Predatory bacteria, like the model endoperiplasmic bacterium Bdellovibrio bacteriovorus , show several adaptations relevant to their requirements for locating, entering and killing other bacteria. The mechanisms underlying prey recognition and handling remain obscure. Here we use complementary genetic, microscopic and structural methods to address this deficit. During invasion, the B. bacteriovorus protein CpoB concentrates into a vesicular compartment that is deposited into the prey periplasm. Proteomic and structural analyses of vesicle contents reveal several fibre-like proteins, which we name the mosaic adhesive trimer (MAT) superfamily, and show localization on the predator surface before prey encounter. These dynamic proteins indicate a variety of binding capabilities, and we confirm that one MAT member shows specificity for surface glycans from a particular prey. Our study shows that the B. bacteriovorus MAT protein repertoire enables a broad means for the recognition and handling of diverse prey epitopes encountered during bacterial predation and invasion.

The bacterium Bdellovibrio bacteriovorus is a predator of other Gram-negative bacteria. The predator invades the prey's periplasm and modifies the prey's cell wall, forming a rounded killed prey, or bdelloplast, containing a live B. bacteriovorus. Redundancy in adhesive processes makes invasive mutants rare. Here, we identify a MIDAS adhesin family protein, Bd0875, that is expressed at the predator-prey invasive junction and is important for successful invasion of prey. A mutant strain lacking bd0875 is still able to form round, dead bdelloplasts; however, 10% of the bdelloplasts do not contain B. bacteriovorus, indicative of an invasion defect. Bd0875 activity requires the conserved MIDAS motif, which is linked to catch-and-release activity of MIDAS proteins in other organisms. A proteomic analysis shows that the uninvaded bdelloplasts contain B. bacteriovorus proteins, which are likely secreted into the prey by the Δbd0875 predator during an abortive invasion period. Thus, secretion of proteins into the prey seems to be sufficient for prey killing, even in the absence of a live predator inside the prey periplasm.

Antimicrobial resistance is a serious and rapidly growing threat to global health. Bdellovibrio bacteriovorus can prey upon an extensive range of Gram-negative bacterial pathogens and thus has promising potential as a novel antibacterial therapeutic and is a source of antibacterial enzymes. Here, we elucidate the role of a unique secreted lytic transglycosylase from B. bacteriovorus which acts on the septal peptidoglycan of its prey.

Histone proteins bind DNA and organize the genomes of eukaryotes and most archaea, whereas bacteria rely on different nucleoid-associated proteins. Homology searches have detected putative histone-fold domains in a few bacteria, but whether these function like archaeal/eukaryotic histones is unknown. Here we report that histones are major chromatin components in the bacteria Bdellovibrio bacteriovorus and Leptospira interrogans . Patterns of sequence evolution suggest important roles for histones in additional bacterial clades. Crystal structures (<2.0 Å) of the B. bacteriovorus histone (Bd0055) dimer and the histone–DNA complex confirm conserved histone-fold topology but indicate a distinct DNA-binding mode. Unlike known histones in eukaryotes, archaea and viruses, Bd0055 binds DNA end-on, forming a sheath of dimers encasing straight DNA rather than wrapping DNA around their outer surface. Our results demonstrate that histones are present across the tree of life and highlight potential evolutionary innovation in how they associate with DNA.

ABSTRACT Histones are the principal constituents of chromatin in eukaryotes and most archaea, while bacteria generally rely on an orthogonal set of proteins to organize their chromosomes. However, several bacterial genomes encode proteins with putative histone fold domains. Whether these proteins are structurally and functionally equivalent to archaeal and eukaryotic histones is unknown. Here, we demonstrate that histones are essential and are major components of chromatin in the bacteria Bdellovibrio bacteriovorus and Leptospira interrogans . Patterns of sequence evolution suggest important roles in several additional bacterial clades. Structural analysis of the B. bacteriovorus histone (Bd0055) dimer shows that histone fold topology is conserved between bacteria, archaea, and eukaryotes. Yet, unexpectedly, Bd0055 binds DNA end-on and forms a sheath of tightly packed histone dimers to encase straight DNA. This binding mode is in stark contrast to archaeal, eukaryotic, and viral histones, which invariably bend and wrap DNA around their outer surface. Our results demonstrate that histones are integral chromatin components across the tree of life and highlight organizational innovation in the domain Bacteria.

