in soil. In vitro, the studied interspecific interactions allowed
utilization of growth substrates that could not be utilized by monocultures, suggesting
that biochemical interactions between substantially different sized soil bacteria
may contribute to the symbiosis of soil bacterial communities.
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The sequence of the Helicoverpa armigera single nucleocapsid
When Spodoptera exigua multicapsid nucleopolyhedrovirus (SeMNPV) is grown in insect cell culture, defective viruses are generated. These viruses lack about 25 kbp of sequence information and are no longer infectious for insects. This... more
When Spodoptera exigua multicapsid nucleopolyhedrovirus (SeMNPV) is grown in insect cell culture, defective viruses are generated. These viruses lack about 25 kbp of sequence information and are no longer infectious for insects. This makes the engineering of SeMNPV for improved insecticidal activity or as expression vectors difficult to achieve. Recombinants of Autographa californica MNPV have been generated in insects after lipofection with viral DNA and a transfer vector into the haemocoel. In the present study a novel procedure to isolate SeMNPV recombinants was adopted by alternate cloning between insect larvae and cultured cells. The S. exigua cell line Se301 was used to select the putative recombinants by following a green fluorescent protein marker inserted in the p10 locus of SeMNPV. Polyhedra from individual plaques were fed to larvae to select for biological activity. In this way an SeMNPV recombinant (SeXD1) was obtained with the speed of kill improved by about 25%. This ...
Research Interests: Western blotting, Recombinant DNA Technology, Biological Sciences, Cell line, General, and 8 moreAnimals, Quantitative Structure Activity Relationship, Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis, Green Fluorescent Protein, Biological activity, Site-directed Mutagenesis, Spodoptera exigua, and Medical and Health Sciences
Spodoptera exigua multicapsid nucleopolyhedrovirus (SeMNPV) contains a number of genes with a homologue found so far only in a distantly related baculovirus. One of these, SeMNPV ORF17/18 (Se17/18) shares 55 % amino acid similarity to... more
Spodoptera exigua multicapsid nucleopolyhedrovirus (SeMNPV) contains a number of genes with a homologue found so far only in a distantly related baculovirus. One of these, SeMNPV ORF17/18 (Se17/18) shares 55 % amino acid similarity to ORF129 of Xestia c-nigrum granulovirus (XcGV). Se17/18 was transcribed in cultured S. exigua 301 cells, as a polyadenylated transcript of 1�1 kb. 5�-RACE analysis demonstrated that Se17/18 transcripts started at 134, 131 and 126 nt upstream of the putative translational start codon. These sites overlap with a baculovirus consensus early promoter motif. Se17/18 transcripts were detected by Northern blot analysis and RT–PCR with increasing abundance from 8 h to 24 h post infection (p.i.) and still present until 72 h p.i. A C-terminal GFP-fusion protein of Se17/18 was primarily localized in the cytoplasm of Se301 and Sf21 cells. A chicken polyclonal antiserum was raised that reacted specifically to Se17/18 protein produced in E. coli. However, no immunore...
Tracheoesophageal Fistula (TOF) is a congenital anomaly for which the cause is unknown in the majority of patients. OA/TOF is a variable feature in many (often mono-) genetic syndromes. Research using animal models targeting genes... more
Tracheoesophageal Fistula (TOF) is a congenital anomaly for which the cause is unknown in the majority of patients. OA/TOF is a variable feature in many (often mono-) genetic syndromes. Research using animal models targeting genes involved in candidate pathways often result in tracheoesophageal phenotypes. However, there is limited overlap in the genes implicated by animal models and those found in OA/TOF-related syndromic anomalies. Knowledge on affected pathways in animal models is accumulating, but our understanding on these pathways in patients lags behind. If an affected pathway is associated with both animals and patients, the mechanisms linking the genetic mutation, affected cell types or cellular defect, and the phenotype are often not well understood. The locus heterogeneity and the uncertainty of the exact heritability of OA/TOF results in a relative low diagnostic yield. OA/TOF is a sporadic finding with a low familial recurrence rate. As parents are usually unaffected, d...
