Malaria protection due to sickle haemoglobin depends on parasite genotype.

Host genetic factors can confer resistance against malaria1, raising the question of whether this has led to evolutionary adaptation of parasite populations. Here we searched for association between candidate host and parasite genetic variants in 3,346 Gambian and Kenyan children with severe malaria caused by Plasmodium falciparum. We identified a strong association between sickle haemoglobin (HbS) in the host and three regions of the parasite genome, which is not explained by population structure or other covariates, and which is replicated in additional samples. The HbS-associated alleles include nonsynonymous variants in the gene for the acyl-CoA synthetase family member2-4 PfACS8 on chromosome 2, in a second region of chromosome 2, and in a region containing structural variation on chromosome 11. The alleles are in strong linkage disequilibrium and have frequencies that covary with the frequency of HbS across populations, in particular being much more common in Africa than other parts of the world. The estimated protective effect of HbS against severe malaria, as determined by comparison of cases with population controls, varies greatly according to the parasite genotype at these three loci. These findings open up a new avenue of enquiry into the biological and epidemiological significance of the HbS-associated polymorphisms in the parasite genome and the evolutionary forces that have led to their high frequency and strong linkage disequilibrium in African P. falciparum populations.

Non-O ABO blood group genotypes differ in their associations with Plasmodium falciparum rosetting and severe malaria.

Blood group O is associated with protection against severe malaria and reduced size and stability of P. falciparum-host red blood cell (RBC) rosettes compared to non-O blood groups. Whether the non-O blood groups encoded by the specific ABO genotypes AO, BO, AA, BB and AB differ in their associations with severe malaria and rosetting is unknown. The A and B antigens are host RBC receptors for rosetting, hence we hypothesized that the higher levels of A and/or B antigen on RBCs from AA, BB and AB genotypes compared to AO/BO genotypes could lead to larger rosettes, increased microvascular obstruction and higher risk of malaria pathology. We used a case-control study of Kenyan children and in vitro adhesion assays to test the hypothesis that "double dose" non-O genotypes (AA, BB, AB) are associated with increased risk of severe malaria and larger rosettes than "single dose" heterozygotes (AO, BO). In the case-control study, compared to OO, the double dose genotypes consistently had higher odds ratios (OR) for severe malaria than single dose genotypes, with AB (OR 1.93) and AO (OR 1.27) showing most marked difference (p = 0.02, Wald test). In vitro experiments with blood group A-preferring P. falciparum parasites showed that significantly larger rosettes were formed with AA and AB host RBCs compared to OO, whereas AO and BO genotypes rosettes were indistinguishable from OO. Overall, the data show that ABO genotype influences P. falciparum rosetting and support the hypothesis that double dose non-O genotypes confer a greater risk of severe malaria than AO/BO heterozygosity.

Novel risk loci in LGI1-antibody encephalitis: genome-wide association study discovery and validation cohorts.

Encephalitis with antibodies to leucine-rich glioma-inactivated 1 (LGI1-Ab-E) is a common form of autoimmune encephalitis, presenting with seizures and neuropsychiatric changes, predominantly in older males. More than 90% of patients carry the human leucocyte antigen (HLA) class II allele, HLA-DRB1*07:01. However, this is also present in 25% of healthy controls. Therefore, we hypothesised the presence of additional genetic predispositions. In this genome-wide association study and meta-analysis, we studied a discovery cohort of 131 French LGI1-Ab-E and a validation cohort of 126 American, British and Irish LGI1-Ab-E patients, ancestry-matched to 2613 and 2538 European controls, respectively. Outside the known major HLA signal, we found two single nucleotide polymorphisms (SNPs) at genome-wide significance (p < 5 x 10-8), implicating PTPRD, a protein tyrosine phosphatase, and LINC00670, a non-protein coding RNA gene. Meta-analysis defined four additional non-HLA loci, including the protein coding COBL gene. Polygenic risk scores with and without HLA variants proposed a contribution of non-HLA loci. In silico network analyses suggested LGI1 and PTPRD mediated interactions via the established receptors of LGI1, ADAM22 and ADAM23. Our results identify new genetic loci in LGI1-Ab-E. These findings present opportunities for mechanistic studies and offer potential markers of susceptibility, prognostics and therapeutic responses.