Antibiotics have revolutionized modern medicine, but their effectiveness is threatened by the spread of multi- drugresistant bacteria for which there is no available treatment [1-3]. Antimicrobial resistance (AMR), often driven by unnecessary antibiotic use, is a serious, global health and economic threat [4-7]. Of particular concern is the declining antibiotic pipeline with limited antibiotics in development (in both number and diversity) to meet current and anticipated patient demands [8-11]. Moreover, scientific and economic challenges have contributed to many large global pharmaceu Antimicrobial resistance (AMR) is a global health and economic crisis. With too few antibiotics in development to meet current and anticipated needs, there is a critical need for new therapies to treat Gram- negative infections. One potential approach is the use of living predatory bacteria, such as Bdellovibrio bacteriovorus (small Gram- negative bacteria that naturally invade and kill Gram- negative pathogens of humans, animals and plants). Moving toward the use of Bdellovibrio as a 'living antibiotic' demands the investigation and characterization of these bacterial predators in biologically relevant systems. We review the fundamental science supporting the feasibility of predatory bacteria as alternatives to antibiotics.

Antimicrobial resistance (AMR) is a global health and economic crisis. With too few antibiotics in development to meet current and anticipated needs, there is a critical need for new therapies to treat Gram-negative infections. One potential approach is the use of living predatory bacteria, such as Bdellovibrio bacteriovorus (small Gram-negative bacteria that naturally invade and kill Gram-negative pathogens of humans, animals and plants). Moving toward the use of Bdellovibrio as a 'living antibiotic' demands the investigation and characterization of these bacterial predators in biologically relevant systems. We review the fundamental science supporting the feasibility of predatory bacteria as alternatives to antibiotics.

Bacteria are preyed upon by diverse microbial predators, including bacteriophage and predatory bacteria, such as Bdellovibrio bacteriovorus. While bacteriophage are used as antimicrobial therapies in Eastern Europe and are being applied for compassionate use in the United States, predatory bacteria are only just beginning to reveal their potential therapeutic uses. However, predation by either predator type can falter due to different adaptations arising in the prey bacteria. When testing poultry farm wastewater for novel Bdellovibrio isolates on Escherichia coli prey lawns, individual composite plaques were isolated containing both an RTP (rosette-tailed-phage)-like-phage and a B. bacteriovorus strain and showing central prey lysis and halos of extra lysis. Combining the purified phage with a lab strain of B. bacteriovorus HD100 recapitulated haloed plaques and increased killing of the E. coli prey in liquid culture, showing an effective side-by-side action of these predators compared to their actions alone. Using approximate Bayesian computation to select the best fitting from a variety of different mathematical models demonstrated that the experimental data could be explained only by assuming the existence of three prey phenotypes: (i) sensitive to both predators, (ii) genetically resistant to phage only, and (iii) plastic resistant to B. bacteriovorus only. Although each predator reduces prey availability for the other, high phage numbers did not abolish B. bacteriovorus predation, so both predators are competent to coexist and are causing different selective pressures on the bacterial surface while, in tandem, controlling prey bacterial numbers efficiently. This suggests that combinatorial predator therapy could overcome problems of phage resistance. IMPORTANCE With increasing levels of antibiotic resistance, the development of alternative antibacterial therapies is urgently needed. Two potential alternatives are bacteriophage and predatory bacteria. Bacteriophage therapy has been used, but prey/host specificity and the rapid acquisition of bacterial resistance to bacteriophage are practical considerations. Predatory bacteria are of interest due to their broad Gram-negative bacterial prey range and the lack of simple resistance mechanisms. Here, a bacteriophage and a strain of Bdellovibrio bacteriovorus, preyed side by side on a population of E. coli, causing a significantly greater decrease in prey numbers than either alone. Such combinatorial predator therapy may have greater potential than individual predators since prey surface changes selected for by each predator do not protect prey against the other predator.

