Herbicides with novel modes of action are urgently needed to safeguard global agricultural industries against the damaging effects of herbicide-resistant weeds. We recently developed the first herbicidal inhibitors of lysine biosynthesis, which provided proof-of-concept for a promising novel herbicide target. In this study, we expanded upon our understanding of the mode of action of herbicidal lysine biosynthesis inhibitors. We previously postulated that these inhibitors may act as proherbicides. Here, we show this is not the case. We report an additional mode of action of these inhibitors, through their inhibition of a second lysine biosynthesis enzyme, and investigate the molecular determinants of inhibition. Furthermore, we extend our herbicidal activity analyses to include a weed species of global significance. Effective herbicides are critical for sustainable agriculture. However, our current options are dwindling as the prevalence of herbicide-resistant weeds continues to rise . Weeds hZ)-2-(5-(4-methoxybenzylidene)-2,4-dioxothiazolidin-3-yl)acetic acid (MBDTA-2) (Despite the success of targeting amino acid biosynthesis enzymes for the development of herbicides (e.g. glyphosate and chlorsulfuron), the inhibition of plant lysine biosynthesis has never been explored commercially. Our group was the first to discover inhibitors of lysine biosynthesis with herbicidal activity . We showMBDTA-2)  targets MBDTA-2) . InteresMBDTA-2) , which eMBDTA-2) .in vitro potency of MBDTA-2 against recombinant Arabidopsis thaliana (At) DHDPS enzymes was similar to the activity against agar-grown A. thaliana . Static docking and biochemical assays revealed that in contrast to the allosteric binding of these inhibitors to DHDPS, active site binding is responsible for their inhibition of DHDPR. Additionally, we extended our previous in vivo activity studies on A. thaliana to include one of the most agriculturally problematic weeds in the world, rigid ryegrass (Lolium rigidum) (The present study sought to extend our understanding of the mode of action of our previously developed DHDPS inhibitors . Specifirigidum) .in vivo to produce compounds with greater potency at the target site, it can be assumed that the metabolised form will have greater activity than the proherbicidal form against the target in vitro. As such, to assess whether MBDTA-2 is a proherbicide that is demethylated in vivo, we measured the inhibitory activity of the demethylated analogue, (Z)-2-(5-(4-hydroxybenzylidene)-2,4-dioxothiazolidin-3-yl)acetic acid (HBDTA), against both recombinant A. thaliana DHDPS enzymes  = 63.3 ± 1.80 µM, IC50 (AtDHDPS2) = 64.0 ± 1.00 µM) (in vivo.Given that proherbicides are metabolised  enzymes . The dosectively . These v1.00 µM) . These din vitro and in vivo activity of MBDTA-2 could not be explained by enhanced target site activity of the demethylated compound, we sought to explore other mechanisms that may explain this observation.Given that the similarity in the in vivo potency relative to the in vitro activity against the target enzyme. It is well established that proteins catalysing sequential reactions within a metabolic pathway often have conserved binding site features  for generation of the DHDP substrate. The relevant DHDPR isoform (2.6 µg ml−1) was added to initiate the reaction, and substrate turnover measured spectrophotometrically at 340 nm via the associated oxidation of the cofactor NADPH. Experiments were performed in technical triplicates.DHDPS enzyme activity was measured using methods previously described . DHDPR eThe AtDHDPR2 crystal structure was retrieved from the Protein Data Bank and hydrogens added using AutoDock Tools. Three-dimensional MBDTA-2 was docked with AtDHDPR2 using an unlimited search space in the PyRX interface using AutoDock Vina with default parameters. The resulting ligand poses were visualised in PyMol.L. rigidum was assessed using methods similar to those reported previously (2O containing 0.01% (vol/vol) Agral  to a final DMSO concentration of 2% (vol/vol). Treatments were given by pipetting 2.0 ml of MBDTA-2, vehicle control or positive control  directly onto seeds upon sowing and on each of the subsequent 2 days. Plants were grown in a chamber at 22℃ under a 16 hr light (100 µmol m−2 s−1)/8 hr dark schedule for 14 days before photos were taken. Roots and shoots were separated prior to drying at 70℃ for 72 hr. Experiments were performed in biological triplicates.The herbicidal efficacy of MBDTA-2 against eviously . Pre-wet This paper presents the highly interesting and novel finding that the investigated compound inhibits two targets in lysine synthesis. Further, the first enzyme has allosteric inhibition, while the second enzyme is a competitive inhibitor. The authors do a good job explaining why this is high interest for herbicide resistance management in a new compound. The authors demonstrated that the compound is not a pro-herbicide, and propose that the higher in vivo activity relative to in vitro activity is due to the simultaneous in vivo inhibition of two separate steps in lysine synthesis. public reviews designed to be posted alongside the preprint for the benefit of readers; (ii) feedback on the manuscript for the authors, including requests for revisions, shown below. We also include an acceptance summary that explains what the editors found interesting or important about the work.Our editorial process produces two outputs: (i) Decision letter after peer review:eLife. Your article has been reviewed by 3 peer reviewers, including Todd Gaines as Reviewing Editor and Reviewer #1, and the evaluation has been overseen by Detlef Weigel as the Senior Editor. The following individual involved in review of your submission has agreed to reveal their identity: Paul Schmitzer (Reviewer #2).Thank you for submitting your article "A dual-target herbicidal inhibitor of lysine biosynthesis" for consideration by The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.Essential revisions:1) Please provide the chlorsulfuron positive control data for the Lolium rigidum bioassay figure. Alter the claims for biological activity on Lolium rigidum and remove the extrapolated application rate, or conduct a new bioassay with methods suggested in the reviews.2) The question arose in the reviews as to whether the compound is a pro-herbicide with respect to DHPDR. Please alter the discussion to raise this question, or consider conducting the experiment to add the demethylated compound to the DHPDR inhibition assay to test its activity.Reviewer #1 (Recommendations for the authors):Excellent paper. The authors could include a reference to work in fungicides to provide further evidence that multi-site inhibitors are lower risk for resistance evolution than single-site inhibitors. The bioassay method used for Lolium rigidum is unusual. The authors pipetted 2 mL of a 1200 mg/L solution directly onto seeds when sowing. Alternatives that could be used would include soaking seeds for 24 hour in herbicide solution; using an agarose based assay; spraying post-emergence on emerged seedlings; or spraying directly onto seeds or seedlings in soil. The authors report a calculated application equivalent of 48 kg/ha. It is not clear how this extrapolated number is obtained, nor is it realistic. No pesticide could be applied at such a high rate , and the sheer volume of water application required to achieve that would also represent drenching a field. I suggest that the authors remove this extrapolated calculation, and perhaps instead discuss the relative activity/toxicity of their compound in comparison to other known herbicides based on in vitro inhibition of the two enzymes. I don't think that more experiments are needed for this paper, but future work could conduct bioassays using methods that would provide more accurate ways to extrapolate to potential field use rates. Future work could also conduct biochemical measurements to ask whether lysine is reduced in Lolium rigidum following treatment with this herbicide, supporting the prediction that the mode of action is two-site inhibition of lysine synthesis. I also suggest that the authors provide more methods in the figure caption for Figure 5 so that it stand alone. Currently the caption only states the application concentration of 1200 mg/L, but it does not provide the application volume of 2 mL pipetted onto each seed, so in the current presentation the reader must examine the methods to understand the total dose applied to the plants. Suggest adding the 2 mL pipetting onto each seed to the caption.Reviewer #2 (Recommendations for the authors):The paper was a joy to read as it represents many processes in the design of new herbicides. I don't like creating additional work but through my review of the paper I kept wondering if the analogs presented in your previous publications (FBDTA and MBDTA-1), as well as any other available analogs, could build the knowledge of the inhibition of DHDPR and show a limited SAR. This may be a topic for a future publication so please disregard it if not appropriate for this paper.Reviewer #3 (Recommendations for the authors):This is a well-written description of experiments to further determine the mode of action of novel lysine synthesis inhibitor. The expansion of the mode of action to two consecutive enzymes of the lysine synthesis pathway is a surprising result, especially with different binding modes for the two enzymes. Again, the in vitro activity MBDTA-2 is rather poor for an effective herbicide. Perhaps inhibition of two enzymes makes up for the poor activity on each of the enzymes. Was the demethylated form of the compound tested in an in vitro assay of AtDHDPR1 and AtDHDP2? This must be done. If HBDTA is much more active than MBDTA-2 on DHDPR, the results would make more sense to me.The bioassay method is very weak, as it is impossible to determine whether MBDTA-2 is an effective herbicide from these results. Herbicides are not pipetted onto weed seeds in the field. MBDTA-2 was applied three times, on three consecutive days. In the field, a herbicide is applied once. I suggest that the compounds be applied once to the soil containing weeds seeds and watered into soil, as soil applied herbicide are used, or applied to foliage as a foliar spray with an adjuvant. I would try both methods, as some herbicides that are excellent soil-applied herbicides are poor foliar herbicides and vice versa. This way, you can compare the efficacy in g/ha with the positive control. Finally, herbicides are expected to kill weeds. Figure 5 shows only growth stunting. Where are the chlorsulfuron results? Essential revisions:1) Please provide the chlorsulfuron positive control data for the Lolium rigidum bioassay figure. Alter the claims for biological activity on Lolium rigidum and remove the extrapolated application rate, or conduct a new bioassay with methods suggested in the reviews.