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Research Interests: Genetics, Pregnancy, Humans, Mutation, Haplotypes, and 4 moreFemale, Prenatal Diagnosis, Beta Thalassemia, and alleles
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4234 Anemia of chronic disease (ACD) is a complication accompanying many inflammatory diseases. The pro-inflammatory cytokine IFNγ has been implicated in this form of anemia, but the underlying mechanism remains unclear. Using a novel... more
4234 Anemia of chronic disease (ACD) is a complication accompanying many inflammatory diseases. The pro-inflammatory cytokine IFNγ has been implicated in this form of anemia, but the underlying mechanism remains unclear. Using a novel model for ACD, we demonstrate that IFNγ reduces both the lifespan and formation of red blood cells. Molecular analysis revealed that IFNγ induces expression of the transcription factors IRF-1 and PU.1 in both murine and human erythroid precursors. We found that IRF-1 binds to the promoter of PU.1 and induces its expression, leading to inhibition of erythropoiesis. Notably, downregulation of either IRF-1 or PU.1 expression is sufficient to overcome IFNγ-induced inhibition of erythropoiesis. PU.1 is a key transcription factor in hematopoietic lineage determination and its expression is sufficient to block erythroid differentiation and enhance myeloid differentiation. We therefore postulate that expression of IFNγ during infection can briefly shift the ba...
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The genetic bases of PD in sub-Saharan African (SSA) populations remain poorly characterized, and analysis of SSA families with PD might lead to the discovery of novel disease-related genes. To investigate the clinical features and... more
The genetic bases of PD in sub-Saharan African (SSA) populations remain poorly characterized, and analysis of SSA families with PD might lead to the discovery of novel disease-related genes. To investigate the clinical features and identify the disease-causing gene in a black South African family with 3 members affected by juvenile-onset parkinsonism and intellectual disability. Clinical evaluation, neuroimaging studies, whole-exome sequencing, homozygosity mapping, two-point linkage analysis, and Sanger sequencing of candidate variants. A homozygous 28-nucleotide frameshift deletion in the PTRHD1 coding region was identified in the 3 affected family members and linked to the disease with genome-wide significant evidence. PTRHD1 was recently nominated as the disease-causing gene in two Iranian families, each containing 2 siblings with similar phenotypes and homozygous missense mutations. Together with the previous reports, we provide conclusive evidence that loss-of-function mutations in PTRHD1 cause autosomal-recessive juvenile parkinsonism and intellectual disability. © 2018 International Parkinson and Movement Disorder Society.
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Carotid intima-media thickness (cIMT) is an established heritable marker for subclinical atherosclerosis. In this study, we aim to identify rare variants with large effects driving differences in cIMT by performing genome-wide linkage... more
Carotid intima-media thickness (cIMT) is an established heritable marker for subclinical atherosclerosis. In this study, we aim to identify rare variants with large effects driving differences in cIMT by performing genome-wide linkage analysis of individuals in the extremes of cIMT trait distribution (>90th percentile) in a large family-based study from a genetically isolated population in the Netherlands. Linked regions were subsequently explored by fine-mapping using exome sequencing. We observed significant evidence of linkage on chromosomes 2p16.3 [rs1017418, heterogeneity LOD (HLOD) = 3.35], 19q13.43 (rs3499, HLOD = 9.09), 20p13 (rs1434789, HLOD = 4.10), and 21q22.12 (rs2834949, HLOD = 3.59). Fine-mapping using exome sequencing data identified a non-coding variant (rs62165235) in gene under the linkage peak at chromosome 2 that is likely to have a regulatory function. The variant was associated with quantitative cIMT in the family-based study population (effect = 0.27, -valu...
Most patients with Parkinson's disease, Parkinson's disease dementia, and dementia with Lewy bodies do not carry mutations in known disease-causing genes. The aim of this study was to identify a novel gene implicated in the... more
Most patients with Parkinson's disease, Parkinson's disease dementia, and dementia with Lewy bodies do not carry mutations in known disease-causing genes. The aim of this study was to identify a novel gene implicated in the development of these disorders. Our study was done in three stages. First, we did genome-wide linkage analysis of an Italian family with dominantly inherited Parkinson's disease to identify the disease locus. Second, we sequenced the candidate gene in an international multicentre series of unrelated probands who were diagnosed either clinically or pathologically with Parkinson's disease, Parkinson's disease dementia, or dementia with Lewy bodies. As a control, we used gene sequencing data from individuals with abdominal aortic aneurysms (who were not examined neurologically). Third, we enrolled an independent series of patients diagnosed clinically with Parkinson's disease and controls with no signs or family history of Parkinson's dis...
