Fatou Sisay-Joof

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.

Genome-wide association analyses identifies a susceptibility locus for tuberculosis on chromosome 18q11.2

We combined two tuberculosis genome-wide association studies from Ghana and The Gambia with subsequent replication in a combined 11,425 individuals. rs4331426, located in a gene-poor region on chromosome 18q11.2, was associated with disease (combined P = 6.8 × 10−9, odds ratio = 1.19, 95% CI = 1.13–1.27). Our study demonstrates that genome-wide association studies can identify new susceptibility loci for infectious diseases, even in African populations, in which levels of linkage disequilibrium are particularly low.

Allelic heterogeneity of G6PD deficiency in West Africa and severe malaria susceptibility

Several lines of evidence link glucose-6-phosphate dehydrogenase (G6PD) deficiency to protection from severe malaria. Early reports suggested most G6PD deficiency in sub-Saharan Africa was because of the 202A/376G G6PD A− allele, and recent association studies of G6PD deficiency have employed genotyping as a convenient way to determine enzyme status. However, further work has suggested that other G6PD deficiency alleles are relatively common in some regions of West Africa. To investigate the consequences of unrecognized allelic heterogeneity on association studies, in particular studies of G6PD deficiency and malaria, we carried out a case–control analysis of 2488 Gambian children with severe malaria and 3875 controls. No significant association was found between severe malaria and the 202A/376G G6PD A− allele when analyzed alone, but pooling 202A/376G with other deficiency alleles revealed the signal of protection (male odds ratio (OR) 0.77, 95% CI 0.62–0.95, P=0.016; female OR 0.71, 95% CI 0.56–0.89, P=0.004). We have identified the 968C mutation as the most common G6PD A− allele in The Gambia. Our results highlight some of the consequences of allelic heterogeneity, particularly the increased type I error. They also suggest that G6PD-deficient male hemizygotes and female heterozygotes are protected from severe malaria.

Lack of Association of Interferon Regulatory Factor 1 with Severe Malaria in Affected Child-Parental Trio Studies across Three African Populations

Interferon Regulatory Factor 1 (IRF-1) is a member of the IRF family of transcription factors, which have key and diverse roles in the gene-regulatory networks of the immune system. IRF-1 has been described as a critical mediator of IFN-gamma signalling and as the major player in driving TH1 type responses. It is therefore likely to be crucial in both innate and adaptive responses against intracellular pathogens such as Plasmodium falciparum. Polymorphisms at the human IRF1 locus have been previously found to be associated with the ability to control P. falciparum infection in populations naturally exposed to malaria. In order to test whether genetic variation at the IRF1 locus also affects the risk of developing severe malaria, we performed a family-based test of association for 18 Single Nucleotide Polymorphisms (SNPs) across the gene in three African populations, using genotype data from 961 trios consisting of one affected child and his/her two parents (555 from The Gambia, 204 from Kenya and 202 from Malawi). No significant association with severe malaria or severe malaria subphenotypes (cerebral malaria and severe malaria anaemia) was observed for any of the SNPs/haplotypes tested in any of the study populations. Our results offer no evidence that the molecular pathways regulated by the transcription factor IRF-1 are involved in the immune-based pathogenesis of severe malaria.

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-α 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-1Kilifi’) 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-1Kilifi 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-1Kilifi to severe malaria susceptibility.

TLR9 polymorphisms in African populations: no association with severe malaria, but evidence of cis-variants acting on gene expression

BackgroundDuring malaria infection the Toll-like receptor 9 (TLR9) is activated through induction with plasmodium DNA or another malaria motif not yet identified. Although TLR9 activation by malaria parasites is well reported, the implication to the susceptibility to severe malaria is not clear. The aim of this study was to assess the contribution of genetic variation at TLR9 to severe malaria.MethodsThis study explores the contribution of TLR9 genetic variants to severe malaria using two approaches. First, an association study of four common single nucleotide polymorphisms was performed on both family- and population-based studies from Malawian and Gambian populations (n>6000 individual). Subsequently, it was assessed whether TLR9 expression is affected by cis-acting variants and if these variants could be mapped. For this work, an allele specific expression (ASE) assay on a panel of HapMap cell lines was carried out.ResultsNo convincing association was found with polymorphisms in TLR9 for malaria severity, in either Gambian or Malawian populations, using both case-control and family based study designs. Using an allele specific expression assay it was observed that TLR9 expression is affected by cis-acting variants, these results were replicated in a second experiment using biological replicates.ConclusionBy using the largest cohorts analysed to date, as well as a standardized phenotype definition and study design, no association of TLR9 genetic variants with severe malaria was found. This analysis considered all common variants in the region, but it is remains possible that there are rare variants with association signals. This report also shows that TLR9 expression is potentially modulated through cis-regulatory variants, which may lead to differential inflammatory responses to infection between individuals.

