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.

Novel genotyping approaches to easily detect genomic admixture between the major Afrotropical malaria vector species, Anopheles coluzzii and An. gambiae.

The two most efficient and most recently radiated Afrotropical vectors of human malaria - Anopheles coluzzii and An. gambiae - are identified by single-locus diagnostic PCR assays based on species-specific markers in a 4 Mb region on chromosome-X centromere. Inherently, these diagnostic assays cannot detect interspecific autosomal admixture shown to be extensive at the westernmost and easternmost extremes of the species range. The main aim of this study was to develop novel, easy-to-implement tools for genotyping An. coluzzii and An. gambiae-specific ancestral informative markers (AIMs) identified from the Anopheles gambiae 1000 genomes (Ag1000G) project. First, we took advantage of this large set of data in order to develop a multilocus approach to genotype 26 AIMs on all chromosome arms valid across the species range. Second, we tested the multilocus assay on samples from Guinea Bissau, The Gambia and Senegal, three countries spanning the westernmost hybridization zone, where conventional species diagnostic is problematic due to the putative presence of a novel "hybrid form". The multilocus assay was able to capture patterns of admixture reflecting those revealed by the whole set of AIMs and provided new original data on interspecific admixture in the region. Third, we developed an easy-to-use, cost-effective PCR approach for genotyping two AIMs on chromosome-3 among those included in the multilocus approach, opening the possibility for advanced identification of species and of admixed specimens during routine large scale entomological surveys, particularly, but not exclusively, at the extremes of the range, where WGS data highlighted unexpected autosomal admixture.

Evaluating the Performance of Malaria Genetics for Inferring Changes in Transmission Intensity Using Transmission Modeling.

Substantial progress has been made globally to control malaria, however there is a growing need for innovative new tools to ensure continued progress. One approach is to harness genetic sequencing and accompanying methodological approaches as have been used in the control of other infectious diseases. However, to utilize these methodologies for malaria, we first need to extend the methods to capture the complex interactions between parasites, human and vector hosts, and environment, which all impact the level of genetic diversity and relatedness of malaria parasites. We develop an individual-based transmission model to simulate malaria parasite genetics parameterized using estimated relationships between complexity of infection and age from five regions in Uganda and Kenya. We predict that cotransmission and superinfection contribute equally to within-host parasite genetic diversity at 11.5% PCR prevalence, above which superinfections dominate. Finally, we characterize the predictive power of six metrics of parasite genetics for detecting changes in transmission intensity, before grouping them in an ensemble statistical model. The model predicted malaria prevalence with a mean absolute error of 0.055. Different assumptions about the availability of sample metadata were considered, with the most accurate predictions of malaria prevalence made when the clinical status and age of sampled individuals is known. Parasite genetics may provide a novel surveillance tool for estimating the prevalence of malaria in areas in which prevalence surveys are not feasible. However, the findings presented here reinforce the need for patient metadata to be recorded and made available within all future attempts to use parasite genetics for surveillance.

High-throughput genotyping assays for identification of glycophorin B deletion variants in population studies.

Glycophorins are the most abundant sialoglycoproteins on the surface of human erythrocyte membranes. Genetic variation in glycophorin region of human chromosome 4 (containing GYPA, GYPB, and GYPE genes) is of interest because the gene products serve as receptors for pathogens of major public health interest, including Plasmodiumsp., Babesiasp., Influenza virus, Vibrio cholerae El Tor Hemolysin, and Escherichia coli. A large structural rearrangement and hybrid glycophorin variant, known as Dantu, which was identified in East African populations, has been linked with a 40% reduction in risk for severe malaria. Apart from Dantu, other large structural variants exist, with the most common being deletion of the whole GYPB gene and its surrounding region, resulting in multiple different deletion forms. In West Africa particularly, these deletions are estimated to account for between 5 and 15% of the variation in different populations, mostly attributed to the forms known as DEL1 and DEL2. Due to the lack of specific variant assays, little is known of the distribution of these variants. Here, we report a modification of a previous GYPB DEL1 assay and the development of a novel GYPB DEL2 assay as high-throughput PCR-RFLP assays, as well as the identification of the crossover/breakpoint for GYPB DEL2. Using 393 samples from three study sites in Ghana as well as samples from HapMap and 1000 G projects for validation, we show that our assays are sensitive and reliable for genotyping GYPB DEL1 and DEL2. To the best of our knowledge, this is the first report of such high-throughput genotyping assays by PCR-RFLP for identifying specific GYPB deletion types in populations. These assays will enable better identification of GYPB deletions for large genetic association studies and functional experiments to understand the role of this gene cluster region in susceptibility to malaria and other diseases.

