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Although circumstantial evidence supports enhanced Toll-like receptor 7 (TLR7) signalling as a mechanism of human systemic autoimmune disease1–7, evidence of lupus-causing TLR7 gene variants is lacking. Here we describe human systemic lupus erythematosus caused by a TLR7 gain-of-function variant. TLR7 is a sensor of viral RNA8,9 and binds to guanosine10–12. We identified a de novo, previously undescribed missense TLR7Y264H variant in a child with severe lupus and additional variants in other patients with lupus. The TLR7Y264H variant selectively increased sensing of guanosine and 2',3'-cGMP10–12, and was sufficient to cause lupus when introduced into mice. We show that enhanced TLR7 signalling drives aberrant survival of B cell receptor (BCR)-activated B cells, and in a cell-intrinsic manner, accumulation of CD11c+ age-associated B cells and germinal centre B cells. Follicular and extrafollicular helper T cells were also increased but these phenotypes were cell-extrinsic. Deficiency of MyD88 (an adaptor protein downstream of TLR7) rescued autoimmunity, aberrant B cell survival, and all cellular and serological phenotypes. Despite prominent spontaneous germinal-centre formation in Tlr7Y264H mice, autoimmunity was not ameliorated by germinal-centre deficiency, suggesting an extrafollicular origin of pathogenic B cells. We establish the importance of TLR7 and guanosine-containing self-ligands for human lupus pathogenesis, which paves the way for therapeutic TLR7 or MyD88 inhibition.The missense TLR7Y264H gain-of-function genetic variation causes systemic lupus erythematosus in humans and mice. [ABSTRACT FROM AUTHOR]

Most humans harbor both CD177neg and CD177pos neutrophils but 1–10% of people are CD177null, placing them at risk for formation of anti-neutrophil antibodies that can cause transfusion-related acute lung injury and neonatal alloimmune neutropenia. By deep sequencing the CD177 locus, we catalogued CD177 single nucleotide variants and identified a novel stop codon in CD177null individuals arising from a single base substitution in exon 7. This is not a mutation in CD177 itself, rather the CD177null phenotype arises when exon 7 of CD177 is supplied entirely by the CD177 pseudogene (CD177P1), which appears to have resulted from allelic gene conversion. In CD177 expressing individuals the CD177 locus contains both CD177P1 and CD177 sequences. The proportion of CD177hi neutrophils in the blood is a heritable trait. Abundance of CD177hi neutrophils correlates with homozygosity for CD177 reference allele, while heterozygosity for ectopic CD177P1 gene conversion correlates with increased CD177neg neutrophils, in which both CD177P1 partially incorporated allele and paired intact CD177 allele are transcribed. Human neutrophil heterogeneity for CD177 expression arises by ectopic allelic conversion. Resolution of the genetic basis of CD177null phenotype identifies a method for screening for individuals at risk of CD177 isoimmunisation. [ABSTRACT FROM AUTHOR]

A diversity of tools is available for identification of variants from genome sequence data. Given the current complexity of incorporating external software into a genome analysis infrastructure, a tendency exists to rely on the results from a single tool alone. The quality of the output variant calls is highly variable however, depending on factors such as sequence library quality as well as the choice of short-read aligner, variant caller, and variant caller filtering strategy. Here we present a two-part study first using the high quality ‘genome in a bottle’ reference set to demonstrate the significant impact the choice of aligner, variant caller, and variant caller filtering strategy has on overall variant call quality and further how certain variant callers outperform others with increased sample contamination, an important consideration when analyzing sequenced cancer samples. This analysis confirms previous work showing that combining variant calls of multiple tools results in the best quality resultant variant set, for either specificity or sensitivity, depending on whether the intersection or union, of all variant calls is used respectively. Second, we analyze a melanoma cell line derived from a control lymphocyte sample to determine whether software choices affect the detection of clinically important melanoma risk-factor variants finding that only one of the three such variants is unanimously detected under all conditions. Finally, we describe a cogent strategy for implementing a clinical variant detection pipeline; a strategy that requires careful software selection, variant caller filtering optimizing, and combined variant calls in order to effectively minimize false negative variants. While implementing such features represents an increase in complexity and computation the results offer indisputable improvements in data quality. [ABSTRACT FROM AUTHOR]