Genome-wide association study of leprosy in Malawi and Mali.

Leprosy is a chronic infection of the skin and peripheral nerves caused by Mycobacterium leprae. Despite recent improvements in disease control, leprosy remains an important cause of infectious disability globally. Large-scale genetic association studies in Chinese, Vietnamese and Indian populations have identified over 30 susceptibility loci for leprosy. There is a significant burden of leprosy in Africa, however it is uncertain whether the findings of published genetic association studies are generalizable to African populations. To address this, we conducted a genome-wide association study (GWAS) of leprosy in Malawian (327 cases, 436 controls) and Malian (247 cases, 368 controls) individuals. In that analysis, we replicated four risk loci previously reported in China, Vietnam and India; MHC Class I and II, LACC1 and SLC29A3. We further identified a novel leprosy susceptibility locus at 10q24 (rs2015583; combined p = 8.81 × 10-9; OR = 0.51 [95% CI 0.40 - 0.64]). Using publicly-available data we characterise regulatory activity at this locus, identifying ACTR1A as a candidate mediator of leprosy risk. This locus shows evidence of recent positive selection and demonstrates pleiotropy with established risk loci for inflammatory bowel disease and childhood-onset asthma. A shared genetic architecture for leprosy and inflammatory bowel disease has been previously described. We expand on this, strengthening the hypothesis that selection pressure driven by leprosy has shaped the evolution of autoimmune and atopic disease in modern populations. More broadly, our data highlights the importance of defining the genetic architecture of disease across genetically diverse populations, and that disease insights derived from GWAS in one population may not translate to all affected populations.

The UK Biobank resource with deep phenotyping and genomic data.

The UK Biobank project is a prospective cohort study with deep genetic and phenotypic data collected on approximately 500,000 individuals from across the United Kingdom, aged between 40 and 69 at recruitment. The open resource is unique in its size and scope. A rich variety of phenotypic and health-related information is available on each participant, including biological measurements, lifestyle indicators, biomarkers in blood and urine, and imaging of the body and brain. Follow-up information is provided by linking health and medical records. Genome-wide genotype data have been collected on all participants, providing many opportunities for the discovery of new genetic associations and the genetic bases of complex traits. Here we describe the centralized analysis of the genetic data, including genotype quality, properties of population structure and relatedness of the genetic data, and efficient phasing and genotype imputation that increases the number of testable variants to around 96 million. Classical allelic variation at 11 human leukocyte antigen genes was imputed, resulting in the recovery of signals with known associations between human leukocyte antigen alleles and many diseases.

Using de novo assembly to identify structural variation of eight complex immune system gene regions.

Driven by the necessity to survive environmental pathogens, the human immune system has evolved exceptional diversity and plasticity, to which several factors contribute including inheritable structural polymorphism of the underlying genes. Characterizing this variation is challenging due to the complexity of these loci, which contain extensive regions of paralogy, segmental duplication and high copy-number repeats, but recent progress in long-read sequencing and optical mapping techniques suggests this problem may now be tractable. Here we assess this by using long-read sequencing platforms from PacBio and Oxford Nanopore, supplemented with short-read sequencing and Bionano optical mapping, to sequence DNA extracted from CD14+ monocytes and peripheral blood mononuclear cells from a single European individual identified as HV31. We use this data to build a de novo assembly of eight genomic regions encoding four key components of the immune system, namely the human leukocyte antigen, immunoglobulins, T cell receptors, and killer-cell immunoglobulin-like receptors. Validation of our assembly using k-mer based and alignment approaches suggests that it has high accuracy, with estimated base-level error rates below 1 in 10 kb, although we identify a small number of remaining structural errors. We use the assembly to identify heterozygous and homozygous structural variation in comparison to GRCh38. Despite analyzing only a single individual, we find multiple large structural variants affecting core genes at all three immunoglobulin regions and at two of the three T cell receptor regions. Several of these variants are not accurately callable using current algorithms, implying that further methodological improvements are needed. Our results demonstrate that assessing haplotype variation in these regions is possible given sufficiently accurate long-read and associated data. Continued reductions in the cost of these technologies will enable application of these methods to larger samples and provide a broader catalogue of germline structural variation at these loci, an important step toward making these regions accessible to large-scale genetic association studies.

