Genomic View Research Articles

Genome-wide off-target analyses of CRISPR/Cas9-mediated T-cell receptor engineering in primary human T cells.

ObjectivesExploiting the forces of human T cells for treatment has led to the current paradigm of emerging immunotherapy strategies. Genetic engineering of the T‐cell receptor (TCR) redirects specificity, ablates alloreactivity and brings significant progress and off‐the‐shelf options to emerging adoptive T‐cell transfer (ACT) approaches. Targeted CRISPR/Cas9‐mediated double‐strand breaks in the DNA enable knockout or knock‐in engineering.MethodsHere, we perform CRISPR/Cas9‐mediated TCR knockout using a therapeutically relevant ribonucleoprotein (RNP) delivery method to assess the safety of genetically engineered T‐cell products. Whole‐genome sequencing was performed to analyse whether CRISPR/Cas9‐mediated DNA double‐strand break at the TCR locus is associated with off‐target events in human primary T cells.ResultsTCRα chain and TCRβ chain knockout leads to high on‐target InDel frequency and functional knockout. None of the predicted off‐target sites could be confirmed experimentally, whereas whole‐genome sequencing and manual Integrative Genomics Viewer (IGV) review revealed 9 potential low‐frequency off‐target events genome‐wide. Subsequent amplification and targeted deep sequencing in 7 of 7 evaluable loci did not confirm these low‐frequency InDels. Therefore, off‐target events are unlikely to be caused by the CRISPR/Cas9 engineering.ConclusionThe combinatorial approach of whole‐genome sequencing and targeted deep sequencing confirmed highly specific genetic engineering using CRISPR/Cas9‐mediated TCR knockout without potentially harmful exonic off‐target effects.

The landscape of plant genomics after 20 years

Many letters of the plant genetic code have been read since the first plant genome was published in 2000 for Arabidopsis. In a recent paper, Marks et al. examined 798 plant genomes and provide a current view of plant genomics in terms of the quality of the assemblies, coupled with their taxonomical and geographical distribution.

Representation and participation across 20 years of plant genome sequencing

The field of plant genome sequencing has grown rapidly in the past 20 years, leading to increases in the quantity and quality of publicly available genomic resources. The growing wealth of genomic data from an increasingly diverse set of taxa provides unprecedented potential to better understand the genome biology and evolution of land plants. Here we provide a contemporary view of land plant genomics, including analyses on assembly quality, taxonomic distribution of sequenced species and national participation. We show that assembly quality has increased dramatically in recent years, that substantial taxonomic gaps exist and that the field has been dominated by affluent nations in the Global North and China, despite a wide geographic distribution of study species. We identify numerous disconnects between the native range of focal species and the national affiliation of the researchers studying them, which we argue are rooted in colonialism—both past and present. Luckily, falling sequencing costs, widening availability of analytical tools and an increasingly connected scientific community provide key opportunities to improve existing assemblies, fill sampling gaps and empower a more global plant genomics community.

Individual Oligogenic Background in p.D91A-SOD1 Amyotrophic Lateral Sclerosis Patients.

The p.D91A is one of the most common ALS-causing SOD1 mutations and is known to be either recessive or dominant. The homozygous phenotype is characterized by prolonged survival and slow progression of disease, whereas the affected heterozygous phenotypes can vary. To date, no genetic protective factors located close to SOD1 have been associated with the mild progressive homozygous phenotype. Using Next Generation Sequencing (NGS), we characterized a small cohort of sporadic and familial p.D91A-SOD1 heterozygous (n = 2) or homozygous (n = 5) ALS patients, to reveal any additional contributing variant in 39 ALS-related genes. We detected unique sets of non-synonymous variants, four of which were of uncertain significance and several in untranslated regions of ALS-related genes. Our results supported an individual oligogenic background underlying both sporadic and familial p.D91A cases irrespective of their p.D91A mutant alleles. We suggest that a comprehensive genomic view of p.D91A-SOD1 ALS patients may be useful in identifying emerging variants and improving disease diagnosis as well as guiding precision medicine.

ProChIPdb: a chromatin immunoprecipitation database for prokaryotic organisms.

The transcriptional regulatory network in prokaryotes controls global gene expression mostly through transcription factors (TFs), which are DNA-binding proteins. Chromatin immunoprecipitation (ChIP) with DNA sequencing methods can identify TF binding sites across the genome, providing a bottom-up, mechanistic understanding of how gene expression is regulated. ChIP provides indispensable evidence toward the goal of acquiring a comprehensive understanding of cellular adaptation and regulation, including condition-specificity. ChIP-derived data's importance and labor-intensiveness motivate its broad dissemination and reuse, which is currently an unmet need in the prokaryotic domain. To fill this gap, we present proChIPdb (prochipdb.org), an information-rich, interactive web database. This website collects public ChIP-seq/-exo data across several prokaryotes and presents them in dashboards that include curated binding sites, nucleotide-resolution genome viewers, and summary plots such as motif enrichment sequence logos. Users can search for TFs of interest or their target genes, download all data, dashboards, and visuals, and follow external links to understand regulons through biological databases and the literature. This initial release of proChIPdb covers diverse organisms, including most major TFs of Escherichia coli, and can be expanded to support regulon discovery across the prokaryotic domain.

