Concomitant diagnosis of immune deficiency and Pseudomonas sepsis in a 19 month old with ecthyma gangrenosum by host whole-genome sequencing.

X-linked agammaglobulinemia (XLA, OMIM#300300) is a rare monogenic primary immunodeficiency caused by mutations in the Bruton tyrosine kinase (BTK) gene. XLA is characterized by insufficient immunoglobulin levels and susceptibility to life-threatening bacterial infections. We report on a patient that presented with ecthyma gangrenosum and septicemia. Rapid trio whole-genome sequencing (rWGS) revealed an apparently de novo hemizygous pathogenic variant (c.726dupT; p.Ile243TyrfsTer15) in the BTK gene. Metagenomic analysis of rWGS sequences that did not align to the human genome revealed 770 aligned to the Pseudomonas aeruginosa PAO1 genome. The patient was diagnosed with XLA and pseudomonal sepsis

Identification of synthetic lethality of PRKDC in MYC-dependent human cancers by pooled shRNA screening

BACKGROUND: MYC family members are among the most frequently deregulated oncogenes in human cancers, yet direct therapeutic targeting of MYC in cancer has been challenging thus far. Synthetic lethality provides an opportunity for therapeutic intervention of MYC-driven cancers. METHODS: A pooled kinase shRNA library screen was performed and next-generation deep sequencing efforts identified that PRKDC was synthetically lethal in cells overexpressing MYC. Genes and proteins of interest were knocked down or inhibited using RNAi technology and small molecule inhibitors, respectively. Quantitative RT-PCR using TaqMan probes examined mRNA expression levels and cell viability was assessed using CellTiter-Glo (Promega). Western blotting was performed to monitor different protein levels in the presence or absence of RNAi or compound treatment. Statistical significance of differences among data sets were determined using unpaired t test (Mann-Whitney test) or ANOVA. RESULTS: Inhibition of PRKDC using RNAi (RNA interference) or small molecular inhibitors preferentially killed MYC-overexpressing human lung fibroblasts. Moreover, inducible PRKDC knockdown decreased cell viability selectively in high MYC-expressing human small cell lung cancer cell lines. At the molecular level, we found that inhibition of PRKDC downregulated MYC mRNA and protein expression in multiple cancer cell lines. In addition, we confirmed that overexpression of MYC family proteins induced DNA double-strand breaks; our results also revealed that PRKDC inhibition in these cells led to an increase in DNA damage levels. CONCLUSIONS: Our data suggest that the synthetic lethality between PRKDC and MYC may in part be due to PRKDC dependent modulation of MYC expression, as well as MYC-induced DNA damage where PRKDC plays a key role in DNA damage repair

Genome-Wide and Functional Annotation of Human E3 Ubiquitin Ligases Identifies MULAN, a Mitochondrial E3 that Regulates the Organelle's Dynamics and Signaling

Specificity of protein ubiquitylation is conferred by E3 ubiquitin (Ub) ligases. We have annotated ∼617 putative E3s and substrate-recognition subunits of E3 complexes encoded in the human genome. The limited knowledge of the function of members of the large E3 superfamily prompted us to generate genome-wide E3 cDNA and RNAi expression libraries designed for functional screening. An imaging-based screen using these libraries to identify E3s that regulate mitochondrial dynamics uncovered MULAN/FLJ12875, a RING finger protein whose ectopic expression and knockdown both interfered with mitochondrial trafficking and morphology. We found that MULAN is a mitochondrial protein – two transmembrane domains mediate its localization to the organelle's outer membrane. MULAN is oriented such that its E3-active, C-terminal RING finger is exposed to the cytosol, where it has access to other components of the Ub system. Both an intact RING finger and the correct subcellular localization were required for regulation of mitochondrial dynamics, suggesting that MULAN's downstream effectors are proteins that are either integral to, or associated with, mitochondria and that become modified with Ub. Interestingly, MULAN had previously been identified as an activator of NF-κB, thus providing a link between mitochondrial dynamics and mitochondria-to-nucleus signaling. These findings suggest the existence of a new, Ub-mediated mechanism responsible for integration of mitochondria into the cellular environment

