The LOVD3 platform: efficient genome-wide sharing of genetic variants.

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 online databases, we have entirely redesigned the LOVD software. LOVD3 is genome-centered and can be used to store summary variant data, as well as full case-level data with information on individuals, phenotypes, screenings, and variants. While built on a standard core, the software is highly flexible and allows personalization to cope with the largely different demands of gene/disease database curators. LOVD3 follows current standards and includes tools to check variant descriptions, generate HTML files of reference sequences, predict the consequences of exon deletions/duplications on the reading frame, and link to genomic views in the different genomes browsers. It includes APIs to collect and submit data. The software is used by about 100 databases, of which 56 public LOVD instances are registered on our website and together contain 1,000,000,000 variant observations in 1,500,000 individuals. 42 LOVD instances share data with the federated LOVD data network containing 3,000,000 unique variants in 23,000 genes. This network can be queried directly, quickly identifying LOVD instances containing relevant information on a searched variant.

Dutch genome diagnostic laboratories accelerated and improved variant interpretation and increased accuracy by sharing data.

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 labs decided to publicly share their classifications. Variant classifications of nearly 100,000 unique variants were catalogued and compared in a centralized MOLGENIS database. Variants classified by more than one center were labeled as "consensus" when classifications agreed, and shared internationally with LOVD and ClinVar. When classifications opposed (LB/B vs. LP/P), they were labeled "conflicting", while other nonconsensus observations were labeled "no consensus". We assessed our classifications using the InterVar software to compare to ACMG 2015 guidelines, showing 99.7% overall consistency with only 0.3% discrepancies. Differences in classifications between Dutch labs or between Dutch labs and ACMG were mainly present in genes with low penetrance or for late onset disorders and highlight limitations of the current 5-tier classification system. The data sharing boosted the quality of DNA diagnostics in Dutch labs, an initiative we hope will be followed internationally. Recently, a positive match with a case from outside our consortium resulted in a more definite disease diagnosis.

Ribosome profiling uncovers selective mRNA translation associated with eIF2 phosphorylation in erythroid progenitors.

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 transcripts such as Atf4, however, is enhanced. Upstream open reading frames (uORFs) are key to this regulation. The aim of this study is to investigate how tunicamycin treatment, that induces eIF2 phosphorylation, affects mRNA translation in erythroblasts. Ribosome profiling combined with RNA sequencing was used to determine translation initiation sites and ribosome density on individual transcripts. Treatment of erythroblasts with Tunicamycin (Tm) increased phosphorylation of eIF2 2-fold. At a false discovery rate of 1%, ribosome density was increased for 147 transcripts, among which transcriptional regulators such as Atf4, Tis7/Ifrd1, Pnrc2, Gtf2h, Mbd3, JunB and Kmt2e. Translation of 337 transcripts decreased more than average, among which Dym and Csde1. Ribosome profiling following Harringtonine treatment uncovered novel translation initiation sites and uORFs. Surprisingly, translated uORFs did not predict the sensitivity of transcripts to altered ribosome recruitment in presence or absence of Tm. The regulation of transcription and translation factors in reponse to eIF2 phosphorylation may explain the large overall response to iron deficiency in erythroblasts.

Assessing the translational landscape of myogenic differentiation by ribosome profiling.

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, using a simplified protocol for ribosome footprint profiling. Comparison of ribosome footprints to total RNA showed that gene expression is mostly regulated at the transcriptional level. However, a subset of transcripts, enriched for mRNAs encoding for ribosomal proteins, was regulated at the level of translation. Enrichment was also found for specific pathways known to regulate muscle biology. We developed a dedicated pipeline to identify translation initiation sites (TISs) and discovered 5333 unannotated TISs, providing a catalog of upstream and alternative open reading frames used during myogenesis. We identified 298 transcripts with a significant switch in TIS usage during myogenesis, which was not explained by alternative promoter usage, as profiled by DeepCAGE. Also these transcripts were enriched for ribosomal protein genes. This study demonstrates that differential mRNA translation controls protein expression of specific subsets of genes during myogenesis. Experimental protocols, analytical workflows, tools and data are available through public repositories (http://lumc.github.io/ribosome-profiling-analysis-framework/).

The Finnish disease heritage database (FinDis) update-a database for the genes mutated in the Finnish disease heritage brought to the next-generation sequencing era.

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 data, but since 1994, a large amount of new information has emerged, making the necessity to update the database evident. We collected information and updated the database to contain genes and causative variants for 35 diseases, including six more genes and more than 1,400 additional disease-causing variants. Information for causative variants for each gene is collected under the LOVD 3.0 platform, enabling easy updating. The FinDis portal provides a centralized resource and user interface to link information on each disease and gene with variant data in the LOVD 3.0 platform. The software written to achieve this has been open-sourced and made available on GitHub (http://github.com/findis-db), allowing biomedical institutions in other countries to present their national data in a similar way, and to both contribute to, and benefit from, standardized variation data. The updated FinDis portal provides a unique resource to assist patient diagnosis, research, and the development of new cures.