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.

Investigating the drivers of the spatio-temporal patterns of genetic differences between Plasmodium falciparum malaria infections in Kilifi County, Kenya.

Knowledge of how malaria infections spread locally is important both for the design of targeted interventions aiming to interrupt malaria transmission and the design of trials to assess the interventions. A previous analysis of 1602 genotyped Plasmodium falciparum parasites in Kilifi, Kenya collected over 12 years found an interaction between time and geographic distance: the mean number of single nucleotide polymorphism (SNP) differences was lower for pairs of infections which were both a shorter time interval and shorter geographic distance apart. We determine whether the empiric pattern could be reproduced by a simple model, and what mean geographic distances between parent and offspring infections and hypotheses about genotype-specific immunity or a limit on the number of infections would be consistent with the data. We developed an individual-based stochastic simulation model of households, people and infections. We parameterized the model for the total number of infections, and population and household density observed in Kilifi. The acquisition of new infections, mutation, recombination, geographic location and clearance were included. We fit the model to the observed numbers of SNP differences between pairs of parasite genotypes. The patterns observed in the empiric data could be reproduced. Although we cannot rule out genotype-specific immunity or a limit on the number of infections per individual, they are not necessary to account for the observed patterns. The mean geographic distance between parent and offspring malaria infections for the base model was 0.5 km (95% CI 0.3-1.5), for a distribution with 68% of distances shorter than the mean. Very short mean distances did not fit well, but mixtures of distributions were also consistent with the data. For a pathogen which undergoes meiosis in a setting with moderate transmission and a low coverage of infections, analytic methods are limited but an individual-based model can be used with genotyping data to estimate parameter values and investigate hypotheses about underlying processes.

A high throughput multi-locus insecticide resistance marker panel for tracking resistance emergence and spread in Anopheles gambiae.

The spread of resistance to insecticides in disease-carrying mosquitoes poses a threat to the effectiveness of control programmes, which rely largely on insecticide-based interventions. Monitoring mosquito populations is essential, but obtaining phenotypic measurements of resistance is laborious and error-prone. High-throughput genotyping offers the prospect of quick and repeatable estimates of resistance, while also allowing resistance markers to be tracked and studied. To demonstrate the potential of highly-mulitplexed genotypic screening for measuring resistance-association of mutations and tracking their spread, we developed a panel of 28 known or putative resistance markers in the major malaria vector Anopheles gambiae, which we used to screen mosquitoes from a wide swathe of Sub-Saharan Africa (Burkina Faso, Ghana, Democratic Republic of Congo (DRC) and Kenya). We found resistance association in four markers, including a novel mutation in the detoxification gene Gste2 (Gste2-119V). We also identified a duplication in Gste2 combining a resistance-associated mutation with its wild-type counterpart, potentially alleviating the costs of resistance. Finally, we describe the distribution of the multiple origins of kdr resistance, finding unprecedented diversity in the DRC. This panel represents the first step towards a quantitative genotypic model of insecticide resistance that can be used to predict resistance status in An. gambiae.

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.

Geographic-genetic analysis of Plasmodium falciparum parasite populations from surveys of primary school children in Western Kenya.