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Hirschsprung disease (HSCR) is an inherited congenital disorder characterized by absence of enteric ganglia in the distal part of the gut. Variants in ret proto-oncogene (RET) have been associated with up to 50% of familial and 35% of... more
Hirschsprung disease (HSCR) is an inherited congenital disorder characterized by absence of enteric ganglia in the distal part of the gut. Variants in ret proto-oncogene (RET) have been associated with up to 50% of familial and 35% of sporadic cases. We searched for variants that affect disease risk in a large, multigenerational family with history of HSCR in a linkage region previously associated with the disease (4q31.3-q32.3) and exome wide. We performed exome sequencing analyses of a family in the Netherlands with 5 members diagnosed with HSCR and 2 members diagnosed with functional constipation. We initially focused on variants in genes located in 4q31.3-q32.3; however, we also performed an exome-wide analysis in which known HSCR or HSCR-associated gene variants predicted to be deleterious were prioritized for further analysis. Candidate genes were expressed in HEK293, COS-7, and Neuro-2a cells and analyzed by luciferase and immunoblot assays. Morpholinos were designed to targe...
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To identify the clinical characteristics and genetic etiology of a family affected with hereditary spastic paraplegia (HSP). Clinical, genetic, and functional analyses involving genome-wide linkage coupled to whole-exome sequencing in a... more
To identify the clinical characteristics and genetic etiology of a family affected with hereditary spastic paraplegia (HSP). Clinical, genetic, and functional analyses involving genome-wide linkage coupled to whole-exome sequencing in a consanguineous family with complicated HSP. A homozygous missense mutation was identified in the gene (c.1240T>G p.Phe414Val) that segregated with HSP complicated by intellectual disability and microcephaly. Lymphoblastoid cell lines of homozygous carrier patients revealed significantly decreased activity of the mitochondrial aconitase enzyme and defective mitochondrial respiration. encodes mitochondrial aconitase, an essential enzyme in the Krebs cycle. Recessive mutations in this gene have been previously associated with cerebellar ataxia. Our findings nominate as a disease-causing gene for autosomal recessive complicated HSP and provide further support for the central role of mitochondrial defects in the pathogenesis of HSP.
Group 2 innate lymphoid cells (ILC2s) are major producers of the cytokines driving allergic asthma, and increased ILC2 numbers have been detected in blood and sputum of asthmatic patients. Asthma susceptibility has a strong genetic... more
Group 2 innate lymphoid cells (ILC2s) are major producers of the cytokines driving allergic asthma, and increased ILC2 numbers have been detected in blood and sputum of asthmatic patients. Asthma susceptibility has a strong genetic component, but the underlying mechanisms and whether asthma genetics affect ILC2 biology remain unclear. We sought to study the ILC2 transcriptome and epigenome during airway inflammation (AI) to couple these to genes and genetic variants associated with asthma pathogenesis. Mice harboring a reporter for the key ILC2 transcription factor GATA-3 were subjected to IL-33-driven AI, and ILC2s were isolated from bronchoalveolar lavage fluid and mediastinal lymph nodes. Human ILC2s were purified from peripheral blood and activated in vitro. We used RNA sequencing, genome-wide identification of histone-3 lysine-4 dimethylation-marked chromatin, and computational approaches to study the ILC2 transcriptome and epigenome. Activated ILC2s in mice displayed a tissue-...
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The immune system produces a diverse repertoire of immunoglobulins in response to foreign antigens. During B-cell development, VDJ recombination and somatic mutations generate diversity, whereas selection processes remove it. Using both... more
The immune system produces a diverse repertoire of immunoglobulins in response to foreign antigens. During B-cell development, VDJ recombination and somatic mutations generate diversity, whereas selection processes remove it. Using both proteomic and NGS approaches, we characterized the immune repertoires in groups of rats after immunization with purified antigens. Proteomics and NGS data on the repertoire are in qualitative agreement, but did show quantitative differences that may relate to differences between the biological niches that were sampled for these approaches. Both methods contributed complementary information in the characterization of the immune repertoire. It was found that the immune repertoires resulting from each antigen had many similarities that allowed samples to cluster together, and that mutated immunoglobulin peptides were shared among animals with a response to the same antigen significantly more than for different antigens. However, the number of shared seq...
DNA methylation is a well-known epigenetic modification that plays a crucial role in gene regulation, but genome-wide analysis of DNA methylation remains technically challenging and costly. DNA methylation-dependent restriction enzymes... more
DNA methylation is a well-known epigenetic modification that plays a crucial role in gene regulation, but genome-wide analysis of DNA methylation remains technically challenging and costly. DNA methylation-dependent restriction enzymes can be used to restrict CpG methylation analysis to methylated regions of the genome only, which significantly reduces the required sequencing depth and simplifies subsequent bioinformatics analysis. Unfortunately, this approach has been hampered by complete digestion of DNA in CpG methylation-dense regions, resulting in fragments that are too small for accurate mapping. Here, we show that the activity of DNA methylation-dependent enzyme, LpnPI, is blocked by a fragment size smaller than 32 bp. This unique property prevents complete digestion of methylation-dense DNA and allows accurate genome-wide analysis of CpG methylation at single-nucleotide resolution. Methylated DNA sequencing (MeD-seq) of LpnPI digested fragments revealed highly reproducible g...