Splicing Factor SRSF1 Promotes Tumorigenesis Via Oncogenic Splice-Switching and Predicts Poor Prognosis in Multiple Myeloma

ObjectiveDysregulation of alternative splicing (AS) triggers many tumors, yet its contribution to multiple myeloma (MM) has not been elucidated. Our study aims to reveal the role of AS in MM progression, and to explore the molecular mechanisms of the candidate splicing factor (SF), thus valuates its feasibility as a treatment target and prognostic factor.Design and results 1.Gene set enrichment analysis (GSEA) revealed a significant difference in the expression pattern of the alternative splicing pathway genes, notably enriched in MM patients (Figure 1A). And, the splicing factor SRFS1 expression showed concordant differential expression in MM versus normal donors in two datasets (GSE6477, GSE13591). Kaplan-Meier analysis showed that high SRSF1 expression in CD138 + MM cells was associated with decreased PFS and poor OS (GSE9782) (Figure 1B). Moreover, the SRSF1 levels appeared a progressive increase in the progression of plasma cell dyscrasias (MGUS, SMM, MM and PCL) (Figure 1C). A cohort of 57 newly diagnosed MM patients was uniformly treated and followed. Univariate and multivariate analyses indicated that high SRSF1 expression was correlated with decreased PFS and OS. The c-indices of the Cox model fit with the combination of RISS and SRSF1 were higher than those of models fit without SRSF1, suggesting that SRSF1 improved the prognostic stratification of patients with MM.2.In vitro, SRSF1 downregulation in MM cells resulted in decreased proliferation, increased apoptosis and cell cycle arrest in G1 phase. In orthotopic MM xenograft models, bioluminescent imaging monitoring revealed a decreased burden of disease and a prolongation in overall survival in mice injected with SRSF1-shRNA cells versus the control (Figure 1D-F).3.Pearson correlation analysis suggested that the expression of TF YY1 was positively correlated with SRSF1 in five sets of GEO (Figure 1G). CHIP-seq BigWig files were downloaded from GSE31477 and Integrative Genomics Viewer (IGV) tracks displayed the binding site of YY1 at SRSF1 promoter locus from chr17: 56084421 to 56084751 (Figure 1H). CHIP-qPCR assay confirmed that endogenous YY1 directly interacted with the core element of the SRSF1 promoter in myeloma cells. Silencing of YY1 expression decreased the luciferase activity of the reporter construct carrying wild-type SRSF1 promoter region but not mutant in the YY1 binding site. Consistent with the effects of SRSF1, downregulation YY1 displayed anticancer activity in myeloma cells. Kaplan-Meier analysis showed that high YY1 expression in CD138 + myeloma cells was associated with decreased PFS and poor OS.4.RNA-seq analysis revealed a total of 7678 SRSF1-regulated AS events in LP-1 cell with an FDR cut-off of <0.05. Among them, the index of percent spliced in percentage-spliced-in (PSI) was upregulated in 4098 AS events and downregulated in 3580 AS via the downregulation of SRSF1. Changes in alternative splicing events were analyzed by replicate multivariate analysis of transcript splicing (rMATS) to identify downstream key genes regulated by SRSF1. Various types of AS events, including skipped exons (SEs), alternative 5‘ ss exons (A5SSs), alternative 3‘ ss exons (A3SSs), retained introns (RIs), and mutually exclusive exons (MXEs), can be regulated by SRSF1, and of these, SE is the most common type of SRSF1-regulated AS events (Figure 1I-O). The KEGG Pathway and GO analysis revealed that SRSF1-regulated AS events were involved in spliceosome, cell cycle and cellular process. Among the most significant AS events, we noted RBBP6, which isoforms have been reported to exhibit opposite effect on oncogenic activity.5.RNA-seq and subsequent validation indicated that SRSF1 knockdown caused a significantly decreased iso1 and increased iso3 of RBBP6. An opposite effect on myeloma proliferation between iso1 and iso3 was observed upon overexpression of individual proteins. Moreover, the level of RBBP6 iso1 is associated with poor prognosis, whereas iso3 expression is associated with favorable prognosis in MM patientsConclusionOur study uncovered that SRSF1-regulated aberrant AS facilitated myeloma progression through creating reducing normal isoforms and increasing oncogenic isoforms (Figure 1P). Moreover, SRSF1 and its critical splicing target could act as an independent prognostic factor for MM patients. Thus, SRSF1 might be considered as a valuable therapeutic target and a potential prognostic biomarker for MM. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Detection of Mosaic Variants in Mothers of MPS II Patients by Next Generation Sequencing.

