Malaria Phenotypes Research Articles

Modulation of Malaria Phenotypes by Pyruvate Kinase (PKLR) Variants in a Thai Population.

Pyruvate kinase (PKLR) is a critical erythrocyte enzyme that is required for glycolysis and production of ATP. We have shown that Pklr deficiency in mice reduces the severity (reduced parasitemia, increased survival) of blood stage malaria induced by infection with Plasmodium chabaudi AS. Likewise, studies in human erythrocytes infected ex vivo with P. falciparum show that presence of host PK-deficiency alleles reduces infection phenotypes. We have characterized the genetic diversity of the PKLR gene, including haplotype structure and presence of rare coding variants in two populations from malaria endemic areas of Thailand and Senegal. We investigated the effect of PKLR genotypes on rich longitudinal datasets including haematological and malaria-associated phenotypes. A coding and possibly damaging variant (R41Q) was identified in the Thai population with a minor allele frequency of ~4.7%. Arginine 41 (R41) is highly conserved in the pyruvate kinase family and its substitution to Glutamine (R41Q) affects protein stability. Heterozygosity for R41Q is shown to be associated with a significant reduction in the number of attacks with Plasmodium falciparum, while correlating with an increased number of Plasmodium vivax infections. These results strongly suggest that PKLR protein variants may affect the frequency, and the intensity of malaria episodes induced by different Plasmodium parasites in humans living in areas of endemic malaria.

Genome-wide Analysis of Host-Plasmodium yoelii Interactions Reveals Regulators of the Type I Interferon Response.

Invading pathogens trigger specific host responses, an understanding of which might identify genes that function in pathogen recognition and elimination. In this study, we performed trans-species expression quantitative trait locus (ts-eQTL) analysis using genotypes of the Plasmodium yoelii malaria parasite and phenotypes of mouse gene expression. We significantly linked 1,054 host genes to parasite genetic loci (LOD score ≥ 3.0). Using LOD score patterns, which produced results that differed from direct expression-level clustering, we grouped hostgenes that function in related pathways, allowing functional prediction of unknown genes. As aproof of principle, 14 of 15 randomly selected genespredicted to function in type I interferon (IFN-I) responses were experimentally validated using overexpression, small hairpin RNA knockdown, viral infection, and/or infection of knockout mice. This study demonstrates an effective strategy for studying gene function, establishes a functional gene database, and identifies regulators in IFN-I pathways.

The genetic control of immunity to Plasmodium infection.

BackgroundMalaria remains a major worldwide public health problem with ~207 million cases and ~627,000 deaths per year, mainly affecting children under five years of age in Africa. Recent efforts at elaborating a genetic architecture of malaria have focused on severe malaria, leading to the identification of two new genes and confirmation of previously known variants in HBB, ABO and G6PD, by exploring the whole human genome in genome-wide association (GWA) studies. Molecular pathways controlling phenotypes representing effectiveness of host immunity, notably parasitemia and IgG levels, are of particular interest given the current lack of an efficacious vaccine and the need for new treatment options.ResultsWe propose a global causal framework of malaria phenotypes implicating progression from the initial infection with Plasmodium spp. to the development of the infection through liver and blood-stage multiplication cycles (parasitemia as a quantitative trait), to clinical malaria attack, and finally to severe malaria. Genetic polymorphism may control any of these stages, such that preceding stages act as mediators of subsequent stages. A biomarker of humoral immunity, IgG levels, can also be integrated into the framework, potentially mediating the impact of polymorphism by limiting parasitemia levels. Current knowledge of the genetic basis of parasitemia levels and IgG levels is reviewed through key examples including the hemoglobinopathies, showing that the protective effect of HBB variants on malaria clinical phenotypes may partially be mediated through parasitemia and cytophilic IgG levels. Another example is the IgG receptor FcγRIIa, encoded by FCGR2A, such that H131 homozygotes displayed higher IgG2 levels and were protective against high parasitemia and onset of malaria symptoms as shown in a causal diagram.ConclusionsWe thus underline the value of parasitemia and IgG levels as phenotypes in the understanding of the human genetic architecture of malaria, and the need for applying GWA approaches to these phenotypes.

Endothelial protein C receptor gene variants not associated with severe malaria in ghanaian children.