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Group 2 innate lymphoid cells (ILC2) are implicated in allergic asthma as an early innate source of the type 2 cytokines IL-5 and IL-13. However, their induction in house dust mite (HDM)-mediated airway inflammation additionally requires... more
Group 2 innate lymphoid cells (ILC2) are implicated in allergic asthma as an early innate source of the type 2 cytokines IL-5 and IL-13. However, their induction in house dust mite (HDM)-mediated airway inflammation additionally requires T cell activation. It is currently unknown whether phenotypic differences exist between ILC2s that are activated in a T cell-dependent or T cell-independent fashion. Here, we compared ILC2s in IL-33- and HDM-driven airway inflammation. Using flow cytometry, we found that surface expression levels of various markers frequently used to identify ILC2s were dependent on their mode of activation, highly variable over time, and differed between tissue compartments, including bronchoalveolar lavage (BAL) fluid, lung, draining lymph nodes, and spleen. WhereasIL-33-activated BAL fluid ILC2s exhibited an almost uniform CD25CD127T1/ST2ICOSKLRG1phenotype, at a comparable time point after HDM exposure BAL fluid ILC2s had a very heterogeneous surface marker pheno...
Exploration and visualization of next-generation sequencing data are crucial for clinical diagnostics. Software allowing simultaneous visualization of multiple regions of interest coupled with dynamic heuristic filtering of genetic... more
Exploration and visualization of next-generation sequencing data are crucial for clinical diagnostics. Software allowing simultaneous visualization of multiple regions of interest coupled with dynamic heuristic filtering of genetic aberrations is, however, lacking. Therefore, the authors developed the web application SNPitty that allows interactive visualization and interrogation of variant call format files by using B-allele frequencies of single-nucleotide polymorphisms and single-nucleotide variants, coverage metrics, and copy numbers analysis results. SNPitty displays variant alleles and allelic imbalances with a focus on loss of heterozygosity and copy number variation using genome-wide heterozygous markers and somatic mutations. In addition, SNPitty is capable of generating predefined reports that summarize and highlight disease-specific targets of interest. SNPitty was validated for diagnostic interpretation of somatic events by showcasing a serial dilution series of glioma t...
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Blepharocheilodontic syndrome (BCDS) consists of lagophthalmia, ectropion of the lower eyelids, distichiasis, euryblepharon, cleft lip/palate and dental anomalies and has autosomal dominant inheritance with variable expression. We... more
Blepharocheilodontic syndrome (BCDS) consists of lagophthalmia, ectropion of the lower eyelids, distichiasis, euryblepharon, cleft lip/palate and dental anomalies and has autosomal dominant inheritance with variable expression. We identified heterozygous variants in two genes of the cadherin-catenin complex, CDH1, encoding E-cadherin, and CTNND1, encoding p120 catenin delta1 in 15 of 17 BCDS index patients, as was recently described in a different publication. CDH1 plays an essential role in epithelial cell adherence; CTNND1 binds to CDH1 and controls the stability of the complex. Functional experiments in zebrafish and human cells showed that the CDH1 variants impair the cell adhesion function of the cadherin-catenin complex in a dominant-negative manner. Variants in CDH1 have been linked to familial hereditary diffuse gastric cancer and invasive lobular breast cancer; however, no cases of gastric or breast cancer have been reported in our BCDS cases. Functional experiments reporte...
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Despite the well-recognized clinical features due to insufficient or excessive thyroid hormone (TH) levels in humans, it is largely unknown which genes are regulated by TH in human tissues. To study the effect of TH on human gene... more
Despite the well-recognized clinical features due to insufficient or excessive thyroid hormone (TH) levels in humans, it is largely unknown which genes are regulated by TH in human tissues. To study the effect of TH on human gene expression profiles in whole blood, mainly consisting of TRα-expressing cells. We performed next-generation RNA sequencing on whole blood samples from 8 athyroid patients (4 females) on and after 4 weeks off levothyroxine replacement. Gene expression changes were analyzed through paired differential expression analysis and confirmed in a validation cohort. Weighted gene co-expression network analysis (WGCNA) was applied to identify thyroid state-related networks. We detected 486 differentially expressed (DE) genes (fold-change above 1.5; multiple testing corrected P-value <0.05), of which 76 % were positively and 24 % were negatively regulated. Gene ontology (GO) enrichment analysis revealed that 3 biological processes were significantly overrepresented ...
