Johan T den Dunnen

Leiden University Medical Centre, Leyden, South Holland, Netherlands

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Publications (324)2169.91 Total impact

  • [Show abstract] [Hide abstract]
    ABSTRACT: We describe an open-source kPAL package that facilitates an alignment-free assessment of the quality and comparability of sequencing datasets by analysing k-mer frequencies. We show that kPAL can detect technical artifacts such as high duplication rates, library chimeras, contamination, and differences in library preparation protocols. kPAL also successfully captures the complexity and diversity of microbiomes and provides a powerful means to study changes in microbial communities. Together, these features make kPAL an attractive and broadly applicable tool to determine the quality and comparability of sequence libraries even in the absence of a reference sequence. kPAL is freely available at https://github.com/LUMC/kPAL.
    Genome biology. 12/2014; 15(12):555.
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    ABSTRACT: A recent review identified 60 common inherited renal diseases caused by DNA variants in 132 different genes. These diseases can be diagnosed with DNA sequencing, but each gene probably also has a thousand normal variants. Many more normal variants have been characterised by individual laboratories than are reported in the literature or found in publicly accessible collections. At present, testing laboratories must assess each novel change they identify for pathogenicity, even when this has been done elsewhere previously, and the distinction between normal and disease-associated variants is particularly an issue with the recent surge in exomic sequencing and gene discovery projects. The Human Variome Project recommends the establishment of gene-specific DNA variant databases to facilitate the sharing of DNA variants and decisions about likely disease causation. Databases improve diagnostic accuracy and testing efficiency, and reduce costs. They also help with genotype-phenotype correlations and predictive algorithms. The Human Variome Project advocates databases that use standardised descriptions, are up-to-date, include clinical information and are freely available. Currently, the genes affected in the most common inherited renal diseases correspond to 350 different variant databases, many of which are incomplete or have insufficient clinical details for genotype-phenotype correlations. Assistance is needed from nephrologists to maximise the usefulness of these databases for the diagnosis and management of inherited renal disease.
    Pediatric nephrology (Berlin, Germany). 11/2014;
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    ABSTRACT: Aerobic methanotrophs can grow in hostile volcanic environments and use methane as their sole source of energy. The discovery of three verrucomicrobial Methylacidiphilum strains has revealed diverse metabolic pathways used by these methanotrophs, including mechanisms through which methane is oxidized. The basis of a complete understanding of these processes and of how these bacteria evolved and are able to thrive in such extreme environments partially resides in the complete characterization of their genome and its architecture.
    BMC genomics. 10/2014; 15(1):914.
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    ABSTRACT: Huntington disease is caused by expansion of a CAG repeat in the huntingtin gene that is translated into an elongated polyglutamine stretch within the N-terminal domain of the huntingtin protein. The mutation is thought to introduce a gain-of-toxic function in the mutant huntingtin protein, and blocking this toxicity by antibody binding could alleviate Huntington disease pathology. Llama single domain antibodies (VHH) directed against mutant huntingtin are interesting candidates as therapeutic agents or research tools in Huntington disease because of their small size, high thermostability, low cost of production, possibility of intracellular expression, and potency of blood-brain barrier passage. We have selected VHH from llama phage display libraries that specifically target the N-terminal domain of the huntingtin protein. Our VHH are capable of binding wild-type and mutant human huntingtin under native and denatured conditions and can be used in Huntington disease studies as a novel antibody that is easy to produce and manipulate.
