Acute seizures are common in children admitted with falciparum malaria to hospitals in sub-Saharan Africa (Taylor et al., 2006; Idro et al., 2008). Some malarial seizures are focal, repetitive, or prolonged (complex), which are associated with the development of subsequent neurocognitive impairments or epilepsy (Carter et al., 2005; Birbeck et al., 2010). It is not clear if these seizures are febrile seizures or acute symptomatic seizures caused by the sequestration of the parasites within the brain (Idro et al., 2005). Furthermore, it is difficult to attribute seizures to malaria in a malaria-endemic area, since a proportion of children will have sequestered parasites and be asymptomatic, and malaria is the most common cause of fever in children aged 6 months to 6 years (Idro et al., 2005). We have determined that most seizures in children admitted to hospital with malaria parasitemia are attributable to malaria using logistic regression techniques (Kariuki et al., 2011), and that children admitted with seizures are more likely to have relatives with seizure disorders than controls (Versteeg et al., 2003).
There is a strong genetic predisposition for febrile seizures, investigated through twin studies, family history, and a number of genetic polymorphisms (DNA sequence variation occurring when a single nucleotide—A, T, C, or G—n the genome differs between members of a biologic species; Rich et al., 1987; Kugler & Johnson, 1998; Kjeldsen et al., 2002). The risk of febrile seizures has been associated with interleukin-1β (IL-1B; Virta et al., 2002; Kira et al., 2005) and interleukin-1 receptor antagonist (IL-1RA; Tsai, 1987). Furthermore, a number of polymorphisms associated with falciparum malaria infections have been identified, which may be protective for example, sickle cell trait (Williams et al., 2005), glucose-6-phosphate dehydrogenase (G6PD; Clark et al., 2009), interadhesion molecule (ICAM-1; Kun et al., 1999), and cluster of differentiation ligand (CD-40LG; Sabeti et al., 2002), or susceptible, for example, complement receptor (CR-1; Nagayasu et al., 2001) and IL-10, -12, and -18 (Chaisavaneeyakorn et al., 2003; Wilson et al., 2005). However, the role of these genes in the genesis of seizures has not been examined.
We examined a number of different polymorphisms associated with malaria-associated seizures (MAS), and the phenotypes of MAS using 4,472 children admitted to hospitals with falciparum malaria in four African countries (Ghana, Kenya, Malawi, and Tanzania) as part of a case–control study for MalariaGEN (Malaria Genetic Epidemiological Network) consortial project (MalariaGEN, 2008). The malaria candidate polymorphisms in the MalariaGEN consortial project (Table S1) were chosen for this analysis because they reflect a pathogenic mechanism for severe malaria that could be involved in the genesis of acute seizures in falciparum malaria. Malaria pathogenesis is considered in the context of malaria with impaired consciousness and/or respiratory distress (Marsh et al., 1996), although the former (of which cerebral malaria is the most important severe neurologic complication) is important as seizures occur in >80% of the cases (Idro et al., 2005).
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This is the first study to investigate the effect of candidate genes for malaria infection on the risk of acute seizures across multiple sites in Africa. Using established definitions for seizure phenotypes (Table 1), our data show that a significant proportion (>70%) of children admitted to hospitals in sub-Saharan Africa with severe malaria exhibit seizures with complex features (prolonged, focal, or repetitive), but the proportions of the different phenotypes vary between sites. A number of different polymorphisms (including the X-chromosome) were associated with the risk of the main phenotypes of MAS at each site, and in a pooled analysis. Polymorphisms of four genes were identified as significant in most of the subgroups and sites (CR1-rs1704660, IL10-rs1800896, and EMR1-rs373533/rs461645 and CD40-rs3092945 [females]), although only CR1 showed an overall significant association across the sites in the prolonged and repetitive MAS phenotypes. Other polymorphisms did show positive association but mostly for one phenotype and site (Table 3). Of the positive association polymorphisms several are thought to code for molecules that could have a putative role in the pathogenesis of seizures in falciparum malaria.
The classification of seizures is largely based on phenotypes found in febrile seizures (Baram & Shinnar, 2002), but some cases may have been misclassified, since generalized seizures may have had a focal origin with rapid secondary generalization (Gwer et al., 2012). We were not able to determine relatedness of the study participants, which may affect the associations, but adjusting for ethnicity did in part account for this. Electroencephalography and neuroimaging were not routinely performed at the time of the study due to logistic reasons. The strength of our study lies in the large sample size from combining studies across four countries, which allowed us to use a control group that comprised the severe malaria cases without seizures, thus ensuring that any associations identified represent the risk of developing acute seizures in malaria. The genotypic tests p-values are reported as they were generated from the analyzing software, so are easier to interpret.
Polymorphisms demonstrated heterogeneities in associations with MAS across the sites. These heterogeneities may be ascribed to sample size differences and the resulting power (Muheza had the smallest samples), possible differences in documenting the three phenotypes of complex MAS, haplotypic (polymorphisms on one or several loci that may be associated) differences between populations, and/or the effects of population structure. We tried to minimize the latter by adjusting for ethnicity within sites. Some of the heterogeneous associations in the sites may be due to linkage disequilibrium. Because we adjusted for multiple testing, such site-specific associations could be due to strong linkage disequilibrium with actual causal polymorphisms not investigated in this study. The finding that a gene was protective in one site and increased risk in another can be explained by the different inheritance models in the affected sites, or could suggest that the associations are related to a gene region rather than the effect of the particular polymorphism.