Mucopolysaccharidosis type II is an X-linked lysosomal storage disorder caused by mutations in the IDS gene that encodes the iduronate-2-sulfatase enzyme. The IDS gene is located on the long arm of the X-chromosome, comprising 9 exons, spanning approximately 24 kb. The analysis of carriers, in addition to detecting mutations in patients, is essential for genetic counseling, since the risk of recurrence for male children is 50%. Mosaicism is a well-known phenomenon described in many genetic disorders caused by a variety of mechanisms that occur when a mutation arises in the early development of an embryo. Sanger sequencing is limited in detecting somatic mosaicism and sequence change levels of less than 20% may be missed. The Next Generation Sequencing (NGS) has been increasingly used in diagnosis. It is a sensitive and fast method for the detection of somatic mosaicism. Compared to Sanger sequencing, which represents a cumulative signal, NGS technology analyzes the sequence of each DNA read in a sample. NGS might therefore facilitate the detection of mosaicism in mothers of MPS II patients. The aim of this study was to reanalyze, by NGS, all MPS II mothers that showed to be non-carriers by Sanger analysis. Twelve non-carriers were selected for the reanalysis on the Ion PGM and Ion Torrent S5 platform, using a custom panel that includes the IDS gene. Results were visualized in the Integrative Genomics Viewer (IGV). We were able to detected the presence of the variant previously found in the index case in three of the mothers, with frequencies ranging between 13 and 49% of the reads. These results suggest the possibility of mosaicism in the mothers. The use of a more sensitive technology for detecting low-level mosaic mutations is essential for accurate recurrence-risk estimates. In our study, the NGS analysis showed to be an effective methodology to detect the mosaic event.

Whole-Exome Sequencing in Multiplex Families to Identify Novel AYA Classical Hodgkin Lymphoma Predisposition Genes

Background:Hodgkin Lymphoma (HL) is a B-cell malignancy that mainly affects young adults in economically developed countries. Classical HL (cHL) is the most common type, comprising >95% of cases. We demonstrated strong heritability of cHL in adolescents and young adults (AYA) with a high risk to the unaffected co-twins of affected monozygotic (MZ) twins. We and others identified common risk variants by GWAS, and sequencing of familial and sporadic HL patients previously identified rare pathogenic germline variants in genes with varying functions. Few have replicated in other families. A greater understanding of the genes involved in cHL predisposition is needed. We performed germline whole-exome sequencing (WES) for 48 individuals in 14 multiplex families with AYA cHL patients to identify novel cHL predisposition genes.Methods:Index cases from multiplex families were ascertained from the USC twin registries (International Twin Registry or California Twin Program, 7 index cases, 2 additional cases, and 7 unaffected relatives) or from the USC Cancer Surveillance Program (7 index cases, 4 additional cases, and 21 unaffected relatives). Among the twin pairs, 5 were concordant MZ pairs, one was a concordant dizygotic pair and one was an MZ discordant pair with a second case occurring in the daughter of the unaffected MZ co-twin. The other 7 families included 4 with two affected siblings, one with a child/father pair, and two with child/uncle pairs; the latter 3 families had specimens available for the index child case only. All index cases were diagnosed under 50 years old, with the median age 27.DNA samples were extracted from blood or saliva of 48 individuals in 14 multiplex families, with specimens from 20 of 28 reported cases. Germline WES was performed using the Nextera® Rapid Capture Exome kit, with data pre-processing performed based on Genome Analysis Toolkit best practices guidelines, and additional filtering applied using BCFtools. Variants were annotated using ANNOVAR. We filtered out variants with an allele frequency >0.001 in population databases (gnomAD or TOPMed), and variants documented as benign/likely benign in ClinVar. We retained splicing/ncRNA variants and exonic variants with loss-of-function/missense/unknown functional consequences, and with a CADD score >10. We further limited variants to those in 1,073 candidate predisposition genes, including cancer/immunodeficiency/hematological-related genes in the Pediatric Cancer Variant Pathogenicity Information Exchange (PeCanPIE) and genes previously identified in sequencing studies or GWAS for cHL. Variants in candidate genes were visually inspected in the Integrative Genomics Viewer and were analyzed for their potential pathogenicity through the PeCanPIE MedalCeremony pipeline. Variants predicted to be tolerated by PeCanPIE were removed. Variants found only in HL patients and not in their sibling controls were considered to be potentially causal.Results:In 45 subjects that passed sequencing QC (3 controls were removed due to low mean coverage) and following variant filtering, there were 33 variants in 33 genes found only in cHL patients but not in their sibling controls, among 9 of 14 families. None of the variants appeared in more than one family.One variant in PGK1 was previously reported as pathogenic/likely pathogenic in ClinVar in patients with phosphoglycerate kinase 1 deficiency associated with hemolytic anemia. Nine variants were reported in ClinVar as variants of uncertain significance, of which 3 were previously reported in patients with immunodeficiency disorders (in genes IL2RA, MALT1, and PRKDC) and 2 were reported in patients with dyskeratosis congenita (TERT) and other anemia-associated disorders (EPB42). Of 22 variants not reported in ClinVar, one was a missense variant in NPAT, a gene previously associated with familial lymphoma; 4 were in genes associated with immune disorders (PLCG2, LIG1, C1QB, and NOD2) and 3 were in genes associated with anemia (SPTB, ALDOA, and GATA1). To our knowledge, none of these genes other than NPAT have previously been implicated in predisposition to HL.Conclusions:WES in 20 AYA cHL patients among multiplex families identified putative novel predisposition genes for HL, including genes implicated in anemia and immune function. Assessment of these genes in sequencing studies of independent HL families is required to understand their role in HL predisposition. DisclosuresWadé: Flatiron Health: Current Employment; Roche: Current equity holder in publicly-traded company.

