Publications by authors named "Wyeth W Wasserman"

148 Publications

Cross-species examination of X-chromosome inactivation highlights domains of escape from silencing.

Epigenetics Chromatin 2021 Feb 17;14(1):12. Epub 2021 Feb 17.

Department of Medical Genetics, The University of British Columbia, Vancouver, Canada.

Background: X-chromosome inactivation (XCI) in eutherian mammals is the epigenetic inactivation of one of the two X chromosomes in XX females in order to compensate for dosage differences with XY males. Not all genes are inactivated, and the proportion escaping from inactivation varies between human and mouse (the two species that have been extensively studied).

Results: We used DNA methylation to predict the XCI status of X-linked genes with CpG islands across 12 different species: human, chimp, bonobo, gorilla, orangutan, mouse, cow, sheep, goat, pig, horse and dog. We determined the XCI status of 342 CpG islands on average per species, with most species having 80-90% of genes subject to XCI. Mouse was an outlier, with a higher proportion of genes subject to XCI than found in other species. Sixteen genes were found to have discordant X-chromosome inactivation statuses across multiple species, with five of these showing primate-specific escape from XCI. These discordant genes tended to cluster together within the X chromosome, along with genes with similar patterns of escape from XCI. CTCF-binding, ATAC-seq signal and LTR repeats were enriched at genes escaping XCI when compared to genes subject to XCI; however, enrichment was only observed in three or four of the species tested. LINE and DNA repeats showed enrichment around subject genes, but again not in a consistent subset of species.

Conclusions: In this study, we determined XCI status across 12 species, showing mouse to be an outlier with few genes that escape inactivation. Inactivation status is largely conserved across species. The clustering of genes that change XCI status across species implicates a domain-level control. In contrast, the relatively consistent, but not universal correlation of inactivation status with enrichment of repetitive elements or CTCF binding at promoters demonstrates gene-based influences on inactivation state. This study broadens enrichment analysis of regulatory elements to species beyond human and mouse.
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http://dx.doi.org/10.1186/s13072-021-00386-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7890635PMC
February 2021

Human MiniPromoters for ocular-rAAV expression in ON bipolar, cone, corneal, endothelial, Müller glial, and PAX6 cells.

Gene Ther 2021 Feb 2. Epub 2021 Feb 2.

Centre for Molecular Medicine and Therapeutics at BC Children's Hospital, University of British Columbia, Vancouver, BC, Canada.

Small and cell-type restricted promoters are important tools for basic and preclinical research, and clinical delivery of gene therapies. In clinical gene therapy, ophthalmic trials have been leading the field, with over 50% of ocular clinical trials using promoters that restrict expression based on cell type. Here, 19 human DNA MiniPromoters were bioinformatically designed for rAAV, tested by neonatal intravenous delivery in mouse, and successful MiniPromoters went on to be tested by intravitreal, subretinal, intrastromal, and/or intravenous delivery in adult mouse. We present promoter development as an overview for each cell type, but only show results in detail for the recommended MiniPromoters: Ple265 and Ple341 (PCP2) ON bipolar, Ple349 (PDE6H) cone, Ple253 (PITX3) corneal stroma, Ple32 (CLDN5) endothelial cells of the blood-retina barrier, Ple316 (NR2E1) Müller glia, and Ple331 (PAX6) PAX6 positive. Overall, we present a resource of new, redesigned, and improved MiniPromoters for ocular gene therapy that range in size from 784 to 2484 bp, and from weaker, equal, or stronger in strength relative to the ubiquitous control promoter smCBA. All MiniPromoters will be useful for therapies involving small regulatory RNA and DNA, and proteins ranging from 517 to 1084 amino acids, representing 62.9-90.2% of human proteins.
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http://dx.doi.org/10.1038/s41434-021-00227-zDOI Listing
February 2021

Exome sequencing enables diagnosis of X-linked hypohidrotic ectodermal dysplasia in patient with eosinophilic esophagitis and severe atopy.

Allergy Asthma Clin Immunol 2021 Jan 14;17(1). Epub 2021 Jan 14.

BC Children's Hospital, University of British Columbia, 950 W 28th Ave, Vancouver, BC, V5Z 4H4, Canada.

X-linked hypohidrotic ectodermal dysplasia (XLHED) is the most common form of ectodermal dysplasia. Clinical and genetic heterogeneity between different ectodermal dysplasia types and evidence of incomplete penetrance and variable expressivity increase the potential for misdiagnosis. We describe a family with X-linked hypohidrotic ectodermal dysplasia (XLHED) presenting with variable expressivity of symptoms between affected siblings. In addition to the classical signs of hypohidrosis, hypotrichosis and hypodontia, the index patient-a 5 year old boy, also presented with a severe atopy phenotype that was not observed in the other two affected brothers. Exome sequencing in the index and the mother identified a pathogenic nonsense variant in EDA (NM_001399.4: c.766 C>T; p. Gln256Ter). This study highlights how exome sequencing was crucial in establishing a precise molecular diagnosis of XLHED by enabling us to rule out other differential diagnoses including NEMO deficiency syndrome, that was initially presented as a clinical diagnosis to the family.
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http://dx.doi.org/10.1186/s13223-021-00510-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7809757PMC
January 2021

The Genome Atlas: Navigating a New Era of Reference Genomes.

Trends Genet 2021 Jan 5. Epub 2021 Jan 5.

Centre for Molecular Medicine and Therapeutics, BC Children's Hospital Research Institute, University of British Columbia, Vancouver, BC, Canada. Electronic address:

The reference genome serves two distinct purposes within the field of genomics. First, it provides a persistent structure against which findings can be reported, allowing for universal knowledge exchange between users. Second, it reduces the computational costs and time required to process genomic data by creating a scaffold that can be relied upon by analysis software. Here, we posit that current efforts to extend the linear reference to a graph-based structure while trying to fulfil both of these purposes concurrently will face a trade-off between comprehensiveness and computational efficiency. In this article, we explore how the reference genome is used and suggest an alternative structure, The Genome Atlas (TGA), to fulfil the bipartite role of the reference genome.
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http://dx.doi.org/10.1016/j.tig.2020.12.002DOI Listing
January 2021

GeneBreaker: Variant simulation to improve the diagnosis of Mendelian rare genetic diseases.

