Publications by authors named "Robi D Mitra"

63 Publications

From karyotypes to precision genomics in 9p deletion and duplication syndromes.

HGG Adv 2022 Jan 24;3(1):100081. Epub 2021 Dec 24.

Department of Genetics, Washington University School of Medicine, St. Louis, MO 63110, USA.

While 9p deletion and duplication syndromes have been studied for several years, small sample sizes and minimal high-resolution data have limited a comprehensive delineation of genotypic and phenotypic characteristics. In this study, we examined genetic data from 719 individuals in the worldwide 9p Network Cohort: a cohort seven to nine times larger than any previous study of 9p. Most breakpoints occur in bands 9p22 and 9p24, accounting for 35% and 38% of all breakpoints, respectively. Bands 9p11 and 9p12 have the fewest breakpoints, with each accounting for 0.6% of all breakpoints. The most common phenotype in 9p deletion and duplication syndromes is developmental delay, and we identified eight known neurodevelopmental disorder genes in 9p22 and 9p24. Since it has been previously reported that some individuals have a secondary structural variant related to the 9p variant, we examined our cohort for these variants and found 97 events. The top secondary variant involved 9q in 14 individuals (1.9%), including ring chromosomes and inversions. We identified a gender bias with significant enrichment for females (p = 0.0006) that may arise from a sex reversal in some individuals with 9p deletions. Genes on 9p were characterized regarding function, constraint metrics, and protein-protein interactions, resulting in a prioritized set of genes for further study. Finally, we achieved precision genomics in one child with a complex 9p structural variation using modern genomic technologies, demonstrating that long-read sequencing will be integral for some cases. Our study is the largest ever on 9p-related syndromes and provides key insights into genetic factors involved in these syndromes.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.xhgg.2021.100081DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8756500PMC
January 2022

Brd4-bound enhancers drive cell-intrinsic sex differences in glioblastoma.

Proc Natl Acad Sci U S A 2021 04;118(16)

Department of Pediatrics, School of Medicine, Washington University in St. Louis, St. Louis, MO 63110;

Sex can be an important determinant of cancer phenotype, and exploring sex-biased tumor biology holds promise for identifying novel therapeutic targets and new approaches to cancer treatment. In an established isogenic murine model of glioblastoma (GBM), we discovered correlated transcriptome-wide sex differences in gene expression, H3K27ac marks, large Brd4-bound enhancer usage, and Brd4 localization to Myc and p53 genomic binding sites. These sex-biased gene expression patterns were also evident in human glioblastoma stem cells (GSCs). These observations led us to hypothesize that Brd4-bound enhancers might underlie sex differences in stem cell function and tumorigenicity in GBM. We found that male and female GBM cells exhibited sex-specific responses to pharmacological or genetic inhibition of Brd4. Brd4 knockdown or pharmacologic inhibition decreased male GBM cell clonogenicity and in vivo tumorigenesis while increasing both in female GBM cells. These results were validated in male and female patient-derived GBM cell lines. Furthermore, analysis of the Cancer Therapeutic Response Portal of human GBM samples segregated by sex revealed that male GBM cells are significantly more sensitive to BET (bromodomain and extraterminal) inhibitors than are female cells. Thus, Brd4 activity is revealed to drive sex differences in stem cell and tumorigenic phenotypes, which can be abrogated by sex-specific responses to BET inhibition. This has important implications for the clinical evaluation and use of BET inhibitors.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1073/pnas.2017148118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8072233PMC
April 2021

High-resolution HLA typing by long reads from the R10.3 Oxford nanopore flow cells.

Hum Immunol 2021 Apr 19;82(4):288-295. Epub 2021 Feb 19.

Department of Genetics, Center for Genome Sciences and Systems Biology, Washington University School of Medicine, St. Louis, MO, USA.

Nanopore sequencing has been investigated as a rapid and cost-efficient option for HLA typing in recent years. Despite the lower raw read accuracy, encouraging typing accuracy has been reported, and long reads from the platform offer additional benefits of the improved phasing of distant variants. The newly released R10.3 flow cells are expected to provide higher read-level accuracy than previous chemistries. We examined the performance of R10.3 flow cells on the MinION device in HLA typing after enrichment of target genes by multiplexed PCR. We also aimed to mimic a 1-day workflow with 8-24 samples per sequencing run. A diverse collection of 102 unique samples were typed for HLA-A, -B, -C, -DPA1, -DPB1, -DQA1, -DQB1, -DRB1, -DRB3/4/5 loci. The concordance rates at 2-field and 3-field resolutions were 99.5% (1836 alleles) and 99.3% (1710 alleles). We also report important quality metrics from these sequencing runs. Continued research and independent validations are warranted to increase the robustness of nanopore-based HLA typing for broad clinical application.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.humimm.2021.02.005DOI Listing
April 2021

Rapid and Extraction-Free Detection of SARS-CoV-2 from Saliva by Colorimetric Reverse-Transcription Loop-Mediated Isothermal Amplification.

Clin Chem 2021 01;67(2):415-424

Department of Genetics, Washington University School of Medicine, St. Louis, MO.

Background: Rapid, reliable, and widespread testing is required to curtail the ongoing COVID-19 pandemic. Current gold-standard nucleic acid tests are hampered by supply shortages in critical reagents including nasal swabs, RNA extraction kits, personal protective equipment, instrumentation, and labor.