Germline DDX41 mutations cause ineffective hematopoiesis and myelodysplasia.

DDX41 mutations are the most common germline alterations in adult myelodysplastic syndromes (MDSs). The majority of affected individuals harbor germline monoallelic frameshift DDX41 mutations and subsequently acquire somatic mutations in their other DDX41 allele, typically missense R525H. Hematopoietic progenitor cells (HPCs) with biallelic frameshift and R525H mutations undergo cell cycle arrest and apoptosis, causing bone marrow failure in mice. Mechanistically, DDX41 is essential for small nucleolar RNA (snoRNA) processing, ribosome assembly, and protein synthesis. Although monoallelic DDX41 mutations do not affect hematopoiesis in young mice, a subset of aged mice develops features of MDS. Biallelic mutations in DDX41 are observed at a low frequency in non-dominant hematopoietic stem cell clones in bone marrow (BM) from individuals with MDS. Mice chimeric for monoallelic DDX41 mutant BM cells and a minor population of biallelic mutant BM cells develop hematopoietic defects at a younger age, suggesting that biallelic DDX41 mutant cells are disease modifying in the context of monoallelic DDX41 mutant BM.

P-058: The dynamics of nucleotide variants in the progression from myeloma precursor conditions to multiple myeloma using targeted sequencing of serial bone marrow samples

<h3>Background</h3> Multiple myeloma (MM) is known to evolve from the premalignant precursor conditions Monoclonal Gammopathy of Undetermined Significance (MGUS) and Smoldering Multiple Myeloma (SMM). The exact mechanisms driving this progression are still not completely understood. The continuum between MGUS-SMM-MM provides the opportunity to investigate its evolution. However, collecting paired/serial samples from low and intermediate risk precursors progressing to MM are significantly hindered by the (i) only incidental diagnosis of asymptomatic precursors, (ii) invasive bone marrow (BM) sampling, (iii) very low number of aberrant BM plasma cells (PCs), and (iv) rather irregular disease follow-up. <h3>Methods</h3> In this study, a unique retrospective collection of paired BM samples was available because of an effective biobanking effort. We had access to 68 archival diagnostic May-Grünwald–Giemsa (MGG)-stained BM smears from 21 progressing low to intermediate risk myeloma precursor patients, 19 MGUS and 2 SMM, with a median time to progression of 6 years. DNA was extracted from these BM smears and Next Generation Sequencing (NGS) was performed using a custom targeted capture-based sequencing panel (Illumina) including coding exons or hotspots of 81 selected myeloma-related genes with the aim to study the evolution of single nucleotide variants (SNVs) and short insertions and deletions (indels). The pooled libraries were paired-end sequenced on a MiSeq instrument (Illumina). <h3>Results</h3> Data was analyzed with Local Run Manager and annotated with VariantStudio. After filtering, only exonic nonsynonymous and loss-of-function variants with a coverage >30 and a variant allele frequency (AF) >1% were retrieved. A variant detected in the MM phase but not in the prestage of that patient was manually inspected in the data visualization tool of Integrated Genome Viewer (IGV) to assess its presence. With a mean coverage of 636x of the targeted captured region the sequencing depth was sufficiently high to detect low burden variants down to an AF of 1%. A median of 2 variants per patient (range 0 to 5 with a total of 38 variants) was detected. While in 19 patients at least one variant was detected in the MM phase, in 2 patients not a single variant was detected in any of the 81 genes. Interestingly, the majority of variants in MM could already be detected at low AFs in the BM smears sampled in the precursor phase, even many years before progression. The reason why some variants were not detected in a prestage is likely due to sensitivity issues (BM PCs ≤2.5%). The median time of detection of a variant in the precursor stage BM was 49 months (>4 years; range 10 to 105 months) prior to MM progression. <h3>Conclusion</h3> In conclusion, targeted sequencing of unsorted BM smears from myeloma precursor conditions can already provide relevant insights into the behavior of mutant clones. Paired sample analysis can reveal the genetic architecture of somatic variation in the prestage even with low proliferative PCs in the BM.