A novel locus of resistance to severe malaria in a region of ancient balancing selection.

The high prevalence of sickle haemoglobin in Africa shows that malaria has been a major force for human evolutionary selection, but surprisingly few other polymorphisms have been proven to confer resistance to malaria in large epidemiological studies. To address this problem, we conducted a multi-centre genome-wide association study (GWAS) of life-threatening Plasmodium falciparum infection (severe malaria) in over 11,000 African children, with replication data in a further 14,000 individuals. Here we report a novel malaria resistance locus close to a cluster of genes encoding glycophorins that are receptors for erythrocyte invasion by P. falciparum. We identify a haplotype at this locus that provides 33% protection against severe malaria (odds ratio = 0.67, 95% confidence interval = 0.60-0.76, P value = 9.5 × 10(-11)) and is linked to polymorphisms that have previously been shown to have features of ancient balancing selection, on the basis of haplotype sharing between humans and chimpanzees. Taken together with previous observations on the malaria-protective role of blood group O, these data reveal that two of the strongest GWAS signals for severe malaria lie in or close to genes encoding the glycosylated surface coat of the erythrocyte cell membrane, both within regions of the genome where it appears that evolution has maintained diversity for millions of years. These findings provide new insights into the host-parasite interactions that are critical in determining the outcome of malaria infection.

Bayesian meta-analysis across genome-wide association studies of diverse phenotypes.

Genome-wide association studies (GWAS) are a powerful tool for understanding the genetic basis of diseases and traits, but most studies have been conducted in isolation, with a focus on either a single or a set of closely related phenotypes. We describe MetABF, a simple Bayesian framework for performing integrative meta-analysis across multiple GWAS using summary statistics. The approach is applicable across a wide range of study designs and can increase the power by 50% compared with standard frequentist tests when only a subset of studies have a true effect. We demonstrate its utility in a meta-analysis of 20 diverse GWAS which were part of the Wellcome Trust Case Control Consortium 2. The novelty of the approach is its ability to explore, and assess the evidence for a range of possible true patterns of association across studies in a computationally efficient framework.

Polymorphism in a lincRNA Associates with a Doubled Risk of Pneumococcal Bacteremia in Kenyan Children.

Bacteremia (bacterial bloodstream infection) is a major cause of illness and death in sub-Saharan Africa but little is known about the role of human genetics in susceptibility. We conducted a genome-wide association study of bacteremia susceptibility in more than 5,000 Kenyan children as part of the Wellcome Trust Case Control Consortium 2 (WTCCC2). Both the blood-culture-proven bacteremia case subjects and healthy infants as controls were recruited from Kilifi, on the east coast of Kenya. Streptococcus pneumoniae is the most common cause of bacteremia in Kilifi and was thus the focus of this study. We identified an association between polymorphisms in a long intergenic non-coding RNA (lincRNA) gene (AC011288.2) and pneumococcal bacteremia and replicated the results in the same population (p combined = 1.69 × 10(-9); OR = 2.47, 95% CI = 1.84-3.31). The susceptibility allele is African specific, derived rather than ancestral, and occurs at low frequency (2.7% in control subjects and 6.4% in case subjects). Our further studies showed AC011288.2 expression only in neutrophils, a cell type that is known to play a major role in pneumococcal clearance. Identification of this novel association will further focus research on the role of lincRNAs in human infectious disease.

Environmental Correlation Analysis for Genes Associated with Protection against Malaria.