-1 of chlorsulfuron.’ The claims for biological activity on Lolium rigidum have been altered as follows: Line 216: ‘as we have demonstrated that MBDTA-2 possesses herbicidal activity against one of the most problematic weed species to global agriculture’ deleted; Line 81:The chlorsulfuron positive control data has been added to Figure 5, panel A. The Figure 5 legend has been updated accordingly with the addition of ‘or 1200 mg·L-1)”.“we successfully extended our previous herbicidal activity studies’ changed to ‘we extended our previous in vivo activity studies’; Line 271: ‘herbicidal’ deleted. Line 211: the extrapolated application rate has been removed by deleting ‘ The question arose in the reviews as to whether the compound is a pro-herbicide with respect to DHPDR. Please alter the discussion to raise this question, or consider conducting the experiment to add the demethylated compound to the DHPDR inhibition assay to test its activity.50 values of 169 ± 0.92 µM and 155.3 ± 0.90 µM for AtDHDPR1 and AtDHDPR2, respectively . HBDTA is therefore >18-fold less potent at the DHDPR target site relative to MBDTA-2, suggesting that the MBDTA-2 methyl group is beneficial for DHDPR inhibition. As such, MBDTA-2 is unlikely to be a proherbicide.The inhibition of DHDPR by the demethylated compound has now been measured and added to Figure 3B and described in the Results section starting at Line 134: Given that these results revealed a new target site for MBDTA-2, we assessed whether the compound could be proherbicidal with respect to DHDPR. To do so, we assessed the inhibitory activity of HBDTA against both AtDHDPR isoforms to determine the ICReviewer #1 (Recommendations for the authors):Excellent paper. The authors could include a reference to work in fungicides to provide further evidence that multi-site inhibitors are lower risk for resistance evolution than single-site inhibitors.We thank the reviewer for the feedback. Line 263: ‘and fungicides’ and Gullino et al., 2010 reference added to provide further evidence that multi-site inhibitors are lower risk for resistance evolution than single-site inhibitors. Line 262: ‘dual-target’ changed to ‘multi-target’ to encompass the use of multi-site fungicides.The bioassay method used for Lolium rigidum is unusual. The authors pipetted 2 mL of a 1200 mg/L solution directly onto seeds when sowing. Alternatives that could be used would include soaking seeds for 24 hour in herbicide solution; using an agarose based assay; spraying post-emergence on emerged seedlings; or spraying directly onto seeds or seedlings in soil.We thank the reviewer for the insightful comment. Direct pipetting onto seeds on soil, as employed in this study, is similar to soaking the seeds prior to planting, but is more relevant to herbicide use in the field. Pipetting directly onto seeds also functions similarly to spraying directly onto the seeds, however it provides a more consistent and controlled dosage as over-spray can occur with spray application. Experiments with Arabidopsis indicated that these inhibitors are more effective when applied pregermination. The value of further bioassays, as highlighted by the reviewer, has been acknowledged with the addition of the following at Line 287:“Whilst the in vivo assays conducted here provide proof-of concept for the potential of dual-target lysine biosynthesis inhibitors as herbicide active ingredients, controlled-dosage spraying experiments with the formulated compound will be pertinent to assess the application rate required for herbicidal efficacy in comparison to commercial herbicides.”The authors report a calculated application equivalent of 48 kg/ha. It is not clear how this extrapolated number is obtained, nor is it realistic. No pesticide could be applied at such a high rate , and the sheer volume of water application required to achieve that would also represent drenching a field. I suggest that the authors remove this extrapolated calculation, and perhaps instead discuss the relative activity/toxicity of their compound in comparison to other known herbicides based on in vitro inhibition of the two enzymes.-1)’. The relative in vitro activity of our compound in comparison to known herbicides is now discussed with the following addition at Line 252:We thank the reviewer for the comment. Line 211: The extrapolated application rate has been removed by deleting ‘”.I don't think that more experiments are needed for this paper, but future work could conduct bioassays using methods that would provide more accurate ways to extrapolate to potential field use rates. Future work could also conduct biochemical measurements to ask whether lysine is reduced in Lolium rigidum following treatment with this herbicide, supporting the prediction that the mode of action is two-site inhibition of lysine synthesis.We thank the reviewer for their feedback. The raised points about future work have now been added to the Discussion as follows: Line 287: the value of further bioassays has been acknowledged with the addition of ‘Whilst the in vivo assays conducted here provide proof-of-concept for the potential of dual-target lysine biosynthesis inhibitors as herbicide active ingredients, controlled-dosage spraying experiments with the formulated compound will be pertinent to assess the application rate required for herbicidal efficacy in comparison to commercial herbicides.’ Line 291:“quantification of lysine levels following treatment with these compounds has been added as a future direction by adding ‘Furthermore, metabolomics experiments to quantify changes in lysine and other aspartate-derived amino acid levels in response to treatment with dual-target lysine biosynthesis inhibitors would be of interest to validate the mode of action at the target site, as well as elucidate whether pathway deregulation contributes to the in vivo activity.”I also suggest that the authors provide more methods in the figure caption for Figure 5 so that it stand alone. Currently the caption only states the application concentration of 1200 mg/L, but it does not provide the application volume of 2 mL pipetted onto each seed, so in the current presentation the reader must examine the methods to understand the total dose applied to the plants. Suggest adding the 2 mL pipetting onto each seed to the caption.We thank the reviewer for the comment. Line 221: ‘Treatments were given by pipetting 2.0 mL per pot directly onto seeds’ added to Figure 5 legend.Reviewer #2 (Recommendations for the authors):The paper was a joy to read as it represents many processes in the design of new herbicides. I don't like creating additional work but through my review of the paper I kept wondering if the analogs presented in your previous publications (FBDTA and MBDTA-1), as well as any other available analogs, could build the knowledge of the inhibition of DHDPR and show a limited SAR. This may be a topic for a future publication so please disregard it if not appropriate for this paper.Indeed, future work will involve the synthesis of a comprehensive series of analogues to understand the structure-activity relationship of dual-target DHDPS/DHDPR inhibitors. This has been added to the Discussion at Line 281 as follows:“Additionally, exploring the structure-activity relationship of MBDTA-2 through the screening of analogues against whole plants may also provide insights into modifications that are advantageous to in vivo potency.”Reviewer #3 (Recommendations for the authors):This is a well-written description of experiments to further determine the mode of action of novel lysine synthesis inhibitor. The expansion of the mode of action to two consecutive enzymes of the lysine synthesis pathway is a surprising result, especially with different binding modes for the two enzymes. Again, the in vitro activity MBDTA-2 is rather poor for an effective herbicide. Perhaps inhibition of two enzymes makes up for the poor activity on each of the enzymes. Was the demethylated form of the compound tested in an in vitro assay of AtDHDPR1 and AtDHDP2? This must be done. If HBDTA is much more active than MBDTA-2 on DHDPR, the results would make more sense to me.50 values of MBDTA-2 against AtDHDPR1 and AtDHDPR2 are 6.9 and 8.6 µM, respectively. These values are similar to the IC50 of the herbicide active ingredient glyphosate against its enzyme target. The potency of MBDTA-2 relative to current herbicides is now discussed with the addition of Line 252: ‘Furthermore, the low-μM potency of MBDTA-2 at the DHDPR target site is comparable to the potency of glyphosate, the most successful commercial herbicide active ingredient, at its enzyme target 5-enolpyruvylshikimate-3-phosphate synthase.’ We have now tested the demethylated compound against AtDHDPR1 and AtDHDPR2 and included the results in the revised manuscript as described in Response 2 above.We thank the reviewer for raising an interesting point. The ICThe bioassay method is very weak, as it is impossible to determine whether MBDTA-2 is an effective herbicide from these results. Herbicides are not pipetted onto weed seeds in the field. MBDTA-2 was applied three times, on three consecutive days. In the field, a herbicide is applied once. I suggest that the compounds be applied once to the soil containing weeds seeds and watered into soil, as soil applied herbicide are used, or applied to foliage as a foliar spray with an adjuvant. I would try both methods, as some herbicides that are excellent soil-applied herbicides are poor foliar herbicides and vice versa. This way, you can compare the efficacy in g/ha with the positive control. Finally, herbicides are expected to kill weeds. Figure 5 shows only growth stunting.Lolium rigidum have been altered as discussed in Response 1 above.The bioassays conducted in this study provide proof-of-concept work for the potential utility of dual-target lysine biosynthesis inhibitors for development as herbicides. Future work will focus on optimisation of the inhibitors presented in this study in order to develop them as herbicides. This point is now included in the Discussion at line 281 as follows: ‘Additionally, exploring the structure activity relationship of MBDTA-2 through the screening of analogues against whole plants may also provide insights into modifications that are advantageous to in vivo potency.’ Future work will also optimise the formulation of these compounds to improve efficacy and this is included in the Discussion at Line 285 as follows: ‘Although these methods offer opportunities to optimise inhibitor potency, formulation also has a substantial impact on the performance of herbicide active ingredients and is therefore an important avenue to pursue for potency maximisation . Whilst the in vivo assays conducted here provide proof-of-concept for the potential of dual-target lysine biosynthesis inhibitors as herbicide active ingredients, controlled-dosage spraying experiments with the formulated compound will be pertinent to assess the application rate required for herbicidal efficacy in comparison to commercial herbicides.’ The compounds presented demonstrate that they inhibit the germination and growth of a weed species and therefore could potentially be developed into herbicides with future optimisation. Accordingly, the claims for biological activity on Where are the chlorsulfuron results?The chlorsulfuron positive control data has been added to Figure 5, panel A. Line 220:-1 of chlorsulfuron.”“The Figure 5 legend has been updated accordingly with the addition of ‘or 1200 mg·L


Plasmodium falciparum parasitaemia. We construct a joint dataset including 1445 bacteraemia cases and 1143 severe malaria cases, and population controls, among critically unwell Kenyan children that have previously been genotyped for human genetic variation. Using these data, we perform a cross-trait genome-wide association study of invasive bacterial infection, weighting cases according to their probability of bacterial disease. In doing so, we identify and validate a novel risk locus for invasive infection secondary to multiple bacterial pathogens, that has no apparent effect on malaria risk. The locus identified modifies splicing of BIRC6 in stimulated monocytes, implicating regulation of apoptosis and autophagy in the pathogenesis of sepsis in Kenyan children.Invasive bacterial disease is a major cause of morbidity and mortality in African children. Despite being caused by diverse pathogens, children with sepsis are clinically indistinguishable from one another. In spite of this, most genetic susceptibility loci for invasive infection that have been discovered to date are pathogen specific and are not therefore suggestive of a shared genetic architecture of bacterial sepsis. Here, we utilise probabilistic diagnostic models to identify children with a high probability of invasive bacterial disease among critically unwell Kenyan children with  Bacterial infections are a major cause of severe illness and death in African children. Understanding which children are at risk of life-threatening infection and why, is key to designing new tools to help protect them. Some risk is likely inherited, but scientists do not know which genes are responsible. Genome-wide association studies (GWAS) may be one way to identify bacterial infection risk genes. GWAS look for genetic differences associated with a particular disease. But previous GWAS studies have failed to find genes linked with bacterial infections in African children because they were too small.Malaria is another frequent cause of life-threatening illness in African children. It can be hard for clinicians to determine if a child's illness is caused by malaria, a bacterial infection, or both. Many children in Africa have malaria parasites in their blood, but they do not always cause disease. Most children with suspected severe malaria are treated with antibiotics in case of bacterial infection. Clinicians may then conduct further testing to determine the illness’s actual cause. Scientists may be able to use this data on children with suspected malaria to study bacterial infections.Gilchrist et al. show that children with an unusual alteration in the BIRC6 gene are at increased risk of bacterial infections. In the experiments, Gilchrist et al. used computer modeling to identify a subset of children with likely bacterial infections among 2,200 children admitted to a hospital in Kenya with a high fever and malaria parasites. By combining information on this subset of children with data on children with confirmed bacterial infections and healthy children, Gilchrist created a sample of 5,400 children for a GWAS. The analyses found that children with a variation in the BIRC6 gene on chromosome 2 had a higher risk of bacterial infections.This genetic change is linked with the production of a modified form of BIRC6 in infection-fighting immune cells called monocytes. More studies will help scientists understand how this change might contribute to severe bacterial infections. Learning more may help scientists develop new treatment strategies and identify children most at risk. Invasive bacterial diseases are a major cause of child morbidity and mortality in Africa . AlthougSalmonella (HBB (sickle haemoglobin), which is associated with raised and lowered risks of infection secondary to a broad range of pathogens among homozygotes , between 11 June 1995 and 12 June 2008 were included in the study. While this definition is sensitive it is not specific, meaning that our study will have included some children with sepsis accompanied by incidental parasitaemia  or white blood cell and platelet counts , to determine the likelihood of ‘true’ severe malaria among these children. The estimated probabilities of ‘true’ severe malaria using each model were well correlated  we used PfHRP2 and platelet count to determine Following quality control measures , we included 1445 cases of culture-confirmed bacteraemia, 1143 cases of severe malaria, and 2812 control children in our current analysis . To acco64×10-9) , Table 3To address the possibility that the observed association at this locus is driven by confounding secondary to population structure, we used ABERRANT  to definovariate . The min samples , which tTo assess whether our analysis could be affected by our choice of model to define severe malaria case weights, we restricted our analysis to samples with data available to calculate estimates for analysis . MoreoveWe sought to replicate evidence of association in our discovery analysis through use of an independent case-control collection of Kenyan children with bacteraemia (n=434) and healthy controls (n=1258) conducted in the same population. The peak trait-associated variants in the discovery analysis were well imputed in the replication data . Genotype at rs183868412 similarly modified risk of bacteraemia in the neonatal period and in older children . Within Africa, rs183868412:T is present in all nine African populations included in the MalariaGEN consortium project   in sevenWAS data  of 33 cahttps://www.ebi.ac.uk/eqtl) mapping data  of the canonical BIRC6 transcript, ENST00000421745.6, is 135 bp long and terminates immediately before the 12 bp sequence associated with rs183868412:T genotype. The 22nd exon  of an alternative BIRC6 transcript, ENST00000648282.1, is 147 bp long, having the same start site but including the 12 bp sequence at its 3’ end. Thus, increased risk of invasive bacterial disease may be associated with decreased expression of an alternative BIRC6 transcript in TLR1/2-stimulated monocytes.Trait-associated genetic variation identified by GWAS is highly enriched for regulatory variation. The African specificity of the trait-associated variation identified here makes annotation with publicly available regulatory mapping data challenging. To investigate the regulatory function of rs183868412 in immune cells in African populations, we used eQTL Catalogue , also known as BRUCE (BIR repeat containing ubiquitin-conjugating enzyme), encodes a large member of the inhibitor of apoptosis protein (IAP) family  family . Members) family . A propo) family . BIRC6 ctagonist . BIRC6 ae fusion , and ubiutophagy .BIRC6 could determine susceptibility to invasive infection through either its regulation of apoptosis or autophagy (or both). Sepsis induces marked changes in apoptosis across a range of immune cells  and gram-negative  pathogens, including intracellular and extracellular bacteria, and enteric and respiratory pathogens. Moreover, rs183868412 modified risk of bacteraemia in both the neonatal period, when infection is likely to be maternally derived, and in older children, when sources of community acquired infection will be more diverse. This modulation of invasive bacterial disease risk, despite diverse sources and routes of infection and diverse mechanisms of invasion, suggests a mechanism in which genetic variation at BIRC6 modifies risk of invasive infection downstream of initial mechanisms of infection and invasion. In an interesting parallel to this, common genetic variation at another ubiquitin-conjugating enzyme, UBE2U, has been shown to modify outcome in meningitis secondary to diverse pathogens in individuals of European ancestry , evidence of respiratory distress, hypoglycaemia, or hyperparasitaemia were eligible for recruitment as cases of severe malaria. During the study period, all children admitted to hospital, with the exception of elective surgical admissions and minor trauma, had a blood sample taken for bacterial culture . Children under 14 years in whom a pathogenic organism was identified in blood were eligible for study inclusion . Control children were recruited between 3 and 12 months of age from consecutive live births from the population which Kilifi County Hospital serves. Control children have been subject to longitudinal