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Indigenous Australians face a disproportionately severe burden of chronic disease relative to other Australians, with elevated rates of morbidity and mortality. While genomics technologies are slowly gaining momentum in personalised treatments for many, a lack of pharmacogenomic research in Indigenous peoples could delay adoption. Appropriately implementing pharmacogenomics in clinical care necessitates an understanding of the frequencies of pharmacologically relevant genetic variants within Indigenous populations. We analysed whole-genome sequence data from 187 individuals from the Tiwi Islands and characterised the pharmacogenomic landscape of this population. Specifically, we compared variant profiles and allelic distributions of previously described pharmacologically significant genes and variants with other population groups. We identified 22 translationally relevant pharmacogenomic variants and 18 clinically actionable guidelines with implications for drug dosing and treatment of conditions including heart disease, diabetes and cancer. We specifically observed increased poor and intermediate metabolizer phenotypes in the CYP2C9 (PM:19%, IM:44%) and CYP2C19 (PM:18%, IM:44%) genes.

The mycoparasite Clonostachys rosea ACM941 is under development as a biocontrol organism against Fusarium graminearum, the causative agent of Fusarium head blight in cereals. To identify molecular factors associated with this interaction, the transcriptomic and exometabolomic profiles of C. rosea and F. graminearum GZ3639 were compared during coculture. Prior to physical contact, the antagonistic activity of C. rosea correlated with a response heavily dominated by upregulation of polyketide synthase gene clusters, consistent with the detected accumulation of corresponding secondary metabolite products. Similarly, prior to contact, trichothecene gene clusters were upregulated in F. graminearum, while those responsible for fusarielin and fusarin biosynthesis were downregulated, correlating with an accumulation of trichothecene products in the interaction zone over time. A concomitant increase in 15-acetyl deoxynivalenol-3-glucoside in the interaction zone was also detected, with C. rosea established as the source of this detoxified mycotoxin. After hyphal contact, C. rosea was found to predominantly transcribe genes encoding cell wall–degradation enzymes, major facilitator superfamily sugar transporters, anion:cation symporters, as well as alternative carbon source utilization pathways, together indicative of a transition to necrotropism at this stage. F. graminearum notably activated the transcription of phosphate starvation pathway signature genes at this time. Overall, a number of signature molecular mechanisms likely contributing to antagonistic activity by C. rosea against F. graminearum, as well as its mycotoxin tolerance, are identified in this report, yielding several new testable hypotheses toward understanding the basis of C. rosea as a biocontrol agent for continued agronomic development and application.

The bacteria in the genus Cellulomonas are known for their ability to degrade plant cell wall biomass. Cellulomonas fimi ATCC 484 and C. flavigena ATCC 482 have been the subject of much research into secreted cellulases and hemicellulases. Recently the genome sequences of both C. fimi ATCC 484 and C. flavigena ATCC 482 were published, and a genome comparison has revealed their full spectrum of possible carbohydrate-active enzymes (CAZymes). Using mass spectrometry, we have compared the proteins secreted by C. fimi and C. flavigena during growth on the soluble cellulose substrate, carboxymethylcellulose (CMC), as well as a soluble xylan fraction. Many known C. fimi CAZymes were detected, which validated our analysis, as were a number of new CAZymes and other proteins that, though identified in the genome, have not previously been observed in the secretome of either organism. Our data also shows that many of these are co-expressed on growth of either CMC or xylan. This analysis provides a new perspective on Cellulomonas enzymes and provides many new CAZyme targets for characterization.

Indigenous Australians face a disproportionately severe burden of chronic disease relative to other Australians, with elevated rates of morbidity and mortality. While genomics technologies are slowly gaining momentum in personalised treatments for many, a lack of pharmacogenomic research in Indigenous peoples could delay adoption. Appropriately implementing pharmacogenomics in clinical care necessitates an understanding of the frequencies of pharmacologically relevant genetic variants within Indigenous populations. We analysed whole-genome sequence data from 187 individuals from the Tiwi Islands and characterised the pharmacogenomic landscape of this population. Specifically, we compared variant profiles and allelic distributions of previously described pharmacologically significant genes and variants with other population groups. We identified 22 translationally relevant pharmacogenomic variants and 18 clinically actionable guidelines with implications for drug dosing and treatment of conditions including heart disease, diabetes and cancer. We specifically observed increased poor and intermediate metabolizer phenotypes in the CYP2C9 (PM:19%, IM:44%) and CYP2C19 (PM:18%, IM:44%) genes.

An important component in host resistance to malaria infection are inherited mutations that give rise to abnormalities and deficiencies in erythrocyte proteins and enzymes. Understanding how such mutations confer protection against the disease may be useful for developing new treatment strategies. A mouse ENU-induced mutagenesis screen for novel malaria resistance-conferring mutations identified a novel nonsense mutation in the gene encoding porphobilinogen deaminase (PBGD) in mice, denoted here as PbgdMRI58155. Heterozygote PbgdMRI58155 mice exhibited approximately 50% reduction in cellular PBGD activity in both mature erythrocytes and reticulocytes, although enzyme activity was approximately 10 times higher in reticulocytes than erythrocytes. When challenged with blood-stage P. chabaudi, which preferentially infects erythrocytes, heterozygote mice showed a modest but significant resistance to infection, including reduced parasite growth. A series of assays conducted to investigate the mechanism of resistance indicated that mutant erythrocyte invasion by P. chabaudi was normal, but that following intraerythrocytic establishment a significantly greater proportions of parasites died and therefore affected their ability to propagate. The Plasmodium resistance phenotype was not recapitulated in Pbgd-deficient mice infected with P. berghei, which prefers reticulocytes, or when P. falciparum was cultured in erythrocytes from patients with acute intermittent porphyria (AIP), which had modest (20-50%) reduced levels of PBGD. Furthermore, the growth of Pbgd-null P. falciparum and Pbgd-null P. berghei parasites, which grew at the same rate as their wild-type counterparts in normal cells, were not affected by the PBGD-deficient background of the AIP erythrocytes or Pbgd-deficient mice. Our results confirm the dispensability of parasite PBGD for P. berghei infection and intraerythrocytic growth of P. falciparum, but for the first time identify a requirement for host erythrocyte PBGD by P. chabaudi during in vivo blood stage infection.

