Novel genetic markers for chronic kidney disease in a geographically isolated population of Indigenous Australians: Individual and multiple phenotype genome-wide association study.

BACKGROUND: Chronic kidney disease (CKD) is highly prevalent among Indigenous Australians, especially those in remote regions. The Tiwi population has been isolated from mainland Australia for millennia and exhibits unique genetic characteristics that distinguish them from other Indigenous and non-Indigenous populations. Notably, the rate of end-stage renal disease is up to 20 times greater in this population compared to non-Indigenous populations. Despite the identification of numerous genetic loci associated with kidney disease through GWAS, the Indigenous population such as Tiwi remains severely underrepresented and the increased prevalence of CKD in this population may be due to unique disease-causing alleles/genes. METHODS: We used albumin-to-creatinine ratio (ACR) and estimated glomerular filtration rate (eGFR) to estimate the prevalence of kidney disease in the Tiwi population (N = 492) in comparison to the UK Biobank (UKBB) (N = 134,724) database. We then performed an exploratory factor analysis to identify correlations among 10 CKD-related phenotypes and identify new multi-phenotype factors. We subsequently conducted a genome-wide association study (GWAS) on all single and multiple phenotype factors using mixed linear regression models, adjusted for age, sex, population stratification, and genetic relatedness between individuals. RESULTS: Based on ACR, 20.3% of the population was at severely increased risk of CKD progression and showed elevated levels of ACR compared to the UKBB population independent of HbA1c. A GWAS of ACR revealed novel association loci in the genes MEG3 (chr14:100812018:T:A), RAB36 (rs11704318), and TIAM2 (rs9689640). Additionally, multiple phenotypes GWAS of ACR, eGFR, urine albumin, and serum creatinine identified a novel variant that mapped to the gene MEIS2 (chr15:37218869:A:G). Most of the identified variants were found to be either absent or rare in the UKBB population. CONCLUSIONS: Our study highlights the Tiwi population's predisposition towards elevated ACR, and the collection of novel genetic variants associated with kidney function. These associations may prove valuable in the early diagnosis and treatment of renal disease in this underrepresented population. Additionally, further research is needed to comprehensively validate the functions of the identified variants/genes.

Platelets mediate the clearance of senescent red blood cells by forming prophagocytic platelet-cell complexes.

ABSTRACT: In humans, ∼0.1% to 0.3% of circulating red blood cells (RBCs) are present as platelet-RBC (P-RBC) complexes, and it is 1% to 2% in mice. Excessive P-RBC complexes are found in diseases that compromise RBC health (eg, sickle cell disease and malaria) and contribute to pathogenesis. However, the physiological role of P-RBC complexes in healthy blood is unknown. As a result of damage accumulated over their lifetime, RBCs nearing senescence exhibit physiological and molecular changes akin to those in platelet-binding RBCs in sickle cell disease and malaria. Therefore, we hypothesized that RBCs nearing senescence are targets for platelet binding and P-RBC formation. Confirming this hypothesis, pulse-chase labeling studies in mice revealed an approximately tenfold increase in P-RBC complexes in the most chronologically aged RBC population compared with younger cells. When reintroduced into mice, these complexes were selectively cleared from the bloodstream (in preference to platelet-free RBC) through the reticuloendothelial system and erythrophagocytes in the spleen. As a corollary, patients without a spleen had higher levels of complexes in their bloodstream. When the platelet supply was artificially reduced in mice, fewer RBC complexes were formed, fewer erythrophagocytes were generated, and more senescent RBCs remained in circulation. Similar imbalances in complex levels and senescent RBC burden were observed in humans with immune thrombocytopenia (ITP). These findings indicate that platelets are important for binding and clearing senescent RBCs, and disruptions in platelet count or complex formation and clearance may negatively affect RBC homeostasis and may contribute to the known risk of thrombosis in ITP and after splenectomy.

The landscape of genomic structural variation in Indigenous Australians.

