Genomics comes of age: From research tool to routine care.
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Editorial↗
Biomedical subjects
Edwin Cuppen
Publications and source records attributed to Edwin Cuppen.
CONREAL web server: identification and visualization of conserved transcription factor binding sites.
The use of orthologous sequences and phylogenetic footprinting approaches have become popular for the recognition of conserved and potentially functional sequences. Several algorithms have been developed for the identification of conserved transcription factor binding sites (TFBSs), which are characterized by their relatively short and degenerative recognition sequences. The CONREAL (conserved regulatory elements anchored alignment) web server provides a versatile interface to CONREAL-, LAGAN-, BLASTZ- and AVID-based predictions of conserved TFBSs in orthologous promoters. Comparative analysis using different algorithms can be started by keyword without any prior sequence retrieval. The interface is available at http://conreal.niob.knaw.nl.
Algorithms↗
Identification of a rat model for usher syndrome type 1B by N-ethyl-N-nitrosourea mutagenesis-driven forward genetics.
The rat is the most extensively studied model organism and is broadly used in biomedical research. Current rat disease models are selected from existing strains and their number is thereby limited by the degree of naturally occurring variation or spontaneous mutations. We have used ENU mutagenesis to increase genetic variation in laboratory rats and identified a recessive mutant, named tornado, showing aberrant circling behavior, hyperactivity, and stereotypic head shaking. More detailed analysis revealed profound deafness due to disorganization and degeneration of the organ of Corti that already manifests at the onset of hearing. We set up a single nucleotide polymorphism (SNP)-based mapping strategy to identify the affected gene, revealing strong linkage to the central region of chromosome 1. Candidate gene resequencing identified a point mutation that introduces a premature stopcodon in Myo7a. Mutations in human MYO7A result in Usher syndrome type 1B, a severe autosomal inherited recessive disease that involves deafness and vestibular dysfunction. Here, we present the first characterized rat model for this disease. In addition, we demonstrate proof of principle for the generation and cloning of human disease models in rat using ENU mutagenesis, providing good perspectives for systematic phenotypic screens in the rat.
Animals↗
CASCAD: a database of annotated candidate single nucleotide polymorphisms associated with expressed sequences.
BACKGROUND: With the recent progress made in large-scale genome sequencing projects a vast amount of novel data is becoming available. A comparative sequence analysis, exploiting sequence information from various resources, can be used to uncover hidden information, such as genetic variation. Although there are enormous amounts of SNPs for a wide variety of organisms submitted to NCBI dbSNP and annotated in most genome assembly viewers like Ensembl and the UCSC Genome Browser, these platforms do not easily allow for extensive annotation and incorporation of experimental data supporting the polymorphism. However, such information is very important for selecting the most promising and useful candidate polymorphisms for use in experimental setups. DESCRIPTION: The CASCAD database is designed for presentation and query of candidate SNPs that are retrieved by in silico mining of high-throughput sequencing data. Currently, the database provides collections of laboratory rat (Rattus norvegicus) and zebrafish (Danio rerio) candidate SNPs. The database stores detailed information about raw data supporting the candidate, extensive annotation and links to external databases (e.g. GenBank, Ensembl, UniGene, and LocusLink), verification information, and predictions of a potential effect for non-synonymous polymorphisms in coding regions. The CASCAD website allows search based on an arbitrary combination of 27 different parameters related to characteristics like candidate SNP quality, genomic localization, and sequence data source or strain. In addition, the database can be queried with any custom nucleotide sequences of interest. The interface is crosslinked to other public databases and tightly coupled with primer design and local genome assembly interfaces in order to facilitate experimental verification of candidates. CONCLUSIONS: The CASCAD database discloses detailed information on rat and zebrafish candidate SNPs, including the raw data underlying its discovery. An advanced web-based search interface http://cascad.niob.knaw.nl allows universal access to the database content and allows various queries supporting many types of research utilizing single nucleotide polymorphisms.
Animals↗
Phylogenetic shadowing and computational identification of human microRNA genes.
We sequenced 122 miRNAs in 10 primate species to reveal conservation characteristics of miRNA genes. Strong conservation is observed in stems of miRNA hairpins and increased variation in loop sequences. Interestingly, a striking drop in conservation was found for sequences immediately flanking the miRNA hairpins. This characteristic profile was employed to predict novel miRNAs using cross-species comparisons. Nine hundred and seventy-six candidate miRNAs were identified by scanning whole-genome human/mouse and human/rat alignments. Most of the novel candidates are conserved also in other vertebrates (dog, cow, chicken, opossum, zebrafish). Northern blot analysis confirmed the expression of mature miRNAs for 16 out of 69 representative candidates. Additional support for the expression of 179 novel candidates can be found in public databases, their presence in gene clusters, and literature that appeared after these predictions were made. Taken together, these results suggest the presence of significantly higher numbers of miRNAs in the human genome than previously estimated.