BackgroundTwo recent reports have identified the Endothelial Protein C Receptor (EPCR) as a key molecule implicated in severe malaria pathology. First, it was shown that EPCR in the human microvasculature mediates sequestration of Plasmodium falciparum-infected erythrocytes. Second, microvascular thrombosis, one of the major processes causing cerebral malaria, was linked to a reduction in EPCR expression in cerebral endothelial layers. It was speculated that genetic variation affecting EPCR functionality could influence susceptibility to severe malaria phenotypes, rendering PROCR, the gene encoding EPCR, a promising candidate for an association study.MethodsHere, we performed an association study including high-resolution variant discovery of rare and frequent genetic variants in the PROCR gene. The study group, which previously has proven to be a valuable tool for studying the genetics of malaria, comprised 1,905 severe malaria cases aged 1–156 months and 1,866 apparently healthy children aged 2–161 months from the Ashanti Region in Ghana, West Africa, where malaria is highly endemic. Association of genetic variation with severe malaria phenotypes was examined on the basis of single variants, reconstructed haplotypes, and rare variant analyses.ResultsA total of 41 genetic variants were detected in regulatory and coding regions of PROCR, 17 of which were previously unknown genetic variants. In association tests, none of the single variants, haplotypes or rare variants showed evidence for an association with severe malaria, cerebral malaria, or severe malaria anemia.ConclusionHere we present the first analysis of genetic variation in the PROCR gene in the context of severe malaria in African subjects and show that genetic variation in the PROCR gene in our study population does not influence susceptibility to major severe malaria phenotypes.

Impairment of executive function in Kenyan children exposed to severe falciparum malaria with neurological involvement

BackgroundPersistent neurocognitive impairments occur in a fifth of children hospitalized with severe falciparum malaria. There is little data on the association between different neurological phenotypes of severe malaria (seizures, impaired consciousness and prostration) and impairments in executive function.MethodsExecutive functioning of children exposed to severe malaria with different neurological phenotypes (N = 58) and in those unexposed (N = 56) was examined using neuropsychological tests such as vigilance test, test for everyday attention test for children (TEA-Ch), contingency naming test (CNT) and self-ordered pointing test (SOPT). Linear regression was used to determine the association between neurological phenotypes of severe malaria and executive function performance scores, accounting for potential confounders.ResultsChildren with complex seizures in severe malaria performed more poorly than unexposed controls in the vigilance (median efficiency scores (interquartile range) = 4.84 (1.28-5.68) vs. 5.84 (4.71-6.42), P = 0.030) and SOPT (mean errors (standard deviation) = 29.50 (8.82) vs. 24.80 (6.50), P = 0.029) tests, but no differences were observed in TEA-Ch and CNT tests. Performance scores for other neurological phenotypes of severe malaria were similar with those of unexposed controls. After accounting for potential confounders, such as child’s age, sex, schooling; maternal age, schooling and economic activity; perinatal factors and history of seizures, complex seizures remained associated with efficiency scores in the vigilance test (beta coefficient (β) (95% confidence interval (CI)) = -0.40 (-0.67, -0.13), P = 0.006) and everyday attention scores of the TEA-Ch test (β (95% CI) = -0.57 (-1.04, -0.10), P = 0.019); the association with SOPT error scores was weak (β (95% CI) = 4.57 (-0.73-9.89), P = 0.089). Combined neurological phenotypes were not significantly associated with executive function performance scores.ConclusionExecutive function impairment in children with severe malaria is associated with specific neurological phenotypes, particularly complex seizures. Effective prophylaxis and management of malaria-associated acute seizures may improve executive functioning performance scores of children.

Association of candidate gene polymorphisms and TGF-beta/IL-10 levels with malaria in three regions of Cameroon: a case-control study.