    10/2014;
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    Johan den Dunnen
    Human Mutation 10/2014; 35(10). · 5.21 Impact Factor
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    ABSTRACT: A mutation update on the nebulin gene (NEB) is necessary because of recent developments in analysis methodology, the identification of increasing numbers and novel types of variants and a widening in the spectrum of clinical and histological phenotypes associated with this gigantic, 183 exons containing gene. Recessive pathogenic variants in NEB are the major cause of nemaline myopathy (NM), one of the most common congenital myopathies. Moreover, pathogenic NEB variants have been identified in core-rod myopathy and in distal myopathies. In this update, we present the disease-causing variants in NEB in 159 families, 143 families with NM and 16 families with NM-related myopathies. Eighty-eight families are presented here for the first time. We summarize 86 previously published and 126 unpublished variants identified in NEB. Furthermore, we have analyzed the NEB variants deposited in the Exome Variant Server (http://evs.gs.washington.edu/EVS/), identifying that pathogenic variants are a minor fraction of all coding variants (∼7%). This indicates that nebulin tolerates substantial changes in its amino acid sequence, providing an explanation as to why variants in such a large gene result in relatively rare disorders. Lastly, we discuss the difficulties of drawing reliable genotype-phenotype correlations in NEB-associated disease.This article is protected by copyright. All rights reserved
    Human Mutation 09/2014; · 5.21 Impact Factor
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    ABSTRACT: A r t i c l e s Current methods in genetic diagnostics and research are limited in their ability to uncover all possible genetic variation in genes of inter-est 1 . Clinical genetic tests, for example, often focus only on exons and therefore miss variants in the noncoding regulatory sequences (pro-moters and enhancers) of genes that can also disrupt gene function 2 . In addition, structural variants—such as copy number variants, trans-locations, insertions and inversions—are difficult to detect, particu-larly those that are balanced (i.e., inversions and translocations that are not accompanied by a loss or gain of sequences). Also, balanced structural variants can cause disease through the disruption of genes, creation of gene fusions or position effects—i.e., when a gene is placed under the control of different regulatory DNA elements 3,4 . Reliable detection of structural variants is hampered by the hypothesis-driven nature of current methods for targeted re-sequencing, in which the sequences to be analyzed are determined by the set of probes used in hybridization-based capture methods 5 or the primers in polymerase or ligase-based re-sequencing approaches 6 . Unknown sequences, such as those introduced by chromosomal rearrangements, are difficult to capture and re-sequence with these methods 7,8 . In addition, none of the existing targeted sequencing methods allow haplotyping, the allelic phasing of genetic variation. This information is useful, for example, when a person carries multiple recessive genetic variants that may coexist on one allele (giving carrier status) or be divided over both alleles (giving disease status). In chromosome-conformation capture (3C) 9 and related methods such as 3C on chip or combined with sequencing 10,11 (4C, which ena-bles searching of the genome for sequences contacting a site of inter-est), chromatin is crosslinked, fragmented and re-ligated to identify, on the basis of their ligation efficiency, genomic loci that are in close spatial proximity in the nucleus. Although these methods are used to detect genome folding inside cells, the resulting contact profiles typically show high enrichment of sequences directly neighboring targeted sequencing by proximity ligation for comprehensive variant detection and local haplotyping
    Nature Biotechnology 08/2014; · 32.44 Impact Factor
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    ABSTRACT: Auriculocondylar syndrome is a rare craniofacial disorder comprising core features of micrognathia, condyle dysplasia and question mark ear. Causative variants have been identified in PLCB4, GNAI3 and EDN1, which are predicted to function within the EDN1-EDNRA pathway during early pharyngeal arch patterning. To date, two GNAI3 variants in three families have been reported. Here we report three novel GNAI3 variants, one segregating with affected members in a family previously linked to 1p21.1-q23.3 and two de novo variants in simplex cases. Two variants occur in known functional motifs, the G1 and G4 boxes, and the third variant is one amino acid outside of the G1 box. Structural modeling shows that all five altered GNAI3 residues identified to date cluster in a region involved in GDP/GTP binding. We hypothesize that all GNAI3 variants lead to dominant negative effects.European Journal of Human Genetics advance online publication, 16 July 2014; doi:10.1038/ejhg.2014.132.
    European journal of human genetics: EJHG 07/2014; · 3.56 Impact Factor
  • H P J Buermans, J T den Dunnen
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    ABSTRACT: Impressive progress has been made in the field of Next Generation Sequencing (NGS). Through advancements in the fields of molecular biology and technical engineering, parallelization of the sequencing reaction has profoundly increased the total number of produced sequence reads per run. Current sequencing platforms allow for a previously unprecedented view into complex mixtures of RNA and DNA samples. NGS is currently evolving into a molecular microscope finding its way into virtually every fieldsof biomedical research. In this chapter we review the technical background of the different commercially available NGS platforms with respect to template generation and the sequencing reaction and take a small step towards what the upcoming NGS technologies will bring. We close with an overview of different implementations of NGS into biomedical research. This article is part of a Special Issue entitled: From Genome to Function.