Methods: To overcome these challenges, we developed a rapid colorimetric assay using reverse-transcription loop-mediated isothermal amplification (RT-LAMP) optimized on human saliva samples without an RNA purification step. We describe the optimization of saliva pretreatment protocols to enable analytically sensitive viral detection by RT-LAMP. We optimized the RT-LAMP reaction conditions and implemented high-throughput unbiased methods for assay interpretation. We tested whether saliva pretreatment could also enable viral detection by conventional reverse-transcription quantitative polymerase chain reaction (RT-qPCR). Finally, we validated these assays on clinical samples.

Results: The optimized saliva pretreatment protocol enabled analytically sensitive extraction-free detection of SARS-CoV-2 from saliva by colorimetric RT-LAMP or RT-qPCR. In simulated samples, the optimized RT-LAMP assay had a limit of detection of 59 (95% confidence interval: 44-104) particle copies per reaction. We highlighted the flexibility of LAMP assay implementation using 3 readouts: naked-eye colorimetry, spectrophotometry, and real-time fluorescence. In a set of 30 clinical saliva samples, colorimetric RT-LAMP and RT-qPCR assays performed directly on pretreated saliva samples without RNA extraction had accuracies greater than 90%.

Conclusions: Rapid and extraction-free detection of SARS-CoV-2 from saliva by colorimetric RT-LAMP is a simple, sensitive, and cost-effective approach with broad potential to expand diagnostic testing for the virus causing COVID-19.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/clinchem/hvaa267DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7665435PMC
January 2021

The qBED track: a novel genome browser visualization for point processes.

Bioinformatics 2021 05;37(8):1168-1170

Department of Genetics, St. Louis, MO 63110, USA.

Summary: Transposon calling cards is a genomic assay for identifying transcription factor binding sites in both bulk and single cell experiments. Here, we describe the qBED format, an open, text-based standard for encoding and analyzing calling card data. In parallel, we introduce the qBED track on the WashU Epigenome Browser, a novel visualization that enables researchers to inspect calling card data in their genomic context. Finally, through examples, we demonstrate that qBED files can be used to visualize non-calling card datasets, such as Combined Annotation-Dependent Depletion scores and GWAS/eQTL hits, and thus may have broad utility to the genomics community.

Availability And Implementation: The qBED track is available on the WashU Epigenome Browser (http://epigenomegateway.wustl.edu/browser), beginning with version 46. Source code for the WashU Epigenome Browser with qBED support is available on GitHub (http://github.com/arnavm/eg-react and http://github.com/lidaof/eg-react). A complete definition of the qBED format is available as part of the WashU Epigenome Browser documentation (https://eg.readthedocs.io/en/latest/tracks.html#qbed-track). We have also released a tutorial on how to upload qBED data to the browser (http://dx.doi.org/10.17504/protocols.io.bca8ishw).
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/bioinformatics/btaa771DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8150125PMC
May 2021

High-throughput single-cell functional elucidation of neurodevelopmental disease-associated genes reveals convergent mechanisms altering neuronal differentiation.

Genome Res 2020 09 4;30(9):1317-1331. Epub 2020 Sep 4.

Department of Genetics, Washington University in St. Louis School of Medicine, St. Louis, Missouri 63110, USA.

The overwhelming success of exome- and genome-wide association studies in discovering thousands of disease-associated genes necessitates developing novel high-throughput functional genomics approaches to elucidate the molecular mechanisms of these genes. Here, we have coupled multiplexed repression of neurodevelopmental disease-associated genes to single-cell transcriptional profiling in differentiating human neurons to rapidly assay the functions of multiple genes in a disease-relevant context, assess potentially convergent mechanisms, and prioritize genes for specific functional assays. For a set of 13 autism spectrum disorder (ASD)-associated genes, we show that this approach generated important mechanistic insights, revealing two functionally convergent modules of ASD genes: one that delays neuron differentiation and one that accelerates it. Five genes that delay neuron differentiation (, , , , and ) mechanistically converge, as they all dysregulate genes involved in cell-cycle control and progenitor cell proliferation. Live-cell imaging after individual ASD-gene repression validated this functional module, confirming that these genes reduce neural progenitor cell proliferation and neurite growth. Finally, these functionally convergent ASD gene modules predicted shared clinical phenotypes among individuals with mutations in these genes. Altogether, these results show the utility of a novel and simple approach for the rapid functional elucidation of neurodevelopmental disease-associated genes.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1101/gr.262295.120DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7545139PMC
September 2020

Human L1 Transposition Dynamics Unraveled with Functional Data Analysis.

Mol Biol Evol 2020 12;37(12):3576-3600

The Huck Institutes of the Life Sciences, Center for Medical Genomics, The Pennsylvania State University, University Park, PA.