Hum Mutat 2020 Dec 25. Epub 2020 Dec 25.

Department of Medical Genetics, Center for Molecular Medicine and Therapeutics, BC Children's Hospital Research Institute, University of British Columbia, Vancouver, British Columbia, Canada.

Mendelian rare genetic diseases affect 5%-10% of the population, and with over 5300 genes responsible for ∼7000 different diseases, they are challenging to diagnose. The use of whole-genome sequencing (WGS) has bolstered the diagnosis rate significantly. The effective use of WGS relies on the ability to identify the disrupted gene responsible for disease phenotypes. This process involves genomic variant calling and prioritization, and is the beneficiary of improvements to sequencing technology, variant calling approaches, and increased capacity to prioritize genomic variants with potential pathogenicity. As analysis pipelines continue to improve, careful testing of their efficacy is paramount. However, real-life cases typically emerge anecdotally, and utilization of clinically sensitive and identifiable data for testing pipeline improvements is regulated and limiting. We identified the need for a gene-based variant simulation framework that can create mock rare disease scenarios, utilizing known pathogenic variants or through the creation of novel gene-disrupting variants. To fill this need, we present GeneBreaker, a tool that creates synthetic rare disease cases with utility for benchmarking variant calling approaches, testing the efficacy of variant prioritization, and as an educational mechanism for training diagnostic practitioners in the expanding field of genomic medicine. GeneBreaker is freely available at http://GeneBreaker.cmmt.ubc.ca.
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http://dx.doi.org/10.1002/humu.24163DOI Listing
December 2020

Linkage analysis identifies an isolated strabismus locus at 14q12 overlapping with FOXG1 syndrome region.

J Med Genet 2020 Nov 30. Epub 2020 Nov 30.

Centre for Molecular Medicine and Therapeutics, The University of British Columbia, Vancouver, British Columbia, Canada

Strabismus is a common condition, affecting 1%-4% of individuals. Isolated strabismus has been studied in families with Mendelian inheritance patterns. Despite the identification of multiple loci via linkage analyses, no specific genes have been identified from these studies. The current study is based on a seven-generation family with isolated strabismus inherited in an autosomal dominant manner. A total of 13 individuals from a common ancestor have been included for linkage analysis. Among these, nine are affected and four are unaffected. A single linkage signal has been identified at an 8.5 Mb region of chromosome 14q12 with a multipoint LOD (logarithm of the odds) score of 4.69. Disruption of this locus is known to cause FOXG1 syndrome (or congenital Rett syndrome; OMIM #613454 and *164874), in which 84% of affected individuals present with strabismus. With the incorporation of next-generation sequencing and in-depth bioinformatic analyses, a 4 bp non-coding deletion was prioritised as the top candidate for the observed strabismus phenotype. The deletion is predicted to disrupt regulation of , which encodes a transcription factor of the Forkhead family. Suggestive of an autoregulation effect, the disrupted sequence matches the consensus FOXG1 and Forkhead family transcription factor binding site and has been observed in previous ChIP-seq studies to be bound by Foxg1 in early mouse brain development. Future study of this specific deletion may shed light on the regulation of expression and may enhance our understanding of the mechanisms contributing to strabismus and FOXG1 syndrome.
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http://dx.doi.org/10.1136/jmedgenet-2020-107226DOI Listing
November 2020

The variability conundrum in neurometabolic degenerative diseases.

Mol Genet Metab 2020 12 6;131(4):367-369. Epub 2020 Nov 6.

Department of Pediatric Neurology, Amsterdam Leukodystrophy Center, Emma Children's Hospital, Amsterdam Neuroscience, Amsterdam University Medical Centers, University of Amsterdam, the Netherlands; Department of Clinical Chemistry, Laboratory Genetic Metabolic Diseases, Amsterdam University Medical Centers, Amsterdam Gastroenterology & Metabolism, University of Amsterdam, Amsterdam, the Netherlands.

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http://dx.doi.org/10.1016/j.ymgme.2020.11.002DOI Listing
December 2020

Multi-Omic Approach to Identify Phenotypic Modifiers Underlying Cerebral Demyelination in X-Linked Adrenoleukodystrophy.

Front Cell Dev Biol 2020 25;8:520. Epub 2020 Jun 25.

Center for Molecular Medicine and Therapeutics, BC Children's Hospital Research Institute, University of British Columbia, Vancouver, BC, Canada.

X-linked adrenoleukodystrophy (ALD) is a peroxisomal metabolic disorder with a highly complex clinical presentation. ALD is caused by mutations in the gene, and is characterized by the accumulation of very long-chain fatty acids in plasma and tissues. Disease-causing mutations are 'loss of function' mutations, with no prognostic value with respect to the clinical outcome of an individual. All male patients with ALD develop spinal cord disease and a peripheral neuropathy in adulthood, although age of onset is highly variable. However, the lifetime prevalence to develop progressive white matter lesions, termed cerebral ALD (CALD), is only about 60%. Early identification of transition to CALD is critical since it can be halted by allogeneic hematopoietic stem cell therapy only in an early stage. The primary goal of this study is to identify molecular markers which may be prognostic of cerebral demyelination from a simple blood sample, with the hope that blood-based assays can replace the current protocols for diagnosis. We collected six well-characterized brother pairs affected by ALD and discordant for the presence of CALD and performed multi-omic profiling of blood samples including genome, epigenome, transcriptome, metabolome/lipidome, and proteome profiling. In our analysis we identify discordant genomic alleles present across all families as well as differentially abundant molecular features across the omics technologies. The analysis was focused on univariate modeling to discriminate the two phenotypic groups, but was unable to identify statistically significant candidate molecular markers. Our study highlights the issues caused by a large amount of inter-individual variation, and supports the emerging hypothesis that cerebral demyelination is a complex mix of environmental factors and/or heterogeneous genomic alleles. We confirm previous observations about the role of immune response, specifically auto-immunity and the potential role of PFN1 protein overabundance in CALD in a subset of the families. We envision our methodology as well as dataset has utility to the field for reproducing previous or enabling future modifier investigations.
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http://dx.doi.org/10.3389/fcell.2020.00520DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7330173PMC
June 2020

Deregulated Regulators: Disease-Causing cis Variants in Transcription Factor Genes.