The mycoparasite

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The Gypsum Hill (GH) springs on Axel Heiberg Island in the Canadian high Arctic are host to chemolithoautotrophic, sulfur-oxidizing streamers that flourish in the high Arctic winter in water temperatures from −1.3 to 7°C with ~8% salinity in a high Arctic winter environment with air temperatures commonly less than −40°C and an average annual air temperature of −15°C. Metagenome sequencing and binning of streamer samples produced a 96% complete Thiomicrorhabdus sp. metagenome-assembled genome representing a possible new species or subspecies. This is the most cold- and salt-extreme source environment for a Thiomicrorhabdus genome yet described. Metaproteomic and metatranscriptomic analysis attributed nearly all gene expression in the streamers to the Thiomicrorhabdus sp. and suggested that it is active in CO2 fixation and oxidation of sulfide to elemental sulfur. In situ geochemical and isotopic analyses of the fractionation of multiple sulfur isotopes determined the biogeochemical transformation of sulfur from its source in Carboniferous evaporites to biotic processes occurring in the sediment and streamers. These complementary molecular tools provided a functional link between the geochemical substrates and the collective traits and activity that define the microbial community's interactions within a unique polar saline habitat where Thiomicrorhabdus-dominated streamers form and flourish.

Over the last 17 years, a large amount of knowledge has been accumulated on various aspects of major histocompatibility complex (MHC) molecules. In conjunction, numerous algorithms and tools have been developed to screen protein molecules for these MHC receptor sites. By combining these computational tools and databases with genomic sequence information that is now widely available for a vast range of organisms, it is possible to screen whole genomes for MHC epitopes. By pre-screening these genomes, it allows the researcher to narrow down possible protein targets for further analysis by traditional tools such as gene knockouts and animal efficacy studies.

An important component in host resistance to malaria infection are inherited mutations that give rise to abnormalities and deficiencies in erythrocyte proteins and enzymes. Understanding how such mutations confer protection against the disease may be useful for developing new treatment strategies. A mouse ENU-induced mutagenesis screen for novel malaria resistance-conferring mutations identified a novel nonsense mutation in the gene encoding porphobilinogen deaminase (PBGD) in mice, denoted here as PbgdMRI58155. Heterozygote PbgdMRI58155 mice exhibited approximately 50% reduction in cellular PBGD activity in both mature erythrocytes and reticulocytes, although enzyme activity was approximately 10 times higher in reticulocytes than erythrocytes. When challenged with blood-stage P. chabaudi, which preferentially infects erythrocytes, heterozygote mice showed a modest but significant resistance to infection, including reduced parasite growth. A series of assays conducted to investigate the mechanism of resistance indicated that mutant erythrocyte invasion by P. chabaudi was normal, but that following intraerythrocytic establishment a significantly greater proportions of parasites died and therefore affected their ability to propagate. The Plasmodium resistance phenotype was not recapitulated in Pbgd-deficient mice infected with P. berghei, which prefers reticulocytes, or when P. falciparum was cultured in erythrocytes from patients with acute intermittent porphyria (AIP), which had modest (20-50%) reduced levels of PBGD. Furthermore, the growth of Pbgd-null P. falciparum and Pbgd-null P. berghei parasites, which grew at the same rate as their wild-type counterparts in normal cells, were not affected by the PBGD-deficient background of the AIP erythrocytes or Pbgd-deficient mice. Our results confirm the dispensability of parasite PBGD for P. berghei infection and intraerythrocytic growth of P. falciparum, but for the first time identify a requirement for host erythrocyte PBGD by P. chabaudi during in vivo blood stage infection.IMPORTANCEThe causative agent of malaria, Plasmodium, adopts a parasitic lifestyle during erythrocyte infection, and as such relies on host cell factors for its survival and growth. Host-encoded mutations that alter the availability of these factors confer disease resistance, including several well-known genetic erythrocyte abnormalities that have arisen due to the historical evolutionary pressure of malaria. This study identified in mice a novel malaria resistance-conferring host mutation in the heme biosynthesis enzyme, porphobilinogen deaminase (PBGD), and compared the relative requirements by Plasmodium for the host versus parasite-encoded forms of PBGD in both in vivo and in vitro settings. The findings demonstrated that parasite PBGD was dispensable, but that the host enzyme was important specifically during in vivo infection by P. chabaudi, and collectively suggest that Plasmodium requires a certain threshold of the enzyme to sustain its intraerythrocytic growth. Plasmodium may therefore be vulnerable to other interventions that limit host PBGD activity.