Long INterspersed Elements-1 (L1s) constitute >17% of the human genome and still actively transpose in it. Characterizing L1 transposition across the genome is critical for understanding genome evolution and somatic mutations. However, to date, L1 insertion and fixation patterns have not been studied comprehensively. To fill this gap, we investigated three genome-wide data sets of L1s that integrated at different evolutionary times: 17,037 de novo L1s (from an L1 insertion cell-line experiment conducted in-house), and 1,212 polymorphic and 1,205 human-specific L1s (from public databases). We characterized 49 genomic features-proxying chromatin accessibility, transcriptional activity, replication, recombination, etc.-in the ±50 kb flanks of these elements. These features were contrasted between the three L1 data sets and L1-free regions using state-of-the-art Functional Data Analysis statistical methods, which treat high-resolution data as mathematical functions. Our results indicate that de novo, polymorphic, and human-specific L1s are surrounded by different genomic features acting at specific locations and scales. This led to an integrative model of L1 transposition, according to which L1s preferentially integrate into open-chromatin regions enriched in non-B DNA motifs, whereas they are fixed in regions largely free of purifying selection-depleted of genes and noncoding most conserved elements. Intriguingly, our results suggest that L1 insertions modify local genomic landscape by extending CpG methylation and increasing mononucleotide microsatellite density. Altogether, our findings substantially facilitate understanding of L1 integration and fixation preferences, pave the way for uncovering their role in aging and cancer, and inform their use as mutagenesis tools in genetic studies.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/molbev/msaa194DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7743743PMC
December 2020

Self-Reporting Transposons Enable Simultaneous Readout of Gene Expression and Transcription Factor Binding in Single Cells.

Cell 2020 08 24;182(4):992-1008.e21. Epub 2020 Jul 24.

Department of Genetics, Washington University School of Medicine in St. Louis, St. Louis, MO 63110, USA; Edison Family Center for Genome Sciences and Systems Biology, Washington University School of Medicine in St. Louis, St. Louis, MO 63110, USA. Electronic address:

Cellular heterogeneity confounds in situ assays of transcription factor (TF) binding. Single-cell RNA sequencing (scRNA-seq) deconvolves cell types from gene expression, but no technology links cell identity to TF binding sites (TFBS) in those cell types. We present self-reporting transposons (SRTs) and use them in single-cell calling cards (scCC), a novel assay for simultaneously measuring gene expression and mapping TFBS in single cells. The genomic locations of SRTs are recovered from mRNA, and SRTs deposited by exogenous, TF-transposase fusions can be used to map TFBS. We then present scCC, which map SRTs from scRNA-seq libraries, simultaneously identifying cell types and TFBS in those same cells. We benchmark multiple TFs with this technique. Next, we use scCC to discover BRD4-mediated cell-state transitions in K562 cells. Finally, we map BRD4 binding sites in the mouse cortex at single-cell resolution, establishing a new method for studying TF biology in situ.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.cell.2020.06.037DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7510185PMC
August 2020

Rapid and extraction-free detection of SARS-CoV-2 from saliva with colorimetric LAMP.

medRxiv 2020 Aug 6. Epub 2020 Aug 6.

Rapid, reliable, and widespread testing is required to curtail the ongoing COVID-19 pandemic. Current gold standard nucleic acid tests are hampered by supply shortages in critical reagents including nasal swabs, RNA extraction kits, personal protective equipment (PPE), instrumentation, and labor. Here we present an approach to overcome these challenges with the development of a rapid colorimetric assay using reverse-transcription loop-mediated isothermal amplification (RT-LAMP) optimized on human saliva samples without an RNA purification step. We describe our optimizations of the LAMP reaction and saliva pretreatment protocols that enabled rapid and sensitive detection of < 102 viral genomes per reaction in contrived saliva controls. Moreover, our saliva pretreatment protocol enabled sensitive viral detection by conventional quantitative reverse transcription polymerase chain reaction (qRT-PCR) without RNA extraction. We validated the high performance of these assays on clinical samples and demonstrate a promising approach to overcome the current bottlenecks limiting widespread testing.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1101/2020.05.07.20093542DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7273276PMC
August 2020

A viral toolkit for recording transcription factor-DNA interactions in live mouse tissues.

Proc Natl Acad Sci U S A 2020 05 16;117(18):10003-10014. Epub 2020 Apr 16.

Department of Genetics, Washington University School of Medicine, St. Louis, MO 63110;

Transcription factors (TFs) enact precise regulation of gene expression through site-specific, genome-wide binding. Common methods for TF-occupancy profiling, such as chromatin immunoprecipitation, are limited by requirement of TF-specific antibodies and provide only end-point snapshots of TF binding. Alternatively, TF-tagging techniques, in which a TF is fused to a DNA-modifying enzyme that marks TF-binding events across the genome as they occur, do not require TF-specific antibodies and offer the potential for unique applications, such as recording of TF occupancy over time and cell type specificity through conditional expression of the TF-enzyme fusion. Here, we create a viral toolkit for one such method, calling cards, and demonstrate that these reagents can be delivered to the live mouse brain and used to report TF occupancy. Further, we establish a Cre-dependent calling cards system and, in proof-of-principle experiments, show utility in defining cell type-specific TF profiles and recording and integrating TF-binding events across time. This versatile approach will enable unique studies of TF-mediated gene regulation in live animal models.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1073/pnas.1918241117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7211997PMC
May 2020

Quantitative analysis of transcription factor binding and expression using calling cards reporter arrays.

Nucleic Acids Res 2020 05;48(9):e50

Department of Genetics, Washington University School of Medicine in St. Louis, St. Louis, MO 63108, USA.