Objective Systemic lupus erythematosus (SLE) is a chronic and heterogeneous autoimmune disease. Both twin and sibling studies indicate a strong genetic contribution to lupus, but in the majority of cases the pathogenic variant remains to be identified. The genetic contribution to disease is likely to be greatest in cases with early onset and severe phenotypes. Whole-exome sequencing now offers the possibility of identifying rare alleles responsible for disease in such cases. This study was undertaken to identify genetic causes of SLE using whole-exome sequencing. Methods We performed whole-exome sequencing in a 4-year-old girl with early-onset SLE and conducted biochemical analysis of the putative defect. Results Whole-exome sequencing in a 4-year-old girl with cerebral lupus identified a rare, homozygous mutation in the three prime repair exonuclease 1 gene ( TREX1) that was predicted to be highly deleterious. The TREX1 R97H mutant protein had a 20-fold reduction in exonuclease activity and was associated with an elevated interferon-α (IFNα) signature in the patient. The discovery and characterization of a pathogenic TREX1 variant in our proband has therapeutic implications. The patient is now a candidate for neutralizing anti-IFNα therapy. Conclusion Our study is the first to demonstrate that whole-exome sequencing can be used to identify rare or novel deleterious variants as genetic causes of SLE and, through a personalized approach, improve therapeutic options. [ABSTRACT FROM AUTHOR]

Forkhead box O (FoxO) transcription factors (TFs) are key drivers of complex transcriptional programmes that determine animal lifespan. FoxOs regulate a number of other TFs, but how these TFs in turn might mediate the anti-ageing programmes orchestrated by FoxOs in vivo is unclear. Here, we identify an E-twenty six (ETS)-family transcriptional repressor, Anterior open (Aop), as regulated by the single Drosophila melanogaster FoxO (dFOXO) in the adult gut. AOP, the functional orthologue of the human Etv6/Tel protein, binds numerous genomic sites also occupied by dFOXO and counteracts the activity of an ETS activator, Pointed (Pnt), to prevent the lifespan-shortening effects of co-activation of dFOXO and PNT. This detrimental synergistic effect of dFOXO and PNT appears to stem from a mis-regulation of lipid metabolism. At the same time, AOP activity in another fly organ, the fat body, has further beneficial roles, regulating genes in common with dfoxo, such as the secreted, non-sensory, odorant binding protein (Obp99b), and robustly extending lifespan. Our study reveals a complex interplay between evolutionarily conserved ETS factors and dFOXO, the functional significance of which may extend well beyond animal lifespan. [ABSTRACT FROM AUTHOR]

IgD and IgM are produced by alternative splicing of long primary RNA transcripts from the Ig heavy chain (Igh) locus and serve as the receptors for antigen on naïve mature B lymphocytes. IgM is made selectively in immature B cells, whereas IgD is coexpressed with IgM when the cells mature into follicular or marginal zone B cells, but the transacting factors responsible for this regulated change in splicing have remained elusive. Here, we use a genetic screen in mice to identify ZFP318, a nuclear protein with two U1-type zinc fingers found in RNA-binding proteins and no known role in the immune system, as a critical factor for IgD expression. A point mutation in an evolutionarily conserved lysine-rich domain encoded by the alternatively spliced Zfp318 exon 10 abolished IgD expression on marginal zone B cells, decreased IgD on follicular B cells, and increased IgM, but only slightly decreased the percentage of B cells and did not decrease expression of other maturation markers CD21, CD23, or CD62L. A targeted Zfp318 null allele extinguished IgD expression on mature B cells and increased IgM. Zfp318 mRNA is developmentally regulated in parallel with IgD, with little in pro-B cells, moderate amounts in immature B cells, and high levels selectively in mature follicular B cells. These findings identify ZFP318 as a crucial factor regulating the expression of the two major antibody isotypes on the surface of most mature B cells. [ABSTRACT FROM AUTHOR]

FoxO transcription factors, inhibited by insulin/insulin‐like growth factor signalling (IIS), are crucial players in numerous organismal processes including lifespan. Using genomic tools, we uncover over 700 direct dFOXO targets in adult female Drosophila. dFOXO is directly required for transcription of several IIS components and interacting pathways, such as TOR, in the wild‐type fly. The genomic locations occupied by dFOXO in adults are different from those observed in larvae or cultured cells. These locations remain unchanged upon activation by stresses or reduced IIS, but the binding is increased and additional targets activated upon genetic reduction in IIS. We identify the part of the IIS transcriptional response directly controlled by dFOXO and the indirect effects and show that parts of the transcriptional response to IIS reduction do not require dfoxo. Promoter analyses revealed GATA and other forkhead factors as candidate mediators of the indirect and dfoxo‐independent effects. We demonstrate genome‐wide evolutionary conservation of dFOXO targets between the fly and the worm Caenorhabditis elegans, enriched for a second tier of regulators including the dHR96/daf‐12 nuclear hormone receptor. [ABSTRACT FROM AUTHOR]