Over the last 17 years, a large amount of knowledge has been accumulated on various aspects of major histocompatibility complex (MHC) molecules. In conjunction, numerous algorithms and tools have been developed to screen protein molecules for these MHC receptor sites. By combining these computational tools and databases with genomic sequence information that is now widely available for a vast range of organisms, it is possible to screen whole genomes for MHC epitopes. By pre-screening these genomes, it allows the researcher to narrow down possible protein targets for further analysis by traditional tools such as gene knockouts and animal efficacy studies., Series: Methods in Molecular Biology

The factors that determine red blood cell (RBC) lifespan and the rate of RBC aging have not been fully elucidated. In several genetic conditions, including sickle cell disease, thalassemia, and G6PD deficiency, erythrocyte lifespan is significantly shortened. Many of these diseases are also associated with protection from severe malaria, suggesting a role for accelerated RBC senescence and clearance in malaria resistance. Here, we report a novel, N-ethyl-N-nitrosourea-induced mutation that causes a gain of function in adenosine 5'-monophosphate deaminase (AMPD3). Mice carrying the mutation exhibit rapid RBC turnover, with increased erythropoiesis, dramatically shortened RBC lifespan, and signs of increased RBC senescence/eryptosis, suggesting a key role for AMPD3 in determining RBC half-life. Mice were also found to be resistant to infection with the rodent malaria Plasmodium chabaudi. We propose that resistance to P. chabaudi is mediated by increased RBC turnover and higher rates of erythropoiesis during infection.

Platelets are understood to assist host innate immune responses against infection, although direct evidence of this function in any human disease, including malaria, is unknown. Here we characterized platelet–erythrocyte interactions by microscopy and flow cytometry in patients with malaria naturally infected with Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, or Plasmodium knowlesi. Blood samples from 376 participants were collected from malaria-endemic areas of Papua, Indonesia, and Sabah, Malaysia. Platelets were observed binding directly with and killing intraerythrocytic parasites of each of the Plasmodium species studied, particularly mature stages, and was greatest in P vivax patients. Platelets preferentially bound to the infected more than to the uninfected erythrocytes in the bloodstream. Analysis of intraerythrocytic parasites indicated the frequent occurrence of platelet-associated parasite killing, characterized by the intraerythrocytic accumulation of platelet factor-4 and terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick-end labeling of parasite nuclei (PF4+TUNEL+ parasites). These PF4+TUNEL+ parasites were not associated with measures of systemic platelet activation. Importantly, patient platelet counts, infected erythrocyte-platelet complexes, and platelet-associated parasite killing correlated inversely with patient parasite loads. These relationships, taken together with the frequency of platelet-associated parasite killing observed among the different patients and Plasmodium species, suggest that platelets may control the growth of between 5% and 60% of circulating parasites. Platelet–erythrocyte complexes made up a major proportion of the total platelet pool in patients with malaria and may therefore contribute considerably to malarial thrombocytopenia. Parasite killing was demonstrated to be platelet factor-4-mediated in P knowlesi culture. Collectively, our results indicate that platelets directly contribute to innate control of Plasmodium infection in human malaria.

Neoantigen-based therapeutic vaccines have a high potential impact on tumor eradication and patient survival. Mass spectrometry (MS)-based immunopeptidomics has the capacity to identify tumor-associated epitopes and pinpoint mutation-bearing major histocompatibility complex (MHC)-binding peptides. This approach presents several challenges, including the identification of low-abundance peptides. In addition, MHC peptides have much lower MS/MS identification rates than tryptic peptides due to their shorter sequence and lack of basic amino acid at C-termini. In this study, we report the development and application of a novel chemical derivatization strategy that combines the analysis of native, dimethylated, and alkylamidated peptides by liquid chromatography–tandem mass spectrometry (LC–MS/MS) to expand the coverage of the MHC peptidome. The results revealed that dimethylation increases hydrophobicity and ionization efficiency of MHC class I peptides, while alkylamidation significantly improves the fragmentation by producing more y-ions during MS/MS fragmentation. Thus, the combination of dimethylation and alkylamidation enabled the identification of peptides that could not be identified from the analysis of their native form. Using this strategy, we identified 3148 unique MHC I peptides from HCT 116 cell lines, compared to only 1388 peptides identified in their native form. Among these, 10 mutation-bearing peptides were identified with high confidence, indicating that this chemical derivatization strategy is a promising approach for neoantigen discovery in clinical applications.

Clonostachys rosea strain ACM941 is a fungal bio-control agent patented against the causative agent of Fusarium Head Blight, Fusarium graminearum. Although the molecular details remain enigmatic, previous studies have suggested that C. rosea may secrete F. graminearum growth inhibitors. Further toward this, experiments described herein show that induction of C. rosea cultures by the addition of an aliquot of F. graminearum(Fg)-spent media (including macroconidia), yield C. rosea (Cr)-spent media that elicited higher anti-F. graminearum activity than either control or deoxynivalenol (DON)-induced Cr-spent media. To gain additional insight into the genetic and metabolic factors modulating this interaction, transcriptomic (RNAseq) profiles of C. rosea in response to DON and Fg-spent media treatment, were developed. This analysis revealed 24,112 C. rosea unigenes, of which 5,605 and 6,285 were differentially regulated by DON and F-spent media, respectively. More than half of these unigenes were up-regulated, with annotations, most notably in the Fg-spent media treatment data, suggesting enhancement of polyketide (PK) and non-ribosomal peptide (NRP) secondary metabolite precursor synthesis, and PK/NRP-like synthases. Four ABC transporters were also up-regulated in response to Fg-spent media. Further analysis showed that the PK and NRP-like synthases belong to three gene clusters that also include ABC transporters, and other genes known to tailor secondary metabolite biosynthesis. The RNAseq data was further validated using quantitative RT-qPCR. Taken together, these results show that C. rosea responds to the presence of Fg-spent media (and to a lesser extent, DON-alone) by up-regulating unique aspects of its secondary metabolism-related genetic repertoire. The identities and roles of C. rosea secondary metabolites produced by the targeted gene clusters are now under investigation.

