Flexible and cost-effective genomic surveillance of P. falciparum malaria with targeted nanopore sequencing.

Genomic surveillance of Plasmodium falciparum malaria can provide policy-relevant information about antimalarial drug resistance, diagnostic test failure, and the evolution of vaccine targets. Yet the large and low complexity genome of P. falciparum complicates the development of genomic methods, while resource constraints in malaria endemic regions can limit their deployment. Here, we demonstrate an approach for targeted nanopore sequencing of P. falciparum from dried blood spots (DBS) that enables cost-effective genomic surveillance of malaria in low-resource settings. We release software that facilitates flexible design of amplicon sequencing panels and use this software to design two target panels for P. falciparum. The panels generate 3-4 kbp reads for eight and sixteen targets respectively, covering key drug-resistance associated genes, diagnostic test antigens, polymorphic markers and the vaccine target csp. We validate our approach on mock and field samples, demonstrating robust sequencing coverage, accurate variant calls within coding sequences, the ability to explore P. falciparum within-sample diversity and to detect deletions underlying rapid diagnostic test failure.

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.

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.

Host genetic polymorphisms and serological response against malaria in a selected population in Sri Lanka.

BACKGROUND: Antibodies against the merozoite surface protein 1-19 (MSP1-19) and the apical membrane antigen 1 (AMA1) of the malaria parasite (Plasmodium vivax) are proven to be important in protection against clinical disease. Differences in the production/maintenance of antibodies may be due to many factors including host genetics. This paper discusses the association of 4 anti-malarial antibodies with selected host genetic markers. METHODS: Blood was collected from individuals (n = 242) with a history of malaria within past 15 years for DNA and serum. ELISA was carried out for serum to determine the concentration of anti-malarial antibodies MSP1-19 and AMA1 for both vivax and falciparum malaria. 170 SNPs related to malaria were genotyped. Associations between seropositivity, antibody levels and genetic, non-genetic factors were determined. RESULTS: Age ranged 13-74 years (mean age = 40.21 years). Majority were females. Over 90% individuals possessed either one or more type(s) of anti-malarial antibodies. Five SNPs were significantly associated with seropositivity. One SNP was associated with MSP1-19_Pv(rs739718); 4 SNPs with MSP1-19_Pf (rs6874639, rs2706379, rs2706381 and rs2075820) and1 with AMA1_Pv (rs2075820). Eleven and 7 genotypes (out of 15) were significantly associated with either presence or absence of antibodies. Three SNPs were found to be significantly associated with the antibody levels viz. rs17411697 with MSP1-19_Pv, rs2227491 with AMA1_Pv and rs229587 with AMA1_Pf. Linkage of the markers in the two groups was similar, but lower LOD scores were observed in seropositives compared to seronegatives. DISCUSSION AND CONCLUSIONS: The study suggests that several SNPs in the human genome that exist in Sri Lankan populations are significantly associated with anti-malarial antibodies, either with generation and/or maintenance of antibodies for longer periods, which can be due to either individual polymorphisms or most probably a combined effect of the markers.

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.

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.

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.

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.

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.

Conjunctival fibrosis and the innate barriers to Chlamydia trachomatis intracellular infection: a genome wide association study.

Chlamydia trachomatis causes both trachoma and sexually transmitted infections. These diseases have similar pathology and potentially similar genetic predisposing factors. We aimed to identify polymorphisms and pathways associated with pathological sequelae of ocular Chlamydia trachomatis infections in The Gambia. We report a discovery phase genome-wide association study (GWAS) of scarring trachoma (1090 cases, 1531 controls) that identified 27 SNPs with strong, but not genome-wide significant, association with disease (5 × 10(-6) > P > 5 × 10(-8)). The most strongly associated SNP (rs111513399, P = 5.38 × 10(-7)) fell within a gene (PREX2) with homology to factors known to facilitate chlamydial entry to the host cell. Pathway analysis of GWAS data was significantly enriched for mitotic cell cycle processes (P = 0.001), the immune response (P = 0.00001) and for multiple cell surface receptor signalling pathways. New analyses of published transcriptome data sets from Gambia, Tanzania and Ethiopia also revealed that the same cell cycle and immune response pathways were enriched at the transcriptional level in various disease states. Although unconfirmed, the data suggest that genetic associations with chlamydial scarring disease may be focussed on processes relating to the immune response, the host cell cycle and cell surface receptor signalling.

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.

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.

Candidate human genetic polymorphisms and severe malaria in a Tanzanian population.