Indigenous Australians harbour rich and unique genomic diversity. However, Aboriginal and Torres Strait Islander ancestries are historically under-represented in genomics research and almost completely missing from reference datasets1-3. Addressing this representation gap is critical, both to advance our understanding of global human genomic diversity and as a prerequisite for ensuring equitable outcomes in genomic medicine. Here we apply population-scale whole-genome long-read sequencing4 to profile genomic structural variation across four remote Indigenous communities. We uncover an abundance of large insertion-deletion variants (20-49 bp; n = 136,797), structural variants (50 b-50 kb; n = 159,912) and regions of variable copy number (>50 kb; n = 156). The majority of variants are composed of tandem repeat or interspersed mobile element sequences (up to 90%) and have not been previously annotated (up to 62%). A large fraction of structural variants appear to be exclusive to Indigenous Australians (12% lower-bound estimate) and most of these are found in only a single community, underscoring the need for broad and deep sampling to achieve a comprehensive catalogue of genomic structural variation across the Australian continent. Finally, we explore short tandem repeats throughout the genome to characterize allelic diversity at 50 known disease loci5, uncover hundreds of novel repeat expansion sites within protein-coding genes, and identify unique patterns of diversity and constraint among short tandem repeat sequences. Our study sheds new light on the dimensions and dynamics of genomic structural variation within and beyond Australia.

The pharmacogenomic landscape of an Indigenous Australian population.

Background: Population genomic studies of individuals of Indigenous ancestry have been extremely limited comprising <0.5% of participants in international genetic databases and genome-wide association studies, contributing to a "genomic gap" that limits their access to personalised medicine. While Indigenous Australians face a high burden of chronic disease and associated medication exposure, corresponding genomic and drug safety datasets are sorely lacking. Methods: To address this, we conducted a pharmacogenomic study of almost 500 individuals from a founder Indigenous Tiwi population. Whole genome sequencing was performed using short-read Illumina Novaseq6000 technology. We characterised the pharmacogenomics (PGx) landscape of this population by analysing sequencing results and associated pharmacological treatment data. Results: We observed that every individual in the cohort carry at least one actionable genotype and 77% of them carry at least three clinically actionable genotypes across 19 pharmacogenes. Overall, 41% of the Tiwi cohort were predicted to exhibit impaired CYP2D6 metabolism, with this frequency being much higher than that for other global populations. Over half of the population predicted an impaired CYP2C9, CYP2C19, and CYP2B6 metabolism with implications for the processing of commonly used analgesics, statins, anticoagulants, antiretrovirals, antidepressants, and antipsychotics. Moreover, we identified 31 potentially actionable novel variants within Very Important Pharmacogenes (VIPs), five of which were common among the Tiwi. We further detected important clinical implications for the drugs involved with cancer pharmacogenomics such as thiopurines and tamoxifen, immunosuppressants like tacrolimus and certain antivirals used in the hepatitis C treatment due to potential differences in their metabolic processing. Conclusion: The pharmacogenomic profiles generated in our study demonstrate the utility of pre-emptive PGx testing and have the potential to help guide the development and application of precision therapeutic strategies tailored to Tiwi Indigenous patients. Our research provides valuable insights on pre-emptive PGx testing and the feasibility of its use in ancestrally diverse populations, emphasizing the need for increased diversity and inclusivity in PGx investigations.

Development of Antiplasmodial Peptide-Drug Conjugates Using a Human Protein-Derived Cell-Penetrating Peptide with Selectivity for Infected Cells.

Malaria continues to impose a global health burden. Drug-resistant parasites have emerged to each introduced small-molecule therapy, highlighting the need for novel treatment approaches for the future eradication of malaria. Herein, targeted drug delivery with peptide-drug conjugates (PDCs) was investigated as an alternative antimalarial therapy, inspired by the success of emerging antibody-drug conjugates utilized in cancer treatment. A synthetic peptide derived from an innate human defense molecule was conjugated to the antimalarial drug primaquine (PQ) to produce PDCs with low micromolar potency toward Plasmodium falciparum in vitro. A suite of PDCs with different design features was developed to identify optimal conjugation site and investigate linker length, hydrophilicity, and cleavability. Conjugation within a flexible spacer region of the peptide, with a cleavable linker to liberate the PQ cargo, was important to retain activity of the peptide and drug.

Interactions with Asialo-Glycoprotein Receptors and Platelets Are Dispensable for CD8+ T Cell Localization in the Murine Liver.