follow-up. Following explanation of the study, written informed consent was obtained from the parent or guardian of each child included in the study. Ethical approval was obtained from the Kenya Medical Research Institute (KEMRI) National Scientific Steering and Research Committees  and the Oxford Tropical Research Ethics Committee .Recruitment of the severe malaria cases, bacteraemia cases, and healthy controls have been described in detail elsewhere . In briePfHRP2 concentrations to derive the probabilistic model. In cases where PfHRP2 concentration was not measured (n=800), we used white blood cell counts and platelet counts as input data to the model.Among cases of severe malaria recruited to the study, we used probabilistic models to assign a probability of that child’s clinical presentation being mediated by parasite sequestration, that is ‘true’ severe malaria, as described previously . Where aPfHRP2/platelet counts; Model 2: platelet counts/white blood cell counts), the probabilities of ‘true’ severe malaria were derived by fitting parametric latent class models. These assumed that each patient had a binary latent state . For Model 1, we assumed that in each latent state the data were distributed as a single bivariate normal distribution. For Model 2, the data did not fit well to a two-component bivariate normal  so we assumed that the data had bivariate Student’s t-distribution for the severe malaria state, and a flexible mixture of bivariates for the not severe malaria state.For both models , observing high levels of genotype concordance . In addition to the sample quality control applied above, we additionally excluded one individual from each sample pair with relatedness coefficient >0.05, leaving a final sample size of 2559 . We estimated SNP heritability using GCTA-GREML . For traglm function in R. As described previously , re-weighting the contribution of a case to the log-likelihood according to its probability of representing invasive bacterial infection. Control samples were assigned a weight of 1. Our regression thus assumes that the lower the probability of ‘true’ severe malaria, the greater the probability that a case represents culture-negative invasive bacterial disease. To control for confounding variation, we included the six major PCs of genotyping data and genotyping platform as covariates in the model. Weighted logistic regression was implemented using the eviously , standarWe used a Bayesian approach to identify a set of SNPs with 95% probability of containing the causal variant at the trait-associated locus. Approximate Bayes factors  were calTo replicate our findings from the discovery analysis, we used a second sample set, recruited from the same population as the discovery samples. Replication case samples were cases of bacteraemia only, and did not include cases of severe malaria without culture-confirmed bacterial infection. Case samples were recruited between 1 August 1998 and 30 October 2010. As for the discovery case samples, children under 14 years with a bacterial pathogen isolated from blood on admission to hospital were eligible for recruitment to the study. As above, control samples were recruited as part of a birth cohort from the same population, with children recruited between the ages of 3 and 12 months. Genotyping and QC procedures for these samples have been described previously. In brief, study samples were genotyped using the Immunochip Consortium  array I. SNP QC SHAPEIT2  and IMPUSHAPEIT2  with 100Following imputation, we further excluded poorly imputed (imputation info score < 0.5) and rare (MAF < 1%) variants and variants with HWE T v2.5.6 . To exclP. falciparum parasitaemia, and bacteraemia presenting at different time periods across a period of declining malaria transmission intensity. For these analyses we used only samples with culture-confirmed bacteraemia. In each case we used control status as the baseline stratum, and included the six major PCs of genotyping data to control for confounding variation as above.At the locus of interest, we used multinomial logistic regression, implemented in SNPTEST v2.5.6  to estimAcinetobacter, n=118; β-haemolytic streptococci, n=130; E. coli, n=141; Haemophilus influenzae type b, n=113; nontyphoidal Salmonella, n=159; pneumococci, n=390; S. aureus, n=152) and one stratum for the remaining other organisms (n=242). For the neonatal/non-neonatal disease analysis, we stratified cases as presenting in the first 28 days of life (n=195) or beyond that (n=1245). For the analysis comparing bacteraemia with and without malaria, we stratified cases with (n=204) and without (n=1236) P. falciparum on their admission blood film. For each of these analysis, case strata were compared to Affymetrix-genotyped discovery control samples (n=1895) as a baseline stratum.For the pathogen-stratified analysis, we defined eight case strata among the discovery cases, one for each of the seven most commonly isolated organisms , 2001–2002 (n=349), 2003–2004 (n=467), 2005–2006 (n=310), 2007–2008 (n=237), 2009–2010 (n=111). For this analysis we used both discovery (Affymetrix) and replication (Immunochip) case and control samples. This allowed better coverage of the years later in the study, which were underrepresented in the discovery samples . In that analysis we used multinomial logistic regression in each cohort to estimate stratum-specific effects, combining these results in a fixed effects meta-analysis using BINGWA .We then compared models of association using a Bayesian approach , conside"Null": effect size = i.e. no association with bacteraemia."Same": effect size Additional models consider each possible combination of a fixed effect size for associated strata and no association at other strata. For each model, we calculated approximate Bayes factors  and posthttps://github.com/eQTL-Catalogue/, We used the eQTL Catalogue  mapping coloc.susie command to allow colocalisation of multiple independent signals at a single locus for each trait. We considered evidence for colocalisation for each gene and exon within a 250 kb window of the peak association (rs183868412). We considered a posterior probability >0.8 supporting a shared causal locus to be significant.We then used the R package coloc v5.1.0  to ident Gilchrist et al. present evidence that a genetic variant in the BIRC6 gene increases the risk of invasive bacterial infection. This paper will be of interest to researchers working in areas relating to invasive bacterial infections, malaria, sepsis, and immunogenetics. The authors find evidence that the risk allele affects the splicing of BIRC6 in stimulated monocytes and that this may explain the association signal they found. Further investigations in African ancestry samples will be needed to elucidate the mechanism. public reviews designed to be posted alongside the preprint for the benefit of readers; ii) feedback on the manuscript for the authors, including requests for revisions, shown below. We also include an acceptance summary that explains what the editors found interesting or important about the work.Our editorial process produces two outputs: i) Decision letter after peer review:BIRC6 modifies risk of invasive bacterial infection in Kenyan children" for consideration by eLife. Your article has been reviewed by 3 peer reviewers, including Alexander Young as the Reviewing Editor and Reviewer #1, and the evaluation has been overseen by Bavesh Kana as the Senior Editor. The following individual involved in review of your submission has agreed to reveal their identity: Diederik van de Beek (Reviewer #3).Thank you for submitting your article "The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.Essential revisions:The manuscript describes the identification of a novel risk locus for bacteraemia. The risk locus identified is biologically interesting and the reviewers generally thought that the methods used were appropriate and conclusions supported by the data and analyses. However, the reviewers raised some points that need addressing:1) Please answer reviewer #1's questions pertaining to population stratification2) In relation to reviewer #3's comments, it would help readers of the manuscript to include a figure/table giving an overview of the cohorts used in the manuscript: what data was collected and when.3) Provide a more detailed justification for using model 2 over model 1, or provide sensitivity analyses comparing results using the two models.4) Comment on whether there is evidence for selection on the risk variantPlease also address other comments related to improving clarity and presentation.Reviewer #1 (Recommendations for the authors):I have some questions about the controls for the population structure that were implemented.Looking at Figure 2 Supplements 2 and 3, it looks to me like PC1 is driven by the presence of ~20 individuals with 'other' ethnicity genotyped on Affymetrix. Do the authors know what is atypical about the ancestry of these samples? Could the authors perform a sensitivity analysis where individuals with outlying values on PC1 are removed from the discovery sample?Why were 6 principal components used for the discovery sample but only 4 for the replication sample?To give further confidence that the main result is not a result of population stratification, it would be helpful to see a table that gives the case-control counts as well as the counts of the rs183868412 alleles stratified by self-reported ethnicity. Related to this, is there any evidence for differentiation between ethnic groups on rs183868412?Line 322: 'excluded SNPs outside the following QC thresholds'. I found the language here very confusing, and is 'genotype probability (info) >0.975' the wrong way around?Line 336: what is IBD>0.2? Do you mean relatedness coefficient or kinship coefficient or something else? How many individuals were excluded based on this threshold?Line 399: 'the major principal components' -- please specify the numberFor the analysis of eQTL data from Quach et al., were both European and African ancestries used? I found this somewhat unclear. What was the count of individuals with the rs183868412:T allele? And were any of them non-African? If both European and African ancestry individuals were used, I would