    Biochimica et biophysica acta. 07/2014;
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    ABSTRACT: Whole-genome sequencing enables complete characterization of genetic variation, but geographic clustering of rare alleles demands many diverse populations be studied. Here we describe the Genome of the Netherlands (GoNL) Project, in which we sequenced the whole genomes of 250 Dutch parent-offspring families and constructed a haplotype map of 20.4 million single-nucleotide variants and 1.2 million insertions and deletions. The intermediate coverage (~13×) and trio design enabled extensive characterization of structural variation, including midsize events (30–500 bp) previously poorly catalogued and de novo mutations. We demonstrate that the quality of the haplotypes boosts imputation accuracy in independent samples, especially for lower frequency alleles. Population genetic analyses demonstrate fine-scale structure across the country and support multiple ancient migrations, consistent with historical changes in sea level and flooding. The GoNL Project illustrates how single-population whole-genome sequencing can provide detailed characterization of genetic variation and may guide the design of future population studies.
    Nature Genetics 06/2014; · 35.21 Impact Factor
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    ABSTRACT: Pollen monitoring is an important and widely used tool in allergy research and creation of awareness in pollen-allergic patients. Current pollen monitoring methods are microscope-based, labor-intensive and cannot identify pollen to the genus level in some relevant allergenic plant groups. Therefore a more efficient, cost-effective and sensitive method is needed. Here, we present a method for identification and quantification of airborne pollen using DNA sequencing. Pollen is collected from ambient air using standard techniques. DNA is extracted from the collected pollen and a fragment of the chloroplast gene trnL is amplified using PCR. The PCR product is subsequently sequenced on a next-generation sequencing platform (Ion Torrent). Amplicon molecules are sequenced individually, allowing identification of different sequences from a mixed sample. We show that this method provides an accurate qualitative and quantitative view of the species composition of samples of airborne pollen grains. We also show that it correctly identifies the individual grass genera present in a mixed sample of grass pollen, which cannot be achieved using microscopic pollen identification. We conclude that our method is more efficient and sensitive than current pollen monitoring techniques and therefore has the potential to increase the throughput of pollen monitoring. This article is protected by copyright. All rights reserved.
    Molecular Ecology Resources 06/2014; · 7.43 Impact Factor
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    ABSTRACT: Hemoglobinopathies, such as sickle cell disease (SCD) and beta-thalassemia major (TM), are severe diseases and the most common autosomal recessive condition worldwide and in particular in Oman. Early screening and diagnosis of carriers are the key for primary prevention. Once a country-wide population screening program is mandated by law, a sequencing technology that can rapidly confirm or identify disease-causing mutations for a large number of patients in a short period of time will be necessary. While Sanger sequencing is the standard protocol for molecular diagnosis, next generation sequencing starts to become available to reference laboratories. Using the Ion Torrent PGM sequencer, we have analyzed a cohort of 297 unrelated Omani cases and reliably identified mutations in the beta-globin (HBB) gene. Our model study has shown that Ion Torrent PGM can rapidly sequence such a small gene in a large number of samples using a barcoded uni-directional or bi-directional sequence methodology, reducing cost, workload and providing accurate diagnosis. Based on our results we believe that the Ion Torrent PGM sequencing platform, able to analyze hundreds of patients simultaneously for a single disease gene can be a valid molecular screening alternative to ABI sequencing in the diagnosis of hemoglobinopathies and other genetic disorders in the near future.
    Blood Cells Molecules and Diseases 05/2014; · 2.26 Impact Factor
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    Eleonora de Klerk, Johan T den Dunnen, Peter A C 't Hoen
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    ABSTRACT: Technological advances in the sequencing field support in-depth characterization of the transcriptome. Here, we review genome-wide RNA sequencing methods used to investigate specific aspects of gene expression and its regulation, from transcription to RNA processing and translation. We discuss tag-based methods for studying transcription, alternative initiation and polyadenylation events, shotgun methods for detection of alternative splicing, full-length RNA sequencing for the determination of complete transcript structures, and targeted methods for studying the process of transcription and translation. With the ensemble of technologies available, it is now possible to obtain a comprehensive view on transcriptome complexity and the regulation of transcript diversity.