Clinical Features of Severe Malaria Associated with Death: A 13-Year Observational Study in The Gambia

Background Severe malaria (SM) is a major cause of death in sub-Saharan Africa. Identification of both specific and sensitive clinical features to predict death is needed to improve clinical management. Methods A 13-year observational study was conducted from 1997 through 2009 of 2,901 children with SM enrolled at the Royal Victoria Teaching Hospital in The Gambia to identify sensitive and specific predictors of poor outcome in Gambian children with severe malaria between the ages 4 months to 14 years. We have measured the sensitivity and specificity of clinical features that predict death or development of neurological sequelae. Findings Impaired consciousness (odds ratio {OR} 4.4 [95% confidence interval {CI}, 2.7–7.3]), respiratory distress (OR 2.4 [95%CI, 1.7–3.2]), hypoglycemia (OR 1.7 [95%CI, 1.2–2.3]), jaundice (OR 1.9 [95%CI, 1.2–2.9]) and renal failure (OR 11.1 [95%CI, 3.3–36.5]) were independently associated with death in children with SM. The clinical features that showed the highest sensitivity and specificity to predict death were respiratory distress (area under the curve 0.63 [95%CI, 0.60–0.65]) and impaired consciousness (AUC 0.61[95%CI, 0.59–0.63]), which were comparable to the ability of hyperlactatemia (blood lactate>5 mM) to predict death (AUC 0.64 [95%CI, 0.55–0.72]). A Blantyre coma score (BCS) of 2 or less had a sensitivity of 74% and specificity of 67% to predict death (AUC 0.70 [95% C.I. 0.68–0.72]), and sensitivity and specificity of 74% and 69%, respectively to predict development of neurological sequelae (AUC 0.72 [95% CI, 0.67–0.76]).The specificity of this BCS threshold to identify children at risk of dying improved in children less than 3 years of age (AUC 0.74, [95% C.I 0.71–0.76]). Conclusion The BCS is a quantitative predictor of death. A BCS of 2 or less is the most sensitive and specific clinical feature to predict death or development of neurological sequelae in children with SM.

Further Evidence Supporting a Role for Gs Signal Transduction in Severe Malaria Pathogenesis

With the functional demonstration of a role in erythrocyte invasion by Plasmodium falciparum parasites, implications in the aetiology of common conditions that prevail in individuals of African origin, and a wealth of pharmacological knowledge, the stimulatory G protein (Gs) signal transduction pathway presents an exciting target for anti-malarial drug intervention. Having previously demonstrated a role for the G-alpha-s gene, GNAS, in severe malaria disease, we sought to identify other important components of the Gs pathway. Using meta-analysis across case-control and family trio (affected child and parental controls) studies of severe malaria from The Gambia and Malawi, we sought evidence of association in six Gs pathway candidate genes: adenosine receptor 2A (ADORA2A) and 2B (ADORA2B), beta-adrenergic receptor kinase 1 (ADRBK1), adenylyl cyclase 9 (ADCY9), G protein beta subunit 3 (GNB3), and regulator of G protein signalling 2 (RGS2). Our study amassed a total of 2278 cases and 2364 controls. Allele-based models of association were investigated in all genes, and genotype and haplotype-based models were investigated where significant allelic associations were identified. Although no significant associations were observed in the other genes, several were identified in ADORA2A. The most significant association was observed at the rs9624472 locus, where the G allele (∼20% frequency) appeared to confer enhanced risk to severe malaria [OR = 1.22 (1.09–1.37); P = 0.001]. Further investigation of the ADORA2A gene region is required to validate the associations identified here, and to identify and functionally characterize the responsible causal variant(s). Our results provide further evidence supporting a role of the Gs signal transduction pathway in the regulation of severe malaria, and request further exploration of this pathway in future studies.