Purpose. While some micro‐organisms, such as Staphylococcus aureus, are clearly implicated in causing tissue damage in diabetic foot ulcers (DFUs), our knowledge of the contribution of the entire microbiome to clinical outcomes is limited. We profiled the microbiome of a longitudinal sample series of 28 people with diabetes and DFUs of the heel in an attempt to better characterize the relationship between healing, infection and the microbiome. Methodology. In total, 237 samples were analysed from 28 DFUs, collected at fortnightly intervals for 6 months or until healing. Microbiome profiles were generated by 16S rRNA gene sequence analysis, supplemented by targeted nanopore sequencing. Result/Key findings. DFUs which failed to heal during the study period (20/28, 71.4 %) were more likely to be persistently colonized with a heterogeneous community of micro‐organisms including anaerobes and Enterobacteriaceae (log‐likelihood ratio 9.56, P=0.008). During clinically apparent infection, a reduction in the diversity of micro‐organisms in a DFU was often observed due to expansion of one or two taxa, with recovery in diversity at resolution. Modelling of the predicted species interactions in a single DFU with high diversity indicated that networks of metabolic interactions may exist that contribute to the formation of stable communities. Conclusion. Longitudinal profiling is an essential tool for improving our understanding of the microbiology of chronic wounds, as community dynamics associated with clinical events can only be identified by examining changes over multiple time points. The development of complex communities, particularly involving Enterobacteriaceae and strict anaerobes, may be contributing to poor outcomes in DFUs and requires further investigation.

In assessing the potential of predatory bacteria, such as Bdellovibrio bacteriovorus, to become live therapeutic agents against bacterial infections, it is crucial to understand and quantify Bdellovibrio host cell interactions at a molecular level. Here, we quantify the interactions of live B. bacteriovorus with human phagocytic cells, determining the uptake mechanisms, persistence, associated cytokine responses and intracellular trafficking of the non-growing B. bacteriovorus in PMA-differentiated U937 cells. B. bacteriovorus are engulfed by U937 cells and persist for 24 h without affecting host cell viability and can be observed microscopically and recovered and cultured post-uptake. The uptake of predators is passive and depends on the dynamics of the host cell cytoskeleton; the engulfed predators are eventually trafficked through the phagolysosomal pathway of degradation. We have also studied the prevalence of B. bacteriovorus specific antibodies in the general human population. Together, these results quantify a period of viable persistence and the ultimate fate of B. bacteriovorus inside phagocytic cells. They provide new knowledge on predator availability inside hosts, plus potential longevity and therefore potential efficacy as a treatment in humans and open up future fields of work testing if predators can prey on host-engulfed pathogenic bacteria.

In worldwide conditions of increasingly antibiotic-resistant hospital infections, it is important to research alternative therapies. Bdellovibrio bacteriovorus bacteria naturally prey on Gram-negative pathogens, including antibiotic-resistant strains and so B. bacteriovorus have been proposed as "living antibiotics" to combat antimicrobially-resistant pathogens. Predator-prey interactions are complex and can be altered by environmental components. To be effective B. bacteriovorus predation needs to work in human body fluids such as serum where predation dynamics may differ to that studied in laboratory media. Here we combine mathematical modelling and lab experimentation to investigate the predation of an important carbapenem-resistant human pathogen, Klebsiella pneumoniae, by B. bacteriovorus in human serum versus buffer. We show experimentally that B. bacteriovorus is able to reduce prey numbers in each environment, on different timescales. Our mathematical model captures the underlying dynamics of the experimentation, including an initial predation-delay at the predator-prey-serum interface. Our research shows differences between predation in buffer and serum and highlights both the potential and limitations of B. bacteriovorus acting therapeutically against K. pneumoniae in serum, informing future research into the medicinal behaviours and dosing of this living antibacterial.

