Malaria is a major health burden in sub-Saharan African countries, including Mali. The disease is complex, with multiple genetic determinants influencing the observed variation in response to infection, progression, and severity. We assess the influence of sixty-four candidate loci, including the sickle cell polymorphism (HbS), on severe malaria in a case-control study consisting of over 900 individuals from Bamako, Mali. We confirm the known protective effects of the blood group O and the HbS AS genotype on life-threatening malaria. In addition, our analysis revealed a marginal susceptibility effect for the CD40 ligand (CD40L)+220C allele. The lack of statistical evidence for other candidates may demonstrate the need for large-scale genome-wide association studies in malaria to discover new polymorphisms. It also demonstrates the need for establishing the region-specific repertoire of functional variation in important genes, including the glucose-6-phosphatase deficiency gene, before embarking on focused genotyping.
Citation: Toure O, Konate S, Sissoko S, Niangaly A, Barry A, Sall AH, et al. (2012) Candidate Polymorphisms and Severe Malaria in a Malian Population. PLoS ONE 7(9): e43987. https://doi.org/10.1371/journal.pone.0043987
Editor: David Joseph Diemert, The George Washington University Medical Center, United States of America
Received: April 15, 2012; Accepted: July 26, 2012; Published: September 5, 2012
Copyright: © Toure et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: The MalariaGEN Project is supported by the Wellcome Trust (WT077383/Z/05/Z) and by the Foundation for the National Institutes of Health (566) as part of the Bill & Melinda Gates' Grand Challenges in Global Health Initiative. The Resource Centre for Genomic Epidemiology of Malaria is supported by the Wellcome Trust (090770/Z/09/Z). This research was supported by the Medical Research Council (G0600718; G0600230). The Wellcome Trust also provided core awards to the Wellcome Trust Centre for Human Genetics (075491/Z/04; 090532/Z/09/Z) and the Wellcome Trust Sanger Institute (077012/Z/05/Z). G0600230), the Wellcome Trust Biomedical ethics Enhancement Award (087285) and Strategic Award (096527). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Malaria is a life-threatening parasitic disease transmitted by mosquitoes. Despite the concerted and renewed efforts to control the disease, it still persists as a major health burden, being responsible for 655,000 deaths in 2010, mainly children in Sub-Sahara Africa . In Mali, there are over 800,000 recorded cases of malaria among its ∼14 million people every year, and it accounts for 17 percent of child deaths . Malaria is a complex disease with many genetic and environmental determinants influencing the observed variation in response to infection, progression and severity. Several factors are important for these different phenotypes observed, such as parasite genetic make-up, and host age, state of immunity and genetic background . It has been estimated that 25% of the total variation in mild and severe malaria in a Kenya cohort was explained by host genes . The different geographic distributions of sickle-cell disease, α thalassemia, glucose-6-phosphatase deficiency (G6PD), ovalocytosis, and the Duffy-negative blood group are examples of the general principle that different populations have evolved different genetic variants to protect against malaria (see , for a review). The most striking example is the beta-globin HBB gene, in which three different coding SNPs confer protection against malaria: Glu6Val (HbS), Glu6Lys (HbC), and Glu26Lys (HbE). The HbS allele is common in Africa but rare in Southeast Asia, whereas the opposite is true for the HbE allele. However, a more complex picture emerges at the local level, exemplified by the Dogon people of Mali, who have a much lower frequency of the HbS allele than do most other West African groups and instead have a high frequency of the HbC allele . Striking differences in response to malaria infection have also been observed among ethnic groups who live in the same geographical region. For example, it has been observed that the Fulani of Burkina Faso  and of Mali  have a significantly lower prevalence of malaria parasitaemia and fewer malaria clinical attacks, when compared to other ethnic groups living in neighbouring villages. In addition to the sickle polymorphism (HbS) , G6PD (reviewed in ), and ABO blood group , , a number of candidate polymorphisms have been proposed for the reduced risk of severe malaria. For example, these include genes that are relevant to immunity and inflammation such as the tumour necrosis factor (TNF, MHC class III region, reviewed in ), Toll-like receptors (TLR-4,9) , CD40 ligand (CD40L) , the interferon gamma (IFNG) (reviewed in , and the Nitric oxide synthase type 2 (NOS2A) genes (reviewed in . Here we investigate whether a number malaria candidate SNPs, including the HbC, HbS and ABO, are associated with severe malaria. Our study is the first to survey malaria candidate SNPs in a Malian population, and we seek to confirm genetic associations found in other studies. We consider a cohort of over 900 individuals recruited in Bamako, predominantly from the Bambara ethnicity, which is under-represented in other genetic epidemiological studies in Western Africa.