VarioML framework for comprehensive variation data representation and exchange.

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 developing a comprehensive data model for capturing biomedical observations, Observ-OM, and building the VarioML format around it. VarioML pairs a simplified open specification for describing variants, with a toolkit for adapting the specification into one's own research workflow. Straightforward variant data can be captured, federated, and exchanged with no overhead; more complex data can be described, without loss of compatibility. The open specification enables push-button submission to gene variant databases (LSDBs) e.g., the Leiden Open Variation Database, using the Cafe Variome data publishing service, while VarioML bidirectionally transforms data between XML and web-application code formats, opening up new possibilities for open source web applications building on shared data. A Java implementation toolkit makes VarioML easily integrated into biomedical applications. VarioML is designed primarily for LSDB data submission and transfer scenarios, but can also be used as a standard variation data format for JSON and XML document databases and user interface components. CONCLUSIONS: VarioML is a set of tools and practices improving the availability, quality, and comprehensibility of human variation information. It enables researchers, diagnostic laboratories, and clinics to share that information with ease, clarity, and without ambiguity.

The KAT6B-related disorders genitopatellar syndrome and Ohdo/SBBYS syndrome have distinct clinical features reflecting distinct molecular mechanisms.

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 proximal portion of the last exon and lead to the expression of a protein without a C-terminal domain. Mutations leading to SBBYSS occur either throughout the gene, leading to nonsense-mediated decay, or more distally in the last exon. Features present only in GPS are contractures, anomalies of the spine, ribs and pelvis, renal cysts, hydronephrosis, and agenesis of the corpus callosum. Features present only in SBBYSS include long thumbs and long great toes and lacrimal duct abnormalities. Several features occur in both, such as intellectual disability, congenital heart defects, and genital and patellar anomalies. We propose that haploinsufficiency or loss of a function mediated by the C-terminal domain causes the common features, whereas gain-of-function activities would explain the features unique to GPS. Further molecular studies and the compilation of mutations in a database for genotype-phenotype correlations (www.LOVD.nl/KAT6B) might help tease out answers to these questions and understand the developmental programs dysregulated by the different truncations.

LOVD v.2.0: the next generation in gene variant databases.

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 maintain and follows the Human Genome Variation Society (HGVS) recommendations. Here we describe LOVD v.2.0, which adds enhanced flexibility and functionality and has the capacity to store sequence variants in multiple genes per patient. To reduce redundancy, patient and sequence variant data are stored in separate tables. Tables are linked to generate connections between sequence variant data for each gene and every patient. The dynamic structure allows database managers to add custom columns. The database structure supports fast queries and allows storage of sequence variants from high-throughput sequence analysis, as demonstrated by the X-chromosomal Mental Retardation LOVD installation. LOVD contains measures to ensure database security from unauthorized access. Currently, the LOVD Website (http://www.LOVD.nl/) lists 71 public LOVD installations hosting 3,294 gene variant databases with 199,000 variants in 84,000 patients. To promote LSDB standardization and thereby database interoperability, we offer free server space and help to establish an LSDB on our Leiden server.

Leiden Open Variation Database of the MUTYH gene.

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).However, for a substantial fraction of the detected variants, the clinical significance remains uncertain,compromising molecular diagnostics and thereby genetic counseling. We have established an interactive MUTYH gene sequence variant database (www.lovd.nl/MUTYH) with the aim of collecting and sharing MUTYH genotype and phenotype data worldwide. To support standard variant description, we chose NM_001128425.1 as the reference sequence. The database includes records with variants per individual, linked to available phenotype and geographic origin data as well as records with in vitro functional and in silico test data. As of April 2010, the database contains 1968 published and 423 unpublished submitted entries, and 230 and 61 unique variants,respectively. This open-access repository allows all involved to quickly share all variants encountered and communicate potential consequences, which will be especially useful to classify variants of uncertain significance.

Therapeutic exon skipping for dysferlinopathies?

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 of internally deleted, but partially functional proteins. It thus relies on the characteristic of the dystrophin protein, which has essential N- and C-terminal domains, whereas the central rod domain is largely redundant. This approach may also be applicable to limb-girdle muscular dystrophy type 2B (LGMD2B), Myoshi myopathy (MM) and distal myopathy with anterior tibial onset (DMAT), which are caused by mutations in the dysferlin-encoding DYSF gene. Dysferlin has a function in repairing muscle membrane damage. Dysferlin contains calcium-dependent C2 lipid binding (C2) domains and an essential transmembrane domain. However, mildly affected patients in whom one or a large number of DYSF exons were missing have been described, suggesting that internally deleted dysferlin proteins can be functional. Thus, exon skipping might also be applicable as a LGMD2B, MM and DMAT therapy. In this study we have analyzed the dysferlin protein domains and DYSF mutations and have described what exons are promising targets with regard to applicability and feasibility. We also show that DYSF exon skipping seems to be as straightforward as DMD exon skipping, as AONs to induce efficient skipping of four DYSF exons were readily identified.