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Obstructive sleep apnea (OSA) is a common sleep breathing disorder associated with an increased risk of cardiovascular and cerebrovascular diseases and mortality. Although OSA is fairly heritable (~40%), there have been only few studies... more
Obstructive sleep apnea (OSA) is a common sleep breathing disorder associated with an increased risk of cardiovascular and cerebrovascular diseases and mortality. Although OSA is fairly heritable (~40%), there have been only few studies looking into the genetics of OSA. In the present study, we aimed to identify genetic variants associated with symptoms of sleep apnea by performing a whole-exome sequence meta-analysis of symptoms of sleep apnea in 1,475 individuals of European descent. We identified 17 rare genetic variants with at least suggestive evidence of significance. Replication in an independent dataset confirmed the association of a rare genetic variant (rs2229918; minor allele frequency = 0.3%) with symptoms of sleep apnea (p-valuemeta = 6.98 × 10-9, βmeta = 0.99). Rs2229918 overlaps with the 3' untranslated regions of ERCC1 and CD3EAP genes on chromosome 19q13. Both genes are expressed in tissues in the neck area, such as the tongue, muscles, cartilage and the trachea...
The development and widespread implementation of chromosome conformation capture (3C) technology has allowed unprecedented new insight into how chromosomes are folded in three-dimensional (3D) space. 3C and its derivatives have... more
The development and widespread implementation of chromosome conformation capture (3C) technology has allowed unprecedented new insight into how chromosomes are folded in three-dimensional (3D) space. 3C and its derivatives have contributed tremendously to the now widely accepted view that genome topology plays an important role in many major cellular processes, at a chromosome-wide scale, but certainly also at the level of individual genetic loci. A particularly popular application of 3C technology is to study transcriptional regulation, allowing researchers to draw maps of gene regulatory connections beyond the linear genome through addition of the third dimension. In this chapter, we provide a highly detailed protocol describing 3C coupled to high-throughput sequencing (referred to as 3C-Seq or more commonly 4C-Seq), allowing the unbiased interrogation of genome-wide chromatin interactions with specific genomic regions of interest. Interactions between spatially clustered DNA frag...
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Chronic lymphocytic leukemia (CLL) is characterized by the accumulation of mature CD5+ B cells in blood. Spontaneous apoptosis of CLL cells in vitro has hampered in-depth investigation of CLL pathogenesis. Here we describe the generation... more
Chronic lymphocytic leukemia (CLL) is characterized by the accumulation of mature CD5+ B cells in blood. Spontaneous apoptosis of CLL cells in vitro has hampered in-depth investigation of CLL pathogenesis. Here we describe the generation of three monoclonal mouse cell lines, EMC2, EMC4 and EMC6, from the IgH.TEμ CLL mouse model based on sporadic expression of SV40 large T antigen. The cell lines exhibit a stable CD5+CD43+IgM+CD19+ CLL phenotype in culture and can be adoptively transferred into Rag1-/- mice. RNA-seq analysis revealed only minor differences between the cell lines and their primary tumors and suggested that NF-κB and mTOR signaling pathways were involved in cell line outgrowth. In vitro survival and proliferation was dependent on constitutive phosphorylation of Bruton's tyrosine kinase (Btk) at Y551/Y223, and Akt(S473). Treatment of the cell lines with small molecule inhibitors specific for Btk (ibrutinib) or PI3K (idelalisib), which is upstream of Akt, resulted in...
Prenatal diagnostics has been impacted by technological changes in the past decade, which have affected the diagnostic yield. The aim of this study was to evaluate the impact of SNP array and noninvasive prenatal testing (NIPT) on the... more
Prenatal diagnostics has been impacted by technological changes in the past decade, which have affected the diagnostic yield. The aim of this study was to evaluate the impact of SNP array and noninvasive prenatal testing (NIPT) on the diagnostic yield and the number of invasive tests in our center. The frequency of pathogenic fetal unbalanced chromosome aberrations was studied in 10,005 cases referred for prenatal testing in 2009-2015. Chromosomal SNP microarray analysis replaced karyotyping in all invasively tested pregnancies and since 2014 a choice between NIPT and diagnostic testing with microarray was offered to women with an increased risk for common aneuploidy. The introduction of microarray led to an additional yield of submicroscopic pathogenic chromosome aberrations: 3.6% in fetuses with ultrasound anomalies and 1.9% in fetuses without ultrasound anomalies. The introduction of NIPT led to a decrease of invasive tests and of diagnostic yield. Moreover, a diagnostic delay in...