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

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

Two distinct skeletal muscle microRNA signatures revealing the complex mechanism of sporadic ALS

Skeletal muscle pathology is thought to have an important role in the onset and/or progression of amyotrophic lateral sclerosis (ALS), which is a neurodegenerative disorder characterized by progressive muscle weakness. Since miRNAs are recognized as important regulatory factors of essential biological processes, we aimed to identify differentially expressed miRNAs in the skeletal muscle of sporadic ALS patients through the combination of molecular-omic technologies and bioinformatic tools. We analyzed the miRnome profiles of skeletal muscle biopsies acquired from ten sALS patients and five controls with Affymetrix GeneChip miRNA 4.0 Array. To find out differentially expressed miRNAs in patients, data were analyzed by The Institute for Genomic Research-Multi Experiment Viewer (MeV) and miRNAs whose expression difference were statistically significant were identified as candidates. The potential target genes of these miRNAs were predicted by miRWalk 2.0 and were functionally enriched by gene ontology (GO) analysis. The expression level of priority candidates was validated by quantitative real-time PCR (qRT-PCR) analysis. We identified ten differentially expressed miRNAs in patients with a fold change threshold ≥ 2.0, FDR = 0. We identified ten differentially expressed miRNAs in patients with a fold change threshold ≥ 2.0, FDR = 0. Nine out of the ten miRNAs were found to be related to top three enriched ALS-related terms. Based on the qRT-PCR validation of candidate miRNAs, patients were separated into two groups: those with upregulated miR-4429 and miR-1825 expression and those with downregulated miR-638 expression. The different muscle-specific miRNA profiles in sALS patients may indicate the involvement of etiologic heterogeneity, which may allow the development of novel therapeutic strategies.

A Novel Mutant Wasp in X-Linked Neutropenia Partially Recapitulates Defective Neutrophil Production through Its Expression in WASP-Deficient NB4 Cells