Liver-resident CD8+ T cells can play critical roles in the control of pathogens, including Plasmodium and hepatitis B virus. Paradoxically, it has also been proposed that the liver may act as the main place for the elimination of CD8+ T cells at the resolution of immune responses. We hypothesized that different adhesion processes may drive residence versus elimination of T cells in the liver. Specifically, we investigated whether the expression of asialo-glycoproteins (ASGPs) drives the localization and elimination of effector CD8+ T cells in the liver, while interactions with platelets facilitate liver residence and protective function. Using murine CD8+ T cells activated in vitro, or in vivo by immunization with Plasmodium berghei sporozoites, we found that, unexpectedly, inhibition of ASGP receptors did not inhibit the accumulation of effector cells in the liver, but instead prevented these cells from accumulating in the spleen. In addition, enforced expression of ASGP on effector CD8+ T cells using St3GalI-deficient cells lead to their loss from the spleen. We also found, using different mouse models of thrombocytopenia, that severe reduction in platelet concentration in circulation did not strongly influence the residence and protective function of CD8+ T cells in the liver. These data suggest that platelets play a marginal role in CD8+ T cell function in the liver. Furthermore, ASGP-expressing effector CD8+ T cells accumulate in the spleen, not the liver, prior to their destruction.

The genomic landscape of blood groups in Indigenous Australians in remote communities.

BACKGROUND: Red blood cell (RBC) membrane-associated blood group systems are clinically significant. Alloimmunisation is a persistent risk associated with blood transfusion owing to the antigen polymorphisms among these RBC-associated blood groups. Next-generation sequencing (NGS) offers an opportunity to characterize the blood group variant profile of a given individual. Australia comprises a large multiethnic population where most blood donors are Caucasian and blood group variants remain poorly studied among Indigenous Australians. In this study, we focused on the Tiwi Islanders, who have lived in relative isolation for thousands of years. METHODS AND MATERIALS: We predicted the blood group phenotype profiles in the Tiwi (457) and 1000 Genomes Phase 3 (1KGP3-2504) cohort individuals using RBCeq (https://www.rbceq.org/). The predicted phenotype prevalence was compared with the previous literature report. RESULTS: We report, for the first time, comprehensive blood group profiles corresponding to the 35 known blood group systems among the Indigenous Tiwi islander population and identify possible novel antigen variants therein. Our results demonstrate that the genetic makeup of the Tiwi participants is distinct from that of other populations, with a low prevalence of LU (Au[a-b+]) and ABO (A2) and D+C+c+E+e- phenotype, an absence of Diego blood group variants, and a unique RHD (DIII type4) variant. CONCLUSION: Our results may contribute to the development of a database of predicted phenotype donors among the Tiwi population and aid in improving transfusion safety for the ~2800 Tiwi people and the ~800,000 other Indigenous Australians throughout the nation.

Pharmacogenomic analysis of a genetically distinct Indigenous population.

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.

Host Porphobilinogen Deaminase Deficiency Confers Malaria Resistance in Plasmodium chabaudi but Not in Plasmodium berghei or Plasmodium falciparum During Intraerythrocytic Growth.

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 non-sense mutation in the gene encoding porphobilinogen deaminase (PBGD) in mice, denoted here as PbgdMRI58155. Heterozygote PbgdMRI58155 mice exhibited ~50% reduction in cellular PBGD activity in both mature erythrocytes and reticulocytes, although enzyme activity was ~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.

Equitable Expanded Carrier Screening Needs Indigenous Clinical and Population Genomic Data.

Expanded carrier screening (ECS) for recessive monogenic diseases requires prior knowledge of genomic variation, including DNA variants that cause disease. The composition of pathogenic variants differs greatly among human populations, but historically, research about monogenic diseases has focused mainly on people with European ancestry. By comparison, less is known about pathogenic DNA variants in people from other parts of the world. Consequently, inclusion of currently underrepresented Indigenous and other minority population groups in genomic research is essential to enable equitable outcomes in ECS and other areas of genomic medicine. Here, we discuss this issue in relation to the implementation of ECS in Australia, which is currently being evaluated as part of the national Government's Genomics Health Futures Mission. We argue that significant effort is required to build an evidence base and genomic reference data so that ECS can bring significant clinical benefit for many Aboriginal and/or Torres Strait Islander Australians. These efforts are essential steps to achieving the Australian Government's objectives and its commitment "to leveraging the benefits of genomics in the health system for all Australians." They require culturally safe, community-led research and community involvement embedded within national health and medical genomics programs to ensure that new knowledge is integrated into medicine and health services in ways that address the specific and articulated cultural and health needs of Indigenous people. Until this occurs, people who do not have European ancestry are at risk of being, in relative terms, further disadvantaged.