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Megacystis microcolon intestinal hypoperistalsis syndrome (MMIHS) is a congenital disorder characterized by loss of smooth muscle contraction in the bladder and intestine. To date, three genes are known to be involved in MMIHS... more
Megacystis microcolon intestinal hypoperistalsis syndrome (MMIHS) is a congenital disorder characterized by loss of smooth muscle contraction in the bladder and intestine. To date, three genes are known to be involved in MMIHS pathogenesis: ACTG2, MYH11, and LMOD1. However, for approximately 10% of affected individuals, the genetic cause of the disease is unknown, suggesting that other loci are most likely involved. Here, we report on three MMIHS-affected subjects from two consanguineous families with no variants in the known MMIHS-associated genes. By performing homozygosity mapping and whole-exome sequencing, we found homozygous variants in myosin light chain kinase (MYLK) in both families. We identified a 7 bp duplication (c.3838_3844dupGAAAGCG [p.Glu1282_Glyfs(∗)51]) in one family and a putative splice-site variant (c.3985+5C>A) in the other. Expression studies and splicing assays indicated that both variants affect normal MYLK expression. Because MYLK encodes an important ki...
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Poised enhancers marked by H3K27me3 in pluripotent stem cells have been implicated in the establishment of somatic expression programs during embryonic stem cell (ESC) differentiation. However, the functional relevance and mechanism of... more
Poised enhancers marked by H3K27me3 in pluripotent stem cells have been implicated in the establishment of somatic expression programs during embryonic stem cell (ESC) differentiation. However, the functional relevance and mechanism of action of poised enhancers remain unknown. Using CRISPR/Cas9 technology to engineer precise genetic deletions, we demonstrate that poised enhancers are necessary for the induction of major anterior neural regulators. Interestingly, circularized chromosome conformation capture sequencing (4C-seq) shows that poised enhancers already establish physical interactions with their target genes in ESCs in a polycomb repressive complex 2 (PRC2)-dependent manner. Loss of PRC2 does not activate poised enhancers or induce their putative target genes in undifferentiated ESCs; however, loss of PRC2 in differentiating ESCs severely and specifically compromises the induction of major anterior neural genes representing poised enhancer targets. Overall, our work illumin...
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The accumulation of irreparable cellular damage restricts healthspan after acute stress or natural aging. Senescent cells are thought to impair tissue function, and their genetic clearance can delay features of aging. Identifying how... more
The accumulation of irreparable cellular damage restricts healthspan after acute stress or natural aging. Senescent cells are thought to impair tissue function, and their genetic clearance can delay features of aging. Identifying how senescent cells avoid apoptosis allows for the prospective design of anti-senescence compounds to address whether homeostasis can also be restored. Here, we identify FOXO4 as a pivot in senescent cell viability. We designed a FOXO4 peptide that perturbs the FOXO4 interaction with p53. In senescent cells, this selectively causes p53 nuclear exclusion and cell-intrinsic apoptosis. Under conditions where it was well tolerated in vivo, this FOXO4 peptide neutralized doxorubicin-induced chemotoxicity. Moreover, it restored fitness, fur density, and renal function in both fast aging Xpd(TTD/TTD) and naturally aged mice. Thus, therapeutic targeting of senescent cells is feasible under conditions where loss of health has already occurred, and in doing so tissue...
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Hirschsprung disease (HSCR), which is congenital obstruction of the bowel, results from a failure of enteric nervous system (ENS) progenitors to migrate, proliferate, differentiate, or survive within the distal intestine. Previous studies... more
Hirschsprung disease (HSCR), which is congenital obstruction of the bowel, results from a failure of enteric nervous system (ENS) progenitors to migrate, proliferate, differentiate, or survive within the distal intestine. Previous studies that have searched for genes underlying HSCR have focused on ENS-related pathways and genes not fitting the current knowledge have thus often been ignored. We identify and validate novel HSCR genes using whole exome sequencing (WES), burden tests, in silico prediction, unbiased in vivo analyses of the mutated genes in zebrafish, and expression analyses in zebrafish, mouse, and human. We performed de novo mutation (DNM) screening on 24 HSCR trios. We identify 28 DNMs in 21 different genes. Eight of the DNMs we identified occur in RET, the main HSCR gene, and the remaining 20 DNMs reside in genes not reported in the ENS. Knockdown of all 12 genes with missense or loss-of-function DNMs showed that the orthologs of four genes (DENND3, NCLN, NUP98, and ...
