Ivo Fokkema

Leiden University Medical Centre | LUMC · Department of Human Genetics

Gene variant databases are the backbone of DNA-based diagnostics. These databases, also called Locus-Specific DataBases (LSDBs), store information on variants in the human genome and the observed phenotypic consequences. The largest collection of public databases uses the free, open-source LOVD software platform. To cope with the current demand for...

DNA diagnostics is based on sharing information on genes, variants, and phenotypes. Without sharing, DNA diagnostics would not be possible. Sharing is the cheapest way to classify variants. In the ideal situation, diagnostic laboratories automatically share their data with public repositories before they use them to classify their variants. Besides...

The human genome, over 3 billion nucleotides in size, collects changes over time that become part of the naturally existing variation in the population. In the case of a genetic disorder, of all of these variants usually only one or two are associated with the presented condition. Finding these variants in such large datasets is truly like searchin...

Each year diagnostic laboratories in the Netherlands profile thousands of individuals for heritable disease using next generation sequencing (NGS). This requires pathogenicity classification of millions of DNA variants on the standard 5‐tier scale. To reduce time spent on data interpretation and increase data quality and reliability, the nine Dutch...

The regulation of translation initiation factor 2 (eIF2) is important for erythroid survival and differentiation. Lack of iron, a critical component of heme and hemoglobin, activates Heme Regulated Inhibitor (HRI). This results in phosphorylation of eIF2 and reduced eIF2 availability, which inhibits protein synthesis. Translation of specific transc...

Tm treatment causes a reduction of translation. (A) Protein synthesis was measured by Click-it technology. Incorporated methionine analogue AHA was coupled to Alexa Fluor 488, and measured by flow cytometry (BD LSR-II). (average values, n = 3, for every pair untreated cells were set to 1, error bar indicated StDev, star indicates p<0.05). (B-C) Cel...

Ribosome profiling data quality. (A) Ribosomes were stabilised with CHX. Shown is the fitted line through the average values of three biological replicates harvested following Tm treatment or three control replicates. Error bars indicate standard deviation. (B) RFP fragments were mapped to the genome and the number of reads (all experiments combine...

Web browser snapshot of Tfcp2. Aggregate Ht- and CHX-stabilized RFP reads from Tm-treated and untreated (Unt) cells are mapped to the genome and visualized in the UCSC web browser. Numbers on the right-hand side indicate maximum read counts in the respective lane. Arrows indicate Ht peaks. Gray lines indicate introns. Part of the 3’UTR is cropped....

Transcripts with differential use of TIS in absence and presence of tunicamycin. Peak intensity ratio between TIS peaks in stressed cells were compared to untreated cells for specific transcripts. At a p-value less than 0.01 few transcripts revealed differentially employed TISs in their 5’UTR Coverage: cumulative reads of the peak. Statistics: two...

Web browser snapshot of ATP-binding cassette sub-family E member 1 (Abce1). Cumulative Ht- and CHX-stabilized RFP counts from Tm-treated and untreated (Unt) cells are mapped to the genome and visualized in the UCSC web browser. Numbers on the right-hand side indicate maximum read counts in the respective lane. Gray lines indicate introns. The arrow...

Web browser snapshot of Ranbp1. Cumulative Ht- and CHX-stabilized RFP counts from Tm-treated and untreated (Unt) cells are mapped to the genome and visualized in the UCSC web browser. Numbers on the right-hand side indicate maximum read counts in the respective lane. Gray lines indicate introns. The uORFs in the 5’UTR and the protein coding ORF (CD...

Overview of ribosome footprint reads mapped with STAR. Ribosome reads were mapped with STAR to the genome. This table gives an overview of read length and how many reads mapped to the genome for each sample. Note: Multi-mapped reads were not discarded, unless they mapped to more than 20 locations. (XLSX)

List of upregulated transcripts during Tm treatment. Upregulated targets were uploaded on Genetrail2 to investigate enrichment of cellular component, biological processes and molecular function. (XLSX)

Harringtonine-induced RFP are mostly translated in frame 3. We used STAR to map Ht-stabilized RFP to the genome, and used our previously described script to map the first nucleotide relative to the annotated reading frame. Shades of blue (a2, b2, c2) represent RFP from untreated cells, shades of orange (a4, b4, c4) represent RFP from Tm-treated cel...

List of downregulated transcripts during Tm treatment. Downregulated targets were uploaded on Genetrail2 to investigate enrichment of cellular component, biological processes and molecular function. (XLSX)

Harringtonine preferentially stalls ribosomes at R and K codons. Mapped Ht RFPs were analysed with a peak calling program to define potential TISs in the 5’UTR (top) or CDS (bottom) in cells treated with Tm (right side) or untreated (left side). In the 5’UTR almost half of the detected TIS represented canonical (AUG) and noncanonical (CUG, UUG, GUG...

Comparison of ribosome occupancy in response to eIF2 phosphorylation in HEK293 cells (Andreev et al.) and mouse erythroblasts (this study). Triangles indicate transcripts of which translation is similarly upregulated upon eIF2 phosphorylation in both studies. White circles represent transcripts with enhanced translation (Z-score >3) in HEK293 cells...

Normalised sequence counts for ribosome footprints (RFP) and pA+ RNA sequencing (counts per million; cpm). 2Log normalized RFP reads (cpm) of the CDS of all transcripts in Tm-treated cells were compared to untreated cells. List of significantly altered transcripts during Tm treatment in erythroblasts, cpm values are given for each sample for riboso...