Human genetic background strongly influences susceptibility to malaria infection and progression to severe disease and death. Classical genetic studies identified haemoglobinopathies and erythrocyte-associated polymorphisms, as protective against severe disease. High throughput genotyping by mass spectrometry allows multiple single nucleotide polymorphisms (SNPs) to be examined simultaneously. We compared the prevalence of 65 human SNP's, previously associated with altered risk of malaria, between Tanzanian children with and without severe malaria. Five hundred children, aged 1-10 years, with severe malaria were recruited from those admitted to hospital in Muheza, Tanzania and compared with matched controls. Genotyping was performed by Sequenom MassArray, and conventional PCR was used to detect deletions in the alpha-thalassaemia gene. SNPs in two X-linked genes were associated with altered risk of severe malaria in females but not in males: heterozygosity for one or other of two SNPs in the G6PD gene was associated with protection from all forms of severe disease whilst two SNPs in the gene encoding CD40L were associated with respiratory distress. A SNP in the adenyl cyclase 9 (ADCY9) gene was associated with protection from acidosis whilst a polymorphism in the IL-1α gene (IL1A) was associated with an increased risk of acidosis. SNPs in the genes encoding IL-13 and reticulon-3 (RTN3) were associated with increased risk of cerebral malaria. This study confirms previously known genetic associations with protection from severe malaria (HbS, G6PD). It identifies two X-linked genes associated with altered risk of severe malaria in females, identifies mutations in ADCY9, IL1A and CD40L as being associated with altered risk of severe respiratory distress and acidosis, both of which are characterised by high serum lactate levels, and also identifies novel genetic associations with severe malaria (TRIM5) and cerebral malaria(IL-13 and RTN3). Further studies are required to test the generality of these associations and to understand their functional consequences.

Analysis of Plasmodium falciparum diversity in natural infections by deep sequencing.

Malaria elimination strategies require surveillance of the parasite population for genetic changes that demand a public health response, such as new forms of drug resistance. Here we describe methods for the large-scale analysis of genetic variation in Plasmodium falciparum by deep sequencing of parasite DNA obtained from the blood of patients with malaria, either directly or after short-term culture. Analysis of 86,158 exonic single nucleotide polymorphisms that passed genotyping quality control in 227 samples from Africa, Asia and Oceania provides genome-wide estimates of allele frequency distribution, population structure and linkage disequilibrium. By comparing the genetic diversity of individual infections with that of the local parasite population, we derive a metric of within-host diversity that is related to the level of inbreeding in the population. An open-access web application has been established for the exploration of regional differences in allele frequency and of highly differentiated loci in the P. falciparum genome.

Host candidate gene polymorphisms and clearance of drug-resistant Plasmodium falciparum parasites.

BACKGROUND: Resistance to anti-malarial drugs is a widespread problem for control programmes for this devastating disease. Molecular tests are available for many anti-malarial drugs and are useful tools for the surveillance of drug resistance. However, the correlation of treatment outcome and molecular tests with particular parasite markers is not perfect, due in part to individuals who are able to clear genotypically drug-resistant parasites. This study aimed to identify molecular markers in the human genome that correlate with the clearance of malaria parasites after drug treatment, despite the drug resistance profile of the protozoan as predicted by molecular approaches. METHODS: 3721 samples from five African countries, which were known to contain genotypically drug resistant parasites, were analysed. These parasites were collected from patients who subsequently failed to clear their infection following drug treatment, as expected, but also from patients who successfully cleared their infections with drug-resistant parasites. 67 human polymorphisms (SNPs) on 17 chromosomes were analysed using Sequenom's mass spectrometry iPLEX gold platform, to identify regions of the human genome, which contribute to enhanced clearance of drug resistant parasites. RESULTS: An analysis of all data from the five countries revealed significant associations between the phenotype of ability to clear drug-resistant Plasmodium falciparum infection and human immune response loci common to all populations. Overall, three SNPs showed a significant association with clearance of drug-resistant parasites with odds ratios of 0.76 for SNP rs2706384 (95% CI 0.71-0.92, P = 0.005), 0.66 for SNP rs1805015 (95% CI 0.45-0.97, P = 0.03), and 0.67 for SNP rs1128127 (95% CI 0.45-0.99, P = 0.05), after adjustment for possible confounding factors. The first two SNPs (rs2706384 and rs1805015) are within loci involved in pro-inflammatory (interferon-gamma) and anti-inflammatory (IL-4) cytokine responses. The third locus encodes a protein involved in the degradation of misfolded proteins within the endoplasmic reticulum, and its role, if any, in the clearance phenotype is unclear. CONCLUSIONS: The study showed significant association of three loci in the human genome with the ability of parasite to clear drug-resistant P. falciparum in samples taken from five countries distributed across sub-Saharan Africa. Both SNP rs2706384 and SNP1805015 have previously been reported to be associated with risk of malaria infection in African populations. The loci are involved in the Th1/Th2 balance, and the association of SNPs within these genes suggests a key role for antibody in the clearance of drug-resistant parasites. It is possible that patients able to clear drug-resistant infections have an enhanced ability to control parasite growth.

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.

Genome-wide and fine-resolution association analysis of malaria in West Africa.

We report a genome-wide association (GWA) study of severe malaria in The Gambia. The initial GWA scan included 2,500 children genotyped on the Affymetrix 500K GeneChip, and a replication study included 3,400 children. We used this to examine the performance of GWA methods in Africa. We found considerable population stratification, and also that signals of association at known malaria resistance loci were greatly attenuated owing to weak linkage disequilibrium (LD). To investigate possible solutions to the problem of low LD, we focused on the HbS locus, sequencing this region of the genome in 62 Gambian individuals and then using these data to conduct multipoint imputation in the GWA samples. This increased the signal of association, from P = 4 × 10(-7) to P = 4 × 10(-14), with the peak of the signal located precisely at the HbS causal variant. Our findings provide proof of principle that fine-resolution multipoint imputation, based on population-specific sequencing data, can substantially boost authentic GWA signals and enable fine mapping of causal variants in African populations.