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Megacystis microcolon intestinal hypoperistalsis syndrome (MMIHS) is a congenital visceral myopathy characterized by severe dilation of the urinary bladder and defective intestinal motility. The genetic basis of MMIHS has been ascribed to... more
Megacystis microcolon intestinal hypoperistalsis syndrome (MMIHS) is a congenital visceral myopathy characterized by severe dilation of the urinary bladder and defective intestinal motility. The genetic basis of MMIHS has been ascribed to spontaneous and autosomal dominant mutations in actin gamma 2 (ACTG2), a smooth muscle contractile gene. However, evidence suggesting a recessive origin of the disease also exists. Using combined homozygosity mapping and whole exome sequencing, a genetically isolated family was found to carry a premature termination codon in Leiomodin1 (LMOD1), a gene preferentially expressed in vascular and visceral smooth muscle cells. Parents heterozygous for the mutation exhibited no abnormalities, but a child homozygous for the premature termination codon displayed symptoms consistent with MMIHS. We used CRISPR-Cas9 (CRISPR-associated protein) genome editing of Lmod1 to generate a similar premature termination codon. Mice homozygous for the mutation showed los...
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Mammalian cells have developed intricate mechanisms to interpret, integrate, and respond to extra-cellular stimuli. For example, tumor necrosis factor (TNF) rapidly activates proinflammatory genes, but our understanding of how this occurs... more
Mammalian cells have developed intricate mechanisms to interpret, integrate, and respond to extra-cellular stimuli. For example, tumor necrosis factor (TNF) rapidly activates proinflammatory genes, but our understanding of how this occurs against the ongoing transcriptional program of the cell is far from complete. We monitor the early phase of this cascade at high spatio-temporal resolution in TNFα-stimulated human endothelial cells. The main driver of the response, NF-κB, hijacks the regulatory machinery by binding active enhancers already-interacting with gene promoters. Notably, a large fraction of these enhancers do not encode the canonical NF-κB recognition motif. Using a combination of genomics tools we find that site selection is a key determinant of NF-κΒ participation in both transcriptional activation and repression, and we exemplify the repressive NF-κΒ function via its synergy with the JDP2 co-repressor. Finally, detailed analysis of a 3-Mbp locus using both targeted ch...
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The dynamic three-dimensional chromatin architecture of genomes and its co-evolutionary connection to its function - the storage, expression, and replication of genetic information - is still one of the central issues in biology. Here, we... more
The dynamic three-dimensional chromatin architecture of genomes and its co-evolutionary connection to its function - the storage, expression, and replication of genetic information - is still one of the central issues in biology. Here, we describe the much debated 3D-architecture of the human and mouse genomes from the nucleosomal to the megabase pair level by a novel approach combining selective high-throughput high-resolution chromosomal interaction capture (T2C), polymer simulations, and scaling analysis of the 3D-architecture and the DNA sequence: The genome is compacted into a chromatin quasi-fibre with ~5+-1 nucleosomes/11nm, folded into stable ~30-100 kbp loops forming stable loop aggregates/rosettes connected by similar sized linkers. Minor but significant variations in the architecture are seen between cell types/functional states. The architecture and the DNA sequence show very similar fine-structured multi-scaling behaviour confirming their co-evolution and the above. Thi...
Research Interests:
Despite high heritability, little success was achieved in mapping genetic determinants of depression-related traits by means of genome-wide association studies. To identify genes associated with depressive symptomology, we performed a... more
Despite high heritability, little success was achieved in mapping genetic determinants of depression-related traits by means of genome-wide association studies. To identify genes associated with depressive symptomology, we performed a gene-based association analysis of nonsynonymous variation captured using exome-sequencing and exome-chip genotyping in a genetically isolated population from the Netherlands (n = 1999). Finally, we reproduced our significant findings in an independent population-based cohort (n = 1604). We detected significant association of depressive symptoms with a gene NKPD1 (p = 3.7 × 10(-08)). Nonsynonymous variants in the gene explained 0.9% of sex- and age-adjusted variance of depressive symptoms in the discovery study, which is translated into 3.8% of the total estimated heritability (h(2) = 0.24). Significant association of depressive symptoms with NKPD1 was also observed (n = 1604; p = 1.5 × 10(-03)) in the independent replication sample despite little overlap with the discovery cohort in the set of nonsynonymous genetic variants observed in the NKPD1 gene. Meta-analysis of the discovery and replication studies improved the association signal (p = 1.0 × 10(-09)). Our study suggests that nonsynonymous variation in the gene NKPD1 affects depressive symptoms in the general population. NKPD1 is predicted to be involved in the de novo synthesis of sphingolipids, which have been implicated in the pathogenesis of depression.