BackgroundSevere congenital neutropenia (SCN) is characterized by prolonged severe neutropenia (absolute neutrophil count < 500/µL) commencing in early infancy, and recurrent bacterial and/or fungal infections. X-linked neutropenia (XLN) is a very rare type of SCN caused by a gain-of-function mutation in the Wiskott-Aldrich syndrome gene (WAS) located on the short arm of the X chromosome. To the best of our knowledge, only three WAS protein (WASp) mutations (L270P, S272P, and I294T) have been described to date. Herein, we report an identification and functional analysis of novel WASP mutation (I290T), which was identified in the first Asian XLN case by phenotype-based gene analysis of next-generation sequencing (NGS) data.CasePatient was 29-year-old male who had been clinically diagnosed with SCN at 6 years of age, however the cause of SCN remained unknown. We performed targeted exome sequencing of 4813 disease-relevant genes using a TruSight One Sequencing Panel (Illumina, San Diego, CA). The sequencing data were analyzed in terms of somatic mutations using our in-house pipeline, “Genomon” (http://genomon.hgc.jp/exome/en/). The resulting gene list, including 925 single nucleotide variants (SNVs), was subjected to examination by Phenolyzer (http://phenolyzer.usc.edu/), a web-based computational tool that analyzes clinical phenotypes on the basis of free-text term(s) and predicts the most likely candidate gene(s) by integrating information from multiple databases. The terms entered were ‘Congenital neutropenia’, ‘Familial’, ‘Myelodysplastic syndrome’, and ‘Autosomal’. Phenolyzer prioritized the WAS gene as the most probable candidate. Additionally, we identified a missense mutation in exon 9 of the patient's WAS gene (c.T869C, p.I290T) using the Integrative Genomics Viewer (IGV).Materials and MethodsTo investigate the molecular mechanisms of WASp-I290T mutation in XLN, we cloned WASp to a human cell line that can differentiate to neutrophils (NB4). Endogenous WASp in NB4 cells was fully knocked out by utilizing CRISPR/Cas9 system. We constructed a lentiviral AcGFP-tagged WASp overexpression plasmid controlled by CMV promoter, and AcGFP-tagged WASp was induced to NB4 cells. Cell viability evaluation, immunofluorescence imaging, and gene expression analysis were performed to compare NB4 cells treated with/without ATRA harboring wild type and mutant WASp.ResultsWestern blot analysis of AcGFP-WASp clearly showed a molecular mass of 84 kD for both wild type and mutant proteins. The viable cell growth assay showed no significant differences between mutant and wild type WASp-transduced NB4ΔWASPcells (NB4WASp-WT and NB4WASp-I290T cells, respectively) (Figure 1A). In contrast, upon ATRA-induced neutrophilic differentiation, the viable cell number significantly decreased in NB4WASp-I290T cells after 72 hours, compared to NB4, NB4ΔWASP and NB4WASp-WT cells. This result was further confirmed by FACS analysis, whereas CD11b expression was induced in surviving NB4WASp-I290T cells, comparably to other sublines of NB4 cells. Confocal laser microscopy revealed that WASp-I290T preferentially localized in nucleus rather than cytoplasm, compared to wild-type WASP, which was compatible with the previous findings reported for other WASp mutants in XLN. Expression of myeloid differentiation-related genes including C/EBP-alpha, -beta, -epsilon, CSF3R and PU.1 was examined at regular intervals by quantitative RT-PCR, indicating that C/EBP-epsilon transcripts were upregulated in WASp-I290T cells to higher extent than other sublines of NB4 cells after 24 hours of ATRA treatment, and that this difference was more prominent after 72 hours (Figure 1B).DiscussionIn this study, a novel WASp-I290T mutant identified in an adult XLN patient partially recapitulated defective neutrophil production through its expression in ATRA-treated NB4 cells lacking endogenous WASp. Our results suggest that premature upregulation of C/EBP-epsilon may be involved in impaired growth of ATRA-treated NB4WASp-I290T cells. A number of reports suggest the putative role of nuclear WASp in transcriptional regulation. Considering the preferential nuclear localization of WASp-I290T, further investigation of its interaction with nuclear proteins such as histone modifying enzymes is required. [Display omitted] DisclosuresTojo:AMED grant: Research Funding.

Gene-Specific Lineage Involvement of Age-Related Clonal Hematopoiesis

INTRODUCTIONAge-related clonal hematopoiesis (ARCH), which was initially suggested by X-chromosome inactivation (XCI) studies, is associated with acquired mutations in driver and non-driver genes. ARCH is highly prevalent after the age of 65, and infers an increased risk of hematological cancers and cardiovascular complications. Quantification of these risks in individual subjects is not possible at this time, leading to the creation of a clinical entity named Clonal Hematopoiesis of Indeterminate Potential, or CHIP (Steensma et al., Blood 2015). There is a need to determine factors that modulate the oncogenic penetrance of these mutations. Identifying the origin within the hematopoietic stem cell (HSC) hierarchy of the mutations responsible for ARCH is a crucial initial step. We recently documented that 93% of driver mutations in ARCH occurred in DNMT3A or TET2 (Buscarlet et al., Blood. 2017). We now report the comparative hematopoietic lineage involvement of these two genes in a cohort of 86 individuals with ARCH.METHODSWe have previously identified driver mutations in polymorphonuclear cells (PMN) of 347 individuals from a cohort of 2350 individuals. We have selected 86 individuals on the basis of having a single DNMT3A or TET2 mutation and the availability of DNA from PMN (granulocytes), CD14+ (monocytes), CD19+ (B-cells) and CD3+ (T-cells) allowing lineage involvement analysis. The lineage specific cells were obtained by Fluorescence-activated cell sorting (FACS) from the mononuclear cell fraction of peripheral blood samples. All samples were sequenced at high coverage (95% > 500x) on an Ion Proton sequencing using the AML Ampliseq panel. All mutations were visually inspected on Integrative Genomics Viewer (IGV). Samples were considered mutated if the mutation was detected above 2% VAF (variant allele frequency).RESULTSThe study cohort comprised 86 individuals from 55 to 96 years-old (mean age: 69.0). Sixty two (62) had a single DNMT3A mutation (mean VAF in PMN = 11.2%) and 24 had a single TET2 mutation (mean VAF in PMN = 10.5%). TET2 mutated individuals where older than those with DNMT3A mutations (72.3 vs 67.7, P < .05). From the 62 DNMT3A mutations initially detected in PMN, 32.3% were present in all cell types analysed (PMN, CD14+, CD19+ and CD3+), 37.1% were detected in PMN, CD14+ and CD19+ cells, and 21.0% were found only in PMN and CD14+ cells. The remaining mutations were detected in PMN, CD14+ and CD3+ cells (4.8%), PMN and CD19+ cells (4.8%), or only in PMN (3.2%). For TET2, 45.8% of the mutations were detected in PMN and CD14+ cells, 45.8% were found in PMN, CD14+ and CD19+ cells, and 8.3% only detected in PMN. No mutations were detected in CD3+ cells. The different lineage involvements were statistically different between DNMT3A and TET2 for myeloid restriction (PMN or PMN and CD14+ cells): 21% (13/62) vs 54.2%, (13/24), P < .05; and for mutation in CD3+ cells: 37% (23/62) vs 0% (0/24), P < .001. Although the proportion of subjects with myeloid and B-cell involvement was similar between the two genes, the CD14+/CD19+ VAF ratio was significantly higher for TET2 (3.13, n = 11) than for DNMT3A (1.94, n = 43) (P < .005). Selecting subjects who had a VAF > 10% in PMN (suggesting a longer evolution) had no impact on the myeloid lineage restriction for TET2, but almost all (21/22) DNMT3A samples showed mutations in both myeloid and lymphoid compartments.CONCLUSIONThese results demonstrate a significantly different lineage involvement between DNMT3A and TET2 mutations in ARCH. DNMT3A mutations predominantly affect both the myeloid and lymphoid compartment, including T-cells in a third of subjects. This strongly suggests that DNMT3A mutations occur in a HSC with multipotent proliferative potential. In contrast, we never documented TET2 mutations in T-cells, suggesting the involvement of more committed HSC. This is paradoxical since both DNMT3A and TET2 mutations have been documented in T-cell lymphoma. This could be explained by a different HSC origin for TET2 mutation in myeloid malignancies vs T-cell lymphomas. Alternatively, it could be that in normal aging individuals, the multipotent mutated HSC has a stronger myeloid proliferation bias than DNMT3A . This bias may be in response to homeostatic stimuli as we have documented a slight neutropenia in subjects with ARCH and TET2 mutations (Buscarlet et al., Blood 2017). [Display omitted] DisclosuresBusque:Bristol Myer Squibb: Honoraria; Novartis Canada Inc.: Honoraria; Paladin: Honoraria; Pfizer: Honoraria.