In this study, we performed a genome-wide N-ethyl-N-nitrosourea (ENU) mutagenesis screen in mice to identify novel genes or alleles that regulate erythropoiesis. Here, we describe a recessive mouse strain, called RBC19, harbouring a point mutation within the housekeeping gene, Tpi1, which encodes the glycolysis enzyme, triosephosphate isomerase (TPI). A serine in place of a phenylalanine at amino acid 57 severely diminishes enzyme activity in red blood cells and other tissues, resulting in a macrocytic haemolytic phenotype in homozygous mice, which closely resembles human TPI deficiency. A rescue study was performed using bone marrow transplantation of wild-type donor cells, which restored all haematological parameters and increased red blood cell enzyme function to wild-type levels after 7 weeks. This is the first study performed in a mammalian model of TPI deficiency, demonstrating that the haematological phenotype can be rescued., Summary: In a novel ENU mutagenesis mouse model of TPI deficiency, bone marrow transplantation was conducted to demonstrate that haemolytic and red blood cell glycolytic defects can be effectively rescued.

The malaria parasite hijacks host erythrocytes to shield itself from the immune system and proliferate. Red blood cell abnormalities can provide protection from malaria by impeding parasite invasion and growth within the cell or by compromising the ability of parasites to avoid host clearance. Here, we describe 2 N-ethyl-N-nitrosourea-induced mouse lines, SptbMRI26194 and SptbMRI53426 , containing single-point mutations in the erythrocyte membrane skeleton gene, β spectrin (Sptb), which exhibit microcytosis but retain a relatively normal ratio of erythrocyte surface area to volume and are highly resistant to rodent malaria. We propose the major factor responsible for malaria protection is the specific clearance of mutant erythrocytes, although an enhanced clearance of uninfected mutant erythrocytes was also observed (ie, the bystander effect). Using an in vivo erythrocyte tracking assay, we established that this phenomenon occurs irrespective of host environment, precluding the involvement of nonerythrocytic cells in the resistance mechanism. Furthermore, we recapitulated this phenotype by disrupting the interaction between ankyrin-1 and β spectrin in vivo using CRISPR/Cas9 genome editing technology, thereby genetically validating a potential antimalarial target. This study sheds new light on the role of β spectrin during Plasmodium infection and highlights how changes in the erythrocyte cytoskeleton can substantially influence malaria susceptibility with minimal adverse consequences for the host.

Malaria remains a deadly parasitic disease caused by Plasmodium, claiming almost half a million lives every year. While parasite genetics and biology are often the major targets in many studies, it is becoming more evident that host genetics plays a crucial role in the outcome of the infection. Similarly, Plasmodium infections in mice also rely heavily on the genetic background of the mice, and often correlate with observations in human studies, due to their high genetic homology with humans. As such, murine models of malaria are a useful tool for understanding host responses during Plasmodium infections, as well as dissecting host-parasite interactions through various genetic manipulation techniques. Reverse genetic approach such as quantitative trait loci studies and random mutagenesis screens have been employed to discover novel host genes that affect malaria susceptibility in mouse models, while other targeted studies utilize mouse models to validate observation from human studies. Herein, we review the findings from the past and present studies on murine models of hepatic and erythrocytic stages of malaria and speculate on how the current mouse models benefit from the recent development in CRISPR/Cas9 gene editing technology.

Plasmodium falciparum malaria causes half a million deaths per year, with up to 9% of this mortality caused by cerebral malaria (CM). One of the major processes contributing to the development of CM is an excess of host inflammatory cytokines. Recently K+ signaling has emerged as an important mediator of the inflammatory response to infection; we therefore investigated whether mice carrying an ENU induced activation of the electroneutral K+ channel KCC1 had an altered response to Plasmodium berghei. Here we show that Kcc1M935K/M935K mice are protected from the development of experimental cerebral malaria, and that this protection is associated with an increased CD4+ T cells and TNF-α response. This is the first description of a K+ channel affecting the development of experimental cerebral malaria.

Allelic heterogeneity is a common phenomenon where a gene exhibit different phenotype depending on the nature of its genetic mutations. In the context of genes affecting malaria susceptibility, it allowed us to explore and understand the intricate host-parasite interactions during malaria infections. In this study, we described a gene encoding erythrocytic ankyrin-1 (Ank-1) which exhibits allelic-dependent heterogeneous phenotypes during malaria infections. We conducted an ENU mutagenesis screen on mice and identified twoAnk-1mutations, one resulted in an amino acid substitution (MRI95845), and the other a truncatedAnk-1protein (MRI96570). Both mutations caused hereditary spherocytosis-like phenotypes and confer differing protection againstPlasmodium chabaudiinfections. Upon further examination, theAnk-1(MRI96570)mutation was found to inhibit intra-erythrocytic parasite maturation, whereasAnk-1(MW95845)caused increased bystander erythrocyte clearance during infection. This is the first description of allelic heterogeneity in ankyrin-1 from the direct comparison between twoAnk-1mutations. Despite the lack of direct evidence from population studies, this data further supported the protective roles of ankyrin-1 mutations in conferring malaria protection. This study also emphasized the importance of such phenomenon to achieve a better understanding of host-parasite interactions, which could be the basis of future studies.

Griseofulvin, an orally active antifungal drug used to treat dermatophyte infections, has a secondary effect of inducing cytochrome P450-mediated production of N-methyl protoporphyrin IX (N-MPP). N-MPP is a potent competitive inhibitor of the heme biosynthetic-enzyme ferrochelatase, and inhibits the growth of cultured erythrocyte stage Plasmodium falciparum. Novel drugs against Plasmodium are needed to achieve malaria elimination. Thus, we investigated whether griseofulvin shows anti-plasmodial activity. We observed that the intraerythrocytic growth of P. falciparum is inhibited in red blood cells pretreated with griseofulvin in vitro. Treatment with 100 μM griseofulvin was sufficient to prevent parasite growth and induce the production of N-MPP. Inclusion of the ferrochelatase substrate PPIX blocked the inhibitory activity of griseofulvin, suggesting that griseofulvin exerts its activity through the N-MPP-dependent inhibition of ferrochelatase. In an ex-vivo study, red blood cells from griseofulvin-treated subjects were refractory to the growth of cultured P. falciparum. However, in a clinical trial griseofulvin failed to show either therapeutic or prophylactic effect in subjects infected with blood stage P. falciparum. Although the development of griseofulvin as an antimalarial is not warranted, it represents a novel inhibitor of P. falciparum growth and acts via the N-MPP-dependent inhibition of ferrochelatase.