We report a tool, Calling Cards Reporter Arrays (CCRA), that measures transcription factor (TF) binding and the consequences on gene expression for hundreds of synthetic promoters in yeast. Using Cbf1p and MAX, we demonstrate that the CCRA method is able to detect small changes in binding free energy with a sensitivity comparable to in vitro methods, enabling the measurement of energy landscapes in vivo. We then demonstrate the quantitative analysis of cooperative interactions by measuring Cbf1p binding at synthetic promoters with multiple sites. We find that the cooperativity between Cbf1p dimers varies sinusoidally with a period of 10.65 bp and energetic cost of 1.37 KBT for sites that are positioned 'out of phase'. Finally, we characterize the binding and expression of a group of TFs, Tye7p, Gcr1p and Gcr2p, that act together as a 'TF collective', an important but poorly characterized model of TF cooperativity. We demonstrate that Tye7p often binds promoters without its recognition site because it is recruited by other collective members, whereas these other members require their recognition sites, suggesting a hierarchy where these factors recruit Tye7p but not vice versa. Our experiments establish CCRA as a useful tool for quantitative investigations into TF binding and function.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/nar/gkaa141DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7229839PMC
May 2020

Homotypic cooperativity and collective binding are determinants of bHLH specificity and function.

Proc Natl Acad Sci U S A 2019 08 24;116(32):16143-16152. Epub 2019 Jul 24.

Department of Genetics, Washington University School of Medicine in St. Louis, St. Louis, MO 63108;

Eukaryotic cells express transcription factor (TF) paralogues that bind to nearly identical DNA sequences in vitro but bind at different genomic loci and perform different functions in vivo. Predicting how 2 paralogous TFs bind in vivo using DNA sequence alone is an important open problem. Here, we analyzed 2 yeast bHLH TFs, Cbf1p and Tye7p, which have highly similar binding preferences in vitro, yet bind at almost completely nonoverlapping target loci in vivo. We dissected the determinants of specificity for these 2 proteins by making a number of chimeric TFs in which we swapped different domains of Cbf1p and Tye7p and determined the effects on in vivo binding and cellular function. From these experiments, we learned that the Cbf1p dimer achieves its specificity by binding cooperatively with other Cbf1p dimers bound nearby. In contrast, we found that Tye7p achieves its specificity by binding cooperatively with 3 other DNA-binding proteins, Gcr1p, Gcr2p, and Rap1p. Remarkably, most promoters (63%) that are bound by Tye7p do not contain a consensus Tye7p binding site. Using this information, we were able to build simple models to accurately discriminate bound and unbound genomic loci for both Cbf1p and Tye7p. We then successfully reprogrammed the human bHLH NPAS2 to bind Cbf1p in vivo targets and a Tye7p target intergenic region to be bound by Cbf1p. These results demonstrate that the genome-wide binding targets of paralogous TFs can be discriminated using sequence information, and provide lessons about TF specificity that can be applied across the phylogenetic tree.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1073/pnas.1818015116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6689977PMC
August 2019

Transposase mapping identifies the genomic targets of BAP1 in uveal melanoma.

BMC Med Genomics 2018 Nov 6;11(1):97. Epub 2018 Nov 6.

Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, 660 S. Euclid Ave., St. Louis, MO, 63110, USA.

Background: BAP1 is a histone deubiquitinase that acts as a tumor and metastasis suppressor associated with disease progression in human cancer. We have used the "Calling Card System" of transposase-directed transposon insertion mapping to identify the genomic targets of BAP1 in uveal melanoma (UM). This system was developed to identify the genomic loci visited by transcription factors that bind directly to DNA; our study is the first use of the system with a chromatin-remodeling factor that binds to histones but does not interact directly with DNA.

Methods: The transposase piggyBac (PBase) was fused to BAP1 and expressed in OCM-1A UM cells. The insertion of transposons near BAP1 binding sites in UM cells were identified by genomic sequencing. We also examined RNA expression in the same OCM-1A UM cells after BAP1 depletion to identify BAP1 binding sites associated with BAP1-responsive genes. Sets of significant genes were analyzed for common pathways, transcription factor binding sites, and ability to identify molecular tumor classes.

Results: We found a strong correlation between multiple calling-card transposon insertions targeted by BAP1-PBase and BAP1-responsive expression of adjacent genes. BAP1-bound genomic loci showed narrow distributions of insertions and were near transcription start sites, consistent with recruitment of BAP1 to these sites by specific DNA-binding proteins. Sequence consensus analysis of BAP1-bound sites showed enrichment of motifs specific for YY1, NRF1 and Ets transcription factors, which have been shown to interact with BAP1 in other cell types. Further, a subset of the BAP1 genomic target genes was able to discriminate aggressive tumors in published gene expression data from primary UM tumors.

Conclusions: The calling card methodology works equally well for chromatin regulatory factors that do not interact directly with DNA as for transcription factors. This technique has generated a new and expanded list of BAP1 targets in UM that provides important insight into metastasis pathways and identifies novel potential therapeutic targets.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/s12920-018-0424-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6219186PMC
November 2018

Single-Cell RNA-Seq Uncovers a Robust Transcriptional Response to Morphine by Glia.

Cell Rep 2018 09;24(13):3619-3629.e4

Department of Genetics, Washington University, School of Medicine, St. Louis, MO 63110, USA; Center for Genome Sciences and Systems Biology, Washington University, School of Medicine, St. Louis, MO 63110, USA. Electronic address:

Molecular and behavioral responses to opioids are thought to be primarily mediated by neurons, although there is accumulating evidence that other cell types play a prominent role in drug addiction. To investigate cell-type-specific opioid responses, we performed single-cell RNA sequencing (scRNA-seq) of the nucleus accumbens of mice following acute morphine treatment. Differential expression analysis uncovered unique morphine-dependent transcriptional responses by oligodendrocytes and astrocytes. We examined the expression of selected genes, including Cdkn1a and Sgk1, by FISH, confirming their induction by morphine in oligodendrocytes. Further analysis using RNA-seq of FACS-purified oligodendrocytes revealed a large cohort of morphine-regulated genes. The affected genes are enriched for roles in cellular pathways intimately linked to oligodendrocyte maturation and myelination, including the unfolded protein response. Altogether, our data illuminate the morphine-dependent transcriptional response by oligodendrocytes and offer mechanistic insights into myelination defects associated with opioid abuse.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.celrep.2018.08.080DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6357782PMC
September 2018

Accurate Typing of Human Leukocyte Antigen Class I Genes by Oxford Nanopore Sequencing.