Participants, Materials and Methods
This study was approved by the Faculty of Medicine, Pharmacy and Dentistry (University of Bamako) Ethics Review Committee. All clinical and biological samples were collected and DNA was genotyped following approval by this committee. Written informed consent was obtained from the next of kin, carers or guardians on the behalf of the minors/children participants involved in this study.
Patient samples were collected as part of ongoing epidemiological studies of severe malaria at the Centre Hospitalier Universitaire Gabriel Toure, Bamako, Mali (malaria cases 541 (57.9%); healthy controls 393 (42.1%)). They had a median age of ∼3 years, and were predominantly from the Bambara ethnic group (53%) (see Table 1).
All cases were children admitted to hospital with evidence of P. falciparum on blood film and clinical features of uncomplicated and severe malaria , . Subjects were defined as having had cerebral malaria (CM) if their Blantyre coma score was less than or equal to 2 on presentation or early during admission. A second phenotypic subset of severe malarial anaemia (SMA) was defined as those subjects having had a haemoglobin concentration of less than 5 g/dl or a haematocrit less than 15%. Participants with co-existing severe or chronic medical conditions (e.g. bacterial pneumonia, kwashiorkor) unrelated to a severe malarial infection were excluded. Most cases had severe malaria (n = 458, 84.7%), but a minority had uncomplicated conditions (n = 83, 15.3%) (see Table 1). Controls were healthy individuals matched for age, ethnicity and residence to severe malaria cases (Table 1). For the purpose of analysis, we either excluded uncomplicated malaria cases or included them as part of the control group. Because of restrictions in sample size, we do not present an analysis on different ethnic groups or specific sub-clinical phenotypes.
Sample preparation and genotyping.
Genomic DNA samples underwent whole genome amplification through Primer Extension Pre-amplification (PEP) , before genotyping on a Sequenom MassArray genotyping platform , . Sixty-four malaria candidate SNPs were genotyped, including: Haemoglobin variants C (HbC, rs33930165) and S (HbS, rs334), plus two SNPs that allow an estimate of the ABO blood group . The rs8176719 derived allele results in a non-functional enzyme, and group O individuals are DD, while non-O Individuals are either II or ID. In addition, rs8176746 is involved in the enzyme's substrate selection and therefore defines either the A or B blood groups. The two Sequenom iPLEX reactions designed also included gender-typing SNPs. The selection of SNPs for genotyping was undertaken by the MalariaGEN Consortium, and were selected by interrogation of the literature and ongoing consortial experiments for evidence of association with severe malaria. Full details of polymorphisms can be found at www.malariagen.net, and a list of SNPs typed can also be found in Tables 2 and S1.
Genotypic deviations from Hardy-Weinberg equilibrium (HWE) were assessed using a chi-square statistical test. SNPs were excluded from analysis if they had at least 10% of genotype calls missing or there was significant deviation from HWE (p<0.001). Case-control association analysis using SNP alleles/genotypes was undertaken by logistic regression and included the covariates: ethnic group and the HbS polymorphism. In this approach we modelled the SNP of interest assuming several related genotypic mechanisms (additive, dominant, recessive, heterozygous advantage and general models) and reported the minimum p-value from these correlated tests. All analyses were performed using the R statistical package (http://www.r-project.org). Performing multiple statistical tests leads to inflation in the occurrence of false positives and using a permutation approach that accounted for correlation between markers and tests, we estimated a p-value cut-off of 0.002 to be statistically significant. Allele frequency differences were estimated using the Fst metric, with values potentially varying from zero (no population differentiation) to one (complete differentiation) .