    Cellular and Molecular Life Sciences CMLS 05/2014; · 5.62 Impact Factor
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    Johan T. den Dunnen
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    ABSTRACT: Currently, we use the term “pathogenicity” to indicate whether a variant in a gene has an influence on the phenotype or not. Since this term is confusing, not neutral, and incorrectly refers to a disease state, it seems better to use a more neutral term. The suggestion is to use “affects function”. This article is protected by copyright. All rights reserved
    Human Mutation 03/2014; · 5.21 Impact Factor
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    ABSTRACT: The growing number of biotech crops with novel genetic elements increasingly complicates the detection of genetically modified organisms (GMOs) in food and feed samples using conventional screening methods. Unauthorized GMOs (UGMOs) in food and feed are currently identified through combining GMO element screening with sequencing the DNA flanking these elements. In this study, a specific and sensitive qPCR assay was developed for vip3A element detection based on the vip3Aa20 coding sequences of the recently marketed MIR162 maize and COT102 cotton. Furthermore, SiteFinding-PCR in combination with Sanger, Illumina or Pacific BioSciences (PacBio) sequencing was performed targeting the flanking DNA of the vip3Aa20 element in MIR162. De novo assembly and Basic Local Alignment Search Tool searches were used to mimic UGMO identification. PacBio data resulted in relatively long contigs in the upstream (1,326 nucleotides (nt); 95 % identity) and downstream (1,135 nt; 92 % identity) regions, whereas Illumina data resulted in two smaller contigs of 858 and 1,038 nt with higher sequence identity (>99 % identity). Both approaches outperformed Sanger sequencing, underlining the potential for next-generation sequencing in UGMO identification.
    Analytical and Bioanalytical Chemistry 02/2014; · 3.66 Impact Factor
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    ABSTRACT: Advances in sequencing technologies and computational algorithms have enabled the study of genomic variants to dissect their functional consequence. Despite this unprecedented progress, current tools fail to reliably detect and characterize more complex allelic variants, such as short tandem repeats (STRs). We developed TSSV as an efficient and sensitive tool to specifically profile all allelic variants present in targeted loci. Based on its design, requiring only two short flanking sequences, TSSV can work without the use of a complete reference sequence to reliably profile highly polymorphic, repetitive, or uncharacterized regions. We show that TSSV can accurately determine allelic STR structures in mixtures with 10% representation of minor alleles or complex mixtures in which a single STR allele is shared. Furthermore, we show the universal utility of TSSV in two other independent studies: characterizing de novo mutations introduced by TALENs and profiling the noise and systematic errors in an IonTorrent sequencing experiment. TSSV complements the existing tools by aiding the study of highly polymorphic and complex regions and provides a high-resolution map that can be used in a wide range of applications, from personal genomics to forensic analysis and clinical diagnostics. We have implemented TSSV as a Python-package that can be installed through the command-line using pip install tssv command. Its source code and documentation are available at https://pypi.python.org/pypi/tssv and http://www.lgtc.nl/tssv.
    Bioinformatics 02/2014; · 5.47 Impact Factor
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  • [Show abstract] [Hide abstract]
    ABSTRACT: Hemoglobinopathies, such as sickle cell disease (SCD) and beta-thalassemia major (TM), are severe diseases and the most common autosomal recessive condition worldwide and in particular in Oman. Early screening and diagnosis of carriers are the key for primary prevention. Once a country-wide population screening program is mandated by law, a sequencing technology that can rapidly confirm or identify disease-causing mutations for a large number of patients in a short period of time will be necessary. While Sanger sequencing is the standard protocol for molecular diagnosis, next generation sequencing starts to become available to reference laboratories. Using the Ion Torrent PGM sequencer, we have analyzed a cohort of 297 unrelated Omani cases and reliably identified mutations in the beta-globin (HBB) gene. Our model study has shown that Ion Torrent PGM can rapidly sequence such a small gene in a large number of samples using a barcoded uni-directional or bi-directional sequence methodology, reducing cost, workload and providing accurate diagnosis. Based on our results we believe that the Ion Torrent PGM sequencing platform, able to analyze hundreds of patients simultaneously for a single disease gene can be a valid molecular screening alternative to ABI sequencing in the diagnosis of hemoglobinopathies and other genetic disorders in the near future.