Computational Biology↗
Haplotype-based genetics in mice and rats.
Haplotype blocks are conceptually defined as genomic segments harbouring sets of coupled polymorphisms that reflect a common ancestral origin. Experimentally, however, haplotype blocks are characterized using computational algorithms based on incomplete inventories of polymorphisms. Haplotype blocks and their deduced strain-distribution patterns are considered to be extremely powerful for use in genetic association and mapping experiments in laboratory mice and rats. However, recent high-density SNP screening in commonly used mouse inbred strains reveals a complex pattern, suggesting that the current expectations for the use of haplotype blocks in genetic mapping will have to be revisited.
Algorithms↗
Target-selected mutagenesis of the rat.
The rat is one of the most extensively studied model organisms, and with its genome being sequenced, tools to manipulate gene function in vivo have become increasingly important. We here report proof of principle for target-selected mutagenesis as a reverse genetic or knockout approach for the rat.
Animals↗
Single nucleotide polymorphisms associated with rat expressed sequences.
Single nucleotide polymorphisms (SNPs) are the most common source of genetic variation in populations and are thus most likely to account for the majority of phenotypic and behavioral differences between individuals or strains. Although the rat is extensively studied for the latter, data on naturally occurring polymorphisms are mostly lacking. We have used publicly available sequences consisting of whole-genome shotgun (WGS), expressed sequence tag (EST), and mRNA data as a source for the in silico identification of SNPs in gene-coding regions and have identified a large collection of 33,305 high-quality candidate SNPs. Experimental verification of 471 candidate SNPs using a limited set of rat isolates revealed a confirmation rate of approximately 50%. Although the majority of SNPs were identified between Sprague-Dawley (EST data) and Brown Norway (WGS data) strains, we found that 66% of the verified variations are common among different rat strains. All SNPs were extensively annotated, including chromosomal and genetic map information, and nonsynonymous SNPs were analyzed by SIFT and PolyPhen prediction programs for their potential deleterious effect on protein function. Interestingly, we retrieved three SNPs from the database that result in the introduction of a premature stop codon and that could be confirmed experimentally. Two of these "in silico-identified knockouts" reside in interesting QTL regions. Data are publicly available via a Web interface (http://cascad.niob.knaw.nl), allowing simple and advanced search queries.
Animals↗
Genetic variation in coding regions between and within commonly used inbred rat strains.
Single nucleotide polymorphisms (SNPs) are the most common genetic variation in mammalian populations. Their significance is illustrated by their potential contribution to common disease but also by their potential for use in genetic association and mapping experiments. We have examined the genetic variation between commonly used inbred rat strains by using an efficient SNP discovery and typing assay based on enzyme-based (CEL I) heteroduplex cleavage. Screening of a panel of 96 different rat (sub-)strains for 100 genomic loci in 55 genes, whose human homologs are implicated in clinically relevant diseases like neurological disorder, cancer, schizophrenia, and obesity, resulted in the identification of 103 novel polymorphisms. As all strains are simultaneously genotyped in this setup, this allowed us to make an estimate of the genetic variation between and within commonly used rat inbred strains. Interestingly, we observed substantial genetic variation between colonies of the same inbred strain, maintained at different locations. Furthermore, we identified 17 non-synonymous SNPs that may have an effect on protein function and contribute to phenotypic differences between different laboratory strains.
Animals↗
CONREAL: conserved regulatory elements anchored alignment algorithm for identification of transcription factor binding sites by phylogenetic footprinting.
Prediction of transcription-factor target sites in promoters remains difficult due to the short length and degeneracy of the target sequences. Although the use of orthologous sequences and phylogenetic footprinting approaches may help in the recognition of conserved and potentially functional sequences, correct alignment of the short transcription-factor binding sites can be problematic for established algorithms, especially when aligning more divergent species. Here, we report a novel phylogenetic footprinting approach, CONREAL, that uses biologically relevant information, that is, potential transcription-factor binding sites as represented by positional weight matrices, to establish anchors between orthologous sequences and to guide promoter sequence alignment. Comparison of the performance of CONREAL with the global alignment programs LAGAN and AVID using a reference data set, shows that CONREAL performs equally well for closely related species like rodents and human, and has a clear added value for aligning promoter elements of more divergent species like human and fish, as it identifies conserved transcription-factor binding sites that are not found by other methods. CONREAL is accessible via a Web interface at http://conreal.niob.knaw.nl/.