Genome-Wide Analysis of MYB Gene Family in Chinese Bayberry (Morella rubra) and Identification of Members Regulating Flavonoid Biosynthesis.

Chinese bayberry (Morella rubra), the most economically important fruit tree in the Myricaceae family, is a rich source of natural flavonoids. Recently the Chinese bayberry genome has been sequenced, and this provides an opportunity to investigate the organization and evolutionary characteristics of MrMYB genes from a whole genome view. In the present study, we performed the genome-wide analysis of MYB genes in Chinese bayberry and identified 174 MrMYB transcription factors (TFs), including 122 R2R3-MYBs, 43 1R-MYBs, two 3R-MYBs, one 4R-MYB, and six atypical MYBs. Collinearity analysis indicated that both syntenic and tandem duplications contributed to expansion of the MrMYB gene family. Analysis of transcript levels revealed the distinct expression patterns of different MrMYB genes, and those which may play important roles in leaf and flower development. Through phylogenetic analysis and correlation analyses, nine MrMYB TFs were selected as candidates regulating flavonoid biosynthesis. By using dual-luciferase assays, MrMYB12 was shown to trans-activate the MrFLS1 promoter, and MrMYB39 and MrMYB58a trans-activated the MrLAR1 promoter. In addition, overexpression of 35S:MrMYB12 caused a significant increase in flavonol contents and induced the expression of NtCHS, NtF3H, and NtFLS in transgenic tobacco leaves and flowers and significantly reduced anthocyanin accumulation, resulting in pale-pink or pure white flowers. This indicates that MrMYB12 redirected the flux away from anthocyanin biosynthesis resulting in higher flavonol content. The present study provides valuable information for understanding the classification, gene and motif structure, evolution and predicted functions of the MrMYB gene family and identifies MYBs regulating different aspects of flavonoid biosynthesis in Chinese bayberry.