Theoretic applicability of antisense-mediated exon skipping for Duchenne muscular dystrophy mutations.

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 large numbers of patients. To assess the actual applicability of the exon skipping approach, we here determined for deletions, duplications and point mutations reported in the Leiden DMD mutation database, which exon(s) should be skipped to restore the open reading frame. In theory, single and double exon skipping would be applicable to 79% of deletions, 91% of small mutations, and 73% of duplications, amounting to 83% of all DMD mutations. Exon 51 skipping, which is being tested in clinical trials, would be applicable to the largest group (13%) of all DMD patients. Further research is needed to determine the functionality of different in-frame dystrophins and a number of hurdles has to be overcome before this approach can be applied clinically.

Novel synonymous substitution in POMGNT1 promotes exon skipping in a patient with congenital muscular dystrophy.

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 underlying defect is hypoglycosylation of alfa-dystroglycan. Currently, six different genes are known to be implicated in these diseases: POMT1, POMT2, POMGNT1, FCMD, FKRP, and LARGE. We report the molecular characterization of a patient presenting clinical features of CMD and reduced immunostaining for alfa-dystroglycan in muscle. Three candidate genes (FCMD, POMT1 and POMGNT1) were analyzed, and a total of 18 sequence variants were detected: 15 polymorphisms in POMT1 [including three unreported single nucleotide polymorphisms (SNPs)], two polymorphisms in FCMD, and the exonic silent mutation c.636C > T in POMGNT1. Expression analysis revealed that this apparently silent mutation compromises correct premessenger RNA (mRNA) splicing, promoting skipping of the entire exon 7, with a consequent frameshift. In silico analysis of this mutation did not predict alterations in the canonical splice sequences, but rather the creation of a new exonic splice silencer. The recognition of such disease-causing elements may contribute to the further understanding of RNA processing and assist mutation screening in routine diagnosis, where such changes may be underestimated. To aid clinical diagnosis, we generated publicly available LOVD-powered Locus Specific Databases for these three genes and recorded all known sequence variants ( http://www.dmd.nl ).

Entries in the Leiden Duchenne muscular dystrophy mutation database: an overview of mutation types and paradoxical cases that confirm the reading-frame rule.

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-frame rule: In Duchenne patients, mutations induce a shift in the reading frame leading to prematurely truncated, dysfunctional dystrophins. In Becker patients, in-frame mutations allow the synthesis of internally deleted, but largely functional dystrophins. Currently, over 4700 mutations have been reported in the Leiden DMD mutation database, of which 91% are in agreement with this rule. In this study we provide an update of the mutational variability in the DMD gene, particularly focusing on genotype-phenotype correlations and mutations that appear to be exceptions to the reading-frame rule.

LOVD: easy creation of a locus-specific sequence variation database using an "LSDB-in-a-box" approach.

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 developed the Leiden Open (source) Variation Database (LOVD) software approaching the "LSDB-in-a-Box" idea for the easy creation and maintenance of a fully web-based gene sequence variation database. LOVD is platform-independent and uses PHP and MySQL open source software only. The basic gene-centered and modular design of the database follows the recommendations of the Human Genome Variation Society (HGVS) and focuses on the collection and display of DNA sequence variations. With minimal effort, the LOVD platform is extendable with clinical data. The open set-up should both facilitate and promote functional extension with scripts written by the community. The LOVD software is freely available from the Leiden Muscular Dystrophy pages (www.DMD.nl/LOVD/). To promote the use of LOVD, we currently offer curators the possibility to set up an LSDB on our Leiden server.

beta-Globin mutation detection by tagged single-base extension and hybridization to universal glass and flow-through microarrays.

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 abnormal hemoglobins. In the SBE reaction, the primers were elongated at the 3'site with a fluorescently labeled dideoxyribonucleotide triphosphate (ddNTP) complementary to the mutation, following tag hybridization to a glass or flow-through microarray. We compared the performance of a generic glass array and a porous system, by testing each mutation separately using heterozygous carriers and by screening a cohort of 40 unknown beta-thalassemia carriers and patients. The results were verified by direct sequencing. The microarray system was able to detect 17 beta-globin mutations simultaneously with >95% accuracy in a single SBE reaction. The flow-through array performed slightly better (96%), but the main advantages of the system included real-time data recording and a considerable time saving achieved through a reduced hybridization time.