suggest performing an equivalent analysis using only African ancestry individuals to avoid potential confounding with ancestry.Reviewer #2 (Recommendations for the authors):It seems surprising that a genotype with such an obviously deleterious effect can reach moderate frequency across multiple African populations. A comment on this seems appropriate. Might there be some favorable effect of the phenotype? Is there any evidence of recent selection? A full investigation of this question Obviously goes beyond the scope of this work but it should obviously help in interpreting the phenotype.Reviewer #3 (Recommendations for the authors):1. The authors make good arguments for lumping up data of malaria and bacteremia cases but it still feels a bit artificial. Please present sensitivity analyses for culture proven bacteremia cases only vs. controls .2. Weighted analysis of malaria cases. This is an interesting approach. Differences between "model 1" and "model 2" should be better explained. Also, just stating we used model 1 for down analyses and model 2 for all other cases is clearly insufficient. Please provide data in more detail to back this up. Alternatively, provide sensitivity analyses using both models – if the outcome is robust using either model 1 and 2 will provide similar results. Please show and discuss.3. Heritability. The authors state in the intro that "..suggests that a significant portion of this variability is heritable." Could the authors include an analysis of human SNP heritability for bacteremia in the manuscript? Essential revisions:The manuscript describes the identification of a novel risk locus for bacteraemia. The risk locus identified is biologically interesting and the reviewers generally thought that the methods used were appropriate and conclusions supported by the data and analyses. However, the reviewers raised some points that need addressing:1) Please answer reviewer #1's questions pertaining to population stratification.We now perform sensitivity analysis excluding the additional population outliers identified by reviewer #1, and present the association analysis at rs183868412 stratified by self-reported ethnicity (Table 4). The association at rs183868412 is unchanged by additional outlier exclusion, and the effect estimates seen in the two major sub-populations (Giriama and Chonyi) are consistent with that seen in the overall analysis.2) In relation to reviewer #3's comments, it would help readers of the manuscript to include a figure/table giving an overview of the cohorts used in the manuscript: what data was collected and when.We now include this information as a new Figure (2).3) Provide a more detailed justification for using model 2 over model 1, or provide sensitivity analyses comparing results using the two models.We now present sensitivity analysis, restricting malaria case samples to children for whom we have data to calculate both models. In that analysis the observed association at rs183868412 is robust to the choice of model .4) Comment on whether there is evidence for selection on the risk variant.We now expand on our reporting of within-Africa differentiation at the locus, reporting evidence for directional selection in 1000 Genomes Project African ancestry populations at the locus and examining evidence for pleiotropy at the locus with cardiometabolic traits in large-scale GWAS in African ancestry populations. We find no evidence for selection or pleiotropy at the locus and outline the limitations of these analyses in the discussion.Reviewer #1 (Recommendations for the authors):I have some questions about the controls for the population structure that were implemented.Looking at Figure 2 Supplements 2 and 3, it looks to me like PC1 is driven by the presence of ~20 individuals with 'other' ethnicity genotyped on Affymetrix. Do the authors know what is atypical about the ancestry of these samples? Could the authors perform a sensitivity analysis where individuals with outlying values on PC1 are removed from the discovery sample?We now include the following sensitivity analysis (lines 144-153):“To address the possibility that the observed association at this locus is driven by confounding secondary to population structure, we used ABERRANT  to define a set of outlier samples on the first two PCs of genotyping data . These individuals are all genotyped on the Illumina Omni 2.5M array and are both cases and controls (15 and 7 respectively). While they are predominantly individuals with less common self-reported ethnicities in our study population , they are not representative of a single self-reported ethnicity . Excluding these samples from the association analysis at rs183868412:T did not significantly alter the association with invasive bacterial infection .”Why were 6 principal components used for the discovery sample but only 4 for the replication sample?We had used 4 principal components in the replication analysis primarily for convenience: these had been computed as part of the original WTCCC2 GWAS of bacteraemia, and the samples used for replication here were identical. To be consistent with the discovery analysis we have recomputed the principal components for these samples and updated our replication analysis to include the first 6 PCs. The effect estimate  and significance (p=8.90x10-4) of replication is in essence unaltered. We have updated Figure 4-supplement 1 with plots of the first 6 principal components and updated the replication and meta-analysis statistics throughout .To give further confidence that the main result is not a result of population stratification, it would be helpful to see a table that gives the case-control counts as well as the counts of the rs183868412 alleles stratified by self-reported ethnicity. Related to this, is there any evidence for differentiation between ethnic groups on rs183868412?We have included these data in a new Table 4. There is no evidence for differentiation between the study subpopulations. We have added the following to the text (lines 153-166):“We further estimated the effect of rs183868412:T on invasive bacterial disease risk in four subpopulations defined by self-reported ethnicity . Within each subpopulation we tested for association between genotype and case status in a weighted logistic regression model, including platform as a categorial covariate (Table 4). The minor allele frequency at rs183868412 ranged from 0.016 (Giriama) to 0.037 (Kauma), with no evidence of differentiation between subpopulations (FST = 0.001). We observed consistent effect sizes in both of the major study subpopulations; Giriama  and Chonyi  samples (Table 4), which together make up 83% of the study samples. Genotype at rs183868412 was also associated with less common self-reported ethnicities grouped together . Genotype was not associated with invasive bacterial disease risk in the Kauma subpopulation, however the sample size in the stratum is very limited  and may simply reflect insufficient power to detect an association.”Line 322: 'excluded SNPs outside the following QC thresholds'. I found the language here very confusing, and is 'genotype probability (info) >0.975' the wrong way around?Apologies that this was not clear. We have reworded this sentence. You are also right the genotype probability exclusion is the wrong way round – this has been corrected.Line 336: what is IBD>0.2? Do you mean relatedness coefficient or kinship coefficient or something else? How many individuals were excluded based on this threshold?Apologies that this also wasn’t clear. By IBD we meant relatedness coefficient. This has been changed throughout.Line 399: 'the major principal components' -- please specify the numberWe have corrected this.For the analysis of eQTL data from Quach et al., were both European and African ancestries used? I found this somewhat unclear. What was the count of individuals with the rs183868412:T allele? And were any of them non-African? If both European and African ancestry individuals were used, I would suggest performing an equivalent analysis using only African ancestry individuals to avoid potential confounding with ancestry.-5). We have updated Figure 6 and our results to reflect these new mapping data (lines 240-252) and have amended the methods to reflect this (lines 516-530).Many thanks for this. The available mapping data from eQTL Catalogue included both European and African individuals. We have now re-mapped eQTL in that dataset restricting the analysis to individuals of African ancestry (n=100). As previously, the only evidence of colocalization with our risk locus is with an eQTL for a 12bp exonic sequence in BIRC6  in the eQTL study. We now include this information in the Results section (line 247) and have added the following to the discussion (lines 333-339):“The eQTL mapping data that we utilise here  is limited in that it allows us only to consider a single immune cell type. It is also important to note that there are relatively few African-ancestry individuals in the eQTL mapping data we utilise here, and the splicing signal at BIRC6 is based on only 10 individuals heterogygous for rs183868412:T. A more complete understanding of the role played by genetic variation at BIRC6 plays in the pathogenesis of sepsis in African children will require more detailed expression and functional studies in African populations.”Reviewer #2 (Recommendations for the authors):It seems surprising that a genotype with such an obviously deleterious effect can reach moderate frequency across multiple African populations. A comment on this seems appropriate. Might there be some favorable effect of the phenotype? Is there any evidence of recent selection? A full investigation of this question Obviously goes beyond the scope of this work but it should obviously help in interpreting the phenotype.We very much agree. To address this we have added the following to the manuscript (lines 217-237):“Common genetic variation associated with a two-fold increased risk of invasive bacterial infection in children, in particular across a broad range of pathogens, will be subject to considerable negative selection pressure. This raises the question of whether this polymorphism might be associated with a positive advantage with respect to