Background. Malaria control, and finally malaria elimination, requires the identification and targeting of residual foci or hotspots of transmission. However, the level of parasite mixing within and between geographical locations is likely to impact the effectiveness and durability of control interventions and thus should be taken into consideration when developing control programs. Methods. In order to determine the geographic-genetic patterns of Plasmodium falciparum parasite populations at a sub-national level in Kenya, we used the Sequenom platform to genotype 111 genome-wide distributed single nucleotide polymorphic (SNP) positions in 2486 isolates collected from children in 95 primary schools in western Kenya. We analysed these parasite genotypes for genetic structure using principal component analysis and assessed local and global clustering using statistical measures of spatial autocorrelation. We further examined the region for spatial barriers to parasite movement as well as directionality in the patterns of parasite movement. Results. We found no evidence of population structure and little evidence of spatial autocorrelation of parasite genotypes (correlation coefficients <0.03 among parasite pairs in distance classes of 1km, 2km and 5km; p value<0.01). An analysis of the geographical distribution of allele frequencies showed weak evidence of variation in distribution of alleles, with clusters representing a higher than expected number of samples with the major allele being identified for 5 SNPs. Furthermore, we found no evidence of the existence of spatial barriers to parasite movement within the region, but observed directional movement of parasites among schools in two separate sections of the region studied. Conclusions. Our findings illustrate a pattern of high parasite mixing within the study region. If this mixing is due to rapid gene flow, then "one-off" targeted interventions may not be currently effective at the sub-national scale in Western Kenya, due to the high parasite movement that is likely to lead to re-introduction of infection from surrounding regions. However repeated targeted interventions may reduce transmission in the surrounding regions.

Micro-epidemiological structuring of Plasmodium falciparum parasite populations in regions with varying transmission intensities in Africa.

Background: The first models of malaria transmission assumed a completely mixed and homogeneous population of parasites. Recent models include spatial heterogeneity and variably mixed populations. However, there are few empiric estimates of parasite mixing with which to parametize such models. Methods: Here we genotype 276 single nucleotide polymorphisms (SNPs) in 5199 P. falciparum isolates from two Kenyan sites (Kilifi county and Rachuonyo South district) and one Gambian site (Kombo coastal districts) to determine the spatio-temporal extent of parasite mixing, and use Principal Component Analysis (PCA) and linear regression to examine the relationship between genetic relatedness and distance in space and time for parasite pairs. Results: Using 107, 177 and 82 SNPs that were successfully genotyped in 133, 1602, and 1034 parasite isolates from The Gambia, Kilifi and Rachuonyo South district, respectively, we show that there are no discrete geographically restricted parasite sub-populations, but instead we see a diffuse spatio-temporal structure to parasite genotypes. Genetic relatedness of sample pairs is predicted by relatedness in space and time. Conclusions: Our findings suggest that targeted malaria control will benefit the surrounding community, but unfortunately also that emerging drug resistance will spread rapidly through the population.

Malaria Host Candidate Genes Validated by Association With Current, Recent, and Historical Measures of Transmission Intensity.

Background: Human malaria susceptibility is determined by multiple genetic factors. It is unclear, however, which genetic variants remain important over time. Methods: Genetic associations of 175 high-quality polymorphisms within several malaria candidate genes were examined in a sample of 8096 individuals from northeast Tanzania using altitude, seroconversion rates, and parasite rates as proxies of historical, recent, and current malaria transmission intensity. A principal component analysis was used to derive 2 alternative measures of overall malaria propensity of a location across different time scales. Results: Common red blood cell polymorphisms (ie, hemoglobin S, glucose-6-phosphate dehydrogenase, and α-thalassemia) were the only ones to be associated with all 3 measures of transmission intensity and the first principal component. Moderate associations were found between some immune response genes (ie, IL3 and IL13) and parasite rates, but these could not be reproduced using the alternative measures of malaria propensity. Conclusions: We have demonstrated the potential of using altitude and seroconversion rate as measures of malaria transmission capturing medium- to long-term time scales to detect genetic associations that are likely to persist over time. These measures also have the advantage of minimizing the deleterious effects of random factors affecting parasite rates on the respective association signals.

Resistance to malaria through structural variation of red blood cell invasion receptors.

The malaria parasite Plasmodium falciparum invades human red blood cells by a series of interactions between host and parasite surface proteins. By analyzing genome sequence data from human populations, including 1269 individuals from sub-Saharan Africa, we identify a diverse array of large copy-number variants affecting the host invasion receptor genes GYPA and GYPB We find that a nearby association with severe malaria is explained by a complex structural rearrangement involving the loss of GYPB and gain of two GYPB-A hybrid genes, which encode a serologically distinct blood group antigen known as Dantu. This variant reduces the risk of severe malaria by 40% and has recently increased in frequency in parts of Kenya, yet it appears to be absent from west Africa. These findings link structural variation of red blood cell invasion receptors with natural resistance to severe malaria.

THE REAL McCOIL: A method for the concurrent estimation of the complexity of infection and SNP allele frequency for malaria parasites.