A Systematic Map of Genetic Variation in Plasmodium falciparum

Discovering novel genes involved in immune evasion and drug resistance in the human malaria parasite, Plasmodium falciparum, is of critical importance to global health. Such knowledge may assist in the development of new effective vaccines and in the appropriate use of antimalarial drugs. By performing a full-genome scan of allelic variability in 14 field and laboratory strains of P. falciparum, we comprehensively identified ≈500 genes evolving at higher than neutral rates. The majority of the most variable genes have paralogs within the P. falciparum genome and may be subject to a different evolutionary clock than those without. The group of 211 variable genes without paralogs contains most known immunogens and a few drug targets, consistent with the idea that the human immune system and drug use is driving parasite evolution. We also reveal gene-amplification events including one surrounding pfmdr1, the P. falciparum multidrug-resistance gene, and a previously uncharacterized amplification centered around the P. falciparum GTP cyclohydrolase gene, the first enzyme in the folate biosynthesis pathway. Although GTP cyclohydrolase is not the known target of any current drugs, downstream members of the pathway are targeted by several widely used antimalarials. We speculate that an amplification of the GTP cyclohydrolase enzyme in the folate biosynthesis pathway may increase flux through this pathway and facilitate parasite resistance to antifolate drugs

Whole genome functional analysis identifies novel components required for mitotic spindle integrity in human cells

A loss-of-function screen for siRNAs that arrest human cells in metaphase reveals genes involved in mitotic spindle integrity

A Gene Wiki for Community Annotation of Gene Function

This manuscript describes the creation of comprehensive gene wiki, seeded with data from public domain sources, which will enable and encourage community annotation of gene function

Complex Loci in Human and Mouse Genomes

Mammalian genomes harbor a larger than expected number of complex loci, in which multiple genes are coupled by shared transcribed regions in antisense orientation and/or by bidirectional core promoters. To determine the incidence, functional significance, and evolutionary context of mammalian complex loci, we identified and characterized 5,248 cis–antisense pairs, 1,638 bidirectional promoters, and 1,153 chains of multiple cis–antisense and/or bidirectionally promoted pairs from 36,606 mouse transcriptional units (TUs), along with 6,141 cis–antisense pairs, 2,113 bidirectional promoters, and 1,480 chains from 42,887 human TUs. In both human and mouse, 25% of TUs resided in cis–antisense pairs, only 17% of which were conserved between the two organisms, indicating frequent species specificity of antisense gene arrangements. A sampling approach indicated that over 40% of all TUs might actually be in cis–antisense pairs, and that only a minority of these arrangements are likely to be conserved between human and mouse. Bidirectional promoters were characterized by variable transcriptional start sites and an identifiable midpoint at which overall sequence composition changed strand and the direction of transcriptional initiation switched. In microarray data covering a wide range of mouse tissues, genes in cis–antisense and bidirectionally promoted arrangement showed a higher probability of being coordinately expressed than random pairs of genes. In a case study on homeotic loci, we observed extensive transcription of nonconserved sequences on the noncoding strand, implying that the presence rather than the sequence of these transcripts is of functional importance. Complex loci are ubiquitous, host numerous nonconserved gene structures and lineage-specific exonification events, and may have a cis-regulatory impact on the member genes

Transcript Annotation in FANTOM3: Mouse Gene Catalog Based on Physical cDNAs

The international FANTOM consortium aims to produce a comprehensive picture of the mammalian transcriptome, based upon an extensive cDNA collection and functional annotation of full-length enriched cDNAs. The previous dataset, FANTOM2, comprised 60,770 full-length enriched cDNAs. Functional annotation revealed that this cDNA dataset contained only about half of the estimated number of mouse protein-coding genes, indicating that a number of cDNAs still remained to be collected and identified. To pursue the complete gene catalog that covers all predicted mouse genes, cloning and sequencing of full-length enriched cDNAs has been continued since FANTOM2. In FANTOM3, 42,031 newly isolated cDNAs were subjected to functional annotation, and the annotation of 4,347 FANTOM2 cDNAs was updated. To accomplish accurate functional annotation, we improved our automated annotation pipeline by introducing new coding sequence prediction programs and developed a Web-based annotation interface for simplifying the annotation procedures to reduce manual annotation errors. Automated coding sequence and function prediction was followed with manual curation and review by expert curators. A total of 102,801 full-length enriched mouse cDNAs were annotated. Out of 102,801 transcripts, 56,722 were functionally annotated as protein coding (including partial or truncated transcripts), providing to our knowledge the greatest current coverage of the mouse proteome by full-length cDNAs. The total number of distinct non-protein-coding transcripts increased to 34,030. The FANTOM3 annotation system, consisting of automated computational prediction, manual curation, and final expert curation, facilitated the comprehensive characterization of the mouse transcriptome, and could be applied to the transcriptomes of other species

BioGPS: an extensible and customizable portal for querying and organizing gene annotation resources

BioGPS is a community based customisable gene annotation portal bringing together gene annotation resources on to a single platform