another life-threatening illness. The derived allele rs183868412:T, associated with increased risk of invasive bacterial disease in Kenyan children, is absent in non-African populations (https://gnomad.broadinstitute.org). Within Africa, rs183868412:T is present in all 9 African populations included in the MalariaGEN consortium project  (Table 6), minor allele frequencies ranging from 0.011 in The Gambia to 0.034 in Malawi. There is no evidence for within-Africa differentiation at rs183868412 (p = 0.601) providing no support for a selective sweep at the locus. We further evaluated evidence for recent directional selection pressure, examining integrate haplotype scores (iHS)  in 7 African populations included in the 1000 Genomes Project. In those data, there is no evidence to support recent selection at the locus . Finally, to understand whether variation at this locus may be maintained in populations through beneficial effects on other traits, we examined evidence for pleiotropy at rs183868412 in a meta-analysis of GWAS data  of 33 cardio-metabolic traits from Uganda, Ghana, Nigeria, South Africa and Kenya . In keeping with the MalariaGEN consortium project data, minor allele frequencies at rs183868412 range from 0.015 to 0.028. In these data there is no evidence for pleiotropy at the locus, with no evidence for an effect of rs183868412 on any of the 33 traits tested . The explanation for the persistence of this polymorphism, therefore, remains an open question”It is noteworthy, however, that the modest allele frequencies of the candidate risk variants make any assessment of selection signature at the locus underpowered. We also now address this in the discussion (lines 342-343).Reviewer #3 (Recommendations for the authors):1. The authors make good arguments for lumping up data of malaria and bacteremia cases but it still feels a bit artificial. Please present sensitivity analyses for culture proven bacteremia cases only vs. controls .We now present this on lines 175-178:“Moreover, restricting our analysis to cases of culture-confirmed bacteraemia, the effect estimate for bacteraemia risk observed in the discovery analysis  is consistent with that seen in the main model.”2. Weighted analysis of malaria cases. This is an interesting approach. Differences between "model 1" and "model 2" should be better explained. Also, just stating we used model 1 for down analyses and model 2 for all other cases is clearly insufficient. Please provide data in more detail to back this up. Alternatively, provide sensitivity analyses using both models – if the outcome is robust using either model 1 and 2 will provide similar results. Please show and discuss.We have performed sensitivity analysis as suggested. The association is robust to the choice of model. This is detailed on lines 167-175 and shown in Figure 3-supplement 3:“To assess whether our analysis could be affected by our choice of model to define severe malaria case weights, we restricted our analysis to samples with data available to calculate estimates for P(SM|data) using both Model 1 and Model 2 . We recalculated effect estimates for the rs183868412 association with invasive bacterial disease using each model alone. The association with invasive bacterial disease at rs183868412:T is robust to the choice of the model for case weights, with effect estimates derived using Model 1 alone  and Model 2 alone  being entirely consistent with those seen in the main analysis .”3. Heritability. The authors state in the intro that "..suggests that a significant portion of this variability is heritable." Could the authors include an analysis of human SNP heritability for bacteremia in the manuscript?Many thanks for this suggestion. Using GCTA we derive an estimate for SNP heritability of 19% (95% CI 3-35%) for bacteraemia in this population. We report this estimate in lines 116-120, and detail the methods in lines 426-435.


Paroxysmal nocturnal hemoglobinuria (PNH) is a rare, chronic hematologic disorder associated with inappropriate terminal complement activity on blood cells that can result in intravascular hemolysis (IVH), thromboembolic events (TEs), and organ damage. Untreated individuals with PNH have an increased risk of morbidity and mortality. Patients with PNH experiencing IVH often present with an elevated lactate dehydrogenase  level which is associated with a significantly higher risk of TEs, one of the leading causes of death in PNH. LDH is therefore used as a biomarker for IVH in PNH. The main objective of PNH treatment should therefore be prevention of morbidity and mortality due to terminal complement activation, with the aim of improving patient outcomes. Approval of the first terminal complement inhibitor, eculizumab, greatly changed the treatment landscape of PNH by giving patients an effective therapy and demonstrated the critical role of terminal complement and the possibility of modulating it therapeutically. The current mainstays of treatment for PNH are the terminal complement component 5 (C5) inhibitors, eculizumab and ravulizumab, which have shown efficacy in controlling terminal complement-mediated IVH, reducing TEs and organ damage, and improving health-related quality of life in patients with PNH since their approval by the United States Food and Drug Administration in 2007 and 2018, respectively. Moreover, the use of eculizumab has been shown to reduce mortality due to PNH. More recently, interest has arisen in developing additional complement inhibitors with different modes of administration and therapeutics targeting other components of the complement cascade. This review focuses on the pathophysiology of clinical complications in PNH and explores why sustained inhibition of terminal complement activity through the use of complement inhibitors is essential for the management of patients with this chronic and debilitating disease. Incidence has been reported to be around 1 per million and prevalence estimated to around 8 per million in the United States agreeing with previous estimates.6 In a recently published UK-based study, the incidence of PNH was estimated to be 0.35 per 100,000 and overall prevalence was 3.81 per 100,000.Paroxysmal nocturnal hemoglobinuria (PNH) is a rare, chronic hematologic disorder associated with inappropriate terminal complement activity on blood cells, resulting in intravascular hemolysis (IVH), a risk of thromboembolic events (TEs), and organ damage (e.g. kidney impairment and pulmonary hypertension). This translates directly into increased morbidity and mortality if PNH is untreated. Elevated LDH levels ⩾ 1.5 × the upper limit of normal (ULN) are associated with a significantly higher risk of TEs, one of the leading causes of death in PNH. Early diagnosis and treatment are essential for managing the wide-ranging symptoms and life-threatening consequences of PNH. However, diagnosis is often delayed by the diversity and nonspecific nature of its clinical manifestations (e.g. anemia and fatigue), compounded by its low prevalence.10 At present, the only curative therapy for PNH is hematopoietic stem cell transplantation. However, high rates of morbidity and mortality remain so, this is only considered feasible in select patients. Stem cell transplantation is therefore commonly reserved for management of the underlying bone marrow failure and not for the management of PNH because complement inhibitors are available.12 The current mainstays of treatment for PNH are the terminal complement component 5 (C5) inhibitors eculizumab and ravulizumab, which have demonstrated efficacy in controlling terminal complement-mediated IVH, reducing TEs and morbidity (including organ damage) in patients with PNH since their approval by the United States Food and Drug Administration (FDA) in 2007 and 2018, respectively.1320 Eculizumab treatment has also been shown to reduce mortality in patients with PNH. Beyond C5 inhibition, the first complement component 3 (C3)-targeted therapy, pegcetacoplan, was approved for use in adults with PNH (May 2021) by the FDA22 and by the European Medicines Agency  as a second-line treatment in adult patients with PNH who are anemic after treatment with a C5 inhibitor for at least 3 months in Europe.Patients with PNH experiencing IVH often present with elevated lactate dehydrogenase (LDH) levels.This review focuses on the pathophysiology of complications in PNH and why sustained inhibition of terminal complement activity is essential for the management of patients with this disease.PIGA) in hematopoietic stem cells, giving rise to red blood cells (RBCs), platelets, and white blood cells (WBCs) with the PNH phenotype.25 The disease starts with the expansion of the hematopoietic stem cell that has the PIGA mutation conferring lack of glycophosphatidylinositol (GPI) anchors. How clonal expansion occurs has been a source of great debate and several hypotheses have been proposed. These include, for example, the selective advantage of mutant cells due to an immune attack to the hematopoietic stem cells or the presence of a second mutation that grants fitness advantage. Mutations in PIGA lead to absence of the complement regulatory proteins CD55 and CD59, resulting in uncontrolled terminal complement activity. CD59 directly inhibits membrane attack complex (MAC) formation, preventing lytic pore development, and so acts as a vital inhibitor of the terminal complement pathway.13 Uncontrolled terminal complement activity causes IVH and complement-mediated activation of platelets and WBCs, with the associated increased risk of TEs, vasoconstriction, anemia, and fatigue. These all contribute to organ damage and an increased risk of morbidity and mortality.PNH is a monogenic disease caused by acquired mutations in the phosphatidylinositol glycan anchor biosynthesis class A gene , which plays a key role in vasodilation and inhibition of coagulation.9 Therefore, IVH and the downstream NO scavenging generate a vasoconstrictive and procoagulant state that is associated with thrombophilia in PNH.9 Crosstalk between the activated/dysregulated complement and coagulation pathways is also thought to play a role in the mechanism of thrombosis in PNH, and multiple factors are likely to contribute to it. Platelet