Platelets play an ambiguous role in a malarial infection. On the one hand, platelets have been implicated adversely in cerebral malaria. They stick to the cerebral endothelium and mediate the adhesion of infected erythrocytes, with the postulated outcome of increasing severity or of even mediating this disease. On the other hand, platelets bind to infected red cells in the periphery and activate and release the cytokine PF4. This, in turn, binds to the Duffy antigen on the red cell and is thought to be internalised and enters the parasite food vacuole which it then destroys, killing the parasites. The control of platelet levels during an infection is also discussed with a view to understanding the thrombocytopenia that frequently accompanies a malarial infection.

Telomere length has a biological link to cancer, with excessive telomere shortening leading to genetic instability and resultant malignant transformation. Telomere length is heritable and genetic variants determining telomere length have been identified. Telomere biology has been implicated in the development of hematological malignancies (HMs), therefore, closer examination of telomere length in HMs may provide further insight into genetic etiology of disease development and support for telomere length as a prognostic factor in HMs. We retrospectively examined mean relative telomere length in the Tasmanian Familial Hematological Malignancies Study using a quantitative PCR method on genomic DNA from peripheral blood samples. Fifty-five familial HM cases, 191 unaffected relatives of familial HM cases and 75 non-familial HM cases were compared with 758 population controls. Variance components modeling was employed to identify factors influencing variation in telomere length. Overall, HM cases had shorter mean relative telomere length (p=2.9×10-6) and this was observed across both familial and non-familial HM cases (p=2.2x10-4 and 2.2x10-5, respectively) as well as additional subgroupings of HM cases according to broad subtypes. Mean relative telomere length was also significantly heritable (62.6%; p=4.7x10-5) in the HM families in the present study. We present new evidence of significantly shorter mean relative telomere length in both familial and non-familial HM cases from the same population adding further support to the potential use of telomere length as a prognostic factor in HMs. Whether telomere shortening is the cause of or the result of HMs is yet to be determined, but as telomere length was found to be highly heritable in our HM families this suggests that genetics driving the variation in telomere length is related to HM disease risk.

The proliferation of large biorepositories and the staggering advances in our ability to analyse large numbers of human genomes relatively quickly and cost-effectively have been important drivers in the move towards personalised medicine. However, our advances in the development of these tools have outstripped our performance in addressing the issues of ethics and consent surrounding health policy and governance of such repositories, the implications of proliferation of genomic information for the individual and its potential for misuse. Public consultation is urgently needed in the development of ethical guidelines for these emergent issues; however, effective strategies for facilitating community engagement and informed debate have been lacking. Public consultation through deliberative democracy is bringing an essential new dimension to public engagement in the genomic medicine era.

Genetic defects in various red blood cell (RBC) cytoskeletal proteins have been long associated with changes in susceptibility towards malaria infection. In particular, while ankyrin (Ank-1) mutations account for approximately 50% of hereditary spherocytosis (HS) cases, an association with malaria is not well-established, and conflicting evidence has been reported. We describe a novel N-ethyl-N-nitrosourea (ENU)-induced ankyrin mutation MRI61689 that gives rise to two different ankyrin transcripts: one with an introduced splice acceptor site resulting a frameshift, the other with a skipped exon. Ank-1(MRI61689/+) mice exhibit an HS-like phenotype including reduction in mean corpuscular volume (MCV), increased osmotic fragility and reduced RBC deformability. They were also found to be resistant to rodent malaria Plasmodium chabaudi infection. Parasites in Ank-1(MRI61689/+) erythrocytes grew normally, but red cells showed resistance to merozoite invasion. Uninfected Ank-1(MRI61689/+) erythrocytes were also more likely to be cleared from circulation during infection; the “bystander effect”. This increased clearance is a novel resistance mechanism which was not observed in previous ankyrin mouse models. We propose that this bystander effect is due to reduced deformability of Ank-1(MRI61689/+) erythrocytes. This paper highlights the complex roles ankyrin plays in mediating malaria resistance.

Multiple sclerosis is a common disease of the central nervous system in which the interplay between inflammatory and neurodegenerative processes typically results in intermittent neurological disturbance followed by progressive accumulation of disability. Epidemiological studies have shown that genetic factors are primarily responsible for the substantially increased frequency of the disease seen in the relatives of affected individuals, and systematic attempts to identify linkage in multiplex families have confirmed that variation within the major histocompatibility complex (MHC) exerts the greatest individual effect on risk. Modestly powered genome-wide association studies (GWAS) have enabled more than 20 additional risk loci to be identified and have shown that multiple variants exerting modest individual effects have a key role in disease susceptibility. Most of the genetic architecture underlying susceptibility to the disease remains to be defined and is anticipated to require the analysis of sample sizes that are beyond the numbers currently available to individual research groups. In a collaborative GWAS involving 9,772 cases of European descent collected by 23 research groups working in 15 different countries, we have replicated almost all of the previously suggested associations and identified at least a further 29 novel susceptibility loci. Within the MHC we have refined the identity of the HLA-DRB1 risk alleles and confirmed that variation in the HLA-A gene underlies the independent protective effect attributable to the class I region. Immunologically relevant genes are significantly overrepresented among those mapping close to the identified loci and particularly implicate T-helper-cell differentiation in the pathogenesis of multiple sclerosis.