    Blood Cells Molecules and Diseases 01/2014; · 2.26 Impact Factor
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    ABSTRACT: Huntington's disease (HD) is a progressive autosomal dominant disorder, caused by a CAG repeat expansion in the HTT gene, which results in expansion of a polyglutamine stretch at the N-terminal end of the huntingtin protein. Several studies have implicated the importance of proteolytic cleavage of mutant huntingtin in HD pathogenesis and it is generally accepted that N-terminal huntingtin fragments are more toxic than full-length protein. Important cleavage sites are encoded by exon 12 of HTT. Here we report proof of concept using antisense oligonucleotides to induce skipping of exon 12 in huntingtin pre-mRNA, thereby preventing the formation of a 586 amino acid N-terminal huntingtin fragment implicated in HD toxicity. In vitro studies showed successful exon skipping and appearance of a shorter huntingtin protein. Cleavage assays showed reduced 586 amino acid N-terminal huntingtin fragments in the treated samples. In vivo studies revealed exon skipping after a single injection of antisense oligonucleotides in the mouse striatum. Recent advances to inhibit the formation of mutant huntingtin using oligonucleotides seem promising therapeutic strategies for HD. Nevertheless, huntingtin is an essential protein and total removal has been shown to result in progressive neurodegeneration in vivo. Our proof of concept shows a completely novel approach to reduce mutant huntingtin toxicity not by reducing its expressing levels, but by modifying the huntingtin protein.
    Nucleic acid therapeutics. 12/2013;

Publication Stats

11k Citations
2,169.91 Total Impact Points

Institutions

  • 1995–2014
    • Leiden University Medical Centre
      • • Department of Human Genetics
      • • Department of Clinical Genetics
      Leyden, South Holland, Netherlands
  • 2013
    • Netherlands Bioinformatics Centre
      Nymegen, Gelderland, Netherlands
  • 2010–2012
    • National Institute of Health Dr. Ricardo Jorge
      Oporto, Porto, Portugal
    • University College London
      • Department of Genetics, Evolution and Environment (GEE)
      London, ENG, United Kingdom
    • Garvan Institute of Medical Research
      • Cancer Research Program
      Darlinghurst, New South Wales, Australia
  • 1987–2012
    • Leiden University
      • Molecular Cell Biology Group
      Leyden, South Holland, Netherlands
  • 2011
    • The Human Variome Project
      Melbourne, Victoria, Australia
    • University of Tampere
      • Institute of Biomedical Technology
      Tampere, Western Finland, Finland
  • 2009–2011
    • Erasmus MC
      • • Department of Clinical Genetics
      • • Genetic Epidemiology Unit
      Rotterdam, South Holland, Netherlands
  • 2008
    • INSA
      Альтамира, Tamaulipas, Mexico
    • St. Vincent's Hospital Melbourne
      Melbourne, Victoria, Australia
    • Erasmus Universiteit Rotterdam
      • Department of Forensic Molecular Biology
      Rotterdam, South Holland, Netherlands
    • Universitair Ziekenhuis Leuven
      • Department of Neurology
      Louvain, Flanders, Belgium
  • 1985–2008
    • Radboud University Nijmegen
      • Department of Molecular Biology
      Nijmegen, Provincie Gelderland, Netherlands
  • 2006
    • Utrecht University
      Utrecht, Utrecht, Netherlands
    • Howard Hughes Medical Institute
      Ashburn, Virginia, United States
  • 2005
    • Institute for Health Protection of Mother and Child of Serbia
      Beograd, Central Serbia, Serbia
  • 1995–2004
    • University of Groningen
      • Department of Medical Genetics
      Groningen, Province of Groningen, Netherlands
  • 1992–1998
    • University of São Paulo
      • • Instituto de Biociências (IB) (São Paulo)
      • • Departamento de Psicologia
      São Paulo, Estado de Sao Paulo, Brazil
  • 1993
    • The Newcastle upon Tyne Hospitals NHS Foundation Trust
      Newcastle-on-Tyne, England, United Kingdom