J Mol Diagn 2018 07 3;20(4):428-435. Epub 2018 Apr 3.

Division of Laboratory and Genomic Medicine, and the Department of Genetics, Center for Genome Sciences and Systems Biology, Washington University School of Medicine, St. Louis, Missouri. Electronic address:

Oxford Nanopore Technologies' MinION has expanded the current DNA sequencing toolkit by delivering long read lengths and extreme portability. The MinION has the potential to enable expedited point-of-care human leukocyte antigen (HLA) typing, an assay routinely used to assess the immunologic compatibility between organ donors and recipients, but the platform's high error rate makes it challenging to type alleles with accuracy. We developed and validated accurate typing of HLA by Oxford nanopore (Athlon), a bioinformatic pipeline that i) maps nanopore reads to a database of known HLA alleles, ii) identifies candidate alleles with the highest read coverage at different resolution levels that are represented as branching nodes and leaves of a tree structure, iii) generates consensus sequences by remapping the reads to the candidate alleles, and iv) calls the final diploid genotype by blasting consensus sequences against the reference database. Using two independent data sets generated on the R9.4 flow cell chemistry, Athlon achieved a 100% accuracy in class I HLA typing at the two-field resolution.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.jmoldx.2018.02.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6039791PMC
July 2018

Massively parallel single-nucleotide mutagenesis using reversibly terminated inosine.

Nat Methods 2016 Nov 3;13(11):923-924. Epub 2016 Oct 3.

Department of Orthopaedic Surgery, Washington University, St. Louis, Missouri, USA.

Large-scale mutagenesis of target DNA sequences allows researchers to comprehensively assess the effects of single-nucleotide changes. Here we demonstrate the construction of a systematic allelic series (SAS) using massively parallel single-nucleotide mutagenesis with reversibly terminated deoxyinosine triphosphates (rtITP). We created a mutational library containing every possible single-nucleotide mutation surrounding the active site of the TEM-1 β-lactamase gene. When combined with high-throughput functional assays, SAS mutational libraries can expedite the functional assessment of genetic variation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/nmeth.4015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5327618PMC
November 2016

A role for genetic susceptibility in sporadic focal segmental glomerulosclerosis.

J Clin Invest 2016 Mar 22;126(3):1067-78. Epub 2016 Feb 22.

Focal segmental glomerulosclerosis (FSGS) is a syndrome that involves kidney podocyte dysfunction and causes chronic kidney disease. Multiple factors including chemical toxicity, inflammation, and infection underlie FSGS; however, highly penetrant disease genes have been identified in a small fraction of patients with a family history of FSGS. Variants of apolipoprotein L1 (APOL1) have been linked to FSGS in African Americans with HIV or hypertension, supporting the proposal that genetic factors enhance FSGS susceptibility. Here, we used sequencing to investigate whether genetics plays a role in the majority of FSGS cases that are identified as primary or sporadic FSGS and have no known cause. Given the limited number of biopsy-proven cases with ethnically matched controls, we devised an analytic strategy to identify and rank potential candidate genes and used an animal model for validation. Nine candidate FSGS susceptibility genes were identified in our patient cohort, and three were validated using a high-throughput mouse method that we developed. Specifically, we introduced a podocyte-specific, doxycycline-inducible transactivator into a murine embryonic stem cell line with an FSGS-susceptible genetic background that allows shRNA-mediated targeting of candidate genes in the adult kidney. Our analysis supports a broader role for genetic susceptibility of both sporadic and familial cases of FSGS and provides a tool to rapidly evaluate candidate FSGS-associated genes.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1172/JCI82592DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4767358PMC
March 2016

Transposon Calling Cards.

Cold Spring Harb Protoc 2016 Feb 1;2016(2):pdb.top077776. Epub 2016 Feb 1.

Department of Genetics, Center for Genome Sciences & Systems Biology, Washington University School of Medicine, St. Louis, Missouri 63110.

Identifying the genomic targets of transcription factors is an important step in understanding the regulatory networks of gene transcription in yeast. We have developed a method that utilizes what we refer to as transposon "calling cards," in which a transcription factor directs the Ty5 retrotransposase to insert transposons into the genome adjacent to where the transcription factor binds. This method is designed to be multiplexed with many barcoded transcription factors and has the potential to decrease the labor required for the study of large numbers of transcription factors.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1101/pdb.top077776DOI Listing
February 2016

Calling Card Analysis in Budding Yeast.

Cold Spring Harb Protoc 2016 Feb 1;2016(2):pdb.prot086918. Epub 2016 Feb 1.

Department of Genetics, Center for Genome Sciences and Systems Biology, Washington University School of Medicine, St. Louis, Missouri 63110.