BackgroundPlasmodium falciparum malaria is one of the most widespread and deadliest infectious diseases in children under five years in endemic areas. The disease has been a strong force for evolutionary selection in the human genome, and uncovering the critical host genetic factors that confer resistance to the disease would provide clues to the molecular basis of protective immunity and improve vaccine development initiatives.MethodsThe effect of single nucleotide polymorphisms (SNPs) and plasma transforming growth factor beta (TGF-β) and interleukin 10 (IL-10) levels on malaria pathology was investigated in a case–control study of 1862 individuals from two major ethnic groups in three regions with intense perennial P. falciparum transmission in Cameroon. Thirty-four malaria candidate polymorphisms, including the sickle cell trait (HbS), were assayed on the Sequenom iPLEX platform while plasma TGF-β and IL-10 levels were measured by sandwich ELISA.ResultsThe study confirms the known protective effect of HbS against severe malaria and also reveals a protective effect of SNPs in the nitrogen oxide synthase 2 (NOS2) gene against malaria infection, anaemia and uncomplicated malaria. Furthermore, ADCY9 rs10775349 (additive G) and ABO rs8176746 AC individuals were associated with protection from hyperpyrexia and hyperparasitaemia, respectively. Meanwhile, individuals with the EMR1 rs373533 GT, EMR1 rs461645 CT and RTN3 rs542998 (additive C) genotypes were more susceptible to hyperpyrexia while both females and males with the rs1050828 and rs1050829 SNPs of G6PD, respectively, were more vulnerable to anaemia. Plasma TGF-β levels were strongly correlated with heterozygosity for the ADCY9 rs2230739 and HBB rs334 SNPs while individuals with the ABO rs8176746 AC genotype had lower IL-10 levels.ConclusionTaken together, this study suggests that some rare polymorphisms in candidate genes may have important implications for the susceptibility of Cameroonians to severe malaria. Moreover using the uncomplicated malaria phenotype may permit the identification of novel pathways in the early development of the disease.

Distribution of erythrocyte binding antigen 175 (EBA-175) gene dimorphic alleles in Plasmodium falciparum field isolates from Sudan.

BackgroundThe Erythrocyte Binding Antigen (EBA) 175 has been considered as one of the most important Plasmodium falciparum (P. falciparum) merozoite ligands that mediate invasion of the erythrocytes through their sialated receptor: Glycophorin A (GPA). The effect of the EBA 175 dimorphic alleles (F and C) on the severity of the disease is not yet fully understood. Therefore this study was designed to assess the distribution of the divergent dimorphic alleles of P. falciparum EBA-175 (F and C) in three different geographical areas in Sudan and the possible association of this dimorphism with the severity of the disease.MethodsA sum of 339 field isolates of P. falciparum obtained from patients in three different geographical areas in Sudan were screened for the dimorphic alleles (F, C) of the EBA-175 using nested PCR.ResultsThe percentage of F, C, and mixed F/C alleles were; 41%, 51%, and 8% respectively. F and C alleles showed significantly different distributions in the various geographic areas (p = 0.00). There was no significant association between malaria clinical manifestation and P. falciparum EBA-175 F and C alleles frequencies.ConclusionsThis study showed a significant differential distribution of F and C alleles in different geographical malaria endemic areas. No significant association was observed between F and C alleles and different malaria phenotypes.

Biomarker Discovery by Sparse Canonical Correlation Analysis of Complex Clinical Phenotypes of Tuberculosis and Malaria

Biomarker discovery aims to find small subsets of relevant variables in ‘omics data that correlate with the clinical syndromes of interest. Despite the fact that clinical phenotypes are usually characterized by a complex set of clinical parameters, current computational approaches assume univariate targets, e.g. diagnostic classes, against which associations are sought for. We propose an approach based on asymmetrical sparse canonical correlation analysis (SCCA) that finds multivariate correlations between the ‘omics measurements and the complex clinical phenotypes. We correlated plasma proteomics data to multivariate overlapping complex clinical phenotypes from tuberculosis and malaria datasets. We discovered relevant ‘omic biomarkers that have a high correlation to profiles of clinical measurements and are remarkably sparse, containing 1.5–3% of all ‘omic variables. We show that using clinical view projections we obtain remarkable improvements in diagnostic class prediction, up to 11% in tuberculosis and up to 5% in malaria. Our approach finds proteomic-biomarkers that correlate with complex combinations of clinical-biomarkers. Using the clinical-biomarkers improves the accuracy of diagnostic class prediction while not requiring the measurement plasma proteomic profiles of each subject. Our approach makes it feasible to use omics' data to build accurate diagnostic algorithms that can be deployed to community health centres lacking the expensive ‘omics measurement capabilities.