Copy number variants (CNVs) account for a major proportion of human genetic polymorphism and have been predicted to have an important role in genetic susceptibility to common disease. To address this we undertook a large, direct genome-wide study of association between CNVs and eight common human diseases. Using a purpose-designed array we typed ∼19,000 individuals into distinct copy-number classes at 3,432 polymorphic CNVs, including an estimated ∼50% of all common CNVs larger than 500 base pairs. We identified several biological artefacts that lead to false-positive associations, including systematic CNV differences between DNAs derived from blood and cell lines. Association testing and follow-up replication analyses confirmed three loci where CNVs were associated with disease—IRGM for Crohn’s disease, HLA for Crohn’s disease, rheumatoid arthritis and type 1 diabetes, and TSPAN8 for type 2 diabetes—although in each case the locus had previously been identified in single nucleotide polymorphism (SNP)-based studies, reflecting our observation that most common CNVs that are well-typed on our array are well tagged by SNPs and so have been indirectly explored through SNP studies. We conclude that common CNVs that can be typed on existing platforms are unlikely to contribute greatly to the genetic basis of common human diseases. [ABSTRACT FROM AUTHOR]

Structural variations of DNA greater than 1 kilobase in size account for most bases that vary among human genomes, but are still relatively under-ascertained. Here we use tiling oligonucleotide microarrays, comprising 42 million probes, to generate a comprehensive map of 11,700 copy number variations (CNVs) greater than 443 base pairs, of which most (8,599) have been validated independently. For 4,978 of these CNVs, we generated reference genotypes from 450 individuals of European, African or East Asian ancestry. The predominant mutational mechanisms differ among CNV size classes. Retrotransposition has duplicated and inserted some coding and non-coding DNA segments randomly around the genome. Furthermore, by correlation with known trait-associated single nucleotide polymorphisms (SNPs), we identified 30 loci with CNVs that are candidates for influencing disease susceptibility. Despite this, having assessed the completeness of our map and the patterns of linkage disequilibrium between CNVs and SNPs, we conclude that, for complex traits, the heritability void left by genome-wide association studies will not be accounted for by common CNVs. [ABSTRACT FROM AUTHOR]

Multicellular animals match costly activities, such as growth and reproduction, to the environment through nutrient-sensing pathways. The insulin/IGF signaling (IIS) pathway plays key roles in growth, metabolism, stress resistance, reproduction, and longevity in diverse organisms including mammals. Invertebrate genomes often contain multiple genes encoding insulinlike ligands, including seven Drosophila insulin-like peptides (DILPs). We investigated the evolution, diversification, redundancy, and functions of the DILPs, combining evolutionary analysis, based on the completed genome sequences of 12 Drosophila species, and functional analysis, based on newly-generated knock-out mutations for all 7 dilp genes in D. melanogaster. Diversification of the 7 DILPs preceded diversification of Drosophila species, with stable gene diversification and family membership, suggesting stabilising selection for gene function. Gene knock-outs demonstrated both synergy and compensation of expression between different DILPs, notably with DILP3 required for normal expression of DILPs 2 and 5 in brain neurosecretory cells and expression of DILP6 in the fat body compensating for loss of brain DILPs. Loss of DILP2 increased lifespan and loss of DILP6 reduced growth, while loss of DILP7 did not affect fertility, contrary to its proposed role as a Drosophila relaxin. Importantly, loss of DILPs produced in the brain greatly extended lifespan but only in the presence of the endosymbiontic bacterium Wolbachia, demonstrating a specific interaction between IIS and Wolbachia in lifespan regulation. Furthermore, loss of brain DILPs blocked the responses of lifespan and fecundity to dietary restriction (DR) and the DR response of these mutants suggests that IIS extends lifespan through mechanisms that both overlap with those of DR and through additional mechanisms that are independent of those at work in DR. Evolutionary conservation has thus been accompanied by synergy, redundancy, and functional differentiation between DILPs, and these features may themselves be of evolutionary advantage. [ABSTRACT FROM AUTHOR]