The 2010s have seen a full recognition of the scourge of antimicrobial resistance (AMR) where antibiotic-resistance genes, many transferred from environmental bacteria, spread rapidly through hospital and farm populations of pathogens when selected for by applied antibiotics. Many of these resistance traits allow survival of bacteria in the very soil environments where other organisms naturally produce antibiotics. In a hospital or farm setting, they nullify the therapeutic effects of prescribed antibiotics. Along with this realization has come a return to natural environments to seek different new solutions. Although these settings are both the source of genes encoding the antibiotics we have relied upon and of the resistance genes that circumvent individual antibiotic agents or their actions, Nature offers much more than just that. Diverse molecular tactics are employed, between different micro-organisms, in natural environments, in a dazzling array of changing combinations to gain advantage in conflicts for territory and food. The war strategist in these processes is evolution and the armies and vigilantes are bacteria, bacteriophage, protozoa and fungi. The iChip, a novel method for growing previously-unculturable bacteria, in their natural environment, developed by Kim Lewis and colleagues (Nichols et al., 2010) has been used to access untapped environmental bacteria for potential antibiotics (Ling et al., 2015). Indeed this may lead to the next generation of antibiotics that can be downstream processed into the pharmaceuticals of tomorrow, and combined with better stewardship may cause AMR to recede for future decades. In these new antibiotic discovery approaches, we humans pick up the individual weapons of micro-organisms, from their battlefields, and fire them ourselves, against pathogens. Certainly this will rearm humans to fight infection using the principles first applied by Fleming, Florey and Chain. Armed with advances in microscopy, 3rd generation sequencing technologies and greater understanding of the microbiota of different environments, we are now able to ask, more holistically, how do whole micro-organisms kill each other? From this fundamental knowledge we will be able to employ them whole in the fight against AMR infection. The advantage of this is that multiple weapons may be used in a regulated way, by whole micro-organisms, against their foes. This more complex approach may be hard to resist by single gene mutations in the pathogens. Elegant microscopic studies by Melanie Blokesch and others (Borgeaud et al., 2015) studying the Type VI secretion apparatus have shown how environmental bacteria, such as Vibrio cholerae, use that system to stab and poison adjacent bacteria in a contact dependent manner wherein the killer senses the 'prey'. The Type VI secretion apparatus shares conserved structures with the injective machinery of the larger bacteriophage viruses of bacteria. Using phage allows a multi-enzyme approach to bacterial killing but because a single receptor is often the portal of phage attack, selection for phage-resistant mutants is possible. On the plus side, given the enormous number and diversity of phage on earth, cocktails of assorted phage can be useful to delay the effect of such single genetic events (Merabishvili et al., 2009). Another, but very different, whole organism approach to bacterial killing, comes in the shape of predatory bacteria, including Bdellovibrio bacteriovorus. This invasive predatory bacterium enters and kills a wide range of Gram-negative bacteria and was, (akin to the discovery of penicillin by Alexander Fleming), isolated in an environmentally 'infiltrated' experiment that went unexpectedly. Stolp and Petzold isolated such predatory Bdellovibrio from plaques on a bacterial lawn in an experiment where they were studying phage. In fact, miniature predatory soil bacteria, too small to cause opacity were also able to invade and kill the larger bacteria on the lawn and produce plaques. (Stolp and Petzold, 1962; Stolp and Starr, 1963). Bacterial killing, by their predator 'cousins', does not rely on receptor binding, and so does not select for simple prey-resistance; but involves outer-membrane contacts and Type IV pilus activity for prey-invasion. After this comes peptidoglycan modification, allowing colonization of the prey periplasm; death of the invaded bacterium and its consumption by waves of secreted predator products. Recently, transcriptomics and transposon mutagenesis studies of predatory bacteria have shown the arsenal of some 200 bacterially destructive enzymes that predators employ to kill prey and transporters involved in their secretion or prey-derived nutrient uptake (Medina et al., 2008; Tudor et al. 2008; Lambert et al., 2010). This sometimes makes the Bdellovibrio scientist think that using a single 'molecular weapon' to combat pathogens might seem like a single 'pop-gun' compared to the '1812 Overture' crescendo of multiple enzymes that living predators employ to dismember their prey! As a study in 2011 showed that Bdellovibrio are safe orally ingested by animals, the way was open for more work using whole live predatory bacteria as anti-bacterials (Atterbury et al., 2011). Also the observation by Iebba and colleagues (2013) that children have DNA indicative of predatory bacteria in their gut microbiota suggested that live predators, possibly ingested accidentally in 'a peck of muck', (soil), during play in the outdoor environment, may be not harmful in the normal microbiota. Encouraged by a US Government Defence Advanced Research Projects