Genome-wide searches for loci involved in human resistance to malaria are currently being conducted on a large scale in Africa using case-control studies. Here, we explore the utility of an alternative approach-"environmental correlation analysis, ECA," which tests for clines in allele frequencies across a gradient of an environmental selection pressure-to identify genes that have historically protected against death from malaria. We collected genotype data from 12,425 newborns on 57 candidate malaria resistance loci and 9,756 single nucleotide polymorphisms (SNPs) selected at random from across the genome, and examined their allele frequencies for geographic correlations with long-term malaria prevalence data based on 84,042 individuals living under different historical selection pressures from malaria in coastal Kenya. None of the 57 candidate SNPs showed significant (P < 0.05) correlations in allele frequency with local malaria transmission intensity after adjusting for population structure and multiple testing. In contrast, two of the random SNPs that had highly significant correlations (P < 0.01) were in genes previously linked to malaria resistance, namely, CDH13, encoding cadherin 13, and HS3ST3B1, encoding heparan sulfate 3-O-sulfotransferase 3B1. Both proteins play a role in glycoprotein-mediated cell-cell adhesion which has been widely implicated in cerebral malaria, the most life-threatening form of this disease. Other top genes, including CTNND2 which encodes δ-catenin, a molecular partner to cadherin, were significantly enriched in cadherin-mediated pathways affecting inflammation of the brain vascular endothelium. These results demonstrate the utility of ECA in the discovery of novel genes and pathways affecting infectious disease.

Genetic risk and a primary role for cell-mediated immune mechanisms in multiple sclerosis.

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

Imputation-based meta-analysis of severe malaria in three African populations.

Combining data from genome-wide association studies (GWAS) conducted at different locations, using genotype imputation and fixed-effects meta-analysis, has been a powerful approach for dissecting complex disease genetics in populations of European ancestry. Here we investigate the feasibility of applying the same approach in Africa, where genetic diversity, both within and between populations, is far more extensive. We analyse genome-wide data from approximately 5,000 individuals with severe malaria and 7,000 population controls from three different locations in Africa. Our results show that the standard approach is well powered to detect known malaria susceptibility loci when sample sizes are large, and that modern methods for association analysis can control the potential confounding effects of population structure. We show that pattern of association around the haemoglobin S allele differs substantially across populations due to differences in haplotype structure. Motivated by these observations we consider new approaches to association analysis that might prove valuable for multicentre GWAS in Africa: we relax the assumptions of SNP-based fixed effect analysis; we apply Bayesian approaches to allow for heterogeneity in the effect of an allele on risk across studies; and we introduce a region-based test to allow for heterogeneity in the location of causal alleles.

Genetic determinants of anti-malarial acquired immunity in a large multi-centre study.

BACKGROUND: Many studies report associations between human genetic factors and immunity to malaria but few have been reliably replicated. These studies are usually country-specific, use small sample sizes and are not directly comparable due to differences in methodologies. This study brings together samples and data collected from multiple sites across Africa and Asia to use standardized methods to look for consistent genetic effects on anti-malarial antibody levels. METHODS: Sera, DNA samples and clinical data were collected from 13,299 individuals from ten sites in Senegal, Mali, Burkina Faso, Sudan, Kenya, Tanzania, and Sri Lanka using standardized methods. DNA was extracted and typed for 202 Single Nucleotide Polymorphisms with known associations to malaria or antibody production, and antibody levels to four clinical grade malarial antigens [AMA1, MSP1, MSP2, and (NANP)4] plus total IgE were measured by ELISA techniques. Regression models were used to investigate the associations of clinical and genetic factors with antibody levels. RESULTS: Malaria infection increased levels of antibodies to malaria antigens and, as expected, stable predictors of anti-malarial antibody levels included age, seasonality, location, and ethnicity. Correlations between antibodies to blood-stage antigens AMA1, MSP1 and MSP2 were higher between themselves than with antibodies to the (NANP)4 epitope of the pre-erythrocytic circumsporozoite protein, while there was little or no correlation with total IgE levels. Individuals with sickle cell trait had significantly lower antibody levels to all blood-stage antigens, and recessive homozygotes for CD36 (rs321198) had significantly lower anti-malarial antibody levels to MSP2. CONCLUSION: Although the most significant finding with a consistent effect across sites was for sickle cell trait, its effect is likely to be via reducing a microscopically positive parasitaemia rather than directly on antibody levels. However, this study does demonstrate a framework for the feasibility of combining data from sites with heterogeneous malaria transmission levels across Africa and Asia with which to explore genetic effects on anti-malarial immunity.