Copy number variation (CNV) of DNA sequences is functionally significant but has yet to be fully ascertained. We have constructed a first-generation CNV map of the human genome through the study of 270 individuals from four populations with ancestry in Europe, Africa or Asia (the HapMap collection). DNA from these individuals was screened for CNV using two complementary technologies: single-nucleotide polymorphism (SNP) genotyping arrays, and clone-based comparative genomic hybridization. A total of 1,447 copy number variable regions (CNVRs), which can encompass overlapping or adjacent gains or losses, covering 360 megabases (12% of the genome) were identified in these populations. These CNVRs contained hundreds of genes, disease loci, functional elements and segmental duplications. Notably, the CNVRs encompassed more nucleotide content per genome than SNPs, underscoring the importance of CNV in genetic diversity and evolution. The data obtained delineate linkage disequilibrium patterns for many CNVs, and reveal marked variation in copy number among populations. We also demonstrate the utility of this resource for genetic disease studies. [ABSTRACT FROM AUTHOR]

Recent work has shown that copy number polymorphism is an important class of genetic variation in human genomes. Here we report a new method that uses SNP genotype data from parent-offspring trios to identify polymorphic deletions. We applied this method to data from the International HapMap Project to produce the first high-resolution population surveys of deletion polymorphism. Approximately 100 of these deletions have been experimentally validated using comparative genome hybridization on tiling-resolution oligonucleotide microarrays. Our analysis identifies a total of 586 distinct regions that harbor deletion polymorphisms in one or more of the families. Notably, we estimate that typical individuals are hemizygous for roughly 30–50 deletions larger than 5 kb, totaling around 550–750 kb of euchromatic sequence across their genomes. The detected deletions span a total of 267 known and predicted genes. Overall, however, the deleted regions are relatively gene-poor, consistent with the action of purifying selection against deletions. Deletion polymorphisms may well have an important role in the genetics of complex traits; however, they are not directly observed in most current gene mapping studies. Our new method will permit the identification of deletion polymorphisms in high-density SNP surveys of trio or other family data. [ABSTRACT FROM AUTHOR]

The human X chromosome has a unique biology that was shaped by its evolution as the sex chromosome shared by males and females. We have determined 99.3% of the euchromatic sequence of the X chromosome. Our analysis illustrates the autosomal origin of the mammalian sex chromosomes, the stepwise process that led to the progressive loss of recombination between X and Y, and the extent of subsequent degradation of the Y chromosome. LINE1 repeat elements cover one-third of the X chromosome, with a distribution that is consistent with their proposed role as way stations in the process of X-chromosome inactivation. We found 1,098 genes in the sequence, of which 99 encode proteins expressed in testis and in various tumour types. A disproportionately high number of mendelian diseases are documented for the X chromosome. Of this number, 168 have been explained by mutations in 113 X-linked genes, which in many cases were characterized with the aid of the DNA sequence. [ABSTRACT FROM AUTHOR]

We present an analysis of the evolutionary rates of the cytochrome c oxidase subunit I genes of primates and other mammals. Five primate genes were sequenced, and this information was combined with published data from other species. The sequences from simian primates show approximately twofold increases in their nonsynonymous substitution rate compared to those from other primates and other mammals. The species range and the overall magnitude of this rate increase are similar to those previously identified for the cytochrome c oxidase subunit II and cytochrome b genes. [ABSTRACT FROM AUTHOR]

Motivation: Increasingly, cost-effective high-throughput DNA sequencing technologies are being utilized to sequence human pedigrees to elucidate the genetic cause of a wide variety of human diseases. While numerous tools exist for variant prioritization within a single genome, the ability to concurrently analyze variants within pedigrees remains a challenge, especially should there be no prior indication of the underlying genetic cause of the disease. Here, we present a tool, variant analysis of sequenced pedigrees (VASP), a flexible data integration environment capable of producing a summary of pedigree variation, providing relevant information such as compound heterozygosity, genome phasing and disease inheritance patterns. Designed to aggregate data across a sequenced pedigree, VASP allows both powerful filtering and custom prioritization of both single nucleotide variants (SNVs) and small indels. Hence, clinical and research users with prior knowledge of a disease are able to dramatically reduce the variant search space based on a wide variety of custom prioritization criteria. [ABSTRACT FROM AUTHOR]