Agency (DARPA) 'Pathogen Predators Research Programme', the vision of Dr. Barry Pallotta and colleagues, researchers have taken, and published, the next steps with therapeutic tests, using whole live predatory bacteria. Shatzkes and colleagues have shown that repeatedly-applied aerosols of predatory Bdellovibrio restrict the bacterial burden of Klebsiella pneumoniae in rat lungs (Shatzkes et al., 2016). Willis and colleagues (2016) showed that injected Bdellovibrio kill injected Shigella pathogens in the hindbrain of zebrafish embryos and that this treatment significantly improves the survival of the zebrafish from c30% to 67% at 72 h post infection. Moreover, the innate immune system of the zebrafish synergises with the action of the predators in clearing the pathogen (Willis et al., 2016). Again this observation may relate back to complex actions of protozoa and predatory bacteria in soil environment. On the early earth, before evolution of higher eukaryotes, simpler mechanisms of such eukaryotic–prokaryotic cooperativity in soil may have contributed to the evolution of the white blood cell (WBC)-predator interactions seen in the zebrafish treatment experiments. Although, as the zebrafish studies show, the immune system will eventually clear live therapeutic predatory bacteria, they may dwell in the bodily environment long enough to be beneficial as an applied emergency dose to an infected wound or surgery site (Willis et al., 2016). Also the work of several laboratories shows that experimental injection/ingestion/application of Bdellovibrio alone to live animals or tissues, while causing some inflammation, are not pathogenic (Atterbury et al., 2011; Shatzkes et al., 2015; Gupta et al., 2016; Monnappa et al., 2016; Willis et al., 2016). These experiments make us think that natural live predatory bacterial remedies may be beneficial treatments for AMR infections, even though the idea of introducing more bacteria into an already pathogen-infected host may sound unconventional. The need for research into new antibacterial treatments is great; a speech by John Rex MD, [at a meeting convened by The Pew Charitable Trust, The Wellcome trust and American Society for Microbiology, following publication of The Pew Charitable Trusts report 'A Scientific Roadmap for Antibiotic Discovery' (June 16, 2016)], put this into sharp relief. John Rex suggested we think of antibiotics as fire extinguishers (Rex and Outterson, 2016). Fire extinguishers are present in certified buildings and available at all times. You may never have had to use one, but they are always there. In the event of a fire, there's no time to start to build a fire service. In the same way anti-bacterials need to be in reserve, ready and available in hospitals, even if rarely used, except for new AMR infections. Scientific challenges need to be addressed now to ensure that the supply of such new anti-bacterials is there in advance of their requirement as AMR infections increase. We feel that whole predatory bacteria may be the contents of future 'extinguishers', discharged into infected compartments of the body to extinguish Gram-negative infections, with bacteriophage additives to combat Gram-positive co-infectors. Predatory bacteria may not be suitable for systemic blood stream infections; but they may play a role decolonizing lungs, guts or wounds from problematic pathogens that resist conventional antibiotics. This approach would bring with it a new challenges; production of whole organisms rather than chemically defined drugs for treatment will need to draw upon the food and brewing industry as much as the pharmaceutical. Supporting the patient's physiology and immune status, during the administration of the predators, and monitoring predator clearance at the end of treatment will be vital. Modern microscopic methods now make it more possible to follow and enumerate the predators in the new environments of the living animal or human body. How to assure the safety of a living predatory bacterium with over 3,500 gene products, rather than a single chemical entity like an antibiotic will be new and difficult legislative – and safety – territory, but the advance of untreatable AMR will certainly focus the minds of clinicians, scientists, patients and legislators on relative risks. Further animal studies will be very important and helpful. Indeed test-treating farm livestock with predators for their conventionally AMR infections and monitoring, (in contained conditions), the longevity and environmental fate of the predators and pathogens (Ibrahim et al., 2016), as well as the treatment outcome, will be a useful way ahead. So in summary, although some of the genes encoding AMR have come from soil bacteria, returning to Nature to source solutions using the multi-faceted approaches of live micro-organisms, including predatory bacteria, will give new possibilities that take longer for pathogens to resist. The natural properties of prokaryotic predators, studied and applied by environmental as well as clinical and fundamental microbiologists working together, can bring new solutions to AMR. Research by the authors was sponsored by the BBSRC UK via grants G003092/1 and BBJ015229/1 and also by the U.S. Army Research Office and the Defense Advanced Research Projects Agency, accomplished under Cooperative Agreement Number W911NF-15-2-0028. The views and conclusions contained in this document are those of the authors and should not be interpreted as representing the official policies, either expressed or implied, of the Army Research Office, DARPA, or the U.S. Government.