Calling card analysis is a high-throughput method for identifying the genomic binding sites of multiple transcription factors in a single experiment in budding yeast. By tagging a DNA-binding protein with a targeting domain that directs the insertion of the Ty5 retrotransposon, the genomic binding sites for that transcription factor are marked. The transposition locations are then identified en masse by Illumina sequencing. The calling card protocol allows for simultaneous analysis of multiple transcription factors. By cloning barcodes into the Ty5 transposon, it is possible to pair a unique barcode with every transcription factor in the experiment. The method presented here uses expression of transcription factors from their native loci; however, it can also be altered to measure binding sites of transcription factors overexpressed from a plasmid.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1101/pdb.prot086918DOI Listing
February 2016

Redesign of the monomer-monomer interface of Cre recombinase yields an obligate heterotetrameric complex.

Nucleic Acids Res 2015 Oct 13;43(18):9076-85. Epub 2015 Sep 13.

Department of Biochemistry and Molecular Biophysics, Washington University in St Louis, St Louis, MO 63110, USA

Cre recombinase catalyzes the cleavage and religation of DNA at loxP sites. The enzyme is a homotetramer in its functional state, and the symmetry of the protein complex enforces a pseudo-palindromic symmetry upon the loxP sequence. The Cre-lox system is a powerful tool for many researchers. However, broader application of the system is limited by the fixed sequence preferences of Cre, which are determined by both the direct DNA contacts and the homotetrameric arrangement of the Cre monomers. As a first step toward achieving recombination at arbitrary asymmetric target sites, we have broken the symmetry of the Cre tetramer assembly. Using a combination of computational and rational protein design, we have engineered an alternative interface between Cre monomers that is functional yet incompatible with the wild-type interface. Wild-type and engineered interface halves can be mixed to create two distinct Cre mutants, neither of which are functional in isolation, but which can form an active heterotetramer when combined. When these distinct mutants possess different DNA specificities, control over complex assembly directly discourages recombination at unwanted half-site combinations, enhancing the specificity of asymmetric site recombination. The engineered Cre mutants exhibit this assembly pattern in a variety of contexts, including mammalian cells.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/nar/gkv901DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4605323PMC
October 2015

Sequencing of idiopathic pulmonary fibrosis-related genes reveals independent single gene associations.

BMJ Open Respir Res 2014 10;1(1):e000057. Epub 2014 Dec 10.

Division of Newborn Medicine, Edward Mallinckrodt Department of Pediatrics , Washington University School of Medicine , St. Louis, Missouri , USA ; Division of Neonatology, Department of Pediatrics , Ann and Robert H. Lurie Children's Hospital of Chicago, Northwestern University Feinberg School of Medicine , Chicago, Illinois , USA.

Background: Previous studies investigating a genetic basis for idiopathic pulmonary fibrosis (IPF) have focused on resequencing single genes in IPF kindreds or cohorts to determine the genetic contributions to IPF. None has investigated interactions among the candidate genes.

Objective: To compare the frequencies and interactions of mutations in six IPF-associated genes in a cohort of 132 individuals with IPF with those of a disease-control cohort of 192 individuals with chronic obstructive pulmonary disease (COPD) and the population represented in the Exome Variant Server.

Methods: We resequenced the genes encoding surfactant proteins A2 (SFTPA2), and C (SFTPC), the ATP binding cassette member A3 (ABCA3), telomerase (TERT), thyroid transcription factor (NKX2-1) and mucin 5B (MUC5B) and compared the collapsed frequencies of rare (minor allele frequency <1%), computationally predicted deleterious variants in each cohort. We also genotyped a common MUC5B promoter variant that is over-represented in individuals with IPF.

Results: We found 15 mutations in 14 individuals (11%) in the IPF cohort: (SFTPA2 (n=1), SFTPC (n=5), ABCA3 (n=4) and TERT (n=5)). No individual with IPF had two different mutations, but one individual with IPF was homozygous for p.E292V, the most common ABCA3 disease-causing variant. We did not detect an interaction between any of the mutations and the MUC5B promoter variant.

Conclusions: Rare mutations in SFTPA2, SFTPC and TERT are collectively over-represented in individuals with IPF. Genetic analysis and counselling should be considered as part of the IPF evaluation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1136/bmjresp-2014-000057DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4265083PMC
January 2015

Toying with fate: Redirecting the differentiation of adrenocortical progenitor cells into gonadal-like tissue.

Mol Cell Endocrinol 2015 Jun 8;408:165-77. Epub 2014 Dec 8.

Department of Pediatrics, Washington University School of Medicine, St. Louis Children's Hospital, St. Louis, MO 63110, USA; Department of Developmental Biology, Washington University School of Medicine, St. Louis Children's Hospital, St. Louis, MO 63110, USA. Electronic address:

Cell fate decisions are integral to zonation and remodeling of the adrenal cortex. Animal models exhibiting ectopic differentiation of gonadal-like cells in the adrenal cortex can shed light on the molecular mechanisms regulating steroidogenic cell fate. In one such model, prepubertal gonadectomy (GDX) of mice triggers the formation of adrenocortical neoplasms that resemble luteinized ovarian stroma. Transcriptomic analysis and genome-wide DNA methylation mapping have identified genetic and epigenetic markers of GDX-induced adrenocortical neoplasia. Members of the GATA transcription factor family have emerged as key regulators of cell fate in this model. Expression of Gata4 is pivotal for the accumulation of gonadal-like cells in the adrenal glands of gonadectomized mice, whereas expression of Gata6 limits the spontaneous and GDX-induced differentiation of gonadal-like cells in the adrenal cortex. Additionally, Gata6 is essential for proper development of the adrenal X-zone, a layer analogous to the fetal zone of the human adrenal cortex. The relevance of these observations to developmental signaling pathways in the adrenal cortex, to other animal models of altered adrenocortical cell fate, and to human diseases is discussed.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.mce.2014.12.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4417465PMC
June 2015

Amyotrophic lateral sclerosis onset is influenced by the burden of rare variants in known amyotrophic lateral sclerosis genes.