Molecular Genetic Dissection of Inflammatory Linear Verrucous Epidermal Naevus Leads to Successful Targeted Therapy

Molecular Genetic Dissection of Inflammatory Linear Verrucous Epidermal Naevus Leads to Successful Targeted Therapy

Genome-wide association study of myrtle rust (Austropuccinia psidii) resistance in Eucalyptus obliqua (subgenus Eucalyptus)

Myrtle rust (caused by Austropuccinia psidii Beenken) is exotic to Australia, yet specific resistance is present in a range of naive myrtaceous host plants. Resistance to myrtle rust is primarily quantitative in nature and controlled by multiple interacting loci. We undertook a genome-wide association study (GWAS) to discover relationships between nuclear genomic sequence variation and multiple aspects of resistance to A. psidii in Eucalyptus obliqua, a representative species from subgenus Eucalyptus. Sequence variation was assessed with respect to numeric severity, binary symptomatic, hypersensitive, and pustulation responses to inoculation of seedlings with A. psidii. A total of 1.13 million, single-nucleotide polymorphisms (SNPs) were tested for association with the rust resistance responses of 637 phenotyped E. obliqua seedlings, each from different, single mother tree seedlots. Thirty-three highly significant SNP-trait associations were detected, of which 26 associated with the binary symptomatic/asymptomatic response to A. psidii. Comparison of the genomic position of these SNPs with rust resistance loci (Ppr1-5), previously reported in species from subgenus Symphyomyrtus, suggested that several were positioned near the major Ppr1 locus and other Ppr loci. This study provides the first integrated genomic view of A. psidii resistance across the Eucalyptus subgenera and provides the foundation for discovering key resistance genes for use in marker-based resistance breeding.

Identification of Genomic Islands in Synechococcus sp. WH8102 Using Genomic Barcode and Whole-Genome Microarray Analysis

Background: Synechococcus sp. WH8102 is one of the most abundant photosynthetic organisms in many ocean regions. Objective: The aim of this study is to identify genomic islands (GIs) in Synechococcus sp. WH8102 with integrated methods. Methods: We have applied genomic barcode to identify the GIs in Synechococcus sp. WH8102, which could make genomic regions of different origins visually apparent. The gene expression data of the predicted GIs was analyzed through microarray data which was collected for functional analysis of the relevant genes. Results: Seven GIs were identified in Synechococcus sp. WH8102. Most of them are involved in cell surface modification, photosynthesis and drug resistance. In addition, our analysis also revealed the functions of these GIs, which could be used for in-depth study on the evolution of this strain. Conclusion: Genomic barcodes provide us with a comprehensive and intuitive view of the target genome. We can use it to understand the intrinsic characteristics of the whole genome and identify GIs or other similar elements.

Chitin Synthesis and Degradation in Crustaceans: A Genomic View and Application.

Chitin is among the most important components of the crustacean cuticular exoskeleton and intestinal peritrophic matrix. With the progress of genomics and sequencing technology, a large number of gene sequences related to chitin metabolism have been deposited in the GenBank database in recent years. Here, we summarized the genes and pathways associated with the biosynthesis and degradation of chitins in crustaceans based on genomic analyses. We found that chitin biosynthesis genes typically occur in single or two copies, whereas chitin degradation genes are all multiple copies. Moreover, the chitinase genes are significantly expanded in most crustacean genomes. The gene structure and expression pattern of these genes are similar to those of insects, albeit with some specific characteristics. Additionally, the potential applications of the chitin metabolism genes in molting regulation and immune defense, as well as industrial chitin degradation and production, are also summarized in this review.

Long-read assembly of a Great Dane genome highlights the contribution of GC-rich sequence and mobile elements to canine genomes

Technological advances have allowed improvements in genome reference sequence assemblies. Here, we combined long- and short-read sequence resources to assemble the genome of a female Great Dane dog. This assembly has improved continuity compared to the existing Boxer-derived (CanFam3.1) reference genome. Annotation of the Great Dane assembly identified 22,182 protein-coding gene models and 7,049 long noncoding RNAs, including 49 protein-coding genes not present in the CanFam3.1 reference. The Great Dane assembly spans the majority of sequence gaps in the CanFam3.1 reference and illustrates that 2,151 gaps overlap the transcription start site of a predicted protein-coding gene. Moreover, a subset of the resolved gaps, which have an 80.95% median GC content, localize to transcription start sites and recombination hotspots more often than expected by chance, suggesting the stable canine recombinational landscape has shaped genome architecture. Alignment of the Great Dane and CanFam3.1 assemblies identified 16,834 deletions and 15,621 insertions, as well as 2,665 deletions and 3,493 insertions located on secondary contigs. These structural variants are dominated by retrotransposon insertion/deletion polymorphisms and include 16,221 dimorphic canine short interspersed elements (SINECs) and 1,121 dimorphic long interspersed element-1 sequences (LINE-1_Cfs). Analysis of sequences flanking the 3' end of LINE-1_Cfs (i.e., LINE-1_Cf 3'-transductions) suggests multiple retrotransposition-competent LINE-1_Cfs segregate among dog populations. Consistent with this conclusion, we demonstrate that a canine LINE-1_Cf element with intact open reading frames can retrotranspose its own RNA and that of a SINEC_Cf consensus sequence in cultured human cells, implicating ongoing retrotransposon activity as a driver of canine genetic variation.