Bdellovibrio bacteriovorus is a small deltaproteobacterial predator that has evolved to invade, reseal, kill, and digest other gram-negative bacteria in soils and water environments. It has a broad host range and kills many antibiotic-resistant, clinical pathogens in vitro, a potentially useful capability if it could be translated to a clinical setting. We review relevant mechanisms of B. bacteriovorus predation and the physiological properties that would influence its survival in a mammalian host. Bacterial pathogens increasingly display conventional antibiotic resistance by expressing and varying surface and soluble biomolecules. Predators coevolved alongside prey bacteria and so encode diverse predatory enzymes that are hard for pathogens to resist by simple mutation. Predators do not replicate outside pathogens and thus express few transport proteins and thus few surface epitopes for host immune recognition. We explain these features, relating them to the potential of predatory bacteria as cellular medicines.

Emulsion-fusion PCR recovers long-range sequence information by combining products in cis from individual genomic DNA molecules. Emulsion droplets act as very numerous small reaction chambers in which different PCR products from a single genomic DNA molecule are condensed into short joint products, to unite sequences in cis from widely separated genomic sites. These products can therefore provide information about the arrangement of sequences and variants at a larger scale than established long-read sequencing methods. The method has been useful in defining the phase of variants in haplotypes, the typing of inversions, and determining the configuration of sequence variants in multiallelic CNVs. In this description we outline the rationale for the application of emulsion-fusion PCR methods to the analysis of multiallelic CNVs, and give practical details for our own implementation of the method in that context.

The human salivary amylase genes display extensive copy number variation (CNV), and recent work has implicated this variation in adaptation to starch-rich diets, and in association with body mass index. In this work, we use paralogue ratio tests, microsatellite analysis, read depth and fibre-FISH to demonstrate that human amylase CNV is not a smooth continuum, but is instead partitioned into distinct haplotype classes. There is a fundamental structural distinction between haplotypes containing odd or even numbers of AMY1 gene units, in turn coupled to CNV in pancreatic amylase genes AMY2A and AMY2B. Most haplotypes have one copy each of AMY2A and AMY2B and contain an odd number of copies of AMY1; consequently, most individuals have an even total number of AMY1. In contrast, haplotypes carrying an even number of AMY1 genes have rearrangements leading to CNVs of AMY2A/AMY2B. Read-depth and experimental data show that different populations harbour different proportions of these basic haplotype classes. In Europeans, the copy numbers of AMY1 and AMY2A are correlated, so that phenotypic associations caused by variation in pancreatic amylase copy number could be detected indirectly as weak association with AMY1 copy number. We show that the quantitative polymerase chain reaction (qPCR) assay previously applied to the high-throughput measurement of AMY1 copy number is less accurate than the measures we use and that qPCR data in other studies have been further compromised by systematic miscalibration. Our results uncover new patterns in human amylase variation and imply a potential role for AMY2 CNV in functional associations.

BACKGROUND:The determination of structural haplotypes at copy number variable regions can indicate the mechanisms responsible for changes in copy number, as well as explain the relationship between gene copy number and expression. However, obtaining spatial information at regions displaying extensive copy number variation, such as the DEFA1A3 locus, is complex, because of the difficulty in the phasing and assembly of these regions. The DEFA1A3 locus is intriguing in that it falls within a region of high linkage disequilibrium, despite its high variability in copy number (n = 3-16); hence, the mechanisms responsible for changes in copy number at this locus are unclear.RESULTS:In this study, a region flanking the DEFA1A3 locus was sequenced across 120 independent haplotypes with European ancestry, identifying five common classes of DEFA1A3 haplotype. Assigning DEFA1A3 class to haplotypes within the 1000 Genomes project highlights a significant difference in DEFA1A3 class frequencies between populations with different ancestry. The features of each DEFA1A3 class, for example, the associated DEFA1A3 copy numbers, were initially assessed in a European cohort (n = 599) and replicated in the 1000 Genomes samples, showing within-class similarity, but between-class and between-population differences in the features of the DEFA1A3 locus. Emulsion haplotype fusion-PCR was used to generate 61 structural haplotypes at the DEFA1A3 locus, showing a high within-class similarity in structure.CONCLUSIONS:Structural haplotypes across the DEFA1A3 locus indicate that intra-allelic rearrangement is the predominant mechanism responsible for changes in DEFA1A3 copy number, explaining the conservation of linkage disequilibrium across the locus. The identification of common structural haplotypes at the DEFA1A3 locus could aid studies into how DEFA1A3 copy number influences expression, which is currently unclear.