Algorithms↗
Efficient target-selected mutagenesis in zebrafish.
One of the most powerful methods available to assign function to a gene is to inactivate or knockout the gene. Recently,we described the first target-selected knockout in zebrafish. Here,we report on the further improvements of this procedure,resulting in a highly efficient and easy method to do target-selected mutagenesis in zebrafish. A library of 4608 ENU-mutagenized F1 animals was generated and kept as a living stock. The DNA of these animals was screened for mutations in 16 genes by use of CEL-I-mediated heteroduplex cleavage (TILLING) and subsequent resequencing. In total,255 mutations were identified,of which 14 resulted in a premature stop codon,7 in a splice donor/acceptor site mutation,and 119 in an amino acid change. By this method,we potentially knocked out 13 different genes in a few months time. Furthermore,we show that TILLING can be used to detect the full spectrum of ENU-induced mutations in a vertebrate genome with the presence of many naturally occurring polymorphisms.
Animals↗
The Wnt/beta-catenin pathway regulates cardiac valve formation.
Truncation of the tumour suppressor adenomatous polyposis coli (Apc) constitutively activates the Wnt/beta-catenin signalling pathway. Apc has a role in development: for example, embryos of mice with truncated Apc do not complete gastrulation. To understand this role more fully, we examined the effect of truncated Apc on zebrafish development. Here we show that, in contrast to mice, zebrafish do complete gastrulation. However, mutant hearts fail to loop and form excessive endocardial cushions. Conversely, overexpression of Apc or Dickkopf 1 (Dkk1), a secreted Wnt inhibitor, blocks cushion formation. In wild-type hearts, nuclear beta-catenin, the hallmark of activated canonical Wnt signalling, accumulates only in valve-forming cells, where it can activate a Tcf reporter. In mutant hearts, all cells display nuclear beta-catenin and Tcf reporter activity, while valve markers are markedly upregulated. Concomitantly, proliferation and epithelial-mesenchymal transition, normally restricted to endocardial cushions, occur throughout the endocardium. Our findings identify a novel role for Wnt/beta-catenin signalling in determining endocardial cell fate.
Adenomatous Polyposis Coli Protein↗
The microRNA-producing enzyme Dicer1 is essential for zebrafish development.
MicroRNAs (miRNAs) are produced by the Dicer1 enzyme; the role of Dicer1 in vertebrate development is unknown. Here we report target-selected inactivation of the dicer1 gene in zebrafish. We observed an initial build-up of miRNA levels, produced by maternal Dicer1, in homozygous dicer1 mutants, but miRNA accumulation stopped after a few days. This resulted in developmental arrest around day 10. These results indicate that miRNA-producing Dicer1 is essential for vertebrate development.
Animals↗
Hyperactivation of the G12-mediated signaling pathway in Caenorhabditis elegans induces a developmental growth arrest via protein kinase C.
The G(12) type of heterotrimeric G-proteins play an important role in development and behave as potent oncogenes in cultured cells. However, little is known about the molecular nature of the components that act in the G(12)-signaling pathway in an organism. We characterized a C. elegans Galpha subunit gene, gpa-12, which is a homolog of mammalian G(12)/G(13)alpha, and found that animals defective in gpa-12 are viable. Expression of activated GPA-12 (G(12)QL) results in a developmental growth arrest caused by a feeding behavior defect that is due to a dramatic reduction in pharyngeal pumping. To elucidate the molecular nature of the signaling pathways in which G(12) participates, we screened for suppressors of the G(12)QL phenotype. We isolated 50 suppressors that contain mutations in tpa-1, which encodes two protein kinase C isoforms, TPA-1A and TPA-1B, most similar to PKCtheta/delta. TPA-1 mediates the action of the tumor promoter PMA. Expression of G(12)QL and treatment of wild-type animals with PMA induce an identical growth arrest caused by inhibition of larval feeding, which is dependent on TPA-1A and TPA-1B function. These results suggest that TPA-1 is a downstream target of both G(12) signaling and PMA in modulating feeding and growth in C. elegans. Taken together, our findings provide a potential molecular mechanism for the transforming capability of G(12) proteins.
Animals↗
GENOTRACE: cDNA-based local GENOme assembly from TRACE archives.
UNLABELLED: GENOTRACE identifies the genomic organization for a cDNA using raw data from genome sequencing projects in progress (trace archives). Local genomic contigs are generated, allowing for example the design of PCR primers in intronic sequences to amplify coding regions of a gene, needed for example for mutation or SNP detection. AVAILABILITY: The package and examples of output files can be downloaded from http://rat.niob.knaw.nl/GENOTRACE
Archives↗