Ann Neurol 2015 Jan 27;77(1):100-13. Epub 2014 Nov 27.

Department of Neurology, Washington University, St, Louis, MO.

Objective: To define the genetic landscape of amyotrophic lateral sclerosis (ALS) and assess the contribution of possible oligogenic inheritance, we aimed to comprehensively sequence 17 known ALS genes in 391 ALS patients from the United States.

Methods: Targeted pooled-sample sequencing was used to identify variants in 17 ALS genes. Fragment size analysis was used to define ATXN2 and C9ORF72 expansion sizes. Genotype-phenotype correlations were made with individual variants and total burden of variants. Rare variant associations for risk of ALS were investigated at both the single variant and gene level.

Results: A total of 64.3% of familial and 27.8% of sporadic subjects carried potentially pathogenic novel or rare coding variants identified by sequencing or an expanded repeat in C9ORF72 or ATXN2; 3.8% of subjects had variants in >1 ALS gene, and these individuals had disease onset 10 years earlier (p = 0.0046) than subjects with variants in a single gene. The number of potentially pathogenic coding variants did not influence disease duration or site of onset.

Interpretation: Rare and potentially pathogenic variants in known ALS genes are present in >25% of apparently sporadic and 64% of familial patients, significantly higher than previous reports using less comprehensive sequencing approaches. A significant number of subjects carried variants in >1 gene, which influenced the age of symptom onset and supports oligogenic inheritance as relevant to disease pathogenesis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/ana.24306DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4293318PMC
January 2015

Novel markers of gonadectomy-induced adrenocortical neoplasia in the mouse and ferret.

Mol Cell Endocrinol 2015 Jan 5;399:122-30. Epub 2014 Oct 5.

Department of Pediatrics, Washington University School of Medicine, St. Louis Children's Hospital, St. Louis, MO 63110, USA; Department of Developmental Biology, Washington University School of Medicine, St. Louis Children's Hospital, St. Louis, MO 63110, USA. Electronic address:

Gonadectomy (GDX) induces sex steroid-producing adrenocortical tumors in certain mouse strains and in the domestic ferret. Transcriptome analysis and DNA methylation mapping were used to identify novel genetic and epigenetic markers of GDX-induced adrenocortical neoplasia in female DBA/2J mice. Markers were validated using a combination of laser capture microdissection, quantitative RT-PCR, in situ hybridization, and immunohistochemistry. Microarray expression profiling of whole adrenal mRNA from ovariectomized vs. intact mice demonstrated selective upregulation of gonadal-like genes including Spinlw1 and Insl3 in GDX-induced adrenocortical tumors of the mouse. A complementary candidate gene approach identified Foxl2 as another gonadal-like marker expressed in GDX-induced neoplasms of the mouse and ferret. That both "male-specific" (Spinlw1) and "female-specific" (Foxl2) markers were identified is noteworthy and implies that the neoplasms exhibit mixed characteristics of male and female gonadal somatic cells. Genome-wide methylation analysis showed that two genes with hypomethylated promoters, Igfbp6 and Foxs1, are upregulated in GDX-induced adrenocortical neoplasms. These new genetic and epigenetic markers may prove useful for studies of steroidogenic cell development and for diagnostic testing.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.mce.2014.09.029DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4262703PMC
January 2015

PDE7B is a novel, prognostically significant mediator of glioblastoma growth whose expression is regulated by endothelial cells.

PLoS One 2014 9;9(9):e107397. Epub 2014 Sep 9.

Department of Pediatrics, Washington University School of Medicine, St. Louis, Missouri, United States of America; Department of Anatomy and Neurobiology, Washington University School of Medicine, St. Louis, Missouri, United States of America.

Cell-cell interactions between tumor cells and constituents of their microenvironment are critical determinants of tumor tissue biology and therapeutic responses. Interactions between glioblastoma (GBM) cells and endothelial cells (ECs) establish a purported cancer stem cell niche. We hypothesized that genes regulated by these interactions would be important, particularly as therapeutic targets. Using a computational approach, we deconvoluted expression data from a mixed physical co-culture of GBM cells and ECs and identified a previously undescribed upregulation of the cAMP specific phosphodiesterase PDE7B in GBM cells in response to direct contact with ECs. We further found that elevated PDE7B expression occurs in most GBM cases and has a negative effect on survival. PDE7B overexpression resulted in the expansion of a stem-like cell subpopulation in vitro and increased tumor growth and aggressiveness in an in vivo intracranial GBM model. Collectively these studies illustrate a novel approach for studying cell-cell interactions and identifying new therapeutic targets like PDE7B in GBM.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0107397PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4159344PMC
May 2015

Mutation and expression analysis in medulloblastoma yields prognostic variants and a putative mechanism of disease for i17q tumors.

Acta Neuropathol Commun 2014 Jul 17;2:74. Epub 2014 Jul 17.