BACKGROUND:Multi-allelic copy number variants include examples of extensive variation between individuals in the copy number of important genes, most notably genes involved in immune function. The definition of this variation, and analysis of its impact on function, has been hampered by the technical difficulty of large-scale but accurate typing of genomic copy number. The copy-variable alpha-defensin locus DEFA1A3 on human chromosome 8 commonly varies between 4 and 10 copies per diploid genome, and presents considerable challenges for accurate high-throughput typing.RESULTS:In this study, we developed two paralogue ratio tests and three allelic ratio measurements that, in combination, provide an accurate and scalable method for measurement of DEFA1A3 gene number. We combined information from different measurements in a maximum-likelihood framework which suggests that most samples can be assigned to an integer copy number with high confidence, and applied it to typing 589 unrelated European DNA samples. Typing the members of three-generation pedigrees provided further reassurance that correct integer copy numbers had been assigned. Our results have allowed us to discover that the SNP rs4300027 is strongly associated with DEFA1A3 gene copy number in European samples.CONCLUSIONS:We have developed an accurate and robust method for measurement of DEFA1A3 copy number. Interrogation of rs4300027 and associated SNPs in Genome-Wide Association Study SNP data provides no evidence that alpha-defensin copy number is a strong risk factor for phenotypes such as Crohn's disease, type I diabetes, HIV progression and multiple sclerosis.

BACKGROUND:Genotyping and massively-parallel sequencing projects result in a vast amount of diploid data that is only rarely resolved into its constituent haplotypes. It is nevertheless this phased information that is transmitted from one generation to the next and is most directly associated with biological function and the genetic causes of biological effects. Despite progress made in genome-wide sequencing and phasing algorithms and methods, problems assembling (and reconstructing linear haplotypes in) regions of repetitive DNA and structural variation remain. These dynamic and structurally complex regions are often poorly understood from a sequence point of view. Regions such as these that are highly similar in their sequence tend to be collapsed onto the genome assembly. This is turn means downstream determination of the true sequence haplotype in these regions poses a particular challenge. For structurally complex regions, a more focussed approach to assembling haplotypes may be required.RESULTS:In order to investigate reconstruction of spatial information at structurally complex regions, we have used an emulsion haplotype fusion PCR approach to reproducibly link sequences of up to 1kb in length to allow phasing of multiple variants from neighbouring loci, using allele-specific PCR and sequencing to detect the phase. By using emulsion systems linking flanking regions to amplicons within the CNV, this led to the reconstruction of a 59kb haplotype across the DEFA1A3 CNV in HapMap individuals.CONCLUSION:This study has demonstrated a novel use for emulsion haplotype fusion PCR in addressing the issue of reconstructing structural haplotypes at multiallelic copy variable regions, using the DEFA1A3 locus as an example.

For most cases of colorectal cancer that arise without a family history of the disease, it is proposed that an appreciable heritable component of predisposition is the result of contributions from many loci. Although progress has been made in identifying single nucleotide variants associated with colorectal cancer risk, the involvement of low-penetrance copy number variants is relatively unexplored. We have used multiplex amplifiable probe hybridization (MAPH) in a fourfold multiplex (QuadMAPH), positioned at an average resolution of one probe per 2 kb, to screen a total of 1.56 Mb of genomic DNA for copy number variants around the genes APC, AXIN1, BRCA1, BRCA2, CTNNB1, HRAS, MLH1, MSH2, and TP53. Two deletion events were detected, one upstream of MLH1 in a control individual and the other in APC in a colorectal cancer patient, but these do not seem to correspond to copy number polymorphisms with measurably high population frequencies. In summary, by means of our QuadMAPH assay, copy number measurement data were of sufficient resolution and accuracy to detect any copy number variants with high probability. However, this study has demonstrated a very low incidence of deletion and duplication variants within intronic and flanking regions of these nine genes, in both control individuals and colorectal cancer patients.

Atopic dermatitis (AD) and psoriasis are common skin diseases characterized by cutaneous inflammation and disturbed epidermal differentiation. Genome-wide analyses have shown overlapping susceptibility loci, such as the epidermal differentiation complex on chromosome 1q21. Recently, a deletion on 1q21 (LCE3C_LCE3B-del), comprising LCE3B and LCE3C, two members of the late cornified envelope (LCE) gene cluster, was found to be associated with psoriasis. Although the mechanistic role of LCE proteins in psoriasis has not been identified, these proteins are putatively involved in skin barrier formation and repair. Considering the potential genetic overlap between the two diseases and the recent finding that mutations in the skin barrier protein filaggrin are associated with AD, we investigated a possible association between LCE3C_LCE3B-del and AD. Evaluation of four different cohorts of European ancestry, containing a total of 1075 AD patients and 1658 controls, did not provide evidence for such an association. Subgroup analysis did not reveal an association with concomitant asthma. Our data suggest that the potential roles of skin barrier defects in the pathogenesis of AD and psoriasis are based on distinct genetic causes.