Gene-Gene Interaction and Functional Impact of Polymorphisms on Innate Immune Genes in Controlling Plasmodium falciparum Blood Infection Level

Genetic variations in toll-like receptors and cytokine genes of the innate immune pathways have been implicated in controlling parasite growth and the pathogenesis of Plasmodium falciparum mediated malaria. We previously published genetic association of TLR4 non-synonymous and TNF-α promoter polymorphisms with P.falciparum blood infection level and here we extend the study considerably by (i) investigating genetic dependence of parasite-load on interleukin-12B polymorphisms, (ii) reconstructing gene-gene interactions among candidate TLRs and cytokine loci, (iii) exploring genetic and functional impact of epistatic models and (iv) providing mechanistic insights into functionality of disease-associated regulatory polymorphisms. Our data revealed that carriage of AA (P = 0.0001) and AC (P = 0.01) genotypes of IL12B 3′UTR polymorphism was associated with a significant increase of mean log-parasitemia relative to rare homozygous genotype CC. Presence of IL12B+1188 polymorphism in five of six multifactor models reinforced its strong genetic impact on malaria phenotype. Elevation of genetic risk in two-component models compared to the corresponding single locus and reduction of IL12B (2.2 fold) and lymphotoxin-α (1.7 fold) expressions in patients'peripheral-blood-mononuclear-cells under TLR4Thr399Ile risk genotype background substantiated the role of Multifactor Dimensionality Reduction derived models. Marked reduction of promoter activity of TNF-α risk haplotype (C-C-G-G) compared to wild-type haplotype (T-C-G-G) with (84%) and without (78%) LPS stimulation and the loss of binding of transcription factors detected in-silico supported a causal role of TNF-1031. Significantly lower expression of IL12B+1188 AA (5 fold) and AC (9 fold) genotypes compared to CC and under-representation (P = 0.0048) of allele A in transcripts of patients' PBMCs suggested an Allele-Expression-Imbalance. Allele (A+1188C) dependent differential stability (2 fold) of IL12B-transcripts upon actinomycin-D treatment and observed structural modulation (P = 0.013) of RNA-ensemble were the plausible explanations for AEI. In conclusion, our data provides functional support to the hypothesis that de-regulated receptor-cytokine axis of innate immune pathway influences blood infection level in P. falciparum malaria.

The repertoire diversity of the Plasmodium falciparum stevor multigene family in complicated and uncomplicated malaria in India

Background The deep vascular sequestration of parasitized erythrocytes is a central pathological event in falciparum malaria. Variant surface antigens are encoded mainly by three multi-copy gene families, namely, var, stevor ,a ndrifin. Var is one of the most important families that plays a crucial role in antigenic variation and immune evasion. Clinical and epidemiological studies have shown that severe or complicated malaria is manifested in a limited number of patients. This indicates that a subset of these multigene families could be determinants in the manifestation of different malaria phenotypes. Recent studies have indicated the possible role of stevor (sub-telomeric variable open read), a multi-gene family, in erythrocyte invasion, antigenic variation and host cell modification, of infected erythrocytes. In this study, we describe the repertoire and diversity of members of the stevor multigene family in patients with complicated and uncomplicated malaria in India.

The rs150311303 Polymorphism in FcγRIIa Enhances IgG Binding Capacity

Fc gamma receptor (FcγR) provides an important link between humoral and cellular immune responses. FcγRIIa-H131R polymorphism has been associated with differential binding to IgG subclasses and susceptibility to severe malaria phenotypes among different populations in the malaria endemic world. In this study, the effect of FCGR2A gene polymorphisms on susceptibility to symptomatic malaria among Ghanaian cohort children was investigated. Blood samples from four hundred and 29 (429) healthy Ghanaian children were genotyped for FCGR2A polymorphisms by direct DNA sequencing. Attributable and relative risks to symptomatic malaria were calculated for the polymorphic variants. Two major FCGR2A polymorphisms, rs1801274A/G (FcγRIIa-H131R) and rs150311303 (FcγRIIa-ins170L), were identified in the study population, and assessment of their risks did not show significant association with susceptibility to symptomatic malaria. The functional significance of these polymorphisms was also examined by evaluating their binding abilities to IgG subclasses using flow cytometric analysis of HEK cells transfected with the FcγRIIa haplotype variants. The binding assay revealed the rs150311303, which was observed only among carriers of the FcγRIIa-131RR genotype for the rs1801274 to consistently enhance binding capacities to all IgG subclasses. Thus, of the three FcγRIIa haplotype variants observed in this study population, the FcγRIIa(RL) haplotype variant was observed to have the highest binding ability to IgG1, IgG3 and IgG4.