Trends Genet 2020 07 22;36(7):523-539. Epub 2020 May 22.

Centre for Molecular Medicine and Therapeutics, BC Children's Hospital Research Institute, Department of Medical Genetics, The University of British Columbia, Vancouver, BC, Canada.. Electronic address:

Whole-genome sequencing is accelerating identification of noncoding variants that disrupt gene expression, although reports of such regulatory variants implicated in disease remain rare. A notable subset of described variants affect transcription factor (TF) genes and other master regulators in cis through dosage effects. From the literature, we compiled 46 regulatory variants linked to 40 TF genes implicated in rare diseases. We discuss the genomic geography of these variants and the evidence presented for their potential pathogenicity. To help advance research on candidate disease variants into the literature, we introduce an evidence framework specific to regulatory variants, which are under-represented in current variant classification guidelines. The clinical research interpretation of patient genomes may be advanced by considering regulatory variants, particularly those that deregulate TF genes.
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http://dx.doi.org/10.1016/j.tig.2020.04.006DOI Listing
July 2020

Indigenous Genomic Databases: Pragmatic Considerations and Cultural Contexts.

Front Public Health 2020 24;8:111. Epub 2020 Apr 24.

Department of Mathematics & Statistics, University of Otago, Dunedin, New Zealand.

The potential to grow genomic knowledge and harness the subsequent clinical benefits has escalated the building of background variant databases (BVDs) for genetic diagnosis across the globe. Alongside the upsurge of this precision medicine, potential benefits have been highlighted for both rare genetic conditions and other diagnoses. However, with the ever-present "genomic divide," Indigenous peoples globally have valid concerns as they endure comparatively greater health disparities but stand to benefit the least from these novel scientific discoveries and progress in healthcare. The paucity of Indigenous healthcare providers and researchers in these fields contributes to this genomic divide both in access to, and availability of culturally safe, relevant and respectful healthcare using this genetic knowledge. The vital quest to provide equitable clinical research, and provision and use of genomic services and technologies provides a strong rationale for building BVDs for Indigenous peoples. Such tools would ground their representation and participation in accompanying genomic health research and benefit acquisition. We describe two, independent but highly similar initiatives-the "Silent Genomes" in Canada and the "Aotearoa Variome" in New Zealand-as exemplars that have had to address the aforementioned issues and work to create Indigenous BVDs with these populations. Taking into account the baseline inequities in genomic medicine for Indigenous populations and the ongoing challenges of implementing genomic research with Indigenous communities, we provide a rationale for multiple changes required that will assure communities represented in BVDs, as well as Indigenous researchers, that their participation will maximize benefits and minimize risk.
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http://dx.doi.org/10.3389/fpubh.2020.00111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7193324PMC
April 2020

ExpansionHunter Denovo: a computational method for locating known and novel repeat expansions in short-read sequencing data.

Genome Biol 2020 04 28;21(1):102. Epub 2020 Apr 28.

Illumina Inc., 5200 Illumina Way, San Diego, CA, 92122, USA.

Repeat expansions are responsible for over 40 monogenic disorders, and undoubtedly more pathogenic repeat expansions remain to be discovered. Existing methods for detecting repeat expansions in short-read sequencing data require predefined repeat catalogs. Recent discoveries emphasize the need for methods that do not require pre-specified candidate repeats. To address this need, we introduce ExpansionHunter Denovo, an efficient catalog-free method for genome-wide repeat expansion detection. Analysis of real and simulated data shows that our method can identify large expansions of 41 out of 44 pathogenic repeats, including nine recently reported non-reference repeat expansions not discoverable via existing methods.
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http://dx.doi.org/10.1186/s13059-020-02017-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7187524PMC
April 2020

JASPAR 2020: update of the open-access database of transcription factor binding profiles.

Nucleic Acids Res 2020 01;48(D1):D87-D92

Centre for Molecular Medicine Norway (NCMM), Nordic EMBL Partnership, University of Oslo, 0318 Oslo, Norway.

JASPAR (http://jaspar.genereg.net) is an open-access database of curated, non-redundant transcription factor (TF)-binding profiles stored as position frequency matrices (PFMs) for TFs across multiple species in six taxonomic groups. In this 8th release of JASPAR, the CORE collection has been expanded with 245 new PFMs (169 for vertebrates, 42 for plants, 17 for nematodes, 10 for insects, and 7 for fungi), and 156 PFMs were updated (125 for vertebrates, 28 for plants and 3 for insects). These new profiles represent an 18% expansion compared to the previous release. JASPAR 2020 comes with a novel collection of unvalidated TF-binding profiles for which our curators did not find orthogonal supporting evidence in the literature. This collection has a dedicated web form to engage the community in the curation of unvalidated TF-binding profiles. Moreover, we created a Q&A forum to ease the communication between the user community and JASPAR curators. Finally, we updated the genomic tracks, inference tool, and TF-binding profile similarity clusters. All the data is available through the JASPAR website, its associated RESTful API, and through the JASPAR2020 R/Bioconductor package.
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http://dx.doi.org/10.1093/nar/gkz1001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7145627PMC
January 2020

Introduction to Genomic Analysis Workshop: A catalyst for engaging life-science researchers in high throughput analysis.

F1000Res 2019 30;8:1221. Epub 2019 Jul 30.

Centre for Molecular Medicine and Therapeutics, BC Children's Hospital Research Institute, Vancouver, British Columbia, V5Z 4H4, Canada.

Researchers in the life sciences are increasingly faced with the task of obtaining compute resources and training to analyze large, high-throughput technology generated datasets. As demand for compute resources has grown, high performance computing (HPC) systems have been implemented by research organizations and international consortiums to support academic researchers. However, life science researchers lack effective time-of-need training resources for utilization of these systems. Current training options have drawbacks that inhibit the effective training of researchers without experience in computational analysis. We identified the need for flexible, centrally-organized, easily accessible, interactive, and compute resource specific training for academic HPC use.  In our delivery of a modular workshop series, we provided foundational training to a group of researchers in a coordinated manner, allowing them to further pursue additional training and analysis on compute resources available to them. Efficacy measures indicate that the material was effectively delivered to a broad audience in a short time period, including both virtual and on-site students. The practical approach to catalyze academic HPC use is amenable to diverse systems worldwide.
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http://dx.doi.org/10.12688/f1000research.19320.1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6774052PMC
June 2020

Identification of novel cerebellar developmental transcriptional regulators with motif activity analysis.