As many malaria-endemic countries move towards elimination of Plasmodium falciparum, the most virulent human malaria parasite, effective tools for monitoring malaria epidemiology are urgent priorities. P. falciparum population genetic approaches offer promising tools for understanding transmission and spread of the disease, but a high prevalence of multi-clone or polygenomic infections can render estimation of even the most basic parameters, such as allele frequencies, challenging. A previous method, COIL, was developed to estimate complexity of infection (COI) from single nucleotide polymorphism (SNP) data, but relies on monogenomic infections to estimate allele frequencies or requires external allele frequency data which may not available. Estimates limited to monogenomic infections may not be representative, however, and when the average COI is high, they can be difficult or impossible to obtain. Therefore, we developed THE REAL McCOIL, Turning HEterozygous SNP data into Robust Estimates of ALelle frequency, via Markov chain Monte Carlo, and Complexity Of Infection using Likelihood, to incorporate polygenomic samples and simultaneously estimate allele frequency and COI. This approach was tested via simulations then applied to SNP data from cross-sectional surveys performed in three Ugandan sites with varying malaria transmission. We show that THE REAL McCOIL consistently outperforms COIL on simulated data, particularly when most infections are polygenomic. Using field data we show that, unlike with COIL, we can distinguish epidemiologically relevant differences in COI between and within these sites. Surprisingly, for example, we estimated high average COI in a peri-urban subregion with lower transmission intensity, suggesting that many of these cases were imported from surrounding regions with higher transmission intensity. THE REAL McCOIL therefore provides a robust tool for understanding the molecular epidemiology of malaria across transmission settings.

Characterisation of the opposing effects of G6PD deficiency on cerebral malaria and severe malarial anaemia.

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is believed to confer protection against Plasmodium falciparum malaria, but the precise nature of the protective effecthas proved difficult to define as G6PD deficiency has multiple allelic variants with different effects in males and females, and it has heterogeneous effects on the clinical outcome of P. falciparum infection. Here we report an analysis of multiple allelic forms of G6PD deficiency in a large multi-centre case-control study of severe malaria, using the WHO classification of G6PD mutations to estimate each individual's level of enzyme activity from their genotype. Aggregated across all genotypes, we find that increasing levels of G6PD deficiency are associated with decreasing risk of cerebral malaria, but with increased risk of severe malarial anaemia. Models of balancing selection based on these findings indicate that an evolutionary trade-off between different clinical outcomes of P. falciparum infection could have been a major cause of the high levels of G6PD polymorphism seen in human populations.

Genomic analysis of local variation and recent evolution in Plasmodium vivax.

The widespread distribution and relapsing nature of Plasmodium vivax infection present major challenges for the elimination of malaria. To characterize the genetic diversity of this parasite in individual infections and across the population, we performed deep genome sequencing of >200 clinical samples collected across the Asia-Pacific region and analyzed data on >300,000 SNPs and nine regions of the genome with large copy number variations. Individual infections showed complex patterns of genetic structure, with variation not only in the number of dominant clones but also in their level of relatedness and inbreeding. At the population level, we observed strong signals of recent evolutionary selection both in known drug resistance genes and at new loci, and these varied markedly between geographical locations. These findings demonstrate a dynamic landscape of local evolutionary adaptation in the parasite population and provide a foundation for genomic surveillance to guide effective strategies for control and elimination of P. vivax.

Heterogeneous alleles comprising G6PD deficiency trait in West Africa exert contrasting effects on two major clinical presentations of severe malaria.