Multiple Sclerosis (MS) is a chronic inflammatory demyelinating disease of the central nervous system. The risk of developing MS is strongly influenced by genetic predisposition, and over 100 loci have been established as associated with susceptibility. However, the biologically relevant variants underlying disease risk have not been defined for the vast majority of these loci, limiting the power of these genetic studies to define new avenues of research for the development of MS therapeutics. It is therefore crucial that candidate MS susceptibility loci are carefully investigated to identify the biological mechanism linking genetic polymorphism at a given gene to the increased chance of developing MS. MERTK has been established as an MS susceptibility gene and is part of a family of receptor tyrosine kinases known to be involved in the pathogenesis of demyelinating disease. In this study we have refined the association of MERTK with MS risk to independent signals from both common and low frequency variants. One of the associated variants was also found to be linked with increased expression of MERTK in monocytes and higher expression of MERTK was associated with either increased or decreased risk of developing MS, dependent upon HLA-DRB1*15:01 status. This discordant association potentially extended beyond MS susceptibility to alterations in disease course in established MS. This study provides clear evidence that distinct polymorphisms within MERTK are associated with MS susceptibility, one of which has the potential to alter MERTK transcription, which in turn can alter both susceptibility and disease course in MS patients., Author Summary Multiple sclerosis (MS) is the most common neurological disease of young Caucasian adults. Oligodendrocytes are the key cell type damaged in MS, a process that is accompanied by loss of the myelin sheath that these cells produce, resulting in demyelination and ultimately in secondary damage to nerve cells. Susceptibility to MS is strongly influenced by genes, and over 100 genes have now been linked with the risk of developing MS. However, surprisingly little is known about the biological mechanism by which any one of these genes increases the probability of developing MS. In this study we have explored in detail the links between one known MS risk gene, MERTK, and MS susceptibility. We found that a number of different alterations in the MERTK gene are independently associated with the risk of developing MS. One these changes was also linked with changes in the level of expression of MERTK in monocytes, an immune cell type known to be involved in the etiology of MS. In an unexpected result, we found this expression-linked alteration in MERTK was either protective or risk-associated, depending on the genotype of the individual at another well known MS risk gene known as HLA-DRB1. In addition, we found that not only were alterations in MERTK associated with MS susceptibility, but potentially with ongoing disease course, indicating that MERTK may be a good target for the development of novel MS therapeutics.

We show in this study that toxin production in Clostridium difficile is altered in cells which can no longer form flagellar filaments. The impact of inactivation of fliC , CD0240 , fliF , fliG , fliM , and flhB-fliR flagellar genes upon toxin levels in culture supernatants was assessed using cell-based cytotoxicity assay, proteomics, immunoassay, and immunoblotting approaches. Each of these showed that toxin levels in supernatants were significantly increased in a fliC mutant compared to that in the C. difficile 630 parent strain. In contrast, the toxin levels in supernatants secreted from other flagellar mutants were significantly reduced compared with that in the parental C. difficile 630 strain. Transcriptional analysis of the pathogenicity locus genes ( tcdR , tcdB , tcdE , and tcdA ) revealed a significant increase of all four genes in the fliC mutant strain, while transcription of all four genes was significantly reduced in fliM , fliF , fliG , and flhB-fliR mutants. These results demonstrate that toxin transcription in C. difficile is modulated by the flagellar regulon. More significantly, mutant strains showed a corresponding change in virulence compared to the 630 parent strain when tested in a hamster model of C. difficile infection. This is the first demonstration of differential flagellum-related transcriptional regulation of toxin production in C. difficile and provides evidence for elaborate regulatory networks for virulence genes in C. difficile .

We aimed to refine the phenotypic spectrum and map the causative gene in two families with familial focal epilepsy with variable foci (FFEVF). A new five-generation Australian FFEVF family (A) underwent electroclinical phenotyping, and the original four-generation Australian FFEVF family (B) (Ann Neurol, 44, 1998, 890) was re-analyzed, including new affected individuals. Mapping studies examined segregation at the chromosome 22q12 FFEVF region. In family B, the original whole genome microsatellite data was reviewed. Five subjects in family A and 10 in family B had FFEVF with predominantly awake attacks and active EEG studies with a different phenotypic picture from other families. In family B, reanalysis excluded the tentative 2q locus reported. Both families mapped to chromosome 22q12. Our results confirm chromosome 22q12 as the solitary locus for FFEVF. Both families show a subtly different phenotype to other published families extending the clinical spectrum of FFEVF.

Mouse models of allergic asthma are characterized by airway hyperreactivity (AHR), Th2-driven eosinophilic airway inflammation, high allergen-specific IgE (anti-OVA IgE) levels in serum, and airway remodeling. Because asthma susceptibility has a strong genetic component, we aimed to identify new asthma susceptibility genes in the mouse by analyzing the asthma phenotypes of the Leishmania major resistant (lmr) recombinant congenic (RC) strains. The lmr RC strains are derived from C57BL/6 and BALB/c intercrosses and carry congenic loci on chromosome 17 (lmr1) and 9 (lmr2) in both backgrounds. Whereas the lmr2 locus on chromosome 9 contributes to a small background-specific effect on anti-OVA IgE and AHR, the lmr1 locus on chromosome 17 mediates a strong effect on Th2-driven eosinophilic airway inflammation and background-specific effects on anti-OVA IgE and AHR. The lmr1 locus contains almost 600 polymorphic genes. To narrow down this number of candidate genes, we performed genome-wide transcriptional profiling on lung tissue from C. lmr1 RC mice and BALB/c control mice. We identified a small number of differentially expressed genes located within the congenic fragment, including a number of Mhc genes, polymorphic between BALB/c and C57Bl/6. The analysis of asthma phenotypes in the C.B10-H2b RC strain, carrying the C57Bl/6 haplotype of the Mhc locus in a BALB/c genetic background, reveals a strikingly similar asthma phenotype compared with C. lmr1, indicating that the differentially expressed genes located within the C.B10-H2b congenic fragment are the most likely candidate genes to contribute to the reduced asthma phenotypes associated with the C57Bl/6 allele of lmr1.