BMC Genomics 2019 Sep 18;20(1):718. Epub 2019 Sep 18.

Centre for Molecular Medicine and Therapeutics at the BC Children's Hospital Research Institute, Department of Medical Genetics, University of British Columbia, Vancouver, BC, Canada.

Background: The work of the FANTOM5 Consortium has brought forth a new level of understanding of the regulation of gene transcription and the cellular processes involved in creating diversity of cell types. In this study, we extended the analysis of the FANTOM5 Cap Analysis of Gene Expression (CAGE) transcriptome data to focus on understanding the genetic regulators involved in mouse cerebellar development.

Results: We used the HeliScopeCAGE library sequencing on cerebellar samples over 8 embryonic and 4 early postnatal times. This study showcases temporal expression pattern changes during cerebellar development. Through a bioinformatics analysis that focused on transcription factors, their promoters and binding sites, we identified genes that appear as strong candidates for involvement in cerebellar development. We selected several candidate transcriptional regulators for validation experiments including qRT-PCR and shRNA transcript knockdown. We observed marked and reproducible developmental defects in Atf4, Rfx3, and Scrt2 knockdown embryos, which support the role of these genes in cerebellar development.

Conclusions: The successful identification of these novel gene regulators in cerebellar development demonstrates that the FANTOM5 cerebellum time series is a high-quality transcriptome database for functional investigation of gene regulatory networks in cerebellar development.
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http://dx.doi.org/10.1186/s12864-019-6063-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6751898PMC
September 2019

Strabismus in Children With Intellectual Disability: Part of a Broader Motor Control Phenotype?

Pediatr Neurol 2019 11 11;100:87-91. Epub 2019 Apr 11.

Department of Medical Genetics, University of British Columbia, Vancouver, BC, Canada; BC Children's Hospital and BC Women's Hospital, Vancouver, BC, Canada. Electronic address:

Purpose: Intellectual disability (ID) results from a heterogeneous group of disorders and affects 1% to 2% of children. ID frequently occurs in association with other clinical features such as seizures or malformations. We suspected that strabismus might also be unusually frequent in this population and that it might be associated with ID groups affecting motor control.

Methods: We reviewed phenotypic descriptors, extracted from medical records, for a heterogeneous series of 222 probands with ID who had been enrolled in a study of clinical application of exome sequencing. We estimated the frequency of strabismus and other common clinical features and explored statistical associations between them. Data from Population Data British Columbia and Online Mendelian Inheritance in Man were also examined for confirmation of our observations.

Results: Strabismus had a higher prevalence among probands with ID than in the general population (odds ratio = 5.46). Moreover, probands with both ID and strabismus were more likely to have problems affecting motor control than those with ID and no strabismus (odds ratio = 2.84). Hypotonia was one of the most common motor control subgroups affecting the ID probands, and a frequent co-occurrence of strabismus and hypotonia was also observed (odds ratio = 2.51) and supported by related gene literature review. There was no evidence for associations between strabismus and other frequent clinical features.

Conclusion: Strabismus is a frequent feature in individuals with ID. The frequent co-occurrence of strabismus and motor control phenotypes, in particular hypotonia, suggests that a common cerebellar mechanism or pathway may underlie these phenotypes.
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http://dx.doi.org/10.1016/j.pediatrneurol.2019.04.002DOI Listing
November 2019

Bi-allelic GOT2 Mutations Cause a Treatable Malate-Aspartate Shuttle-Related Encephalopathy.

Am J Hum Genet 2019 09 15;105(3):534-548. Epub 2019 Aug 15.

On behalf of "United for Metabolic Diseases," 1105AZ Amsterdam, the Netherlands; Department of Laboratory Medicine, Translational Metabolic Laboratory, Radboud University Medical Centre, 6525 GA Nijmegen, the Netherlands. Electronic address:

Early-infantile encephalopathies with epilepsy are devastating conditions mandating an accurate diagnosis to guide proper management. Whole-exome sequencing was used to investigate the disease etiology in four children from independent families with intellectual disability and epilepsy, revealing bi-allelic GOT2 mutations. In-depth metabolic studies in individual 1 showed low plasma serine, hypercitrullinemia, hyperlactatemia, and hyperammonemia. The epilepsy was serine and pyridoxine responsive. Functional consequences of observed mutations were tested by measuring enzyme activity and by cell and animal models. Zebrafish and mouse models were used to validate brain developmental and functional defects and to test therapeutic strategies. GOT2 encodes the mitochondrial glutamate oxaloacetate transaminase. GOT2 enzyme activity was deficient in fibroblasts with bi-allelic mutations. GOT2, a member of the malate-aspartate shuttle, plays an essential role in the intracellular NAD(H) redox balance. De novo serine biosynthesis was impaired in fibroblasts with GOT2 mutations and GOT2-knockout HEK293 cells. Correcting the highly oxidized cytosolic NAD-redox state by pyruvate supplementation restored serine biosynthesis in GOT2-deficient cells. Knockdown of got2a in zebrafish resulted in a brain developmental defect associated with seizure-like electroencephalography spikes, which could be rescued by supplying pyridoxine in embryo water. Both pyridoxine and serine synergistically rescued embryonic developmental defects in zebrafish got2a morphants. The two treated individuals reacted favorably to their treatment. Our data provide a mechanistic basis for the biochemical abnormalities in GOT2 deficiency that may also hold for other MAS defects.
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http://dx.doi.org/10.1016/j.ajhg.2019.07.015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6732527PMC
September 2019

TFEA.ChIP: a tool kit for transcription factor binding site enrichment analysis capitalizing on ChIP-seq datasets.

Bioinformatics 2019 12;35(24):5339-5340

Departamento de Bioquímica, Universidad Autónoma de Madrid (UAM) and Instituto de Investigaciones Biomédicas 'Alberto Sols' (CSIC-UAM), Madrid, Spain.