Analysis of the F2LR3 (PAR4) Single Nucleotide Polymorphism (rs773902) in an Indigenous Australian Population.

The F2RL3 gene encoding protease activated receptor 4 (PAR4) contains a single nucleotide variant, rs773902, that is functional. The resulting PAR4 variants, Thr120, and Ala120, are known to differently affect platelet reactivity to thrombin. Significant population differences in the frequency of the allele indicate it may be an important determinant in the ethnic differences that exist in thrombosis and hemostasis, and for patient outcomes to PAR antagonist anti-platelet therapies. Here we determined the frequency of rs773902 in an Indigenous Australian group comprising 467 individuals from the Tiwi Islands. These people experience high rates of renal disease that may be related to platelet and PAR4 function and are potential recipients of PAR-antagonist treatments. The rs773902 minor allele frequency (Thr120) in the Tiwi Islanders was 0.32, which is similar to European and Asian groups and substantially lower than Melanesians and some African groups. Logistic regression and allele distortion testing revealed no significant associations between the variant and several markers of renal function, as well as blood glucose and blood pressure. These findings suggest that rs773902 is not an important determinant for renal disease in this Indigenous Australian group. However, the relationships between rs773902 genotype and platelet and drug responsiveness in the Tiwi, and the allele frequency in other Indigenous Australian groups should be evaluated.

Author Correction: KCC1 Activation protects Mice from the Development of Experimental Cerebral Malaria.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

KCC1 Activation protects Mice from the Development of Experimental Cerebral Malaria.

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+ and TNFa response. This is the first description of a K+ channel affecting the development of experimental cerebral malaria.

Defense Peptides Engineered from Human Platelet Factor 4 Kill Plasmodium by Selective Membrane Disruption.

Malaria is a serious threat to human health and additional classes of antimalarial drugs are greatly needed. The human defense protein, platelet factor 4 (PF4), has intrinsic antiplasmodial activity but also undesirable chemokine properties. We engineered a peptide containing the isolated PF4 antiplasmodial domain, which through cyclization, retained the critical structure of the parent protein. The peptide, cPF4PD, killed cultured blood-stage Plasmodium falciparum with low micromolar potency by specific disruption of the parasite digestive vacuole. Its mechanism of action involved selective penetration and accumulation inside the intraerythrocytic parasite without damaging the host cell or parasite membranes; it did not accumulate in uninfected cells. This selective activity was accounted for by observations of the peptide's specific binding and penetration of membranes with exposed negatively charged phospholipid headgroups. Our findings highlight the tremendous potential of the cPF4PD scaffold for developing antimalarial peptide drugs with a distinct and selective mechanism of action.

Platelets kill circulating parasites of all major Plasmodium species in human malaria.

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.

Host genetics in malaria: lessons from mouse studies.

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.

Ankyrin-1 Gene Exhibits Allelic Heterogeneity in Conferring Protection Against Malaria.

Allelic heterogeneity is a common phenomenon where a gene exhibits a 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 two Ank-1 mutations, one resulting in an amino acid substitution (MRI95845), and the other a truncated Ank-1 protein (MRI96570). Both mutations caused hereditary spherocytosis-like phenotypes and confer differing protection against Plasmodium chabaudi infections. Upon further examination, the Ank-1(MRI96570) mutation was found to inhibit intraerythrocytic parasite maturation, whereas Ank-1(MRI95845) caused increased bystander erythrocyte clearance during infection. This is the first description of allelic heterogeneity in ankyrin-1 from the direct comparison between two Ank-1 mutations. 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 phenomena in achieving a better understanding of host-parasite interactions, which could be the basis of future studies.

Griseofulvin impairs intraerythrocytic growth of Plasmodium falciparum through ferrochelatase inhibition but lacks activity in an experimental human infection study.

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.

Erythrocyte ß spectrin can be genetically targeted to protect mice from malaria.

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, ß 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 ß 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 ß spectrin during Plasmodium infection and highlights how changes in the erythrocyte cytoskeleton can substantially influence malaria susceptibility with minimal adverse consequences for the host.

A novel ENU-induced ankyrin-1 mutation impairs parasite invasion and increases erythrocyte clearance during malaria infection in mice.

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.