Plasmodium vivax lineages: geographical distribution, tandem repeat polymorphism, and phylogenetic relationship

BackgroundMulti-drug resistance and severe/complicated cases are the emerging phenotypes of vivax malaria, which may deteriorate current anti-malarial control measures. The emergence of these phenotypes could be associated with either of the two Plasmodium vivax lineages. The two lineages had been categorized as Old World and New World, based on geographical sub-division and genetic and phenotypical markers. This study revisited the lineage hypothesis of P. vivax by typing the distribution of lineages among global isolates and evaluated their genetic relatedness using a panel of new mini-satellite markers.Methods18S SSU rRNA S-type gene was amplified from 420 Plasmodium vivax field isolates collected from different geographical regions of India, Thailand and Colombia as well as four strains each of P. vivax originating from Nicaragua, Panama, Thailand (Pak Chang), and Vietnam (ONG). A mini-satellite marker panel was then developed to understand the population genetic parameters and tested on a sample subset of both lineages.Results18S SSU rRNA S-type gene typing revealed the distribution of both lineages (Old World and New World) in all geographical regions. However, distribution of Plasmodium vivax lineages was highly variable in every geographical region. The lack of geographical sub-division between lineages suggests that both lineages are globally distributed. Ten mini-satellites were scanned from the P. vivax genome sequence; these tandem repeats were located in eight of the chromosomes. Mini-satellites revealed substantial allelic diversity (7-21, AE = 14.6 ± 2.0) and heterozygosity (He = 0.697-0.924, AE = 0.857 ± 0.033) per locus. Mini-satellite comparison between the two lineages revealed high but similar pattern of genetic diversity, allele frequency, and high degree of allele sharing. A Neighbour-Joining phylogenetic tree derived from genetic distance data obtained from ten mini-satellites also placed both lineages together in every cluster.ConclusionsThe global lineage distribution, lack of genetic distance, similar pattern of genetic diversity, and allele sharing strongly suggested that both lineages are a single species and thus new emerging phenotypes associated with vivax malaria could not be clearly classified as belonging to a particular lineage on basis of their geographical origin.

Impact of Changing Drug Treatment and Malaria Endemicity on the Heritability of Malaria Phenotypes in a Longitudinal Family-Based Cohort Study

Despite considerable success of genome wide association (GWA) studies in identifying causal variants for many human diseases, their success in unraveling the genetic basis to complex diseases has been more mitigated. Pathogen population structure may impact upon the infectious phenotype, especially with the intense short-term selective pressure that drug treatment exerts on pathogens. Rigorous analysis that accounts for repeated measures and disentangles the influence of genetic and environmental factors must be performed. Attempts should be made to consider whether pathogen diversity will impact upon host genetic responses to infection.We analyzed the heritability of two Plasmodium falciparum phenotypes, the number of clinical malaria episodes (PFA) and the proportion of these episodes positive for gametocytes (Pfgam), in a family-based cohort followed for 19 years, during which time there were four successive drug treatment regimes, with documented appearance of drug resistance. Repeated measures and variance components analyses were performed with fixed environmental, additive genetic, intra-individual and maternal effects for each drug period. Whilst there was a significant additive genetic effect underlying PFA during the first drug period of study, this was lost in subsequent periods. There was no additive genetic effect for Pfgam. The intra-individual effect increased significantly in the chloroquine period.The loss of an additive genetic effect following novel drug treatment may result in significant loss of power to detect genes in a GWA study. Prior genetic analysis must be a pre-requisite for more detailed GWA studies. The temporal changes in the individual genetic and the intra-individual estimates are consistent with those expected if there were specific host-parasite interactions. The complex basis to the human response to malaria parasite infection likely includes dominance/epistatic genetic effects encompassed within the intra-individual variance component. Evaluating their role in influencing the outcome of infection through host genotype by parasite genotype interactions warrants research effort.

Genetic polymorphisms linked to susceptibility to malaria

The influence of host genetics on susceptibility to Plasmodium falciparum malaria has been extensively studied over the past twenty years. It is now clear that malaria parasites have imposed strong selective forces on the human genome in endemic regions. Different genes have been identified that are associated with different malaria related phenotypes. Factors that promote severity of malaria include parasitaemia, parasite induced inflammation, anaemia and sequestration of parasitized erythrocytes in brain microvasculature.Recent advances in human genome research technologies such as genome-wide association studies (GWAS) and fine genotyping tools have enabled the discovery of several genetic polymorphisms and biomarkers that warrant further study in host-parasite interactions. This review describes and discusses human gene polymorphisms identified thus far that have been shown to be associated with susceptibility or resistance to P. falciparum malaria. Although some polymorphisms play significant roles in susceptibility to malaria, several findings are inconclusive and contradictory and must be considered with caution. The discovery of genetic markers associated with different malaria phenotypes will help elucidate the pathophysiology of malaria and enable development of interventions or cures. Diversity in human populations as well as environmental effects can influence the clinical heterogeneity of malaria, thus warranting further investigations with a goal of developing new interventions, therapies and better management against malaria.