Summary: The computational identification of the transcription factors (TFs) [more generally, transcription regulators, (TR)] responsible for the co-regulation of a specific set of genes is a common problem found in genomic analysis. Herein, we describe TFEA.ChIP, a tool that makes use of ChIP-seq datasets to estimate and visualize TR enrichment in gene lists representing transcriptional profiles. We validated TFEA.ChIP using a wide variety of gene sets representing signatures of genetic and chemical perturbations as input and found that the relevant TR was correctly identified in 126 of a total of 174 analyzed. Comparison with other TR enrichment tools demonstrates that TFEA.ChIP is an highly customizable package with an outstanding performance.

Availability And Implementation: TFEA.ChIP is implemented as an R package available at Bioconductor https://www.bioconductor.org/packages/devel/bioc/html/TFEA.ChIP.html and github https://github.com/LauraPS1/TFEA.ChIP_downloads. A web-based GUI to the package is also available at https://www.iib.uam.es/TFEA.ChIP/.

Supplementary Information: Supplementary data are available at Bioinformatics online.
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http://dx.doi.org/10.1093/bioinformatics/btz573DOI Listing
December 2019

Glutaminase Deficiency Caused by Short Tandem Repeat Expansion in .

N Engl J Med 2019 04;380(15):1433-1441

From Amsterdam University Medical Centers, University of Amsterdam, Departments of Clinical Chemistry, Pediatrics, and Clinical Genetics, Emma Children's Hospital, Amsterdam Gastroenterology and Metabolism (A.B.P.K., R.L., J.K., J. Meijer, L.A.T., M.T., M.W., R.J.A.W., H.R.W., C.D.M.K.), and United for Metabolic Diseases (A.B.P.K., R.J.A.W., H.R.W., C.D.M.K.), Amsterdam, and the Department of Neurology, Brain Center Rudolf Magnus, University Medical Center Utrecht (J.J.F.A.V., J.H.V.), and the Project MinE ALS Sequencing Consortium (J.J.F.A.V., J.H.V.), Utrecht - all in the Netherlands; the Departments of Biochemistry and Molecular Biology and Medical Genetics, Cumming School of Medicine, and Alberta Children's Hospital Research Institute, University of Calgary, Calgary (M.T.-G.), Centre for Molecular Medicine and Therapeutics, BC Children's Hospital Research Institute (P.A.R., M.J.J., M.S.K., J. MacIsaac, W.W.W., C.D.M.K.), the Faculty of Pharmaceutical Sciences (B.I.D., G.E.B.W., C.J.R.), and the Departments of Medical Genetics (C.M., I.-S.R.-B., W.W.W.) and Pediatrics (C.D.M.K.), University of British Columbia, Vancouver, the Zebrafish Centre for Advanced Drug Discovery, St. Michael's Hospital and University of Toronto (K.B.-A., F.K., M.L., Y.W., X.-Y.W.), the Centre for Applied Genomics, Genetics and Genome Biology, the Hospital for Sick Children (C.N., S.W.S., B.T., R.K.C.Y.), and the Department of Molecular Genetics (C.N., S.W.S., R.K.C.Y.), the McLaughlin Centre (S.W.S.), and the Departments of Medicine, Physiology, and Laboratory Medicine and Pathobiology, Institute of Medical Science (X.-Y.W.), University of Toronto, Toronto, and the Division of Medical Genetics, Department of Pediatrics, Children's Hospital Eastern Ontario, University of Ottawa, Ottawa (J.S.W., M.T.G.) - all in Canada; the Departments of Medicine and Physiology, National University of Singapore (M.A.P.), and the Translational Laboratory in Genetic Medicine, Agency for Science, Technology, and Research (M.A.P., B.S., X.X., J.Z.) - both in Singapore; Uppsala University, Department of Chemistry-Biomedical Center, Uppsala, Sweden (D.D.); Illumina, San Diego, CA (E.D., M.A.E.); Gene Structure and Disease Section, Laboratory of Cell and Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD (B.H., D.K., K.U.); and the Department of Clinical Inherited Metabolic Disorders, Birmingham Children's Hospital, Birmingham, United Kingdom (S.S.).

We report an inborn error of metabolism caused by an expansion of a GCA-repeat tract in the 5' untranslated region of the gene encoding glutaminase () that was identified through detailed clinical and biochemical phenotyping, combined with whole-genome sequencing. The expansion was observed in three unrelated patients who presented with an early-onset delay in overall development, progressive ataxia, and elevated levels of glutamine. In addition to ataxia, one patient also showed cerebellar atrophy. The expansion was associated with a relative deficiency of messenger RNA transcribed from the expanded allele, which probably resulted from repeat-mediated chromatin changes upstream of the repeat. Our discovery underscores the importance of careful examination of regions of the genome that are typically excluded from or poorly captured by exome sequencing.
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http://dx.doi.org/10.1056/NEJMoa1806627DOI Listing
April 2019

Curation and bioinformatic analysis of strabismus genes supports functional heterogeneity and proposes candidate genes with connections to RASopathies.

Gene 2019 May 15;697:213-226. Epub 2019 Feb 15.

Centre for Molecular Medicine and Therapeutics, BC Children's Hospital Research Institute, University of British Columbia, 950 West 28th Avenue, Vancouver, BC V5Z 4H4, Canada; Department of Medical Genetics, University of British Columbia, Vancouver, BC, Canada. Electronic address:

Strabismus refers to the misalignment of the eyes and occurs in 2-4% of individuals. The low-resolution label "strabismus" covers a range of heterogeneous defects, which makes it challenging to unravel this condition. Consequently a coherent understanding of the causes is lacking. Here, we attempt to gain a better understanding of the underlying genetics by combining gene curation, diverse bioinformatic analyses (including gene ontology, pathway mapping, expression and network-based methods) and literature review. Through a phenotype-based curation process, we identify high-confidence and permissive sets of 54 and 233 genes potentially involved in strabismus. These genes can be grouped into 10 modules that together span a heterogeneous set of biological and molecular functions, and can be linked to clinical sub-phenotypes. Multiple lines of evidence associate retina and cerebellum biology with the strabismus genes. We further highlight a potential role of the Ras-MAPK pathway. Independently, sets of 11 genes and 15 loci tied to strabismus with definitive genetic basis have been compiled from the literature. We identify strabismus candidate genes for 5 of the 15 reported loci (CHD7; SLC9A6; COL18A1, COL6A2; FRY, BRCA2, SPG20; PARK2). Finally, we synthesize a Strabismus Candidate Gene Collection, which together with our curated gene sets will serve as a resource for future research. The results of this informatics study support the heterogeneity and complexity of strabismus and point to specific biological pathways and brain regions for future focus.
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http://dx.doi.org/10.1016/j.gene.2019.02.020DOI Listing
May 2019

Twenty-Seven Tamoxifen-Inducible iCre-Driver Mouse Strains for Eye and Brain, Including Seventeen Carrying a New Inducible-First Constitutive-Ready Allele.