Absence-like seizures in the Genetic Absence Epilepsy Rats from Strasbourg (GAERS) model are believed to arise in hyperexcitable somatosensory cortical neurons, however the cellular basis of this increased excitability remains unknown. We have previously shown that expression of the Transmembrane AMPA receptor Regulatory Protein (TARP), stargazin, is elevated in the somatosensory cortex of GAERS. TARPs are critical regulators of the trafficking and function of AMPA receptors. Here we examine the developmental expression of stargazin and the impact this may have on AMPA receptor trafficking in the GAERS model. We show that elevated stargazin in GAERS is associated with an increase in AMPA receptor proteins, GluA1 and GluA2 in the somatosensory cortex plasma membrane of adult epileptic GAERS. Elevated stargazin expression is not seen in the epileptic WAG/Rij rat, which is a genetically distinct but phenotypically similar rat model also manifesting absence seizures, indicating that the changes seen in GAERS are unlikely to be a secondary consequence of the seizures. In juvenile (6 week old) GAERS, at the age when seizures are just starting to be expressed, there is elevated stargazin mRNA, but not protein expression for stargazin or the AMPA receptor subunits. In neonatal (7 day old) pre-epileptic GAERS there was no alteration in stargazin mRNA expression in any brain region examined. These data demonstrate that stargazin and AMPA receptor membrane targeting is altered in GAERS, potentially contributing to hyperexcitability in somatosensory cortex, with a developmental time course that would suggest a pathophysiological role in the epilepsy phenotype.

Malaria is a disease that infects over 500 million people, causing at least 1 million deaths every year, with the majority occurring in developing countries. The current antimalarial arsenal is becoming dulled due to the rapid rate of resistance of the parasite. However, in populations living in malaria-endemic regions there are many examples of genetic-based resistance to the severe effects of the parasite Plasmodium. Defining the genetic factors behind host resistance has been an area of great scientific interest over the last few decades; this review summarizes the current knowledge of the genetic loci involved. Perhaps the lessons learned from the natural variation in both the human populations and experimental mouse models of infection may pave the way for novel resistance-proof antimalarials.

Genetic linkage studies of the host response to Leishmania major , the causative agent of cutaneous leishmaniasis, have identified significant genetic complexity in humans and mice. In the mouse model, multiple loci have been implicated in susceptibility to infection, but to date, the genes underlying these loci have not been identified. We now describe the contribution of a novel candidate gene, Fli1 , to both L. major resistance and enhanced wound healing. We have previously mapped the L. major response locus, lmr2 , to proximal chromosome 9 in a genetic cross between the resistant C57BL/6 strain and the susceptible BALB/c strain. We now show that the presence of the resistant C57BL/6 lmr2 allele in susceptible BALB/c mice confers an enhanced L. major resistance and wound healing phenotype. Fine mapping of the lmr2 locus permitted the localization of the lmr2 quantitative trait locus to a 5-Mb interval comprising 21 genes, of which microarray analysis was able to identify differential expression in 1 gene— Fli1 . Analysis of Fli1 expression in wounded and L. major -infected skin and naïve and infected lymph nodes validated the importance of Fli1 in lesion resolution and wound healing and identified 3 polymorphisms in the Fli1 promoter, among which a GA repeat element may be the important contributor.

The risk for development of multiple sclerosis has been associated with human leukocyte antigen-DRB1*1501-DQB1*0602 (HLA-DR15) genotype, low infant sibling exposure, and high Epstein-Barr nuclear antigen IgG levels. In a population-based case-control study (Tasmania, Australia), we found that the combined effect of HLA-DR15 positivity and low infant sibling exposure on multiple sclerosis (odds ratio, 7.88; 95% confidence interval, 3.43-18.11) was 3.9-fold greater than expected (test for interaction, p = 0.019) This interaction was observed irrespective of Epstein-Barr nuclear antigen IgG levels. This suggests that immune mechanisms involving HLA class II molecules are susceptible to modulation in early life. Ann Neurol 2009;66:261-265 ANN NEUROL 2010;67:259-263.

Background: The influence of APOE allelic heterogeneity on multiple sclerosis (MS) disease severity has been reported in multiple datasets with conflicting results. Several studies have reported an unfavorable association of APOE e4 with more severe clinical disease course while, in contrast, APOE e2 has been associated with a more benign disease course. In this study, we examine the influence of heterogeneity of the APOE gene on disease severity in a large, Australian, population-based MS cohort. Methods: Associations between APOE allele status, 2 promoter region single nucleotide polymorphisms (−219 G/T and +113 C/G), and 4 measures of disease severity were tested in 1,006 patients with relapsing-remitting MS and secondary progressive MS: 1) Multiple Sclerosis Severity Score; 2) Progression Index (Expanded Disability Status Scale/disease duration); 3) age at first symptom; and 4) interval between the first and second attack. The Symbol Digit Modalities Test was used as a single cognitive marker in 889 patients. Brain atrophy was measured in 792 patients using the intercaudate ratio. APOE e4 and e3 carriers were stratified by −219 G/T or +113 C/G to investigate haplotypic heterogeneity in the APOE gene region. Results: In this MS study, neither APOE allele status nor promoter region heterogeneity at positions −219 G/T or +113 C/G influenced the clinical disease severity, cognition, or cerebral atrophy. Conclusions: Allelic and haplotypic heterogeneity of the APOE gene region does not influence multiple sclerosis disease course in this well-defined Australian multiple sclerosis cohort.