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Overexpression of LMO2 is known to be one of the causes of T-cell acute lymphoblastic leukemia (T-ALL) development; however, the mechanisms behind its oncogenic activity are incompletely understood. LMO2-overexpressing transgenic mouse... more
Overexpression of LMO2 is known to be one of the causes of T-cell acute lymphoblastic leukemia (T-ALL) development; however, the mechanisms behind its oncogenic activity are incompletely understood. LMO2-overexpressing transgenic mouse models suggest an accumulation of immature T-cell progenitors in the thymus as the main preleukemic event. The effects of LMO2 overexpression on human T-cell development in vivo are unknown. Here, we report studies of a humanized mouse model transplanted with LMO2-transduced human hematopoietic stem/progenitor cells. The effects of LMO2 overexpression were confined to the T-cell lineage; however, initially, multipotent cells were transduced. Three effects of LMO2 on human T-cell development were observed: (1) a block at the double-negative/immature single-positive stage, (2) an accumulation of CD4(+)CD8(+) double-positive CD3(-) cells, and (3) an altered CD8/CD4 ratio with enhanced peripheral T lymphocytes. Microarray analysis of sorted double-positiv...
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Alveolarization of the developing lung is an important step toward the switch from intrauterine life to breathing oxygen-rich air after birth. The distal airways structurally change to minimize the gas exchange path, and Type II... more
Alveolarization of the developing lung is an important step toward the switch from intrauterine life to breathing oxygen-rich air after birth. The distal airways structurally change to minimize the gas exchange path, and Type II pneumocytes increase the production of surfactants, which are required to reduce surface tension at the air-liquid interface in the alveolus. Hypoxia-inducible factor 2α (Hif2α) is an oxygen-regulated transcription factor expressed in endothelial and Type II cells, and its expression increases toward the end of gestation. We investigated the role of Hif2α in Type II cells by conditionally expressing an oxygen-insensitive mutant of Hif2α in airway epithelial cells during development. Newborn mice expressing the mutant Hif2α were born alive but quickly succumbed to respiratory distress. Subsequent analysis of the lungs revealed dilated alveoli covered with enlarged, aberrant Type II cells and a diminished number of Type I cells. The Type II cells accumulated glycogen in part by increased glucose uptake via the up-regulation of the glucose transporter 1. Furthermore, the cells lacked two crucial enzymes involved in the metabolism of glycogen into surfactant lipids, lysophosphatidylcholine acyltransferase and ATP-binding cassette sub-family A member 3. We conclude that Hif2α is a key regulator in alveolar maturation and the production of phospholipids by Type II cells.
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Establishing the age of unknown persons, or persons with unknown age, can provide important leads in police investigations, disaster victim identification, fraud cases, and in other legal affairs. Previous methods mostly relied on... more
Establishing the age of unknown persons, or persons with unknown age, can provide important leads in police investigations, disaster victim identification, fraud cases, and in other legal affairs. Previous methods mostly relied on morphological features available from teeth or skeletal parts. The development of molecular methods for age estimation allowing to use human specimens that possess no morphological age information, such as bloodstains, is extremely valuable as this type of samples is commonly found at crime scenes. Recently, we introduced a DNA-based approach for human age estimation from blood based on the quantification of T-cell specific DNA rearrangements (sjTRECs), which achieves accurate assignment of blood DNA samples to one of four 20-year-interval age categories. Aiming at improving the accuracy of molecular age estimation from blood, we investigated different types of biomarkers. We started out by systematic genome-wide surveys for new age-informative mRNA and DN...
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The underlying relation between Parkinson&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;s disease (PD) etiopathology... more
The underlying relation between Parkinson&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;s disease (PD) etiopathology and its major risk factor, aging, is largely unknown. In light of the causative link between genome stability and aging, we investigate a possible nexus between DNA damage accumulation, aging, and PD by assessing aging-related DNA repair pathways in laboratory animal models and humans. We demonstrate that dermal fibroblasts from PD patients display flawed nucleotide excision repair (NER) capacity and that Ercc1 mutant mice with mildly compromised NER exhibit typical PD-like pathological alterations, including decreased striatal dopaminergic innervation, increased phospho-synuclein levels, and defects in mitochondrial respiration. Ercc1 mouse mutants are also more sensitive to the prototypical PD toxin MPTP, and their transcriptomic landscape shares important similarities with that of PD patients. Our results demonstrate that specific defects in DNA repair impact the dopaminergic system and are associated with human PD pathology and might therefore constitute an age-related risk factor for PD.