Genetics 2019 04 14;211(4):1155-1177. Epub 2019 Feb 14.

Centre for Molecular Medicine and Therapeutics at BC Children's Hospital, University of British Columbia, Vancouver, British Columbia V5Z 4H4, Canada

To understand gene function, the cre/loxP conditional system is the most powerful available for temporal and spatial control of expression in mouse. However, the research community requires more cre recombinase expressing transgenic mouse strains (cre-drivers) that restrict expression to specific cell types. To address these problems, a high-throughput method for large-scale production that produces high-quality results is necessary. Further, endogenous promoters need to be chosen that drive cell type specific expression, or we need to further focus the expression by manipulating the promoter. Here we test the suitability of using knock-ins at the docking site 5' of for rapid development of numerous cre-driver strains focused on expression in adulthood, using an improved cre tamoxifen inducible allele (icre/ERT2), and testing a novel inducible-first, constitutive-ready allele (icre/f3/ERT2/f3). In addition, we test two types of promoters either to capture an endogenous expression pattern (MaxiPromoters), or to restrict expression further using minimal promoter element(s) designed for expression in restricted cell types (MiniPromoters). We provide new cre-driver mouse strains with applicability for brain and eye research. In addition, we demonstrate the feasibility and applicability of using the locus 5' of for the rapid generation of substantial numbers of cre-driver strains. We also provide a new inducible-first constitutive-ready allele to further speed cre-driver generation. Finally, all these strains are available to the research community through The Jackson Laboratory.
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http://dx.doi.org/10.1534/genetics.119.301984DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6456315PMC
April 2019

PLPHP deficiency: clinical, genetic, biochemical, and mechanistic insights.

Brain 2019 03;142(3):542-559

British Columbia Children's Hospital Research Institute, Vancouver, BC, Canada.

Biallelic pathogenic variants in PLPBP (formerly called PROSC) have recently been shown to cause a novel form of vitamin B6-dependent epilepsy, the pathophysiological basis of which is poorly understood. When left untreated, the disease can progress to status epilepticus and death in infancy. Here we present 12 previously undescribed patients and six novel pathogenic variants in PLPBP. Suspected clinical diagnoses prior to identification of PLPBP variants included mitochondrial encephalopathy (two patients), folinic acid-responsive epilepsy (one patient) and a movement disorder compatible with AADC deficiency (one patient). The encoded protein, PLPHP is believed to be crucial for B6 homeostasis. We modelled the pathogenicity of the variants and developed a clinical severity scoring system. The most severe phenotypes were associated with variants leading to loss of function of PLPBP or significantly affecting protein stability/PLP-binding. To explore the pathophysiology of this disease further, we developed the first zebrafish model of PLPHP deficiency using CRISPR/Cas9. Our model recapitulates the disease, with plpbp-/- larvae showing behavioural, biochemical, and electrophysiological signs of seizure activity by 10 days post-fertilization and early death by 16 days post-fertilization. Treatment with pyridoxine significantly improved the epileptic phenotype and extended lifespan in plpbp-/- animals. Larvae had disruptions in amino acid metabolism as well as GABA and catecholamine biosynthesis, indicating impairment of PLP-dependent enzymatic activities. Using mass spectrometry, we observed significant B6 vitamer level changes in plpbp-/- zebrafish, patient fibroblasts and PLPHP-deficient HEK293 cells. Additional studies in human cells and yeast provide the first empirical evidence that PLPHP is localized in mitochondria and may play a role in mitochondrial metabolism. These models provide new insights into disease mechanisms and can serve as a platform for drug discovery.
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http://dx.doi.org/10.1093/brain/awy346DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6391652PMC
March 2019

Sialic acid catabolism by N-acetylneuraminate pyruvate lyase is essential for muscle function.

JCI Insight 2018 12 20;3(24). Epub 2018 Dec 20.

Department of Neurology, Donders Institute for Brain, Cognition, and Behavior, Radboud University Medical Center, Nijmegen, Netherlands.

Sialic acids are important components of glycoproteins and glycolipids essential for cellular communication, infection, and metastasis. The importance of sialic acid biosynthesis in human physiology is well illustrated by the severe metabolic disorders in this pathway. However, the biological role of sialic acid catabolism in humans remains unclear. Here, we present evidence that sialic acid catabolism is important for heart and skeletal muscle function and development in humans and zebrafish. In two siblings, presenting with sialuria, exercise intolerance/muscle wasting, and cardiac symptoms in the brother, compound heterozygous mutations [chr1:182775324C>T (c.187C>T; p.Arg63Cys) and chr1:182772897A>G (c.133A>G; p.Asn45Asp)] were found in the N-acetylneuraminate pyruvate lyase gene (NPL). In vitro, NPL activity and sialic acid catabolism were affected, with a cell-type-specific reduction of N-acetyl mannosamine (ManNAc). A knockdown of NPL in zebrafish resulted in severe skeletal myopathy and cardiac edema, mimicking the human phenotype. The phenotype was rescued by expression of wild-type human NPL but not by the p.Arg63Cys or p.Asn45Asp mutants. Importantly, the myopathy phenotype in zebrafish embryos was rescued by treatment with the catabolic products of NPL: N-acetyl glucosamine (GlcNAc) and ManNAc; the latter also rescuing the cardiac phenotype. In conclusion, we provide the first report to our knowledge of a human defect in sialic acid catabolism, which implicates an important role of the sialic acid catabolic pathway in mammalian muscle physiology, and suggests opportunities for monosaccharide replacement therapy in human patients.
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http://dx.doi.org/10.1172/jci.insight.122373DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6338320PMC
December 2018

Atypical cerebral palsy: genomics analysis enables precision medicine.