Six SNPs were removed from the analysis because they were monomorphic (rs33950507, rs5743611, rs2814778), deviated from HWE in controls (rs2227478, rs2535611) or had high rates of missing genotype calls (rs1801274). Allelic-based tests revealed potential associations of HbS polymorphism (rs334, HBB gene), the O blood group (and its components rs8176746 and rs8176719), and rs1126535 (CD40L+220) with severe malaria (P<0.002) (Table 2). Figure 1 shows the minimum p-values from the genotypic tests applied to the autosomal SNPs, and confirms that the sickle cell (HbS) and ABO polymorphisms (rs8176746, rs8176719) are significantly associated with severe malaria. This genotypic analysis adjusting for the potentially confounding effects of ethnicity (and where appropriate HbS) supports the heterozygous advantage effect of the HbS-AS genotype (Odds ratio AS vs. AA: 0.03, P<6e-10), and the reduced risk in the O blood group (Odds ratio O vs. A/B/AB: 0.58, P = 0.0003) (Table 3). An increased risk from the CD40L+220-C allele was observed for males (Odds ratio C vs. A 2.12, P = 0.05) and females (Odds ratio additive C model 1.67, P = 0.03), and the pooled result was marginally non-significant (Odds ratio additive C model 1.79, P = 0.0045) (Table 3). The G6PD-202 polymorphism has been used as a molecular surrogate for the A- deficiency (see ,  for a review). We found no strong evidence of a G6PD association with severe malaria risk in males, females or overall (Tables 2 and 3, P>0.06); the direction of the odds ratios suggests those with the 202-A (A-) allele were at increased (rather than a decreased) risk of the disease. All our results were insensitive to the inclusion or exclusion of uncomplicated malaria cases in the control group. There were no major allele frequency differences between the Bambara and other ethnic groups (median Fst 0.001, only 3 Fst values greater than 0.01, maximum 0.015).
*Genotypic tests of dominant, recessive, general, heterozygous advantage, and additive models, adjusted for HbS and ethnicity; in this analysis controls include uncomplicated malaria cases; the dashed line represents a p-value of 0.002.
In our study, we set out to investigate the role of candidate malaria polymorphisms on severe malaria risk in a predominantly Bambara population in and around Bamako. To minimise errors we standardised procedures using case report forms, pre-defined definitions of severe malaria and sub-clinical phenotypes, and ensured all samples underwent genotyping on the same Sequenom MassArray platform with resulting low rates of missing data (<5%). In addition, all analyses were adjusted for ethnicity, minimising the confounding effects and potential false positives arising from population stratification.
Data analysis confirmed the known (∼90%) reduced severe malaria risk from the sickle cell AS genotype . The low frequency of the S allele in the controls (∼3.8%) is in keeping with other populations (see http://www.map.ox.ac.uk/) in West (Burkina Faso 5.2%, Cameroon 6.5%, Gambia 7.6%, Ghana 6.5%) and East (Kenya 6.4%, Malawi 2.7%, Tanzania 7.8%) Africa. The frequency of the HbC allele was 5% in cases and 6% in controls, both greater than the HbS allele, but there was no strong evidence of association (P>0.3). The higher frequency of the HbC allele has been observed in other West African populations .