Malaria endemicity linked to shorter telomeres in leukocytes.

Leukocyte telomere length is a highly polygenic trait that has been associated with a complex range of lifestyle factors and disease risk. McQuillan et al.'s results comparing telomere length to malaria incidence rates suggest that infections may be another important factor, possibly through permanent shortening of telomeres in hematopoietic progenitor cells.

Dissection of the genetics of Parkinson's disease identifies an additional association 5' of SNCA and multiple associated haplotypes at 17q21.

We performed a genome-wide association study (GWAS) in 1705 Parkinson's disease (PD) UK patients and 5175 UK controls, the largest sample size so far for a PD GWAS. Replication was attempted in an additional cohort of 1039 French PD cases and 1984 controls for the 27 regions showing the strongest evidence of association (P< 10(-4)). We replicated published associations in the 4q22/SNCA and 17q21/MAPT chromosome regions (P< 10(-10)) and found evidence for an additional independent association in 4q22/SNCA. A detailed analysis of the haplotype structure at 17q21 showed that there are three separate risk groups within this region. We found weak but consistent evidence of association for common variants located in three previously published associated regions (4p15/BST1, 4p16/GAK and 1q32/PARK16). We found no support for the previously reported SNP association in 12q12/LRRK2. We also found an association of the two SNPs in 4q22/SNCA with the age of onset of the disease.

BIRC6 modifies risk of invasive bacterial infection 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 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.

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.

Evidence of the interplay of genetics and culture in Ethiopia.

The rich linguistic, ethnic and cultural diversity of Ethiopia provides an unprecedented opportunity to understand the level to which cultural factors correlate with-and shape-genetic structure in human populations. Using primarily new genetic variation data covering 1,214 Ethiopians representing 68 different ethnic groups, together with information on individuals' birthplaces, linguistic/religious practices and 31 cultural practices, we disentangle the effects of geographic distance, elevation, and social factors on the genetic structure of Ethiopians today. We provide evidence of associations between social behaviours and genetic differences among present-day peoples. We show that genetic similarity is broadly associated with linguistic affiliation, but also identify pronounced genetic similarity among groups from disparate language classifications that may in part be attributable to recent intermixing. We also illustrate how groups reporting the same culture traits are more genetically similar on average and show evidence of recent intermixing, suggesting that shared cultural traits may promote admixture. In addition to providing insights into the genetic structure and history of Ethiopia, we identify the most important cultural and geographic predictors of genetic differentiation and provide a resource for designing sampling protocols for future genetic studies involving Ethiopians.

Haplotype heterogeneity and low linkage disequilibrium reduce reliable prediction of genotypes for the ­α 3.7I form of α-thalassaemia using genome-wide microarray data.

Background: The -α 3.7I-thalassaemia deletion is very common throughout Africa because it protects against malaria. When undertaking studies to investigate human genetic adaptations to malaria or other diseases, it is important to account for any confounding effects of α-thalassaemia to rule out spurious associations. Methods: In this study we have used direct α-thalassaemia genotyping to understand why GWAS data from a large malaria association study in Kilifi Kenya did not identify the α-thalassaemia signal. We then explored the potential use of a number of new approaches to using GWAS data for imputing α-thalassaemia as an alternative to direct genotyping by PCR. Results: We found very low linkage-disequilibrium of the directly typed data with the GWAS SNP markers around α-thalassaemia and across the haemoglobin-alpha ( HBA) gene region, which along with a complex haplotype structure, could explain the lack of an association signal from the GWAS SNP data. Some indirect typing methods gave results that were in broad agreement with those derived from direct genotyping and could identify an association signal, but none were sufficiently accurate to allow correct interpretation compared with direct typing, leading to confusing or erroneous results. Conclusions: We conclude that going forwards, direct typing methods such as PCR will still be required to account for α-thalassaemia in GWAS studies.

The ferroportin Q248H mutation protects from anemia, but not malaria or bacteremia.