Genome Wide Linkage Study, Using a 250K SNP Map, of Plasmodium falciparum Infection and Mild Malaria Attack in a Senegalese Population

Multiple factors are involved in the variability of host's response to P. falciparum infection, like the intensity and seasonality of malaria transmission, the virulence of parasite and host characteristics like age or genetic make-up. Although admitted nowadays, the involvement of host genetic factors remains unclear. Discordant results exist, even concerning the best-known malaria resistance genes that determine the structure or function of red blood cells. Here we report on a genome-wide linkage and association study for P. falciparum infection intensity and mild malaria attack among a Senegalese population of children and young adults from 2 to 18 years old. A high density single nucleotide polymorphisms (SNP) genome scan (Affimetrix GeneChip Human Mapping 250K-nsp) was performed for 626 individuals: i.e. 249 parents and 377 children out of the 504 ones included in the follow-up. The population belongs to a unique ethnic group and was closely followed-up during 3 years. Genome-wide linkage analyses were performed on four clinical and parasitological phenotypes and association analyses using the family based association tests (FBAT) method were carried out in regions previously linked to malaria phenotypes in literature and in the regions for which we identified a linkage peak. Analyses revealed three strongly suggestive evidences for linkage: between mild malaria attack and both the 6p25.1 and the 12q22 regions (empirical p-value = 5×10−5 and 9×10−5 respectively), and between the 20p11q11 region and the prevalence of parasite density in asymptomatic children (empirical p-value = 1.5×10−4). Family based association analysis pointed out one significant association between the intensity of plasmodial infection and a polymorphism located in ARHGAP26 gene in the 5q31–q33 region (p-value = 3.7×10−5). This study identified three candidate regions, two of them containing genes that could point out new pathways implicated in the response to malaria infection. Furthermore, we detected one gene associated with malaria infection in the 5q31–q33 region.

Interaction of malaria with a common form of severe thalassemia in an Asian population

In many Asian populations, the commonest form of severe thalassemia results from the coinheritance of HbE and beta thalassemia. The management of this disease is particularly difficult because of its extreme clinical diversity; although some genetic and adaptive factors have been identified as phenotypic modifiers, the reasons remain unclear. Because the role of the environment in the course of severe thalassemia has been neglected completely and because malaria due to both Plasmodium falciparum and Plasmodium vivax has been prevalent in Sri Lanka, we carried out a pilot study of patients with HbE beta thalassemia that showed high frequencies of antibodies to both parasite species and that 28.6% of the children had DNA-based evidence of current infection with P. vivax. Malarial antibodies then were assessed in patients with HbE beta thalassemia compared with those in age-matched controls. There was a significant increase in the frequency of antibodies in the thalassemic patients, particularly against P. vivax and in young children. There was also a higher frequency in those who had been splenectomized compared with those with intact spleens, although in the latter it was still higher than that in the controls. The thalassemic patients showed significant correlations between malaria antibody status and phenotype. Patients with HbE beta thalassemia may be more prone to malaria, particularly P. vivax, which is reflected in their clinical severity. Because P. vivax malaria is widespread in Asia, further studies of its interaction with HbE beta thalassemia and related diseases are required urgently as a part of ongoing thalassemia control programs.

Positive selection of a CD36 nonsense variant in sub-Saharan Africa, but no association with severe malaria phenotypes

The prevalence of CD36 deficiency in East Asian and African populations suggests that the causal variants are under selection by severe malaria. Previous analysis of data from the International HapMap Project indicated that a CD36 haplotype bearing a nonsense mutation (T1264G; rs3211938) had undergone recent positive selection in the Yoruba of Nigeria. To investigate the global distribution of this putative selection event, we genotyped T1264G in 3420 individuals from 66 populations. We confirmed the high frequency of 1264G in the Yoruba (26%). However, the 1264G allele is less common in other African populations and absent from all non-African populations without recent African admixture. Using long-range linkage disequilibrium, we studied two West African groups in depth. Evidence for recent positive selection at the locus was demonstrable in the Yoruba, although not in Gambians. We screened 70 variants from across CD36 for an association with severe malaria phenotypes, employing a case–control study of 1350 subjects and a family study of 1288 parent–offspring trios. No marker was significantly associated with severe malaria. We focused on T1264G, genotyping 10 922 samples from four African populations. The nonsense allele was not associated with severe malaria (pooled allelic odds ratio 1.0; 95% confidence interval 0.89–1.12; P = 0.98). These results suggest a range of possible explanations including the existence of alternative selection pressures on CD36, co-evolution between host and parasite or confounding caused by allelic heterogeneity of CD36 deficiency.