In these associations, we studied polymorphisms that were genotyped as part of MalariaGEN consortium project (MalariaGEN, 2008), and could not investigate some polymorphisms associated with febrile seizures such as the six susceptibility febrile seizure loci (FEB)1–6, voltage-gated sodium channel genes (SCN1A, SCN1B, or SCN2A), γ-amino-butyric acids genes (GABA(A) or GABRG2) (Nakayama & Arinami, 2006). We did, however, include polymorphisms of the interleukin family (IL-1A, 1L-1B, 1L-4, 1L-20RA), some of which have been previously investigated in febrile seizures studies (Tsai, 1987; Virta et al., 2002; Kira et al., 2005; Ishizaki et al., 2009).
Some polymorphisms had stronger associations with acute seizures with complex phenotypes, suggesting that these three phenotypes may provide insights into the epileptogenesis of malaria. The finding that specific polymorphisms were replicated across the different complex phenotypes supports the role of these genes in the epileptogenesis of malaria. The complex phenotypes are particularly associated with malaria (Kariuki et al., 2011) and are also important risk factors for development of epilepsy following severe malaria (Birbeck et al., 2010). Those polymorphisms that are found in more than one site are likely to be linked to genes that may play a significant role in the pathogenesis of acute seizures in these children.
The biologic functions of some polymorphisms have not been fully investigated, and can only be speculated. EMR1 is sometimes elevated in placental malaria (Muehlenbachs et al., 2007; Sevastianova et al., 2008), which is characterized by sequestration of parasites, similar to that found in the brain. EMR1 is a membrane receptor that also activates G protein receptor subunit alpha (GNAS) (Auburn et al., 2008), whose subclass C receptors bind glutamate and GABA, the two important neurotransmitters of epileptogenesis. GNAS did not reach the 0.009 significance level in this study but reached the 0.05 level. In addition, EMR1 is the human homologue of F4/80 in mice that defines macrophages, whose elevation in malaria infection precedes leucocyte sequestration into the brain (Wozencraft et al., 1984).
Many of the polymorphisms genotyped in this study have previous published associations with severe malaria, and we have analyzed them in the context of association with seizures. CR1 is postulated to be involved in the adhesion of P. falciparum infected erythrocytes to uninfected erythrocytes (rosetting; Nagayasu et al., 2001; Idro et al., 2005) and is therefore thought to play a role in the pathogenesis of seizures in severe malaria. Polymorphisms of the ABO blood system are associated with rosetting of erythrocytes among people of blood types A and B (Cserti & Dzik, 2007), resulting in impaired tissue perfusion that could increase the risk for seizures. Similarly, the association between ICAM1 polymorphisms seizures with severe malaria (Kun et al., 1999) could be explained by the binding of ICAM1 to P. falciparum–infected erythrocytes onto brain microvasculature (Berendt et al., 1992; Kun et al., 1999) causing sequestration. CD36 facilitates this sequestration (Cserti & Dzik, 2007). Interleukins are usually increased in falciparum malaria infections, and may directly precipitate seizures because they are inflammatory and pyrogenic in nature (Haysa et al., 1999; Chaisavaneeyakorn et al., 2003; Wilson et al., 2005). G6PD enhances parasite clearance by phagocytosis, a process that can culminate in release of inflammatory molecules often associated with seizures (Sabeti et al., 2002; Wilson et al., 2005). Activation of microglial CD40 results in inflammation-induced seizures (Sun et al., 2008) and underpins the association between this polymorphism and MAS in coma/cerebral malaria.
Our findings have demonstrated that children admitted with falciparum malaria may have a genetic predisposition to acute seizures, but the polymorphisms associated with seizures in malaria vary with the phenotype and sites across Africa. These polymorphisms were particularly associated with the three complex phenotypes of MAS, which could also be important in epileptogenesis of severe malaria. Studies from other malaria-endemic settings are required to confirm these findings, and genome-wide association studies are required to study if other seizures-specific genes determine the risk of acute seizures.
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We would like to thank the all the study participants and their families for their help with this study. Carolyne Ndila, George Mtove, and Tony Enimil were invaluable in assisting with the clinical database and/or the clinical notes. This study was supported by Wellcome Trust through a Senior Clinical Fellowship awarded to CRJCN (083744) and a Research Training Fellowship (099782/Z/12/Z) awarded to SMK. TNW, HR, and TET are MalariaGEN principal investigators and SMK is a MalariaGEN data fellow. TNW is funded by the Wellcome Trust and the European Union EVIMalR Network 7 Programme.
The MalariaGEN Project is supported by the Wellcome Trust (WT077383/Z/05/Z) and the Bill and Melinda Gates Foundation through the Foundations of the National Institutes of Health (566) as part of the Grand Challenges in Global Health Initiative. This research was supported by the Medical Research Council (G0600230). The Wellcome Trust also provides core awards to Wellcome Trust Centre for Human Genetics (075491/Z/04; 090532/Z/09/Z) and the Wellcome Trust Sanger Institute (077012/Z/05/Z).
This work was presented as a poster in the Genomics of Malaria Conference, Cambridge, United Kingdom in June 2010 and in the 29th International Epilepsy Congress, Rome, Italy in August, 2011.