Genet Med 2019 07 13;21(7):1621-1628. Epub 2018 Dec 13.

BC Children's Hospital Research Institute, Vancouver, BC, Canada.

Purpose: The presentation and etiology of cerebral palsy (CP) are heterogeneous. Diagnostic evaluation can be a prolonged and expensive process that might remain inconclusive. This study aimed to determine the diagnostic yield and impact on management of next-generation sequencing (NGS) in 50 individuals with atypical CP (ACP).

Methods: Patient eligibility criteria included impaired motor function with onset at birth or within the first year of life, and one or more of the following: severe intellectual disability, progressive neurological deterioration, other abnormalities on neurological examination, multiorgan disease, congenital anomalies outside of the central nervous system, an abnormal neurotransmitter profile, family history, brain imaging findings not typical for cerebral palsy. Previous assessment by a neurologist and/or clinical geneticist, including biochemical testing, neuroimaging, and chromosomal microarray, did not yield an etiologic diagnosis.

Results: A precise molecular diagnosis was established in 65% of the 50 patients. We also identified candidate disease genes without a current OMIM disease designation. Targeted intervention was enabled in eight families (~15%).

Conclusion: NGS enabled a molecular diagnosis in ACP cases, ending the diagnostic odyssey, improving genetic counseling and personalized management, all in all enhancing precision medicine practices.
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http://dx.doi.org/10.1038/s41436-018-0376-yDOI Listing
July 2019

Gene expression models based on transcription factor binding events confer insight into functional cis-regulatory variants.

Bioinformatics 2019 08;35(15):2610-2617

Department of Medical Genetics, Centre for Molecular Medicine and Therapeutics, BC Children's Hospital Research Institute, University of British Columbia, Vancouver, BC, Canada.

Motivation: Deciphering the functional roles of cis-regulatory variants is a critical challenge in genome analysis and interpretation. It has been hypothesized that altered transcription factor (TF) binding events are a central mechanism by which cis-regulatory variants impact gene expression levels. However, we lack a computational framework to understand and quantify such mechanistic contributions.

Results: We present TF2Exp, a gene-based framework to predict the impact of altered TF-binding events on gene expression levels. Using data from lymphoblastoid cell lines, TF2Exp models were applied successfully to predict the expression levels of 3196 genes. Alterations within DNase I hypersensitive, CTCF-bound and tissue-specific TF-bound regions were the greatest contributing features to the models. TF2Exp models performed as well as models based on common variants, both in cross-validation and external validation. Combining TF alteration and common variant features can further improve model performance. Unlike variant-based models, TF2Exp models have the unique advantage to evaluate the functional impact of variants in linkage disequilibrium and uncommon variants. We find that adding TF-binding events altered only by uncommon variants could increase the number of predictable genes (R2 > 0.05). Taken together, TF2Exp represents a key step towards interpreting the functional roles of cis-regulatory variants in the human genome.

Availability And Implementation: The code and model training results are publicly available at https://github.com/wqshi/TF2Exp.

Supplementary Information: Supplementary data are available at Bioinformatics online.
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http://dx.doi.org/10.1093/bioinformatics/bty992DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6662294PMC
August 2019

Development and user evaluation of a rare disease gene prioritization workflow based on cognitive ergonomics.

J Am Med Inform Assoc 2019 02;26(2):124-133

Centre for Molecular Medicine and Therapeutics, BC Children's Hospital Research Institute, University of British Columbia, Vancouver, British Columbia, Canada.

Objective: The clinical diagnosis of genetic disorders is undergoing transformation, driven by whole exome sequencing and whole genome sequencing (WES/WGS). However, such nucleotide-level resolution technologies create an interpretive challenge. Prior literature suggests that clinicians may employ characteristic cognitive processes during WES/WGS investigations to identify disruptions in genes causal for the observed disease. Based on cognitive ergonomics, we designed and evaluated a gene prioritization workflow that supported these cognitive processes.

Materials And Methods: We designed a novel workflow in which clinicians recalled known genetic diseases with similarity to patient phenotypes to inform WES/WGS data interpretation. This prototype-based workflow was evaluated against the common computational approach based on physician-specified sets of individual patient phenotypes. The evaluation was conducted as a web-based user study, in which 18 clinicians analyzed 2 simulated patient scenarios using a randomly assigned workflow. Data analysis compared the 2 workflows with respect to accuracy and efficiency in diagnostic interpretation, efficacy in collecting detailed phenotypic information, and user satisfaction.

Results: Participants interpreted genetic diagnoses faster using prototype-based workflows. The 2 workflows did not differ in other evaluated aspects.

Discussion: The user study findings indicate that prototype-based approaches, which are designed to model experts' cognitive processes, can expedite gene prioritization and provide utility in synergy with common phenotype-driven variant/gene prioritization approaches. However, further research of the extent of this effect across diverse genetic diseases is required.

Conclusion: The findings demonstrate potential for prototype-based phenotype description to accelerate computer-assisted variant/gene prioritization through complementation of skills and knowledge of clinical experts via human-computer interaction.
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http://dx.doi.org/10.1093/jamia/ocy153DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6339516PMC
February 2019

Computational Analysis of Transcriptional Regulation Sites at the HTT Gene Locus.

J Huntingtons Dis 2018 ;7(3):223-237

Department of Medical Genetics, Centre for Molecular Medicine and Therapeutics, BC Children's Hospital, University of British Columbia, Vancouver, BC, Canada.

Background: Huntington's disease is a late onset neurological disorder caused by a trinucleotide CAG repeat expansion mutation in the HTT gene encoding for the protein huntingtin. Despite considerable ongoing research, the wild-type function of huntingtin is not yet fully understood.

Objective: To improve knowledge of HTT gene regulation at the transcriptional level and inform future studies aimed at uncovering the HTT gene's normal function.