Tumor necrosis factor and lymphotoxin-alpha polymorphisms and severe malaria in African populations.

The tumor necrosis factor gene (TNF) and lymphotoxin-alpha gene (LTA) have long attracted attention as candidate genes for susceptibility traits for malaria, and several of their polymorphisms have been found to be associated with severe malaria (SM) phenotypes. In a large study involving >10,000 individuals and encompassing 3 African populations, we found evidence to support the reported associations between the TNF -238 polymorphism and SM in The Gambia. However, no TNF/LTA polymorphisms were found to be associated with SM in cohorts in Kenya and Malawi. It has been suggested that the causal polymorphisms regulating the TNF and LTA responses may be located some distance from the genes. Therefore, more-detailed mapping of variants across TNF/LTA genes and their flanking regions in the Gambian and allied populations may need to be undertaken to find any causal polymorphisms.

Variation in the ICAM1 gene is not associated with severe malaria phenotypes.

Evidence from autopsy and in vitro binding studies suggests that adhesion of erythrocytes infected with Plasmodium falciparum to the human host intercellular adhesion molecule (ICAM)-1 receptor is important in the pathogenesis of severe malaria. Previous association studies between polymorphisms in the ICAM1 gene and susceptibility to severe malarial phenotypes have been inconclusive and often contradictory. We performed genetic association studies with 15 single nucleotide polymorphisms (SNPs) around the ICAM1 locus. All SNPs were screened in a family study of 1071 trios from The Gambia, Malawi and Kenya. Two key non-synonymous SNPs with previously reported associations, rs5491 (K56M or 'ICAM-1(Kilifi)') and rs5498 (K469E), were tested in an additional 708 Gambian trios and a case-control study of 4058 individuals. None of the polymorphisms were associated with severe malaria phenotypes. Pooled results across our studies for ICAM-1(Kilifi) were, in severe malaria, odds ratio (OR) 1.02, 95% confidence interval (CI) 0.96-1.09, P=0.54, and cerebral malaria OR 1.07, CI 0.97-1.17, P=0.17. We assess the available epidemiological, population genetic and functional evidence that links ICAM-1(Kilifi) to severe malaria susceptibility.

Common variation in the ABO glycosyltransferase is associated with susceptibility to severe Plasmodium falciparum malaria.

There is growing epidemiological and molecular evidence that ABO blood group affects host susceptibility to severe Plasmodium falciparum infection. The high frequency of common ABO alleles means that even modest differences in susceptibility could have a significant impact on the health of people living in malaria endemic regions. We performed an association study, the first to utilize key molecular genetic variation underlying the ABO system, genotyping >9000 individuals across three African populations. Using population- and family-based tests, we demonstrated that alleles producing functional ABO enzymes are associated with greater risk of severe malaria phenotypes (particularly malarial anemia) in comparison with the frameshift deletion underlying blood group O: case-control allelic odds ratio (OR), 1.2; 95% confidence interval (CI), 1.09-1.32; P = 0.0003; family-studies allelic OR, 1.19; 95% CI, 1.08-1.32; P = 0.001; pooled across all studies allelic OR, 1.18; 95% CI, 1.11-1.26; P = 2 x 10(-7). We found suggestive evidence of a parent-of-origin effect at the ABO locus by analyzing the family trios. Non-O haplotypes inherited from mothers, but not fathers, are significantly associated with severe malaria (likelihood ratio test of Weinberg, P = 0.046). Finally, we used HapMap data to demonstrate a region of low F(ST) (-0.001) between the three main HapMap population groups across the ABO locus, an outlier in the empirical distribution of F(ST) across chromosome 9 (approximately 99.5-99.9th centile). This low F(ST) region may be a signal of long-standing balancing selection at the ABO locus, caused by multiple infectious pathogens including P. falciparum.