Iron acquisition is critical for life. Ferroportin (FPN) exports iron from mature erythrocytes, and deletion of the Fpn gene results in hemolytic anemia and increased fatality in malaria-infected mice. The FPN Q248H mutation (glutamine to histidine at position 248) renders FPN partially resistant to hepcidin-induced degradation and was associated with protection from malaria in human studies of limited size. Using data from cohorts including over 18,000 African children, we show that the Q248H mutation is associated with modest protection against anemia, hemolysis, and iron deficiency, but we found little evidence of protection against severe malaria or bacteremia. We additionally observed no excess Plasmodium growth in Q248H erythrocytes ex vivo, nor evidence of selection driven by malaria exposure, suggesting that the Q248H mutation does not protect from malaria and is unlikely to deprive malaria parasites of iron essential for their growth.

Human candidate gene polymorphisms and risk of severe malaria in children in Kilifi, Kenya: a case-control association study.

BACKGROUND: Human genetic factors are important determinants of malaria risk. We investigated associations between multiple candidate polymorphisms-many related to the structure or function of red blood cells-and risk for severe Plasmodium falciparum malaria and its specific phenotypes, including cerebral malaria, severe malaria anaemia, and respiratory distress. METHODS: We did a case-control study in Kilifi County, Kenya. We recruited as cases children presenting with severe malaria to the high-dependency ward of Kilifi County Hospital. We included as controls infants born in the local community between Aug 1, 2006, and Sept 30, 2010, who were part of a genetics study. We tested for associations between a range of candidate malaria-protective genes and risk for severe malaria and its specific phenotypes. We used a permutation approach to account for multiple comparisons between polymorphisms and severe malaria. We judged p values less than 0·005 significant for the primary analysis of the association between candidate genes and severe malaria. FINDINGS: Between June 11, 1995, and June 12, 2008, 2244 children with severe malaria were recruited to the study, and 3949 infants were included as controls. Overall, 263 (12%) of 2244 children with severe malaria died in hospital, including 196 (16%) of 1233 with cerebral malaria. We investigated 121 polymorphisms in 70 candidate severe malaria-associated genes. We found significant associations between risk for severe malaria overall and polymorphisms in 15 genes or locations, of which most were related to red blood cells: ABO, ATP2B4, ARL14, CD40LG, FREM3, INPP4B, G6PD, HBA (both HBA1 and HBA2), HBB, IL10, LPHN2 (also known as ADGRL2), LOC727982, RPS6KL1, CAND1, and GNAS. Combined, these genetic associations accounted for 5·2% of the variance in risk for developing severe malaria among individuals in the general population. We confirmed established associations between severe malaria and sickle-cell trait (odds ratio [OR] 0·15, 95% CI 0·11-0·20; p=2·61 × 10-58), blood group O (0·74, 0·66-0·82; p=6·26 × 10-8), and -α3·7-thalassaemia (0·83, 0·76-0·90; p=2·06 × 10-6). We also found strong associations between overall risk of severe malaria and polymorphisms in both ATP2B4 (OR 0·76, 95% CI 0·63-0·92; p=0·001) and FREM3 (0·64, 0·53-0·79; p=3·18 × 10-14). The association with FREM3 could be accounted for by linkage disequilibrium with a complex structural mutation within the glycophorin gene region (comprising GYPA, GYPB, and GYPE) that encodes for the rare Dantu blood group antigen. Heterozygosity for Dantu was associated with risk for severe malaria (OR 0·57, 95% CI 0·49-0·68; p=3·22 × 10-11), as was homozygosity (0·26, 0·11-0·62; p=0·002). INTERPRETATION: Both ATP2B4 and the Dantu blood group antigen are associated with the structure and function of red blood cells. ATP2B4 codes for plasma membrane calcium-transporting ATPase 4 (the major calcium pump on red blood cells) and the glycophorins are ligands for parasites to invade red blood cells. Future work should aim at uncovering the mechanisms by which these polymorphisms can result in severe malaria protection and investigate the implications of these associations for wider health. FUNDING: Wellcome Trust, UK Medical Research Council, European Union, and Foundation for the National Institutes of Health as part of the Bill & Melinda Gates Grand Challenges in Global Health Initiative.