Methods: The HTT gene region was functionally characterized through an in silico analysis using publicly available data sets. ChIP-seq data sets and the online STRING database were used to identify putative transcription factor binding sites (TFBSs) and protein-protein interactions within the HTT promoter region. siRNA-mediated knockdown and ChIP-qPCR of STAT1, a TF identified from the in silico analysis, were used to validate the bioinformatics screen.

Results: 16 regions containing potential regulatory genomic markers were identified. TFBSs for 59 transcription factors (TFs) were detected in one or more of the 16 candidate regions. Using these TFs, 15 clusters of protein-protein interactions were identified using STRING. siRNA-mediated knockdown of STAT1 resulted in an increase in HTT expression, and ChIP-qPCR detected enrichment of STAT1 binding at one of the predicted regions. These assays confirmed the utility of the bioinformatic analysis.

Conclusions: Putative regulatory regions outside of the immediate HTT promoter region have been identified with specific protein-protein interactions. Future work will focus on in vitro and in vivo studies to examine the effect of modulating identified TFBSs and altering the levels of specific TFs of interest in regulating HTT gene expression.
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http://dx.doi.org/10.3233/JHD-170272DOI Listing
October 2019

New MiniPromoter Ple345 (NEFL) Drives Strong and Specific Expression in Retinal Ganglion Cells of Mouse and Primate Retina.

Hum Gene Ther 2019 03 2;30(3):257-272. Epub 2018 Oct 2.

1 Centre for Molecular Medicine and Therapeutics at BC Children's Hospital, University of British Columbia, Vancouver, British Columbia, Canada.

Retinal gene therapy is leading the neurological gene therapy field, with 32 ongoing clinical trials of recombinant adeno-associated virus (rAAV)-based therapies. Importantly, over 50% of those trials are using restricted promoters from human genes. Promoters that restrict expression have demonstrated increased efficacy and can limit the therapeutic to the target cells thereby reducing unwanted off-target effects. Retinal ganglion cells are a critical target in ocular gene therapy; they are involved in common diseases such as glaucoma, rare diseases such as Leber's hereditary optic neuropathy, and in revolutionary optogenetic treatments. Here, we used computational biology and mined the human genome for the best genes from which to develop a novel minimal promoter element(s) designed for expression in restricted cell types (MiniPromoter) to improve the safety and efficacy of retinal ganglion cell gene therapy. Gene selection included the use of the first available droplet-based single-cell RNA sequencing (Drop-seq) dataset, and promoter design was bioinformatically driven and informed by a wide range of genomics datasets. We tested seven promoter designs from four genes in rAAV for specificity and quantified expression strength in retinal ganglion cells in mouse, and then the single best in nonhuman primate retina. Thus, we developed a new human-DNA MiniPromoter, Ple345 (NEFL), which in combination with intravitreal delivery in rAAV9 showed specific and robust expression in the retinal ganglion cells of the nonhuman-primate rhesus macaque retina. In mouse, we also developed MiniPromoters expressing in retinal ganglion cells, the hippocampus of the brain, a pan neuronal pattern in the brain, and peripheral nerves. As single-cell transcriptomics such as Drop-seq become available for other cell types, many new opportunities for additional novel restricted MiniPromoters will present.
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http://dx.doi.org/10.1089/hum.2018.118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6437624PMC
March 2019

MANTA2, update of the Mongo database for the analysis of transcription factor binding site alterations.

Sci Data 2018 07 24;5:180141. Epub 2018 Jul 24.

Centre for Molecular Medicine Norway (NCMM), Nordic EMBL Partnership, University of Oslo, 0318 Oslo, Norway.

Interpreting the functional impact of noncoding variants is an ongoing challenge in the field of genome analysis. With most noncoding variants associated with complex traits and disease residing in regulatory regions, altered transcription factor (TF) binding has been proposed as a mechanism of action. It is therefore imperative to develop methods that predict the impact of noncoding variants at TF binding sites (TFBSs). Here, we describe the update of our MANTA database that stores: 1) TFBS predictions in the human genome, and 2) the potential impact on TF binding for all possible single nucleotide variants (SNVs) at these TFBSs. TFBSs were predicted by combining experimental ChIP-seq data from ReMap and computational position weight matrices (PWMs) derived from JASPAR. Impact of SNVs at these TFBSs was assessed by means of PWM scores computed on the alternate alleles. The updated database, MANTA2, provides the scientific community with a critical map of TFBSs and SNV impact scores to improve the interpretation of noncoding variants in the human genome.
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http://dx.doi.org/10.1038/sdata.2018.141DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6057437PMC
July 2018

c-Myc is a novel virulence factor by proxy that targets the host miRNA system and is essential for survival in human macrophages.

J Biol Chem 2018 08 22;293(33):12805-12819. Epub 2018 Jun 22.

From the Division of Infectious Diseases, Department of Medicine, Vancouver Coastal Health Research Institute, Vancouver, British Columbia V5Z 1M9 and

species are intracellular protozoan pathogens that have evolved to successfully infect and deactivate host macrophages. How this deactivation is brought about is not completely understood. Recently, microRNAs (miRNAs) have emerged as ubiquitous regulators of macrophage gene expression that contribute to shaping the immune responses to intracellular pathogens. Conversely, several pathogens have evolved the ability to exploit host miRNA expression to manipulate host-cell phenotype. However, very little is known about the mechanisms used by intracellular pathogens to drive changes in host-cell miRNA abundance. Using miRNA expression profiling of -infected human macrophages, we show here that infection induced a genome-wide down-regulation of host miRNAs. This repression occurred at the level of miRNA gene transcription, because the synthesis rates of primary miRNAs were significantly decreased in infected cells. miRNA repression depended on the host macrophage transcription factor c-Myc. Indeed, the expression of host c-Myc was markedly up-regulated by infection, and c-Myc silencing reversed the miRNA suppression. Furthermore, c-Myc silencing significantly reduced intracellular survival of , demonstrating that c-Myc is essential for pathogenesis. Taken together, these findings identify c-Myc not only as being responsible for miRNA repression in -infected macrophages but also as a novel and essential virulence factor by proxy that promotes survival.
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http://dx.doi.org/10.1074/jbc.RA118.002462DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6102154PMC
August 2018