Our analysis also confirms the known protective effects of the blood group O on life threatening malaria, which is thought to act on malaria pathogenesis through the mechanism of reduced P. falciparum rosetting , . An insight into an underlying molecular mechanism could lead to the development of a new anti-malarial therapy. Our inability to detect associations found in other studies may be due to previously reported false-positives, and methodological issues such as variation in phenotype definition, choice of controls, village surveys versus hospital-based studies, possible heterogeneity in the parasite population, immune status of subjects, and study location. There may also be issues with sample size (statistical power). At the present sample size, we have 90% §power to detect a dominant odds ratio effect of 1.5 with a minor allele frequency of 0.20 and type I error of 5%. Reducing the odds ratio to 1.4 or MAF to 0.13 would lead to 80% power. It is clear that much larger studies are required to detect more modest effects, especially if relying on markers in LD with a causal untyped polymorphism. In this setting, dense SNP genome-wide association strategies are required to discover new candidate genes.
Our analysis revealed a marginal susceptibility effect for the CD40 ligand (CD40L)+220C allele. CD40L is a glycoprotein involved in B cell proliferation, antigen presenting cell activation, and Ig class switching, and therefore important in the immune response to infection . Previous work in the Gambia found the CD40L+220C allele had a non-significant susceptibility effect, and instead identified the genotyped CD40L–726 polymorphism with a significant reduction in risk for severe malaria in males-hemizygous . Similarly, we expected to see a reduced malaria risk with G6PD-202A (A- deficiency), as shown in another Malian population  with similar allele frequencies in controls to our study. These discrepancies in CD40L and G6PD results may be due to allelic heterogeneity. For example, it has been shown that the G6PD-202 may not be a good marker of A- deficiency, and other polymorphisms are required to confirm the protective effect . In addition, discrepancies may arise due to differences in linkage disequilibrium (LD) patterns between populations. For example, it is known there are at least five classical haplotypes surrounding the sickle polymorphism in the HBB gene, and the resulting differences in LD can make it difficult to localise HbS using indirect associations . In addition, some functional polymorphisms may be distal from candidate genes and polymorphisms genotyped. For example, it has been suggested that the causal polymorphisms regulating TNF and LTA response may be some distance from the genes , .
In conclusion, our work reinforces in a Malian (mainly Bambara) population the importance of the sickle cell polymorphism and ABO blood group on severe malaria susceptibility. It may also demonstrate the need for establishing the region-specific repertoire of functional variation in important genes such as G6PD, before embarking on focused genotyping. Proposed large-scale genomewide association studies and resequencing of important genes in a number of Malian (and other African) populations is likely to generate insights into how P. falciparum has shaped our genome. These approaches could expose new candidate protective polymorphisms, resulting in functional work to understand underlying molecular mechanisms, leading to the development of therapies and vaccines for malaria control.
We thank the participants and Bamako communities who made this study possible; we thank Professor Mamadou Marouf Keita and his team of healthcare workers at the Centre Hospitalier Universitaire Gabriel Toure who assisted with this work.
Conceived and designed the experiments: OT KR TC MT OD. Performed the experiments: OT SK SS AN AB AS ED BP. Analyzed the data: TC NS SC OT. Contributed reagents/materials/analysis tools: OD MalariaGen TC. Wrote the paper: TC OT OD MT KR SC.
- 1. World Health Organisation (2011) World Malaria Report 2011. Available: http://who.int/malaria/world_malaria_report_2011/en/index.html. Accessed 2012 Aug 14.
- 2. Mackinnon MJ, Mwangi TW, Snow RW, Marsh K, Williams TN (2005) Heritability of malaria in Africa. PLoS Med 2: e340.
- 3. Campino S, Kwiatkowski D, Dessein A (2006) Mendelian and complex genetics of susceptibility and resistance to parasitic infections. Semin Immunol 18: 411–22.
- 4. Agarwal A, Guindo A, Cissoko Y, Taylor JG, Coulibaly D, et al. (2000) Hemoglobin C associated with protection from severe malaria in the Dogon of Mali, a West African population with a low prevalence of hemoglobin S. Blood 96: 2358–63.
- 5. Modiano D, Petrarca V, Sirima BS, Bosman A, Nebie I, et al. (1995) Plasmodium falciparum malaria in sympatric ethnic groups of Burkina Faso, west Africa. Parassitologia 37: 255–9.