activation, complement-mediated hemolysis, impairment of the fibrinolytic system, and the presence of inflammatory mediators are all pertinent factors thought to be responsible for the increased TE risk observed in patients with PNH.Thromboembolism is the most common cause of death in patients with PNH not treated with C5 inhibitors, accounting for approximately 40–67% of deaths for which the cause is known,29 This fatigue is more likely to be associated with NO depletion resulting from IVH.31 The free hemoglobin release from IVH, and therefore the NO depletion, causes smooth muscle dystonia, resulting in abdominal pain, dysphagia, and erectile dysfunction.33 Pulmonary hypertension is reported in 36–60% of patients with PNH and has two likely principal causes: NO depletion, resulting in pulmonary vasculature vasoconstriction, and, less commonly, silent pulmonary emboli; both are due to the activity of terminal complement.35 The chronic kidney disease seen in approximately 65% of patients with PNH involves similar mechanisms.36 All of these factors highlight how PNH is a disorder of dysregulated terminal complement activity, having an impact on not only RBCs but also platelets and WBCs.Uncontrolled terminal complement activity has a role in other complications and symptoms of PNH. The fatigue experienced by patients with PNH is not always associated with anemia and appears to be out of proportion with the hemoglobin level.38 Understanding that the significant symptomology of PNH, including increased morbidity and mortality, arises from dysregulated or uncontrolled terminal complement activity led to the C5 inhibition approach nearly 20 years ago. Disease management for patients with PNH should therefore seek to achieve complete and sustained terminal complement inhibition,38 which in itself will lead to (i) reduction in the incidence of TEs, (ii) reduction in IVH as indicated by lowered LDH levels, (iii) reduction in (or prevention of progression of) pulmonary pressures and renal impairment, (iv) improvement in PNH symptoms, and (v) improvement in patient health-related quality of life (HRQoL).The treatment of patients with PNH can be complex and challenging, given the heterogeneity and plethora of clinical symptoms, and the rare and chronic nature of the condition.41 Patients with inherited C5 deficiency have near-normal life expectancy compared with, for example, those with inherited C3 deficiency, who may have recurrent pyogenic infections, autoimmune complications, and a much earlier death, often in childhood. However, as the majority of patients receiving a diagnosis of PNH are adults with an already mature immune system, therapeutic inhibition is expected to result in a lower infection risk  in these patients than in those with a congenital deficiency.Targeting C5 was an attractive option because it impairs MAC formation while allowing the proximal complement system to function normally.41 Eculizumab was the first complement inhibitor therapy and the first disease-specific treatment to be approved for patients with PNH by the FDA, EMA, and other regulatory agencies. Eculizumab is administered by intravenous (IV) infusion and has a terminal half-life of approximately 11 days, which translates to a 2-week dosing interval after an initial loading phase of weekly doses for 5 weeks. Ravulizumab, an analog of eculizumab that binds to the same epitope on C5, is a second-generation C5 inhibitor that differs from eculizumab by the substitution of four amino acids. These substitutions alter its pharmacokinetic (PK) and pharmacodynamic (PD) profiles, resulting in a substantially longer terminal half-life (50 days) permitting a longer dosing interval (8 weeks), while retaining the clinical benefits of eculizumab.43 Based on a series of pivotal trials43 and a 10-year pharmacovigilance study, eculizumab and ravulizumab are the current standard of care in patients with PNH in countries where they are commercially available.40The C5 inhibitors eculizumab and ravulizumab are humanized monoclonal antibodies designed to target the complement protein C5, thereby preventing formation of the MAC, release of anaphylatoxin C5a, activation of WBCs and platelets, and lysis of RBCs with the PNH phenotype.43 involving 581 patients. These trials had placebo-controlled, non-placebo-controlled, and active-controlled study designs, and included patients with PNH who were either naïve to complement inhibitor therapy or had clinically stable disease following on average 5.8 years of eculizumab therapy  in patients with PNH,43 suggesting that these drugs were effective at lowering the incidence of one of the primary causes of death in PNH. In addition, a post hoc analysis found that 2 years of ravulizumab treatment resulted in fewer TEs and MAVEs than were reported in the 2 years before ravulizumab therapy was initiated, adding to the growing body of evidence that C5 inhibitors help to reduce the incidence of TEs and MAVEs.It is paramount to reduce the risk of TEs in patients with PNH because these are the most common known cause of death.29 a retrospective analysis of data from both study populations demonstrated a statistically significant 92% reduction (p < 0.001) in TE rate during the period of eculizumab treatment compared with that in the same time period before treatment was started.Although the pivotal phase 3 eculizumab studies were not designed to establish the effect of this drug on TEs, and are associated with a higher prevalence of TE and PNH symptoms, such as abdominal pain, chest pain, and hemoglobinuria. Patients with PNH experiencing persistent IVH can be identified on the basis of their LDH levels being ⩾ 1.5 × ULN.51 For example, in complement inhibitor-naïve patients with PNH recruited into one of the pivotal studies, baseline LDH levels were ⩾ 3 × ULN in 86.2% and 1.5 to < 3 × ULN in the remaining 13.8%. Achieving and maintaining an LDH level ⩽ 1.5 × ULN in patients with PNH is a clinically relevant threshold based on the receiver operating characteristic analysis of TEs and LDH level conducted by Lee et al. , in addition to clinically meaningful improvements in dyspnea, were observed in eculizumab-treated patients compared with individuals receiving placebo.PNH pathophysiology is also associated with high levels of N-terminal pro-brain natriuretic peptide (NT-proBNP), a biomarker of ventricular and cardiac dysfunction, and are now understood to be a direct consequence of IVH and the release of free hemoglobin. Reduction in IVH with eculizumab treatment resulted in considerable improvements in abdominal pain and severe dysphagia in patients with PNH who had a prior history of these symptoms.Abdominal pain and dysphagia, attributed to smooth muscle dystonia, are commonly reported in patients with hemolytic PNH,55 It has been suggested that the presence of IVH may be the causative etiology of low HRQoL in patients with PNH, and therefore treatment goals should be focused on the reduction of LDH levels. In the pivotal phase 3 clinical trials of eculizumab and ravulizumab in individuals with PNH, both treatments were associated with statistically significant improvements in PNH-related symptoms and HRQoL  and the Functional Assessment of Chronic Illness Therapy–Fatigue (FACIT-F) scale).5658 In addition, in an analysis of International PNH Registry data, eculizumab was better associated with clinically meaningful improvements in both FACIT-F and EORTC QLQ-C30 scores compared with no treatment, regardless of transfusion dependence. Although hemoglobin may not return to normal levels in all patients, a significant and clinically meaningful improvement in fatigue can be demonstrated by improvement in FACIT-F scores. These data suggest that NO depletion secondary to free hemoglobin from IVH may result in the fatigue reported in patients with PNH; therefore, controlling IVH is likely to help improve fatigue.Patients with PNH often report poor HRQoL as a result of symptoms such as pain, anemia, erectile dysfunction, and fatigue.A multivariate analysis also showed that a reduction in IVH was more predictive of improvement in fatigue than an increase in hemoglobin level.27 An analysis comparing patients with PNH treated with eculizumab with an historical cohort of individuals with PNH before approval of the drug and with a sex-matched healthy population found that those with PNH receiving eculizumab had mortality that was significantly lower than that of the historical cohort and comparable with that in the sex-matched healthy population. Long-term data on survival in patients with PNH treated with ravulizumab are not available due to its more recent regulatory approval.Before the approval of C5 inhibitors, the median survival of an individual following a PNH diagnosis was 10–15 years.61 BTH is defined as the return of IVH and reappearance of classic PNH symptoms  in the presence of an LDH level ⩾ 2 × ULN after prior reduction to < 1.5 × ULN on treatment.50If terminal complement inhibition is suboptimal in a patient with PNH, there is a risk of breakthrough IVH (BTH), which represents temporary loss of disease control. Suboptimal C5 inhibition may lead to PK BTH, which typically occurs in the final 24–48 h before the next planned infusion of eculizumab (when trough plasma levels of eculizumab are at their lowest) without any obvious trigger. PK BTH is identified by elevated LDH levels, free C5 levels, or detectable 50% hemolytic complement (CH50).62 Shortening the dosing interval or increasing the dose of eculizumab is usually effective for managing PK BTH. Conversely, PD BTH is more unpredictable in that it appears to be independent of the time from the last eculizumab infusion. PD BTH is typically associated with complement-amplifying conditions such as infection, surgery, and other events that may trigger inflammation and complement activation.63 Of the five BTH events occurring in ravulizumab-treated patients across the studies, none was temporally associated with suboptimal C5 inhibition (free C5 ⩾ 0.5 mg/mL); four (80%) were temporally associated with complement-amplifying conditions.Approximately, 10–15% of patients with PNH treated with eculizumab have been found to have suboptimal C5 inhibition at some point in time. When