Leishmaniasis is one of the world's important infectious diseases. It is prevalent in tropical and subtropical regions of the world and endemic in 88 countries, with two million new cases of leishmaniasis reported annually. As a complex disease, the pathology of leishmaniasis varies and is determined by factors such as the environment, the insect vector, and parasite and host genetics. The contributing host genetics involve multiple genes; thus, the mouse model of leishmaniasis has been exploited extensively in an attempt to identify and dissect the contribution of disease modifier genes to pathogenesis. This review summarizes recent advances in the identification of genetic loci involved in the host response to Leishmania spp. in the mouse model and in the human situation.

Human leucocyte antigen (HLA)-DRB1*1501 and other class II alleles influence susceptibility to multiple sclerosis (MS), but their contribution if any to the clinical course of MS remains uncertain. Here, we have investigated DRB1 alleles in a large sample of 1230 Australian MS cases, with some enrichment for subjects with primary progressive (PPMS) disease (n = 246) and 1210 healthy controls. Using logistic regression, we found that DRB1*1501 was strongly associated with risk (P = 7 x 10(-45)), as expected, and after adjusting for DRB1*1501, a predisposing effect was also observed for DRB1*03 (P = 5 x 10(-7)). Individuals homozygous for either DRB1*15 or DRB1*03 were considerably more at risk of MS than heterozygotes and non-carriers. Both the DRB1*04 and the DRB1*01/DRB1*15 genotype combination, respectively, protected against PPMS in comparison to subjects with relapsing disease. Together, these data provide further evidence of heterogeneity at the DRB1 locus and confirm the importance of HLA variants in the phenotypic expression of MS.

Insights into the role of ankyrin-1 (ANK-1) in the formation and stabilization of the red cell cytoskeleton have come from studies on the nb/nb mice, which carry hypomorphic alleles of Ank-1. Here, we revise several paradigms established in the nb/nb mice through analysis of an N-ethyl-N-nitrosourea (ENU)–induced Ank-1–null mouse. Mice homozygous for the Ank-1 mutation are profoundly anemic in utero and most die perinatally, indicating that Ank-1 plays a nonredundant role in erythroid development. The surviving pups exhibit features of severe hereditary spherocytosis (HS), with marked hemolysis, jaundice, compensatory extramedullary erythropoiesis, and tissue iron overload. Red cell membrane analysis reveals a complete loss of ANK-1 protein and a marked reduction in β-spectrin. As a consequence, the red cells exhibit total disruption of cytoskeletal architecture and severely altered hemorheologic properties. Heterozygous mutant mice, which have wild-type levels of ANK-1 and spectrin in their RBC membranes and normal red cell survival and ultrastructure, exhibit profound resistance to malaria, which is not due to impaired parasite entry into RBC. These findings provide novel insights into the role of Ank-1, and define an ideal model for the study of HS and malarial resistance.

Genetic heterogeneity is a difficulty frequently encountered in the search for genes conferring susceptibility to prostate cancer. To circumvent this issue, we selected a large prostate cancer pedigree for genome-wide linkage analysis from a population that is genetically homogeneous. Selected cases and first-degree relatives were genotyped with Affymetrix 10K SNP arrays, identifying a 14 Mb haplotype on chromosome 5 (5p13-q12) inherited identical-by-descent (IBD) by multiple cases. Microsatellite genotyping of additional deceased case samples confirmed that a total of eight cases inherited the common haplotype (P=0.0017). Re-sequencing of eight prioritised candidate genes in the region in six selected individuals identified 15 SNPs segregating with the IBD haplotype, located within the ITGA2 gene. Three of these polymorphisms were selected for genotyping in an independent Tasmanian data set comprising 127 cases with familial prostate cancer, 412 sporadic cases and 319 unaffected controls. Two were associated with prostate cancer risk: rs3212649 (OR=1.67 (1.07-2.6), P=0.0009) and rs1126643 (OR=1.52 (1.01-2.28), P=0.0088). Significant association was observed in both familial and sporadic prostate cancer. Although the functional SNP remains to be identified, considerable circumstantial evidence, provided by in vivo and in vitro studies, supports a role for ITGA2 in tumour development.

PURPOSE: Studies in animal models and patients have implicated changes in hyperpolarization-activated cyclic nucleotide-gated cation channel (HCN) expression in the pathogenesis of temporal lobe epilepsy (TLE). However, the nature of HCN changes during the epileptogenic process and their commonality across different TLE models is unknown. Here HCN1 and HCN2 mRNA expression was quantitatively measured at different time points during epileptogenesis in two distinct animal models of TLE; the kainic acid (KA)-induced status epilepticus (SE) and amygdala kindling models. METHODS: Hippocampal subregions (CA1, CA3, and dentate gyrus [DG]) and entorhinal cortex were dissected at different time-points. For KA-induced SE animals this was 24 h, 7 days (preepileptic), and 6 weeks (epileptic) post status. For amygdala kindling animals this was 2 weeks after reaching either "partially kindled" (one class II/III seizure) or "fully kindled" (five class V seizures) states. Quantification of regional hippocampal neuronal loss in the KA-treated animals was done using NeuN immunofluorescence and confocal microscopy. RESULTS: HCN mRNA levels decreased in an isoform and region specific manner at all time points after KA-induced SE. The decrease in neuronal number could not account for all reductions in HCN mRNA levels post-KA insult, implicating transcriptional changes. A reduction in HCN2 mRNA levels was also observed in fully kindled animals in the CA3 region. CONCLUSIONS: A reduction in HCN mRNA levels is present in two different models of TLE. This supports the case that a reduction in HCN channel expression is an accompaniment of epileptogenesis in different adult models of TLE.