- 6. Dolo A, Modiano D, Maiga B, Daou M, Dolo G, et al. (2005) Difference in susceptibility to malaria between two sympatric ethnic groups in Mali. Am J Trop Med Hyg 72: 243–8.
- 7. Jallow M, Teo YY, Small KS, Rockett KA, Deloukas P, et al. (2009) Genome-wide and fine-resolution association analysis of malaria in West Africa. Nat Genet 41(6): 657–665.
- 8. Guindo A, Fairhurst RM, Doumbo OK, Wellems TE, Diallo DA (2007) X-linked G6PD deficiency protects hemizygous males but not heterozygous females against severe malaria. PLoS Med 4: e66.
- 9. Rowe JA, Handel IG, Thera MA, Deans AM, Lyke KE, et al. (2007) Blood group O protects against severe Plasmodium falciparum malaria through the mechanism of reduced rosetting. . Proc Natl Acad Sci U S A 104: 17471–17476.
- 10. Fry AE, Griffiths MJ, Auburn S, Diakite M, Forton JT, et al. (2008) Common variation in the ABO glycosyltransferase is associated with susceptibility to severe Plasmodium falciparum malaria. Hum Mol Genet 17: 567–76.
- 11. Clark TG, Diakite M, Auburn S, Campino S, Fry AE (2009) Tumor necrosis factor and lymphotoxin-alpha polymorphisms and severe malaria in African populations. J Infect Dis 199: 569–75.
- 12. Mockenhaupt FP, Hamann L, von Gaertner C, Bedu-Addo G, von Kleinsorgen C (2006) Common polymorphisms of toll-like receptors 4 and 9 are associated with the clinical manifestation of malaria during pregnancy. J Infect Dis 194: 184–8.
- 13. Sabeti P, Usen S, Farhadian S, Jallow M, Doherty T, et al. (2002) CD40L association with protection from severe malaria. Genes Immun 5: 286–291.
- 14. Stevenson MM, Riley EM (2004) Innate immunity to malaria. Nat Rev Immunol 4: 169–80.
- 15. Clark IA, Rockett KA (1996) Nitric oxide and parasitic disease. Adv Parasitol 37: 1–56.
- 16. Marsh K, Forster D, Waruiru C, Mwangi I, Winstanley M, et al. (1995) Indicators of life-threatening malaria in African children. N Engl J Med 332: 1399–404.
- 17. World Health Organisation (1990) Severe and complicated malaria. World Health Organization, Division of Control of Tropical Diseases. Trans R Soc Trop Med Hyg 84 Suppl 2 1–65.
- 18. Zhang L, Cui X, Schmitt K, Hubert R, Navidi W, et al. (1992) Whole genome amplification from a single cell: implications for genetic analysis. Proc Natl Acad Sci U S A 89: 5847–51.
- 19. Wilson JN, Rockett K, Jallow M, Pinder M, Sisay-Joof F, et al. (2005) Analysis of IL10 haplotypic associations with severe malaria. Genes Immun 6: 462–6.
- 20. Ross P, Hall L, Smirnov I, Haff L (1998) High level multiplex genotyping by MALDI-TOF mass spectrometry. Nat Biotechnol 16: 1347–51.
- 21. Clark TG, Fry A, Auburn S, Campino S, Diakite M, et al. (2009) G6PD deficiency and severe malaria: unrecognized allelic heterogeneity is confounding association studies in Africa. Eur J Human Genetics 17: 1080–5.
- 22. Weir BS, Cockerham CC (1984) Estimating F-statistics for the analysis of population structure. Evolution 38 (6) 1358–1370.
- 23. Diakite M, Clark TG, Auburn S, Campino S, Fry AE, et al. (2009) A genetic association study in the Gambia using tagging polymorphisms in the major histocompatibility complex (MHC) class III region implicates a BAT2 polymorphism in severe malaria susceptibility. Human Genetics 125: 105–9.