We have sequenced the cytochrome b gene of Horsfield's tarsier, Tarsius bancanus, to complete a data set of sequences for this gene from representatives of each primate infraorder. These primate cytochrome b sequences were combined with those from representatives of three other mammalian orders (cat, whale, and rat) in an analysis of relative evolutionary rates. The nonsynonymous nucleotide substitution rate of the cytochrome b gene has increased approximately twofold along lineages leading to simian primates compared to that of the tarsier and other primate and nonprimate mammalian species. However, the rate of transversional substitutions at fourfold degenerate sites has remained uniform among all lineages. This increase in the evolutionary rate of cytochrome b is similar in character and magnitude to that described previously for the cytochrome c oxidase subunit II gene. We propose that the evolutionary rate increase observed for cytochrome b and cytochrome c oxidase subunit II may underlie an episode of coadaptive evolution of these two proteins in the mitochondria of simian primates. [ABSTRACT FROM AUTHOR]

Background: Short-read resequencing of genomes produces abundant information of the genetic variation of individuals. Due to their numerous nature, these variants are rarely exhaustively validated. Furthermore, low levels of undetected variant miscalling will have a systematic and disproportionate impact on the interpretation of individual genome sequence information, especially should these also be carried through into in reference databases of genomic variation. Results: We find that sequence variation from short-read sequence data is subject to recurrent-yet-intermittent miscalling that occurs in a sequence intrinsic manner and is very sensitive to sequence read length. The miscalls arise from difficulties aligning short reads to redundant genomic regions, where the rate of sequencing error approaches the sequence diversity between redundant regions. We find the resultant miscalled variants to be sensitive to small sequence variations between genomes, and thereby are often intrinsic to an individual, pedigree, strain or human ethnic group. In human exome sequences, we identify 2–300 recurrent false positive variants per individual, almost all of which are present in public databases of human genomic variation. From the exomes of non-reference strains of inbred mice, we identify 3–5000 recurrent false positive variants per mouse – the number of which increasing with greater distance between an individual mouse strain and the reference C57BL6 mouse genome. We show that recurrently miscalled variants may be reproduced for a given genome from repeated simulation rounds of read resampling, realignment and recalling. As such, it is possible to identify more than two-thirds of false positive variation from only ten rounds of simulation. Conclusion: Identification and removal of recurrent false positive variants from specific individual variant sets will improve overall data quality. Variant miscalls arising are highly sequence intrinsic and are often specific to an individual, pedigree or ethnicity. Further, read length is a strong determinant of whether given false variants will be called for any given genome – which has profound significance for cohort studies that pool datasets collected and sequenced at different points in time. [ABSTRACT FROM AUTHOR]

Pyroptosis requires the induction of gasdermin D expression by the transcription factor IRF2. IRF2 induces gasdermin D: In response to activation of canonical and noncanonical inflammasomes, a subset of caspases processes the protein gasdermin D (GSDMD) to release N-terminal fragments that oligomerize and form pores in the plasma membrane. Assembly of the GSDMD pore leads to release of the inflammatory cytokine IL-1β and causes cell death by pyroptosis. Kayagaki et al. found that loss of the transcriptional regulator IRF2 reduced GSDMD mRNA and protein abundance in mice and in human cells, resulting in decreased IL-1β secretion and reduced pyroptosis in response to inflammasome activation. Given that loss of GSDMD in mice results in ameliorated disease in models of inflammasome-driven pathologies, these findings suggest that IRF2 might be a therapeutic target for the treatment of sepsis and other inflammasome-mediated diseases. Gasdermin-D (GSDMD) is cleaved by caspase-1, caspase-4, and caspase-11 in response to canonical and noncanonical inflammasome activation. Upon cleavage, GSDMD oligomerizes and forms plasma membrane pores, resulting in interleukin-1β (IL-1β) secretion, pyroptotic cell death, and inflammatory pathologies, including periodic fever syndromes and septic shock—a plague on modern medicine. Here, we showed that IRF2, a member of the interferon regulatory factor (IRF) family of transcription factors, was essential for the transcriptional activation of GSDMD. A forward genetic screen with N-ethyl-N-nitrosourea (ENU)–mutagenized mice linked IRF2 to inflammasome signaling. GSDMD expression was substantially attenuated in IRF2-deficient macrophages, endothelial cells, and multiple tissues, which corresponded with reduced IL-1β secretion and inhibited pyroptosis. Mechanistically, IRF2 bound to a previously uncharacterized but unique site within the GSDMD promoter to directly drive GSDMD transcription for the execution of pyroptosis. Disruption of this single IRF2-binding site abolished signaling by both the canonical and noncanonical inflammasomes. Together, our data illuminate a key transcriptional mechanism for expression of the gene encoding GSDMD, a critical mediator of inflammatory pathologies. [ABSTRACT FROM AUTHOR]