Current consensus identifies four molecular subtypes of medulloblastoma (MB): WNT, sonic hedgehog (SHH), and groups "3/C" and "4/D". Group 4 is not well characterized, but harbors the most frequently observed chromosomal abnormality in MB, i17q, whose presence may confer a worse outcome. Recent publications have identified mutations in chromatin remodeling genes that may be overrepresented in this group, suggesting a biological role for these genes in i17q. This work seeks to explore the pathology that underlies i17q in MB. Specifically, we examine the prognostic significance of the previously-identified gene mutations in an independent set of MBs as well as to examine biological relevance of these genes and related pathways by gene expression profiling. The previously-implicated p53 signaling pathway is also examined as a putative driver of i17q tumor oncogenesis. The data show gene mutations associated with i17q tumors in previous studies (KMD6A, ZMYM3, MLL3 and GPS2) were correlated with significantly worse outcomes despite not being specific to i17q in this set. Expression of these genes did not appear to underlie the biology of the molecular variants. TP53 expression was significantly reduced in i17q/group 4 tumors; this could not be accounted for by dosage effects alone. Expression of regulators and mediators of p53 signaling were significantly altered in i17q tumors. Our findings support that chromatin remodeling gene mutations are associated with significantly worse outcomes in MB but cannot explain outcomes or pathogenesis of i17q tumors. However, expression analyses of the p53 signaling pathway shows alterations in i17q tumors that cannot be explained by dosage effects and is strongly suggestive of an oncogenic role.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/s40478-014-0074-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4149211PMC
July 2014

Transcription factor regulation and chromosome dynamics during pseudohyphal growth.

Mol Biol Cell 2014 Sep 9;25(17):2669-76. Epub 2014 Jul 9.

Department of Genetics and Center for Genome Sciences and Systems Biology, Washington University School of Medicine, St. Louis, MO 63108

Pseudohyphal growth is a developmental pathway seen in some strains of yeast in which cells form multicellular filaments in response to environmental stresses. We used multiplexed transposon "Calling Cards" to record the genome-wide binding patterns of 28 transcription factors (TFs) in nitrogen-starved yeast. We identified TF targets relevant for pseudohyphal growth, producing a detailed map of its regulatory network. Using tools from graph theory, we identified 14 TFs that lie at the center of this network, including Flo8, Mss11, and Mfg1, which bind as a complex. Surprisingly, the DNA-binding preferences for these key TFs were unknown. Using Calling Card data, we predicted the in vivo DNA-binding motif for the Flo8-Mss11-Mfg1 complex and validated it using a reporter assay. We found that this complex binds several important targets, including FLO11, at both their promoter and termination sequences. We demonstrated that this binding pattern is the result of DNA looping, which regulates the transcription of these targets and is stabilized by an interaction with the nuclear pore complex. This looping provides yeast cells with a transcriptional memory, enabling them more rapidly to execute the filamentous growth program when nitrogen starved if they had been previously exposed to this condition.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1091/mbc.E14-04-0871DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4148256PMC
September 2014

Synonymous ABCA3 variants do not increase risk for neonatal respiratory distress syndrome.

J Pediatr 2014 Jun 20;164(6):1316-21.e3. Epub 2014 Mar 20.

Edward Mallinckrodt Department of Pediatrics, Washington University School of Medicine, St. Louis, MO.

Objective: To determine whether synonymous variants in the adenosine triphosphate-binding cassette A3 transporter (ABCA3) gene increase the risk for neonatal respiratory distress syndrome (RDS) in term and late preterm infants of European and African descent.

Study Design: Using next-generation pooled sequencing of race-stratified DNA samples from infants of European and African descent at ≥34 weeks gestation with and without RDS (n = 503), we scanned all exons of ABCA3, validated each synonymous variant with an independent genotyping platform, and evaluated race-stratified disease risk associated with common synonymous variants and collapsed frequencies of rare synonymous variants.

Results: The synonymous ABCA3 variant frequency spectrum differs between infants of European descent and those of African descent. Using in silico prediction programs and statistical strategies, we found no potentially disruptive synonymous ABCA3 variants or evidence of selection pressure. Individual common synonymous variants and collapsed frequencies of rare synonymous variants did not increase disease risk in term and late-preterm infants of European or African descent.

Conclusion: In contrast to rare, nonsynonymous ABCA3 mutations, synonymous ABCA3 variants do not increase the risk for neonatal RDS among term and late-preterm infants of European or African descent.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.jpeds.2014.02.021DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4035386PMC
June 2014

Clinical genomicist workstation.

AMIA Jt Summits Transl Sci Proc 2013 18;2013:156-7. Epub 2013 Mar 18.

Dept. of Pathology and Immunology, Washington University in St. Louis, St. Louis, MO;

The use of NextGen Sequencing clinically necessitates the need for informatics tools that support the complete workflow from sample accessioning to data analysis and reporting. To address this need we have developed Clinical Genomicist Workstation (CGW). CGW is a secure, n-tiered application where web browser submits requests to application servers that persist the data in a relational database. CGW is used by Washington University Genomic and Pathology Services for clinical genomic testing of many cancers. CGW has been used to accession, analyze and sign out over 409 cases since November, 2011. There are 22 ordering oncologists and 7 clinical genomicists that use the CGW. In summary, CGW a 'soup-to-nuts' solution to track, analyze, interpret, and report clinical genomic diagnostic tests.
View Article and Find Full Text PDF

Download full-text PDF

Source
December 2013
-->