BACKGROUND: Glucose-6-phosphate dehydrogenase (G6PD) deficiency exhibits considerable allelic heterogeneity which manifests with variable biochemical and clinical penetrance. It has long been thought that G6PD deficiency confers partial protection against severe malaria, however prior genetic association studies have disagreed with regard to the strength and specificity of a protective effect, which might reflect differences in the host genetic background, environmental influences, or in the specific clinical phenotypes considered. METHODS: A case-control association study of severe malaria was conducted in The Gambia, a region in West Africa where there is considerable allelic heterogeneity underlying expression of G6PD deficiency trait, evaluating the three major nonsynonymous polymorphisms known to be associated with enzyme deficiency (A968G, T542A, and C202T) in a cohort of 3836 controls and 2379 severe malaria cases. RESULTS: Each deficiency allele exhibited a similar trend toward protection against severe malaria overall (15-26% reduced risk); however, in stratifying severe malaria to two of its constituent clinical subphenotypes, severe malarial anaemia (SMA) and cerebral malaria (CM), the three deficiency alleles exhibited trends of opposing effect, with risk conferred to SMA and protection with respect to CM. To assess the overall effect of G6PD deficiency trait, deficiency alleles found across all three loci were pooled. G6PD deficiency trait was found to be significantly associated with protection from severe malaria overall (OR 0.83 [0.75-0.92], P = 0.0006), but this was limited to CM (OR 0.73 [0.61-0.87], P = 0.0005), with a trend toward increased risk for SMA, especially in fully-deficient individuals (OR 1.43 [0.99-2.08], P = 0.056). Sex-stratified testing largely comported with these results, with evidence suggesting that protection by G6PD deficiency trait is conferred to both males and females, though susceptibility to SMA may be restricted to fully-deficient male hemizygotes. CONCLUSIONS: In a part of Africa where multiple alleles contribute to expression of G6PD deficiency trait, these findings clarify and extend previous work done in populations where a single variant predominates, and taken together suggest a causal role for G6PD deficiency trait itself with respect to severe malaria, with opposing effects seen on two major clinical subphenotypes.

High-throughput genotyping of Anopheles mosquitoes using intact legs by Agena Biosciences iPLEX.

Recent developments in genotyping technologies coupled with the growing desire to characterize genome variation in Anopheles populations open the opportunity to develop more effective genotyping strategies for high-throughput screening. A major bottleneck of this goal is nucleic acid extraction. Here, we examined the feasibility of using intact portions of a mosquito's leg as sources of template DNA for whole-genome amplification (WGA) by primer-extension preamplification. We used the Agena Biosciences MassARRAY(®) platform (formerly Sequenom) to genotype 78 SNPs for 265 WGA leg samples. We performed nucleic acid extraction on 36 mosquito carcasses and compared the genotype call concordance with their corresponding legs and observed full concordance. Using three legs instead of one improved genotyping success rates (96% vs. 89%, respectively), although this difference was not significant. We provide a proof of concept that WGA reactions can be performed directly on mosquito legs, thereby eliminating the need to extract nucleic acid. This approach is straightforward and sensitive and allows both species determination and genotyping of Anopheles mosquitoes to be performed in a high-throughput manner. Our protocol also leaves the mosquito body intact facilitating other experimental analysis to be undertaken on the same sample. Based on our findings, this method would also be suitable for use with other insect species.

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.

Genetic determinants of glucose-6-phosphate dehydrogenase activity in Kenya.

BACKGROUND: The relationship between glucose-6-phosphate dehydrogenase (G6PD) deficiency and clinical phenomena such as primaquine-sensitivity and protection from severe malaria remains poorly defined, with past association studies yielding inconsistent and conflicting results. One possibility is that examination of a single genetic variant might underestimate the presence of true effects in the presence of unrecognized functional allelic diversity. METHODS: We systematically examined this possibility in Kenya, conducting a fine-mapping association study of erythrocyte G6PD activity in 1828 Kenyan children across 30 polymorphisms at or around the G6PD locus. RESULTS: We demonstrate a strong functional role for c.202G>A (rs1050828), which accounts for the majority of variance in enzyme activity observed (P=1.5×10⁻²°°, additive model). Additionally, we identify other common variants that exert smaller, intercorrelated effects independent of c.202G>A, and haplotype analyses suggest that each variant tags one of two haplotype motifs that are opposite in sequence identity and effect direction. We posit that these effects are of biological and possible clinical significance, specifically noting that c.376A>G (rs1050829) augments 202AG heterozygote risk for deficiency trait by two-fold (OR = 2.11 [1.12 - 3.84], P=0.014). CONCLUSIONS: Our results suggest that c.202G>A is responsible for the majority of the observed prevalence of G6PD deficiency trait in Kenya, but also identify a novel role for c.376A>G as a genetic modifier which marks a common haplotype that augments the risk conferred to 202AG heterozygotes, suggesting that variation at both loci merits consideration in genetic association studies probing G6PD deficiency-associated clinical phenotypes.