comparing BTH events occurring in each treatment group, it was found that none of the patients treated with ravulizumab had elevated C5 levels at the time of BTH, suggesting that these were unrelated to suboptimal terminal complement inhibition. However, in patients treated with eculizumab who experienced recurrent BTH, episodes were temporally associated with elevated free C5 levels.The occurrence of clinical BTH events was assessed in patients with PNH included in the two pivotal ravulizumab studies. A retrospective analysis assessing data from the 26-week primary evaluation periods of both studies found that fewer BTH events were reported in patients treated with ravulizumab than in those receiving eculizumab. Some patients require periodic blood transfusions because of anemia.50 Although EVH can limit the hematologic benefit provided by C5 inhibitors, it does not appear to have a clear impact on survival or other outcomes. To best equip physicians for the optimal monitoring and management of patients with PNH receiving C5 inhibitors, a clinical algorithm has been developed to evaluate the causes of residual anemia comprehensively.Patients with PNH receiving a terminal complement (C5) inhibitor are susceptible to extravascular hemolysis (EVH), which occurs when the now-surviving PNH RBCs accumulate C3 fragments on their surface, resulting in their recognition and subsequent removal by hepatosplenic phagocytes.post hoc clinical trial analyses have shown that eculizumab is effective and well tolerated when used concurrently with immunosuppressive therapy to treat underlying bone marrow failure while not diminishing the effectiveness of the immunosuppressive therapy.65C5 inhibitors have proven to be efficacious in a variety of subpopulations of patients with PNH. Registry and  The largest study of ravulizumab use in pediatric patients with PNH who are either complement inhibitor-naïve or complement inhibitor-experienced is ongoing, with results showing that ravulizumab is well tolerated and provides immediate, sustained terminal complement inhibition irrespective of the patient’s prior eculizumab treatment status. Ravulizumab was approved for the treatment of pediatric patients by the FDA in June 2021 and is indicated for use in patients with PNH aged 1 month and older, and by the EMA in September 2021 for adults and children with PNH weighing at least 10 kg.The effectiveness of eculizumab has also been demonstrated in pediatric patients with PNH, with reported improvements in clinical outcomes comparable with those seen in adults with the disease. In a retrospective analysis of 75 pregnancies in 61 women, eculizumab was found to be well tolerated and effective. Maternal outcomes were superior to those seen in historical data. Only two TEs were reported, both occurring shortly after delivery, and there were no maternal deaths. In addition, only low-level eculizumab was detected in a minority of cord blood samples, and no drug was identified in breast milk. At present, no data are available on the safety of ravulizumab in pregnant patients with PNH, but the potential for placental transfer of the drug and its long half-life should be considered when counseling women with PNH.Before the introduction of eculizumab in 2007, maternal mortality in pregnant patients with PNH was as high as 20%, driven primarily by TEs. and ravulizumab ineffective. The prevalence of this polymorphism was reported as 3.2% in a population of Japanese patients with PNH and was comparable with that observed in a healthy Japanese population (3.5%). In addition, when the study investigators screened DNA samples from other ethnic populations, the same C5 polymorphism was identified with a prevalence of 0.8% in a Han Chinese population but was not detected in samples from populations with either British or Mexican ancestry. If a lack of clinical/biochemical response to eculizumab is observed in a patient with PNH (defined as the maintenance of elevated LDH levels and no improvements in PNH symptoms), clinicians should be mindful of this polymorphism. Other therapeutics not affected by the polymorphism are anticipated to be effective in such cases  patient-years. The second study was a pharmacovigilance analysis that evaluated eculizumab use in patients with PNH and atypical hemolytic uremic syndrome over 10 years. The rate of meningococcal infection for patients with PNH over the study period was 0.24 per 100 patient-years, with the rate of fatal meningococcal infection reported as 0.03 per 100 patient-years. The rate in patients with PNH receiving eculizumab during the pharmacovigilance study was approximately 1000–2000 times higher than that seen in a healthy population. However, the investigators concluded that current mitigation strategies were generally effective, and that the few meningococcal infections that resulted in a fatality were largely due to delayed diagnosis and/or treatment. As new broad proximal inhibition therapies that target C3 or complement factors B and D become available, the risk of infection with pathogens, such as Haemophilus influenzae, Proteus species, and Pseudomonas species, as well as N. meningitis, will need to be assessed when more evidence becomes available after greater patient exposure to these treatments and increased access to longer-term safety data.Two real-world studies assessing infection risk in patients with PNH have recently been conducted. An International PNH Registry study investigated prophylactic antibiotic use and the risk of meningococcal infection in patients with PNH who had been given a meningococcal vaccination and were receiving eculizumab.88 and the findings of a 10-year eculizumab pharmacovigilance analysis , both drugs are well tolerated. The most frequently reported adverse events were headache, nasopharyngitis, fatigue, and upper respiratory tract infection, and no new safety risks apart from meningococcal infection were reported.According to the results from pivotal phase 3 clinical trials of eculizumab and ravulizumab in PNH,91 Results from an ongoing phase 3 clinical trial have shown noninferiority of a novel subcutaneous (SC) formulation of ravulizumab compared with IV ravulizumab at day 71 in eculizumab-experienced patients with PNH. Current approved and future C5 treatments are listed in 7584Approval of the first terminal complement inhibitor, eculizumab, changed the treatment landscape of PNH by giving patients an effective therapy and demonstrated the critical role of terminal complement in this disease. Over time, interest has arisen in developing additional C5 inhibitors with different modes of administration and therapeutics targeting other components of the complement cascade, especially broader C3 inhibition and proximal complement cascade factors, such as D and B. Eculizumab biosimilars have also emerged.92 has demonstrated efficacy in achieving terminal complement inhibition in eculizumab-experienced patients with PNH using either IV or SC formulations. Tesidolumab is a monoclonal antibody under development in a proof-of-concept phase 2 study in untreated patients with PNH; preliminary results are not yet available.83 Pozelimab is a monoclonal antibody that demonstrated complement inhibition in healthy volunteers, and an open-label, single-arm, phase 2 study is ongoing in patients with PNH who are either complement-inhibitor-naïve or have not recently received a complement inhibitor. Zilucopan is a synthetic macrocyclic peptide inhibitor of complement C5. Phase 2 studies assessing its efficacy and safety in PNH have been conducted and an extension study is ongoing. Coversin is a broad-acting C5 inhibitory protein that successfully reduced LDH levels to < 1.8 × ULN in untreated patients using an SC self-administered formulation. Cemdisiran is an RNA interference therapeutic that targets hepatic C5 synthesis and has been shown to reduce levels of C5 in patients with PNH.Recent developments in the field of C5 inhibitors include crovalimab, tesidolumab, pozelimab, zilucoplan, coversin, and cemdisiran which all target C5 while having different mechanisms of action. Crovalimab, a monoclonal antibody that targets a different epitope to eculizumab/ravulizumab and utilizes similar novel antibody recycling technology to enhance its half-life,14 In particular, uncontrolled terminal complement activity and its consequences put patients at risk of TEs, pulmonary hypertension, renal failure, smooth muscle dystonia, and symptomatic anemia. Complement activation rapidly amplifies in patients with PNH so terminal complement blockade needs to be effective and sustained. Results from a combination of large clinical trials, real-world studies,94 and analyses of safety data collected over extended follow-up periods have shown that eculizumab and ravulizumab are effective and well tolerated when used for preventing morbidity and mortality related to PNH.PNH is a chronic, life-threatening disorder of impaired terminal complement regulation in hematopoietic cells. Its pathology is driven not only by IVH, but also by direct terminal complement activation of GPI-negative platelets and WBCs. It is associated with a range of systemic clinical complications that can result in significant morbidity and mortality. the need for routine travel to clinic to receive treatment, and dosing duration. Ravulizumab, with its 8-weekly dosing interval, was considered the preferred choice for 93% of clinical trial participants consulted.Because PNH is a chronic disease, improved patient HRQoL is a continuing aim. As evidenced by the number of active and upcoming clinical trials of novel complement inhibitors in PNH, the choice of treatments will most likely expand over the next few years. Therefore, when more than one therapeutic option is available the choice of treatment should be guided by patient preference. Influences on preference include the dosing interval,2123 New treatment strategies will aim to address some of the challenges of current therapies . Future PNH therapy should seek to maintain terminal complement suppression and improve HRQoL in this chronic and potentially devastating disease.Pegcetacoplan, the first C3 inhibitor for the treatment of PNH, was recently approved by the FDA and EMA.