Translation initiation sites detected by stalling of ribosomes in the presence of Harringtonine. Peaks were called with the cumulative reads of each triplicate, with our previously developed peak calling algorithm to identify translation initiation sites (TIS)[26]. Peaks were divided into 5’UTR TISs, annotated start codon TISs, TISs in the CDS, or...

Codons at -13 (P) position of translation initiation sites, measured after ribosome stalling with Harringtonine. Called peaks and triplet codons were compared in untreated and Tm-treated erythroblasts. (XLSX)

The regulation of translation initiation factor 2 (eIF2) is important for erythroid survival and differentiation. Lack of iron, a critical component of heme and hemoglobin, activates Heme Regulated Inhibitor (HRI). This results in phosphorylation of eIF2 and reduced eIF2 availability, which inhibits protein synthesis. Translation of specific transc...

To keep circulating erythrocytes between narrow boundaries, the erythroid progenitor compartment has a large expansion capacity, that is tightly regulated by environmental factors. Erythropoietin (Epo), stem cell factor (SCF) and glucocorticoids (dexamethasone; Dex) can induce a large increase of erythroblasts both in vivo and in vitro. We previous...

With the widespread use of Next Generation Sequencing technologies, the primary bottleneck of genetic research has shifted from data production to data analysis. However, heterogeneous data sets makes comparisons and integration challenging and time consuming. Here, we apply a data interoperability approach that provides unambiguous (machine readab...

The formation of skeletal muscles is associated with drastic changes in protein requirements known to be safeguarded by tight control of gene transcription and mRNA processing. The contribution of regulation of mRNA translation during myogenesis has not been studied so far. We monitored translation during myogenic differentiation of C2C12 myoblasts...

The erythroid progenitor compartment possesses a large expansion capacity, both in vivo and in vitro, which enables a rapid restoration of peripheral erythrocytes following severe blood loss. This expansion is tightly regulated to maintain erythrocyte numbers between narrow boundaries, and to balance expansion of the erythroid compartment against t...

With the widespread use of Next Generation Sequencing (NGS) technologies, the primary bottleneck of genetic research has shifted from data production to data analysis. However, annotated datasets produced by different research groups are often in different formats, making genomic comparisons and integration with other datasets challenging and time...

The Finnish Disease Heritage Database (FinDis) (http://findis.org) was originally published in 2004 as a centralized information resource for rare monogenic diseases enriched in the Finnish population The FinDis database originally contained 405 causative variants for 30 diseases. At the time, the FinDis database was a comprehensive collection of d...

Genitopatellar syndrome (GPS) and Say-Barber-Biesecker-Young-Simpson syndrome (SBBYSS or Ohdo syndrome) have both recently been shown to be caused by distinct mutations in the histone acetyltransferase KAT6B (a.k.a. MYST4/MORF). All variants are de novo dominant mutations that lead to protein truncation. Mutations leading to GPS occur in the proxim...

Background Sharing of data about variation and the associated phenotypes is a critical need, yet variant information can be arbitrarily complex, making a single standard vocabulary elusive and re-formatting difficult. Complex standards have proven too time-consuming to implement. Results The GEN2PHEN project addressed these difficulties by develop...

Locus-Specific DataBases (LSDBs) store information on gene sequence variation associated with human phenotypes and are frequently used as a reference by researchers and clinicians. We developed the Leiden Open-source Variation Database (LOVD) as a platform-independent Web-based LSDB-in-a-Box package. LOVD was designed to be easy to set up and maint...

The MUTYH gene encodes a DNA glycosylase involved in base excision repair (BER). Biallelic pathogenic MUTYH variants have been associated with colorectal polyposis and cancer. The pathogenicity of a few variants is beyond doubt, including c.536A4G/p.Tyr179Cys and c.1187G4A/p.Gly396Asp (previously c.494A4G/p.Tyr165Cys and c.1145G4A/p.Gly382Asp).Howe...

Antisense-mediated exon skipping is a promising therapeutic approach for Duchenne muscular dystrophy (DMD) currently tested in clinical trials. The aim is to reframe dystrophin transcripts using antisense oligonucleotides (AONs). These hide an exon from the splicing machinery to induce exon skipping, restoration of the reading frame and generation...

Antisense-mediated exon skipping aiming for reading frame restoration is currently a promising therapeutic application for Duchenne muscular dystrophy (DMD). This approach is mutation specific, but as the majority of DMD patients have deletions that cluster in hotspot regions, the skipping of a small number of exons is applicable to relatively larg...

Walker-Warburg syndrome, muscle-eye-brain disease, Fukuyama congenital muscular dystrophy, congenital muscular dystrophy type 1C, and congenital muscular dystrophy type 1D are overlapping clinical entities belonging to a subgroup of the congenital muscular dystrophies (CMD), collectively designated dystroglycanopathies, in which the common underlyi...

The severe Duchenne and milder Becker muscular dystrophy are both caused by mutations in the DMD gene. This gene codes for dystrophin, a protein important for maintaining the stability of muscle-fiber membranes. In 1988, Monaco and colleagues postulated an explanation for the phenotypic difference between Duchenne and Becker patients in the reading...

The completion of the human genome project has initiated, as well as provided the basis for, the collection and study of all sequence variation between individuals. Direct access to up-to-date information on sequence variation is currently provided most efficiently through web-based, gene-centered, locus-specific databases (LSDBs). We have develope...

To test the feasibility of developing a diagnostic microarray for a specific disease, we selected all pathogenic changes of the beta-globin gene occurring at a frequency >/=1% in the multi-ethnic Dutch population for analysis. A tagged single-base extension (SBE) approach was used to detect 19 different mutations causing beta-thalassemia or abnorma...